Quest Test Selection

Diagnostics and Interpretation

Nichols 2008
Institute

Nichols Institute
 Quest Test Application
Diagnostics and Interpretation
Nichols 2008
Institute

Nichols Institute
 3

Contents

Section 1. Cardiovascular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2 Markers of Lipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3 Non-Lipid Markers of Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Section 2. Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.1 Cystatin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2 Microalbumin, Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.3 Microalbumin, Intact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Section 3. Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.2 Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.3 Platelet Immune Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4 Thrombocytopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5 Thrombophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.6 von Willebrand Comprehensive Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Section 4. Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.1 Calcium and Bone Metabolism (Including Osteoporosis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2 Diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3 Polycystic Ovary Syndrome (PCOS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.4 Thyroid Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Section 5. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2 Biochemical Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.3 Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
5.4 Molecular Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
5.5 Oncology-Related Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.6 Developmental Delay/Mental Retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Section 6. Hematology/Oncology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

6.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
6.2 Bladder Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
6.3 Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
6.4 Cervical Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
6.5 Gastrointestinal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.6 Hematopoietic/Lymphoid Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.7 Hepatocellular Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
6.8 Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
6.9 Ovarian Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
6.10 Paroxysmal Nocturnal Hemoglobinuria (PNH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
6.11 Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
6.12 Tumor Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4

Contents

Section 7. Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

7.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
7.2 Allergic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
7.3 Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
7.4 Gastrointestinal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
7.5 Hematologic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
7.6 HLA and Transplantation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
7.7 Immunodeficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
7.8 Neurologic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
7.9 Rheumatic and Related Systemic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Section 8. Infectious Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

8.1 Available Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
8.2 Diarrhea/Gastrointestinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
8.3 Hepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
8.4 HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
8.5 Immunocompromised . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
8.6 Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
8.7 Respiratory Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
8.8 STI/Genitourinary Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
8.9 Vector-borne Diseases and Travel Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

Section 9. Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

9.1 Drug Half-Life, Steady State, and Recommended Sample Collection Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
9.2 Sirolimus (Rapamycin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
9.3 Trace Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
 5

Section 1 Cardiovascular Tests

1.1 Available Tests

Lipid Markers Clinical Application
Apolipoprotein A1 Assess CVD risk and diagnose dyslipidemia
Apolipoprotein B Assess CVD risk and diagnose dyslipidemia
Apolipoprotein A1 & B Assess CVD risk and diagnose dyslipidemia
Cholesterol, Direct LDL Assess CVD risk and monitor therapy when triglyceride level is >400 mg/dL
Cholesterol, Pleural Fluid Differentiate between transudate (CHF) and exudate
Cholesterol, Total Assess CVD risk, predict outcome and therapeutic benefit, monitor therapy
HDL Cholesterol Assess CVD risk, predict outcome and therapeutic benefit, monitor therapy
HDL Cholesterol Subclasses Assess CVD risk
Lipid Panel Assess CVD risk
Includes cholesterol, HDL, cholesterol/HDL ratio, LDL (calculated),
and triglycerides.
Lipoprotein (a) [ie, Lp(a)] Assess CVD risk
Lipoprotein Fractionation, Ion Mobility with Reflex to Direct LDL Assess risk of premature CVD
Lipoprotein Fractionation, Ultracentrifugation Comprehensive CVD risk assessment and diagnosis
Metabolic Syndrome Panel Diagnose metabolic syndrome
Includes glucose, triglycerides, and HDL cholesterol.
Phospholipids1 Diagnose and manage lipid metabolism disorders
Triglycerides Assess CVD risk
Triglycerides, Pleural Fluid Differentiation of transudate (CHF) and exudate
VAP™’ Cholesterol Test Comprehensive CVD risk assessment and diagnosis
Includes total cholesterol, LDL-C, HDL-C, triglycerides, LDL pattern size,
HDL subfractions (HDL2 and HDL3), Lp(a), IDL-C, and VLDL.

Hypercoagulation Markers Clinical Application

Activated Partial Thromboplastin Time Assess risk of thrombosis, monitor patients on heparin therapy, and
diagnose patients with hypercoagulable states or bleeding diathesis
Activated Protein C-Resistance Assess risk of CVD, venous thromboembolic disease, and cerebrovascular
disease
C-Glycoprotein I Antibody (IgA) Antiphospholipid antibody screen for acquired thrombophilia
C-Glycoprotein I Antibody (IgG) Antiphospholipid antibody screen for acquired thrombophilia
C-Glycoprotein I Antibody (IgM) Antiphospholipid antibody screen for acquired thrombophilia
C-Glycoprotein I Antibodies (IgA, IgG, IgM) Antiphospholipid antibody screen for acquired thrombophilia
Cardiolipin Antibody (IgA) Assess risk of CVD, thromboembolic disease, and cerebrovascular disease
Cardiolipin Antibody (IgG) Assess risk of CVD, thromboembolic disease, and cerebrovascular disease
Cardiolipin Antibody (IgM) Assess risk of CVD, thromboembolic disease, and cerebrovascular disease
Cardiolipin Antibody Screen w/ Reflex to IgG and IgM Assess risk of CVD, thromboembolic disease, and cerebrovascular disease
Cardiolipin Antibody Screen w/ Reflex to IgG, IgA, IgM Assess risk of CVD, thromboembolic disease, and cerebrovascular disease
D-Dimer Diagnose (rule out) deep vein thrombosis; monitor thrombolytic therapy
Factor V (Leiden) Mutation Analysis2 Assess risk of CVD, venous thromboembolic disease, and cerebrovascular
disease
Factor V HR2 Allele DNA Mutation Analysis2 Assess risk of venous thromboembolic disease in Factor V (Leiden) carriers
Factor V (Leiden) Mutation Analysis w/Reflex to HR2 Mutation Analysis2 Assess risk of CVD, venous thromboembolic disease, and cerebrovascular
disease
Factor VIII Activity, Clotting Assess risk of hereditary thrombophilia
Fibrinogen, Clauss Assess risk of CVD, CVD-related mortality, and cerebrovascular disease
(of limited predictive value for individual patients)
Fibrinogen, Quantitative, Nephelometry Assess risk of CVD, CVD-related mortality, and cerebrovascular disease
(of limited predictive value for individual patients)
Heparin, Low Molecular Weight (Xa Inhibition) Monitor low molecular weight heparin (LMWH) therapy
Heparin, Unfractionated (Xa Inhibition) Monitor heparin therapy
Heparin-Induced Platelet Antibody Diagnose heparin-induced thrombocytopenia
Homocysteine (Cardiovascular), Serum Assess CVD risk
Homocysteine, Total, Urine Assess CVD risk
Human Platelet Antigen 1 Genotype Assess risk of aspirin resistance
Lupus Anticoagulant Evaluation with Reflexes Diagnose acquired arterial or venous thrombophilia
Includes lupus anticoagulant, hexagonal phase neutralization, dRVVT screen,
and phospholipid neutralization.
Methylenetetrahydrofolate Reductase (MTHFR), DNA Mutation Analysis2 Assess risk of CVD and stroke; identify individuals at risk for
hyperhomocysteinemia
6

Section 1 Cardiovascular Tests

Plasminogen Activator Inhibitor (PAI-1) Activity Assess risk of coronary vascular disease
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism2 Assess thrombotic risk
Plasminogen, Activity Diagnose a rare hereditary thrombophilia
Protein C Activity Diagnose hereditary thrombophilia
Protein C Activity with Reflex to Protein C Antigen Diagnose hereditary thrombophilia
Protein C Activity and Antigen Diagnose hereditary thrombophilia
Protein C Antigen Diagnose hereditary thrombophilia
Protein C and S Activity with Reflex to Protein C and/or S Antigen Diagnose hereditary thrombophilia
Protein S Activity Diagnose hereditary thrombophilia
Protein S Activity with Reflex to Protein S Antigen Diagnose hereditary thrombophilia
Protein S Antigen and Protein S, Free Diagnose hereditary thrombophilia
Protein S, Free Diagnose hereditary thrombophilia
Protein S, Total Antigen Diagnose hereditary thrombophilia
Protein S Panel Diagnose hereditary thrombophilia
Includes protein S activity, free and total protein S, and C4 binding protein.
Prothrombin (Factor II) 20210GmA Mutation Analysis2 Diagnose hereditary thrombophilia
Reptilase Clotting Time Assess thrombophilia risk through dysfibrinogenemia
Thrombin Clotting Time Assess thrombophilia risk through dysfibrinogenemia
Thrombophilia Mutation Analysis with Reflex to HR2 Mutation Analysis2 Assess risk of CVD, venous thromboembolic disease, and cerebrovascular
Includes mutation analysis for factor V (Leiden), prothrombin (factor II) 20210GmA, disease
and the factor V HR2 allele.
Tissue Plasminogen Activator (TPA), EIA Assess thrombophilia risk; monitor fibrinolytic therapy

Inflammatory Markers Clinical Application

Cardio CRP® (high-sensitivity C-reactive protein) Determine relative risk of first cardiovascular event in asymptomatic
individuals
Lp-PLA2 (PLAC™’) Assess coronary heart disease risk and stroke risk

Endocrine/Metabolic Markers Clinical Application

Aldosterone, 24-Hour Urine Differential diagnosis of hypertension
Aldosterone, LC/MS/MS, Serum Differential diagnosis of hypertension
Aldosterone, Response to ACTH Stimulation, LC/MS/MS Differential diagnosis of hypertension
Angiotensin Converting Enzyme (ACE) Differential diagnosis of hypertension
Angiotensin Converting Enzyme (ACE) Polymorphism (Insertion/Deletion)2 Assess risk of CVD and restenosis
Angiotensin II Type 1 Receptor (AGTR1) Gene 1166AmC Polymorphism2 Assess risk of hypertension and CVD
B-Type Natriuretic Peptide (BNP) Diagnose CHF, predict morbidity and mortality, and optimize drug therapy
Catecholamines, Fractionated, 24-Hour Urine Differential diagnosis of hypertension
Catecholamines, Fractionated, Plasma Differential diagnosis of hypertension
Catecholamines, Fractionated, Random Urine Differential diagnosis of hypertension
Catecholamines, Fractionated, & VMA, 24-Hour Urine Differential diagnosis of hypertension
Creatine Kinase Isoenzymes (CK Isoenzymes) Assess presence and degree of myocardial damage
Cytochrome P450 2C9 Genotype2 Assess potential for poor therapeutic response to or toxicity from warfarin,
tolbutamide, phenytoin, and certain anti-inflammatory agents
Cytochrome P450 2C9 and VKORC1 Mutation Analysis3 Predict response to warfarin therapy; assist in selection of appropriate
dose in treatment-naïve individuals
Cytochrome P450 2D6 Genotype2 Assess potential for poor therapeutic response to or toxicity from
antihypertensives, antiarrhythmics, antidepressants, neuroleptics,
antianginals, or opioids
Cytochrome P450 2D6/2C19 Genotyping Identify increased risk of cardiac toxicity due to prolonged drug
metabolism
Epinephrine, Plasma Differential diagnosis of hypertension
Folate, Serum Help select appropriate hyperhomocysteinemia therapy
Folate, RBC Help select appropriate hyperhomocysteinemia therapy
Folate & Vitamin B12 Help select appropriate hyperhomocysteinemia therapy
Homocysteine (Cardiovascular), Serum Assess risk of CVD, stroke, and dementia; monitor therapy in patients with
hyperhomocysteinemia
Homocysteine, Total, Urine Assess risk of CVD, stroke, and dementia; monitor therapy in patients
with hyperhomocysteinemia
Lactate Dehydrogenase (LD, LDH) Assess myocardial damage; however, troponin and CK isozymes are
preferred
Lactate Dehydrogenase Isoenzymes (LD Isoenzymes) Assess myocardial damage; however, troponin and CK isozymes are
preferred
LD, Pericardial Fluid Differentiate transudate (CHF) and exudate
 7

Section 1 Cardiovascular Tests

LD, Pleural Fluid Differentiate transudate (CHF) and exudate

Metabolic Syndrome Panel Diagnose metabolic syndrome
Includes glucose, triglycerides, and HDL cholesterol.
Metanephrines, Fractionated, LC/MS/MS, 24-Hour Urine Differential diagnosis of hypertension
Metanephrines, Fractionated, LC/MS/MS, Random Urine Differential diagnosis of hypertension
Metanephrines, Fractionated, Plasma Differential diagnosis of hypertension
Methylenetetrahydrofolate Reductase (MTHFR), DNA Mutation Analysis2 Assess risk of CVD and stroke; identify individuals at risk for
hyperhomocysteinemia
Methylmalonic Acid Determine etiology of increased homocysteine (a CVD risk marker) levels,
investigate potential B12 or folate deficiency
Microalbumin, 24-Hour Urine (with Creatinine) Assess risk of cardiovascular events, including heart failure in individuals
with CVD
Microalbumin, Intact, HPLC, 24-Hour Urine (with Creatinine) Assess risk of cardiovascular events, including heart failure in individuals
with CVD
Microalbumin, Intact, HPLC, 24-Hour Urine (without Creatinine) Assess risk of cardiovascular events, including heart failure in individuals
with CVD
Microalbumin, Intact, HPLC, Random Urine (with Creatinine) Assess risk of cardiovascular events, including heart failure in individuals
with CVD
Microalbumin, Random Urine (with Creatinine) Assess risk of cardiovascular events, including heart failure in individuals
with CVD
Norepinephrine, Plasma Differential diagnosis of hypertension and treatment selection
proBNP, N-terminal Diagnose CHF, predict morbidity and mortality, and optimize drug therapy
Troponin I Assess myocardial damage
Troponin T Assess prognosis in patients with acute coronary syndrome
Vitamin B6 Help select appropriate hyperhomocysteinemia therapy
Vitamin B12 Help select appropriate hyperhomocysteinemia therapy
Vitamin B12 Binding Capacity, Unsaturated (Transcobalamin) Help select appropriate hyperhomocysteinemia therapy
VMA (Vanillylmandelic Acid), Random Urine Differential diagnosis of hypertension
VMA, 24-Hour Urine Differential diagnosis of hypertension

Infectious Disease Markers Clinical Application

Chlamydia pneumoniae Antibodies (IgA, IgG, IgM)2 Assess CVD risk
Cytomegalovirus IgG Antibody Assess CVD risk
Cytomegalovirus IgM Antibody Assess CVD risk
Cytomegalovirus Antibody (IgG) with Reflex to IgM Assess CVD risk
Cytomegalovirus Antibodies (IgG, IgM) Assess CVD risk
Helicobacter pylori Antibody (IgG), Qualitative Assess CVD risk

Therapeutic Drug Monitoring Clinical Application

Amiodarone Monitor treatment response
Digitoxin, Serum/Plasma Monitor treatment response
Digoxin, ICMA Monitor treatment response
Disopyramide Monitor treatment response
Flecainide (Tambocor®’) Monitor treatment response
Heparin, Low Molecular Weight (Xa Inhibition) Monitor treatment response
Heparin, Unfractionated (Xa Inhibition) Monitor treatment response
Lidocaine (Xylocaine®’) Monitor treatment response
Procainamide Monitor treatment response
Propafenone Monitor treatment response
Quinidine Monitor treatment response
CVD, cardiovascular disease; CHF, congestive heart failure; Lp-PLA2, lipoprotein-associated phospholipase A2.
1
This test is performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been determined by Quest Diagnostics
Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means.
2
This test was developed and its performance characteristics have been determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
3
This test was developed and its performance characteristics have been determined by Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical performance of
the test.
Reflex tests are performed at an additional charge.
8

Section 1 Cardiovascular Tests

1.2. Markers of Lipidemia
1.2.1 Markers of Lipidemia Test Guide
Identification and treatment of dyslipidemia can reduce the risk of
coronary heart disease (CHD) events. This laboratory test guide provides
an easy-to-follow overview of the most recent lipidemia-related
guidelines from the National Cholesterol Education Program’s (NCEP’s)
Adult Treatment Panel III (ATP III).1-4 The primary target of these
guidelines is controlling levels of low-density lipoprotein-cholesterol
(LDL), a major modifiable risk factor for the development of CHD.
The overall scheme for identifying and treating elevated LDL levels is
outlined in Figures 1, 2, and 3 with supportive information in Tables 1, 2,
and 3. Once the target LDL level is achieved, therapy should be directed
toward treating metabolic syndrome and maintaining desirable
triglyceride and high-density lipoprotein-cholesterol (HDL) levels, as
outlined in Tables 4-9. In addition, Table 10 presents emerging markers
of lipidemia to assist in global risk assessment and individualized drug
selection. For more detailed information, please visit the National Heart,
Lung, and Blood Institute’s Web site at http://www.nhlbi.nih.gov/
guidelines/cholesterol/index.htm.

#56+0)>.+2+&>241(+.'

55'55>%1410#4;>*'#46>&+5'#5' >4+5-

 " "    ""   !""  " 

1>*+5614;>1(>>>
>1>*+5614;>1(>>
>>14>>4+5->'37+8#.'065

>1>>4+5->'37+8#.'065
>1>>4+5->'37+8#.'065  "

>@ >/#,14>4+5->(#%6145>>
> >14>/14'>/#,14>4+5->(#%6145>
 >14>/14'>/#,14>4+5->(#%6145>
>':%.7&+0)>> ':%.7&+0)>> >>':%.7&+0)>>#0&> @;'#4>
< <

> @;'#4>>4+5- > 
>/1&'4#6'> >> 4+5- > 
>
4+5->14> @ 
>/1&'4#6'.;>*+)*>4+5->
">2.75>%10641.>>
>> >>
'2'#6>6'56+0)> 16*'4>4+5->(#%6145>
 >/)&>  >/)&
+0>
>;'#45> 4'2'#6>6'56+0)>+0>>
>> >>
 >;'#4=
 >/)&  >/)&
>>
'2'#6>6'56+0)> 056+676'>"
@ >/)&> >>
+0>
>;'#45> +)> >2.75>>
'2'#6>6'56+0)> 056+676'>>   /)&
=
>.19'4+0)>&47)>
>> 056+676'>> +0>
>;'#45 ">+)> >= 6*'4#2;>+)> >=
?

 >/)& >">+)> >

>> 105+&'4>  "" "
 "  ""  !"

 >/)& .19'4+0)>&47)>
 +)#4'66'>5/1-+0)> !  "
6*'4#2;

>;2'46'05+10> >//>)>14> ">>'(14'>56#46+0)>@
+)> >=?
>6#-+0)>#06+*;2'46'05+8'>/'&+%#6+10> .19'4+0)>6*'4#2;>2'12.'>9+6*>

>19>> >/)&>>> '.'8#6'&>>.'8'.5>14>16*'4>
/)&>+5>#>0')#6+8'>4+5->(#%614>#0& (14/5>1(>*;2'4.+2+&'/+#>5*17.&>
>57$64#%65> >4+5->(#%614>>(41/>6*'>616#. $'>6'56'&>(14>5'%10&#4;>
""   "" >%1706> &;5.+2+&'/+#>

>!;/261/#6+%>%#416+&>#46'4;>&+5'#5'>
>#/+.;>*+5614;>1(>24'/#674'>>> !'%10&#4;>&;5.+2+&'/+#>%#0>$'>

>'4+2*'4#.>#46'4;>&+5'#5'>> >+0> 56>&')4''>4'.#6+8'>/#.'> >> %#75'&>$;>&+#$'6'5>*;21@

>$&1/+0#.>#146+%> #0'74;5/> >;'#45>1.&>14>('/#.'> >;'#45>1.&> 6*;41+&+5/>1$5647%6+8'>.+8'4>

>)'> >(14>/'0>14  >(14>91/'0> &+5'#5'>%*410+%>4'0#.>(#+.74'>14>

>+#$'6'5>/'..+675
/'&+%#6+105>
LDL, low-density lipoprotein-cholesterol; HDL, high-density lipoprotein-cholesterol; TLC, therapeutic lifestyle changes.
Note: The information in this flowchart is adapted from references 1–3.
*Fasting lipid profile includes total cholesterol (TC), LDL, HDL, and triglyceride levels.
†
To determine the estimated 10-year risk of CHD, see the National Heart, Lung, and Blood Institute’s Web site at http://hin.nhlbi.nih.gov/atpiii/calculator.asp?usertype=prof or use the
Framingham Point Scale in the Appendix.
‡
For moderately high-risk persons, an LDL goal of <100 mg/dL is an option; for patients with very high risk, a goal of <70 mg/dL should be considered. TLC should be considered for all
individuals with moderately high or high CVD risk who have risk factors related to lifestyle, regardless of LDL levels.
§
When LDL lowering drug therapy is initiated, the goal should be a decrease in LDL levels of at least 30% to 40%.3

Figure 1: Strategy for cholesterol testing.


Section 1
#0 0
0"%(/0'*)$#0#0$!()'$!000
00#0()*')0)0#) 0
0#$*'0"$')0-'(0
0$#('0'''!0)$0)#0


%)0!%$%'$)#
#!.((
0$!0 0$!0#$)
+00 +
 *')'0'*0#) 00
0$0()*')0)0#00
$#)#*00#0)')0
0$!()'$!0
$)'0'( 0)$'( 0'%)0
!%$%'$)#0#!.((0+'.0 0$#('0#0%!#)00
0()#$!(0#0()'$!(0
1 0"$#)(0$'0'()0.'0
#0+'.0 10"$#)(0 0#'(0'0#) 0
0$#('0'''!0)$00
)')'0
0))#


0$! %)0!%$%'$)#
+00 #!.((
0$!0#$)0+
0$#('0#0'*00
0)'%.0
0
0)')0)'%.0$'0")$!00
0(.#'$"00%'(#)00
0
!(00#00
0#)#(.0,)0"#"#)0000
000#0%.(!0)+).0
*See Secondary Targets of Therapy. 0$#('0'''!0)$0))#0
Figure 2. Steps in therapeutic lifestyle changes (TLC).
Table 1. Overview of TLC Diet
Diet
Saturated fat <7% of total calories
Polyunsaturated fat b10% of total calories
Monounsaturated fat b20% of total calories
Total fat 25%-35% of total calories
Carbohydrates 50%-60% of total calories
Fiber 20-30 g/day
Protein About 15% of total calories
Cholesterol <200 mg/day
(continued)
9
Cardiovascular Tests
0 #01!$,'#0'*0)'%.0
,)0())#0!00(&*()'#)0
$'0#$)#0†


%)0!%$%'$)#
#!.((
0$! 0$!0#$)
+0 +
$#)#*0)'%.00
#0)')0$)'0!%0
#)#(.00
'( 0)$'(* '%)00
'*0)'%.†
!%$%'$)#0#!.((00
+'.0 1 0"$#)(0


0$! %)0!%$%'$)#
+0 #!.((
0$! #$)0+
#)#(.0'*0)'%.0$'00
''0)$00!%0(%!() 0
'%)0!%$%'$)#0#!.((0
+'.00"$#)(0*#)!00$!
*See Secondary Targets of Therapy.
† +0)#0)')0$)'0
For high-risk individuals with elevated
triglyceride or low HDL levels, consider !%0#0#$#1!%0'( 0)$'(0
combining a fibrate or nicotinic acid with #0"$#)$'0'(%$#(0+'.0
an LDL-lowering drug. 1 0"$#)(0
Figure 3. Drug therapy for CHD prevention.
Table 1. Overview of TLC Diet (continued)
Weight management
Adjust caloric intake to maintain
desirable body weight
Increased physical activity
Enough moderate exercise to expend
at least 200 Kcal/day
Adapted from reference 2.
10

Section 1 Cardiovascular Tests

Table 2. Low-Density Lipoprotein-Cholesterol (LDL) Goals

LDL, mg/dL
Risk Category Goal Start TLC Initiate Drug Therapy
High risk: <100* r100 r100
CHD or CHD risk equivalent
(10-year CHD risk >20%)
Moderately high risk: <130* r130 r130 (optional at 100-129)†
r2 risk factors for CHD
10-year CHD risk 10%-20%
Moderate risk: <130 r130 r160
r2 risk factors for CHD
10-year CHD risk <10%
Low risk: <160§ r160 r190 (optional at 160-189)†
<2 risk factors for CHD
CHD, coronary heart disease; TLC, therapeutic lifestyle changes.
*For moderately high-risk persons, an LDL goal of <100 mg/dL is an option; for patients with very high risk, a goal of <70 mg/dL should be considered. TLC should be considered for all
individuals with moderately high or high CVD risk who have risk factors related to lifestyle, regardless of LDL levels.
†
Factors favoring drug initiation include 1) a severe single risk factor: heavy cigarette smoking, poorly controlled hypertension, a strong family history of premature CHD, or very low high-
density lipoprotein-cholesterol; 2) multiple life-habit risk factors or emerging risk factors; and 3) 10-year CHD risk approaching 10%.
‡
American Heart Association guidelines suggest that drug therapy (preferably with a statin) should be initiated in combination with lifestyle therapy in high-risk women with LDL-cholesterol
levels r100 mg/dL; statin therapy should also be initiated in high-risk women with LDL levels <100 mg/dL unless contraindicated.5
§
American Heart Association guidelines indicate that the optimal level in women is <100 mg/dL.5
Note: Individuals with high or moderately high risk with lifestyle-related risk factors should consider TLC to address those risk factors, regardless of LDL-C level.3
Table adapted from reference 2.

Table 3. Pharmaceutical Options for Treating Dyslipidemia

Therapeutic
Objective Relevant Drugs Comments
Decrease LDL levels 1. Statins Statins are the most potent and are well tolerated. Low-dose resins combined with statins
2. Resins improve effectiveness; however, resins are less well tolerated, leading to poor adherence.
3. Niacin Niacin is less effective and should be used with caution in patients with impaired glucose
metabolism.
Increase HDL levels 1. Statins Statins may be the drugs of choice when lowering LDL is also desired. Niacin is recommended
2. Niacin even in the absence of elevated LDL. Use niacin with caution in patients with impaired glucose
3. Fibrates metabolism. Fibrates may be the drug of choice when lowering triglycerides is also desired.
Decrease triglyceride levels 1. Fibrates Statins are useful to lower LDL and VLDL, but have no direct effect on triglycerides and are not
2. Niacin a first-line treatment if the triglyceride level is >500 mg/dL. Use niacin with caution when
3. Statins administering to those with impaired glucose tolerance. n-3 Fatty acids from fish oils may be
4. Fish oils used in people with very high triglyceride levels. Bile acids (resins) are contraindicated if the
triglyceride level is >500 mg/dL.
Decrease VLDL levels 1. Statins Statins, nicotinic acid, and fibrates lower LDL as well as VLDL. Non-HDL cholesterol (LDL +
2. Nicotinic acid VLDL) becomes a secondary target of therapy when the triglyceride level is 200-499 mg/dL.
3. Fibrates
Decrease lipoprotein (a) 1. Niacin Lp(a)-lowering therapy may be indicated for people with elevated Lp(a) and either premature
levels 2. Neomycin CHD or a strong family history of premature CHD not explained by other dyslipidemias. Clinical
benefit from lowering Lp(a) is still to be demonstrated.
Increase LDL 1. Niacin (and Use niacin with caution when administering to those with impaired glucose tolerance.
subparticle size (ie, dietary therapy)
convert small dense LDL 2. Fibrates
[pattern B] to pattern A)
Decrease oxidation of 1. Vitamin E Studies have shown mixed results; conclusive evidence of clinical benefit is not yet
LDL particles 2. Probucol established.
Treat multiple lipid 1. Statins Multiple lipid abnormalities are most frequently treated with statins; however, combination
abnormalities 2. Niacin therapy may be needed to achieve treatment goals.
3. Fibrates
LDL, low-density lipoprotein-cholesterol; HDL, high-density lipoprotein-cholesterol; VLDL, very low-density lipoprotein-cholesterol; statins, HMG CoA reductase inhibitors; resins, bile acid
sequestrants; niacin, nicotinic acid; fibrates, fibric acid derivatives; Lp(a), lipoprotein(a).
 11

Section 1 Cardiovascular Tests

Secondary Targets of Therapy
Metabolic syndrome, closely linked to insulin resistance, is a
constellation of lipid and non-lipid factors that increase risk for CHD.
Metabolic syndrome is considered a secondary target of therapy and
should be treated, if present, after 3 months of TLC. Tables 4 and 5
outline the diagnosis and treatment of metabolic syndrome while Tables
Table 7. Treating High Triglyceride (TG) Levels (r150 mg/dL)
• LDL should be the first target of therapy.
• Intensify weight management and physical activity.
• If TG level is r200 mg/dL after LDL goal is achieved, direct treatment
at achieving non-HDL goal (Table 5).
O If TG level is 200-499 mg/dL after LDL goal is met, intensify

6-9 describe the identification and treatment of elevated triglyceride therapy with LDL-lowering drugs or add nicotinic acid or fibrate to
and low HDL levels. lower very low-density lipoprotein-cholesterol (VLDL) level.
O If TG concentration is r500 mg/dL after LDL goal is met, lower TG

Table 4. Identifying Metabolic Syndrome* level to avert pancreatitis: very low-fat diet (b15% of calories
from fat); intensify weight management and physical activity; add
Risk Factor Defining Level fibrate or nicotinic acid. Return to LDL-lowering therapy when TG
level drops below 500 mg/dL.
Abdominal obesity Waist circumference Adapted from references 2 and 4.
Men >102 cm (>40 inches)†
Women >88 cm (>35 inches)
Triglycerides r150 mg/dL Table 8. Non-HDL Cholesterol* Goals
HDL cholesterol Non-HDL Goal
Men <40 mg/dL Risk Category mg/dL
Women <50 mg/dL
High risk <130
Blood pressure r130/r85 mm Hg CHD or CHD risk equivalent or
Fasting glucose r110 mg/dL 10-year CHD risk equivalent >20%
*Individuals with any 3 of these risk factors are considered to have metabolic syndrome. Moderate risk <160
†
Men with marginally increased waist circumferences (37-39 inches) who develop multiple r2 risk factors for CHD, 10-year CHD risk b20%
metabolic risk factors may have a strong genetic contribution to insulin resistance. Such
men should benefit from lifestyle changes, similarly to men with abdominal obesity. Low risk <190†
Adapted from references 2 and 4. <2 risk factors for CHD
*Non-HDL cholesterol = total cholesterol minus HDL cholesterol.
†
Table 5. Treating Metabolic Syndrome American Heart Association guidelines indicate a goal of <130 mg/dL for women.5
Table adapted from references 2 and 4.

Treat underlying causes

Intensify weight management Table 9. Treating Low HDL Levels (<40 mg/dL)
Increase physical activity
Treat lipid and non-lipid risk factors • LDL should be the first target of therapy.
Treat hypertension • After LDL goal is reached, intensify weight management and physical
Use aspirin for patients with CHD to reduce prothrombotic state activity.
Treat for elevated triglyceride and low HDL levels as needed • If triglyceride level is 200-499 mg/dL, direct treatment at achieving
non-HDL goal.
Adapted from references 2 and 3.
• If triglyceride level is <200 mg/dL (isolated low HDL) in patients with
CHD or CHD equivalent, consider nicotinic acid or fibrate.
Table 6. ATP III Triglyceride (TG) Level Classification Adapted from references 2 and 4.

Classification
Normal
Borderline
High
Very High
References
1. Third report of the National Cholesterol Education Program (NCEP)
expert panel on detection, evaluation, and treatment of high blood
cholesterol in adults (Adult Treatment Panel III). Available at
http://www.nhlbi.nih.gov/guidelines/cholesterol/atp3_rpt.htm.
Accessed July 25, 2006.
2. National Cholesterol Education Program. Executive summary of the
third report of the National Cholesterol Education Program (NCEP)
expert panel on detection, evaluation, and treatment of high blood
TG mg/dL cholesterol in adults (Adult Treatment Panel III). National Institutes
of Health. National Heart, Lung, and Blood Institute. NIH Publication
<150
No. 01-3670; May, 2001. Available at http://rover2.nhlbi.nih.gov/
150-199 guidelines/cholesterol/atp3xsum.pdf. Accessed July 25, 2006. Also
published in JAMA: JAMA. 2001;285:2486-2509.
200-499
3. Grundy SM, Cleeman JI, Merz CN, et al for the Coordinating
r500 Committee of the National Cholesterol Education Program.
Implications of recent clinical trials for the National Cholesterol
Education Program Adult Treatment Panel III guidelines. Circulation.
2004;110:227-239.
4. National Cholesterol Education Program. ATP III Guidelines At-a-
Glance: Quick Desk Reference. National Institutes of Health.
National Heart, Lung, and Blood Institute. Available at http://rover2.
nhlbi.nih.gov/guidelines/cholesterol/atglance.pdf. Accessed July 25,
2006.
5. Mosca L, Appel LJ, Benjamin EJ, et al. Evidence-based guidelines
for cardiovascular disease prevention in women. Circulation.
2004;109:672-693.
12

Section 1 Cardiovascular Tests

Table 10. Additional Lipidemia Markers

Marker Use Adult Level Interpretation

Apolipoprotein A1 Assess risk for CHD <94 (M) or 101 (F) mg/dL Hypoalphalipoproteinemia, increased
and diagnose CHD risk, Tangier disease
dyslipidemia 94-176 mg/dL M: Normal
101-198 mg/dL F: Normal
Apolipoprotein B Assess risk for CHD and 52-109 mg/dL M: Normal
diagnose dyslipidemia 49-103 mg/dL F: Normal
>109 (M) or 103 (F) mg/dL Hyperapobetalipoproteinemia, increased CHD risk
associated with pattern B
Apolipoprotein B: Assess risk for CHD <0.29 Below-average CHD risk
Apolipoprotein A1 0.29-1.30 Average risk for CHD
ratio >1.30 Above average risk for CHD
Direct LDL Stratify risk and monitor <100 mg/dL Optimal
cholesterol therapy when triglyceride 100-129 mg/dL Near optimal/above optimal
level is >400 mg/dL 130-159 mg/dL Borderline high
160-189 mg/dL High
r190 mg/dL Very high
HDL subclasses Assess risk for CHD HDL2 <9 mg/dL Increased risk for CHD
HDL2 9-38 mg/dL Normal
HDL3 22-35 mg/dL Normal
Lipoprotein Assess risk of Cholesterol: <200 mg/dL Normal
Fractionationa premature CHD LDL: <130 mg/dL Normal
HDL: >40 mg/dL M: Normal
HDL: >50 mg/dL F: Normal
VLDL: <30 mg/dL Normal
Triglycerides: <150 mg/dL Normal
Non-HDL: <160 mg/dL Normal
Lp(a): <75 nmol/L Normal
LDL particles: 508-1279 nmol/L M: Normal
LDL particles: 272-1181 nmol/L F: Normal
LDL size: 215.4-230.9 Angstrom M: Normal
LDL size: 215.4-232.9 Angstrom F: Normal
LDL phenotype: A pattern Normal
LDL I large: 48-164 nmol/L M: Normal
LDL I large: 51-186 nmol/L F: Normal
LDL II large: 200-596 nmol/L M: Normal
LDL II large: 91-574 nmol/L F: Normal
LDL III small: 136-627 nmol/L M: Normal
LDL III small: 82-442 nmol/L F: Normal
LDL IV small: 38-164 nmol/L M: Normal
LDL IV small: 33-129 nmol/L F: Normal
HDL 2b large: 169-1153 nmol/L M: Normal
HDL 2b large: 384-1616 nmol/L F: Normal
HDL 2a intermed: 1174-3744 nmol/L M: Normal
HDL 2a intermed: 903-3779 nmol/L F: Normal
HDL 3 small: 613-3344 nmol/L M: Normal
HDL 3 small: 475-4244 nmol/L F: Normal
IDL 1 large: 11-41 nmol/L M: Normal
IDL 1 large: 10-38 nmol/L F: Normal
IDL 2 small: 12-59 nmol/L M: Normal
IDL 2 small: 11-48 nmol/L F: Normal
VLDL large: 0.2-2.5 nmol/L M: Normal
VLDL large: 0.2-1.8 nmol/L F: Normal
VLDL intermed: 1.1-7.3 nmol/L M: Normal
VLDL intermed: 1.0-5.7 nmol/L F: Normal
VLDL small: 5.0-23.0 nmol/L M: Normal
VLDL small: 5.8-26.6 nmol/L F: Normal
Lipoprotein(a) [Lp(a)] Assess risk for CHD <75 nmol/L Normal
(continued)
 13

Section 1 Cardiovascular Tests

Table 10. Additional Lipidemia Markers (continued)

Marker Use Adult Level Interpretation

Total Assess risk for CHD; predict <5 M: Desirable
cholesterol:HDL outcome and therapeutic <4.4 F: Desirable
cholesterol ratio benefit; monitor therapy
Very low-density Monitor non-HDL–targeted <20 mg/dL Desirable
lipoprotein- therapy in people with elevated 21-39 mg/dL Borderline
cholesterol (VLDL) triglyceride levels r40 mg/dL High
M, male; F, female.
Note: Information in this table is derived from Quest Diagnostics internal data and published literature (references 6-9).
a
LDL particles are measured in 4 fractions: LDL I and II, larger, more buoyant particles, and LDL III and IV, smaller, more dense particles. LDL pattern A and pattern B are characterized by LDL
peak particle diameter greater or less than 215.4 A, respectively.
HDL particles are measured in 3 fractions: HDL 2b, large buoyant particles that are most strongly correlated with HDL cholesterol level, and HDL 2a and 3, small denser HDL particles.
IDL and VLDL fractions: Triglyceride-rich lipoprotein particles are measured in 5 fractions of increasing size (IDL1 and 2, and small, intermediate, and large VLDL). Higher triglyceride levels
are associated with increased numbers of larger particles. The smaller fractions are enriched in remnant particles.

6. Ballantyne CM. Current thinking in lipid lowering. Am J Med. Clinical Background: LDL-cholesterol testing is an important part of
1998;104:33S-41S. a CHD prevention strategy. It is used to assess the risk of CHD and to
7. Criqui MH, Golomb BA. Epidemiologic aspects of lipid monitor patients who are at increased risk. Furthermore, LDL-cholesterol
abnormalities. Am J Med. 1998;105:48S-57S. is used to monitor patients with prior CHD, other atherosclerotic
8. Kinosian B, Glick H, Garland G. Cholesterol and coronary heart disease, or diabetes mellitus (see Markers of Lipidemia, section 1.2.1).
disease: predicting risks by levels and ratios. Ann Intern Med.
1994;121:641-647. LDL-cholesterol is the primary target of therapy, according to NCEP
9. Haffner SM, Lehto S, Ronnemaa T, et al. Mortality from coronary ATPIII. Follow-up LDL-cholesterol determination should be made 4 to 6
heart disease in subjects with type 2 diabetes and in nondiabetic weeks after initiating drug therapy and again at 3 months. A minimum
subjects with and without prior myocardial infarction. N Engl J Med. of 2 lipoprotein determinations is essential for evaluating the efficacy of
1998;339:229-234. a given drug dose. The mean of these 2 determinations and a careful
10. Stein JH, Rosenson RS. Lipoprotein Lp(a) excess and coronary heart assessment of drug adherence should be used to judge the efficacy of
disease. Arch Intern Med. 1997;157:1170-1176. drug treatment. After the target LDL-cholesterol concentration has been
achieved, patients should be followed up every 4 months, or more
Appendix. Estimating 10-Year Risk of CHD: Framingham Point frequently depending on the drug being used, to monitor cholesterol
Scale levels and possible side effects.
(Adapted from reference 2)
Method: The calculated LDL-cholesterol relies on a calculation using 3
Add the points from each risk category (Tables A-E) to derive the separate measurements: total cholesterol, HDL-cholesterol, and
estimated 10-year risk of a hard CHD event (myocardial infarction or triglycerides. The calculated LDL-cholesterol is still considered an
coronary death) in Table F. excellent initial test and is reliable and appropriate in most instances.
However, if the triglycerides are abnormally high (ie, >400 mg/dL) or the
1.2.2 LDL Cholesterol patient has not appropriately fasted (recommended fast is 12 hours),
then the calculated LDL-cholesterol will be artificially low or non-
Clinical Use: This test is used to assess risk of coronary heart disease reportable.
(CHD; primary or secondary) and to monitor nondrug or drug therapy.
In contrast, the direct LDL-cholesterol assay does not rely on a
calculation. When an accurate and precise LDL-cholesterol
Table A. Age Group measurement is required, direct measurement provides an alternative.
Direct measurement provides a reliable result even when triglyceride
Age Men (Points) Women (Points) levels are up to 1000 mg/dL and circumvents the need for patients to
fast, for which compliance is suboptimal. Further, the direct LDL-
20-34 -9 -7 cholesterol assay has been correlated with the CDC-accepted reference
35-39 -4 -3 method. Thus, results can be related to the epidemiologic data that
have been generated for the assessment of CHD risk and the monitoring
40-44 0 0 of therapy to reduce that risk.
45-49 3 3
Reference Range: See Table 11.
50-54 6 6
55-59 8 8 Interpretive Information: The target LDL-cholesterol level varies
according to the risk profile of the patient (Table 12). Typically,
60-64 10 10 therapeutic lifestyle changes (TLC) are the first choice for moderate
65-69 11 12 elevations in LDL-cholesterol. Drug therapy may be the first choice in
individuals with higher cholesterol levels and those who do not respond
70-74 12 14 to TLC.
75-79 13 16
14

Section 1
Cardiovascular Tests Cardiovascular
SectionTests
1

Table B. Total Cholesterol

Men (Points) Women (Points)

Total Cholesterol,
mg/dL 20-39 y 40-49 y 50-59 y 60-69 y 70-79 y 20-39 y 40-49 y 50-59 y 60-69 y 70-79 y
<160 0 0 0 0 0 0 0 0 0 0
160-199 4 3 2 1 0 4 3 2 1 1
200-239 7 5 3 1 0 8 6 4 2 1
240-279 9 6 4 2 1 11 8 5 3 2
280+ 11 8 5 3 1 13 10 7 4 2

Table C. Smoking Status

Men (Points) Women (Points)
20-39 y 40-49 y 50-59 y 60-69 y 70-79 y 20-39 y 40-49 y 50-59 y 60-69 y 70-79 y

Nonsmoker 0 0 0 0 0 0 0 0 0 0
Smoker 8 5 3 1 1 9 7 4 2 1

Table D. HDL Level Table F. 10-Year Risk According to Total Framingham Point
Scores
HDL, mg/dL Men (Points) Women (Points)
60+ -1 -1 Men (Points) Women (Points)
50-59 0 0 Point 10-Year Point 10-Year
Total Risk (%) Total Risk (%)
40-49 1 1
<0 <1 <9 <1
<40 2 2
0-4 1 9-12 1
5-6 2 13-14 2
Table E. Systolic Blood Pressure and Treatment Status
7 3 15 3
Men (Points) Women (Points) 8 4 16 4
Systolic BP Untreated Treated Untreated Treated 9 5 17 5
<120 0 0 0 0 10 6 18 6
120-129 0 1 1 3 11 8 19 8
130-139 1 2 2 4 12 10 20 11
140-159 1 2 3 5 13 12 21 14
160+ 2 3 4 6 14 16 22 17
15 20 23 22
References 16 25 24 27
1. American Academy of Pediatrics. National Cholesterol Education 17 or more ≥30 25 or more ≥30
Program: Report of the Expert Panel on Blood Cholesterol Levels in
Children and Adolescents. Pediatrics. 1992;89:525-584.
2. Executive Summary of The Third Report of The National Cholesterol Clinical Background: Lipoprotein (a) [Lp(a)] is a modified form of low-
Education Program (NCEP) Expert Panel on Detection, Evaluation, and density lipoprotein (LDL) cholesterol in which the glycoprotein
Treatment of High Blood Cholesterol in Adults (Adult Treatment apolipoprotein (a) is covalently linked to the apolipoprotein B (Apo B)
Panel III). JAMA. 2001;285:2486-2509. moiety of LDL. Multiple Lp(a) isoforms exist due to variation in the
number of repeats of kringle 4, a protein domain present in
1.2.3 Lipoprotein (a) plasminogen. Genetically determined isoforms with smaller molecular
weights are associated with higher Lp(a) blood levels.1 Lp(a)’s genetic
Clinical Use: This test is used to assess the risk of cardiovascular linkage to the plasminogen gene leads to prothrombotic activities,
disease. whereas similarity to LDL leads to atherogenic activities.2

498
 15

Section 1 Cardiovascular Tests

Table 11. LDL Cholesterol Reference Ranges1,2 Method: This immunoprecipitin method has no crossreactivity with
Apo B (up to 436 mg/dL of Apo B). The limit of quantification is
LDL mg/dL Interpretation 1.5 mg/dL.
Pediatrics (b19 y) Reference Range
<110 Desirable <75 nmol/L
110-129 Borderline
r130 High Interpretive Information: Normal levels in the African American
Adults (>19 y) population may be 2 to 3 times the values in white and Asian
<100 Optimal populations. Native Americans and Mexican Americans have lower
100-129 Near optimal/above optimal normal levels (no lower than one-half) relative to white and Asian
130-159 Borderline high populations.
160-189 High
r190 Very high Increased levels of Lp(a) are observed in patients with coronary artery
disease, stroke, cerebrovascular, and peripheral vascular disease.
Substantial increases are secondarily (not genetically related) observed
in nephrotic syndrome and end-stage renal disease. Decreased Lp(a)
Elevated Lp(a) blood levels are commonly observed in patients and levels may be seen in several rare disorders (lecithin:cholesterol
families with premature coronary heart disease (CHD). Retrospective acyltransferase [LCAT] deficiency, lipoprotein lipase [LPL] deficiency,
and prospective studies have identified Lp(a) as an independent risk liver disease). Results should be interpreted in conjunction with the
factor for CHD.3-6 Conflicting results attributed to analytical issues, patient history, clinical findings, and other laboratory test results.
sample size, and duration of follow-up, however, have been reported.7,8
More recent studies such as the Framingham Study3,4,9 have addressed Table 13 provides CHD risks attributable to Lp(a) relative to other risk
these limitations. factors.

The treatment of elevated Lp(a) levels has been controversial. Diet,
exercise, and the lipid-lowering statins appear ineffective; whereas,
estrogen replacement therapy, niacin (nicotinic acid), neomycin, and
apheresis are more successful. Nonetheless, the clinical effect of
lowered Lp(a) levels is not yet known.
Individuals Suitable for Testing include those with suspected
premature CHD or cerebrovascular disease, those with a strong family
history of premature CHD, family members of patients with increased
Lp(a) levels, and individuals with CHD but no established risk factors.
References
1. Kronenberg F, Steinmetz A, Kostner GM, et al. Lipoprotein(a) in health
and disease. Crit Rev Clin Lab Sci. 1996;33:495-543.
2. Duriez P, Dallongeville J, Fruchart JC. Lipoprotein(a) as a marker for
coronary heart disease. Br J Clin Pract. 1996;Suppl 77A:54-61.
3. Bostom AG, Cupples LA, Jenner JL, et al. Elevated plasma
lipoprotein(a) and coronary heart disease in men aged 55 years and
younger. A prospective study. JAMA. 1996;276:544-548.
4. Bostom AG, Gagnon DR, Cupples LA, et al. A prospective
investigation of elevated lipoprotein(a) detected by electrophoresis

Table 12. Low-Density Lipoprotein-Cholesterol (LDL) Goals2

LDL, mg/dL
Risk Category Goal Start TLC Initiate Drug Therapy
High risk: <100* r100 r100
CHD or CHD risk equivalent
(10-year CHD risk >20%)
Moderately high risk: <130* r130 r130 (optional at 100-129)†
r2 risk factors for CHD
10-year CHD risk 10%-20%
Moderate risk: <130 r130 r160
r2 risk factors for CHD
10-year CHD risk <10%
Low risk: <160§ r160 r190 (optional at 160-189)†
<2 risk factors for CHD
CHD, coronary heart disease; TLC, therapeutic lifestyle changes
* For moderately high-risk persons, an LDL goal of <100 mg/dL is an option; for patients with very high risk, a goal of <70 mg/dL should be considered. TLC should be considered for all
individuals with moderately high or high CVD risk who have risk factors related to lifestyle, regardless of LDL levels.
†
Factors favoring drug initiation include 1) a severe single risk factor: heavy cigarette smoking, poorly controlled hypertension, a strong family history of premature CHD, or very low high-
density lipoprotein-cholesterol; 2) multiple life-habit risk factors or emerging risk factors; and 3) 10-year CHD risk approaching 10%.
‡
American Heart Association guidelines suggest that drug therapy (preferably with a statin) should be initiated in combination with lifestyle therapy in high-risk women with LDL levels r100
mg/dL; statin therapy should also be initiated in high-risk women with LDL levels <100 mg/dL unless contraindicated.
§
American Heart Association guidelines indicate that the optimal level in women is <100 mg/dL.
Note: Individuals with high or moderately high risk with lifestyle-related risk factors should consider TLC to address those risk factors, regardless of LDL level.
16
Section 1
Table 13. Attributable Risk (%) for CHD Associated with
Various Factors3,4
Men Women
Lp(a), high 9 18
Cholesterol, high 10 19
HDL cholesterol, low 13 19
Smoking 55
and cardiovascular disease in women. The Framingham Heart Study.
Circulation. 1994;90:1688-1695.
5. Cremer P, Nagel D, Labrot B, et al. Lipoprotein Lp(a) as predictor of
myocardial infarction in comparison to fibrinogen, LDL cholesterol
and other risk factors: results from the prospective Gottingen Risk
Incidence and Prevalence Study (GRIPS). Eur J Clin Investig.
1994;24:444-453.
6. Schaefer EJ, Lamon-Fava S, Jenner JL, et al. Lipoprotein(a) levels and
risk of coronary heart disease in men: the Lipid Research Clinics
Coronary Primary Prevention Trial. JAMA. 1994;271:999-1003.
7. Jauhiainen M, Koskinen P, Ehnholm C, et al. Lipoprotein(a) and
coronary heart disease risk: a nested case-control study of the
Helsinki Heart Study participants. Atherosclerosis. 1991;89:59-67.
8. Ridker PM, Hennekens CH, Stampfer MJ. A prospective study of
lipoprotein(a) and the risk of myocardial infarction. JAMA.
1993;270:2195-2199.
9. Contois JH, Lammi-Keefe CJ, Vogel S, et al. Plasma lipoprotein(a)
distribution in the Framingham Offspring Study as determined with a
commercially available immunoturbidimetric assay. Clin Chim Acta.
1996;253:21-35.
1.3 Non-Lipid Markers
1.3.1 Non-Lipid Markers of Cardiovascular Disease Test Guide
A significant percentage of CVD is attributed to non-lipid factors. These
include genetic mutations, inflammation, coagulation disorders,
infection, autoimmune disease, and other unknown factors. The markers
in Table 14 may be useful for assessing cardiovascular risk irrespective
of lipid status.
References
1. Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-
associated phospholipase A2, high-sensitivity C-reactive protein,
and risk for incident coronary heart disease in middle-aged men and
women in the Atherosclerosis Risk in Communities (ARIC) Study.
Circulation. 2004;109:837-842.
2. Bick RL, Kaplan H. Syndromes of thrombosis and hypercoagulability:
congenital and acquired thrombophilias. Clin Appl
Thrombosis/Hemostasis. 1998;4:25-50.
3. Brey RL. Antiphospholipid antibodies and ischemic stroke. Heart Dis
Stroke. 1992;1:379-382.
4. Gensini GF, Comeglio M, Colella A. Classical risk factors and
emerging elements in the risk profile for coronary artery disease.
Eur Heart J. 1998;19:A53-A61.
5. Hillege HL, Fidler V, Diercks G, et al. Urinary albumin excretion
predicts cardiovascular and noncardiovascular mortality in general
population. Circulation. 2002;106:1777-1782.
6. Josefsberg Z, Ross SA, Lev-Ran A, et al. Effects of enalapril and
nitrendipine on the excretion of epidermal growth factor and
albumin in hypertensive NIDDM patients. Diabetes Care.
1995;18:690-693.
Cardiovascular Tests
7. Kannel WB. Influence of fibrinogen on cardiovascular disease.
Drugs. 1997;54:S32-S40.
8. McGee GS, Pearce WH, Sharma L, et al. Antiphospholipid
antibodies and arterial thrombosis. Arch Surg. 1992;127:342-346.
9. Mehta JL, Saldeen TG, Rand K. Interactive role of infection,
inflammation and traditional risk factors in atherosclerosis and
coronary artery disease. J Am Coll Cardiol. 1998;31:1217-1225.
10. Ridker PM, Cushman M, Stampfer MJ, et al. Inflammation, aspirin,
and the risk of cardiovascular disease in apparently healthy men.
N Engl J Med. 1997;336:973-979.
11. Rosendaal FR, Siscovick DS, Schwartz SM, et al. Factor V Leiden
(resistance to activated protein C) increases the risk of myocardial
infarction in young women. Blood. 1997;89:2817-2821.
12. Shionoiri H, Kosaka T, Kita E, et al. Comparison of long-term
therapeutic effect of an ACE inhibitor, temocapril, with that of a
diuretic on microalbuminuria in non-diabetic essential hypertension.
Hypertens Res. 2000;23;593-600.
13. Stein JH, McBride PE. Hyperhomocysteinemia and atherosclerotic
vascular disease: pathophysiology, screening, and treatment. Arch
Intern Med. 1998;158:1301-1306.
14. Thom DH, Grayston JT, Siscovick DS, et al. Association of prior
infection with Chlamydia pneumoniae and angiographically
demonstrated coronary artery disease. JAMA. 1992;268:68-72.
15. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl
J Med. 1998;338:1042-1050.
16. Oei HS, van der Meer IM, Hofman A, et al. Lipoprotein-associated
phospholipase A2 activity is associated with risk of coronary heart
disease and ischemic stroke: The Rotterdam Study. Circulation.
2005;111:570-575.
1.3.2 Cardio CRP®
Clinical Use: This test is used to determine the relative risk of
cardiovascular disease (CVD) and to assess risk of recurrent
cardiovascular event in patients with coronary heart disease (CHD).
Clinical Background: C-reactive protein (CRP) is a non-specific acute-
phase protein produced by the liver in response to tissue injury,
infection, and inflammation. Measurement of serum levels, which rise
as much as 1,000-fold after an acute event, has traditionally been used
to diagnose and monitor acute inflammatory states. However, mild CRP
elevation (within the normal, non-acute-phase range) has recently
emerged as a valuable marker of cardiovascular risk.1
Mildly elevated CRP (eg, b10 mg/L) has been linked with risk for CVD,
including first and recurrent coronary events1 and stroke;2 vascular
events after stroke;3 myocardial infarction or angina in patients with
peripheral vascular disease;4 poor outcome in acute coronary
syndromes1,5 and congestive heart failure;6 restenosis after coronary
angioplasty;7 sudden cardiac death;8 hypertension;9 dementia;10 and type
2 diabetes mellitus.11 Prospective studies with highly sensitive assays
such as Cardio CRP have consistently shown CRP to be a strong
predictor of increased cardiovascular risk in both men and women.1 The
predictive value of CRP is independent of other established risk factors,
including LDL-cholesterol, and screening with both CRP and LDL may
provide a better risk assessment than using either test alone.12
Additionally, evidence suggests patients with high CRP/normal LDL are
at greater risk than those with normal CRP/high LDL.12
Many therapies aimed at reducing cardiovascular risk act through anti-
inflammatory pathways. Aspirin and statins both yield the greatest
preventive effect in patients with the highest CRP levels.1,13 Statin
therapy reduces the risk of first acute coronary events14 and stroke15
associated with elevated CRP, and recent evidence suggests patients

Section 1
Table 14. Non-Lipid Markers of Cardiovascular Disease (CVD)*
Marker
Activated protein C resistance (APC-R)
Angiotensin converting enzyme (ACE)
polymorphism (insertion/deletion)‡
Angiotensin II type 1 receptor (AGTR1)
gene 1166AmC polymorphism‡
B-type natriuretic peptide (BNP)
Beta2-Glycoprotein I Antibodies
Cardio CRP®
(high-sensitivity C-
reactive protein)
Cardiolipin antibody
Chlamydia pneumoniae antibodies
Cytomegalovirus (CMV) antibodies
dRVVT Screen with Reflex to
Phospholipid Neutralization
Factor V Leiden mutation
(1691GmA, producing R506Q) ‡
Factor V HR2 allele DNA mutation
analysis‡
17
Cardiovascular Tests
Adult Level Interpretation Therapeutic Options
†
<2.0 (ratio) Positive; increased risk of venous thromboembolic Anticoagulants
disease, CVD (particularly in women >50 years of
age and in women who smoke), and cerebrovascular
disease; associated with acute phase reactions
r2.0 (ratio)† Negative; desirable
Insertion Normal Not established
Deletion Heterozygous or homozygous: increased risk
of cardiovascular disease and restenosis
Negative Normal Not established
Positive Increased risk of hypertension and
cardiovascular disease
<100 pg/mL Normal; heart failure unlikely ACE inhibitor drugs, angiotensin
r100 pg/mL High probability of heart failure (levels correlate receptor blockers, beta-
with NY heart classification); increased risk of adrenergic blockers
future cardiac events (eg, carvedilol), aldosterone
antagonists, BNP
Negative Normal Anticoagulants, antiplatelet
Positive Increased risk for CVD, cerebrovascular and therapy, corticosteroids
thromboembolic disease; associated with recurrent
fetal loss, thrombocytopenia, and systemic
lupus erythematosus (SLE)
<1.0 mg/L Low cardiovascular risk Anti-inflammatory drugs
1.0-3.0 mg/L Average cardiovascular risk (eg, aspirin), statins
3.1-10.0 mg/L High cardiovascular risk
>10.0 mg/L Persistent elevations may represent
non-cardiovascular inflammation
Negative Desirable Anticoagulants, antiplatelet
Positive Increased risk of CVD, cerebrovascular and therapy, corticosteroids
thromboembolic disease; associated with recurrent
fetal loss, thrombocytopenia, and systemic
lupus erythematosus (SLE)
Negative Probable absence of infection Antibiotics
Positive Active or resolved infection; increased CVD risk
Negative Probable absence of infection Antiviral drugs
Positive Active or resolved infection; increased
CVD risk
Ratio <1.3 Probable absence of phospholipid antibodies/ Anticoagulants, antiplatelet
lupus anticoagulant therapy, corticosteroids
Ratio r1.3 Increased risk for CVD, cerebrovascular and
thromboembolic disease; associated with recurrent
fetal loss, thrombocytopenia, and systemic
lupus erythematosus (SLE)
No mutation Normal (wild type) Anticoagulants
Heterozygote APC-R; 4- to 8-fold increased risk of venous
thrombosis; increased risk of CVD (particularly
in women who smoke) and cerebrovascular
disease
Homozygote APC-R; 80-fold increased risk of venous thrombosis;
increased risk of CVD (particularly in women who
smoke) and cerebrovascular disease
No mutation Normal (wild type) Anticoagulants
Heterozygous Increased risk (3- to 4-fold) of venous thrombosis
if factor V Leiden mutation is also present (no
increased risk if factor V Leiden mutation is absent)
(continued)
18

Section 1 Cardiovascular Tests

Table 14. Non-Lipid Markers of Cardiovascular Disease (CVD)* (continued)

Marker Adult Level Interpretation Therapeutic Options

Fibrinogen, quantitative, nephelometry <180 mg/dL Low risk of CHD even in presence of increased Anti-inflammatory drugs
cholesterol; associated with hypo- and (eg, aspirin)
afibrinogenemia, DIC, systemic fibrinolysis,
pancreatitis, severe hepatic dysfunction, treatment
with L-asparaginase or valproate
180-350 mg/dL Normal
>350 mg/dL Predicts increased incidence of CVD, CVD-related
mortality, and cerebrovascular disease (of limited
predictive value for individual patients); also
elevated in hypercoagulability, pregnancy, oral
contraceptive use, smoking, postmenopausal
status, and systemic inflammation
Helicobacter pylori antibody Negative Probable absence of infection Antibiotics
Positive Active or resolved infection; increased CVD risk
Homocysteine (cardiovascular) <11.4 Nmol/L (M) Normal Folic acid and sometimes
r11.4 Nmol/L (M) Increased risk of CVD, stroke, dementia (including cobalamin (vitamin B12),
Alzheimer disease), B12 and/or folate deficiency, and/or B6
chronic renal disease, and homocystinuria
<10.4 Nmol/L (F) Normal
r10.4 Nmol/L (F) Increased risk of CVD, stroke, dementia (including
Alzheimer disease), B12 and/or folate deficiency,
chronic renal disease, and homocystinuria
Lp-PLA2 115-245 ng/mL (M) Normal Not established; fenofibrate and
85-245 ng/mL (F) atorvastatin under evaluation
Methylenetetrahydrofolate reductase No mutation Normal (wild type) Folic acid and/or cobalamin
(MTHFR) mutation (677CmT, Heterozygote Carrier; absence of phenotypic expression (vitamin B12)
producing A223V) ‡ Homozygote At risk for hyperhomocysteinemia, increased
CVD risk, neural tube defects
Microalbumin, Intact <30 μg/mg Cr Normal ACE inhibitor drugs, angiotensin
30-299 μg/mg Cr Microalbuminuria receptor blockers
r300 μg/mg Cr Clinical albuminuria
<30 mg/24 h Normal
30-299 mg/24 h Microalbuminuria
r300 mg/24 h Clinical albuminuria
<20 μg/min Normal
20-199 μg/min Microalbuminuria
r200 μg/min Clinical albuminuria
ProBNP, N-terminal Age <50 y:
300 pg/mL Normal; heart failure unlikely ACE inhibitor drugs, angiotensin
>450 pg/mL High probability of heart failure (levels correlate receptor blockers, beta-
with NY heart classification); increased risk of adrenergic blockers (eg,
future cardiac events carvedilol), aldosterone
Age >50 y: antagonists, BNP
300 pg/mL Normal; heart failure unlikely
>900 pg/mL High probability of heart failure (levels correlate
with NY heart classification); increased risk of
future cardiac events
Prothrombin (factor II) No mutation Normal (wild type) Anticoagulants
20210GmA mutation analysis‡ Heterozygote Increased risk of venous thrombosis (3- to 6-fold),
obstetrical complications, and possibly
premature coronary heart disease
Homozygote Rare
(continued)
 19

Section 1 Cardiovascular Tests

Table 14. Non-Lipid Markers of Cardiovascular Disease (CVD)* (continued)

Marker Adult Level Interpretation Therapeutic Options

PTT-LA with Reflex to Hexagonal <8 second reduction Probable absence of phospholipid antibodies/ Anticoagulants, antiplatelet
Phase Neutralization of aPTT after lupus anticoagulant therapy, corticosteroids
addition of
hexagonal phase
phospholipid
>8 second reduction Increased risk for CVD, cerebrovascular and
of aPTT after thromboembolic disease; associated with recurrent
addition of fetal loss, thrombocytopenia, and systemic lupus
hexagonal phase erythematosus (SLE)
phospholipid
APC-R, activated protein C resistance; M, male; F, female; DIC, disseminated intravascular coagulation; Cr, creatinine.
*Cardiovascular disease includes atherosclerotic coronary heart disease and arterial thrombosis (peripheral and cerebral).
†
Ratio: aPTT with activated protein C: baseline aPTT.
‡
This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.

who have low CRP levels after statin therapy have better clinical
outcomes regardless of the resultant LDL level.16 Statin therapy also
appears to reduce the risk of major adverse cardiac events after stent
implantation in patients with elevated CRP levels.17 Furthermore,
evidence from multiple studies indicate that intensive statin therapy
leads to an early reduction in cardiac events, sustained for over 2 years,
in patients with acute coronary syndrome.18 Such reduction is likely
related to diminished inflammation as evidenced by greater decreases
in CRP levels observed after statin therapy than observed after
placebo.19 Weight loss20 and regular physical activity,21 both associated
with reduced cardiovascular risk, appear to have anti-inflammatory
effects as well (ie, reduced CRP, fibrinogen, and white blood cell levels).
Individuals Suitable for Testing include those without a previous
history of CHD, especially those with intermediate CHD risk (10-year risk
= 10% to 20% according to Framingham global risk scoring system1,22),
and those with stable or acute coronary disease.
Method: This nephelometric method utilizes latex particles coated with
CRP monoclonal antibodies. The assay is standardized against the
International Federation of Clinical Chemistry and Laboratory Medicine
(IFCC)/ Bureau Communautaire de Référence (BCR)/College of American
Pathologists (CAP) CRP reference preparation. The analytical sensitivity
is 0.2 mg/L. Cardio CRP results are reported in mg/L with an interpretive
comment regarding the risk for CHD.
Reference Range: See Table 15.
Interpretive Information: Ideally, CRP levels should be measured
twice, 2 weeks apart, and the average of the 2 values used for risk
assessment.
Table 15. Cardio CRP Reference Range1
Cardio CRP mg/L Relative Cardiovascular Risk
<1.0 Low
1.0-3.0 Average
3.1-10.0 High
>10.0 Persistent elevations may represent
non-cardiovascular inflammation
The reference ranges listed in Table 15 are derived from a study of more
than 40,000 adults from various populations.1 CRP values in the range of
3.1 to 10 mg/L indicate an approximate 2.0 relative risk of CVD
compared with those in the lowest tertile. Levels persistently above 10
mg/L may indicate an acute inflammatory process; sources of infection
or inflammation should be sought and the test repeated at least 2
weeks later, after the inflammatory response has resolved.1
The following are associated with increased CRP levels: elevated blood
pressure, elevated body mass index, cigarette smoking, metabolic
syndrome, diabetes, low HDL levels, high triglyceride levels, use of
estrogen or progesterone, and chronic infections or inflammation.
Moderate alcohol intake, physical activity, weight loss, and medications
including statins, fibrates, and niacin are associated with decreased
levels.1
References
1. Pearson TA, Mensah GA, Alexander RW, et al. Markers of
inflammation and cardiovascular disease: application to clinical and
public health practice: A statement for healthcare professionals
from the Centers for Disease Control and Prevention and the
American Heart Association. Circulation. 2003;107:499-511.
2. Rost NS, Wolf PA, Kase CS, et al. Plasma concentration of C-
reactive protein and risk of ischemic stroke and transient ischemic
attack: The Framingham Study. Stroke. 2001;32:2575-2579.
3. Di Napoli M, Papa F; for the Villa Pini Stroke Data Bank
Investigators. Inflammation, hemostatic markers, and antithrombotic
agents in relation to long-term risk of new cardiovascular events in
first-ever ischemic stroke patients. Stroke. 2002;33:1763-1771.
4. Rossi E, Biasucci LM, Citterio F, et al. Risk of myocardial infarction
and angina in patients with severe peripheral vascular disease:
predictive role of C-reactive protein. Circulation. 2002;105:800-803.
5. Zebrack JS, Muhlestein JB, Horne BD, et al. C-reactive protein and
angiographic coronary artery disease: independent and additive
predictors of risk in subjects with angina. J Am Coll Cardiol.
2002;39:632-637.
6. Yin WH, Chen JW, Jen HL, et al. Independent prognostic value of
elevated high-sensitivity C-reactive protein in chronic heart failure.
Am Heart J. 2004;147:931-938.
7. Buffon A, Liuzzo G, Biasucci LM, et al. Preprocedural serum levels of
C-reactive protein predict early complications and late restenosis
after coronary angioplasty. J Am Coll Cardiol. 1999;34:1512-1521.
8. Albert CM, Ma J, Rifai N, et al. Prospective study of C-reactive
20

Section 1 Cardiovascular Tests

protein, homocysteine, and plasma lipid levels as predictors of Table 16. Evaluation of BNP and NT-proBNP Clinical
sudden cardiac death. Circulation. 2002;105:2595-2599. Performance
9. Sesso HD, Buring JE, Rifai N, et al. C-reactive protein and the risk
of developing hypertension. JAMA. 2003;290:3000-3002. Sensitivity Specificity PPV NPV
10. Schmidt R, Schmidt H, Curb JD, et al. Early inflammation and Study (%) (%) (%) (%)
dementia: a 25-year follow-up of the Honolulu-Asia Aging Study.
Ann Neurol. 2002;52:168-174. Diagnose impaired LVEF3
11. Pradhan AD, Manson JE, Rifai N, et al. C-reactive protein, BNP 73 77 70 79
interleukin 6, and risk of developing type 2 diabetes mellitus. NT-proBNP 70 73 61 80
JAMA. 2001;286:327-334. Diagnose LV systolic
12. Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein dysfunction after MI2
and low-density lipoprotein cholesterol levels in the prediction of BNP 68 69 56 79
first cardiovascular events. N Engl J Med. 2002;347:1557-1565. NT-proBNP 71 69 56 80
13. Balk E, Lau J, Goudas L, et al. Effects of statins on nonlipid serum
markers associated with cardiovascular disease. Ann Intern Med. Diagnose LV systolic
2003;139:670-682. dysfunction after MI15
14. Ridker PM, Rifai N, Clearfield M, et al. Measurement of C-reactive BNP 94 40 NG 96
protein for the targeting of statin therapy in the primary prevention NT-proBNP 94 37 NG 96
of acute coronary events. N Engl J Med. 2001;344:1959-1965. Prognosis in newly
15. Ridker PM. Inflammatory biomarkers, statins, and the risk of stroke: diagnosed heart failure
cracking a clinical conundrum. Circulation. 2002;105:2583-2585. patients: prediction of
16. Ridker PM, Cannon CP, Morrow D, et al. C-reactive protein levels mortality/survival1
and outcomes after statin therapy. N Engl J Med. 2005;352:20-28. BNP 98 22 42 94
17. Walter DH, Fichtlscherer S, Britten MB, et al. Statin therapy, NT-proBNP 95 37 47 93
inflammation, and recurrent coronary events in patients following
coronary stent implantation. J Am Coll Cardoiol. 2001;38:2006-2012. Prognosis post myocardial
18. Ray KK, and Cannon CP. Intensive statin therapy in acute coronary infarction: prediction of
syndromes: clinical benefits and vascular biology. Curr Opin Lipidol. mortality2
2004;15:637-643. BNP 86 72 39 96
19. Kinlay S, Schwartz GG, Olsson AG, et al. High-dose atorvastatin NT-proBNP 91 72 39 97
enhances the decline in inflammatory markers in patients with Prognosis post myocardial
acute coronary syndromes in the MIRACL study. Circulation. infarction: prediction of
2003;108:1560-1566. heart failure2
20. Tchernof A, Nolan A, Sites CK, et al. Weight loss reduces C-reactive BNP 85 73 54 93
protein levels in obese postmenopausal women. Circulation. NT-proBNP 82 69 50 91
2002;105:564-569.
PPV, positive predictive value; NPV, negative predictive value; NG, not given.
21. Abramson JL, Vaccarino V. Relationship between physical activity
and inflammation among apparently healthy middle-aged and older
US adults. Arch Intern Med. 2002;162:1286-1292. BNP increases glomerular filtration rate, decreases sodium retention,
22. National Cholesterol Education Program. Executive summary of the and inhibits renin and aldosterone secretion. It is a marker of cardiac
third report of the National Cholesterol Education Program (NCEP) dysfunction that correlates with the severity of symptomatic and
expert panel on detection, evaluation, and treatment of high blood asymptomatic left ventricular hypertrophy4 and CHF5 (including the
cholesterol in adults (Adult Treatment Panel III). National Institutes NYHA classification6). BNP can be used to differentiate cardiac failure
of Health. National Heart, Lung, and Blood Institute. NIH Publication from primary lung disease in patients with acute dyspnea7 and to
No. 01-3670; May, 2001. Available at http://rover2.nhlbi.nih.gov/ indicate increased left ventricular mass in patients with essential
guidelines/cholesterol/atp3xsum.pdf. Accessed June 23, 2001. Also hypertension.8 Since NT-proBNP levels also correlate strongly with heart
published in JAMA: JAMA. 2001;285:2486-2509. failure, either can be used to triage symptomatic patients (eg, patients
with dyspnea). When levels are within the normal reference range, the
1.3.3. Congestive Heart Failure (BNP and NT-proBNP) patient’s symptoms are probably not due to heart failure. Conversely,
when levels are elevated, there is an increased probability of heart
Clinical Use: These tests are used to rule out congestive heart failure failure and further cardiac assessment is warranted.
(CHF) in symptomatic individuals; determine prognosis in individuals
with CHF or other cardiac disease; and maximize therapy in individuals The degree of BNP or NT-proBNP elevation can be used to predict future
with heart failure cardiac events and survival. For example, BNP levels predicted heart
failure and death after myocardial infarction.9 Similarly, the relative risk
Clinical Background: B-type, or brain, natriuretic peptide (BNP) was of death based on baseline NT-proBNP levels in patients presenting
first isolated from brain tissue, but is synthesized primarily in the with chest pain and no ST-segment elevation was 4.2, 10.7, and 26.6 for
ventricles of the heart. Cleavage of the 108-amino acid precursor of BNP NT-proBNP quartiles 2, 3, and 4, respectively (relative to the 1st
(proBNP) produces two molecules: (1) BNP, the active C-terminal, 77 to quartile).10
108-amino acid molecule; and (2) N-terminal proBNP (NT-proBNP), the
inactive 1 to 76-amino acid molecule. Studies indicate that NT-proBNP If early studies are confirmed and clinical cut-points are established,
testing has the same clinical utility as BNP testing (Table 16).1,2,3 The these markers may assist in maximizing therapy. Troughton et al showed
assay results, however, are not interchangeable even though levels are improved treatment outcomes when a specific NT-proBNP level (<200
similar in healthy subjects. pmol/L) was used as the therapeutic target rather than clinical criteria.11
 21

Section 1 Cardiovascular Tests

Richards et al showed that NT-proBNP levels above the median
predicted response to carvedilol therapy in a group of patients with
chronic ischemic (LV) dysfunction.12
Investigation into the utility of BNP testing for heart disease screening
in a primary care setting (high-risk individuals and/or the general
population) is also on-going.4,13
Quest Diagnostics offers both BNP and NT-proBNP tests (Table 17).
Individuals Suitable for Testing include patients with suspected or
diagnosed heart failure.
Methods
BNP: This fully automated (ADVIA Centaur®’) immunochemiluminometric
assay (ICMA) utilizes a capture antibody specific for the C-terminal end
of BNP-32 and a detection antibody specific for the intramolecular ring
structure of BNP-32. Bound BNP is separated from free BNP via
streptavidin-coated magnetic particles. Quantitation is based on the
relative light units (RLU) produced in a chemical reaction. The analytical
sensitivity is 4 pg/mL. There is no cross-reactivity with ANP, CNP (7-28),
or NT-proBNP (1-76). The reportable range is 4 to 4,200 pg/mL.
proBNP, N-terminal: This immuno(electro)chemiluminescence assay
(ICMA) utilizes 2 different polyclonal sheep NT-proBNP antibodies (one
labeled with biotin and the other labeled with ruthenium). Bound NT-
proBNP is separated from free NT-proBNP via streptavidin-coated
magnetic microparticles. Quantitation is based on the RLU produced in an
electrochemical reaction between ruthenium and tri-propylamine (TPA).
The analytical sensitivity is 10 pg/mL and there is no cross-reactivity
with ANP, BNP, or CNP; there is no interference from hemoglobin (<1.4
g/dL), triglyceride (<4000 mg/dL), bilirubin (<35 mg/dL), biotin (<30 ng/dL),
rheumatoid factors (<1500 IU/mL), or heterophilic antibodies. The assay’s
reportable range is 10 – 35,000 pg/mL.
Reference Range:
BNP: <100 pg/mL14
proBNP, N-terminal: < 300 pg/mL
The NT-proBNP reference range is based on EDTA plasma. Other sample
types will produce higher values.
Interpretive Information
Symptomatic patients who present with a BNP or NT-proBNP level
within the normal reference range are highly unlikely to have CHF.
Conversely, an elevated baseline level indicates the need for further
cardiac assessment and indicates the patient is at increased risk for
future heart failure and mortality.
BNP levels increase with age in the general population, with the highest
concentrations seen in those greater than 75 years of age.16 Heart
failure is unlikely in individuals with a BNP level <100 pg/mL and
proBNP level < 300 pg/mL. Heart failure is very likely in individuals with
a BNP level >500 pg/mL and proBNP level >450 pg/mL who are <50
years of age, or >900 pg/mL for patients >50 years of age. Patients in
between are either hypertensive or have mild ischemic or valvular
disease and should be observed closely.17

Table 17. Specifications for the Quest Diagnostics BNP and proBNP, N-terminal Assays

BNP proBNP, N-terminal

SAMPLE REQUIREMENTS
Preferred specimen type EDTA plasma in plastic tube EDTA plasma
Specimen volume 1 mL (0.5 mL minimum) 1 mL (0.3 mL minimum)
Handling instructions Collect in plastic tube; transfer plasma to plastic Separate plasma as soon after collection as possible.
screw-cap tube. Ship frozen. Do not use glass tubes. Ship refrigerated.
ASSAY DESCRIPTION
Method Immunochemiluminometric assay Immuno(electro)chemiluminescence
Measures Active, C-terminal, 77-108 amino acids Inactive, N-terminal 1-76 amino acids
Analytic time 1 day 1 day
Automated Yes Yes
FDA status Cleared Cleared
CPT code 83880* 83880*
PERFORMANCE SPECIFICATIONS
Analytical sensitivity 4 pg/mL 10 pg/mL
Analytical specificity No cross-reactivity with ANP, CNP (7-28), or No cross-reactivity with ANP, BNP, or CNP
NT-proBNP (1-76)
Reportable range 4-4200 pg/mL 10-35,000 pg/mL
†
REFERENCE RANGE <100 pg/mL <300 pg/mL
ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide.
*The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions
regarding coding to the payer being billed.
†
Decision threshold based on evaluation of patients with and without heart failure.14
22

Section 1 Cardiovascular Tests

BNP is increased in CHF, left ventricular hypertrophy, acute myocardial
infarction, atrial fibrillation, cardiac amyloidosis, and essential
hypertension. Elevations are also observed in right ventricular
dysfunction, pulmonary hypertension, acute lung injury, subarachnoid
hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.
NT-proBNP levels are increased in CHF, left ventricular dysfunction,
myocardial infarction, valvular disease, hypertensive pregnancy, and
renal failure, even after hemodialysis.
Although levels of BNP and NT-proBNP are similar in normal individuals,
NT-proBNP levels are substantially greater than BNP levels in patients
with cardiac disease due to increased stability (half-life) of NT-proBNP
in circulation. Thus, results from the two tests are not interchangeable.
volume and function. A prospective study of 150 patients. Dtsch
Med Wochenschr. 2002;127:2605-2609.
16. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain
natriuretic peptide concentration: impact of age and gender. J Am
Coll Cardiol. 2002;40:976-982.
17. Weber M, Hamm C. Role of B-type natriuetic peptid (BNP) and NT-
proBNP in clinical routine. Heart. 2006;92:843-849.
1.3.4 Factor V (Leiden) Mutation Analysis
See Coagulation, “Thrombophilia” section 3.5.4.
1.3.5. Heparin-induced Thrombocytopenia: Serotonin Release
Assay (SRA) and Heparin-induced Platelet Antibody
See Coagulation, “Thrombocytopenia” section 3.4.1.

References 1.3.6 Homocysteine

See Genetics, section 5.2.3.
1. Cowie MR and Mendez GF. BNP and congestive heart failure. Prog
Cardiovasc Dis. 2002;44:293-321.
1.3.7 Lp-PLA2 (PLAC™)
2. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-
brain natriuretic peptide and adrenomedullin. New neurohormonal
Clinical Use: This test is used to assess risk of coronary heart disease
predictors of left ventricular function and prognosis after myocardial
and stroke
infarction. Circulation. 1998:97:1921-1929.
3. Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head
Clinical Background: Traditional markers of lipidemia identify only
comparison of N-terminal pro-brain natriuretic peptide, brain
about half of the individuals at risk of cardiovascular disease.
natriuretic peptide and N-terminal pro-atrial natriuretic peptide in
Atherosclerosis is now recognized as an inflammatory disease, and
diagnosing left ventricular dysfunction. Clin Chim Acta.
inflammatory markers, most notably C-reactive protein (CRP), have been
2001;310:193-197.
shown to identify additional individuals who are at risk. Other
4. McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection
inflammatory markers are currently being studied. One of these is
of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as
5. Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as
platelet activating factor acetylhydrolase (PAF-AH). More than 80% of
a novel cardiac hormone in humans: Evidence for an exquisite dual
circulating Lp-PLA2 is bound to LDL cholesterol, preferentially to the
natriuretic peptide system, ANP and BNP. J Clin Invest.
small, dense LDL; the remainder is bound to HDL cholesterol and very
1991;87:1402-1412.
low-density lipoproteins.
6. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-
terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of
Increased Lp-PLA2 activity or blood levels have been seen in cerebral
cardiac impairment. Clin Endocrinol. 1997;47:287-296.
thrombosis1-3 and in various types of CHD.4-12 Three large, prospective
7. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic
studies have evaluated the ability of Lp-PLA2 to predict CHD
peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-
independently of other markers, including lipid markers. Blake et al
444.
found that Lp-PLA2 was not a very useful predictor in women
8. Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a
participating in the Women’s Health Study (WHS).13 Packard et al, on
cardiac hormone in essential hypertension. Am J Med. 1992;92:29-
the other hand, found almost a doubling of risk for CHD in men
34.
participating in the West of Scotland Coronary Prevention Study
9. Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical
(WOSCOPS) when Lp-PLA2 levels were in the highest quintile.8
significance of brain natriuretic peptide in patients with
Ballantyne et al (Atherosclerosis Risk in Communities [ARIC] Study)
postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
showed that both men and women had a 2-fold increase in risk when
10. Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain
their Lp-PLA2 levels were in the 3rd tertile and their LDL cholesterol
natriuretic peptide on admission for early risk stratification of
levels were <130 mg/dL. Moreover, CHD risk was 3 times greater in
patients with chest pain and no ST-segment elevation. J Am Coll
those with the highest levels of both CRP and Lp-PLA2.14
Cardiol. 2002;40:437-445.
11. Troughton RW, Frampton CM, Yandle TG, et al. Treatment of heart
Currently, there are no consensus recommendations from independent
failure guided by plasma aminoterminal brain natriuretic peptide (N-
professional organizations or agreement in published literature on either
BNP) concentrations. Lancet. 2000;355:1126-1130.
subjects with no prior history of CAD or subjects with acute coronary
12. Richards AM, Doughty R, Nicholls MG, et al. Plasma N-terminal pro-
syndrome as to an appropriate upper tertile cutoff with earlier trials
brain natriuretic peptide and adrenomedullin. Prognostic utility and
reporting higher values. For example, in the MONICA trial published in
prediction of benefit from carvedilol in chronic ischemic left
2004, the relative risk was found to be higher in the upper tertile
ventricular dysfunction. J Am Coll Cardiol. 2001;37:1781-1787.
defined as r290.8 ng/mL. In the ARIC trial an association was found at
13. Smith H, Pickering RM, Struthers A, et al. Biochemical diagnosis of
the upper tertile defined as r422 ng/mL only in subjects with LDL levels
ventricular dysfunction in elderly patients in general practice:
<130 mg/dL. These differences may be related to earlier versions of the
observational study. BMJ. 2000;320:906-908.
Lp-PLA2 assay or to differences in study populations.
14. Siemens ADVIA Centaur® BNP directional insert; 2003.
15. Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic
Therapies such as fenofibrate10 and atorvastatin12 are being evaluated
peptides NT-pro-BNP and BNP for the assessment of left-ventricular
for anti-Lp-PLA2 activity. Furthermore, Lp-PLA2 inhibitors are being

Section 1
sought for drug development. Thus, Lp-PLA2 levels may one day be
useful in selecting optimal therapies for individual patients.
Individuals Suitable for Testing include those with a family history
of CHD, borderline LDL levels (100 to 129 mg/dL), optimal LDL levels
(<100 mg/dL) but with other CHD risk factors, and in whom CRP cannot
be performed owing to presence of a known or suspected inflammatory
condition
Method: In this enzyme-linked immunosorbent assay (ELISA) anti-Lp-
PLA2 monoclonal antibodies bound to microtiter wells react with patient
sample. After washing to remove unbound antigen, a second anti-Lp-
PLA2 monoclonal antibody conjugated to horseradish peroxidase is
added. Wells are again washed and a chromogenic substrate is added
and the change in color, which is proportional to the concentration of
Lp-PLA2, is measured.
Reference Range:
Men: 115-245 ng/mL
Women: 85-245 ng/mL
Interpretive Information: Increased Lp-PLA2 levels are associated
with CHD and stroke or with increased risk of CHD and stroke. Levels
may be increased in patients with essential hypertension, hyper-
cholesterolemia, atherosclerosis, myocardial infarct, and cerebral
thrombosis.14
References
1. Hiramoto M, Yoshida H, Imaizumi T, et al. A mutation in plasma
platelet-activating factor acetylhydrolase (Val279mPhe) is a genetic
risk factor for stroke. Stroke. 1997;28:2417-2420.
2. Satoh K, Yoshida H, Imaizumi T, et al. Platelet-activating factor
acetylhydrolase in plasma lipoproteins from patients with ischemic
stroke. Stroke. 1992;23:1090-1092.
3. Satoh K, Imaizumi T, Kawamura Y, et al. Activity of platelet-
activating factor (PAF) acetylhydrolase in plasma from patients with
ischemic cerebrovascular disease. Prostaglandins. 1988;35:685-698.
4. Blankenberg S, Stengel D, Rupprecht HJ, et al. Plasma PAF-
acetylhydrolase in patients with coronary artery disease: results of a
cross-sectional analysis. J Lipid Res. 2003;44:1381-1386.
5. Caslake MJ, Packard CJ, Suckling KE, et al. Lipoprotein-associated
phospholipase A(2), platelet-activating factor acetylhydrolase: a
potential new risk factor for coronary artery disease.
Atherosclerosis. 2000;150:413-419.
6. Karabina SA, Elisaf M, Bairaktari E, et al. Increased activity of
platelet-activating factor acetylhydrolase in low-density lipoprotein
subfractions induces enhanced lysophosphatidylcholine production
during oxidation in patients with heterozygous familial
hypercholesterolaemia. Eur J Clin Invest. 1997;27:595-602.
7. Ostermann G, Lang A, Holtz H, et al. The degradation of platelet-
activating factor in serum and its discriminative value in
atherosclerotic patients. Thromb Res. 1988;52:529-540.
8. Packard CJ, O’Reilly DSJ, Caslake MJ, et al. Lipoprotein-associated
phospholipase A2 as an independent predictor of coronary heart
disease. N Engl J Med. 2000;343:1148-1155.
9. Satoh K, Imaizumi T, Kawamura Y, et al. Increased activity of the
platelet activating factor acetylhydrolase in plasma low density
lipoprotein from patients with essential hypertension.
Prostaglandins. 1989;37:673-682.
10. Tsimihodimos V, Kakafika A, Tambaki AP, et al. Fenofibrate induces
HDL-associated PAF-AH but attenuates enzyme activity associated
with apoB-containing lipoproteins. J Lipid Res. 2003;44:927-934.
11. Tsimihodimos V, Karabina SA, Tambake AP, et al. Altered distribution
23
Cardiovascular Tests
of platelet-activating factor-acetylhydrolase activity between LDL
and HDL as a function of the severity of hypercholesterolemia. J
Lipid Res. 2002;43:256-263.
12. Tsimihodimos V, Karabina SA, Tambaki AP, et al. Atorvastatin
preferentially reduces LDL-associated platelet-activating factor
acetylhydrolase activity in dyslipidemias of type IIA and type IIB.
Aterioscler Thromb Vasc Biol. 2002;22:306-311.
13. Blake GJ, Dada N, Fox JC, et al. A prospective evaluation of
lipoprotein-associated phospholipase A(2) levels and the risk of
future cardiovascular events in women. J Am Coll Cardiol.
2001;38:1302-1306.
14. Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-
associated phospholipase A2, high-sensitivity C-reactive protein, and
risk for incident coronary heart disease in middle-aged men and
women in the Atherosclerosis Risk in Communities (ARIC) Study.
Circulation. 2004;109:837-842.
15. Dada N, Kim NW, Wolfert RL. Lp-PLA2: an emerging biomarker of
coronary heart disease. Expert Rev Mol Diagn. 2002;2:17-22.
1.3.8 Microalbumin, Urine
See Chronic Kidney Disease, “Microalbumin” section 2.3.
1.3.9 Microalbumin, Intact, HPLC
See Chronic Kidney Disease, “Microalbumin” section 2.4.
1.3.10 Plasma Renin Activity (PRA)
Clinical Use: This test is predominantly used to rule out primary
hyperaldosteronism (see The Quest Diagnostics Manual: Endocrinology
Test Selection and Interpretation). It may also be of use in
differentiating low renin (sodium/volume) and renin-mediated idiopathic
primary hypertension.
Clinical Background: Renin plays a central role in maintaining blood
pressure by enzymatically converting angiotensinogen to angiotensin I,
which is then cleaved by angiotensin converting enzyme (ACE) to form
angiotensin II. Angiotensin II increases blood pressure directly through
vasoconstriction and indirectly by stimulating secretion of aldosterone, a
hormone that promotes sodium retention and potassium loss through its
action on the distal nephron of the kidney. Renin secretion is inhibited
by high blood pressure and stimulated by factors that lower blood
pressure, such as upright posture, sodium deprivation, and drugs such
as captopril. PRA is typically used in conjunction with the measurement
of aldosterone, sodium, and potassium levels in plasma and/or urine.
In the work-up of hypertension, renin levels r0.65 ng/mL/h rule out
primary hyperaldosteronism and suggest the hypertension is likely
idiopathic. In untreated hypertension, the likelihood of renovascular
hypertension is low if renin activity levels are b1.6 ng/mL/h. Conversely,
if renin levels are <0.65 ng/mL/h, an aldosterone test should be ordered
to rule out primary hyperaldosteronism.1 If this condition is ruled out,
low renin levels indicate that the hypertension may be caused by
sodium and water retention and that renin plays a negligible role.
Differentiating these underlying causes of hypertension may allow more
rational treatment selection.
Individuals Suitable for Testing include those suspected of having
secondary hypertension and those with idiopathic primary hypertension.
Method: Angiotensinogen in patient plasma is first converted to
angiotensin I via renin enzymatic activity. Next, the amount of
angiotensin I pre and post enzymatic conversion is measured by
radioimmunoassay. The rate of angiotensin I production, which is
directly proportional to the amount of renin, is then calculated.
24

Section 1 Cardiovascular Tests

Reference Range: Table 19. Plasma Renin Activity (PRA) Diagnostic Reference
Table 18 lists reference ranges for apparently healthy, non-medicated, Ranges*
non-pregnant adults and children on unrestricted diets. Values may be
affected by sodium intake, posture, and medications. Condition PRA (ng/mL/h)
Interpretive Information: Patients with primary aldosteronism Sodium/volume hypertension <0.65
typically have low renin concentrations associated with elevated Primary hyperaldosteronism possible
aldosterone levels. If primary hyperaldosteronism is ruled out (see Renovascular hypertension highly unlikely
Clinical Background), a low PRA suggests sodium/volume mediated Renin-mediated hypertension likely r0.65
causes of hypertension. Such causes may be essential or secondary to Primary hyperaldosteronism unlikely†
diet, medication, or certain uncommon endocrine disorders (eg,
*In ambulatory, briefly seated hypertensive patients; values may be affected by sodium
pheochromocytoma, increased mineralocorticoid activity from causes intake, posture, and medications.
other than primary aldosteronism). Patients with low-renin essential †
Rule out primary hyperaldosteronism by using PRA in conjunction with aldosterone. Many
hypertension are candidates for treatment targeting volume and salt non-pregnant adults with primary hyperaldosteronism have an aldosterone/PRA ratio >30.
depletion; a low sodium diet and drugs such as diuretics, alpha
adrenergic blockers, aldosterone receptor blockers, or calcium channel
blockers may be used. Patients with renin-mediated hypertension 4. Fukushige J, Shimomura K, Ueda K. Influence of upright activity on
typically have PRA r0.65 ng/mL/h and generally respond better to beta- plasma renin activity and aldosterone concentration in children. Eur J
blockers, ACE inhibitors, and angiotensin receptor blockers. While Pediatr. 1994;153:284-286.
extremes in PRA levels may either exclude or suggest renovascular 5. Wilcox CS. Functional testing: renin studies. Semin Nephrol.
hypertension, the high degree of overlap with other causes of increased 2000;20:432-436.
PRA (eg, dehydration, diuretic use, etc.) makes static tests of PRA less 6. Textor SC. Renovascular hypertension update. Curr Hypertens Rep.
desirable for diagnosis of renovascular hypertension.5-7 The captopril 2006;8:521-527.
provocation tests that measure either an ACE-stimulated increase in 7. Klassen PS, Svetkey LP. Diagnosis and management of renovascular
PRA or decreased renal perfusion as evidenced on scintigraphy are more hypertension. Cardiol Rev. 2000;8:17-29.
sensitive and specific for detecting renovascular hypertension. The most
reliable diagnostic method is magnetic resonance angiography.7 1.3.11 Prothrombin (Factor II) 20210GmA Mutation Analysis
Additional causes of hypertension (eg, pheochromocytoma and See Coagulation, “Thrombophilia” section 3.5.7.
medications) may need to be ruled out. Table 19 summarizes the clinical
cutoffs for various disorders.

References
1. Sealey JE, Gordon RD, and Mantero F. Plasma renin and aldosterone
measurements in low renin hypertensive states. Trends Endocrinol
Metab. 2005;16:86-91.
2. Stalker HP, Holland NH, Kotchen JM, et al. Plasma renin activity in
healthy children. J Pediatr. 1976;89:256-258.
3. Sulyok E, Nemeth M, Tenyi I, et al. Postnatal development of renin-
angiotensin-aldosterone system, RAAS, in relation to electrolyte
balance in premature infants. Pediatr Res. 1979;13:817-820.

Table 18. Plasma Renin Activity (PRA) Reference Ranges for

Non-hypertensive Adults and Children
PRA (ng/mL/h)
Upright or
Age Supine Sitting
Adults 0.65-5.0
2,3,4
Children
3-12 mo b15.0 NG
1-3 y b10.0 NG
4-6 y b7.5 b15.0
7-9 y b5.9 b17.0
10-12 y b5.3 b16.0
13-15 y b4.4 b16.0
NG, not given.

Section 2
2.1 Cystatin C
Clinical Use: This test is used to assess glomerular filtration rate in
type 1 and 2 diabetes mellitus.
Clinical Background: Cystatin C is a low molecular weight (13,359
Da) protein belonging to the superfamily of cystine protease inhibitors.
It is produced by all nucleated cells at a reasonably constant rate;
production is minimally affected by diet, inflammatory states, lean
body mass, or circadian rhythm. Like creatinine, cystatin C is freely
filtered by the glomeruli. It is reabsorbed and metabolized by renal
tubular cells and does not appear in urine. Tan et al (Diabetes Care.
2002;25:2004-2009) showed serum cystatin C has good correlation with
creatinine clearance (p = 0.74) and iohexol clearance (p = –0.80) and is
a useful marker of renal dysfunction in diabetic patients with minimal
tubular reabsorption. Because creatinine production is relatively
variable, cystatin C has been proposed, but is not yet recommended, as
a better marker of glomerular filtration.
Method: Nephelometry
Interpretive Information: Increased levels are associated with
impaired glomerular filtration rate.
2.2 Glomerular Filtration Rate, Estimated
(eGFR)
Clinical Use: This test is used in adults for early detection of chronic
kidney disease (CKD) and to monitor CKD therapy and/or progression.
Clinical Background: Approximately 20 million people in the United
States are currently affected by CKD, and the incidence and prevalence
of ensuing kidney failure is rising. Since evidence has shown that
treatment at earlier stages is generally effective in preventing or
delaying adverse outcomes, monitoring patients with and at risk of CKD
becomes critically important for decreasing morbidity and mortality.
CKD is defined by the presence of glomerular filtration rate (GFR) <60
mL/min/1.73m2 for r3 months and/or evidence of kidney damage (eg,
structural abnormalities visualized on biopsy, imaging studies, and
proteinuria) for r3 months.1 Thus, monitoring should include tests for
GFR, microalbuminuria, and presence of red and white blood cells in the
urine. In most patients, microalbuminuria becomes evident before GFR is
substantially reduced.
GFR has traditionally been estimated using the 24-hour creatinine
clearance; however, a calculation of estimated glomerular filtration rate
(eGFR) is now recommended by the National Institutes of Health (NIH)
and the National Kidney Foundation. Since a 24-hour urine collection is
not needed for the eGFR, the eGFR is a simpler test.
Individuals Suitable for Testing include those at risk of CKD and
those with CKD. Individuals with diabetes, hypertension, autoimmune
disease, systemic infections, and family history of kidney disease are
some of those at increased risk. Also at risk are African Americans,
Hispanics, Asian Americans, Pacific Islanders, Native Americans, and
those >60 years of age.
Method: The eGFR is calculated using isotope dilution mass
spectrometry (IDMS)-traceable serum creatinine measurements and the
patient’s age (18 to 70 years), gender, and race (African American vs
non-African American) according to the Modification of Diet in Renal
Disease (MDRD) study formula.2,3,4 Alternative calculations have been
proposed for patients under age 18 years, but they are not offered by
25
Chronic Kidney Disease
Table 1. Test Order Codes
Test Name Test Code
BUN/Creatinine Ratio with Glomerular Filtration Rate, 296X
Estimated (eGFR)
Creatinine with Glomerular Filtration Rate, Estimated (eGFR) 375X
Creatinine Clearance with Glomerular Filtration Rate, 7943X
Estimated (eGFR)
Basic Metabolic Panel with Glomerular Filtration Rate, 10165X
Estimated (eGFR)
Includes BUN/creatinine ratio (calculated), calcium, carbon dioxide,
chloride, creatinine, eGFR (calculated), glucose, potassium, sodium,
and urea nitrogen (BUN).
Comprehensive Metabolic Panel with Glomerular 10231X
Filtration Rate, Estimated (eGFR)
Includes albumin, albumin/globulin ratio (calculated), alkaline
phosphatase, ALT, AST, BUN/creatinine ratio, calcium, carbon
dioxide, chloride, creatinine, eGFR (calculated), globulin (calculated),
glucose, potassium, sodium, total bilirubin, total protein, and urea
nitrogen.
Renal Function Panel with Glomerular Filtration Rate, 10314X
Estimated (eGFR)
Includes albumin, BUN/creatinine ratio (calculated), calcium, carbon
dioxide, chloride, creatinine, estimated GFR (calculated), glucose,
phosphate (as phosphorous), potassium, sodium, and urea nitrogen (BUN).
Quest Diagnostics at present. Quest Diagnostics’ test codes that include
eGFR are listed in Table 1.
Interpretive Information: CKD stages are based on laboratory
evaluation of the severity of kidney disease and determine the
probability of complications (eg, loss of kidney function and
development of cardiovascular disease). Table 2 lists the stages
associated with specific eGFR values. Only patients in stages 1 through
5 are considered to have CKD. Recommended clinical actions are listed
for each stage and for individuals without CKD but at risk for CKD.
References:
1. K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease:
Evaluation, Classification, and Stratification. National Kidney
Foundation Web site. Available at:
http://www.kidney.org/professionals/kdoqi/guidelines_ckd/toc.htm.
Accessed September 5, 2007.
2. Levey AS, Bosch JP, Lewis JB, et al. A more accurate method to
estimate glomerular filtration rate from serum creatinine: a new
prediction equation. Modification of Diet in Renal Disease Study
Group. Ann Intern Med. 1999;130:461-470.
3. Levey AS, Coresh J, Balk E, et al. National Kidney Foundation
practice guidelines for chronic kidney disease: evaluation,
classification, stratification. Ann Intern Med. 2003;139:137-147.
4. Levey AS, Coresh J, Greene T, et al for the Chronic Kidney Disease
Epidemiology Collaboration. Using standardized serum creatinine
values in the modification of diet in renal disease study equation for
estimating glomerular filtration rate. Ann Intern Med. 2006;145:247-
254.
2.3 Microalbumin, Urine
Clinical Use: This test is used to detect and monitor early renal
disease in patients with diabetes mellitus, detect and monitor
26

Section 2 Chronic Kidney Disease

Table 2. Classification of Chronic Kidney Disease and Clinical Action Plans1

eGFR
(mL/min/1.73m2) Stage Description Clinical Action Plan*
r90 At increased risk Screening; cardiovascular disease risk reduction
(with CKD risk factors)
r90 1 Kidney damage Diagnosis and treatment; treatment of comorbid conditions; slow progression;
with normal or l eGFR cardiovascular disease risk reduction
60-89 2 Kidney damage Estimating progression
with mild n eGFR
30-59 3 Moderate n eGFR Evaluating and treating complications
15-29 4 Severe n eGFR Preparation for kidney replacement therapy
15 5 Kidney failure Replacement (if uremia present)
(or dialysis)
eGFR, estimated glomerular filtration rate.
*Includes actions from preceding stages.

microalbuminuria in patients with cardiovascular disease, and to detect References

kidney damage in individuals at risk for renal disease.
Clinical Background: Microalbuminuria is frequently one of the first
signs of renal disease and may indicate renal damage which, left
untreated, may progress to overt renal failure.1,2 In patients with diabetes,
early detection of microalbuminuria, followed by improvement of glucose
control and treatment with angiotensin converting enzyme inhibitors or
angiotensin receptor blockers, may slow or prevent progression to overt
albuminuria and end stage renal disease.2 Without specific intervention,
80% of patients with type 1 and 20% to 40% of those with type 2
diabetes who have persistent microalbuminuria will progress to overt
nephropathy.2 Additionally, microalbuminuria is independently associated
with an increased risk of cardiovascular events, heart failure, and an
increased incidence of all-cause mortality in the general population.3
Individuals Suitable for Testing include those with diabetes
mellitus without overt albuminuria, those with cardiovascular disease,
and those at risk for renal disease.
Method: This immunoturbidimetric assay is specific for immunoreactive
albumin. The analytical sensitivity is 2 mg/L.
Reference Range: See Table 3.
Interpretive Information: The American Diabetes Association and the
National Kidney Foundation recommend 2 of 3 specimens collected
within a 3 to 6 month period be abnormal before considering a patient
to have microalbuminuria or clinical albuminuria.1,2
Exercise within 24 hours of collection, infection, fever, congestive heart
failure, marked hyperglycemia, marked hypertension, pyuria, and hematuria
may elevate urinary albumin above normal levels. Results should be
interpreted in conjunction with other laboratory and clinical findings.
1. Levey AS, Coresh J, Balk E, et al. National Kidney Foundation
practice guidelines for chronic kidney disease: evaluation,
classification, stratification. Ann Intern Med. 2003;139:137-147.
2. American Diabetes Association position statement: nephropathy in
diabetes. Diabetes Care. 2004;27:S79-S83.
3. Hillege HL, Fidler V, Diercks G, et al. Urinary albumin excretion
predicts cardiovascular and noncardiovascular mortality in general
population. Circulation. 2002;106:1777-1782.
2.4 Microalbumin, Intact
Clinical Use: This test is used to detect and monitor early renal
disease in patients with diabetes mellitus, detect and monitor
microalbuminuria in patients with cardiovascular disease, and to detect
kidney damage in individuals at risk for renal disease.
Clinical Background: Recent studies have shown renal albumin
excretion is more complex than previously thought.1 In healthy
individuals, approximately 1.3 g of albumin leaks through the glomerular
walls daily. Approximately 13 mg (1%) of this albumin passes into the
urine, while the remainder is degraded by lysosomes in the proximal
tubules with subsequent reuptake of the degradation products. In early
renal disease, the lysosomal degradation and reuptake of albumin is
disrupted, and albumin-derived protein fragments, along with
unmodified, immunochemically reactive albumin, leak into the urine.1
It has also been shown that immunochemically unreactive, intact albumin
molecules leak into the urine in early renal disease.1 These molecules are
immunochemically unreactive, presumably due to changes in their 3-
dimensional structure secondary to disruption of disulfide bonds, and
therefore not detectable by routine immunoassay. They are, however,
detectable by high performance liquid chromatography (HPLC).2 Studies

Table 3. Definitions of Albumin Excretion2

Random Collection 24-Hour Collection Timed Collection

(mg/g creatinine) (mg/24 h) (μg/min)
Normal 30 30 20
Microalbuminuria 30–299 30–299 20–199
Clinical albuminuria r300 r300 r200
 27

Section 2 Chronic Kidney Disease

have shown up to 50% of patients with diabetes who are negative for
microalbuminuria by urine dipstick or routine immunoassay will have
positive HPLC results.3 This translates into detection of renal disease an
average of 4 years earlier in these patients.4

Individuals Suitable for Testing include those with diabetes

mellitus without overt albuminuria, those with cardiovascular disease,
and those at risk for renal disease.

Method: This assay utilizes size-exclusion HPLC. The analytical

sensitivity is 3 mg/L, and the assay is specific for immunochemically
reactive and unreactive albumin. Alias: Accumin™’ Direct Albumin.

Reference Range: See Table 3.

Interpretive Information: The American Diabetes Association and the

National Kidney Foundation recommend 2 of 3 specimens collected
within a 3 to 6 month period be abnormal before considering a patient
to have microalbuminuria or clinical albuminuria.5,6

Exercise within 24 hours of collection, infection, fever, congestive heart

failure, marked hyperglycemia, marked hypertension, pyuria, and hematuria
may elevate urinary albumin above normal levels. Results should be
interpreted in conjunction with other laboratory and clinical findings.
References
1. Peters T. New form of urinary albumin in early diabetes. Clin Chem.
2004;12:2238-2239.
2. Comper WD, Osicka TM, Jerums G. High prevalence of immuno-
unreactive intact albumin in urine of diabetic patients. Am J Kidney
Dis. 2003;41:336-342.
3. Comper WD, Jerums G, Osicka TM. Deficiency in the detection of
microalbuminuria by urinary dipstick in diabetic patients. Diabetes
Care. 2003;26:3135-3136.
4. Comper WD, Osicka TM, Clark M, et al. Earlier detection of
microalbuminuria in diabetic patients using a new urinary albumin
assay. Kidney International. 2004;65:1-6.
5. American Diabetes Association position statement: nephropathy in
diabetes. Diabetes Care. 2004;27:S79-S83.
6. Levey AS, Coresh J, Balk E, et al. National Kidney Foundation
practice guidelines for chronic kidney disease: evaluation,
classification, stratification. Ann Intern Med. 2003;139:137-147.
28

Section 3 Coagulation

3.1 Available Tests Antiphospholipid Syndrome Diagnostic Panel

Includes cardiolipin antibody (IgG, IgA, IgM), beta2-glycoprotein I antibody (IgG,
Coagulation Consultation IgA, IgM), lupus anticoagulant with reflex to hexagonal phase confirmation, and
dRVVT screen with reflexes to dRVVT confirm and dRVVT 1:1 mix.
Hemostasis Disorders (Bleeding and Hypercoagulability) Beta2-Glycoprotein I Antibody (IgA)
Alpha2-Antiplasmin Beta2-Glycoprotein I Antibody (IgG)
Factor II Activity, Clotting Beta2-Glycoprotein I Antibody (IgM)
Factor V Activity, Clotting Beta2-Glycoprotein I Antibodies (IgA, IgG, IgM)
Factor V Activity and Human Inhibitor Cardiolipin Antibody (IgA)
Factor VII Activity, Clotting Cardiolipin Antibody (IgG)
Factor VIII Activity, Clotting Cardiolipin Antibody (IgM)
Factor VIII Activity, Chromogenic Cardiolipin Antibody Screen w/ Reflex to IgG and IgM
Factor VIII Activity and Human Inhibitor with Reflex to Nijmegen Assay Cardiolipin Antibody Screen w/ Reflex to IgG, IgA, IgM
Factor VIII Inhibitor Screen, EIA with Reflexes to Clot-based Assays dRVVT Screen with Reflex to dRVVT Confirm and dRVVT 1:1 Mix
Factor IX Activity, Clotting Hexagonal Phase Neutralization
Factor IX Activity and Human Inhibitor Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (on
Factor X Activity, Clotting Coumadin)
Factor X Activity, Chromogenic Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Factor XI Activity, Clotting dRVVT screen with reflex to phospholipid neutralization, hexagonal phase
Factor XI Activity and Human Inhibitor neutralization, prothrombin time with INR, and thrombin clotting time.
Factor XI Mutation Analysis (Ashkenazi Jewish)1 Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (on
Factor XII Activity, Clotting Coumadin) with Consultation
Factor XIII, Functional Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Fibrinogen Activity, Clauss dRVVT screen with reflex to phospholipid neutralization, hexagonal phase
Fibrinogen Antigen, Nephelometry neutralization, prothrombin time with INR, thrombin clotting time, and coagulation
Fibrinolysis Comprehensive Panel consultation.
Includes B2-antiplasmin, D-dimer, euglobulin clot lysis time, fibrin monomer, Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (no
fibrinogen degradation products, PAI-1 activity, functional plasminogen levels, and Coumadin)
TPA. Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Plasminogen Activator Inhibitor (PAI-1) Activity and prolonged aPTT thrombotic evaluation.
Plasminogen Activator Inhibitor-1 Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (no
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism1 Coumadin) with Consultation
Prekallikrein (Fletcher Factor) Activity Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Prothrombin Fragment 1.2 prolonged aPTT thrombotic evaluation, and coagulation consultation.
Reptilase Clotting Time Lupus Anticoagulant Evaluation with Reflex
Thrombin Clotting Time Includes lupus anticoagulant (with reflex to hexagonal phase neutralization) and
Thrombin Clotting Time with Reflex to Mixing Study dRVVT screen (with reflex to dRVVT confirm and dRVVT mix).
Phosphatidic Acid Antibodies (IgG, IgA, IgM)
Heparin-Induced Thrombocytopenia (HIT) Phosphatidylcholine Antibodies (IgG, IgA, IgM)
Heparin-Induced Platelet Antibody Phosphatidylethanolamine Antibodies (IgG, IgA, IgM)
Heparin-Induced Thrombocytopenia Panel Phosphatidylglycerol Antibodies (IgG, IgA, IgM)
Includes heparin-induced platelet antibody and SRA. Phosphatidylinositol Antibodies (IgG, IgA, IgM)
Serotonin Release Assay (SRA)1 Phosphatidylserine Antibodies (IgA)
Phosphatidylserine Antibodies (IgG, IgA, IgM)
Intravascular Coagulation (Local and Disseminated) Phosphatidylserine Antibodies (IgG, IgM)
Alpha2-Antiplasmin Prothrombin Antibodies (IgG, IgM)
Antithrombin III Activity PTT-LA with Reflex to Hexagonal Phase Confirm
Antithrombin III Antigen
Antithrombin III Activity with Reflex to Antithrombin III Antigen Platelet Immune Disorders
Antithrombin III Activity and Antigen Platelet Antibody, Direct, Flow Cytometry1
D-Dimer Platelet Antibody, Indirect (IgG)
Euglobulin Clot Lysis Time Platelet Glycoprotein Antibody
Fibrin Monomer
Fibrinogen Activity, Clauss Screen for Coagulation Disorders
Fibrinogen Antigen, Nephelometry Activated Partial Thromboplastin Time
Fibrinogen Degradation Products (FDP) Activated Partial Thromboplastin Time Mixing Study
Plasminogen, Antigenic Mixing Study
Plasminogen, Activity Includes prothrombin time, prothrombin time mix, PTT-LA, PTT-LA mix, and
Thrombin-Antithrombin (TAT) Complex incubated PTT-LA mix.
Prolonged aPTT Asymptomatic Evaluation
Phospholipid Antibody Syndrome Includes aPTT and thrombin clotting time (TCT) with potential reflexes to minimally
Annexin V Antibodies (IgG, IgM)1 prolonged aPTT/ normal TCT; prolonged aPTT/normal TCT; aPTT mixing study;
Antiphospholipid Antibody Panel dRVVT screen with reflex to phospholipid neutralization; fibrinogen; hexagonal
Includes C2-glycoprotein I antibodies (IgG, IgA, IgM), phosphatidylserine antibodies phase neutralization; prolonged PT evaluation; reptilase clotting time; and specific
(IgG, IgA, IgM), and cardiolipin antibodies (IgG, IgA, IgM). factor assays.
 29

Section 3 Coagulation

Prolonged aPTT Bleeding Evaluation Hexagonal Phase Neutralization

Includes aPTT with potential reflexes to minimally prolonged aPTT, aPTT progressive Homocysteine (Cardiovascular), Serum
mixing study, dRVVT screen with reflex to phospholipid neutralization, factor XII Human Platelet Antigen 1 Genotype
activity, fibrinogen, low molecular weight heparin, prolonged PT evaluation, PTT-LA Lupus Anticoagulant Evaluation with Reflex
with reflex to hexagonal phase neutralization, and thrombin clotting time. Includes lupus anticoagulant (with reflex to hexagonal phase neutralization) and
Prolonged aPTT Thrombotic Evaluation dRVVT screen (with reflex to dRVVT confirm and dRVVT mix).
Includes PTT-LA with reflex to hexagonal phase neutralization and dRVVT screen Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (on
with reflex to phospholipid neutralization and potential reflexes to D-dimer, Coumadin)
fibrinogen profile, low molecular weight heparin, prothrombin time, specific factor Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
assays, and thrombin clotting time. dRVVT screen with reflex to phospholipid neutralization, hexagonal phase
Prolonged PT Evaluation neutralization, prothrombin time with INR, and thrombin clotting time.
Includes prothrombin time with INR and potential reflexes to minimally prolonged Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (on
PT panel, PT progressive mixing study, D-dimer, dRVVT screen with reflex to Coumadin) with Consultation
phospholipid neutralization, factor II and IX activities, fibrinogen activity, and Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
fibrinogen antigen. dRVVT screen with reflex to phospholipid neutralization, hexagonal phase
Prothrombin Mixing Study neutralization, prothrombin time with INR, thrombin clotting time, and coagulation
Prothrombin Time consultation.
Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (no
Therapeutic Monitoring Coumadin)
Activated Partial Thromboplastin Time Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Warfarin (Coumadin) and prolonged aPTT thrombotic evaluation.
Cytochrome P450 2C9 Genotype1 Lupus Anticoagulant and Antiphospholipid Confirmatory Panel (no
Cytochrome P450 2C9 and VKORC1 Mutation Analysis2 Coumadin) with Consultation
D-Dimer Includes beta2-glycoprotein I antibody (IgG, IgM), cardiolipin antibody (IgG, IgM),
Factor X Activity, Chromogenic prolonged aPTT thrombotic evaluation, and coagulation consultation.
Fibrinogen Degradation Products (FDP) Lipoprotein (a)
Fondaparinux Sodium (Xa Inhibition)1 Methylenetetrahydrofolate Reductase (MTHFR), DNA Mutation
Heparin, Low Molecular Weight (Xa Inhibition) Analysis1
Heparin, Unfractionated (Xa Inhibition) Phosphatidylserine Antibodies (IgA)
Plasminogen, Antigenic Phosphatidylserine Antibodies (IgG, IgM)
Prothrombin Time Phosphatidylserine Antibodies (IgA, IgG, IgM)
Tissue Plasminogen Activator (TPA), EIA Plasminogen Activator Inhibitor (PAI-1) Activity
Vitamin K Plasminogen Activator Inhibitor-1
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism1
Thrombophilic Disorders Prekallikrein (Fletcher Factor) Activity
Activated Protein C-Resistance Protein C Activity
Antiphospholipid Antibody Panel Protein C Antigen
Includes C2-glycoprotein I antibodies (IgA, IgG, IgM), phosphatidylserine antibodies Protein C Activity with Reflex to Protein C Antigen
(IgA, IgG, IgM), and cardiolipin antibodies (IgA, IgG, IgM). Protein C Activity and Antigen
Antithrombin III Activity Protein C and S Activity with Reflex to Protein C and/or S Antigen
Antithrombin III Antigen Protein S Activity
Antithrombin III Activity with Reflex to Antithrombin III Antigen Protein S, Free
Antithrombin III Activity and Antigen Protein S, Total Antigen
Beta2-Glycoprotein I Antibody (IgA) Protein S Activity with Reflex to Protein S Antigen
Beta2-Glycoprotein I Antibody (IgG) Protein S Antigen and Protein S, Free
Beta2-Glycoprotein I Antibody (IgM) Protein S Panel
Beta2-Glycoprotein I Antibodies (IgA, IgG, IgM) Includes protein S activity, free and total protein S, and C4 binding protein.
C4 Binding Protein1 Prothrombin (Factor II) 20210GmA Mutation Analysis1
Cardiolipin Antibody (IgA) Prothrombin Fragment 1.2
Cardiolipin Antibody (IgG) Reptilase Clotting Time
Cardiolipin Antibody (IgM) tPA/PAI-1 Panel
Cardiolipin Antibody Screen w/ Reflex to IgG and IgM Includes PAI-1, PAI-1 activity, and tissue plasminogen activator.
Cardiolipin Antibody Screen w/ Reflex to IgG, IgA, IgM Thrombin Clotting Time
CD55 and CD59 Expression, Red Cells and Granulocytes3 Thrombin Clotting Time with Reflex to Mixing Study
Cytochrome P450 2C9 Genotype1 Thrombin-Antithrombin (TAT) Complex
Cytochrome P450 2C9 and VKORC1 Mutation Analysis2 Thrombophilia DNA Mutation Analysis1
D-Dimer Includes factor V (Leiden) and prothrombin (factor II) 20210GmA mutation analyses.
Factor V (Leiden) Mutation Analysis1 Thrombophilia Mutation Analysis with Reflex to HR2 Mutation Analysis1
Factor V HR2 Allele DNA Mutation Analysis1 Includes factor V (Leiden) and prothrombin (factor II) 20210GmA mutation analyses
Factor V (Leiden) Mutation Analysis w/Reflex to HR2 Mutation Analysis1 with reflex to HR2 mutation analysis.
Fibrin Monomer Thrombophilia Screen, Inherited1
Fibrinogen Activity, Clauss Includes antithrombin III activity, factor V (Leiden) mutation with reflex to factor V
Fibrinogen Antigen, Nephelometry HR2 mutation, protein C activity, free protein S, and prothrombin (factor II)
Fibrinogen Degradation Products (FDP) 20210GmA mutation.
30
Section 3
Thrombotic Marker Panel
Includes D-dimer, fibrin monomer, prothrombin fragment 1.2, and thrombin-
antithrombin complex.
Thrombotic Marker Panel with Consultation
Includes D-dimer, fibrin monomer, prothrombin fragment 1.2, thrombin-antithrombin
complex, and coagulation consultation.
Tissue Factor1
Tissue Plasminogen Activator (TPA), EIA
Venous Thrombosis Panel (no Coumadin)
Includes APCR, antithrombin III activity, beta2-glycoprotein I antibody (IgG, IgM),
cardiolipin antibody (IgG, IgM), factor VIII activity, homocysteine, protein C activity,
free and total protein S, lupus anticoagulation evaluation with reflex, and
prothrombin (factor II) 20210GmA mutation analysis.
Venous Thrombosis Panel (no Coumadin) with Consultation
Includes APCR, antithrombin III activity, beta2-glycoprotein I antibody (IgG, IgM),
cardiolipin antibody (IgG, IgM), factor VIII activity, homocysteine, protein C activity,
free and total protein S, lupus anticoagulation evaluation with reflex, prothrombin
(factor II) 20210GmA mutation analysis, and consultation.
Venous Thrombosis Panel (with Coumadin)
Includes APCR, antithrombin III activity, beta2-glycoprotein I antibody (IgG, IgM),
cardiolipin antibody (IgG, IgM), dRVVT screen with reflex to phospholipid
neutralization, factor VIII activity, hexagonal phase neutralization, homocysteine,
and prothrombin (factor II) 20210GmA mutation analysis.
Venous Thrombosis Panel (with Coumadin) with Consultation
Includes APCR, antithrombin III activity, beta2-glycoprotein I antibody (IgG, IgM),
cardiolipin antibody (IgG, IgM), dRVVT screen with reflex to phospholipid
neutralization, factor VIII activity, hexagonal phase neutralization, homocysteine,
prothrombin (factor II) 20210GmA mutation analysis, and consultation.
von Willebrand’s Disease
Alpha2-Antiplasmin
Factor VIII Activity, Clotting
Factor VIII Activity, Chromogenic
Ristocetin Cofactor
Ristocetin Cofactor with Reflex to von Willebrand Factor Activity (ELISA)
von Willebrand Antigen, Multimeric Analysis
von Willebrand Comprehensive Panel
Includes aPTT, factor VIII activity (clotting), ristocetin cofactor, vWF antigen, and
vWF antigen (multimeric analysis).
von Willebrand Comprehensive Panel with Coagulation Consultation
Includes aPTT, factor VIII activity (clotting), ristocetin cofactor, vWF antigen, vWF
antigen (multimeric analysis), and coagulation consultation.
von Willebrand Factor Activity
von Willebrand Factor Antigen
von Willebrand Factor Collagen Binding Assay
von Willebrand Factor Protease Activity with Reflex to Protease
Inhibitor1
von Willebrand Disease Mutation Analysis2
von Willebrand Disease (vWD) Type 2N Panel
Includes coag factor VIII activity, vWF antigen, and vWF factor VIII binding.
Women’s Health
Menorrhagia Screen
Includes factors VIII and IX activity, prothrombin time, ristocetin cofactor activity,
thromboplastin time, and vWF antigen.
Menorrhagia Screen with Consultation
Includes factors VIII and IX activity, prothrombin time, ristocetin cofactor activity,
thromboplastin time, vWF antigen, and consultation.
1
This test was developed and its performance characteristics have been determined
by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the
U.S. Food and Drug Administration. The FDA has determined that such clearance or
approval is not necessary. Performance characteristics refer to the analytical
performance of the test.
Coagulation
2
This test was developed and its performance characteristics have been determined
by Quest Diagnostics Nichols Institute. Performance characteristics refer to the
analytical performance of the test.
3
This test is performed using a kit that has not been approved or cleared by the FDA.
The analytical performance characteristics of this test have been determined by
Quest Diagnostics Nichols Institute. This test should not be used for diagnosis
without confirmation by other medically established means.
Reflex tests are performed at an additional charge.
3.2 Hemostasis
3.2.1 Fibrinogen Comprehensive Panel
Clinical Use: This test is used to evaluate suspected hypo- or
dysfibrinogenemia.
Individuals Suitable for Testing include those with lifelong or
acquired bruising or bleeding (with or without surgery), a low fibrinogen
level, and no evidence of disseminated intravascular coagulation (DIC).
Method: This panel determines the conversion rate of fibrinogen to
fibrin (based on thrombin and reptilase times and exclusion of
interfering proteins). The panel includes tests for fibrinogen clotting
activity and quantitation, reptilase clotting time, and thrombin clotting
time with reflex to a mixing study. The reflex to a mixing study will
occur when the thrombin clotting time is >23 seconds.
Interpretive Information: Decreased fibrinogen activity (30 to 150
mg/dL) in an asymptomatic person suggests familial hypo- or
hypodysfibrinogenemia. There may be a parallel decrease in fibrinogen
quantitation (antigen). Decreased fibrinogen activity can also occur with
localized fibrinolysis as in Kasabach-Merritt and Klippel-Trenaunay
syndromes. Bruising or hemorrhage may occur at levels of 10 to 75
mg/dL, but the severity is highly variable.
A prolongation of the reptilase and thrombin clotting times indicates
dysfibrinogenemia. An isolated prolongation of the thrombin time may
be due to heparin, direct thrombin inhibitor therapy, or monoclonal
gammopathy. In a thrombin time mixing study, complete correction of
the prolonged thrombin clotting time suggests a fibrinogen deficiency or
a dysfibrinogenemia, whereas a lack of correction suggests the
presence of an inhibitor.
Approximately 30% of hypodysfibrinogenemias may be associated with
a prothrombotic state. Acquired causes of hypofibrinogenemia are
common in both acute and chronic DIC, fibrinolytic therapy, dead fetus
syndrome, and L-asparaginase therapy.
3.2.2 Fibrinolysis Comprehensive Panel
Clinical Use: This test is used to evaluate fibrinolysis when an
abnormality (ie, increased or decreased fibrinolytic activity) is
suspected.
Individuals Suitable for Testing include those with unexplained
thrombosis or bleeding disorders and negative thrombophilic risk panels;
those “resistant” to fibrinolytic therapy; and those who have suffered a
stroke or heart attack.
Method: This panel includes tests for alpha2-antiplasmin, D-dimer,
euglobulin clot lysis time, fibrin monomer, fibrinogen degradation
products, plasminogen activator inhibitor (PAI-1) activity, functional
plasminogen levels, and tissue plasminogen activator (TPA).
 31

Section 3 Coagulation

Interpretive Information: An increase in PAI-1 activity is associated

with an increased risk for post-operative venous thrombosis, myocardial
infarction, and (probably) stroke. A severe deficiency of alpha2- " !( "$&
antiplasmin or PAI-1 activity has been associated with bleeding. A " (
 
shortened euglobulin clot lysis time correlates with a severe deficiency ( ("(  %" !( "$&
of alpha2-antiplasmin or PAI-1 activity. " ( !!#("
" (  !!#(" ( "$&( "
Elevations in fibrin monomer, fibrinogen degradation products, and D- " ( " (
dimer usually indicate vascular thrombosis (mainly venous) but are also
associated with malignancy and infection. Conversely, a normal D-dimer ( "$&
and/or negative fibrin monomer suggests the absence of deep venous " (
thrombosis and pulmonary emboli. " (
" 
 
Marked reduction in plasminogen has been associated with a  
prothrombotic state, and an elevated TPA level is a risk marker of
coronary artery disease.

3.2.3 Menorrhagia Panel " (   !
 &!

Clinical Use: This test is used to uncover a hemostatic disorder in
women with unexplained menorrhagia and to assess suitability of
desmopressin (DDAVP) and other therapies.
Individuals Suitable for Testing include women with menorrhagia
and women with menorrhagia with menarche.
Method: This panel includes tests for activated partial thromboplastin
time (aPTT), factor VIII and XI clotting activities, functional factor XIII,
prothrombin time, ristocetin cofactor, and von Willebrand Factor (vWF)
antigen.
Interpretive Information: A prolonged aPTT suggests a possible
intrinsic factor deficiency. A reduction in factor VIII activity of less than
50% of normal indicates hemophilia carrier state or von Willebrand
disease (vWD). In that case, a von Willebrand comprehensive panel
(test code 11333X) is indicated. A decrease in either vWF antigen or
ristocetin cofactor indicates vWD. A decreased factor XI level combined
with a normal factor VIII level is diagnostic of heterozygous factor XI
deficiency.
When the aPTT is normal, a reduction in factor XIII activity (b10% of
normal) will cause menorrhagia. A prolonged prothrombin time indicates
a deficiency of factor II, V, VII, or X. A prothrombin time >20 seconds
(INR >2.0) may cause menorrhagia.
!"'"  
"(
 
 )
!
 &!!(

"(!!#"
Figure 1. Pathways of coagulation and fibrinolysis.
3.2.6 Prothrombin Time
Clinical Use: This test is used to screen for bleeding disorders and to
monitor anticoagulant (warfarin) therapy.
Clinical Background: The prothrombin time (PT) test assesses the
extrinsic and common coagulation pathways, beginning with factor VII
and ending with fibrin formation. It can be used to screen for
deficiencies in, or inhibitors of, factors II, V, VII, or X as well as to
monitor patients who are receiving anticoagulant therapy.
Interpretive Information: Prolonged PT results suggest a potential
bleeding disorder that may be caused by a deficiency in factor II, V, VII,
or X. Prolonged results are also associated with very low fibrinogen
levels, factor-specific inhibitors, therapeutic anticoagulants, or a lupus
anticoagulant.

When results of all these tests are normal and the patient presents with
“spontaneous” ecchymoses and mucous membrane bleeding, a platelet
%
dysfunction (inherited or acquired) should be considered.
%"% %
DDAVP therapy may be helpful in patients with vWD or platelet
dysfunction disorders. Appropriate replacement therapy for factor XI and  
XIII are available.
%"% %
3.2.4 Pathways of Coagulation and Fibrinolysis  
Figure 1 depicts the intrinsic, extrinsic, and common pathways of
coagulation as well as the pathway of fibrinolysis.    % !#%
$%  %#%%
3.2.5 Prolonged APTT and dRVVT Work-ups %
Figures 2 and 3 depict follow-up testing for prolonged APTT and dRVVT  
test results, respectively.

#%%

%
Figure 2. Follow-up testing for prolonged APTT test results.
32

Section 3 Coagulation

%# #". antigens on the platelet surface. Following preparation of a platelet rich
plasma, platelets are incubated with a phycoerythrin-conjugated
.+".'(&. #%%'#" monoclonal anti-platelet antibody (anti-CD61) and a FITC-conjugated
goat anti-human IgG antibody. Platelets positive for bound antibodies
# & will fluoresce and be detected by flow cytometry. The results are
#&$# $ #!!#".'*,. '#% reported as negative, borderline, or positive.
')
('% -'#". &&,&
. '#%. "'#%& This test was developed and its performance characteristics determined by Quest
'(& &&,&  '#%&... .  Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
#&') necessary. Performance characteristics refer to the analytical performance of the test.

"'$#&$# $.  " (.". ($(&."'#( "'
"'#,. #"%!. #"%!'#"   .#"%!
 ($(&."'#( "'
Figure 3. Follow-up testing for prolonged dRVVT results.
The therapeutic range for oral anticoagulant (warfarin) therapy for most
indications is an international normalized ratio (INR) of between 2.0 and
3.0. The recommended therapeutic INR range for various clinical
situations is listed in Table 1.
3.3 Platelet Immune Disorders
3.3.1 Platelet Antibody, Direct
Clinical Use: This test is used for thrombocytopenia differential
diagnosis.
Clinical Background: Thrombocytopenia is caused by inherited
disorders and immune or nonimmune-related acquired disorders.
Platelet autoantibodies directed against intrinsic platelet antigens
(glycoproteins), immune complexes, drug-protein immune complexes, or
other antigens binding the platelet surface can help differentiate
between immune and nonimmune disorders. Two methods are available
for direct platelet antibody detection. The flow cytometry method is
suggested as an initial screen due to its superior sensitivity; it detects
any platelet-associated IgG immunoglobulins that may be present in
immune or non-immune thrombocytopenia. Positive flow cytometry
results should be confirmed with the more specific enzyme-linked
immunosorbent assay (ELISA), which detects only glycoprotein-specific
platelet antibodies (GP IIb/IIIa, GP Ib/IX, GP Ia/IIa) that are associated
with immune thrombocytopenia.
Method: This flow cytometric procedure utilizes 2-color
immunofluorescence to detect the presence of antibodies bound to
Table 1. Recommended Therapeutic INR Range
Clinical Situation INR Range*
Interpretive Information: A positive result indicates the presence of
antibodies bound to the platelet surface. Positive results are observed in
patients with idiopathic thrombocytopenic purpura (ITP), systemic lupus
erythematosus (SLE), lymphoma, HIV infection, and drug-induced
thrombocytopenia subsequent to quinidine, quinine, sulfonamides, gold
salts, heparin, and other drug therapy. Positive results may also be seen
in neonatal alloimmune thrombocytopenia and post-transfusion purpura;
however, the indirect platelet antibody test is recommended when
evaluating these 2 disorders since it is more likely to yield positive
results. For suspected immune thrombocytopenias, all positive results
should be confirmed by the platelet glycoprotein antibody ELISA
method.
A negative result suggests a nonimmune etiology in patients with
thrombocytopenia.
Results from this test should be interpreted in context with all clinical
and laboratory findings.
3.3.2 Platelet Antibody, Indirect (IgG)
Clinical Use: This test is used to diagnose neonatal alloimmune
thrombocytopenia (NAIT) as well as for differential diagnosis of post-
transfusion purpura and thrombocytopenia.
Clinical Background: Neonatal alloimmune thrombocytopenia results
from the placental transfer of maternal platelet IgG antibodies
(alloantibodies) formed against incompatible fetal platelet antigens. For
example, HPA-1a (PLA-A1; Zw) negative mothers carrying an HPA-1a
positive fetus develop the anti-HPA-1a antibodies that are found in 80%
of neonatal alloimmune thrombocytopenia cases. Additional platelet
antigens involved include HPA-5 (Br; Zv) (10% of cases), HPA-2 (Ko),
HPA-3 (Bak; Lek), HPA-4 (Pen; Yuk), and HPA-6 (Ca; Tu). Involvement of
HLA class I antibodies has been suspected in a few cases, but is not yet
proven.
Post-transfusion purpura (post-transfusion alloimmune
thrombocytopenia) is a rare disorder that occurs 5 to 10 days following
transfusion of platelet-containing blood products having incompatible
HLA or platelet specific antigens. Severe bleeding may occur due to
antibody-induced destruction of the transfused platelets.

Mechanical prosthetic heart valves 2.5–3.5 Method: This enzyme immunoassay (EIA) detects serum or plasma
Prevention of systemic embolism from 2.0–3.0 antibodies directed towards HLA class I antigens and platelet specific
Acute myocardial infarction antigens (HPA-1 through HPA-8). The assay employs a microwell plate
Valvular heart disease coated with a panel of platelet glycoproteins (IIb/IIIa, Ib/IX, and Ia/IIa)
Atrial fibrillation and HLA class I antigens obtained from group O donors. Results are
reported as negative or positive for HLA class I antibodies, platelet
Pulmonary embolism treatment 2.0–3.0 specific antibodies, or both HLA class I and platelet specific antibodies.
Venous thrombosis treatment 2.0–3.0
Interpretive Information: A positive result indicates the presence of
*Patients who are being treated with anticoagulant therapy and who have a lupus circulating antibodies directed to either HLA class I antigens or to
anticoagulant are better monitored with the factor X chromogenic assay (therapeutic platelet specific antigens or directed to both. Positive results are most
range: 11% to 41%).

Section 3
often associated with neonatal alloimmune thrombocytopenia and post-
transfusion purpura, but may also be observed in immune
thrombocytopenia and drug-induced thrombocytopenia, disorders usually
associated with direct (cell bound) platelet antibodies.
A negative result strongly suggests the absence of circulating
antibodies; however, false negative results may occur when the level of
antibody is below the detectable limit or when the antibody is directed
to rare antigens not included as a substrate in the test. A maternal
platelet alloantibody may not be detectable in up to 20% of neonatal
alloimmune thrombocytopenia cases.
Results from this test should be interpreted in context with all clinical
and laboratory findings.
3.3.3 Platelet Glycoprotein Antibody
Clinical Use: This test is used for thrombocytopenia differential
diagnosis.
Clinical Background: Thrombocytopenia is caused by inherited
disorders and immune or nonimmune-related acquired disorders.
Platelet autoantibodies directed against intrinsic platelet antigens
(glycoproteins), immune complexes, drug-protein immune complexes, or
other antigens binding the platelet surface can help differentiate
between immune and nonimmune disorders. Two methods are available
for direct platelet antibody detection. The flow cytometry method is
suggested as an initial screen due to its superior sensitivity; it detects
any platelet-associated IgG immunoglobulins that may be present in
immune or non-immune thrombocytopenia. Positive flow cytometry
results should be confirmed with the more specific enzyme-linked
immunosorbent assay (ELISA) which detects only glycoprotein-specific
platelet antibodies (GP IIb/IIIa, GP Ib/IX, GP Ia/IIa) that are associated
with immune thrombocytopenia.
Method: This enzyme linked immunosorbent assay (ELISA) utilizes
microwell strips that have been coated with specific platelet
glycoproteins (GP IIb/IIIa, GP Ib/IX and GP Ia/IIa). After eluting any
platelet autoantibodies attached to the patient’s platelets, the eluate is
incubated in 6 microwells, 2 for each glycoprotein. Bound antibodies are
then detected colorimetrically. Results are reported as negative or
positive for GP IIb/IIIa, GP Ib/IX, or GP Ia/IIa.
Interpretive Information: A positive result indicates the presence of
platelet antibodies bound to the platelet surface. Positive results are
observed in patients with idiopathic thrombocytopenic purpura (ITP),
systemic lupus erythematosus (SLE), lymphoma, and HIV infection.
Although antiplatelet antibodies are detected in 70% to 90% of patients
with ITP, they are not deemed necessary for routine diagnosis.
A negative result suggests an alternative immune or nonimmune
etiology in patients with thrombocytopenia.
Results from this test should be interpreted in context with all clinical
and laboratory findings.
3.4 Thrombocytopenia
3.4.1 Heparin-Induced Thrombocytopenia: Serotonin Release
Assay (SRA) and Heparin-induced Platelet Antibody
Clinical Use: These tests are used to diagnose heparin-induced
thrombocytopenia (HIT) type II.
33
Coagulation
Clinical Background: HIT, the most common form of drug-induced
thrombocytopenia, may develop in up to 5% of patients treated with
heparin. The clinical course of HIT is varied, but can include catastrophic
outcomes such as amputation and death secondary to sudden arterial or
venous thrombosis. HIT usually results from the formation of antibodies
that react with a trimolecular complex between platelet factor 4 (PF4),
heparin, or other glycosaminoglycans and the platelet membrane
receptor Fc(gamma)RIIa, leading to reduced platelet count and
paradoxically increased thrombosis. Thrombocytopenia typically
develops after 5 days in naive patients and can develop within minutes
to hours post-exposure in those who have received heparin therapy
within the past 6 months.
A presumptive diagnosis of HIT can be based on a >50% reduction in
platelet count more than 5 days after initiation of heparin therapy, a
platelet count <150,000/NL, or the development of thromboembolic
complication(s). Laboratory confirmation is important, but withdrawal of
all heparin sources should not be delayed while awaiting confirmatory
results.
The 14C-serotonin release assay (SRA) is considered the gold standard
for laboratory diagnosis because of its high sensitivity and specificity.
An enzyme-linked immunosorbent assay (ELISA) detecting antibodies to
the platelet factor 4 (PF4)/PVS complex offers rapid and sensitive
detection, but lower specificity than the SRA. Physicians often order the
2 assays together. Quest Diagnostics offers the SRA and ELISA
separately and as a panel.
Method
SRA: This assay is performed using Fc(gamma)RIIa receptor-phenotyped
platelets from highly reactive donors. After incubation of donor platelets
with 14C serotonin, porcine heparin (0.1, 0.5, and 100 U) is added to the
test mixture along with patient serum. False-positive results are
excluded by running a parallel study with anti-Fc(gamma)RIIa. Results
are reported as percent serotonin released and values <20% are
considered negative. Specimens with borderline results are re-tested
the next day with fresh platelets from a different donor. Positive results
are called in to the ordering physician, and consultation is available.
ELISA: This assay utilizes the PF4/PVS complex as the capture antigen
and an alkaline phosphatase-labeled anti-human globulin as the
detection antibody. Results are reported as positive (optical density
>0.41) or negative for the presence of heparin-induced antibodies.
Interpretive Information
SRA: A positive result strongly suggests HIT; consider substituting
heparin with an appropriate direct thrombin inhibitor. A negative result
suggests the absence of HIT.
ELISA: A positive result indicates the presence of heparin-induced
antibodies but is not diagnostic of HIT; other clinical and laboratory
findings should be considered prior to diagnosis. The presence of
immune complexes or other immunoglobulin aggregates may cause
false-positive results. A negative result suggests the absence of
heparin-induced antibodies, although this assay may not detect low-
titer, low-avidity antibodies.
Panel: Although SRA is the gold standard for diagnosis of HIT, caution
may be warranted when the results of the ELISA and SRA are
discrepant. Table 2 is designed to help interpret the results of the 2
assays when ordered together.
34

Section 3 Coagulation

Table 2. Interpretation of Heparin-Induced Platelet Antibody
(ELISA) and SRA Results in the Presence of
Thrombocytopenia
SRA ELISA
+ +
– –
+ –
– +
HIT II, heparin-induced thrombocytopenia type II.
*Look for other causes of thrombocytopenia.
†
Consider repeat SRA if clinically warranted.
3.5 Thrombophilia
3.5.1 Laboratory Support of Diagnosis and Management
Clinical Background
Thrombophilia is characterized by hypercoagulability and an increased
propensity for thrombosis. Almost 2 million Americans succumb
annually to a thromboembolic event,1 with venous thrombosis the third
most common cardiovascular disease after ischemic heart disease and
stroke. Venous thrombosis affects 1 to 2 in 1000 individuals every year
and is associated with life-threatening conditions such as pulmonary
embolism (PE).1 Though less clearly delineated, hypercoagulability is
also believed to play a role in the pathogenesis of arterial thrombosis.2
Conditions associated with an increased risk of venous thrombosis can
be either inherited or acquired (Tables 3 and 4). One or more
predisposing factors are identifiable in 80% of individuals with a first
episode of thrombosis, and an inherited cause of thrombophilia can be
identified in approximately 30% to 35% of individuals with a first
thrombotic episode.3 Manifestations include deep vein thrombosis (DVT)
of the lower limbs, PE, superficial thrombophlebitis, mesenteric or
cerebral vein thrombosis, fetal loss (spontaneous abortion or stillbirth),
preeclampsia, and neonatal purpura fulminans.
Individuals at high risk for venous thrombosis include those with a
personal or family history of thrombosis, inherited coagulation disorders,
homocystinuria, paroxysmal nocturnal hemoglobinuria, essential
thrombocythemia, polycythemia vera, recurrent spontaneous abortion
Interpretation and stillbirth, and malignancy. Additional risk factors include surgery,
trauma, physical inactivity (bed confinement or paralysis), warfarin
HIT II confirmed induced skin necrosis, diabetes, hyperlipidemia, vasculitis,
HIT II unlikely* thrombocytopenia, sepsis, congestive heart failure, and use of purified
prothrombin complex concentrates. Other disorders that may be
HIT II likely associated with increased thrombotic risk are thrombomodulin
HIT II unlikely*† mutations, plasminogen deficiency, and elevations of histidine-rich
glycoprotein, plasminogen activator inhibitor-1, interleukin 8, lipoprotein
(a), D-dimer, and thrombin-activatable fibrinolysis inhibitor.
The risk of thrombosis increases with the number of defects or risk
factors present; ie, individuals with multiple conditions associated with
thrombosis are at greater risk than those with only one condition.1 Risk
factors for thrombophilia are generally not associated with risk of
arterial thrombosis, with the exception of hyperlipidemia,
antiphospholipid syndrome (Appendix 2), elevated homocysteine, and
dysfibrinogenemia.2
The identification of thrombotic risk factors and diagnosis of
thrombophilia contributes to patient management in multiple ways
(Table 5). Such diagnosis is based on personal and family history of
thrombosis (especially during adolescence and young adult years),
clinical manifestations, and laboratory testing.
Clear guidelines how to best manage individuals with a family or
personal history of documented risk factors and who have not
experienced a thrombotic episode have not been established. The
decision for prophylactic therapy should be based on an individual’s
clinical history.12 Screening general populations for inherited disorders
associated with venous thrombosis is not recommended; however, the
clinical utility of global screening assays in high risk populations is
being evaluated.
Patients with acute thrombosis are treated with intravenous heparin or
oral anticoagulants such as warfarin (Coumadin®’). Prophylactic

Table 3. Inherited Conditions Associated with Venous Thrombosis

Frequency (%) Frequency (%) in

in Healthy Patients with Relative Risk (%)
Individuals Venous Thrombosis* of Thrombosis
Activated protein C resistance/factor V mutation†‡4,5 5 21 3–7
6
Antithrombin deficiency 0.02–0.17 1 15–40
§7
Factor VIII excess 11 25 6
|| 8,9
Hyperhomocysteinemia 5–10 10–25 3–4
6
Protein C deficiency 0.3 3 5–12
6
Protein S deficiency 0.7 2 4–10
Prothrombin (factor II) 20210GmA mutation†10,11 2 6 2–3
Other rare inherited conditions that may be associated with inherited venous thrombosis include sticky platelet syndrome, heparin cofactor II deficiency, factor XII deficiency, and dys- and
hyperfibrinogenemia. Deficiency of antithrombin, protein C, and protein S may also be acquired.
*Data is for heterozygotes.
†
Affects primarily Caucasian population.
‡
Homozygosity of the factor V HR2 allele increases the risk of venous thrombosis 3- to 4-fold in the presence of the factor V Leiden mutation (no increase if factor V Leiden mutation is
absent).
§
Can also be acquired.
||
MTHFR C677T or A1298C mutation, dietary deficiency of folate, and/or vitamins B6 and B12.

Section 3
Table 4. Acquired Conditions Associated with Venous
Thrombosis
Antiphospholipid syndrome (most common cause)
Autoimmune disorders (eg, systemic lupus erythematosus)
Combined oral contraceptives
Elevated factor IX, XI
Endocrine disorders (eg, diabetes mellitus, Cushing’s syndrome)
Heparin-induced thrombocytopenia (HIT)*
Hormone replacement therapy
Liver disease
†
Malignancy
Myeloproliferative disorders (eg, polycythemia vera, chronic
myelogenous leukemia)
Nephrotic syndrome
Obesity
Paroxysmal nocturnal hemoglobinuria
Pregnancy and puerperium
Thrombotic thrombocytopenic purpura
*Should be considered in any individual who has received heparin within the 30 days
preceding a thrombotic episode and has a decrease in platelet count to <100,000/NL or
more than 50% of baseline.
†
A thrombotic event can precede the diagnosis of malignancy by months to years.
treatment is provided to diagnosed patients when in high risk situations,
eg, surgery, prolonged immobilization, pregnancy and puerperium.
Lifelong prophylactic therapy may be considered for those with recurrent
thrombotic episodes, high risk disorders, or with multiple risk factors
and may include plasma transfusions (eg, antithrombin concentrates),
oral anticoagulants, low dose aspirin, and heparin.12 Heparin is of
limited benefit post thrombosis in patients with antithrombin deficiency,
however, and heparin selection for pregnant women should be
individualized due to risk of bone fracture.13 Low molecular weight
heparin (LMWH) may be a better option for those at risk of osteoporosis
since LMWH does not cause bone thinning. Individuals with
hyperhomocysteinemia may be treated with vitamin supplementation
(folic acid, cobalamin, pyridoxine).
Table 5. Value of Thrombophilia Diagnosis
Identify pathologic basis of thrombotic event
Aid in selection of appropriate therapy
Determine duration and intensity of treatment
Determine need for prophylaxis
Estimate future thrombotic risk
Determine degree of risk associated with oral contraceptives and
estrogen replacement therapy
Determine need for evaluation of family members
35
Coagulation
Individuals Suitable for Testing
• Symptomatic individuals
• Individuals with a personal or family history of thrombosis or
thrombophilia-associated mutations
• High risk individuals predisposed by surgery, trauma, immobility,
pregnancy, oral contraceptives, etc.
Note: high risk pregnant women include those with a personal or family
history of thrombosis, previous neural tube defect affected fetus,
recurrent spontaneous abortions, severe early onset preeclampsia,
cesarean section, obesity, advanced maternal age, higher parity, and
prolonged immobilization.13
Test Availability
Tests available to assist in diagnosis and management of thrombophilia
disorders are listed in Appendix 1. Additionally, Quest Diagnostics offers
panels that include multiple tests, thereby simplifying the test ordering
process. Refer to the Quest Diagnostics Directory of Services for
information on these panels, which are typically named according to the
medical condition.
Test Selection
A venous thrombosis laboratory work-up for high-risk or symptomatic
individuals begins with a personal and family history. Test selection may
vary for each individual based on his/her history as well as a particular
defect’s prevalence in specific populations. For example, venous
thrombosis in a pediatric patient suggests the likelihood of an inherited
disorder; in an individual with SLE, antiphospholipid syndrome should be
considered; and in an older individual, malignancy. Testing for multiple
etiologies is recommended since venous thrombosis is a polyfactorial
disorder, and presence of multiple etiologies increases the risk for
thrombosis.14,15 Generally accepted testing guidelines suggest the use of
first and second line testing in the thrombophilia diagnosis (Figure 4).14,15
First line testing for an individual with venous thrombosis typically
includes a CBC with smear and APTT; activated protein C resistance
(APCR); functional (activity) assays for antithrombin, proteins C and S,
and factor VIII; prothrombin 20210GmA mutation detection;
homocysteine; and anticardiolipin and antiphospholipid antibodies (see
Appendix 2. Antiphospholipid Syndrome). APCR and prothrombin
20210GmA mutation detection need not be performed initially in non-
Caucasian individuals since these disorders are primarily observed in
Caucasians. Likewise, if a first thrombotic event occurs after the age of
50, testing for protein C, S, and antithrombin deficiency may be
postponed as hypercoagulability due to these disorders usually
manifests as thrombosis earlier than the fifth decade. Testing for
heparin induced thrombocytopenia should be considered for any
individual who has received heparin within the 30 days preceding a
thrombotic episode and has a decrease in platelet count to <100,000/NL
or more than 50% of baseline. Additional testing directed toward
diagnosis of other causes of acquired thrombophilia such as systemic
lupus erythematosus, liver disease, nephrotic syndrome, polycythemia
vera, chronic myelogenous leukemia, diabetes mellitus, Cushing’s
syndrome, etc. may be indicated (see Table 4).
Positive functional assays can be confirmed by genetic testing in some
cases or by demonstration of the abnormality in another family member.
For example, a borderline or positive APCR test can be confirmed with
factor V (Leiden) mutation analysis. Such analysis differentiates
homozygous and heterozygous states, providing additional prognostic
information. Factor V HR2 allele mutation analysis provides even more
prognostic information in factor V (Leiden) carriers. Homocysteine
elevations may be due to an acquired nutritional deficiency (vitamin B12,
 36

Individual with Documented Thrombotic Episode or at High Risk

First Line Testing

Myeloproliferative Antiphospholipid Antithrombin Protein C Protein S Excess Coagulation Prothrombin Hyperhomocysteinemia Fibrinogen
APCR/FVL
Disorders Syndrome Deficiency Deficiency Deficiency Factor VIII Pathway Defects Gene Mutation Deficiency

Rule Out
CBC with Cardiolipin Antithrombin Protein C Free Factor VIII 20210G A Plasma Thrombin
APCR APTT
Smear Antibodies Activity Activity Protein S Activity Mutation Homocysteine Clotting Time*

Test
Abnormal Normal Normal Abnormal Normal Normal Normal Normal Normal Normal Normal

Positive Negative Normal Decreased Decreased Elevated Prolonged Prothrombin >18 M/L Prolonged
20210G A mutation
Medical
Evaluation Factor V Antithrombin Antigen: dRVVT Screen Mixing studies
APS C4 Binding Protein • MTHFR genetic analysis †
Leiden/ Decreased in type I with Reflex to Mixing • Vitamin B6, B12, and
HR2 DNA deficiency; Normal Phospholipid studies folate levels
analysis in type II deficiency Neutralization
Positive Negative Increased Normal
Decreased Not

Interpretation
B2GP Negative Positive corrected Corrected
Protein S Activity
and Antigen:
Positive Negative distinguish type I Confirm presence of LA Corrected Evaluate for Not
Positive Negative and III deficiency factor deficiencies corrected

Repeat in 6 to 8 Acquired APCR or Protein C antigen: Repeat in 6 to 8 PTT-LA with Reflex Evaluate for Confirm presence
weeks to confirm mutation other than Decreased in type I deficiency; weeks to confirm to Hexagonal Phase factor deficiencies of inhibitor
factor V Leiden Normal in type II deficiency Neutralization

Individual with Documented Thrombotic Episode(s), Strong Family History of Thrombosis, and Negative First Line Tests

Second Line Testing ‡

Coagulation factors Fibrinogen Heparin Lipoprotein (a) Plasminogen Plasminogen activator Plasminogen activator Reptilase time Thrombomodulin Tissue plasminogen
V, VII, IX, X, XI cofactor II inhibitor-1 (urokinase-like) activator

Figure 4. Testing algorithm for the diagnosis of thrombophilia in individuals with a history of thrombosis or those at high risk. An individual with a documented thrombotic episode should undergo a complete medical evaluation to rule out
conditions associated with thrombophilia not diagnosed by first line testing, eg, nephrotic syndrome, diabetes mellitus, etc. High risk individuals are those with a strong family history of thrombosis and/or those with acquired risk factors,
eg, obesity, prolonged immobilization, etc. All non-genetic testing should be repeated in 6 to 8 weeks to reduce the likelihood of false-positives. Some assays are affected by anticoagulants or the acute thrombotic process.
APCR/FVL indicates activated protein C resistance/factor V Leiden; APS, antiphosphatidylserine antibody; B2GP, beta2-glycoprotein I; LA, lupus anticoagulant.
*Some authors consider thrombin clotting time to be a second line test.
†
Mutation analysis is considered optional by some authors as treatment is not changed by the presence or absence of the MTHFR mutation.
‡
Second line testing is for the identification of rare causes of thrombophilia and recommended for individuals with a documented thrombotic episode(s), a strong family history of thrombosis, and negative first line tests.
 37

Section 3 Coagulation

B6, or folate), methylenetetrahydrofolate reductase (MTHFR) mutation, or
cystathionine C-synthase mutation. Acquired causes for antithrombin,
protein C, and protein S deficiencies can be ruled out by a liver function
testing, disseminated intravascular coagulation screen (D-dimer, fibrin
degradation product, PT, APTT, fibrinogen, platelet count), and a
proteinuria test (urine albumin).16 Decreased antithrombin and protein C
and S activities (function) can be further characterized as a deficiency
(type I or III) or dysproteinemia (type II) by using antigenic assays;
however, such characterization will not affect treatment decisions.
If all of the aforementioned testing is negative, the patient may have a
rare disorder that can be identified by testing for fibrinogen,
plasminogen activity (function), plasminogen activator inhibitor-1 (PAI-1),
lipoprotein (a) [Lp(a)], tissue plasminogen activator, heparin cofactor II,
etc (Figure 4). Testing for rare disorders is only recommended for
individuals with a strong personal and family history of thrombosis and
negative first line tests or in whom clinical suspicion is high. Since all
thrombophilia etiologies are not yet known, it is possible for all of these
tests to be negative.
Test Interpretation
Acute thrombosis, anticoagulant therapy, drug therapy, and certain
medical conditions can affect the results and interpretation of tests
used to diagnose causes of thrombophilia (Tables 6 and 7). Additional
interpretive information, specific to each test, is provided below.
Activated Partial Thromboplastin Time (APTT)
The APTT will be prolonged if there is deficiency or inhibition of factors
of the intrinsic pathway including high molecular weight kininogen
(HMWK), prekallikrein, factors V, VIII, IX, X, XI and XII, prothrombin, and
fibrinogen. Prolongation is also seen in individuals with lupus
anticoagulant.
Activated Protein C Resistance (APCR)
A decreased ratio of dRVVT clotting times obtained with and without
activation of endogenous protein C is suggestive of an activated protein
C (APC) inhibitor, a factor V (Leiden) mutation, and increased risk of
deep vein thrombosis. Assay sensitivity and specificity approach 100%,
even in the presence of anticoagulants and heparin (b1 IU/mL
plasma).17,18 Falsely decreased ratios (false-positive test) are observed in
patients with lupus anticoagulants and specimens with platelet counts
>20,000/NL. In <5% of cases, an APC inhibitor is found without a
corresponding factor V (Leiden) mutation, perhaps indicative of an
unknown mutation. Such cases are also associated with increased
venous thrombosis risk.19
Antithrombin
Decreased levels of antithrombin are associated with an increased risk
of both arterial and venous thrombosis and are seen in individuals with
hereditary antithrombin deficiency, nephrotic syndrome, colitis, liver
disease, active thrombosis, disseminated intravascular coagulation
(DIC), those receiving L-asparaginase therapy or oral contraceptives, and
individuals who are pregnant or have undergone surgery. Levels are also
decreased in individuals receiving heparin. Levels in neonates are
approximately half of the adult level, which is reached by 6 months of
age. Low levels in both the activity and antigen assays indicate type I
deficiency, whereas low activity levels in the presence of normal
antigen levels indicate type II deficiency (dysproteinemia). Increased
levels may be due to oral anticoagulants or heparin cofactor II.
C4 Binding Protein
Approximately 65% of protein S circulates in plasma bound to C4
binding protein. Increased levels of C4 binding protein may cause
decreased levels of free protein S, and subsequent increased risk of
thrombosis, and are associated with inflammation, pregnancy, diabetes
mellitus, SLE, AIDS, allograft rejection, estrogen and progesterone
administration, and smoking.

Table 6. Confounding Effects of Anticoagulant Therapy and Acute Thrombosis on Testing Used in the Diagnosis of
Thrombophilia

Test Confounding Effect

Warfarin Heparin Acute Thrombosis

Antithrombin None (rarely increases)* Decreases May decrease†
Antiphospholipid antibodies None Potential false positive None
‡ §
APCR/Factor V Leiden Nonsensical results None None
APTT May increase Increases Increases
Homocysteine None None None
Lupus anticoagulant Potential false positive Nonsensical results None
§
Protein C Decreases None May decrease†
Protein S Decreases None§ May decrease†
Prothrombin gene mutation None None None
Reptilase time Increases None Increases
Thrombin time Increases Increases Increases
APCR, activated protein C resistance; APTT, activated partial thromboplastin time.
*Can be increased to normal range in individuals with heterozygous deficiency.
†
Results are inaccurate during acute thrombosis; however, a normal level during an acute thrombotic event essentially excludes deficiency of these proteins.
‡
Mutation analysis not affected.
§
Heparin up to 1 U/mL does not affect results.
38
Section 3
Table 7. Conditions That Affect Testing for Inherited Causes of Thrombophilia
Condition
Lupus anticoagulant
Deficiency of Vitamin B12, B6, or folate
Methotrexate, phenytoin, theophylline
Hypothyroidism, malignancy, menopause
Oral contraceptives
Estrogen replacement
Pregnancy and puerperium
Acute phase reaction, inflammation, infection
Increased factor VIII
Kidney disease/nephrotic syndrome
DIC, liver disease, sepsis, L-asparaginase therapy
Surgery, recent thrombosis
Vitamin K deficiency
APCR; activate protein C resistance, DIC; disseminated intravascular coagulation.
Cardiolipin Antibodies
Anticardiolipin antibodies of the IgA, IgG, and IgM isotype are
associated with the antiphospholipid syndrome and, when >40 GPL
units, increase the risk for venous thrombosis 5- to 8-fold. IgG
antibodies appear to be more predictive of disease activity, while IgM
antibody occurs more often in drug-induced disorders and infectious
disease (eg, syphilis). Higher antibody titers are generally correlated
with greater thrombotic risk (see Appendix 2).
Cytochrome P450 2C9 Genotype
The cytochrome P450 enzyme CYP2C9 participates in the metabolism of
a number of important drugs, including warfarin. Individuals carrying
variants in the CYP2C9 gene have reduced metabolism of warfarin and
those with 2 copies of variant alleles are at high risk of life-threatening
side effects.
D-Dimer
Elevated levels are associated with myocardial infarction, deep vein
thrombosis, pulmonary embolism, DIC and other coagulation disorders,
surgery, trauma, sickle cell disease, liver disease, severe infection,
sepsis, inflammation, malignancy, obstetric complications, and
hyperfibrinolysis.
dRVVT Screen with Reflex to Phospholipid Neutralization
This test evaluates the activity of factors II, V, and X (common clotting
pathway). A confirmed dRVVT test result (ie, increased ratio) is
indicative of a lupus anticoagulant and/or phospholipid antibody since
excess phospholipid shortens (overrides) the prolonged dRVVT. A falsely
prolonged dRVVT test may occur when heparin is >1.0 IU/mL. A false-
negative dRVVT test may be due to platelet contamination of the
plasma. Samples with moderate or severe icterus or lipemia are
contraindicated.
Factor V HR2 Allele DNA Mutation Analysis
The HR2 allele is associated with APCR and increased risk of venous
thrombosis in individuals also heterozygous for the factor V (Leiden)
mutation. Such co-inheritance increases the risk of venous
thromboembolism 3- to 4-fold when compared with factor V (Leiden)
alone. An individual heterozygous positive for the HR2 allele and
Coagulation
Effect
Decreases APCR, protein S, and factor VIII
Increases homocysteine
Decreases antithrombin and protein C and S
Decreases APCR and protein C and S
Increases homocysteine
Decreases protein S
Decreases antithrombin and protein S
Decreases antithrombin and protein C and S
Decreases antithrombin and protein C and S
Increases homocysteine
Decreases protein C and protein S
negative for factor V (Leiden) is not at increased risk of thrombosis
compared to factor V (Leiden) alone. However, homozygosity for factor V
HR2 is associated with increased risk of thrombosis even in the absence
of a factor V (Leiden) mutation.
Factor V (Leiden) Mutation Analysis
The 1691G m A factor V Leiden mutation results in the laboratory
finding of APCR. Factor V (Leiden) confers approximately a 7-fold
increase in venous thromboembolic events in heterozygous individuals
and an 80-fold increase in homozygous subjects.20 When a heterozygous
mutation is coupled with oral contraceptive use, the risk increases
synergistically to 30-fold.21 The mutation is also associated with arterial
thrombosis (especially in smokers), complications of pregnancy
(including fetal loss),22 and increased levels of factor VIII. Although this
test is highly specific, identification of a mutation may occur in the
absence of APCR in rare cases. Sensitivity of this test for APCR is
94%;23 thus, a negative result does not rule out APCR or an increased
risk of venous thrombosis.
Factor VIII
Factor VIII is an acute phase reactant and increased levels are found
during periods of stress, postoperatively, and in inflammatory
conditions. Elevated levels are also found at birth and during pregnancy.
Increased levels are associated with increased risk for venous
thrombosis,24 whereas decreased levels are associated with hemophilia
A. A factor VIII activity:fibrinogen ratio >0.75 is considered diagnostic of
factor VIII excess.
Fibrin Monomer
The presence of soluble fibrin monomer complexes in plasma is
diagnostic of DIC.
Fibrinogen
Increased levels are associated with acute phase reactions, pregnancy,
and increased risk of thrombosis. Low fibrinogen activity levels are
associated with afibrinogenemia, hypofibrinogenemia, or
dysfibrinogenemia (which may be associated with thrombophilia in rare
instances), as well as with DIC, systemic fibrinolysis, pancreatitis,
severe hepatic dysfunction, and L-asparaginase or valproate treatment.

Section 3
Individuals with afibrinogenemia or hypofibrinogenemia will have
decreased activity and antigen levels. Individuals with
dysfibrinogenemia will typically have decreased activity levels and
normal or decreased antigen levels.
Fibrinogen Degradation Products (FDP)
FDP result from the breakdown of fibrinogen, as well as fibrin, by
plasmin. Normally, the fibrinolytic process is localized to fibrin, however,
during conditions such as DIC, fibrinolysis spreads and becomes
systemic. Elevated levels of FDP are seen in many clinical states (eg,
DVT and PE); thus measurement of FDP is useful for their diagnosis.
Persistent elevations indicate that abnormal fibrinolysis and
fibringenolysis are occurring.
C2-Glycoprotein I Antibodies
C2-Glycoprotein I antibodies of the IgA, IgG, and IgM isotype are
associated with the antiphospholipid syndrome, and their presence is
more specific but less sensitive than cardiolipin antibodies for the
diagnosis of antiphospholipid symdrome. Individuals who are positive
for cardiolipin antibodies and negative for C2-glycoprotein I antibodies
are more likely to have an infection (varicella, rubella, adenovirus, HIV)
or drug exposure (amoxicillin, chlorpromazine, hydralazine) than
antiphospholipid syndrome (see Appendix 2).
Heparin, Low Molecular Weight (Xa Inhibition)
LMWH are prepared by the chemical or enzymatic degradation of
unfractionated heparin, and are used in the prevention and treatment of
thromboembolic conditions. Measurement of LMWH in plasma is used
to monitor therapeutic levels. The therapeutic range for LMWH is 0.7 to
1.3 anti-Xa units/mL measured 4 to 6 hours after a subcutaneous dose,
and the prophylactic range is 0.3 to 0.7 anti-Xa units/mL measured 4 to
6 hours after a subcutaneous dose. (This assay was validated using
enoxaprin; thus may not be appropriate for optimal evaluation of
patients receiving other LMWH.)
Heparin, Unfractionated (Xa Inhibition)
Unfractionated heparin is used for the prevention and treatment of
thromboembolic conditions and measurement is used to monitor
therapeutic levels. When administered as an intravenous infusion, the
therapeutic range is 0.35 to 0.7 IU/mL.
Homocysteine
Levels are increased in the following: cardiovascular disease, vitamin
B12 and folate deficiencies, chronic renal disease, homocystinuria,
hypothyroidism, selected malignancies, individuals whose diet is rich in
methionine (high meat intake), cigarette smokers, and in individuals
treated with corticosteroids, methotrexate, cyclosporin, vitamin B6
antagonists (isoniazid, azauridine, penicillamine, procarbazine),
anticonvulsants (phenytoin, carbamazepine), and S-adenosyl-methionine.
When coupled with the factor V (Leiden) mutation, venous thrombosis
risk increases synergistically.25 Falsely increased levels may occur if
serum or plasma is not separated from the red cells within 1 hour of
collection.
Homocysteine is decreased in pregnancy (except in some women
carrying a fetus with a neural tube defect), individuals less than 15
years of age, and individuals taking oral contraceptives or hormone
replacement therapy.
Human Platelet Antigen 1 (HPA-1) Genotype
The HPA-1b platelet antigen polymorphism is associated with increase
platelet thrombogenecity, alloimmune thrombocytopenia, and post-
transfusion purpura.
39
Coagulation
Lipoprotein (a) [Lp(a)]
Increased levels of Lp(a) are observed in patients with coronary artery
disease, stroke, cerebrovascular and peripheral vascular disease, and
venous thrombosis. Substantial increases are secondarily (not
genetically related) observed in nephrotic syndrome and end-stage renal
disease. Decreased Lp(a) levels may be seen in several rare disorders
(lecithin:cholesterol acyltransferase [LCAT] deficiency, lipoprotein lipase
[LPL] deficiency, liver disease). Normal levels in the African American
population may be 2 to 3 times the values in Caucasian and Asian
populations. Native Americans and Mexican Americans have lower
normal levels (no lower than one half) relative to the Caucasian and
Asian populations.
Methylenetetrahydrofolate Reductase (MTHFR) DNA Mutation
Analysis
A homozygous MTHFR mutation (677CmT) may be associated with
hyperhomocysteinemia, an increased risk for arterial and venous
thrombosis, and an increased risk for obstetrical complications (eg,
preeclampsia, abruptio placenta, fetal growth retardation, and
stillbirth).22 Heterozygosity for this mutation, in the absence of vitamin
deficiency, usually is associated with normal plasma homocysteine
levels. When a 677CmT mutation is combined with a 1298AmC
mutation (ie, double heterozygosity), the risk is the same or greater than
that of a 677CmT homozygote.
Mixing/Correction Study
Test results are consistent with an intrinsic factor deficiency when a
prolonged APTT is normalized after mixing patient plasma with normal
plasma and the normalized result does not reverse after incubation of
the mixed sample. A specific factor inhibitor, lupus inhibitor, fibrinolysis,
or fibrinogenolysis is suggested when 1) the PT is normal, and the APTT
is prolonged initially, normalizes after mixing, and reverses to prolonged
after incubation of the mixed sample; 2) the PT is normal, and the APTT
remains prolonged after mixing studies; 3) a prolonged PT is corrected in
the mixing study, and a prolonged APTT remains prolonged; 4) a
prolonged PT remains prolonged, and a prolonged APTT normalizes after
mixing, and reverses to prolonged after incubation. A prolonged PT that
is corrected in the mixing study, along with a normal APTT, suggests a
factor VII deficiency. Test results are consistent with a single or multiple
deficiency of factors II, V, or X (common pathway) when a prolonged PT
is normalized after mixing studies and when a prolonged APTT
normalizes after mixing studies and remains normalized after
incubation.
Phosphatidylserine Antibodies
Antibodies to phosphatidylserine are associated with antiphospholipid
syndrome, an increased risk of venous thrombosis, thrombocytopenia,
and recurrent fetal loss.
Plasminogen Activator Inhibitor-1 (PAI-1)
Increased levels of PAI-1 antigen and PAI-1 activity are associated with
decreased fibrinolytic activity and increased risk for venous thrombosis
and coronary artery disease. Interpretation of increased levels is
confounded by circadian variation (morning values being about 2-fold
higher than afternoon values), increases associated with the acute
phase response (eg, following myocardial infarction, major surgery,
severe trauma, or sepsis), as well as increases associated with normal
pregnancy and disorders such as endotoxemia, liver disease, obesity,
hyperinsulinemia, hypertriglyceridemia, and malignancy. Severely
decreased or undetectable levels may be associated with bleeding
problems. The antigenic, but not the activity test can help distinguish
between a constitutional deficiency of PAI-1 and a dysproteinemia.
40
Section 3
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism
The 4G allele is associated with increased PAI-1 antigen and activity
levels. Similar to PAI-1 antigen and activity levels, data regarding utility
of the 4G/5G polymorphism in predicting venous thrombosis is
conflicting.27 It may be more useful when co-inherited with another
thrombophilia marker. When co-inherited, the 4G allele may further
increase the risk of thrombophilia. For example, Visanji et al found the
risk for venous thrombosis increased approximately 2-fold in patients
with at least 1 copy of the 4G allele (4G/4G or 4G/5G genotypes)
relative to that in patients with the 5G/5G genotype.27 All patients were
heterozygous for factor V (Leiden) mutation and had experienced at
least 1 venous thromboembolic event. Furthermore, Zoller et al reported
an approximate 4-fold increase in the risk of pulmonary embolism
among subjects with hereditary protein S deficiency who were
homozygous for the 4G allele.28
Plasminogen
Decreased levels are associated with liver disease, DIC, thrombolytic
agents, primary fibrinolysis, tissue plasminogen activator and, rarely,
with venous thrombosis and pulmonary embolism (homozygous state
only). Increased levels are associated with trauma, infection, acute
myocardial infarction, surgery, and chronic inflammation. A functional
assay is usually the preferred assay because the presence of a normal
amount of antigen does not exclude a qualitative defect of the protein.
The antigen level is most often obtained to assess a quantitative
abnormality of the protein.
Protein C
Decreases are associated with venous thrombosis, recurrent superficial
thrombophlebitis, neonatal purpura fulminans, arterial thrombosis
(rarely), oral anticoagulant-induced skin necrosis, DIC, infection, acute
illness such as the flu or a gastrointestinal disorder, malignancy, liver
disease, vitamin K deficiency, surgery, and L-asparaginase therapy.
Falsely low values may be obtained in individuals on oral anticoagulant
therapy and those who are APC resistant. Heparin levels up to 1 IU/mL
do not interfere with test results. Protein C levels are significantly
decreased in neonates; adult levels are reached only after 10 years of
age. Low levels in both activity and antigen assays are suggestive of
type I deficiency, whereas low activity levels in the presence of normal
antigen levels indicate type II deficiency (dysproteinemia). Increases are
associated with oral contraceptives, and pregnancy. Although not
affected by increased factor VIII or acute phase reactions, overall
specificity for inherited deficiency is low, and positive results (ie, low
levels) should be confirmed by demonstration of a deficiency in another
family member.
Protein S
Decreases are associated with increased risk for venous, and possibly
arterial, thrombosis, oral anticoagulant-induced skin necrosis, neonatal
purpura fulminans, DIC, acute phase reactions, oral anticoagulants, APC
resistance, vitamin K deficiency, liver disease, surgery, L-asparaginase
therapy, oral contraceptives, estrogen replacement therapy, pregnancy,
nephrotic syndrome, infections (HIV, varicella), Crohn’s disease, and
ulcerative colitis. Levels are significantly decreased in neonates;
however, adult levels are reached by 6 months of age. Low levels in
both activity and antigen (free and total) assays are suggestive of type I
deficiency, whereas low activity in the presence of normal total antigen
levels indicate type II deficiency (dysproteinemia). Type III deficiency is
characterized by low levels in the activity and free antigen assays, but
normal levels in the total antigen assay. Increases may be observed in
lipemic samples. Although protein S levels are not affected by heparin
(up to 1 IU/mL) or factor VIII (up to 250%), overall specificity of protein S
measurement for the diagnosis of inherited deficiency is low, and
Coagulation
positive results (ie, low levels) should be confirmed by demonstration of
a deficiency in another family member.
Prothrombin (Factor II) 20210GmA Mutation
This mutation is associated with increased prothrombin levels,
increased risk for venous thrombosis,11,24 increased risk for obstetric
complications (eg, preeclampsia, abruptio placenta, fetal growth
retardation, and stillbirth),21 and, possibly, premature coronary heart
disease. Venous thrombosis risk increases synergistically in the
presence of oral contraceptive use.29 The combination may also lead to
cerebral sinus and spinal vein thromboses.
Prothrombin Fragment 1.2
Prothrombin fragment 1.2 is the amino terminus fragment of
prothrombin released when prothrombin is converted to thrombin.
Elevated levels are associated with an increased risk of thrombosis and
found in patients with trauma, ecalmpsia, pre-eclampsia, DIC, DVT, and
PE. Levels are also increased in individuals with antithrombin deficiency.
PTT-LA with Reflex to Hexagonal Phase Neutralization
Lupus anticoagulants are non-specific antibodies that extend the
clotting time of phospholipid-dependent clotting assays, and lupus
anticoagulant antibodies bind to hexagonal phase phospholipids. A
reduction of the APTT by more than 8 seconds after the addition of
hexagonal phase phospholipid is considered confirmation of the
presence of a lupus anticoagulant and an increased risk of thrombosis.
The sensitivity and specificity of this test are 96% and >95%,
respectively. While false-negatives are rare, if clinical suspicion of LA is
high, dRVVT with confirm is suggested.
Red Cell CD55 and CD59 Expression
A percentage of CD55 and/or CD59 deficient red blood cells >15% is
diagnostic of paroxysmal nocturnal hemoglobinuria (PNH). Individuals
with PNH are at markedly increased risk of thrombosis, particularly of
the intra-abdominal and cerebral veins.
Reptilase Clotting Time
Unlike thrombin, reptilase is not affected by the presence of heparin or
hirudin; thus, a prolonged thrombin time in an individual with a normal
reptilase time is consistent with contamination or the presence of
heparin. Reptilase clotting time is also prolonged in individuals with a-,
hypo-, and dysfibrinogenemias.
Serotonin Release Assay (SRA)
A value >20% is considered positive and strongly suggests heparin
induced thrombocytopenia. False-positive test results are automatically
excluded by running a parallel study with anti-Fc(gamma)RIIa.
Soluble P-Selectin (SPS)
P-selectin is a cell adhesion molecule found on the surface of activated
platelets after release from platelet alpha granules. A soluble form of
P-selectin, which has a lower molecular mass, is secreted into plasma.
Elevated levels of SPS are found in individuals with thrombotic disease
states, eg, DVT, atherosclerosis.
Thrombin-Antithrombin (TAT) Complex
Elevated TAT complex is a risk factor for thrombosis and found in
individuals with DIC, malignancies, and those receiving heparin and
fibrinolysis therapy.
Thrombin Clotting Time
Prolonged clotting times may indicate abnormal fibrinogen levels
(elevated or decreased), dysfibrinogenemia, the presence of heparin,
hirudin, paraproteins, uremia, or increased levels of fibrin degradation

Section 3
products. Normalization of clotting time after mixing indicates hypo- or
dysfibrinogenemia, whereas continued prolongation indicates the
presence of heparin, hirudin, paraproteins, uremia, or increased levels of
fibrin degradation products.
Thrombin Generation
Thrombin generation is a global test for hypercoagulability and, as such,
is useful for identifying individuals at risk for thrombosis. It may uncover
unexplained clinical thrombophilia when no known causes have been
identified. Factor VIII levels >250% have been associated with increased
thrombin generation potential. This assay is also useful in monitoring
anticoagulant therapy.
Tissue Factor (TF)
TF, released after vascular injury, activates Factor VII, and the TF:Factor
VIIa complex activates the coagulation protease cascade. Elevated
levels of TF are seen in atherosclerosis, cancer, and sepsis and may play
a role in venous thrombosis.
Tissue Plasminogen Activator (TPA)
TPA converts plasminogen to plasmin, which in turn degrades fibrin to
soluble degradation products. TPA is inhibited by plasminogen activator
inhibitor-1. Elevated TPA levels are associated with an increased risk of
atherosclerosis, myocardial infarction, stroke, and recurrent venous
thrombosis.
von Willebrand Factor Protease (ADAMTS-13) Activity with
Reflex to Protease Inhibitor
von Willebrand factor is released into cirulation as high molecular
weight multimeric forms that are broken down into smaller, less active
multimers by von Willebrand factor cleaving protease (ADAMTS-13).
The persistence of high weight multimeres is associated with platelet
aggregates and thrombi. Deficiency of von Willebrand factor cleaving
protease is associated with thrombotic thrombocytopenic purpura.
Sample Collection Considerations
Anticoagulants may interfere with some test results (Table 6). When
clinically indicated, replace an oral anticoagulant with heparin for 7 to
10 days, then stop the heparin and draw the sample 12 to 24 hours
later.6
Certain medicines and medical conditions may also affect some test
results (Table 7).
Platelets significantly decrease the sensitivity of antiphospholipid
antibody testing; thus, the specimen must be centrifuged for 15 minutes
at 1,500 g and/or filtered through a 0.22 micron screen to remove
platelets prior to freezing.30 The final platelet count must be <10,000
platelets/NL of plasma (<5,000 platelets/NL preferred; note that 1 NL=1
mm3).
References
1. Heit JA. Venous thromboembolism: disease burden, outcomes and
risk factors. Thromb Haemost. 2005;3:1611-1617.
2. Prandoni P, Bilora F, Narchiori A, et al. An association between
atherosclerosis and venous thrombosis. N Engl J Med.
2003;348:1435-1441.
3. Cohen AT, Alikhan R, Arcelus J, et al. Assessment of venous
thromboembolism risk and the benefits of thromboprophylaxis in
medical patients. Thromb Haemost. 2005;94:750-759.
4. Ridker PM, Miletich JP, Hennekens CH, et al. Ethnic distribution of
factor V Leiden in 4047 men and women. Implications for venous
thromboembolism screening. JAMA. 1997;277:1305-1307.
41
Coagulation
5. Castoldi E, Rosing J. Factor V Leiden: a disorder of factor V
anticoagulant function. Curr Opin Hematol. 2004;11:176-181.
6. De Stefano V, Finazzi G, Mannucci PM. Inherited thrombophilia:
pathogenesis, clinical syndromes, and management. Blood.
1996;87:3531-3544.
7. Schambeck CM, Hinney K, Haubitz B, et al. Familial clutering of high
factor VIII levels in patients with venous thromboembolism.
Arterioscler Thromb Vasc Biol. 2001;21:289-292.
8. den Heijer M, Koster T, Blom HK, et al. Hyperhomocysteinemia as a
risk factor for deep-vein thrombosis. N Engl J Med. 1996;334:759-
762.
9. Martinelli I, Battaglioli T, Pedotti P, et al. Hyperhomocysteinemia in
cerebral vein thrombosis. Blood. 2003;102:1363-1366.
10. Rosendaal FR, Doggen CJ, Zivelin A, et al. Geographic distribution
of the 20210 G to A prothrombin variant. Thromb Haemost.
1998;79:706-708.
11. Poort SR, Rosendaal FR, Reitsma PH, et al: A common genetic
variation in the 3’-untranslated region of the prothrombin gene is
associated with elevated plasma prothrombin levels and an
increase in venous thrombosis. Blood. 1996;88:3698-3703.
12. Gallus A. Management options for thrombophilias. Semin Thromb
Hemost. 2005;31:118-126.
13. Girling J, de Swiet M. Inherited thrombophilia and pregnancy. Curr
Opin Obstet Gynecol. 1998;10:135-144.
14. Franchini M, Veneri D. Inherited thrombophilia: an update. Clin Lab.
2005;51:357-365.
15. Van Cott EM, Laposata M, Prins M. Laboratory evaluation of
hypercoagulability with venous or arterial thrombosis. Arch Pathol
Lab Med. 2002;126:1281-1295.
16. Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable
states. Hematol Oncol Clin North Am. 1998;12:1141-1166.
17. Jorquera JI, Montoro JM, Fernandez MA, et al. Modified test for
activated protein C resistance. Lancet. 1994;344:1162-1163.
18. Tripodi A, Negri B, Bertina RM, et al. Screening for the FV:Q506
mutation—evaluation of thirteen plasma-based methods for their
diagnostic efficacy in comparison with DNA analysis. Thromb
Haemost. 1997;77:436-439.
19. de Visser MC, Rosendaal FR, Bertina RM. A reduced sensitivity for
activated protein C in the absence of factor V Leiden increases the
risk of venous thrombosis. Blood. 1999;93:1271-1276.
20. Rosendaal FR, Koster T, Vandenbroucke JP, et al. High risk of
thrombosis in patients homozygous for factor V Leiden. Blood.
1995;85:1504-1508.
21. Vandenbroucke JP, Koster T, Briet E, et al. Increased risk of venous
thrombosis in oral-contraceptive users who are carriers of factor V
Leiden mutation. Lancet. 1994;344:1453-1457.
22. Kupferminc MJ, Eldor A, Steinman N, et al. Increased frequency of
genetic thrombophilia in women with complications of pregnancy.
N Engl J Med. 1999;340:9-13.
23. Zoller B, Svensson PJ, He X, Dahlback B. Identification of the same
factor V gene mutation in 47 out of 50 thrombosis-prone families
with inherited resistance to activated protein C. J Clin Invest.
1994;94:2521-2524.
24. Triado I, Mateo J, Soria JM, et al. The ABO blood group genotype
and factor VIII levels as independent risk factors for venous
thromboembolism. Thromb Haemost. 2005;93:468-474.
25. Ridker PM, Hennekens CH, Selhub J, et al. Interrelation of
hyperhomocyst(e)inemia, factor V Leiden, and risk of future venous
thromboembolism. Circulation. 1997;95:1777-1782.
26. Francis CW. Plasminogen activator inhibitor-1 levels and
polymorphisms: association with venous thromboembolism. Arch
Pathol Lab Med. 2002;126:1401-1404.
27. Visanji JM, Seargent J, Tahri D, et al. Influence of the –675 4G/5G
dimorphism of the plasminogen activator inhibitor 1 promoter on
42

Section 3 Coagulation

thrombotic risk in patients with factor V Leiden. Br J Haematol.
2000;110:135-138.
28. Zoller B, Garcia de Frutos P, Dahlback B. A common 4G allele in the
promoter of the plasminogen activator inhibitor-1 (PAI-1) gene as a
risk factor for pulmonary embolism and arterial thrombosis in
hereditary protein S deficiency. Thromb Haemost. 1998;79:802-807.
29. Martinelli I, Taioli E, Bucciarelli P, et al. Interaction between the
G20210A mutation of the prothrombin gene and oral contraceptive
use in deep vein thrombosis. Arterioscler Thromb Vasc Biol.
1999;19:700-703.
30. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of
lupus anticoagulants: an update. On behalf of the Subcommittee on
Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and
Standardization Committee of the ISTH. Thromb Haemost.
1995;74:1185-1190.
31. Exner T, Triplett DA, Taberner D, et al. Guidelines for testing and
revised criteria for lupus anticoagulants. SSC Subcommittee for the
Standardization of Lupus Anticoagulants. Thromb Haemost.
1991;65:320-322.

Appendix 1. Tests Used in The Diagnosis of Thrombophilia

Test Code Test Name Method Description

763X Activated Partial Photo-optical clot A silica-synthetic phospholipid reagent is mixed with patient plasma, and calcium
Thromboplastin detection chloride is then added to initiate clot formation. The time elapsed for formation of
Time (APTT) a fibrin clot is measured.
22X Activated Protein C dRVVT clot-based Endogenous protein C is activated prior to performing a dRVVT test. Results are
Resistance reported as the ratio of dRVVT clotting times obtained with and without activation
of endogenous protein C.
216X Antithrombin, Antithrombin in the patient’s sample binds to thrombin; excess thrombin cleaves a
Activity synthetic thrombin substrate, releasing a fluorescent substance, the amount of
which is inversely proportional to the amount of antithrombin activity in the
plasma.
5158X Antithrombin, Nephelometry Antithrombin antigen is measured in a fixed rate time nephelometry assay.
Antigen
15914X C4 Binding Protein1 Radial immunodiffusion The patient’s sample is mixed with agarose gel containing sheep anti-human C4
binding protein antibodies. Antigen-antibody complexes are detected as a visible
precipitating ring.
36189X Cardiolipin Antibody EIA The level of antibodies directed against various plasma proteins that bind to
Screen with Reflex phospholipid surfaces (eg, damaged endothelial membranes, monocytes, tumor
to IgA, IgG, and IgM cells, etc) is measured. The method is highly sensitive but not specific for
E2-glycoprotein I. Individual tests for IgA, IgG, and IgM antibodies are available
(separate order codes).
11294X Cytochrome P450 PCR Single nucleotide primer extension and detection of fluorescent extension products
2C9 Genotype1 are utilized to detect the 2 most common variants in the CYP2C9 gene, CYP2C9*2
and CYP2C9*3.
8659X D-Dimer Immunoturbidimetric The increase in light absorption caused by the agglutination of D-dimer antibody-
coated latex particles with endogenous D-dimer is measured.
15780X dRVVT Screen with Photo-optical clot The time elapsed for clot formation following Russell viper venom activation of
Reflex to detection factor X is determined. When the dRVVT is prolonged, another dRVVT test is
Phospholipid performed (at an additional charge [additional CPT code]) after adding excess
Neutralization phospholipid in the presence of a heparin neutralizer. The ratio of the 2 dRVVT
tests is calculated, and the result is reported as “confirmed” or “not confirmed”.
17902X Factor V HR2 Allele PCR, oligonucleotide The HR2 allele (4070GmA) in the factor V gene is detected by amplification of the
DNA Mutation ligation, gene region with PCR, followed by oligonucleotide ligation and hybridization to
Analysis2,3 chemiluminescent color-coded microspheres. Reaction products are detected by chemiluminescent
detection detection of the microspheres.
17900X Factor V (Leiden) PCR, luminescent The 1691GmA factor V Leiden mutation is detected using allele specific probes.
Mutation Analysis3 detection Results are reported as negative, heterozygous positive, or homozygous positive.
16049X Factor VIII Activity, Chromogenic Factor VIII in the patient sample is activated by mixing with thrombin and
Chromogenic activated factor IX. Activated factor VIII then accelerates the factor IXa-mediated
conversion of factor X to Xa. Factor Xa activity, which is proportional to factor VIII
activity, is measured by the change in a chromogenic substrate. This assay
provides increased sensitivity (relative to the factor VIII clotting assay) at levels
>150% and is insensitive to the effects of direct thrombin inhibitors and heparin. It
is, therefore, the preferred assay when a lupus anticoagulant is present.

(continued)
 43

Section 3 Coagulation

Appendix 1. Tests Used in The Diagnosis of Thrombophilia (continued)

Test Code Test Name Method Description

347X Factor VIII Activity, Photometric clot Patient plasma is mixed with factor VIII-deficient normal plasma. The APTT
Clotting detection clotting time of the mixed plasma is compared to that of reference plasma. Results
are reported as percent of normal factor VIII activity.
11074X Fibrin Monomer Hemagglutination Patient plasma is mixed with human erythrocytes coated with purified fibrin monomer.
The presence of soluble fibrin monomer complexes in plasma results in agglutination.
461X Fibrinogen Photo-optical clot Fibrinogen in the patient sample is converted to fibrin in the presence of excess
detection thrombin. The clotting time obtained is inversely proportional to the fibrinogen
level (activity).
458X Fibrinogen Agglutination In the presence of corresponding antigens, latex particles coated with monoclonal
Degradation Products anti-FDP antibodies agglutinate to form macroscopic clumps.
37801X Fibrinogen, Nephelometry Patient plasma is reacted with highly specific antiserum to form insoluble antigen-
Quantitative, antibody complexes, which are detected in solution as turbidity.
Nephelometry
30340X C2-Glycoprotein I EIA This enzyme immunoassay semi-quantitatively measures antibodies directed
Antibodies (IgA, against C2-glycoprotein I (C2-GPI, apolipoprotein H). Individual tests for IgA, IgG,
IgG, IgM) and IgM are available (separate order codes).
30292X Heparin, Low Chromogenic The level of low molecular weight heparin in plasma is measured by the inhibition
Molecular Weigh of activated factor X (Xa) activity in the presence of excess antithrombin.
(Xa Inhibition)1
404X Heparin, Chromogenic The level of unfractionated heparin in plasma is measured by inhibition of
Unfractionated activated factor X (Xa) activity in the presence of excess antithrombin.
(Xa Inhibition)
31789X Homocysteine Competitive The level of total homocysteine (ie, protein-bound, oxidized, and free, reduced
immunoassay homocysteine) is measured in a competitive immunochemiluminometric assay.
10707X Human Platelet DNA based capture/ After amplification of the segment of the GPIIIa gene that includes the HPA-1
Antigen 1 Genotype2 binding polymorphism, allele specific probes are used in a chromogenic assay to
determine the genotype.
34604X Lipoprotein (a) Immunoprecipitin Agglutination occurs between lipoprotein (a) in the patient sample and anti-
lipoprotein (a) antibodies adsorbed to latex particles and is measured
photometrically.
17911X Methylenetetra- PCR, oligonucleotide This test detects the 677CmT and 1298AmC point mutations. Results for both
hydrofolate ligation assay mutations are reported as negative, heterozygous positive, or homozygous
Reductase (MTHFR) positive.
DNA Mutation
Analysis3
8922X Mixing/Correction Clot Detection APT and/or APTT is measured after mixing patient plasma and normal plasma to
Study determine the cause (eg, factor deficiency, factor inhibitor, and/or nonspecific
inhibitor) of a prolonged PT and/or APTT test result.
36595X Phosphatidylserine Chromogenic The presence of IgA, IgG, and IgM antibodies to phosphatidylserine is detected in
Antibodies an enzyme immunoassay.
36555X Plasminogen Activator EIA The concentration of plasminogen activator inhibitor-1 (antigen) is measured in an
Inhibitor-1 (Antigen) enzyme immunoassay.
10491X Plasminogen Chromogenic The amount of functional PAI-1 in plasma is detected based on PAI-1 inhibition of
Activator Inhibitor-1 tissue plasminogen activator (tPA), which catalyzes the conversion of plasminogen
Activity to plasmin.
11368X Plasminogen PCR This test employs PCR amplification of a 150-bp fragment of the PAI-1 gene,
Activator Inhibitor-1 followed by restriction enzyme digestion and gel electrophoresis separation of the
4G/5G Polymorphism3 products.
4458X Plasminogen Activity Chromogenic Plasminogen in the patient sample forms a complex with streptokinase. Hydrolysis
of this complex produces a color, the intensity of which is directly proportional to
the percent of normal plasminogen activity.

(continued)
44

Section 3 Coagulation

Appendix 1. Tests Used in The Diagnosis of Thrombophilia (continued)

Test Code Test Name Method Description

5164X Plasminogen, Nephelometry Specific antiserum to plasminogen forms antigen-antibody complexes that are
Antigenic detected in solution as turbidity.
1777X Protein C Activity Chromogenic Snake venom is used to activate protein C in the patient sample. The activated
protein C then stimulates the breakdown of a chromogen inducing production of a
color that is measured photometrically.
4948X Protein C Antigen EIA Protein C antigen is measured via an enzyme immunoassay.
1779X Protein S, Activity Clot detection Protein S in the patient sample enhances the anticoagulant action of activated
protein C, resulting in a prolonged clotting time. The increase in clotting time is
directly proportional to the percent of normal protein S activity.
5165X Protein S, Immunoassay The level of total protein S (bound and free) is measured in a microlatex particle-
Total Antigen mediated immunoassay.
10170X Protein S, Free Immunoturbidimetric The increase in light absorption caused by the agglutination of free protein S
antibody-coated latex particles with endogenous free protein S is measured.
17909X Prothrombin PCR In this single nucleotide polymorphism genotyping system, PCR amplification is
(factor II) 20210Gm followed by hybridization to wild-type and mutation-specific probes in separate
A Mutation1,3 reaction wells. A perfect match between probe and patient DNA sequence is
required for light generation and identification of wild-type and mutant DNA.
Results are reported as negative, heterozygous, or homozygous positive for the
20210GmA mutation.
37674X Prothrombin EIA Anti-prothrombin fragment 1.2 is used to measure the level of prothrombin
Fragment 1.2 fragment 1.2
17408X PTT-LA with Reflex Clot-based PTT Dilute cephalin is used, thus increasing this assay’s sensitivity to weak
to Hexagonal Phase phospholipid antibodies. Prolonged PTT-LA test results are confirmed (at an
Neutralization additional charge [additional CPT code]) with a hexagonal phase neutralization
test.
36737X Red Cell CD55 and Flow cytometry Single color flow cytometry utilizes anti-CD55 and anti-CD59 monoclonal
CD59 Expression2 antibodies to detect the relative amounts of CD55- and CD59-deficient red blood
cells.
37700X Reptilase Clotting Clotting Reptilase, an enzyme derived from the venom of Bothrops atrox, cleaves
Time1 fibrinopeptide A from fibrinogen resulting in the production of fibrin and
subsequent clot formation. Clotting time is determined by measuring the increase
in viscosity after reptilase is added to test plasma.
14627X Serotonin Release Radiobinding Fc(gamma)RIIa receptor-phenotyped platelets from highly reactive donors are
Assay (SRA)1 14
C serotonin incubated with 14C-serotonin. Porcine heparin (0.1, 0.5, and 100 U) is then added
along with patient serum; the presence of antibodies to the heparin-Fc(gamma)RIIa
receptor complex in the sera of patients with HIT causes the release of serotonin.
Results are reported as percent serotonin released.
10296X Soluble P-Selectin, ELISA Soluble P-selectin is quantitatively determined in a solid phase ELISA assay.
ELISA2
10162X Thrombin- ELISA Thrombin is inhibited by antithrombin and the resulting inactive
Antithrombin (TAT) proteinase/inhibitor complex is measured quantitatively by ELISA.
Complex
883X Thrombin Clotting Clot detection Thrombin is mixed with the patient plasma and clotting time is determined
Time photometrically.
19413X Thrombin Generation1 Flourogenic, kinetic The coagulation process is initiated by the addition of tissue factor, anionic
phospholipid membrane, and calcium chloride to plasma. The amount of thrombin
generated is then measured by the fluorescence of a thrombin-specific
chromogenic substrate. The fluorescent intensity is proportional to the
concentration of thrombin and monitored continuously.
10656X Tissue Factor1 EIA This EIA utilizes a murine anti-human tissue factor monoclonal antibody to
determine the level of tissue factor.

(continued)
 45

Section 3 Coagulation

Appendix 1. Tests Used in The Diagnosis of Thrombophilia (continued)

Test Code Test Name Method Description
29816X Tissue Plasminogen EIA This EIA utilizes goat anti-tPA IgG to determine the level of tPA.
Activator (tPA)
14532X von Willebrand Electrophoresis The patient sample is incubated with protease free multimeres and the extent of
Factor Protease degradation is determined by electrophoresis. The presence of inhibitors is
(ADAMTS-13) Activity determined in a mixing assay (at an additional charge [additional CPT code]).
with Reflex to
Protease Inhibitor1
EIA, enzyme immunoassay; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.
1
This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
2
This test is performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been determined by Quest Diagnostics
Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means.
3
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche Molecular Systems, Inc.

Appendix 2. Antiphospholipid Syndrome
Antiphospholipid syndrome, the most common cause of acquired
thrombophilia, is characterized by the presence of antiphospholipid
antibodies such as lupus anticoagulants (LA), anticardiolipin antibodies
(ACA), and phosphatidylserine antibody. The syndrome is associated
with both arterial and venous thrombosis, systemic lupus erythematosus
(SLE), connective tissue and autoimmune disorders, malignancy, HIV
infection, drug ingestion, and obstetric, hematologic, neurologic,
dermatologic, and cardiac complications (Table 8).
Although no single test is 100% sensitive or specific for LA, the
diagnosis usually begins with demonstration of prolonged phospholipid-
dependent clotting times (eg, APTT).30,31 When the APTT is prolonged, a
mixing study is highly recommended to rule in presence of an inhibitor,
signified by lack of clotting time correction. Following an uncorrected
mixing study result, use of the dRVVT and the PTT-LA tests is highly
recommended. When the dRVVT or the PPT-LA is prolonged, a
confirmatory test is automatically performed (at an additional charge
and CPT code). A prolonged dRVVT is confirmed with a phospholipid
neutralization test (dRVVT confirm), while a prolonged PTT-LA test is
confirmed with the hexagonal phase neutralization test. Traditional
mixing studies to confirm prolonged dRVVT and PTT-LA tests are not
needed since normal plasma is included in the confirmation test.
Normal plasma will correct prolonged clotting times if there is an
underlying factor deficiency, whereas in the presence of an inhibitor, the
test remains prolonged.
Although highly sensitive, ACA testing is not specific for thrombotic risk.
Transient LA or ACA may be due to infectious diseases (varicella,
rubella, adenovirus, HIV) or drug exposure (amoxicillin, chlorpromazine,
hydralazine). Since transient LA or ACA are not associated with clinical
complications, 2 positive tests, more than 3 months apart, are
recommended for diagnosis of clinically significant antiphospholipid
antibodies.16
The diagnosis of antiphospholipid syndrome may be improved by using
the C2-glycoprotein I antibody assay. C2-Glycoprotein I antibodies of the
IgA, IgG, and IgM isotype are associated with the antiphospholipid
syndrome, and their presence is more specific but less sensitive than
cardioipin antibodies for the diagnosis of antiphospholipid syndrome.
Individuals who are positive for cardiolipin antibodies and negative for
C2-glycoprotein I antibodies are more likely to have an infection
(varicella, rubella, adenovirus, HIV) or drug exposure (amoxicillin,
chlorpromazine, hydralazine) than antiphospholipid syndrome.

Table 8: Complications Associated with Antiphospholipid Syndrome

Obstetric – Maternal Hematologic Dermatologic
Deep vein thrombosis Thrombocytopenia Skin necrosis
Chorea Hemolytic anemia Ulceration
Postpartum syndrome Evans syndrome Livedo reticularis
Eclampsia/preeclampsia Low complement 3 & 4 Digital gangrene

Obstetric – Fetal Neurologic Cardiac

Intrauterine fetal death Chorea Coronary artery disease
Recurrent spontaneous abortion Stroke Libman-Sacks endocarditis
Fetal growth retardation Multi-infarct dementia Arterial graft occlusion
Prematurity TIA/CVA
Neonatal thrombosis Transverse myelitis
Migraine
46
Section 3
A specific phosphatidylserine antibody test can also be used to identify
an antiphospholipid antibody.
3.5.2 Cardiolipin Antibody Screen with Reflex to IgA, IgG, IgM
Clinical Use: This test is used in the differential diagnosis of
thrombosis and recurrent spontaneous abortions. It is also used to rule
out antiphospholipid syndrome and false-positive VDRL attributable to
cardiolipin antibodies.
Clinical Background: Although their mechanism of action has not yet
been clearly identified, cardiolipin antibodies now have a well-
established causative role in thrombosis. Individuals with the
antiphospholipid syndrome can have cardiolipin antibodies and/or lupus
anticoagulant antibodies. The latter are detected by a phospholipid-
dependent coagulation test (Lupus Anticoagulant). Both tests should be
performed to rule out an antiphospholipid syndrome.
The distribution of values from the normal population is skewed to the
right, so that results within 2 times the cutoff are considered low-
positive and have relatively low positive predictive values. The presence
of persistent high-positive IgG cardiolipin antibody results correlates
well with presence of disease, while IgM antibodies are more transient
and are seen in patients taking medication known to cause drug-
induced lupus. IgA antibodies usually correlate with IgG and are not
commonly found alone in patients with antiphospholipid syndrome. The
diagnosis of an antiphospholipid syndrome may be based on the
detection of cardiolipin antibodies, preferably high levels of IgG on 2
separate occasions, in a patient with a well-documented episode of
unexplained venous-arterial thromboembolism or recurrent miscarriage.
These individuals should then be evaluated for long-term anticoagulant
therapy.
Interpretive Information: Cardiolipin antibodies are associated with
antiphospholipid syndrome manifest by arterial or venous thrombosis or
spontaneous miscarriage. They are also associated with some infectious
diseases (eg, hepatitis C infection), false-positive VDRL test results,
prolonged activated partial thromboplastin time (aPTT), and drug
reactions. Almost half of the patients with systemic lupus
erythematosus have cardiolipin antibodies.
3.5.3 CD55 and CD59 Expression
See Hematology/Oncology, “Paroxysmal Nocturnal Hemoglobinuria
(PNH)” section 6.10.1
3.5.4 Factor V (Leiden) Mutation Analysis
Clinical Use: This test may be used to identify individuals at increased
risk of venous and arterial thrombosis or pregnancy-associated
complications, to identify those at risk from oral contraceptive use, and
to confirm positive activated protein C resistance (APCR) test results,
especially in patients with lupus anticoagulant (LA).
Clinical Background: The factor V (Leiden) mutation (1691GmA)
occurs primarily in white populations and is a major risk factor for
venous thrombosis (21% frequency in affected individuals) and a lesser
risk factor for arterial thrombosis (cardiovascular disease). The mutation
is also highly associated with increased complications during pregnancy
and puerperium. Such complications include severe preeclampsia,
placental abruption, fetal growth retardation, recurrent miscarriage,
stillbirth, and maternal pulmonary embolism and death. Due to a
synergistic increase in venous thrombosis risk, individuals heterozygous
for the factor V mutation are at greater risk when taking oral
contraceptives.
Coagulation
The factor V mutation leads to replacement of Arg506 with Gln (R506Q)
and APCR. Since laboratory tests for APCR are highly sensitive and
specific and simpler to perform, APCR is usually the test of choice;
however, factor V mutation analysis is recommended to confirm positive
APCR tests. It is also recommended in place of APCR for pregnant
patients and those with lupus anticoagulant, since such patients often
have a false-positive APCR test result.
Method: Utilizing polymerase chain reaction (PCR), allele-specific
probes, and luminescent detection, this assay detects the 1691GmA
factor V mutation. Results are reported as negative, heterozygous
positive, or homozygous positive.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The factor V Leiden mutation leads to the
laboratory finding of APCR and is associated with a 4- to 8-fold increase
in venous thromboembolic events in heterozygous individuals and a 50-
to 100-fold increase in homozygous subjects. When a heterozygous
mutation is coupled with oral contraceptive use, risk increases
synergistically to 30-fold. Risk also increases synergistically when the
mutation is coupled with increased homocysteine levels or the factor II
(prothrombin) 20210GmA mutation. Additionally, factor V is associated
with arterial thrombosis (especially in smokers), complications of
pregnancy (including fetal loss), and increased levels of factor VIII.
Although this test is highly specific, identification of a mutation may
occur in the absence of APCR in rare cases. Sensitivity of this test for
APCR is 94%; thus, a negative result does not rule out APCR or an
increased risk of venous thrombosis.
Mutation analysis is not affected by heparin, oral anticoagulants,
pregnancy, oral contraceptives, estrogen replacement therapy, or lupus
anticoagulants.
3.5.5 Protein S Panel
Clinical Use: This test is used to confirm the presence of a protein S
deficiency, subtype a protein S deficiency, and exclude a transient
protein S deficiency secondary to elevation in C4 binding protein.
Individuals Suitable for Testing include individuals <50 years of age
with stroke, superficial or deep vein thrombophlebitis, recurrent
miscarriage, or thrombosis after warfarin withdrawal.
Method: This panel includes tests for C4 binding protein, protein S
activity, free protein S, and total protein S antigen.
The C4 binding protein test was developed and its performance characteristics determined
by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S.
Food and Drug Administration. The FDA has determined that such clearance or approval is
not necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A normal total protein S level, combined
with a reduced free protein S and an increased C4 binding protein
indicates an acquired, rather than inherited, disorder. This is because C4
binding protein is an acute-phase reactant and binds approximately 50%
of protein S, rendering that portion inactive. Protein S activity is
commonly reduced during pregnancy but rebounds to normal 6 weeks
post-partum. Although commonly associated with deep vein thrombosis,
protein S deficiency is also reported in cerebral and arterial thrombosis.
A decrease in free protein S and/or protein S activity may sometimes
occur in association with pregnancy, nephrotic syndrome, lupus
 47

Section 3 Coagulation

Table 9. Subtyping Protein S Deficiency
Type I Type II
Protein S activity Low Low
Free protein S Low Normal
Total protein S Low Normal
anticoagulant, and L-asparaginase therapy as well as after vaccination
(eg, measles vaccine). These patients are prothrombotic and may require
anticoagulation.
Protein S activity results can be used in combination with free and total
levels to subtype protein S deficiency (Table 9).
3.5.6 Prothrombin Time
See Coagulation, “Hemostasis” section 3.2.6
3.5.7 Prothrombin (Factor II) 20210GmA Mutation Analysis
Clinical Use: This test is used to estimate future thrombotic risk,
assess risk of obstetric complications, and assess thrombophilia risk in
family members of affected individuals.
Clinical Background: Prothrombin, or factor II, is the precursor of
thrombin; as such, it plays a key role in the balance between
procoagulation and anticoagulation. A prothrombin genetic variant
known as 20210GmA has been associated with elevated levels of
prothrombin and with thrombophilia. This variant is located at position
20210 of the 3’ untranslated region of the prothrombin gene on
chromosome 11 and leads to substitution of an adenine for a guanine.
Found primarily in whites, the prevalence is approximately 2% in
healthy individuals, 6% to 8% in venous thrombosis cases (18% in
known familial cases1), and 3% to 5% in arterial thrombosis cases
(Table 10). More importantly, the relative risk (RR) in the general
population is 2 to 5 for venous thrombosis and 0.9 to 4 for arterial
thrombosis. The venous thrombosis risk is even higher in selected
populations such as patients with a history of thrombotic episodes
during pregnancy or the puerperium (RR = 15).2 Additionally, in patients
heterozygous for both the factor V Leiden mutation and the 20210GmA
Type III mutation, risk of thrombosis increases synergistically.2 Approximately
11% of individuals positive for factor V Leiden are positive for the
Low prothrombin 20210GmA mutation. The mutation has also been
associated with risk of recurrent miscarriage (RR = 4.6)3 and other
Low
obstetric complications. In summary, the prothrombin 20210GmA
Normal mutation is well established as a risk factor for venous thrombosis, but
much controversy still exists regarding the mutation’s value in assessing
arterial thrombotic risk.
Individuals Suitable for Testing include symptomatic individuals,
individuals with a family history of thrombosis or thrombophilia-
associated mutations, and high-risk individuals predisposed by surgery,
trauma, immobility, pregnancy, oral contraceptives, etc.
Note: Pregnant women with a personal or family history of thrombosis,
recurrent spontaneous abortions, and severe early onset preeclampsia
are at high risk.
Method: In this single nucleotide polymorphism (SNP) genotyping
system, the 3’ untranslated region of the prothrombin gene is amplified
via polymerase chain reaction (PCR), followed by hybridization to wild
type and mutation-specific probes in separate reaction wells. A perfect
match between probe and patient DNA sequence is required for light
generation and identification of wild type and mutant DNA. Results are
reported as negative, heterozygous positive, or homozygous positive for
the 20210GmA mutation.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Negative results indicate the absence of
the prothrombin 20210GmA mutation but do not rule out the presence
of other rare mutations within the prothrombin gene. Heterozygous
positive results are associated with a 2- to 5-fold increased risk for
venous thrombosis, increased risk for obstetric complications (eg,
preeclampsia, abruptio placentae, fetal growth retardation, and
stillbirth), and, possibly, premature coronary heart disease. Thrombosis

Table 10. Prothrombin 20210GmA Prevalence and Associated Relative Risk

Prevalence (%) Venous Thrombosis Arterial Thrombosis*

in Healthy Individuals Prevalence (%) RR Prevalence (%) RR

Poort SR, et al1 2.3 6.2 2.8

Cumming AM, et al4 1.2 5.5 5.4
5
Hillarp A, et al 1.8 7.1 4.2
6
Schobess R, et al 3 8.4 3.0
7
Ridker PM, et al 3.9 6.4 1.7 3.5 0.95
8
Rosendaal FR, et al 1.6 5.1 4.0
9 †
Araujo F, et al 5 3.8 Not provided
Arruda VR, et al10 0.7 3.2‡ Not provided
Martinelli I, et al11 3.2 3.8 1.2
RR, relative risk.
*Includes cerebral ischemia, myocardial infarction, unstable angina.
†
Not significantly different from healthy individuals.
‡
Significantly different from healthy individuals (P 
0.03).
48
Section 3
risk increases synergistically in the presence of oral contraceptive use
(odds ratio is 149.3 for cerebral vein thrombosis12 and 69 for venous
thrombosis2) and the factor V Leiden mutation (estimated odds ratio is
107 for women with a history of venous thromboembolism during
pregnancy and the puerperium2). Homozygous positive results are rare.
All test results should be interpreted in conjunction with clinical and
family data.
References
1. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common
genetic variation in the 3’-untranslated region of the prothrombin
gene is associated with elevated plasma prothrombin levels and an
increase in venous thrombosis. Blood. 1996;88:3698-3703.
2. Gerhardt A, Scharf RE, Beckmann MW, et al. Prothrombin and factor
V mutations in women with a history of thrombosis during
pregnancy and the puerperium. N Engl J Med. 2000;342:374-380.
3. Foka ZJ, Lambropoulos AF, Saravelos H, et al. Factor V Leiden and
prothrombin G20210A mutations, but not methylenetetrahydrofolate
reductase C677T, are associated with recurrent miscarriages. Hum
Reprod. 2000;15:458-462.
4. Cumming AM, Keeney S, Salden A, et al. The prothrombin gene
G20210A variant: prevalence in a U.K. anticoagulant clinic
population. Br J Haematol. 1997;98:353-355.
5. Hillarp A, Zoller B, Svensson PJ, Dahlback B. The 20210 A allele of
the prothrombin gene is a common risk factor among Swedish
outpatients with verified deep venous thrombosis. Thromb Haemost.
1997;78:990-992.
6. Schobess R, Junker R, Auberger K, et al. Factor V G1691A and
prothrombin G20210A in childhood spontaneous venous thrombosis
– Evidence of an age-dependent thrombotic onset in carriers of
factor V G1691A and prothrombin G20210A mutation. Eur J Pediatr.
1999;158(Suppl 3):S105-S108.
7. Ridker PM, Hennekens CH, Miletich JP. G20210A mutation in
prothrombin gene and risk of myocardial infarction, stroke, and
venous thrombosis in a large cohort of US men. Circulation.
1999;99:999-1004.
8. Rosendaal FR, Siscovick DS, Schwartz SM, et al. A common
prothrombin variant (20210 G to A) increases the risk of myocardial
infarction in young women. Blood. 1997;90:1747-1750.
9. Araujo F, Santos A, Araujo V, et al. Genetic risk factors in acute
coronary disease. Haemostasis. 1999;29:212-218.
10. Arruda VR, Siquiera LH, Chiaparini LC, et al. Prevalence of the
prothrombin gene variant 20210 GmA among patients with
myocardial infarction. Cardiovasc Res. 1998;37:42-45.
11. Martinelli I, Franchi F, Akwan S, et al. The transition G to A at
position 20210 in the 3’-untranslated region of the prothrombin
gene is not associated with cerebral ischemia. Blood. 1997;90:3806.
12. Martinelli I, Sacchi E, Landi G, et al. High risk of cerebral-vein
thrombosis in carriers of a prothrombin-gene mutation and in users
of oral contraceptives. N Engl J Med. 1998;338:1793-1797.
3.5.8 Thrombotic Marker Panel
Clinical Use: This test is used to screen for intravascular thrombosis.
Individuals Suitable for Testing include individuals in a
chemotherapy program; individuals with vascular access grafts,
suspected failed vascular grafts, suspected dissecting aneurysm,
suspected intra-atrial or ventricular thrombus formation, suspected
silent venous thrombosis (pelvic veins), or suspected low-grade
disseminated intravascular coagulation (DIC); and those on
anticoagulation therapy.
Coagulation
Method: This panel includes tests for D-dimer, fibrin monomer,
prothrombin fragment 1.2, and thrombin-antithrombin (TAT) complex.
Interpretive Information: When intravascular thrombosis is
suspected in the absence of overt clinical symptoms, an elevated level
of D-dimer, fibrin monomer, prothrombin fragment 1.2, or TAT complex
may be the only indicator of intravascular clot formation and generation
of activated factor X. Marked elevation in only 1 or 2 of these markers
should lead to further investigation of the site of the thrombosis (eg,
early dissecting aortic aneurysm, anomalous arterial/venous fistula).
3.5.9 Thrombophilia Mutation Analysis with Reflex to HR2
Mutation Analysis
Clinical Use: This test is used to detect mutations associated with an
increased risk of thrombophilia.
Individuals Suitable for Testing include those with a personal or
family history of early-onset venous thrombosis, usually before the age
of 50, with or without provocation. The mutations detected in this panel
are more prevalent in individuals of European ancestry.
Method: This panel includes tests for the factor V (Leiden) mutation
and the prothrombin (factor II) 20210GmA mutation. When a factor V
(Leiden) mutation is detected, the test reflexes to a factor V HR2
mutation analysis.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The factor V (Leiden) 1691GmA mutation
lowers resistance to activated protein C (APC) and leads to a
thrombophilic state. Heterozygotes have a 4-8-fold increased risk for
venous thrombosis, and homozygotes have an 80-fold increased risk.
The presence of the HR2 mutation, in combination with a factor V
(Leiden) mutation, lowers the resistance to APC even further and
therefore increases the prothrombotic phenotype.
Presence of the prothrombin 20210GmA mutation is associated with
increased prothrombin levels and 2- to 5-fold increased risk of venous
thrombosis. Both factor V (Leiden) and the prothrombin 20210GmA
mutation are associated with increased risk of obstetric complications
(eg, preeclampsia, abruptio placentae, fetal growth retardation, and
stillbirth), and possibly premature coronary artery disease.
These mutations can be detected even when patients are on warfarin
therapy.
3.5.10 Thrombophilia Screen, Inherited
Clinical Use: This test is used to screen for the more frequent causes
of inherited thrombophilia.
Individuals Suitable for Testing include symptomatic individuals,
individuals with a family history of thrombosis or thrombophilia-
associated mutations, and high-risk individuals predisposed by surgery,
trauma, immobility, pregnancy, oral contraceptives, etc.
Note: high-risk pregnant women include those with a personal or family
history of thrombosis and those with a previous neural tube defect-
affected fetus, recurrent spontaneous abortion, or severe early-onset
pre-eclampsia.
 49

Section 3 Coagulation

Method: This panel includes tests for antithrombin III activity, factor V
(Leiden) mutation with reflex to factor V HR2 mutation,* protein C
activity, free protein S, and prothrombin (factor II) 20210GmA
mutation.* The reflex to factor V HR2 mutation will occur when a factor
V (Leiden) mutation is present.
*This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Decreased antithrombin III activity, protein
C activity, and free protein S, and presence of the factor V (Leiden)
1691GmA, factor V HR2, and/or factor II 20210GmA mutation each
contribute to an increased risk of hereditary thrombophilia.
Approximately 10% of factor V (Leiden) heterozygotes also carry the
prothrombin mutation. Likewise, protein C and/or S deficiency have
been reported with the factor V (Leiden) mutation. Since venous
thrombosis can be a multifactorial disorder, presence of a second risk
factor further increases the thrombotic risk.
3.6 von Willebrand Disease
3.6.1 Laboratory Support of Diagnosis
Clinical Background: von Willebrand disease (vWD), the most
common inherited bleeding disorder, is due to either a quantitative or
qualitative defect of von Willebrand Factor (vWF). vWF is synthesized by
endothelial cells and megakaryocytes and circulates as a high molecular
weight glycoprotein multimer non-covalently bonded to factor VIII and,
as such, functions as a carrier protein that protects factor VIII from
proteolysis. vWF multimers range in size from 500,000 to >20 million
Daltons, and multimer size is correlated with hemostatic activity, ie,
larger multimers are more hemostatically active. vWF is stored in
platelet alpha granules and endothelial cell Wiebel-Palade bodies.
In normal clotting, vWF mediates platelet-platelet and platelet-vessel
wall adhesion via glycoprotein 1b (GP1b) on the platelet surface. When
the level of vWF is decreased, hemostasis is impaired due to
inadequate platelet binding and a reduction in factor VIII activity, which
is caused by decreased levels of factor VIII (ie, lack of protection from
proteolysis).
The incidence of vWD is estimated to be 1% to 3%.1,2 Inherited vWD is
divided into 3 types reflecting pathophysiology (Table 11).2-4 Additionally,
acquired vWD, frequently called von Willebrand syndrome, and platelet-
type vWD have been described.
Type 1 vWD accounts for approximately 70% of cases. It is characterized
by a partial deficiency (10% to 45% of normal) of vWF secondary to
decreased production or release. Additionally, a subset of type 1 vWD
secondary to increased clearance of vWF has recently been identified.5
Individuals with type 1 vWD may be asymptomatic or have mild
symptoms (eg, bleeding from gums or heavy menstrual cycles) until a
severe injury or operation precipitates a significant bleeding episode.1,2
Type 2 vWD accounts for approximately 30% of cases and is
characterized by defective vWF function. Circulating vWF levels are

Table 11. Characteristics and Differential Diagnosis of Inherited von Willebrand Disease
vWF
Ristocetin Factor VIII Collagen Molecular
Frequency Genetic Response vWF Cofactor Clotting Binding Weight
Type (% of vWD) Bleeding Transmissiona to DDAVP Antigen (vWF Activity) Activity Assay Multimer
1 70 Asymptomatic Autosomal Good n n n Normal or n Normal
to moderately dominant
severe
2A 10-15 Moderate to Autosomal Mild to Normal or n Normal or n Normal or n nnn High &
moderately dominant or moderate intermediate
severe recessive missing
2B <5 Moderate to Autosomal DDAVP not Normal or n Normal or n Normal or n nnn High missing
moderately dominant indicatedb
severe
2M 10-15 Significant Autosomal Mild to Normal or n n or nn Normal or n Normal Normal
dominant moderate
2N Uncommon Mild to Autosomal Suboptimal Normal Normal n or nn Normal Normal
moderate recessive
e e e
3 Rare Severe Autosomal No nnn nnn nnn nnn Absent
recessive responsec
Plateletd Uncommon Moderate to Autosomal DDAVP not Normal or n lll Normal or n Normal High often
moderately dominant indicated missing
severe
DDAVP, desamino-8-arginine vasopressin; vWD, von Willebrand disease; vWF, von Willebrand factor.
a
Incomplete penetrance is typically seen.
b
Some patients have a mild to moderate response without untoward events, while others have worsening thrombocytopenia and increased risk of stroke or heart attack.
c
Mild to moderate response in double heterozygotes (ie, type 1 and 3 heterozygote).
d
Also referred to as pseudo-von Willebrand disease.
e
Levels must be <10% to be classified as type 3 vWD.
50
Section 3
normal or marginally decreased. Individuals have bleeding symptoms
similar to those with type 1 vWD.1,2 Type 2 vWD subtypes, reflecting
distinct defects of vWF processing and multimeric composition, have
been identified (Table 11). von Willebrand type 2N (Normandy) is an
unusual variant associated with a defect in factor VIII binding to vWF,
which causes a low factor VIII activity, mimicking hemophilia.2
Type 3 vWD affects approximately 1 in 500,000 individuals and is
characterized by an almost complete deficiency of vWF and very low
levels of factor VIII. Affected individuals have severe bleeding that can
be life-threatening if not recognized and treated.1,2
Acquired vWD is associated with malignancies, immunologic diseases,
circulatory dysfunction such as aortic stenosis, and other medical
conditions (eg, hypothyroidism); it is also frequently associated with
myeloproliferative disorders.6,7 In acquired vWD, vWF is produced
normally but is rapidly removed from circulation by tumor cell adhesion,
vWF antibody mediated disruption of large multimers, or proteolytic
digestion. Some experts believe that up to 20% of individuals diagnosed
with vWD have an acquired form of the disorder.6,7
Platelet-type vWD is uncommon and characterized by a gain of function
of the von Willebrand binding protein located on the surface of
platelets, the loss of large multimers, and thrombocytopenia.8
Classification of vWD is crucial, as risk of bleeding and treatment vary
with type. Treatment for type 1 vWD is desmopressin (DDAVP), which
induces the release of stored vWF from endothelial cells. DDAVP may
also be effective in treating some individuals with type 2A and 2M
vWD, but it is ineffective in type 2N and 3 disease. DDAVP is not
indicated for type 2B and platelet vWD. Exogenous factor VIII and vWF
are used to treat types 2N and 3 vWD and individuals with type 2A or
2M when they don’t respond to DDAVP (Table 11). Some individuals
with acquired vWD respond to DDAVP; those that do not are typically
treated with exogenous factor VIII and vWF.6,7
Individuals Suitable for Testing include those with a history of
unexplained menorrhagia, lifelong bruising, unexplained epistaxis, or
significant bleeding from minor surgical procedures; those with a family
history of vWD; and some individuals with autoimmune disorders and/or
lymphoma/myeloma and new-onset bleeding.
Test Availability: Tests available to assist in diagnosis of von
Willebrand disease are listed in the Appendix. Refer to the Quest
Diagnostics Directory of Services for ordering information.
Test Selection and Interpretation: A laboratory work-up for
individuals suspected of having a bleeding disorder such as vWD begins
with a personal and family history. Individuals with vWD will frequently
have a history of epistaxis, which may be prolonged or unusually severe;
easy bruisability; gingival bleeding; prolonged bleeding from minor
wounds; or prolonged and heavy menstrual bleeding (Table 12). The
severity of bleeding, however, does not always correlate with the
degree of vWF deficiency, and symptoms can vary between patients
with the same type of vWD as well as between family members.1,3,9
Though vWD is inherited in an autosomal dominant or recessive
manner, many affected individuals will have a negative family history
due to incomplete penetrance. Additionally, it is important to rule out
the use of drugs that alter platelet function in individuals suspected of
having vWD, eg, non-steroidal anti-inflammatory drugs (NSAIDs) and
diuretics.10
Testing typically begins with a complete blood count (to assess
hemoglobin, hematocrit, platelet count, and morphology), bleeding time,
Coagulation
Table 12. Bleeding Characteristics of von Willebrand Disease
Bleeding from skin and mucous membranes (gingivae, nares, GI and
genitourinary tract)
Petechiae
Small, superficial ecchymoses
Hemarthroses, muscle hematomas (rare)
Significant bleeding after minor cuts
Immediate, mild bleeding after surgery
prothrombin time (PT), and activated partial thromboplastin time (aPTT).
Measurement of the bleeding time has been largely replaced by
determination of closure time measured with the platelet function
analyzer, PFA-100®’ (Dade Behring, Newark, DE).11 In this document
“bleeding time” refers to a traditionally measured bleeding time and
closure time. Individuals with vWD will usually have a normal PT and
platelet count and an increased bleeding time and aPTT. While an
increased bleeding time and/or aPTT is consistent with vWD, the aPTT
may be normal in untreated mild or moderate disease and alone cannot
exclude vWD.1,12 It will be prolonged in severe disease due to very low
vWF and factor VIII and may be decreased in platelet-type vWD.
Likewise, bleeding time may be normal in mild or moderate vWD.
Consequently, individuals with an increased bleeding time and/or aPTT,
or individuals with a strong family or personal history suggestive of
vWD, should be tested for vWF antigen, ristocetin cofactor (vWF
activity), factor VIII activity, and ABO blood group (Figure 5).
A decreased level of vWF antigen, ristocetin cofactor, and/or factor VIII
clotting activity is consistent with vWD. Additional testing is then
warranted to identify the type of vWD and includes vWF collagen
binding assay, vWF:factor VIII binding activity ratio (von Willebrand
Disease Type 2N Panel), and multimeric analysis.12 vWF collagen
binding assay results are reported as a collagen binding:vWF antigen
ratio; a ratio <0.5 is consistent with types 2A and 2B vWD. A normal
ratio (r0.5) is associated with type 2M and 2N vWD.13 The vWF:factor
VIII binding activity ratio is typically <0.73 in individuals with type 2N.
Table 11 summarizes the expected results of tests used in the diagnosis
and typing of inherited vWD. Though no definitive pattern of test results
are found in individuals with acquired vWD, typically, vWF antigen
levels are decreased and factor VIII activity levels may be decreased or
normal.
von Willebrand mutation analysis may be useful in select cases to
differentiate hemophilia A from type 2N vWD, distinguish the various
type 2 subtypes, and counsel individuals with type 3 vWD.3
Additional information useful in the interpretation of tests used in the
diagnosis of vWD follows. Test results should be interpreted in
conjunction with other laboratory and clinical findings. A full clinical
consultation is available upon request.
Additional Information for Tests Useful in von Willebrand
Disease
Activated Partial Thromboplastin Time (aPTT)
The aPTT will be prolonged if there is deficiency or inhibition of factors
in the intrinsic pathway including high molecular weight kininogen
(HMWK); prekallikrein; factors V, VIII, IX, X, XI, and XII; prothrombin; and
fibrinogen. Levels of factor V or X typically have to be <15% of normal
before a prolongation of aPTT is seen. Prolongation is also seen in

Section 3
individuals with lupus anticoagulant. Most individuals with severe vWD
or type 2N will have an increased aPTT, whereas approximately 25% to
50% of individuals with type 1 vWD will have an aPTT outside the
reference range.
Bleeding Time
Bleeding time is a measure of the interaction of platelets with blood
vessel walls and is increased in individuals with thrombocytopenia,
vWD, vascular purpura, severe fibrinogen deficiency, qualitative platelet
abnormalities (eg, uremia), and the use of certain drugs (eg, NSAIDs). A
normal value does not rule out the presence of vWD; conversely, an
abnormal result in the absence of other causes justifies specific testing
for vWD. Measurement of platelet function by determining closure time
with the PFA-100 has largely replaced bleeding time measurements. The
PFA-100 is an in vitro system that simulates the in vivo function of
platelets in primary hemostasis. Testing is performed using a small
whole blood sample anti-coagulated with citrate.
51
Coagulation
Complete Blood Count (CBC)
Hemoglobin and hematocrit may range from normal to markedly
decreased depending on the type and severity of vWD. See also Platelet
Count.
DDAVP Response
DDAVP (desamino-8-arginine vasopressin) is a synthetic analogue of
antidiuretic hormone. It is considered the primary treatment for bleeding
in individuals with mild vWD (type 1, sometimes types 2A and 2M) and
works by causing the release of vWF from endothelial storage sites. An
individual’s therapeutic response to DDAVP is usually tested before
prescribing the medication. A positive response is an increase of vWF
antigen, factor VIII, and/or ristocetin cofactor of 2- to 5-fold over
baseline 30 to 90 minutes after the administration of DDAVP. DDAVP is
contraindicated in individuals with type 2B vWD due to the possibility of
platelet clumping and subsequent increased risk of thrombosis. It is
ineffective in individuals with type 2N, 3, and platelet vWD.
52
Section 3
Factor VIII
Factor VIII is an acute phase reactant and increased levels are found
during periods of stress, postoperatively, and in inflammatory
conditions. Elevated levels are also found at birth and during pregnancy.
Increased levels are associated with increased risk for venous
thrombosis. vWF binding normally protects factor VIII from proteolysis;
thus, decreased levels of vWF lead to decreased levels of factor VIII.
Levels are typically in the low normal range in mild vWD. Low levels
(6% to 45% of normal) are found in type 2N, and very low levels (<10%)
in type 3 vWD. Decreased levels are also associated with hemophilia A,
disseminated intravascular coagulation (DIC) and with specific factor VIII
inhibitors (antibodies).
Mixing/Correction Study
Test results are consistent with an intrinsic factor deficiency when a
prolonged aPTT is normalized after mixing patient plasma with normal
plasma and the normalized result does not reverse after incubation of
the mixed sample. A specific factor inhibitor, lupus inhibitor, fibrinolysis,
or fibrinogenolysis is suggested when 1) the PT is normal, and the aPTT
is prolonged initially, normalizes after mixing, and reverses to prolonged
after incubation of the mixed sample; 2) the PT is normal, and the aPTT
remains prolonged after mixing; 3) a prolonged PT is corrected in the
mixing study, and a prolonged aPTT remains prolonged; and 4) a
prolonged PT remains prolonged, and a prolonged aPTT normalizes after
mixing and reverses to prolonged after incubation. A prolonged PT that
is corrected in the mixing study, along with a normal aPTT, suggests a
factor VII deficiency. Test results are consistent with a single or multiple
deficiency of factors II, V, or X (common pathway) when a prolonged PT
is normalized after mixing studies and when a prolonged aPTT
normalizes after mixing studies and remains normalized after
incubation.
Platelet Count
The platelet count is normal in most individuals with vWD; it is
decreased, however, in those with type 2B and platelet type vWD. A
normal platelet count, in the presence of an increased bleeding time, is
consistent with vWD, vasculopathies, connective tissue diseases, and
qualitative platelet abnormalties and warrants further testing for vWD.
A normal platelet count and normal bleeding time is consistent with
fibrinolytic disorders and may be seen in individuals with vWD; thus,
further vWD testing is warranted when clinical suspicion is high.
Prothrombin Time (PT)
The PT measures the time for clot formation after the addition of tissue
factor (thromboplastin) and calcium to citrated blood and, as such, is
prolonged with deficiencies of factors II, V, VII, X, and fibrinogen; liver
disease; coumadin use; and vitamin K deficiency. PT is within the
reference range in individuals with vWD.
Ristocetin Cofactor (von Willebrand Factor Activity)
Ristocetin is an antibiotic that causes vWF to bind and subsequently
activate platelets. In plasma of normal individuals, platelets rapidly
agglutinate in response to ristocetin due to the presence of vWF (ie,
ristocetin cofactor). In individuals with vWD, the degree of platelet
agglutination is proportional to the amount of vWF (and ristocetin)
present; thus, the level of ristocetin cofactor (von Willebrand factor
activity) is variably decreased in individuals with vWD. In type 2B and
platelet vWD, a supranormal (exaggerated) response to ristocetin is seen.
von Willebrand Antigen, Multimeric Analysis
The size distribution of vWF multimers is determined by gel
electrophoresis and used in determining the type of vWD present. High
molecular weight multimers are missing in type 2B and platelet vWD,
whereas high and intermediate weight multimers are absent in type 2A.
Coagulation
All multimers are missing in type 3 vWD. In types 2M and 2N vWD, the
multimeric analysis is typically normal.
von Willebrand Disease Mutation Analysis
This assay identifies mutations in the vWF gene associated with types
2A, 2B, 2M, 2N, and some forms of type 1 and 3 vWD. While this assay
has limited clinical utility in the diagnosis of vWD (ie, diagnosis is
typically made based on vWF antigen/activity levels and multimeric
structure), it is useful for differentiating mild hemophilia A from type 2N
vWD, distinguishing type 2A from type 2B vWD (useful due to the
relative contraindication of DDAVP in individuals with type 2B vWD),
and determining the causative mutation in families with type 3 vWD for
the management of future pregnancies.3 Additional information
regarding the use and interpretation of this test may be obtained by
calling Quest Diagnostics’ genetic counselors at 1-866-GENEINFO.
von Willebrand Disease Type 2N Panel
This test is used to identify type 2N vWD. A vWF:factor VIII binding
activity ratio of 0.73 to 1.42 is considered normal. A normal ratio may be
seen in vWD other than type 2N because factor VIII activity and vWF
antigen may be reduced proportionally. In type 2N vWD, factor VIII
activity is reduced, but vWF antigen levels are normal; thus, the ratio is
decreased. A ratio <0.73 is consistent with type 2N vWD.
von Willebrand Factor Activity (GP1b-specific EIA; functional
vWF)
This assay directly measures the functional activity of vWF, and is used
to confirm low (<20%) ristocetin cofactor levels. Levels <35% are
consistent with vWD, while levels above the reference range (35% to
134%) have no known clinical significance.
von Willebrand Factor Antigen
This test measures total amount of the vWF protein. Levels are
decreased in types 1 and 3 vWD. Levels may be normal or decreased in
types 2A, 2B, and 2M due to abnormal multimeric structure. This test is
normal in type 2N vWD.
Levels of vWF vary by blood type and ethnicity. The mean level of vWF
in individuals with blood type O is approximately 30% lower than in
individuals with blood types A, B, or AB.14 Higher mean levels of vWF
are found in African Americans than in other ethnic groups.15 Debate
exists whether reference ranges should be specific for blood group type
or ethnicity; however, bleeding tendency is primarily related to vWF
antigen level and multimeric composition.1,12
Increased levels of vWF may be seen secondary to stress, inflammation,
acute infection, physical exercise, following surgery, during the second
and third trimesters of pregnancy, and in individuals receiving estrogen
therapies; thus, serial testing may be necessary to confirm or rule out
vWD.
von Willebrand Factor Collagen Binding Assay
The activity of vWF is determined by measuring the ability of vWF to
bind collagen. The ability of vWF to bind collagen is a function of large
multimers; thus, collagen binding activity is abnormal in types 2A and
2B vWD, but may be normal in types 2M and 2N. Collagen binding is
compared to the vWF antigen value by calculating a collagen binding
ratio (vWF collagen binding:vWF antigen). Typically, a ratio r0.5 is
considered normal, whereas, a ratio <0.5 is consistent with vWD.
References
1. Federici AB. Diagnosis of inherited von Willebrand disease: a
clinical perspective. Semin Thromb Hemost. 2006;32:555-565.
 53

Section 3 Coagulation

2. Sadler JE, Budde U, Eikenboom CJ, et al. Update on the

pathophysiology and classification of von Willebrand disease: a
report of the Subcommittee on von Willebrand factor. J Thromb
Haemost. 2006;4:2103-2114.
3. Pruthi RK. A practical approach to genetic testing for von Willebrand
disease. Mayo Clin Proc. 2006;81:679-691.
4. Ginsburg D, Bowie EJ. Molecular genetics of von Willebrand
disease. Blood. 1992;79:2507-2519.
5. Haberichter SL, Balistreri M, Christopherson P, et al. Assay of the
von Willebrand factor (VWF) propeptide to identify patients with
type 1 von Willebrand disease with decreased VWF survival. Blood.
2006;108:3344-3351.
6. Franchini M, Lippi G. Acquired von Willebrand syndrome: an update.
Am J Hematol. 2007;82:368-375.
7. Federici AB. Acquired von Willebrand syndrome: an underdiagnosed
and misdiagnosed bleeding complication in patients with
lymphoproliferative and myeloproliferative disorders. Semin
Hematol. 2006;43:S48-S58.
8. Weiss HJ, Meyer D, Rabinowitz R, et al. Pseudo-von Willebrand’s
disease. An intrinsic platelet defect with aggregation by unmodified
human factor VIII/von Willebrand factor and enhanced adsorption of
its high-molecular-weight multimers. N Engl J Med. 1982;306:326-
333.
9. Tosetta A, Rodeghiero F, Castaman G, et al. A quantitative analysis
of bleeding symptoms in type 1 von Willebrand disease: results
from a Multicenter European study (MCMDM-1 VWD). J Thromb
Haemost. 2006;4:766-773.
10. Kottke-Marchant K, Corcoran G. The laboratory diagnosis of platelet
disorders. Arch Pathol Lab Med. 2002;126:133-146.
11. Franchini M. The platelet function analyzer (PFA-100): an update on
its clinical use. Clin Lab. 2005;51:367-372.
12. Favaloro EJ, Lillicrap D, Zazzari MA, et al. von Willebrand disease:
laboratory aspects of diagnosis and treatment. Haemophilia.
2004;10:164-168.
13. Popov J, Zhukov O, Ruden S, et al. Performance and clinical utility of
a commercial von Willebrand factor collagen binding assay for
laboratory diagnosis of von Willebrand disease. Clin Chem.
2006;52:1965-1967.*
14. Gill JC, Endres-Brooks J, Bauer PJ, et al. The effect of ABO blood
group on the diagnosis of von Willebrand disease. Blood.
1987;69:1691-1695.
15. Sukhu K, Poovalingam V, Mahomed R, et al. Ethnic variation in von
Willebrand factor levels can influence the diagnosis of von
Willebrand disease. Clin Lab Haematol. 2003;25:247-249.

Related References
16. Kasper CK. von Willebrand disease: an introduction for young
physicians. 2005. Available at:
www.med.unc.edu/isth/publications/vwd_monograph/VWD_monog
raph_2005.pdf.
17. Mannucci PM. How I treat patients with von Willebrand disease.
Blood. 2001;97:1915-1919.
*This study was funded and performed by Quest Diagnostics Nichols Institute. The
investigators/authors are employees of Quest Diagnostics Incorporated.

Appendix. Tests Used in the Diagnosis of von Willebrand

Disease
The Appendix Table contains a comprehensive list of tests used in the
diagnosis of von Willebrand disease.
54

Section 3 Coagulation

Appendix Table. Tests Used in The Diagnosis of von Willebrand Disease

Test Code Test Name Method Description
763X Activated Partial Photo-optical clot A phospholipid reagent is mixed with patient plasma, and calcium chloride is then
Thromboplastin detection added to initiate clot formation. The time elapsed for formation of a fibrin clot is
Time (aPTT) measured.
8504X Bleeding Time Template (in vivo A standard incision is made by an automated device in the forearm and blood is
clotting) blotted away every 30 seconds. The time until bleeding has ceased is measured.
(Measurement of the bleeding time has been largely replaced by determination of
closure time measured with the platelet function analyzer, PFA-100®’)
6399X CBC with Differential Electronic cell sizing, Flow cytometry with specific gating is used to quantify individual components.
and Platelet Count sorting/cytometry/ Significant abnormalities are reviewed microscopically.
microscopy
See DDAVP Response Perturbation test Baseline vWF antigen, factor VIII clotting activity, and/or ristocetin cofactor are
individual measured followed by administration of DDAVP. Levels are then measured again
tests 30 to 90 minutes after DDAVP administration.
347X Factor VIII Activity, Photo-optical clot Patient plasma is mixed with factor VIII-deficient normal plasma. The APTT clotting
Clotting detection time of the mixed plasma is compared to that of reference plasma. Results are
reported as percent of normal factor VIII activity.
8922X Mixing/Correction Photo-optical clot A PT and/or aPTT is measured after mixing patient plasma and normal plasma to
Study detection determine the cause (eg, factor deficiency, factor inhibitor, and/or nonspecific
inhibitor) of a prolonged PT and/or aPTT test result.
8847X Prothrombin Time Photo-optical clot Thromboplastin and calcium chloride is mixed with patient plasma and the time to
detection clot formation is measured photometrically.
4459X Ristocetin Cofactor Platelet Agglutination Ristocetin cofactor (vWF) in the sample causes agglutination of stabilized platelets
(ie, vWF antigen in the presence of ristocetin. Activity is measured by a change in turbidity.
activity)
16028X vWF Activity ELISA A monoclonal antibody specific to the portion of vWF that binds platelets is used to
(ie, GP1b-specific EIA, capture vWF. This bound vWF antigen is quantified using horseradish peroxidase
functional vWF) conjugated anti-human vWF antibody.
4919X vWF Antigen Immunoturbidimetric Patient plasma is mixed with vWF antibody-coated latex particles. vWF from the
sample causes aggregation of the latex particles, which is measured
photo-optically.
5168X von Willebrand Electrophoresis vWF multimers are separated on agarose gel, the gel is electro-blotted onto a
Antigen, Multimeric PVDF membrane and the multimeric pattern is visualized via chemiluminography.
Analysis
10924X vWF Collagen ELISA vWF in patient plasma binds collagen immobilized on microtiter plates. Bound vWF
Binding Assay is detected using peroxidase conjugated anti-vWF antibody.
15540X vWF Comprehensive See individual tests Panel includes: aPTT; factor VIII activity, clotting; vWF antigen; ristocetin cofactor;
Panel vWF factor collagen binding assay; vWF antigen, multimeric analysis;
interpretation.
19837X von Willebrand PCR Exons 11, 12, 14, 15, 16, 18, 19, 20, 24, 27, 28, and 52 of the vWF gene are
Disease Mutation amplified in a single-plex PCR reaction. Known and novel mutations in the exons
Analysis sequenced are identified.
19735X von Willebrand ELISA, Photo-optical Two assays performed simultaneously are used to determine exogenous
Disease Type 2N clot detection, recombinant factor VIII binding to patient vWF and to quantify patient vWF. The
Panel Immunoturbidimetric vWF:Factor VIII binding activity ratio is calculated.
DDAVP, desamino-8-arginine vasopressin; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; PVDF, polyvinylidene difluoride; vWF, von
Willebrand factor.

Section 4
This section includes information about selected tests that are more
commonly ordered by primary care physicians. See also tests in section
1 (Cardiovascular) and section 2 (Chronic Kidney Disease). For more
complete information about endocrine tests, refer to The Quest
Diagnostics Manual: Endocrinology Test Selection and Interpretation.
4.1 Calcium and Bone Metabolism
(Including Osteoporosis)
4.1.1 Collagen Cross-Linked N-Telopeptide (NTx) and Collagen
Type 1 C-Telopeptide (CTx)
Clinical Use: These tests are used to monitor therapeutic response in
patients with metabolic bone disorders, predict future bone mineral
density (BMD), predict therapeutic response prior to initiation of
antiresorptive therapy, and detect bone metastasis in patients with
various malignancies.
Clinical Background: Healthy levels of bone mineral density (BMD)
are maintained by a balance between bone resorption and bone
formation. N-telopeptide (NTx) and C-telopeptide (CTx) are the amino-
terminal and C-terminal cross-linked peptides of type I collagen,
respectively. They are released during bone resorption and have been
correlated with BMD T-scores. Multiple studies have shown that NTx
and CTx not only correlate inversely with BMD response to therapy but
also are early markers or predictors of BMD response. Thus, therapeutic
response can be determined within 3 to 6 months of therapy rather than
1 to 2 years. Studies have also demonstrated that elevated
pretreatment values predict positive response to therapies such as
hormone replacement therapy in postmenopausal women. In patients
with malignancies, elevated levels may indicate bone metastases.
NTx Method: This enhanced immunochemiluminometric assay (ICMA)
has an analytical sensitivity of 10 nmol BCE/L.
CTx Method: This electrochemiluminescent immunoassay (ECLIA) has
an analytical sensitivity of 30 pg/mL.
Interpretive Information: NTx and CTx test results may be increased
in primary or secondary osteoporosis and osteopenia due to vitamin D
deficiency as well as in growth hormone deficiency, hypogonadism, high
bone turnover states such as hyperparathyroidism, hyperthyroidism, and
Paget’s disease. They may also be increased in inflammatory states
such as rheumatoid arthritis, in bone metastasis, and in patients taking
corticosteroid or immunosuppressive therapy. Decreased results (ie,
relative to pre-treatment baseline) indicate therapeutic response.
4.1.2 Intact PTH and Calcium
Clinical Use: This test is used to discriminate primary hyperparathy-
roidism from tumor hypercalcemia, diagnose hypoparathyroidism, and
monitor the severity of secondary hyperparathyroidism (eg, in chronic
kidney disease).
Clinical Background: Parathyroid hormone (PTH) acts to increase
calcium absorption from the gut and to mobilize calcium from bone.
Additionally, PTH aids in the conversion of 25-hydroxyvitamin D to
1,25-dihydroxyvitamin D via 1B-hydroxylase enzymatic activity. The net
effect is to increase the extracellular concentration of calcium and
prevent hypocalcemia. PTH secretion by the parathyroid gland is
modulated by serum calcium concentration, which is sensed via a
specific calcium receptor. Low calcium stimulates and high calcium
inhibits PTH secretion.
55
Endocrinology
PTH levels are used to assess disorders of calcium metabolism,
including primary and secondary hyperparathyroidism, tumor
hypercalcemia, and hypoparathyroidism.
PTH Method: This immunochemiluminometric assay (ICMA) has an
analytical sensitivity of 3 pg/mL. It has 100% cross-reactivity with intact
PTH (amino acids 1-84) and 45% with the 7-84 C-terminal fragment;
there is no detectable cross-reactivity with other PTH fragments or with
calcitonin.
Total Calcium Method: This spectrophotometry method has an
analytical sensitivity of 0.2 mg/dL.
Interpretive Information: Increased intact PTH and calcium levels are
associated with primary hyperparathyroidism and tertiary hyperparathy-
roidism as seen in renal failure. In secondary hyperparathyroidism, PTH
levels are elevated, but calcium concentrations can be normal or
decreased. Decreased PTH levels are associated with hypoparathy-
roidism, hypercalcemia of malignancy, and hypercalcemia associated
with increased 1,25-dihydroxyvitamin D synthesis as seen in lymphomas
and granulomatous disease.
4.1.3 Osteocalcin
Clinical Use: This test is used to determine efficacy of therapy in
osteoporosis and metastatic bone disease. It is a useful marker of bone
formation and remodeling.
Clinical Background: Osteocalcin is a 6-kd protein constituting 1% to
2% of total bone protein. It contains 3 gamma-carboxyglutamic acid
(Gla) residues that bind hydroxylapatite. Osteocalcin is produced
exclusively by bone osteoblasts, and production is dependent on
1,25-dihydroxyvitamin D, vitamin K, and vitamin C. Measurement
provides a specific biochemical index of bone activity. As assessed by
bone histomorphometry, serum osteocalcin levels correlate with bone
formation and not directly with bone resorption. Serum osteocalcin is an
important bone formation marker.
Method: This immunoradiometric assay (IRMA) has an analytical
sensitivity of 2.0 ng/mL.
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
Interpretive Information: Osteocalcin concentration may be
increased in primary or secondary osteoporosis and osteopenia due to
vitamin D deficiency and in growth hormone deficiency, hypogonadism,
high bone turnover states such as hyperparathyroidism, hyperthyroidism,
and in some, but not all, Paget’s disease cases. It may also be increased
in inflammatory states such as rheumatoid arthritis, in bone metastasis,
and in patients taking corticosteroid or immunosuppressive therapy.
Decreased results (ie, relative to pre-treatment baseline) indicate
therapeutic response.
4.1.4 Pyridinium Collagen Cross-links (PYD and DPYD), Urine
Clinical Use: This test is used to assess the rate of bone collagen
degradation and to monitor response to therapy.
Clinical Background: Collagen fibers are linked together by
interchain molecules referred to as pyridinium cross-links. Collagen
pyridinium cross-links have been identified in all connective tissue
except skin. As collagen is broken down by collagenase, small
56
Section 4
breakdown products are excreted in urine with the attached cross-links,
including pyridinoline (PYD) and deoxypyridinoline (DPYD). Bone
collagen is constantly turning over and is rich in DPYD. Thus, urine PYD
and, especially, DPYD are useful markers for bone matrix degradation
and resorption.
Method: This high-performance liquid chromatography (HPLC) method
has an analytical sensitivity of 20 pmol/mL for PYD and 13 pmol/mL for
DPYD. The creatinine concentration is also reported.
Interpretive Information: PYD and DPYD test results may be
increased in primary or secondary osteoporosis and osteopenia due to
vitamin D deficiency and in growth hormone deficiency, hypogonadism,
high bone turnover states such as hyperparathyroidism, hyperthyroidism,
and Paget’s disease. They may also be increased in inflammatory states
such as rheumatoid arthritis, in bone metastasis, and in patients taking
corticosteroid or immunosuppressive therapy. Decreased results (ie,
relative to pre-treatment baseline) indicate therapeutic response.
4.1.5 Vitamin D, 25-Hydroxy
Clinical Use: This test is used to diagnose vitamin D deficiency or
intoxication.
Clinical Background: 25-Hydroxyvitamin D (25OHD) is the major
circulating form of vitamin D and the precursor of the active form (1,25-
dihydroxyvitamin D). Because of its long half-life, 25OHD measurements
are useful for assessing vitamin D status in patients.
Vitamin D occurs in 2 forms: vitamin D3 (cholecalciferol) and vitamin D2
(ergocalciferol). Vitamin D3 is obtained from foods of animal origin and
from ultraviolet light-stimulated conversion of 7-dehydrocholesterol in
the skin, whereas vitamin D2 is obtained from foods of plant origin.
Vitamin D2 is used in a pharmacologic high potency (50,000 IU)
formulation for weekly (8 weeks duration) treatments of severe vitamin
D deficiency.1 Alternatively, pharmacologic doses of vitamin D3 (1000
to 2000 IU/d or 200,000 IU every 3 mo) may be used.1 Both forms are
used in much lower doses in over-the-counter supplements and
fortified foods and are metabolized to their respective 25OHD forms
(ie, 25OHD3 and 25OHD2). Monitoring vitamin D2 pharmacologic
therapy is easier than monitoring D3 pharmacologic therapy since
almost all of the vitamin D2 measured comes from the therapy; this
may not be true for measurements of vitamin D3. While total vitamin D
levels may approximate the adequacy of therapy, monitoring D2 levels
may identify patients with malabsorptive syndromes and enable
improved dose titration. Thus, analytical methods that can accurately
quantitate both forms are useful for diagnosis and monitoring patients
with vitamin D deficiency as well as differentiating between
intoxication and other hypercalcemic disorders.
Method: This liquid chromatography tandem mass spectrometry
(LC/MS/MS) method has an analytical sensitivity of 4 ng/mL for 25OHD2
and 25OHD3. There is no cross-reactivity with vitamin D2 or D3;
1B,25(OH)2D2; 1B,25(OH)2D3, calcitriol; 25,26(OH)2D3; 1B(OH)D2,
doxercalciferol; or 1B(OH)D3, alfacalcidiol.
Interpretive Information: 25OHD levels are increased in vitamin D
intoxication. In secondary hyperparathyroidism, levels may be within the
normal range or decreased. Decreased levels are also observed in
nutritional rickets, osteomalacia, severe cholestatic or parenchymal liver
disease, patients taking antiepileptics such as theophylline and
rifampin, nephrotic syndrome with marked proteinuria, intestinal
malabsorption, obesity, sarcoidosis, hyperphosphatemic tumoral
Endocrinology
calcinosis, tuberculosis, primary hyperparathyroidism, and type II
vitamin D-dependent rickets.
Levels of 25OHD3 reflect both endogenous production and
supplementation, whereas levels of 25OHD2 reflect only exogenous
sources such as diet or supplementation. 25OHD levels of 20 to 100
ng/mL are considered physiologically normal, but optimal levels are 30-
60 ng/mL.1 Levels <20 ng/mL suggest vitamin D deficiency, while levels
between 20 and 30 ng/mL suggest insufficiency. Thus, there is a need
for intense to moderate supplementation when levels are b30 ng/mL.
Levels vary with exposure to sunlight, peaking in the summer months.
Reference
1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266-281.
4.2 Diabetes
4.2.1 Glucose
Clinical Use: This test is used to assess carbohydrate metabolic
status, diagnose diabetes mellitus, and diagnose hypoglycemia.
Clinical Background: Blood glucose concentrations are narrowly
maintained by balancing hormonal factors. Insulin promotes glucose
utilization and storage, lowering blood levels. Carbohydrate ingestion
raises blood glucose concentrations; peak levels are modulated by
stimulated insulin secretion. Several counter regulatory hormones
(glucagon, growth hormone, cortisol, catecholamines) function to
increase glucose concentrations by stimulating glycogenolysis,
gluconeogenesis and increased hepatic glucose output, raising blood
glucose at the expense of carbohydrate and other substrate stores.
Method: This spectrophotometry (hexokinase) method has an analytical
sensitivity of 2 mg/dL.
Interpretive Information: Glucose levels may be increased in
impaired fasting glucose (IFG), diabetes mellitus, Cushing’s syndrome,
and pheochromocytoma. Decreased levels are associated with islet cell
tumors, glucagon deficiency, Addison’s disease, and hypoglycemic
syndromes.
4.2.2 GlycoMark®’ (1,5-Anhydroglucitol)
Clinical Use: This test is used for intermediate-term monitoring of
glycemic control in patients with diabetes.
Clinical Background: Tight glycemic control is essential for
preventing the complications of diabetes. The clinical standard for
evaluating glycemic control is the measurement of hemoglobin A1c
(HbA1c); elevated levels (>7.0%) indicate significant hyperglycemia.
However, even in patients with well-controlled HbA1c levels,
postprandial glucose levels may be significantly elevated and lowering
these will further improve control.
1,5-Anhydroglucitol (1,5-AG) is a glucose-like monosaccharide contained
in food. Intake is normally balanced with urinary excretion; however,
during periods of hyperglycemia, glucose blocks reabsorption of 1,5-AG
in the renal tubules. Thus, low blood levels of 1,5-AG are associated
with hyperglycemia. 1,5-AG levels change more rapidly than HbA1c
levels and reflect glycemia over the previous 1-2 week period.

Section 4
Individuals Suitable for Testing include patients with type 1 or 2
diabetes mellitus.
Method: 1,5-AG is measured colorimetrically following enzymatic
release of hydrogen peroxide. The analytical sensitivity is 0.2 Ng/mL.
Reference Range
Males 10.7-32.0 Ng/mL
Females 6.8-29.3 Ng/mL
Interpretive Information: In patients with moderate- to well-
controlled diabetes (HbA1c <8.0%), low levels of 1,5-AG primarily reflect
postprandial hyperglycemia, and measures aimed at decreasing the
postprandial blood glucose level are indicated. Increasing levels reflect
a positive short- to intermediate-term response when monitoring
changes in diet or medication. In patients with poorly-controlled
diabetes, in vivo levels of 1,5-AG may be depleted; thus, in this group
1,5-AG measurements may not accurately reflect therapeutic response.
Low levels are also associated with pregnancy, kidney disease,
advanced cirrhosis, prolonged inability to intake food orally, and steroid
therapy. Increased levels are associated with intravenous
hyperalimentation and some Chinese medicines (eg, polygala tenuifolia
and senega syrup).
Reference
1. Dungan KM, Buse JB, Largay J, et al. 1,5-Anhydroglucitol and
postprandial hyperglycemia as measured by continuous glucose
monitoring system in moderately controlled patients with diabetes.
Diabetes Care. 2006;29:1214-1219.
2. McGill JB, Cole TG, Nowatzke W, et al. Circulating 1,5-anhydro-
glucitol levels in adult patients with diabetes reflect longitudinal
changes of glycemia. Diabetes Care. 2004;27:1859-1865.
3. Nguyen TM, Rodriguez LM, Mason KJ, et al. Serum 1,5-anhydro-
glucitol (Glycomark) levels in children with and without type 1
diabetes mellitus. Pediatr Diabetes. 2007;8:214-219.
4.2.2 Hemoglobin A1c
Clinical Use: This test is used to monitor glucose control (long-term,
2-3 months) in patients with diabetes.
Clinical Background: Hemoglobin A1 (HbA1 or glycated hemoglobin)
is structurally related to adult hemoglobin (HbA) but has a glucose
molecule attached to the terminal valine of the beta chain.
Glycosylation is a nonenzymatic, irreversible process dependent on the
glucose concentration and the duration of exposure of the erythrocyte to
glucose. HbA1 is continuously formed during the 120-day life of the
erythrocyte, and a single measurement of HbA1 reflects the average
blood glucose level during the preceding months. HbA1 can be
separated into HbA1a, HbA1b, and HbA1c fractions. HbA1c correlates best
with high glucose concentrations.
Method: This colorimetric, immunoturbidimetric method has an
analytical sensitivity of 3% at a hemoglobin concentration of 8.2
mmol/L (13.2 g/dL).
Interpretive Information: Increased hemoglobin A1c levels are
associated with diabetes mellitus, chronic hyperglycemia, and, in some
methods, hemoglobin S (sickle cell) and hemoglobin C variants. Levels
are relatively decreased by improved diabetic control and in association
with high levels of hemoglobin F.
57
Endocrinology
4.2.3 Microalbumin
See Chronic Kidney Disease, sections 2.2 and 2.3.
4.3 Polycystic Ovary Syndrome (PCOS)
PCOS affects about 1 in 15 women of reproductive age. It is
characterized by 1) clinical hyperandrogenism (eg, hirsutism, acne,
alopecia) and/or hyperandrogenemia (eg, elevated total or free
testosterone); 2) oligo- or anovulation (eg, amenorrhea or
oligomenorrhea); and 3) polycystic ovaries visualized by ultrasound. The
American Society for Human Reproductive Medicine defines PCOS as
the presence of 2 of these 3 conditions in the absence of other
disorders such as congenital adrenal hyperplasia (CAH),
hyperprolactinemia, adrenal virilizing tumors, functional hypothalamic
amenorrhea, and Cushing’s syndrome (the Rotterdam 2003 criteria).1 The
Androgen Excess Society, on the other hand, makes the first criterion
(androgen excess) a necessary condition for the diagnosis and therefore
excludes non-virilized women who are oligomenorrheic and have
polycystic ovaries.2
Women with PCOS are at greater risk for abnormal uterine bleeding,
endometrial cancer, infertility, type 2 diabetes mellitus, and the
metabolic syndrome.
The tests described herein are used to provide evidence of
hyperandrogenemia (total or free testosterone and DHEA sulfate) or to
rule out other disorders such as CAH (17-hydroxyprogesterone) or
pituitary tumors (prolactin).
References
1. The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop
Group. Revised 2003 consensus on diagnostic criteria and long-term
health risks related to polycystic ovary syndrome. Fertil Steril.
2004;81:19-25.
2. Azziz R, Carmina E, Dewailly D, et al. Position statement: Criteria for
defining polycystic ovary syndrome as a predominantly
hyperandrogenic syndrome: an Androgen Excess Society guideline.
J Clin Endocrinol Metab. 2006 Aug 29 [Epub ahead of print].
4.3.1 DHEA Sulfate
Clinical Use: This test is used in the differential diagnosis of PCOS
and/or hirsute or virilized female patients and for the diagnosis of
isolated premature adrenarche and adrenal tumors.
Clinical Background: Dehydroepiandrosterone sulfate (DHEA-S),
which is synthesized almost exclusively by the adrenals, is a weak
androgen, the most abundant C19 plasma steroid, and the major source
of urinary 17-ketosteroids. Measuring serum DHEA-S, therefore, can
replace older urinary 17-ketosteroid determinations.
Since DHEA-S levels reflect adrenal androgen production, the
measurement of serum concentrations serves as an early indicator for
the onset of adrenarche. Serum DHEA-S measurements, however, are
most commonly employed in the differential diagnosis of a virilized
patient. Since about 1 in 10 women with PCOS do not exhibit elevations
of total or free testosterone but do have increased DHEA-S levels,
DHEA-S can be helpful in establishing a diagnosis of PCOS.
Method: This immunochemiluminometric assay (ICMA) has an
analytical sensitivity of 15 Ng/dL.
58
Section 4
Interpretive Information: DHEA-S levels are markedly increased in
congenital adrenal hyperplasia. In patients with adrenal virilizing
tumors, levels usually exceed 7,000 Ng/dL. A few of these tumors are
adrenal adenomas but most are adrenal carcinomas that may also
secrete cortisol. Moderate increases are seen in the majority of patients
with pituitary-dependent Cushing’s disease, whereas patients with
Cushing’s syndrome due to an adrenal adenoma usually exhibit low or
normal levels. Moderate increases are also associated with premature
adrenarche of adrenal, and not gonadal, origin. Increased levels are
seen in PCOS too. Decreased levels are observed in Addison’s disease
and adrenal hypoplasia.
4.3.2 17-Hydroxyprogesterone, LC/MS/MS
Clinical Use: This test is used as a marker for adrenal P450c21
(21-hydroxylase) enzyme deficiency.
Clinical Background: 17-Hydroxyprogesterone (17-OHP) is an adrenal
steroid intermediate in the biosynthesis of cortisol. The enzyme P-450c17
catalyzes its synthesis from progesterone; it is then further metabolized
to cortisol or androstenedione.
17-OHP measurement is appropriate when complete or partial
21-hydroxylase deficiency is suspected in 1) infants with features of
adrenal insufficiency (hypotension, vomiting, fever, hypoglycemia, and
hyperkalemia) or ambiguous genitalia and 2) women with clinical
evidence of possible androgen excess, particularly Ashkenazi Jews who
have a high prevalence of nonclassical 21-hydroxylase deficiency which
may mimic PCOS. Some authorities consider measurement of ACTH-
stimulated 17-OHP levels essential to exclude the diagnosis. Cushing’s
disease may also cause elevated 17-OHP levels and should be excluded
before glucocorticoid treatment of presumed nonclassical 21-hydroxyl-
ase deficiency is considered.
Method: This liquid chromatography, tandem mass spectrometry
(LC/MS/MS) method has an analytical sensitivity of 8 ng/dL.
Interpretive Information: Increased 17-OHP levels are associated
with congenital adrenal hyperplasia (CAH), 21-hydroxylase deficiency,
male pseudohermaphroditism (P-450c17 deficiency), late-onset CAH, and
Cushing’s disease. Levels may be decreased in Addison’s disease and
following steroid treatment (cortisone, hydrocortisone).
4.3.3 Prolactin
Clinical Use: This test is used to diagnose and manage pituitary
adenomas. It is also used in the differential diagnosis of male and
female hypogonadism.
Clinical Background: Prolactin is a protein hormone secreted by the
anterior pituitary gland and the placenta. It may modulate the number of
follicles developing in the follicular phase of each menstrual cycle.
During and following pregnancy, prolactin, in association with other
hormones, stimulates breast development and milk production.
Prolactin secretion is stimulated by sleep, stress (physical and
emotional), and the hypothalamic hormone, thyrotropin releasing
hormone (TRH). Prolactin secretion is inhibited by dopamine and
dopamine analogs such as bromocriptine.
Hypersecretion of prolactin can be caused by pituitary tumors,
hypothalamic disease, breast or chest wall stimulation, hypothyroidism,
renal failure, acute exercise, stress, eating, and several medications (eg,
phenothiazines, metoclopramide). Hyperprolactinemia inhibits
Endocrinology
gonadotropin secretion and can produce hypogonadism in men and
women with accompanying low or inappropriately “low normal” LH and
FSH levels. Hyperprolactinemia may also stimulate adrenal androgen
production contributing to the hirsutism, oligo/amenorrhea, and
polycystic ovarian appearance seen in some hyperprolactinemic
patients.
Method: This immunochemiluminometric assay (ICMA), ADVIA
Centaur®’, has an analytical sensitivity of 1.0 ng/mL.
Interpretive Information: Prolactin concentration is increased in
hypothalamic or pituitary tumors and subsequent to stress (physical and
emotional), antidepressants, and breast-feeding. Levels are decreased
following administration of bromocriptine, a dopamine agonist. Normal
levels rule out pituitary tumor in patients with suspected PCOS.
4.3.4 Testosterone (Free, Bioavailable, and Total)
Clinical Use: These tests are used to assess androgen status in
female hirsutism, virilization, acne, and amenorrhea; in children; and in
male hypogonadism.
Clinical Background: Testosterone is secreted by the testes in the
male and by both the adrenal and the ovary in the female. It is the most
potent of the circulating androgenic hormones and perhaps the most
reliable for clinical assessment of androgenic effects. Most testosterone
is transported in blood by sex hormone binding globulin (SHBG) from the
liver. Free testosterone is the small amount (only 2%) of testosterone
circulating unbound. Total blood testosterone levels are dependent on
rates of production, interconversion, metabolic clearance, and binding
protein concentration. Because SHBG levels are altered by aging,
medications, disease, sex steroids, and insulin, measurement of free
testosterone more accurately reflects the level of bioactive testosterone
than measurements of total serum testosterone.
In general, total testosterone, a less technically demanding and more
economical procedure, is satisfactory for primary screening of hirsute
women [see Testosterone, Total (Women and Children), LC/MS/MS] and
for evaluating potential male hypogonadism. In the former setting,
however, a modest increase in testosterone production may be masked
when the total testosterone concentration alone is assessed, because
androgen excess lowers the level of circulating SHBG. Therefore,
determination of free testosterone concentrations offers greater
sensitivity in the evaluation of mildly hyperandrogenemic women.
Total Testosterone Method: This liquid chromatography tandem
mass spectrometry (LC/MS/MS) method has an analytical sensitivity of
1 ng/dL.
Total and Free Testosterone Method: Total testosterone
measurements are determined using liquid chromatography tandem
mass spectrometry (LC/MS/MS); the analytical sensitivity is 1 ng/dL.
Tracer equilibrium dialysis and calculation are used to determine free
testosterone.
Free, Bioavailable, and Total Testosterone Method: Total
testosterone levels are determined using liquid chromatography tandem
mass spectrometry (LC/MS/MS). SHBG levels are determined using an
immunochemiluminometric assay (ICMA), and albumin levels are
determined using spectrophotometry. Free and bioavailable testosterone
are then calculated.
Interpretive Information: Increased testosterone concentration is
associated with PCOS, Cushing’s syndrome, and congenital adrenal

Section 4
hyperplasia. Very high levels are seen in women with ovarian tumors
(arrhenoblastomas, >150-200 ng/dL) and in hyperthecosis (>200 ng/dL).
Levels may be decreased in primary (increased LH) and secondary
(decreased LH) hypogonadism, CAH (17-hydroxylase deficiency), delayed
puberty in boys, gonadotropin deficiency, and testicular steroidogenetic
defects.
4.4 Thyroid Disease
4.4.1 Free T3
Clinical Use: This test is used to diagnose hyperthyroidism and to
clarify thyroid status in the presence of a possible protein binding
abnormality.
Clinical Background: Most circulating T3 (triiodothyronine) is bound
to plasma proteins. Only 0.3% exists in the free, unbound state and is
available for exchange with intracellular T3 receptors.
T3 measurements are used to diagnose and monitor treatment of
hyperthyroidism. When an increase in circulating thyroxine-binding
proteins is suspected as the cause of an elevated total T3 level, the free
T3 assay can differentiate this condition from true hyperthyroidism.
Non-dialysis Method: This immunochemiluminometric assay (ICMA)
has an analytical sensitivity of 50 pg/dL.
Non-dialysis free T3 immunoassay methods tend to underestimate free
T3 due to nonspecific absorption of free T3 to proteins or matrix
reagents. Although they do provide a useful free T3 index, the most
reliable free T3 measurement is the equilibrium dialysis method.
Dialysis Method: Free T3 is measured using equilibrium dialysis
followed by radioimmunoassay (RIA); the analytical sensitivity is 0.01%.
Total T3 (analytical sensitivity, 25 ng/dL) is measured using
immunochemiluminometric assay (ICMA). Reported free T3 is the
product of the 2: (total T3 x DFT3) where DFT3 is the dialyzable fraction
of T3.
Interpretive Information: Serum free T3 elevation is associated with
Graves disease, T3 thyrotoxicosis, thyroid hormone resistance, and
functional thyroid adenoma (T3-producing). Free T3 is decreased in
nonthyroidal illness and in one-third of hypothyroidism cases.
4.4.2 Free T4
Clinical Use: This test is used to assess thyroid status in patients with
abnormal total T4 concentrations. It can be used to differentiate
euthyroid hyperthyroxinemia from hyperthyroidism and euthyroid
hypothyroxinemia from hypothyroidism.
Clinical Background: Thyroid hormones are transported in blood
bound to several binding proteins. These include thyroxine-binding
globulin, prealbumin, and albumin. Only 0.03% of thyroxine (T4) is
unbound or free, but this free fraction provides the hormone available to
most tissues. Hyper- and hypothyroidism result from abnormal
concentrations of serum free T4. Since free T4 concentrations are never
high enough to influence results of total T4 immunoassays, total T4
assays measure only protein-bound T4.
Non-dialysis Method: This immunochemiluminometric assay (ICMA)
has an analytical sensitivity of 0.1 ng/dL. It is the initial test of thyroid
function in patients with suspected thyroid dysfunction (see also TSH).
This method may provide falsely low free T4 estimates owing to
59
Endocrinology
nonspecific absorption of free T4 to proteins or matrix reagents. This
method does, however, provide a useful free T4 index.
Dialysis Method: This direct equilibrium dialysis, radioimmunoassay
(RIA) method has an analytical sensitivity of 0.2 ng/dL.
The most accurate method for determining free T4 is the direct
equilibrium dialysis method. Direct dialysis separates free T4 from
protein-bound T4. The free T4 is then measured directly from the
protein-free dialysate. Results are independent of T4-binding protein
concentrations and are unaffected by the presence of molecular variants
of these proteins or by circulating thyroid autoantibodies.
Interpretive Information: Serum free T4 elevations are associated
with hyperthyroidism and thyroid hormone resistance. They are
decreased in primary and secondary hypothyroidism.
4.4.3 TSH, 3rd Generation (Thyroid Stimulating Hormone)
Clinical Use: This test is used to diagnose hypo- and hyperthyroidism,
monitor T4-replacement or T4-suppressive therapy, and to quantify TSH
levels in the sub-normal range.
Clinical Background: The serum TSH measurement is 1 of the most
important tools in the diagnosis of thyroid disorders. TSH secretion from
the pituitary gland is controlled by hypothalamic TRH and a negative
feedback effect from circulating, free thyroid hormones. Thus, in
subjects with a normal hypothalamic-pituitary system, there is an
inverse correlation between free thyroid hormone and TSH
concentrations in serum. Increased serum TSH is an early and sensitive
indicator of decreased thyroid reserve and overt primary hypothyroidism.
A third-generation TSH assay using chemiluminescent technology
produces functional sensitivities of 0.01 mU/L, permitting reliable
quantification of serum TSH concentrations in the subnormal range. This
allows differentiation of euthyroid from hyperthyroid patients. This
assay also helps diagnose essentially all patients with TSH-independent
hyperthyroidism (Graves disease, etc.) with a single blood sample. In
addition, the assay allows adjustment of exogenous thyroxine dosage in
hypothyroid patients and in patients on suppressive thyroxine therapy
for thyroid neoplasia.
Method: This immunochemiluminometric assay (ICMA) has an
analytical sensitivity of 0.01 mU/L.
Interpretive Information: Increased levels of TSH are associated with
primary hypothyroidism, decreased thyroid reserve (sub-clinical
hypothyroidism), TSH-dependent hyperthyroidism, and thyroid hormone
resistance. Decreased levels are associated with Graves disease,
autonomous thyroid hormone secretion, and TSH deficiency.
60

Section 5 Genetics

5.1 Available Tests Triple Screen

Tryptophan, LC/MS
Biochemical Genetics Tyrosine
Acetylcholinesterase
Acetylcholinesterase and Fetal Hemoglobin Cytogenetics
Acylcarnitine, Plasma Cell Culture for Possible Additional Prenatal Studies
Alpha-1-Antitrypsin Quantitation Chromosome Analysis & AFP w/Reflex to AchE & Fetal Hgb, Amniotic
Alpha-Fetoprotein, Amniotic Fluid with Reflex to AchE and Fetal Hgb Fluid
Amino Acid Analysis for MSUD, LC/MS, Plasma Chromosome Analysis, Amniotic Fluid
Amino Acid Analysis, LC/MS, CSF Chromosome Analysis, Blood
Amino Acid Analysis, LC/MS, Plasma Chromosome Analysis, Chorionic Villus Sample
Amino Acid Analysis, LC/MS, Urine Chromosome Analysis, Follow-up
Amino Acid Analysis, Limited, LC/MS, Plasma Chromosome Analysis, High Resolution
Arylsulfatase A Chromosome Analysis, Mosaicism
CAH Panel 1 (21-Hydroxylase vs 11Beta-Hydroxylase Deficiency) Chromosome Analysis, Sister Chromatid Exchange
CAH Panel 11, Neonatal Random Urine Chromosome Analysis, Tissue
CAH Panel 3 (Aldosterone Synthase Deficiency) Chromosomes, DEB Assay for Fanconi Anemia
CAH Panel 4 (17-Hydroxylase Deficiency in Females) ER/PR/DNA/HER2 w/Reflex to HER2 FISH (Photomicrograph), Paraffin
CAH Panel 6 (StAR Deficiency) Block2
CAH Panel 6B (Comprehensive Screen) ER/PR/DNA/HER2 w/Reflex to HER2 FISH, Paraffin Block2
CAH Panel 7 (21-Hydroxylase Deficiency Therapeutic Monitoring) ER/PR/HER2 w/Reflex to HER2 FISH, Paraffin Block2
CAH Panel 8 (17-Hydroxylase Deficiency in Males) FISH, Angelman2
CAH Panel 9 (3Beta-Hydroxysteroid Dehydrogenase Deficiency) FISH, Chromosome-Specific Probe2
Carnitine, LC/MS/MS Choose one of the following: chromosome-specific (1-22, X and Y) centromere or
Carnitine, LC/MS/MS and Acylcarnitine chromosome-specific (1-22, X and Y) painting.
Cholinesterase, RBC & Plasma FISH, Cri du chat2
Cholinesterase, Serum FISH, DiGeorge, Velocardiofacial (VCFS)2
Cholinesterase, Serum, with Dibucaine Inhibition FISH, Kallmann2
Cystine, Quantitative, 24-Hour Urine FISH, Microdeletion Syndromes Panel2
Cystine, Quantitative, Random Urine Includes FISH markers for DiGeorge, Kallmann, Prader-Willi/Angelman, Smith-
Erythrocyte Protoporphyrin (EP) Magenis, and Williams syndromes.
Fetal Hemoglobin, Amniotic Fluid FISH, Miller-Dieker2
Fetal Hemoglobin, Whole Blood FISH, Neonatal Screen
First Trimester Screen, hCG1 FISH, Prader Willi2
First Trimester Screen, Hyperglycosylated hCG (h-hCG)1 FISH, Prenatal Screen
Glucose-6-Phosphate Dehydrogenase, Quantitative (G-6-PD) FISH, Product of Conception (POC) Panel2
Hemoglobin A2, Quantitative Includes FISH probes for centromeres X, Y, and 18; and probes for 13q14, 16q11.2,
Hemoglobin S, Quantitative 21q11.2-q22.2, and 22q11.2.
Hemoglobinopathy Evaluation FISH, SKY®’ Marker Chromosome2
Includes RBC, hemoglobin, hematocrit, MCV, MCH, RDW, hemoglobin A1, fetal FISH, Smith-Magenis2
hemoglobin, hemoglobin A2, and abnormal hemoglobins. FISH, SRY/X Centromere2
Homocysteine (Cardiovascular), Serum FISH, Subtelomere Screen2
Homocysteine (Nutritional & Congenital) FISH, Williams2
Homocysteine, Total, Urine FISH, Wolf-Hirschhorn2
Maternal Serum AFP FISH, X-Linked Ichthyosis Steroid Sulfatase Deficiency2
Methylmalonic Acid & Homocysteine (Nutritional & Congenital) Male Infertility Genetic Analysis2
Methylmalonic Acid, Serum
Methylmalonic Acid, Urine Molecular Genetics
Organic Acids, Qualitative, Urine Achondroplasia Mutation Analysis2
Organic Acids, Quantitative, Full Panel, Urine Alpha-1 Antitrypsin (AAT) Mutation Analysis2
Includes minimum of 76 organic acids. Alpha-Globin Complete3
Penta Screen Alpha-Globin Gene Deletion or Duplication3
Phenylalanine Alpha-Thalassemia DNA Mutation Analysis3
Phenylalanine & Tyrosine Angiotensin Converting Enzyme (ACE) Polymorphism
Porphobilinogen, Quantitative, 24-Hour Urine (Insertion/Deletion)2
Porphobilinogen, Quantitative, Random Urine Angiotensin II Type 1 Receptor (AGTR1) Gene 1166AmC Polymorphism2
Porphyrins, Fractionated, Plasma Ashkenazi Jewish Panel2
Porphyrins, Fractionated, Quantitative & Porphobilinogen, 24-Hour Urine Includes mutations associated with cystic fibrosis, Canavan disease, Gaucher
Porphyrins, Fractionated, Quantitative, 24-Hour Urine disease, Fanconi anemia, Bloom syndrome, Tay-Sachs disease, familial
Porphyrins, Fractionated, Quantitative, Random Urine dysautonomia, and Niemann-Pick disease.
Porphyrins, Total, Plasma Beta-Globin Complete2
Quad Screen Bloom Syndrome DNA Mutation Analysis2
Sickle Cell Screen CAH (21-Hydroxylase Deficiency) Common Mutations2
Sickle Cell Screen with Reflex to Hemoglobinopathy Evaluation CAH (21-Hydroxylase Deficiency) Rare Mutations2
 61

Section 5 Genetics

Canavan Disease Mutation Analysis2 Thrombophilia Screen, Inherited2

Central Diabetes Insipidus (CDI) Mutations2 Includes antithrombin III activity, factor V (Leiden) mutation with reflex to factor V
CFTR Intron 8 Poly-T Analysis2 HR2 mutation, protein C activity, free protein S, and prothrombin (factor II)
CKR-5 Gene, DNA Mutation Analysis2 20210GmA mutation.
CYP1B1 Mutation Analysis2 TPMT Genotype3
Cystic Fibrosis Complete Rare Mutation Analysis, Entire Gene Tremor/Ataxia Syndrome (FXTAS)2
Sequence2 Twin Zygosity1
Cystic Fibrosis D1152H Mutation Analysis2 Venous Thrombosis Hypercoagulability Panel2
Cystic Fibrosis DNA Analysis, Fetus2 Includes activated protein C resistance (APCR), antithrombin III activity, cardiolipin
Cystic Fibrosis Gene Deletion or Duplication2 antibody (IgG, IgM), factor V (Leiden) mutation with reflex to factor V HR2
Cystic Fibrosis Rare Mutation Analysis, One Exon2 mutation, homocysteine, lupus anticoagulant dRVVT confirmation, lupus
Cystic Fibrosis Rare Mutation Analysis, Two Exon2 anticoagulant-hexagonal phospholipid neutralization, protein C activity with reflex
Cystic Fibrosis Screen2 to protein C antigen, and free protein S.
Cytochrome P450 2C19 Genotyping2 von Willebrand Disease Mutation Analysis3
Cytochrome P450 2C9 and VKORC1 Mutation Analysis3 XSense™, Fragile X with Reflex3
Cytochrome P450 2C9 Genotype2 XSense™, Fragile X with Reflex and Chromosome Analysis, Blood3
Cytochrome P450 2D6 Genotype2 Y Chromosome Microdeletion, DNA Analysis1
Cytochrome P450 2D6/2C19 Genotyping
Dihydropyrimidine Dehydrogenase (DPD) Gene Mutation Analysis2 Oncology-related Genetics
Factor V (Leiden) Mutation Analysis w/Reflex to HR2 Mutation Analysis2
Factor V (Leiden) Mutation Analysis2 Lymphoid Malignancies
Factor V HR2 Allele DNA Mutation Analysis2 B-Cell Gene Rearrangement, Minimal Residual Disease (MRD)2
Factor XI Mutation Analysis (Ashkenazi Jewish)2 B-Cell Gene Rearrangement, PCR2
Familial Dysautonomia Mutation Analysis2 B-Cell Gene Rearrangement, Qualitative PCR, Plasma-based,
Fanconi’s Anemia DNA Mutation Analysis2 Leumeta™2
Fragile X DNA Analysis, Fetus2 B-Cell Gene Rearrangement, Quantitative PCR, Plasma-based,
Gaucher Disease, DNA Mutation Analysis2 Leumeta™2
Genomic Alterations, Postnatal, ClariSure™ CGH3 bcl-2, IHC2
Glycogen Storage Disease Type Ia Mutation Analysis (Ashkenazi Chromosome Analysis, CLL/LPD
Jewish)2 Chromosome Analysis, Hematologic Malignancy
Hereditary Hemochromatosis DNA Mutation Analysis2 Chromosome Analysis, Lymph Node
Huntington Disease Mutation Analysis2 Chronic Lymphocytic Leukemia, IgVH Mutation Status, Cell-based2
Long Chain Acyl-CoA Dehydrogenase (LCHAD) Mutation Analysis2 Chronic Lymphocytic Leukemia, IgVH Mutation Status, Leumeta™2
Male Infertility Genetic Analysis2 FISH, ALCL, ALK, 2p23 Rearrangements2
Maple Syrup Disease (MSUD) Mutation Analysis (Ashkenazi Jewish)2 FISH, ALL, TEL/AML1 Translocation 12;212
Maternal Cell Contamination Study, STR Analysis1 FISH, ALL/NHL, MYC-BA, 8q24 Rearrangement2
Medium Chain Acyl-CoA Dehydrogenase (MCAD) Mutation Analysis2 FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel2
Methylenetetrahydrofolate Reductase (MTHFR), DNA Mutation Includes FISH probes for 11q22.3, 12 cen, 13q14.3, and 17p13 as well as 13q34
Analysis2 (control probe).
Mucolipidosis Type IV Mutation Analysis2 FISH, B-Cell Malignancy, IGH, 14q32 Rearrangement2
Nephrogenic Diabetes Insipidus (Autosomal) Mutations2 FISH, Burkitt’s/NHL/ALL, IGH/MYC, t(8;14)2
Nephrogenic Diabetes Insipidus (X-Linked) Mutations2 FISH, EGFR2
Niemann-Pick Disease Mutation Analysis2 FISH, Follicular Lymphoma, IGH/BCL2, t(14;18)2
Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G Polymorphism2 FISH, Mantle Cell Lymphoma, IGH/CCND1, t(11;14)2
Prader-Willi/Angelman Syndrome2 FISH, MLL (11q23) Gene Rearrangement2
Prothrombin (Factor II) 20210GmA Mutation Analysis2 FISH, Multiple Myeloma, 13q-, 17p-, rea 14q322
Resistance to Thyroid Hormone (RTH) Mutation Analysis2 FISH, X/Y, Post Bone Marrow Transplant
Rett Syndrome Mutation Analysis2 Follicular Lymphoma, bcl-2/JH t(14;18), Real-time PCR, Cell-based2
Sickle Cell Anemia, DNA Probe Analysis, Fetus2 Follicular Lymphoma, bcl-2/JH t(14;18), Real time-PCR, Leumeta™2
Tay-Sachs Disease Mutation Analysis (Ashkenazi Jewish)2 Mantle Cell Lymphoma, bcl-1/JH t(11;14), Real-time PCR, Cell-based2
Tay-Sachs Mutation Analysis (non-Ashkenazi Jewish)2 Mantle Cell Lymphoma, bcl-1/JH t(11;14), Real-time PCR, Leumeta™2
Thrombophilia Comprehensive Panel2 T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR,
Includes antithrombin III activity; cardiolipin antibody screen with reflex to IgA, IgG, LeumetaTM2
and IgM; factor V (Leiden) mutation with reflex to factor V HR2 mutation; T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR,
homocysteine; lupus anticoagulant-hexagonal phospholipid neutralization; lupus LeumetaTM2
anticoagulant dRVVT confirmation; methylenetetrahydrofolate reductase (MTHFR) T-cell Receptor (TCR) Gene Rearrangement2
mutation; protein C activity; free protein S; and prothrombin (factor II) 20210GmA
mutation. Myeloid Malignancies
Thrombophilia DNA Mutation Analysis2 AML1/ETO t(8;21) Quantitative Real-Time PCR2
Includes factor V (Leiden) and prothrombin (factor II) 20210GmA mutation analyses. bcr/abl Gene Rearrangement, Quantitative PCR with Reflex to Subtype2
Thrombophilia Mutation Analysis with Reflex to HR2 Mutation Analysis2 bcr/abl Gene Rearrangement, Quantitative PCR, Plasma-based,
Includes factor V (Leiden) and prothrombin (factor II) 20210GmA mutation analyses Leumeta™2
with reflex to HR2 mutation analysis bcr/abl Gene Rearrangement, Quantitative PCR2
bcr/abl Protein Quantitation (Total and Phosphorylated), Leumeta™2
62

Section 5 Genetics

CBFB/MYH11 inv(16), Quantitative Real-Time PCR2 retardation, developmental delay, learning disabilities, seizures,
Chromosome Analysis, Hematologic Malignancy lethargy, coma, vomiting, metabolic acidosis or alkalosis, sudden infant
FISH, AML M3, PML/RARA,Translocation 15;172 death syndrome (SIDS), osteomalacia, and osteoporosis. Depending on
FISH, AML, AML1/ETO Translocation 8;212 the natural history of the disorder, symptoms may be minimized or
FISH, AML, CBFB/MYH11, Inversion 162 prevented by early diagnosis and treatment. Treatment may be based on
FISH, Chromosome 20q Deletion2 dietary restrictions and/or supplementation with cofactors (eg, riboflavin
FISH, CML/ALL, bcr/abl Translocation 9;222 or cobalamin) or conjugating agents (eg, carnitine or sodium benzoate).
FISH, MLL (11q23) Gene Rearrangement2
FISH, Myeloid Disorders Profile2 Individuals Suitable for Testing include neonates/infants, children,
Includes FISH probes for 5q31, 7q31, 8 centromere, and 20q12 as well as 5p15.2 and adults.
and 7 centromere (control probes).
FISH, X/Y, Post Bone Marrow Transplant Method: This liquid chromatography, mass spectrometry (LC/MS) has
FLT3 Mutations (ITD and D835)2 an analytical sensitivity of 0.02-4.5 Nmol/L, depending on analyte, and
PML/RARA t(15;17), Quantitative PCR2 has no known cross-reactivity with other substances. The reportable
range is 1.0-25,000 Nmol/L.
Solid Tumor Malignancies
Chromosome Analysis, Solid Tumor Reference Ranges are provided in Tables 1-3 for plasma, urine, and
Colorectal Cancer (CRC) Pharmacogenomic Panel2 CSF, respectively.
Epidermal Growth Factor Receptor (EGFR) Mutation Analysis (TK
Domain)2 Interpretive Information: Elevation of one or more amino acids may
FISH, Bladder Cancer, Bladder Washing be diagnostic of an aminoacidopathy. Elevated amino acid levels are
FISH, EGFR2 also associated with noninherited diseases such as severe liver disease
FISH, Ewing/PNET, EWSR1, 22q12 Rearrangements2 and renal tubular disorders (eg, Fanconi syndrome). Decreased levels of
FISH, HER-2/neu, Paraffin Block amino acids are associated with malnutrition as seen in the elderly or
FISH, Lung Cancer2 those with poor protein intake or gastrointestinal disease.
FISH, N-myc Amplification, Neuroblastoma2
FISH, Oligodendroglioma, 1p/19q2 Additional laboratory testing is required to diagnose other inherited
FISH, Prostate Cancer2 disorders (ie, lactic acidosis, organic aciduria, and some urea cycle
FISH, Vysis®’ UroVysion™’, Bladder Cancer defects). Results should be evaluated in the context of clinical findings
HER2 (HercepTest®’), IHC with Reflex to FISH and/or additional test results.
MEN2 and FMTC Mutations, Exons 10, 11, 13-162
Microsatellite Instability (MSI), HNPCC2 Infant formulas that are supplemented with amino acids (particularly
MLH1 and MSH2 Mutations (Deletion and Duplication), HNPCC2 methionine and homocitrulline) and parenteral nutrition may affect the
MLH1 and MSH2 Mutations, HNPCC2 clinical accuracy of this test. Bacterial contamination of specimens and
MLH1 Mutation, One Exon, HNPCC2 certain medications, such as valproic acid, can also affect the levels of
MSH2 Mutation, One Exon, HNPCC2 specific amino acids. In addition, the absence of a protein-containing
MSH6 Mutation, HNPCC2 diet in newborns may preclude detection of selected aminoacidopathies.
MSH6 Mutation, One Exon, HNPCC2
UGT1A1 Gene Polymorphism (TA Repeat)2 Table 4 lists the amino acids that are elevated in the more common
1
This test is performed using a kit that has not been approved or cleared by the FDA. The
disorders.
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without References
confirmation by other medically established means.
2
This test was developed and its performance characteristics determined by Quest 1. Part 8. Amino Acids. In: Scriver CR, Beaudet AL, Valle D, Sly WS,
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Childs B, Kinzler KW, Vogelstein B, eds. The Metabolic and
Drug Administration. The FDA has determined that such clearance or approval is not Molecular Bases of Inherited Disease. 8th ed. New York, NY:
necessary. Performance characteristics refer to the analytical performance of the test.
3
This test was developed and its performance characteristics have been determined by McGraw-Hill, Inc; 2001;1665-2105.
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical 2. Part IV. Disorders of amino acid metabolism and transport. Fernandes
performance of the test. J, Saudubray J-M, Van den Berghe G, eds. Inborn Metabolic
Reflex tests are performed at an additional charge. Diseases Diagnosis and Treatment. 3rd ed. New York, NY: Springer;
2000;169-273.
5.2 Biochemical Genetics 3. Part 2. Disorders of amino acid metabolism. Nyhan WL, Barshop BA,
Ozand PT, eds. Atlas of Metabolic Diseases. 2nd ed. New York, NY:
5.2.1 Amino Acids Oxford University Press Inc; 2005;109-189.
4. Blau N, Duran M, Blaskovics ME, Gibson KM, eds. Physician’s Guide
Clinical Use: This test is used to diagnose primary aminoacidopathies, to the Laboratory Diagnosis of Metabolic Diseases. 2nd ed. New York,
screen for secondary aminoacidopathies, monitor therapeutic response, NY: Springer; 2003.
and assess nutritional status.
5.2.2 Congenital Adrenal Hyperplasia (CAH)
Clinical Background: Primary aminoacidopathies are typically
autosomal recessive or X-linked inherited disorders resulting from a 5.2.2.1 Laboratory Support of Diagnosis and Management
deficient enzyme or transport protein. Over 30 aminoacidopathies have
been described in the literature. Symptoms range from relatively benign Clinical Background: Congenital adrenal hyperplasia (CAH) is a
to severe and may include, but are not limited to, growth and mental group of autosomal recessive disorders caused by deficiency in one or
 63

Section 5 Genetics

Table 1. Plasma Amino Acid Age-Specific Reference Ranges (μmol/L)

Amino Acid <1 month 1–23 months 2–17 years Adults (r18 years)
Aspartic acid 2-20 2-14 1-8 1-4
Glutamic acid 51-277 32-185 9-109 10-97
Hydroxyproline 13-72 7-63 6-32 4-27
Serine 87-241 83-212 85-185 65-138
Asparagine 12-70 20-77 23-70 31-64
B-Aminoadipic acid b3 b4 b2 b2
Glycine 133-409 103-386 138-349 122-322
Glutamine 240-1194 303-1459 405-923 428-747
Sarcosine b5 b4 b4 b4
C-Alanine b8 b8 b5 b5
Taurine 29-161 26-130 32-114 31-102
Histidine 40-143 42-125 54-113 60-109
Citrulline 3-35 4-50 9-52 16-51
Arginine 14-135 30-147 38-122 43-407
Threonine 56-392 40-428 59-195 67-198
Alanine 83-447 119-523 157-481 200-483
1-Methylhistidine b4 b9 b27 b47
H-Aminobutyric acid 1 1 b2 b3
3-Methylhistidine b10 b8 1-6 2-9
C-Aminoisobutyric acid b9 b8 b6 1
Proline 87-375 104-348 99-351 104-383
Ethanolamine 8-106 5-19 5-15 5-13
B-Aminobutyric acid 1-20 4-30 6-30 7-32
a
Tyrosine 33-160 24-125 31-108 38-96
a,b
Valine 57-250 84-354 130-307 132-313
Methionine 13-45 12-50 14-37 16-34
Cystathionine 1 1 1 1
Isoleucinea,b 12-92 10-109 33-97 34-98
Leucinea,b 23-172 43-181 65-179 73-182
Homocystine 1 1 1 1
a
Phenylalanine 30-79 31-92 38-86 40-74
a
Tryptophan 17-85 16-92 30-94 40-91
Ornithine 29-168 19-139 33-103 27-83
Lysine 66-226 70-258 98-231 119-233
Alloisoleucineb 1 1 1 1
The full panel (test code 767X) includes all amino acids listed except alloisoleucine.
a
Included in the limited panel (test code 1776X).
b
Included in the MSUD panel (test code 19779X).
64

Section 5 Genetics

Table 2. Urine Amino Acid Age-Specific Reference Ranges (mmol/mol creatinine)

Amino Acid <1 month 1–23 months 2–17 years Adults (r18 years)
Aspartic acid b7.0 b11.0 b2.0 b2.0
Glutamic acid 4-19 3-30 b10.0 b3.0
Hydroxyproline 30-485 2-345 b4.0 b2.0
Serine 44-454 39-422 13-127 10-71
Asparagine 8-42 5-132 3-42 2-37
B-Aminoadipic acid b10 b36 b34 b11
Glycine 215-2053 105-413 23-413 b330
Glutamine b355 41-396 18-188 21-182
Sarcosine b18 b19 b2 b69
C-Alanine b9 b15 b5 b10
Taurine b650 b670 b255 b232
Histidine 40-301 56-543 9-425 17-266
Citrulline b4 b13 b4 b2
Arginine b30 b35 b8 b5
Threonine b112 9-158 4-60 4-46
Alanine 45-264 16-294 8-156 9-67
1-Methylhistidine b16 4-71 5-400 b204
H-Aminobutyric acid b1.4 b1.5 b1.6 b1.6
3-Methylhistidine 9-45 14-35 11-40 10-35
C-Aminoisobutyric acid b269 b309 b133 b88
Proline b219 b216 b11 b2
Ethanolamine 87-490 54-176 27-114 21-65
B-Aminobutyric acid b7 b7 b5 b2
Tyrosine 4-59 10-69 3-48 3-19
Valine 2-20 4-21 2-20 2-5
Methionine b7 b7 b5 b2
Cystathionine 2-20 b29 b8 b9
Isoleucine b9 b12 b5 b3
Leucine b23 b24 b13 b6
Homocystine 1.0 b4.0 1.0 1.0
Phenylalanine 3-24 6-39 2-22 2-9
Tryptophan 2-21 5-46 2-27 2-14
Ornithine b39 b11 b5 b4
Lysine 13-284 4-239 3-112 3-59
Cystine 15-48 6-28 3-20 3-13
Hydroxylysine 5-117 2-72 b8 b8

more of the enzymes required for synthesis of cortisol, aldosterone, and
sex steroids in the adrenal gland. In most instances, the levels of
steroids proximal to the enzyme defect (precursors) are elevated, and
the levels of those distal to the defect (products) are decreased (Figure
1). Decreased cortisol production leads to an increase of
adrenocorticotrophic hormone (ACTH), and the resulting adrenal
stimulation leads to a further increase of the steroids, and their
associated metabolites, proximal to the defect. Depending on the level
of the enzymatic defect, the shunting of precursor steroids may then
result in either an increased or decreased production of sex hormones.
 65

Section 5 Genetics

Table 3. CSF Amino Acid Age-Specific Reference Ranges (Nmol/L)

Amino Acid 3 months 3-23 months 2-10 years 10 years

Aspartic acid b2.7 1.0 1.0 b2
Glutamic acid 1-9 b5.1 b10.6 1.1-13.2
Hydroxyproline 0.9-3.9 b1.6 1.0 b1.7
Serine 30-88 22-61 15-62 9-41
Asparagine b27 b13 b25 b24
B-Aminoadipic acid 1.0 1.0 1.0 1.0
Glycine 3-26 b12 b13 b10
Glutamine 525-1583 386-742 377-1738 361-1175
Sarcosine 1.0 1.0 1.0 1.0
C-Alanine 1.0 1.0 1.0 1.0
Taurine 0-18 b8 1-8 1-8
Histidine 8-32 4-25 7-25 7-22
Citrulline 1-4 b3 1-2 b2
Arginine 2-27 7-32 9-31 10-32
Threonine 23-104 10-55 8-85 12-64
Alanine 13-50 8-48 5-62 1-107
H-Aminobutyric acid 1.0 1.0 b2.2 b3.1
C-Aminoisobutyric acid 1.0 1.0 1.0 1.0
Proline b3.9 b2.3 b1.7 b5.9
B-Aminobutyric acid b6 b6 1-11 1-11
Tyrosine 9-41 5-20 5-32 5-18
Valine 11-31 8-19 2-37 7-42
Methionine 2-14 1-7 b9 1-8
Isoleucine 3-11 3-7 2-13 3-10
Leucine 7-22 7-12 8-27 9-32
Homocystine 1.0 1.0 b2.5 b2.1
Phenylalanine 4-31 4-14 b25 6-31
Tryptophan b5.9 b7.7 0.6-4.6 b9.3
Ornithine b25.7 b4.5 b4.7 b14.2
Lysine 6-38 3-29 9-58 19-60

Depending on the position of the enzymatic defect in the biochemical
pathway, the shunting of precursor steroids may then result in either an
increased or decreased production of sex hormones. Additionally,
decreased production of aldosterone can lead to renal salt loss and
hypotension. Genes coding for the enzymes have been identified (Table
5), and nomenclature reflects these findings. For example, 21-hydroxlase
deficiency is coded for by CYP21A2, and the defect is frequently
referred to by the gene name.1,2
those with severe disease may exhibit marked adrenal insufficiency and
salt-wasting, genital ambiguity, and virilization ranging from mild to
complete masculinization of the external genitalia of female (XX)
fetuses.3 Masculinization of females can be prevented by maternal
treatment with dexamethasone beginning before the 7th week of
gestation in at-risk pregnancies.4 Treatment is then discontinued if DNA
testing indicates a low likelihood of CAH or male (XY) genotype is
established.

The clinical manifestations of CAH vary with the enzyme defect present Neonatal screening for 21-hydroxylase deficiency (CYP21A2), the most
(Table 5) and the degree of deficiency. While individuals with nonclassic common cause of CAH, is performed routinely in many states by
disease (ie, partial deficiency) may be minimally symptomatic and measuring 17-hydroxyprogesterone (17-OHP) in filter paper blood
present with symptoms suggestive of polycystic ovarian syndrome, samples obtained from newborns. Diagnosis in infants with an elevated
66
Section 5
Table 4. Common Aminoacidopathies and Associated Amino
Acid Elevations
Common Aminoacidopathies Elevated Amino Acids
Primary Aminoacidopathies
Arginase deficiency Arginine, glutamine
Arginosuccinase deficiency Argininosuccinate, glutamine
Citrullinemia Citrulline, glutamine
Cystinuria Cystine, ornithine, lysine,
arginine (urine only)
Homocystinuria Homocystine
Maple Syrup Urine Disease (MSUD) Valine, isoleucine, leucine,
alloisoleucine
Phenylketonuria (PKU) Phenylalanine
Tyrosinemia Tyrosine
Secondary Aminoacidopathies
Hyperammonemia Glutamine
Lactic acidosis Alanine
Organic acidurias, selected Glycine
Transient tyrosinemia of the newborn Tyrosine
17-OHP or those with clinical manifestations of CAH is accomplished by
measuring blood and/or urine steroid and metabolite levels.
Additionally, DNA analysis can identify CYP21A2 mutations, which is
helpful for determining carrier status, confirming the diagnosis in
affected individuals, and establishing the diagnosis prenatally.
21-Hydroxylase Deficiency
21-Hydroxylase deficiency (CYP21A2) accounts for 90% to 95% of CAH
cases, and classic and nonclassic forms have been described.5 Classic
21-hydroxylase deficiency has an incidence of 1:5,000 to 1:15,000 live
births in Western populations, though higher frequencies have been
identified in certain ethnic groups.5 The disorder is characterized by
markedly diminished or absent 21-hydroxylase activity, which results in
decreased levels of deoxycorticosterone, 11-deoxycortisol,
corticosterone, cortisol, and aldosterone and increased levels of 17-OHP
and androstenedione. Affected individuals typically present at birth or in
the neonatal period with either a virilizing or salt-wasting form. Female
(XX) infants with virilizing 21-hydroxylase deficiency have varying
degrees of masculinization ranging from clitoral enlargement to
complete development of male external genitalia, which may lead to
inaccurate sex assignment at birth. Male infants (XY) have normal male
genitalia. Approximately three-quarters of affected infants also have
mineralocorticoid deficiency that leads to salt-wasting. Symptoms of
hyponatremia, hyperkalemia, volume depletion, and decreased blood
pressure generally appear within the first 2 weeks of life.
Nonclassic 21-hydroxylase deficiency is thought to have an incidence as
high as 1:1,000 and is characterized by marginally decreased
21-hydroxylase activity.5 Affected individuals typically do not have
developmental abnormalities or salt-wasting. Presentation is in
childhood or post puberty with evidence of androgen excess. In boys,
increased androgens typically manifest as sexual precocity, whereas in
girls increased pubic hair growth and/or clitoral enlargement is seen. In
women, nonclassic 21-hydroxylase deficiency is frequently confused
with polycystic ovary syndrome.
The genetic diagnosis of 21-hydroxylase deficiency is complex because
of the large number of unique mutations that may result in decreased
enzyme activity and interactions between CYP21A2 and its pseudogene
CYP21A. The majority of mutations arise from 2 types of recombination
Genetics
between CYP21A2 and CYP21A: 1) deleterious mutations that have been
transferred from the pseudogene to CYP21A2 during mitosis (75% of
cases), and 2) unequal recombinations during meiosis between the gene
and pseudogene that result in deletion of the intervening 30-kb segment
(20% of cases).5 Although over 60 relevant mutations have been
identified, 11 account for approximately 90% of those found in
heterozygous carriers.5 Additionally, in affected individuals, 1% to 2% of
abnormal alleles contain spontaneous mutations that are not carried by
either parent. Enzyme activity varies with the mutations present, and 3
phenotypic categories, which manifest 0%, 1% to 2%, or 20% to 60%
of normal activity, have been identified.5 Although allelic variation
accounts for 80% to 90% of the phenotypic variation, the affected
individual’s unique genetic factors (eg, steroid receptor binding
properties) may influence final expression. 5
11C-Hydroxylase Deficiency
11C-Hydroxlase deficiency (CYP11B1) accounts for 5% to 8% of CAH
cases.6 Deficiency of 11C-hydroxlase leads to decreased levels of
cortisol, corticosterone, and aldosterone and increased levels of
deoxycorticosterone and 11-deoxycortisol. Salt-wasting does not occur
as with 21-hydroxylase deficiency; however, virilization of female (XX)
fetuses can be as severe. Elevated blood pressure manifests early in life
in approximately two-thirds of patients and, along with elevated
deoxycorticosterone and 11-deoxycortisol levels, clinically distinguishes
11C- from 21-hydroxylase deficiency (Table 5). A late-onset form that
typically presents with signs of androgen excess is analogous to
nonclassic 21-hydroxylase deficiency.
17B-Hydroxylase Deficiency
17B-Hydroxylase deficiency (CYP17) accounts for approximately 1% of
all CAH cases, with an estimated incidence of 1:50,000 newborns.7 The
CYP17 gene encodes an enzyme that catalyzes both 17B-hydroxylation
and 17,20-lyase reactions. Isolated deficiency of either activity has been
reported; however, a combined deficiency in which there is failure of
catalysis of both reactions is the most common form. Affected
individuals have decreased levels of cortisol, androgens, and estrogens.
Presentation is typically at puberty; females (XX) have primary
amenorrhea and lack secondary sexual characteristics, and males (XY)
are found to have complete pseudohermaphroditism (ie, female external
genitalia, absence of uterus and fallopian tubes, and intra-abdominal
testes). At the time of diagnosis, individuals are usually found to be
hypertensive and hypokalemic.
3C-Hydroxysteroid Dehydrogenase Deficiency
3C-Hydroxysteroid dehydrogenase deficiency (HSD3B2) is a rare form of
CAH characterized by increased levels of pregnenolone, 17-hydroxy-
pregnenolone, and DHEA and decreased levels of all other adrenal
steroids.8 Affected individuals usually present in infancy with signs of
adrenal insufficiency. Female (XX) infants will typically have mild
virilization. Phenotypic variation in male (XY) infants may range from
hypospadias to complete male pseudohermaphroditism.
Aldosterone Synthase Deficiency
Aldosterone synthase deficiency (CYP11B2) is a rare form of CAH in
which only aldosterone synthesis is affected. Two forms have been
identified; type I is characterized by decreased, and type II by increased,
18-hydroxycorticosterone.9 Infants usually present within the first 5 days
of life with failure to thrive, recurrent dehydration secondary to salt-
wasting, decreased blood pressure, and acidosis.
Steroid Acute Regulatory Protein (StAR) Deficiency
StAR deficiency is responsible for congenital lipoid adrenal hyperplasia,
a defect of cholesterol transport resulting in deficiency of all adrenal
steroids. It is the rarest of the congenital adrenal steroid defects and
 67

Section 5 Genetics

StAR*

Cholesterol

Cholesterol
desmolase
(CYP11A)
17 17,20
(CYP17 ) (CYP17 )
Pregnenolone 17-Hydroxypregnenolone DHEA DHEAS

3ß 3ß 3ß
(HSD3B2 ) (HSD3B2 ) (HSD3B2 )
17 17,20
(CYP17 ) (CYP17 )
Progesterone 17-Hydroxyprogesterone Androstenedione Estrone

21 21
(CYP21A2 ) (CYP21A2 )

Deoxycorticosterone 11-Deoxycortisol Testosterone Estradiol

11ß 11ß
(CYP11B1) (CYP11B1)

Corticosterone Cortisol Dihydrotestosterone

A
(CYP11B2 )

Aldosterone
*StAR, steroid acute regulatory protein: transports cholesterol from the outer to inner mitochondrial membrane.

Figure 1. Pathways of adrenal steroid synthesis. Enzymes and encoding genes are indicated at arrows. When an enzymatic defect occurs, steroids
proximal to the defect increase and are frequently shunted into other products, and steroids distal to the defect decrease (eg, a deficiency of 11C-
hydroxylase will cause increased levels of deoxycorticosterone, progesterone, 11-deoxycortisol, and 17-hydroxyprogesterone and decreased levels of
corticosterone, aldosterone, and cortisol). 17B, 17B-hydroxylase; 17,20, 17,20-lyase; 3C, 3C-hydroxysteroid dehydrogenase; 21, 21-hydroxylase; 11C,
11C-hydroxylase; A, two-step process of aldosterone synthesis: 1) hydroxylation of corticosterone to form 18-hydroxycorticosterone, 2) oxidation of
18-hydroxycorticosterone to form aldosterone; DHEA, dihydroepiandrostenedione; DHEAS, dihydroepiandrostenedione sulfate.

has been fatal in two-thirds of the reported cases. The defect was
thought to reside in CYP11A, the gene that codes for the cholesterol
side-chain cleavage enzyme; however, recent molecular studies indicate
the defect resides on chromosome 8 in the STAR gene, which encodes a
phosphoprotein that enhances cholesterol transport from the outer to
inner mitochondrial membrane.10 Affected individuals present in the
neonatal period with severe adrenal insufficiency manifested by failure
to thrive, vomiting, diarrhea, hyponatremia, and hypokalemia. Males
(XY) typically have normal female external genitalia.
P450 Oxidoreductase Deficiency (ORD)
ORD (CYPOR) is a newly identified cause of CAH.11,12 P450
oxidoreductase contributes electrons to microsomal P450 enzymes, and
its deficiency results in decreased 17B- and 21-hydroxylase activity.
Steroid profiles may show increased levels of 17-OHP, 21-deoxycortisol,
progesterone, and pregnenolone. Baseline cortisol is typically normal;
however, the response to ACTH is decreased. Genital ambiguity is
frequently seen, and the majority of patients have Antley-Bixler
syndrome, a unique constellation of craniofacial and skeletal
68

Section 5 Genetics

Table 5. Characteristics of Congenital Adrenal Hyperplasia Enzyme Deficiencies

Steroid Acute
21-Hydroxylase
3C-Hydroxysteroid Aldosterone Regulatory P450
Classic Nonclassic 11C-Hydroxylase 17B-Hydroxylase Dehydrogenase Synthase Protein (StAR) Oxidoreductase
Gene CYP21A2 CYP21A2 CYP11B1 CYP17 HSD3B2 CYP11B2 STAR CYPOR
Incidence* 1:5,000-15,000 1:1000 1:100,000 Rare Rare Rare Rare Rare
Elevated 17-OHP 17-OHP (post ACTH) DOC DOC DHEA Corticosterone None Pregnenolone
Steroids androstenedione androstenedione 11-deoxycortisol corticosterone 17-OH pregnenolone 18-OHC (type II) progesterone
progesterone pregnenolone DOC
Decreased Aldosterone None Cortisol Cortisol Cortisol Aldosterone All Cortisol (n ACTH
Steroids corticosterone corticosterone aldosterone aldosterone 18-OHC (type I) response)
(salt-wasting) aldosterone (classic) 17-OHP
cortisol (simple
virilizing)
Age at Diagnosis Infancy Childhood/puberty Neonatal to adult Puberty Early infancy (severe) Neonatal Neonatal Infancy/childhood
post puberty (mild)
Genitalia
Females (X,X) Virilized ± Mild virilization Mild-severe No puberty Mild virilization Normal No puberty Ambiguous
virilization
Males (X,Y) Normal Normal Normal Ambiguous Ambiguous Normal Ambiguous Ambiguous
Androgens l l l n n
in males Normal n n
l
in females
Estrogens n n n in females n n Normal n n
+
Na n in salt-wasting Normal l l n n n Normal
K+ l Normal n n l l l Normal
Blood Pressure n Normal l l n n n Normal
17-OHP, 17-hydroxyprogesterone; ACTH, adrenocorticotrophic hormone; DOC, deoxycorticosterone; DHEA, dihydroepiandrostenedione; 18-OHC, 18-hydroxycorticosterone; Na+, sodium;
K+, potassium.
*Incidence found in the general population. Some disorders have higher incidence in certain ethnic groups.1,5

abnormalities. ORD is also considered a potential cause of decreased ratios necessary for diagnosis are available (Table 6). Additionally, a
estriol in mid-trimester Down syndrome screening tests. Compound plasma renin activity (PRA) assay is available for use in monitoring
heterozygotes for ORD and 21 hydroxylase have been reported. treatment of individuals with salt-wasting.

Individuals Suitable for Testing include newborns with a positive
CAH screening test; newborns with ambiguous genitalia; newborns and
infants with evidence of adrenal insufficiency and/or unexplained sodium
and potassium abnormalities; infants or children with evidence of CAH
not attributable to known causes or in whom Antley-Bixler syndrome is
suspected; children with evidence of precocious or delayed puberty or
unexplained hypertension; women with polycystic ovary syndrome,
hirsutism, and/or evidence of estrogen deficit; individuals with suspected
androgen excess; individuals with a family history of CAH and their
partners, who desire carrier screening; and pregnant women at risk for a
fetus affected with CYP21A2 mutations who desire prenatal diagnosis.
Test Availability
Steroid Assays
The diagnosis of enzymatic defects responsible for CAH relies upon
accurate measurement of steroid and steroid metabolite levels and the
calculation of precursor-product ratios. Quest Diagnostics Nichols
Institute utilizes mass spectrometry technology for increased sensitivity
and discrimination in steroid measurement. While assays that measure
steroid and metabolite levels in blood or urine may be ordered
individually, CAH panels that include the analytes and corresponding
DNA Analysis
CAH (21-Hydroxylase Deficiency) Common Mutations
Using 4 polymerase chain reactions to examine the gene, pseudogene,
and recombinant genes, this test detects the most common mutations of
CYP21A2: P30L, In2G, G110del8, I172N, exon 6 cluster mutation (I236N,
V237E, M239K), V281L, F306+1nt, Q318X, R356W, P453S, and a 30-kb
deletion. These 12 mutations and the 30-kb deletion are responsible for
approximately 90% of the CYP21A2 defects that can result in
21-hydroxylase deficiency.
CAH (21-Hydroxylase Deficiency) Rare Mutations
This test provides complete sequencing of CYP21A2.
Chromosome Analysis
Chromosome analysis differentiates XX and XY genotypes in those
presenting with ambiguous genitalia.
Test Selection
Steroid Assays
Blockage of an adrenal biosynthetic pathway due to an enzymatic defect
results in increased levels of the enzyme’s precursors and associated
 69

Section 5 Genetics

metabolites as well as increased precursor-product ratios. In severe 21-
and 11C-hydroxylase and 3C-hydroxysteroid dehydrogenase
deficiencies, baseline steroid and precursor-product ratios are frequently
adequate for diagnosis; however, steroid measurement after ACTH
stimulation is usually necessary for diagnosis when partial deficiency is
suspected. ACTH stimulates adrenal hormone synthesis, which
accentuates precursor steroid levels and diagnostic ratios. For example,
ACTH stimulation followed by 17-OHP measurement is useful for
evaluating newborns with increased 17-OHP levels in the absence of
virilization or adrenal insufficiency.
In newborns with suspected CAH (ie, adrenal insufficiency, virilization,
17-OHP >400 ng/dL, family history), assay selection must be guided by
clinical findings and other laboratory information (Table 5). The
differential diagnosis of virilizing CAH in a newborn includes
21-hydroxylase, 11C-hydroxylase, and 3C-hydroxysteroid dehydrogenase
deficiencies as well as ORD. In those with salt-wasting, aldosterone
synthase and StAR deficiencies must also be considered.
Table 6 specifies the clinical use for the CAH panels offered by Quest
Diagnostics. CAH Panel 1 enables diagnosis of the 2 most common

Table 6. Congenital Adrenal Hyperplasia Diagnostic Panels*

Panel 1 Panel 3 Panel 4 Panel 6 Panel 6b† Panel 7† Panel 8 Panel 9
Monitor
21-OH vs Aldosterone Female 21-OH Male
11C-OH Synthase 17B-OH StAR Comprehensive Deficiency 17B-OH 3C-HSD
Deficiency Deficiency Deficiency Deficiency Screen Treatment Deficiency Deficiency
Analyte
Aldosterone n n n n
Androstenedione l • • n
Cortisol (total) n n n • n n
Corticosterone l l
11-Deoxycortisol l
in 11C Normal •
(Compound S) n
in 21
DOC •
DHEA n • l
Estradiol n
18-Hydroxycorticosterone n in type I
l
in type II
17-Hydroxypregnenolone • l
17-OHP l Normal n • • n n
Pregnenolone n
Progesterone l • l
Testosterone (total) l • • n
Diagnostic Ratio
11-Deoxycortisol >9 in 11C
Cortisol
DHEA >9
Androstenedione
18-Hydroxycorticosterone <10 type I
Aldosterone >100 type II
17-Hydroxypregnenolone >12
17-OHP
17-OHP >3 in 21 >3
11-Deoxycortisol
Progesterone
DOC
Progesterone >4 >4
17-OHP
21-OH, 21-hydroxylase; 11C-OH, 11C-hydroxylase; 17B-OH, 17B-hydroxylase; StAR, steroid acute regulatory protein; 3C-HSD, 3C-hydroxysteroid dehydrogenase; DOC, deoxycorticosterone;
DHEA, dihydroepiandrostenedione; 17-OHP,
17-hydroxyprogesterone.
*Analytes and expected result if enzyme defect is present are indicated for each panel.
†
Results for panels 6b and 7 will be within reference range if no defect is present (6b) or treatment is adequate (7).
Not shown: Panel 11 (Tables 6 and 7) is a comprehensive neonatal profile (urine) that includes 15 steroid analytes and 11 ratios for differential diagnosis of 21-hydroxylase, 11-hydroxylase,
17B-hydroxylase, and 3C-hydroxysteroid dehydrogenase deficiencies.
70

Section 5 Genetics

forms of CAH: 21- and 11C-hydroxylase deficiencies. Panel 6b serves as
a comprehensive screen that is useful when a broader differential
diagnosis is being considered. During the neonatal period, CAH Panel 11
can distinguish between and diagnose 21-, 11C -, 17C-hydroxylase, and
3C-hydroxysteroid dehydrogenase deficiencies.13 This urine-based assay
is an alternative to serum measurements when clinical suspicion for
CAH is high or the 17-OHP screen is positive. Other CAH panels are
used to diagnose the enzyme defects responsible for rare causes of CAH
and should be selected when other laboratory and clinical findings
suggest an uncommon type of CAH.
21-hydroxylase deficiency. An 11-deoxycortisol level >350 ng/dL or
11-deoxycortisol:cortisol ratio >15 is diagnostic of 11C-hydroxylase
deficiency. After ACTH stimulation, a 17-hydroxypregnenolone level
>1500 ng/dL and 17-hydroxypregnenolone:17-OHP and
DHEA:androstenedione ratios >10 are considered diagnostic of 3C-HSD.
However, recent studies utilizing DNA analysis of HSD3B2 to confirm
disease state have questioned these criteria, and new diagnostic
standards based upon 17-hydroxypregnenolone levels and 17-hydroxy-
pregnenolone:cortisol ratios before and after ACTH stimulation have
been proposed.14,15

Treatment of CAH requires monitoring adrenal steroid levels to ensure
correct dosing of glucocorticoid and/or mineralocorticoid replacement.
CAH Panel 7 is designed for monitoring treatment of 21-hydroxylase
deficiency. PRA assays are useful for monitoring mineralocorticoid
replacement in individuals with salt-wasting.
DNA Analysis
CAH (21-Hydroxylase Deficiency) Common Mutations
This test is suitable for diagnosis in infants with a positive newborn
screen and in individuals known or suspected to be 21-hydroxylase
deficient. It is also useful for carrier screening in those with a family
history (especially first degree relatives of affected individuals) and in
spouses of affected individuals or carriers (to identify high-risk
pregnancies). DNA analysis is the preferred method for determining
carrier status in individuals with marginally elevated 17-OHP levels,
because post-ACTH stimulation 17-OHP levels overlap in normal
individuals and carriers.3 This assay is also suitable for prenatal
diagnosis of 21-hydroxylase deficiency when performed on CVS or
amniotic fluid samples.
Table 7 contains pre- and post-ACTH stimulation 17-OHP reference
ranges for infants, children, and adults.
Table 8 contains additional steroid reference ranges for infants and
children pre- and post-ACTH stimulation, and Table 9 contains infant
and children reference ranges for precursor-product ratios pre- and post-
ACTH stimulation. Tables 10 and 11 contain reference ranges for
diagnostic ratios and steroids, respectively, for CAH Panel 11 (neonatal
urine panel).
Steroid and metabolite levels and ratios should be interpreted in
conjunction with other laboratory and clinical findings.
DNA Analysis
The following information will assist in understanding test results. A
complete family history and parental and affected sibling genotypes are
required for the most accurate interpretation of 21-hydroxylase
deficiency DNA testing. Assistance is available from our Genetic
Counselors by calling 1-866-GENE-INFO (1-866-436-3463).

CAH (21-Hydroxylase Deficiency) Rare Mutations
This test is indicated for 21-hydroxylase deficiency-affected individuals
in whom only 1 or none of the common mutations have been identified
and for individuals whose family history includes a rare mutation.
Chromosome Analysis
Karyotyping is suitable to determine the genotype (XX or XY) and
thereby establish gender in infants born with ambiguous genitalia.
CAH (21-Hydroxylase Deficiency) Common Mutations
A negative result indicates none of the 12 common mutations or 30-kb
deletion responsible for 21-hydroxylase deficiency were detected. In an
individual without clinical symptoms of CAH, this lowers, but does not
eliminate the risk of a mutation being present; the residual risk of being
a carrier depends upon the individual’s family history. In an individual
affected with CAH, a negative result suggests the presence of a rare
mutation or a cause other than 21-hydroxylase deficiency.

Testing protocols for CAH work-up vary with treatment centers. Figures
2 and 3 illustrate several testing options (algorithms). These options are Table 7. 17-Hydroxyprogesterone Reference Ranges Pre-
based on accepted diagnostic standards1-7,11-13 and have been reviewed
by Medical Directors at Quest Diagnostics Nichols Institute. and Post-ACTH Stimulation

Test Interpretation 60 Minutes Post

Baseline ACTH Stimulation
Steroid Assays (ng/dL) (ng/dL)
An early morning 17-OHP level <200 ng/dL almost always eliminates the Males* 50-250 42-250
diagnosis of 21- or 11C-hydroxylase deficiency. Though 17-OHP levels
up to 400 ng/dL may be normal, those >200 ng/dL require evaluation. Females (Follicular Phase) * 20-100 42-250
Levels >400 ng/dL are consistent with 21- or 11C-hydroxylase Children†
deficiency.3,5 Prematurity, illness, and stress can cause elevations in 1-12 mo 11-170 85-465
17-OHP levels. 1-5 y 4-115 50-350
6-12 y 7-69 75-220
Elevated precursor-product ratios are diagnostic of enzyme deficiencies
and are used to discriminate between causes of CAH. In normal Tanner Stage II-III
children, adolescents, and adults, precursor-product ratios associated Males 12-130 69-310
with the 3C-HSD, 21-, and 17B-hydroxylase enzymes will not exceed 10, Females 18-220 80-420
while the ratios associated with 11C-hydroxylase and aldosterone Tanner Stage IV-V
synthase will not exceed 15. In unaffected individuals, precursor-product Males 51-190 105-230
ratios are similar before and after ACTH stimulation. Females 36-200 80-225
*Includes data from references 16-19.
After puberty, a post-ACTH stimulation 17-OHP level between 1500 †
Includes data from references 20-22.
ng/dL and 10,000 ng/dL is considered diagnostic of nonclassic
 71

Section 5 Genetics





Abnormal



†

 


Panel 11* 21-OH Deficiency

17-OHP (am) Common Mutations
Neonatal Urine

Abnormal Normal Normal Abnormal Normal Abnormal

CAH CAH Treatment based on CAH 21-OH deficiency

‡
unlikely unlikely type of CAH indicated unlikely

<300 ng/dL CAH unlikely

†

 <1500 ng/dL


300 to 1499 ng/dL Likely heterozygous

17-OHP after
1,500 to 10,000 ng/dL Nonclassic CAH likely
ACTH stimulation

>10,000 ng/dL Classic CAH likely



Normal CAH unlikely
Panel 1
21-OH vs 11ȕ-OH
Deficiency
‡
Abnormal Treatment based on type of CAH indicated

†


Normal CAH unlikely

21-OH Deficiency
Common Mutations

Abnormal 21-OH deficiency

17-OHP, 17-hydroxyprogesterone; 21-OH, 21-hydroxylase; 11C-OH, 11C-hydroxylase.

*Neonates up to 28 days old.
†
Deoxycorticosterone and/or 11-deoxycorticosterone may be indicated to rule out 11C-hydroxylase deficiency.
‡
DNA testing (21-OHD Common Mutations) may be indicated to confirm a diagnosis of 21-hydroxylase deficiency, to assess fetal risk during pregnancy, or for family studies.
§
One allele contains a functional copy of CYP21A2 and the second allele contains a non-functional copy of CYP21A2.

Figure 2. Testing options that can be used when the newborn screen is abnormal and there are no signs or symptoms of CAH.
72

Section 5 Genetics






Normal genitalia Abnormal genitalia Karyotype

Assign genotype


 


†

 


Panel 1 Panel 6b
Panel 11* 21-OH vs 11ȕ-OH 21-OH Deficiency
Comprehensive
Neonatal Urine Deficiency Common Mutations
Screen

Normal Abnormal Normal Abnormal Normal Abnormal Normal Abnormal

Consider non-CAH Rule out non-CAH Consider non-CAH CAH

diagnosis diagnosis diagnosis unlikely

Treatment based on Treatment based on Treatment based on 21-OH deficiency

‡ ‡ ‡
type of CAH indicated type of CAH indicated type of CAH indicated

Consider rare causes

of CAH

Panel 11 or 6b

Normal Abnormal

CAH unlikely Treatment based on

‡
type of CAH indicated

21-OH, 21-hydroxylase; 11C-OH, 11C-hydroxylase.

*Neonates up to 28 days old.
†
Deoxycorticosterone and/or 11-deoxycorticosterone may be indicated to rule out 11C-hydroxylase deficiency.
‡
DNA testing (21-OHD Common Mutations) may be indicated to confirm a diagnosis of 21-hydroxylase deficiency, to assess fetal risk during pregnancy, or for family studies.

Figure 3. Testing options that can be used when CAH is suspected.

 73

Section 5 Genetics

Table 8. Observed Ranges for Serum Adrenal Steroids in Infants and Children. Values Before (B), After (A), and Response (%) to
Rapid ACTH Test

Pubertal Pubertal
Steroid 26-28 wk* 34-36 wk* 1-6 mo <1 y 1-5 y 6-12 y Male Female
Pregnenolone B 260-2100 203-1024 10-150 10-137 10-48 15-45 15-84 24-50
A 962-3179 637-1888 110-359 49-359 34-135 38-104 33-218 37-149
% 70-2673 162-1685 20-282 19-282 4-114 16-73 6-193 9-101
Progesterone B 18-640 – 5-53 5-80 8-64 5-93 6-1286 17-145
A 52-1348 – 74-200 74-200 51-233 38-204 32-1069 35-223
% 29-796 – 35-165 35-192 19-192 22-170 0-104 0-192
17-OH pregnenolone B 375-3559 559-2906 52-828 13-788 9-98 10-177 19-346 50-516
A 2331-11440 831-9760 633-3286 373-3125 43-702 67-624 84-817 239-1525
% 1219-9799 346-8911 229-3104 200-3000 15-680 60-500 65-750 108-1280
17-OH progesterone B 124-841 186-472 13-173 11-173 4-114 7-69 12-190 18-220
A 285-1310 334-1725 85-250 85-466 50-350 75-218 69-313 80-422
% 50-596 18-1253 52-193 50-275 30-300 50-250 7-281 9-287
DHEA B 236-3640 223-3640 26-505 26-500 9-42 11-153 25-400 69-686
A 1320-8952 727-7821 67-1453 18-1100 21-98 34-322 62-509 95-1557
% 408-8610 32-7219 28-1343 5-600 5-70 20-220 22-386 26-1233
Androstenedione B 92-892 90-837 6-78 6-78 5-51 7-68 17-151 43-221
A 145-1248 183-1367 21-114 21-139 12-68 12-98 2-215 58-319
% 40-718 13-1084 9-76 10-75 5-60 5-60 8-121 9-118
11-Deoxycortisol B 110-1376 70-455 10-200 10-200 7-210 14-136 11-151 15-130
A 206-2504 81-645 101-392 80-390 98-360 95-322 87-283 78-250
% 15-1128 40-190 5-366 5-350 50-280 30-180 35-241 34-233
Cortisol B 1-11 3-34 3-22 3-23 5-25 5-23 4-15 4-16
A 6-52 16-76 27-50 32-60 22-40 17-40 15-45 16-35
% 4-41 6-44 19-41 17-40 5-25 5-20 5-32 7-26
Deoxycorticosterone B 20-105 28-78 7-48 7-57 4-49 5-34 2-12 4-30
A 44-320 28-95 40-158 20-157 26-143 19-138 13-63 12-74
% 17-215 1-67 13-144 26-110 23-135 16-130 10-53 7-43
Corticosterone B 235-1108 201-5030 78-2500 78-1750 120-2030 155-1365 111-598 115-1219
A 1667-8251 2240-11900 2225-4974 2225-6505 2150-7540 1775-7500 1723-5100 1472-5060
% 1338-8016 2039-10141 1149-4789 1140-5120 960-7300 1490-7300 1380-4700 1003-4740
18-OH corticosterone B 10-670 38-779 5-300 5-310 7-155 10-74 11-82 5-73
A 35-1500 152-2183 130-465 67-470 49-370 79-360 69-322 73-1472
% 16-830 114-2183 21-394 22-395 33-333 69-310 58-254 22-1467
Aldosterone B 5-635 12-736 2-71 2-130 2-37 3-21 2-32 1-14
A 13-1046 42-1365 5-166 5-167 13-85 14-50 10-34 10-33
% 8-517 28-629 3-123 4-122 7-54 4-40 0-22 7-25
Results in ng/dL except cortisol (Ng/dL); ACTH 1-24 (250 Ng) given as intravenous bolus; data from extraction, chromatography, RIA; References 21-23.
*Premature. Samples obtained on postnatal days 2-4.

Three different positive results may be reported: affected with 21-hydroxylase deficiency, the severity determined by
the mutations present.
1. Positive: 1 copy of CYP21A2 contains at least 1 common mutation,
3. Positive for the heterozygous presence of at least 2 common
while the other copy does not contain any of the 12 common
mutations (ie, mutations are present, but it cannot be determined if
mutations or 30-kb deletion. In most instances the individual is
they are on the same or separate chromosomes). If the mutations all
considered a carrier and may exhibit mild symptoms depending upon
reside on the same chromosome (cis configuration), the individual is
the mutation present. DNA testing of parents and affected siblings
a carrier; if they are on separate chromosomes (trans configuration),
may be necessary to eliminate the possibility of gene duplication (the
the individual will be affected with 21-hydroxylase deficiency. DNA
individual may have a functional gene on one chromosome, and a
testing of parents and affected siblings is frequently necessary to
functional as well as a mutated gene on the second chromosome).
determine if the mutations are in cis or trans configuration.
Unrecognized, the individual may be incorrectly classified as a carrier.
2. Positive: both copies of CYP21A2 contain common mutations (ie, 2
Mutations will be identified in approximately 90% of carriers; the other
distinct copies of CYP21A2 are identified and both contain 1 or more
10% may have a rare mutation not tested for in this assay. Furthermore,
of the 12 common mutations or 30-kb deletion). Individuals will be
74

Section 5 Genetics

Table 9. Normal Adrenal Enzyme Precursor/Product Ratio Ranges in Infants and Children. Values Before (B) and After (A) Rapid
ACTH Test*
Precursor 26-28 wk 34-36 wk 1-6 mo 6 mo – 1 y 1-5 y 6-12 y Pubertal
Ratios
Product B A B A B A B A B A B A B A
21-OH Deficiency
Progesterone 1.6-9.4 1.1-9.8 – – 0.3-7.0 0.9-4.0 0.3-7.0 0.9-4.0 0.5-10 0.6-6.0 0.9-8.4 0.9-3.7 1.3-14 1.2-6.6
Deoxycorticosterone
17-OH progesterone 0.4-2.4 0.3-2.1 0.9-4.8 0.8-4.2 0.4-3.1 0.5-2.0 0.4-3.1 0.5-2.0 0.3-2.1 0.5-1.6 0.2-2.1 0.5-1.6 0.2-3.7 0.4-2.7
11-Deoxycortisol
11C-OH Deficiency
11-Deoxycortisol 25-300 10-189 3-115 3-26 0.8-10 2.4-1.0 0.8-10 2.4-10 1.0-6.8 3.8-11 1.2-9.0 2.8-9.0 1.8-12 3.4-11
Cortisol
17B-OH Deficiency
Pregnenolone 0.3-0.7 0.3-5.0 0.2-0.7 0.2-1.3 0.17-0.7 0.03-0.3 0.1-2.9 0.1-0.5 0.3-3.6 0.2-1.5 0.2-2.8 0.2-0.9 0.1-1.7 0.1-1.0
17-OH pregnenolone
Progesterone 0.2-1.8 0.1-1.5 – – 0.2-2.1 0.4-1.1 0.2-5.2 0.4-1.1 0.2-3.5 0.5-1.5 0.2-2.6 0.2-0.9 0.2-2.2 0.2-1.5
17-OH progesterone
3C-HSD Deficiency
17-OH pregnenolone 1.1.5.2 3.6-11 1.8-6.5 3.6-12 2-22 3-20 2-22 2-20 0.3-3.0 0.5-3.3 0.5-6.0 0.3-5.3 0.4-3.4 0.5-6.3
17-OH progesterone
DHEA 1.0-8.4 3.4-15 1.6-5.0 2.2-7.8 2.2-6.5 2.8-13 0.8-6.5 1.5-13 0.6-9.0 0.7-7.5 2.0-4.5 1.1-5.8 1.5-4.0 1.8-4.9
Androstenedione
Aldosterone Synthase
Deficiency
18-OH corticosterone 1.0-4.5 0.8-2.6 1.1-10 1.2-11 1.3-5.0 2-13 1.3-5.0 2-13 1.2-6.0 1.9-15 2.6-7.1 5-12 2.0-5.7 3.4-13
Aldosterone
21-OH, 21-hydroxylase; 11C-OH, 11C-hydroxylase; 17B-OH, 17B-hydroxylase; 3C-HSD, 3C-hydroxysteroid dehydrogenase.
*Values in ng/dL except 11-deoxycortisol = ng/dL
ng/dL cortisol μg/dL.
Measurements by extraction, chromatography, and radioimmunoassay; data from references 20 and 22, and Quest Diagnostics Nichols Institute Clinical Correlations.
Premature data derived during first week; measurements by extraction, chromatography, and radioimmunoassay; data from references 22 and 24.

this test will detect both relevant mutations in approximately 81% of
affected individuals, only 1 relevant mutation in another 18%, and no
mutations in the final 1% of affected individuals. Thus, rare mutation
analysis may be needed to detect additional mutations in affected
individuals.
Ordering Information
Early morning samples are preferred for steroid assays. Specify age,
sex, and suspected clinical diagnosis on the test request form. Refer to
the Quest Diagnostics Directory of Services for specific test codes, CPT
codes, and specimen collection and handling requirements.

CAH (21-Hydroxylase Deficiency) Rare Mutations References

Negative results reduce, but do not eliminate, the possibility that an
1. Orth DN, Kovacs WJ. The adrenal cortex. In: Wilson, Foster,
unaffected individual is a carrier, because certain sequence alterations
Kronenberg, et al. eds. Williams Textbook of Endocrinology. 9th ed.
(eg, large deletions) may not be detected. The residual risk is influenced
Philadephia, PA: W.B. Saunders Company; 1998:598-607.
by the individual’s family history.
2. Wajnrajch MP, New MI. Defects of adrenal steroidogenesis. In:
DeGroot LJ, Jameson JL, eds. Endocrinology. 4th ed. Philadelphia,
Positive results include: 1) sequence alterations known to be
PA: W.B. Saunders Company; 2001:1721-1739.
pathogenic, 2) sequence alterations predicted to be pathogenic but not
3. Speiser PW, White PC. Congenital adrenal hyperplasia. N Engl J
reported in literature, 3) sequence alterations known to be benign, 4)
Med. 2003;349:776-788.
sequence alterations predicted to be benign, and 5) sequence
4. Speiser PW. Prenatal treatment of congenital adrenal hyperplasia.
alterations of unknown clinical significance. In the presence of positive
J Urol. 1999;162:534-536.
clinical findings, the detection of 2 mutations known or predicted to be
5. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-
pathogenic, or 1 in addition to a previously identified mutation, is
hydroxylase deficiency. Endocr Rev. 2000;21:245-291.
consistent with a diagnosis of 21-hydroxylase deficiency. The detection
6. White PC, Curnow KM, Pascoe L. Disorders of steroid 11C-
of 2 mutations known or predicted to be pathogenic in the absence of
hydroxlase isozymes. Endocr Rev. 1994;15:421-438.
clinical symptoms of CAH suggests cis configuration of the mutations.
7. Santos M, Kater CE, Auchus RJ, et al. Two prevalent CYP17
mutations and genotype-phenotype correlations in 24 Brazilian
Results must be interpreted in light of the individual’s clinical status and
patients with 17-hydroxylase deficiency. J Clin Endocrinol Metab.
family history including genotypes of parents and siblings.
2004;89:49-60.
 75

Section 5 Genetics

Table 10. CAH Panel 11 Diagnostic Ratio Reference Ranges in Normal Neonates and Patients with CAH

21-OH 3C-HSD 11C-OH 17B-OH

Age Normals Deficiency Deficiency Deficiency Deficiency
Steroid Ratio (Days) n = 59 n = 32 n = 2* n = 2† n = 1‡
21-OH Deficiency
15C,17B-(OH)2-pregnanolone x 100 1 3.8-17.4 47.4-400
Denominator 2-4 1.0-14.4 116-498
>16 1.0-2.6 170-1627 67.1; 150 33.6; 21.4 4.4
17B-OH-pregnanolone x 100 1 2.0-22.0 29.9-423
Denominator 2-4 1.0-11.0 103-974
>16 1.0-4.0 248-1470 74.8; 224 36.4; 22.0 8.0
Pregnanetriol x 100 1 2.0-30.0 36.6-648
Denominator 2-4 1.0-13.0 112-286
>16 b3.0 76.6-1344 22.6; 103 16.4; 5.8 4.1
Pregnanetriolone x 100 1 0.7-15.9 40.6-461
Denominator 2-4 b14.5 45.1-331
>16 b0.2 83.3-961 8.3; 4.6 6.4; 3.4 12.9
17B-OH Deficiency
5B-THA x 100 1 b8.0 b55.4
Denominator 2-4 b11.0 b7.8
>16 b34.3 b75.4 ND; 57.0 ND; ND 50.4
6B-OH-THA x 100 1 1.0-28.0 ND
Denominator 2-4 1.0-11.0 ND
>16 1.0-2.0 ND ND; ND NMC; NMC 29 983
16B-OH-pregnenolone 1 0.3-1.3 1.2-4.4
16B-Hydroxy-DHEA 2-4 0.1-2.2 1.9-11.4
>16 0.2-0.7 1.0-11.7 0.9; 0.4 9.5; 8.1 156
11C-OH Deficiency
THS x 100 1 2.9-25.0 13.6-34.0
Denominator 2-4 1.1-26.0 10.4-49.1
>16 0.5-1.4 1.5-136 15.6; 27.0 366; 97.2 14.2
6B-OH-THS x 100 1 b13.0 ND
Denominator 2-4 b11.0 ND
>16 ND ND ND; ND 190 482; ND
117 723
3C-HSD Deficiency
Pregnenetriol x 100 1 b24.4 b27.4
Denominator 2-4 b17.1 b36.6
>16 0.5-2.4 b287.9 356; 149 36.4; 1.2 ND
Pregnenetriol 1 0.1-10.8 b0.4
Pregnanetriolone 2-4 0.2-95.2 b0.4
>16 5.6-28.6 b1.5 42.8; 32.5 2.6; 1.7 ND
21-OH, 21-hydroxylase; 17B-OH, 17B-hydroxylase; 11C-OH, 11C-hydroxylase; 3C-HSD, 3C-hydroxysteroid dehydrogenase; Denominator = sum of tetrahydrocortisone, alpha, and beta
cortolone; THA = tetrahydro-compound A = tetrahydro-11-dehydrocorticosterone; ND = not detected, numerator level below detectable limit of assay; NMC = not measured due to
contamination of numerator component; THS = tetrahydro-substance S = tetrahydro-11-deoxycortisol.
*One patient; samples collected when 7 and 15 days of age.
†
Two patients, 14 and 49 days of age.
‡
One 7 day old patient.

8. Azziz R, Dewailly D, Owerbach D. Clinical review 56: nonclassic
adrenal hyperplasia: current concepts. J Clin Endocrinol Metab.
1994;78:810-815.
9. Peter M, Partsch CJ, Sippell WG. Multisteroid analysis in children
with terminal aldosterone biosynthesis defects. J Clin Endocrinol
Metab. 1995;80:1622-1627.
10. Chen X, Baker BY, Abduljabbar MA, et al. A genetic isolate of
congenital lipoid adrenal hyperplasia with atypical clinical findings.
J Clin Endocrinol Metab. 2005;90:835-840.
11. Arlt W, Walker EA, Draper N, et al. Congenital adrenal hyperplasia
caused by mutant P450 oxidoreductase and human androgen
synthesis: analytical study. Lancet. 2004;363:2128-2135.
12. Fukami M, Horikawa R, Nagai T, et al. Cytochrome P450
oxidoreductase gene mutations and Antley-Bixler syndrome with
abnormal genitalia and/or impaired steroidogenesis: molecular and
clinical studies in 10 patients. J Clin Endocrinol Metab.
2005;90:414-423.
13. Caulfield MP, Lynn TL, Gottschalk ME, et al. The diagnosis of
76

Section 5 Genetics

Table 11. CAH Panel 11: Reference Ranges of Steroid Metabolites Used to Calculate Ratios

Lowest Limit of
Quantitation 1 Day Old 2-4 Days Old >16 Days Old
Steroid Metabolite (μg/g Creat) (μg/g Creat) (μg/g Creat) (μg/g Creat)
17B-Hydroxypregnanolone 10 30-420 15-200 40-90
15C,17B-Dihydroxypregnanolone 20 85-490 25-270 45-80
16B-Hydroxy-dehydroepiandrosterone (DHEA) 100 5000-220,000 2000-180,000 200-16,650
Pregnanetriol 2 45-620 20-280 5-70
Tetrahydro-11-deoxycortisol 10 70-670 30-470 15-60
15C,17B-Dihydroxypregnenolone 100 4900-43,900 1800-59,000 630-3300
Pregnanetriolone 2 20-280 b260 b10
16B-Hydroxypregnenolone 50 4400-98,900 3460-150,840 75-10,910
Pregnenetriol 10 b360 b390 20-180
Tetrahydrocortisone 100 620-13,160 530-7430 1600-5570
6B-Hydroxytetrahydro-11-deoxycortisol 40 b120 b170 <40
Tetrahydro-11-dehydrocorticosterone 150 * * 320-1490
B-Cortolone 10 25-520 20-380 40-600
C-Cortolone 50 120-1360 150-1175 170-1350
6B-Hydroxytetrahydro-11-dehydrocorticosterone 10 30-540 20-250 10-100
Table includes only metabolites used to calculate diagnostic ratios (see Table 10)
*Prior to 16 days of age, measurement of this steroid is compromised by interference from other fetal steroids.

congenital adrenal hyperplasia in the newborn by gas
chromatography/mass spectrometry analysis of random urine
specimens. J Clin Endocrinol Metab. 2002;87:3682-3690.
14. Lutfallah C, Wang W, Mason JI, et al. Newly proposed hormonal
criteria via genotypic proof for type II 3C-hydroxysteroid
dehydrogenase deficiency. J Clin Endocrinol Metab. 2002;87:2611-
2622.
15. Mermejo LM, Elias LL, Marui S, et al. Refining hormonal diagnosis
of type II 3C-hydroxysteroid dehydrogenase deficiency in patients
with premature pubarche and hirsutism based on HSD3B2
genotyping. J Clin Endocrinol Metab. 2005;90:1287-1293.
16. Azziz R, Zacur HA. 21-Hydroxylase deficiency in female
hyperandrogenism: screening and diagnosis. J Clin Endocrinol
Metab. 1989;69:577-584.
17. Siegel SF, Finegold DN, Lanes R, et al. ACTH stimulation tests and
plasma dehydroepiandrosterone sulfate levels in women with
hirsutism. N Engl J Med. 1990;323:849-854.
18. Azziz R, Bradley E, Huth J, et al. Acute adrenocorticotropin-(1-24)
(ACTH) adrenal stimulation in eumonorrheic women: reproducibility
and effect of ACTH dose, subject weight, and sampling time. J Clin
Endocrinol Metab. 1990;70:1273-1279.
19. Bridges NA, Hindmarch PC, Pringle PJ, et al. Cortisol,
androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS)
and 17-hydroxyprogesterone (17OHP) responses to low doses of (1-
24) ACTH. J Clin Endocrinol Metab. 1998;83:3750-3753.
20. Lee MM, Rajagopalan L, Berg GJ, et al. Serum adrenal steroid
concentrations in premature infants. J Clin Endocrinol Metab.
1989;69:1133-1136.
21. Lashansky G, Saenger P, Fishman K, et al. Normative data for
adrenal steroidogenesis in a healthy pediatric population: age- and
sex-related changes after adrenocorticotropin stimulation. J Clin
Endocrinol Metab. 1991;73:674-686.
22. Hingre RV, Gross SJ, Hingre KS, et al. Adrenal steroidogenesis in
very low birth weight preterm infants. J Clin Endocrinol Metab.
1994;78:266-270.
23. Lashansky G, Saenger P, Dimartino-Nardi J, et al. Normative data
for the steroidogenic response of mineralocorticoids and their
precursors to adrenocorticotropin in a healthy pediatric population.
J Clin Endocrinol Metab. 1992;75:1491-1496.
24. Adrenal steroid response to ACTH. Pediatrics, Endocrine Sciences,
Calabasses Hills, CA. May 1991.
5.2.2.2 CAH Panel 11, Neonatal Random Urine
Clinical Use: This test is used to diagnose congenital adrenal
hyperplasia (mild as well as severe forms) in the newborn.
Clinical Background: Congenital adrenal hyperplasia (CAH) is an
autosomal recessive disorder characterized by deficiency of any one of 4
enzymes required for cortisol synthesis in the adrenal gland. Clinical
symptoms vary according to the particular enzyme deficiency and
include developmental abnormalities of the external genitalia and salt
wasting (sodium and potassium imbalance). Differential diagnosis and
treatment in the neonatal period is necessary to minimize morbidity and
mortality.
CAH diagnosis is based on clinical findings, biochemical and hormonal
test results, karyotyping, and sometimes mutation analysis.1 Although
single hormone levels may be useful, ACTH stimulation tests are
frequently required. In some cases, hormonal precursor-product ratios
are needed to establish the definitive diagnosis. The precursor (ie,
substrate of the deficient enzyme) is often elevated, and the product is
decreased, leading to an elevated ratio. The differential diagnosis is
made based on the pattern of precursor-product ratios.
 77

Section 5 Genetics

Current testing necessitates blood sample collections that are invasive
and difficult to obtain in newborn infants. This profile, however,
provides random urine levels for 34 different steroid metabolites as well
as 4 precursor-product ratios for 21-hydroxylase (21-OH) deficiency
diagnosis, 3 for 17-hydroxylase (17-OH) deficiency, 2 for 11C-hydroxylase
(11C-OH) deficiency, and 2 for 3C-hydroxysteroid dehydrogenase
(3C-HSD) deficiency.
Individuals Suitable for Testing include infants with genital
ambiguity, infants with hyponatremia and hyperkalemia, and infants
with a family history of CAH.
Method: This gas chromatography/mass spectrometry (GC/MS) method
includes solid-phase extraction of urinary steroids, enzyme hydrolysis,
chromatography, and GC/MS analysis. The report includes Ng/g
creatinine for each steroid, 11 ratios, and a patient-specific
interpretation. The analytical sensitivity for each analyte is shown in
Table 11.
Reference Range: Steroid pediatric reference ranges are shown in
Table 11. Pediatric reference ranges for the precursor-product ratios are
shown in Table 10.
Interpretive Information: Over 90% of CAH cases are attributed to
21-hydroxylase deficiency, which is associated with increased levels of
the following precursor-product ratios: (1) 15C,17B-dihydroxy pregnano-
lone x 100:denominator; (2) 17B-hydroxypregnanolone x
100:denominator; (3) pregnanetriol x 100:denominator; and (4)
pregnanetriolone x 100:denominator.2 The denominator used is the
summation of cortisol product metabolites including tetrahydrocortisone
(THE), B-cortolone, and C-cortolone.2 A fifth ratio, pregnenetriol x
100:pregnanetriolone, is used to differentiate 21-OH deficiency from the
rare 3C-HSD deficiency. These ratios, as well as those used for the
other forms of CAH, can be viewed in Table 10.
Figures 4-6 illustrate the observed differentiation between 21-OH
deficient individuals and normal individuals, and Figure 7 illustrates the
differentiation between 21-OH deficiency and 3CHSD deficiency.

 







Value
Value








Ratio

Ratio










A B C
A B C

Figure 4. 17B-Hydroxypregnanolone x 100/Denominator.

Figure 4. 17B-Hydroxypregnanolone x 100/Denominator. Figure 6. Pregnanetriolone x 100/Denominator.
A = 21-OH; B = 3CHSD; C = Normal samples.
A = 21-OH; B = 3CHSD; C = Normal samples. A = 21-OH; B = 3CHSD; C = Normal samples.

 







Value

Value








Ratio

Ratio








 

A B C A B C

Figure 5. Pregnanetriol x 100/Denominator. Figure 7. 15C,17B-(OH)2 pregnanolone x 100/Denominator.

A = 21-OH; B = 3CHSD; C = Normal samples. A = 21-OH; B = 3CHSD; C = Normal samples.
78

Section 5 Genetics

Results from each patient are reviewed and interpreted by a Scientific plasma homocysteine levels in some patients with mild or moderate
or Medical Director at Quest Diagnostics Nichols Institute. An Academic homocysteinemia, but this has not yet been proven to reduce the risk of
Associate is consulted on difficult cases. a first coronary event. However, B-vitamin supplementation may reduce
the risk of restenosis after coronary angioplasty,10 improve endothelial
References function in patients with coronary heart disease,11 and reduce the rate
of subclinical atherosclerosis.12 In individuals with elevated
1. Wajnrajch MP, New MI. Defects of adrenal steroidogenesis. In:
homocysteine levels, adequate folate intake should be ensured once
DeGroot LJ, Jameson JL, eds. Endocrinology. 4th ed. Philadelphia,
vitamin B12 deficiency is ruled out.13
PA: W.B. Saunders Company; 2001:1721-1739.
2. Caulfield MP, Lynn T, Gottschalk ME, et al. The diagnosis of
Individuals Suitable for Testing: It is appropriate to test samples
congenital adrenal hyperplasia in the newborn by gas
from individuals with clinical evidence of homocystinuria, elderly
chromatography/mass spectrometry analysis of random urine
individuals with clinical evidence of reduced cobalamin and folate
specimens. J Clin Endocrinol Metab. 2002;87:3682-3690.
intake, individuals with early (<50 years of age) evidence of CVD, and
otherwise low-risk individuals with a family history of premature CVD.
5.2.3 Homocysteine
Method: This competitive immunochemiluminometric assay (ICMA)
Clinical Use: This test is used to diagnose homocystinuria, vitamin B12
measures total homocysteine (ie, protein-bound, oxidized, and free,
deficiency, and folate deficiency; assess risk of cardiovascular disease
reduced homocysteine). The analytical sensitivity is 2.0 mmol/L, and
(CVD), stroke, and dementia (including Alzheimer disease); and monitor
there is no cross-reaction with carbamazepine, phenytoin, 6-azauridine,
therapy in patients with elevated homocysteine levels.
anthopterin, adenosine, L-cysteine, or glutathione. Cross-reaction is
0.6% with S-adenosyl-L-methionine and 6.1% with cystathionine.
Clinical Background: Severe hyperhomocysteinemia (>100 Nmol/L) is
generally caused by homocystinuria, an autosomal recessive inborn
Reference Range: See Table 12.
error of homocysteine metabolism (often involving cystathionine-C-
synthase deficiency). While symptoms are often not apparent at birth,
Interpretive Information: The following are some of the conditions
affected individuals may develop high myopia, ectopia lentis, marfanoid
associated with increased homocysteine levels: homocystinuria
habitus, mental retardation, and thromboembolism. Early diagnosis and
(cystathionine-C-synthase deficiency); vitamin B12 (MMA increased) and
homocysteine-lowering therapy are important to minimize the effects of
folate deficiency (MMA not increased); CVD; chronic renal disease
this metabolic disorder.
(typically 9-50 Nmol/L); increasing age; male sex; MTHFR mutations;
hypothyroidism; selected malignancies (eg, breast, ovarian, and
Mild or moderate homocysteine elevation can be caused by deficiencies
pancreatic cancer); diets rich in methionine (high meat intake); cigarette
of cobalamin (vitamin B12), folate, and vitamin B6 (essential cofactors in
smoking; and treatment with corticosteroids, methotrexate, nitrous
homocysteine metabolism); variations in the methylenetetrahydrofolate
oxide, cyclosporine, vitamin B6 antagonists (isoniazid, azauridine,
reductase (MTHFR) gene; and certain medications, among other factors.
penicillamine, procarbazine), and anticonvulsants (phenytoin,
Vitamin B12 deficiency can lead to irreversible neurologic damage, folate
carbamazepine).
deficiency during pregnancy may cause neural tube defects, and either
can cause megaloblastic anemia. Although folate and vitamin B12 can be
Homocysteine levels are low (typically <9 Nmol/L) in individuals who are
measured directly, homocysteine is a more accurate indicator of
pregnant (except in cases of fetal neural tube defect), <15 years of age,
deficiency at the tissue level.1 Used in combination with the
or taking oral contraceptives or hormone replacement therapy.
methylmalonic acid (MMA) assay, homocysteine measurement can
differentiate between folate and vitamin B12 deficiency (see below). This
Treatment with S-adenosylmethionine may cause falsely elevated
can be crucial because folate supplementation can mask signs of
homocysteine levels, and human anti-mouse antibodies (HAMA) may
vitamin B12 deficiency (such as anemia), allowing neurodegenerative
also interfere with measurement.
processes to continue.
References
Modest elevation in plasma homocysteine has also been reported as a
risk factor for atherosclerotic disease in coronary, cerebral, renal, and 1. Klee GG. Cobalamin and folate evaluation: measurement of
peripheral vessels,2,3 and for arterial and venous thrombosis.3 The methylmalonic acid and homocysteine vs vitamin B(12) and folate.
greater the homocysteine level, the greater the risk.2 There appears to Clin Chem. 2000;46:1277-1283.
be an additive effect with hyperlipidemia4 and a synergistic interaction 2. Boushey C, Beresford S, Omenn G, et al. A quantitative assessment
with hypertension and smoking,4 as well as factor V Leiden.5 In 2 recent of plasma homocysteine as a risk factor for vascular disease:
meta-analyses, the predictive value of homocysteine level for CVD risk probable benefits of increasing folic acid intakes. JAMA.
was lower than in earlier, individual reports.6,7 Homocysteine 1995;274:1049-1057.
concentrations predict the risk of mortality in patients with known 3. Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl
coronary artery disease; mortality ratios across quartiles of J Med. 1998;338:1042-1050.
homocysteine concentrations are 1.0 (<9.0 Nmol/L), 1.9 (9.0-14.9
Nmol/L), 2.8 (15.0-19.9 Nmol/L), and 4.5 (r20 Nmol/L).8 Furthermore,
homocysteine may be an independent predictor of stroke and dementia,
including Alzheimer disease.9 In the Framingham study, involving Table 12. Homocysteine Reference Ranges for Various
primarily people of European descent, a 5 Nmol/L increase in plasma Clinical Applications
homocysteine level was associated with a 40% increase in the 8-year
risk of Alzheimer disease.9 Cardiovascular Disease15 Congenital and Nutritional
Men: <11.4 μmol/L 5.4-11.9 μmol/L
Supplementation with folate, vitamin B12, and vitamin B6 can lower Women: <10.4 μmol/L

Section 5
4. Graham IM, Daly LE, Refsum HM, et al. Plasma homocysteine as a
risk factor for vascular disease: The European Concerted Action
Project. JAMA. 1997. 277:1775-1781.
5. Ridker PM, Hennekens CH, Selhub J, et al: Interrelation of
hyperhomocyst(e)inemia, factor V Leiden, and risk of future venous
thromboembolism. Circulation. 1997. 95:1777-1782.
6. The Homocysteine Studies Collaboration. Homocysteine and risk of
ischemic heart disease and stroke: a meta-analysis. JAMA.
2002;288:2015-2022.
7. Klerk M, Verhoef P, Clarke R, et al. MTHFR 677C—>T polymorphism
and risk of coronary heart disease: a meta-analysis. JAMA.
2002;288:2023-2031.
8. Nygard O, Nordrehaug JE, Refsum H, et al. Plasma homocysteine
levels and mortality in patients with coronary artery disease. N Engl
J Med. 1997;337:230-236.
9. Seshadri S, Beiser A, Selhub J, et al. Plasma homocysteine as a risk
factor for dementia and Alzheimer’s disease. N Engl J Med.
2002;346:476-483.
10. Schnyder G, Roffi M, Pin R, et al. Decreased rate of coronary
restenosis after lowering of plasma homocysteine levels. N Engl J
Med. 2001;345:1593-1600.
11. Chambers JC, Ueland PM, Obeid OA, et al. Improved vascular
endothelial function after oral B vitamins: an effect mediated
through reduced concentrations of free plasma homocysteine.
Circulation. 2000;102:2479-2483.
12. Vermeulen E, Stehouwer C, Twisk J, et al. Effect of homocysteine-
lowering treatment with folic acid plus vitamin B6 on progression of
subclinical atherosclerosis: a randomised, placebo-controlled trial.
Lancet. 2000;355:517-522.
13. Third report of the National Cholesterol Education Program (NCEP)
expert panel on detection, evaluation, and treatment of high blood
cholesterol in adults (Adult Treatment Panel III). Available at
http://www.nhlbi.nih.gov/guidelines/cholesterol/atp3_rpt.htm.
Accessed June 4, 2002.
14. Andersson A, Isaksson A, Hultberg B. Homocysteine export from
erythrocytes and its implication for plasma sampling. Clin Chem.
1992;38:1311-1315.
15. Selhub J, Jacques PF, Rosenberg IH, et al. Serum total
homocysteine concentrations in the third National Health and
Nutrition Examination Survey (1991-1994): population reference
ranges and contribution of vitamin status to high serum
concentrations. Ann Intern Med. 1999;131:331-339.
5.2.4 Methylmalonic Acid, Serum or Urine
Clinical Use: This test is used to diagnose and monitor vitamin B12
(cobalamin) deficiency and to diagnose and monitor patients with
methylmalonic acidemia.
Clinical Background: Methylmalonyl CoA is the catabolic product of
certain amino acids and fatty acids. Methylmalonyl CoA is metabolized
to succinic acid in the presence of the enzyme methylmalonyl-CoA
mutase and the cofactor vitamin B12 (cobalamin). It may also be
hydrolyzed to methylmalonic acid (MMA).
MMA increases when tissue levels of vitamin B12 are decreased, either
due to a defect in the cobalamin metabolic pathway or to deficient
vitamin B12 dietary intake. Elevated MMA is thus a functional indicator
of vitamin B12 deficiency, and is a better indicator of early vitamin B12
deficiency than plasma vitamin B12 levels. MMA is increased in
cobalamin-dependent neurologic disease, including those with normal
hematologic parameters and normal to low-normal B12 levels and those
with megaloblastic anemia. A large accumulation of MMA is also
observed in methylmalonic acidemia, an autosomal recessive inborn
79
Genetics
error of metabolism in which the methylmalonyl CoA mutase enzyme is
deficient. Homocysteine may likewise be elevated due to a defect in the
cobalamin metabolic pathway.
Method: The assays utilize either tandem mass spectrometry (serum) or
gas chromatography/mass spectrometry (urine). Results are reported in
nmol/L (serum) or mmol/mol creatinine (urine). Concentrations of MMA
in urine are higher than in serum.
Interpretive Information: Increased methylmalonic acid levels are
associated with vitamin B12 deficiency, selected neurologic diseases,
and methylmalonic acidemia. Levels are also increased in vegans. Urine
MMA levels increase after meals, but serum MMA is unaffected by
eating. Early vitamin B12 deficiency is best detected by serum MMA
measurement.
5.2.5 Organic Acids, Qualitative, Urine
Clinical Use: This test is used as a screen for organic acidurias.
Clinical Background: Organic acidurias are inherited disorders
resulting from a deficient enzyme or transport protein. Although most
are autosomal recessive disorders, several are X-linked. The more than
60 described organic acidurias affect many metabolic pathways
including amino acid metabolism, lipid metabolism, purine and
pyrimidine metabolism, the urea cycle, the Krebs cycle, and fatty acid
oxidation. These disorders are characterized by a wide variety of
symptoms such as lethargy, coma, hypotonia, seizures, ataxia, vomiting,
failure to thrive, developmental delay, liver disease, neutropenia,
thrombocytopenia, osteomalacia, and osteoporosis. Severity of
presentation is highly variable as is age of onset, and patients may not
present with the most characteristic features. Laboratory results
commonly indicate metabolic acidosis, increased anion gap,
hyperammonemia, hypoglycemia, lactic acidemia, ketosis, or abnormal
lipid patterns. Treatment may be based on dietary restrictions and/or
supplementation with cofactors (eg, riboflavin or cobalamin) or
conjugating agents (eg, carnitine or sodium benzoate); however, there is
no effective therapy for some of the disorders.
Individuals Suitable for Testing include infants, children, or adults.
Method: This gas chromatography/mass spectrometry method
qualitatively assesses 63 organic acids. The results are reported as
normal or increased; a patient specific interpretation is also included in
the report.
Interpretive Information: This test screens for organic acids that are
at least moderately increased. Positive results should be confirmed
using the quantitative urinary organic acid test (test code 38067X). This
screen may miss disorders characterized by minimal or intermittent
metabolite excretion, especially if the patient is asymptomatic at the
time of sample collection. Additionally, the following metabolites are
poorly extracted from urine and may result in a missed diagnosis: 2-
methylacetoacetic acid, 3-hydroxyisovaleric acid, 4-hydroxybutyric acid,
glutaconic acid, glyoxylic acid, mevalonic acid, pyruvic acid, and
suberylglycine.
Table 13 lists selected organic acidurias along with the organic acids
that may be elevated in each disorder. Only those organic acids included
in this test are listed.
80

Section 5 Genetics

Table 13. Selected Organic Acidurias and Associated Organic Acid Elevations

Organic Aciduria Elevated Organic Acid

Methylmalonic acidemia Methylmalonic acid, methylcitric acid, 3-hydroxypropionic acid, propionylglycine,
3-hydroxyvaleric acid
Fatty oxidation defects (medium chain acyl-CoA dehydrogenase Adipic acid, suberic acid, sebacic acid, suberylglycine, hexanoylglycine,
deficiency [MCAD]) phenylpropionylglycine
Propionic acidemia Propionylglycine, methylcitric acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid
Glutaric aciduria, type 1 Glutaric acid, glutaconic acid, 3-hydroxyglutaric acid
Multiple acyl-CoA dehydrogenase deficiency (glutaric aciduria, Glutaric acid, adipic acid, suberic acid, 2-hydroxyglutaric acid, ethylmalonic acid,
type II) isovalerylglycine
Isovaleric acidemia Isovalerylglycine, 3-hydroxyisovaleric acid
Multiple carboxylase deficiency 3-Methylcrotonylglycine, methylcitric acid, lactic acid, 3-hydroxyisovaleric acid,
tiglylglycine, 3-hydroxypropionic acid
Urea cycle defects Orotic acid
Maple syrup urine disease (MSUD) 2-Oxoisocaproic acid, 2-hydroxyisocaproic acid, 2-hydroxyisovaleric acid,
2-oxoisovaleric acid, 2-hydroxy-3-methylvaleric acid, 2-oxo-3-methylvaleric acid
Lactic acidosis Lactic acid, pyruvic acid, 2-hydroxybutyric acid, 4-hydroxyphenyllactic acid
Tyrosinemia 4-Hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpyruvic
acid, N-acetyltyrosine, succinylacetone (type I only)
Canavan disease N-acetylaspartic acid
Ketosis Acetoacetic acid, 3-hydroxybutyric acid, adipic acid, suberic acid, 3-hydroxy-
isobutyric acid, 3-hydroxyisovaleric acid, 3-hydroxy-2-methylbutyric acid
Phenylketonuria (PKU) Phenyllactic acid, phenylpyruvic acid, 2-hydroxyphenylacetic acid
3-Hydroxy-3-methylglutaric aciduria 3-Hydroxy-3-methylglutaric acid, 3-hydroxyisovaleric acid, 3-methylcrotonyl-
glycine, 3-methylglutaconic acid, 3-methylglutaric acid
3-Methylcrotonyl-CoA carboxylase deficiency 3-Hydroxyisovaleric acid, 3-methylcrotonylglycine
3-Methylglutaconic aciduria 3-Methylglutaconic acid, 3-hydroxyisovaleric acid, 3-methylglutaric acid
3-Oxothiolase deficiency 3-Hydroxy-2-methylbutyric acid, tiglylglycine (±), acetoacetic acid, 3-hydroxy-
butyric acid
Alkaptonuria Homogentisic acid
5-Oxoprolinuria 5-Oxoproline
Dihydrolipoyl dehydrogenase deficiency Lactic acid, 2-hydroxyisocaproic acid, 2-hydroxyisovaleric acid, 2-hydroxy-
(lipoamide dehydrogenase, E3) 3-methylvaleric acid, 2-oxoglutaric acid, 2-oxoisocaproic acid, 2-oxoisovaleric
acid, 2-oxo-3-methylvaleric acid

References amino acid metabolism, lipid metabolism, purine and pyrimidine

metabolism, the urea cycle, the Krebs cycle, and fatty acid oxidation.
1. Part 6, Organic Acids. In Scriver CR, Beaudet AL, Sly WS, et al, eds.
These disorders are characterized by a wide variety of symptoms such as
The Metabolic and Molecular Basis of Inherited Disease. 7th ed.
lethargy, coma, hypotonia, seizures, ataxia, vomiting, failure to thrive,
New York, NY: McGraw-Hill, Inc; 1995:1371-1652.
developmental delay, liver disease, neutropenia, thrombocytopenia,
2. Kushnir MM, Komaromy-Hiller G. Rapid screening for urine organic
osteomalacia and osteoporosis. Severity of presentation is highly
acids by GC-MS. Clin Chem. 1999;45:A21.
variable as is age of onset, and patients may not present with the most
characteristic features. Laboratory results may indicate metabolic
5.2.6 Organic Acids, Quantitative
acidosis, increased anion gap, hyperammonemia, hypoglycemia, lactic
acidemia, ketosis, or abnormal lipid patterns. Treatment may be based on
Clinical Use: This test is used to diagnose organic acidurias.
dietary restrictions and/or supplementation with cofactors (eg, riboflavin
or cobalamin) or conjugating agents (eg, carnitine or sodium benzoate);
Clinical Background: Organic acidurias are inherited disorders
however, there is no effective therapy for some of the disorders.
resulting from a deficient enzyme or transport protein. Although most are
autosomal recessive disorders, several are X-linked. The more than 60
Individuals Suitable for Testing include infants, children, or adults.
described organic acidurias affect many metabolic pathways including
 81

Section 5 Genetics

Method: This gas chromatography/mass spectrometry method References

quantitates 76 organic acids (full panel). Results are reported as
mmol/mol creatinine, and a patient-specific interpretation is provided.
Interpretive Information: Elevation of one or more organic acids may
be diagnostic for an organic aciduria. Elevations should be interpreted in
context with clinical findings, a comprehensive organic acid analysis,
and/or other test results.
1. Part 6, Organic Acids. In Scriver CR, Beaudet AL, Sly WS, et al, eds.
The Metabolic and Molecular Basis of Inherited Disease. 7th ed.
New York, NY: McGraw-Hill, Inc; 1995:1371-1652.
2. Sweetman L. Organic Acid Analysis. In Hommes FA, ed. Techniques
in Diagnostic Human Biochemical Genetics: A Laboratory Manual.
New York, NY: Wiley-Liss; 1991:143-176.

Since many organic acidurias are episodic, the diagnostic efficacy is
maximized when the patient is expressing symptoms at the time of
specimen collection.
Table 14 lists selected organic acidurias along with the organic acids
that may be elevated in each disorder. Only those organic acids included
in this test are listed.
5.2.7 Prenatal Screening and Diagnosis of Neural Tube
Defects, Down Syndrome, and Trisomy 18
Clinical Background: Prenatal screening and diagnosis are routinely
offered for neural tube defects (NTDs), Down syndrome, and trisomy 18
detection. The intent of such screening and diagnosis is to enable

Table 14. Selected Organic Acidurias and Associated Organic Acid Elevations (Full Panel)

Organic Aciduria Elevated Organic Acid

Methylmalonic acidemia Methylmalonic acid, methylcitric acid, 3-hydroxypropionic acid, propionylglycine,
3-hydroxyvaleric acid
Fatty oxidation defects (medium chain acyl-CoA dehydrogenase Adipic acid, suberic acid, sebacic acid, octanoic acid, suberylglycine,
deficiency [MCAD]) hexanoylglycine, octenedioic acid, phenylpropionylglycine, 5-hydroxyhexanoic
acid
Propionic acidemia Propionylglycine, methylcitric acid, 3-hydroxypropionic acid, 3-hydroxyvaleric acid
Glutaric aciduria, type 1 Glutaric acid, glutaconic acid, 3-hydroxyglutaric acid
Multiple acyl-CoA dehydrogenase deficiency Glutaric acid, adipic acid, suberic acid, 2-hydroxyglutaric acid, ethylmalonic acid,
(glutaric aciduria, type II) isovalerylglycine
Isovaleric acidemia Isovalerylglycine, 3-hydroxyisovaleric acid
Multiple carboxylase deficiency 3-Methylcrotonylglycine, methylcitric acid, lactic acid, 3-hydroxyisovaleric acid,
tiglylglycine, 3-hydroxypropionic acid
Urea cycle defects Orotic acid
Maple syrup urine disease (MSUD) 2-Oxoisocaproic acid, 2-hydroxyisocaproic acid, 2-hydroxyisovaleric acid,
2-oxoisovaleric acid, 2-hydroxy-3-methylvaleric acid, 2-oxo-3-methylvaleric acid
Lactic acidosis Lactic acid, pyruvic acid, 2-hydroxybutyric acid, 4-hydroxyphenyllactic acid
Tyrosinemia 4-Hydroxyphenyllactic acid, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpyruvic
acid, N-acetyltyrosine, succinylacetone (type I only)
Canavan disease N-acetylaspartic acid
Ketosis Acetoacetic acid, 3-hydroxybutyric acid, adipic acid, suberic acid, 3-hydroxy-
isobutyric acid, 3-hydroxyisovaleric acid, 3-hydroxy-2-methylbutyric acid
Phenylketonuria (PKU) Phenyllactic acid, phenylpyruvic acid, 2-hydroxyphenylacetic acid
2-Oxoadipic aciduria 2-Oxoadipic acid, 2-hydroxyadipic acid
3-Hydroxy-3-methylglutaric aciduria 3-Hydroxy-3-methylglutaric acid, 3-hydroxyisovaleric acid,
3-methylcrotonylglycine, 3-methylglutaconic acid, 3-methylglutaric acid
3-Methylcrotonyl-CoA carboxylase deficiency 3-Hydroxyisovaleric acid, 3-methylcrotonylglycine
3-Methylglutaconic aciduria 3-Methylglutaconic acid, 3-hydroxyisovaleric acid, 3-methylglutaric acid
3-Oxothiolase deficiency 3-Hydroxy-2-methylbutyric acid, tiglylglycine, 2-methylacetoacetic acid,
acetoacetic acid, 3-hydroxybutyric acid
5-Oxoprolinuria 5-Oxoproline
Dihydrolipoyl dehydrogenase deficiency Lactic acid, 2-hydroxyisocaproic acid, 2-hydroxyisovaleric acid, 2-hydroxy-
(lipoamide dehydrogenase, E3) 3-methylvaleric acid, 2-oxoglutaric acid, 2-oxoisocaproic acid, 2-oxoisovaleric
acid, 2-oxo-3-methylvaleric acid
82

Section 5 Genetics

pregnant women to make informed decisions regarding the pregnancy The MoM is calculated for each requested analyte using different
and be better prepared in the event of the birth of an affected infant. medians for white, African American, Hispanic, and Asian populations
(AFP, hCG, uE3, and DIA only). The MoM values for all analytes are
The Disorders adjusted for maternal weight. Adjustment for insulin-dependent
Neural tube defects (anencephaly, open spina bifida, and diabetes status is needed only for the AFP MoM. MoM values for the
encephalocele) are a heterogenous group of congenital malformations tested analytes are combined with the maternal age at the time of
resulting from a failure of fusion of the neural tube. Anencephaly is delivery to determine the risk for Down syndrome. Different
almost always fatal at or within a few hours of birth. The survival rate, mathematical algorithms are used to calculate risk for patients having
degree of handicap (surgically correctable to severely disabling), and ultrasound- vs LMP-based gestational age. The report includes the
intelligence of children with spina bifida or encephalocele vary with the concentration and adjusted MoM for each analyte tested, the Down
location and severity of the lesion and the treatment given. The syndrome risk based on maternal age alone as well as on the
estimated incidence of NTDs (~1 per 1300 live births) ranks second to combination of maternal age and test results, and an interpretation.
cardiac abnormalities as a cause of major congenital malformations in
the United States.1 Trisomy 18 screening is performed as described for Down syndrome
screening. In the first trimester, the risk calculation is based on maternal
Down syndrome (trisomy 21) is a common autosomal aneuploidy age, PAPP-A, and NT. In the second trimester, the risk calculation is
characterized by growth retardation, lack of muscle tone, and moderate to based on maternal age, AFP, hCG, and uE3. The report includes the
severe mental retardation. Fetal demise occurs in about 31% of cases (as concentration and adjusted MoM for each analyte tested, the NT MoM,
assessed from 10 weeks’ gestation to birth).2 The estimated incidence of the risk for trisomy 18, and an interpretation.
Down syndrome in the United States is 1 per 750 live births.3 The risk of
having an affected fetus increases with increasing maternal age. Patient reports are modified as needed following revision of clinical
information (eg, gestational age).
Trisomy 18 (Edwards’ syndrome) is a chromosomal aneuploidy
characterized by severe mental retardation, congenital heart disease, The Diagnostic Programs
renal malformations, lowset ears, and clenched fists. An unknown Amniotic fluid AFP, acetylcholinesterase, and fetal hemoglobin are
number of cases spontaneously abort during the first trimester, and offered for diagnosis of NTD. In the event of an elevated amniotic fluid
approximately 70% of cases spontaneously abort during the second and AFP, acetylcholinesterase and fetal hemoglobin assays are automatically
third trimesters. Thirty percent of those born with trisomy 18 die within performed (at additional charge). Chromosome analysis is offered for
the first month of life and 90% within the first year. The estimated
incidence is 1 per 6000 live births, and the incidence increases with
advanced maternal age. Table 15. Markers for Prenatal Screening and Diagnosis of
NTD and Fetal Aneuploidy
The Screening Programs
NTD screening is optimally performed between 16 and 18 weeks of Marker Method
gestation; it is not performed during the first trimester, as the neural tube
closes at about 10 weeks’ gestation. The concentration of maternal Screening*
serum alpha-fetoprotein (AFP) is determined, and the multiple of the Alpha-fetoprotein (AFP) ICMA
median (MoM) is calculated by dividing the patient’s AFP concentration Dimeric inhibin A (DIA) EIA
by the median AFP concentration for normal singleton pregnancy at the
appropriate day of gestation. Different medians are used for white, Human chorionic gonadotropin (hCG) ICMA specific for intact and
African American, Hispanic, and Asian populations. Adjustments are free beta-hCG
made to the AFP MoM for maternal weight and insulin-dependent Hyperglycosylated hCG (h-hCG) ICMA specific for hyper-
diabetes (type 1 diabetes). These adjustments are required because glycosylated hCG
blood volume varies with maternal weight, and women with type 1
diabetes have lower levels of AFP and a higher incidence of NTD relative Maternal age Age at expected time of
to women without type 1 diabetes. The report includes the AFP delivery (ie, term)
concentration and adjusted MoM, the risk for NTD, and an interpretation. Nuchal translucency (NT) Ultrasound measurement by a
certified ultrasonographer
Down syndrome screening can be performed in the first or second
trimester. In the first trimester, screening is based on maternal age at time Pregnancy-associated plasma EIA
of delivery, either human chorionic gonadotropin (hCG) or hyperglycosylated protein A (PAPP-A)
hCG (h-hCG, invasive trophoblast antigen [ITA]), pregnancy-associated Unconjugated estriol (uE3) ICMA
plasma protein A (PAPP-A), and an ultrasound measurement (nuchal †
translucency, NT). The MoMs are calculated for hCG or h-hCG, PAPP-A, and Diagnosis
NT, and the hCG or h-hCG and PAPP-A MoMs are then adjusted for Acetylcholinesterase Electrophoresis
maternal weight. The report includes the concentration and adjusted MoM Alpha-fetoprotein (AFP) MEIA
for each analyte tested; the NT MoM, which is calculated using the NT
measurement provided by the ordering physician; the Down syndrome risk Chromosome analysis Cell culture, microscopy, and
based on maternal age alone as well as on the combination of maternal karyotyping
age and test results; and an interpretation. Fetal hemoglobin (HbF) Radial immunodiffusion
ICMA, immunochemiluminometric assay; EIA, enzyme immunoassay; MEIA, microparticle
In the second trimester, the concentrations of AFP, hCG, and EIA.
unconjugated estriol (uE3) are determined along with h-hCG and/or *Serum samples are used for all markers except maternal age and NT.
dimeric inhibin A (DIA) levels when requested by the referring physician. †
Sample type: amniotic fluid or fetal cells from amniotic fluid.
 83

Section 5 Genetics

diagnosis of Down syndrome, trisomy 18, and other chromosome Table 17. Down Syndrome Screening Sensitivity and
abnormalities. Specificity in the First Trimester6
Individuals Suitable for Testing include pregnant women, preferably Detection Rate (%)*
between 11.0 and 13.9 weeks’ gestation (first trimester screen) or 16
and 18 weeks’ gestation (second trimester screen). For diagnosis, Age, PAPP-A, total hCG 64
individuals suitable for testing include pregnant women at increased Age, PAPP-A, free-C hCG 67
risk based on clinical history, screening results, or ultrasound findings
and pregnant women r35 years of age. Age, PAPP-A, h-hCG 67
Age, PAPP-A, total hCG, NT 84
Test Availability
Table 15 lists the markers and methods used for screening and Age, PAPP-A, free-C hCG, NT 84
diagnosis. The tests offered for screening and diagnosis at Quest Age, PAPP-A, h-hCG, NT 84
Diagnostics are listed in Table 16.
*At a 5% false-positive rate.
Test Selection
Table 16 lists the multiple marker combinations offered by Quest
Screening Tests
Diagnostics; these include the “triple,” “quad,” and “penta” screens.
Maternal serum AFP is the recommended screening test for open NTD;
First trimester screens that include maternal age, hCG or h-hCG, PAPP-
however, AFP alone is not recommended for Down syndrome screening.
A, and NT are also offered for Down syndrome and trisomy 18
The American College of Obstetricians and Gynecologists (ACOG)
screening. The “single” screen (AFP) and the “double” screen (age, AFP,
recommends multiple markers for detection of chromosome
hCG) are not offered for fetal aneuploidy screening owing to the
abnormalities.4,5 In addition to AFP, such markers include maternal age,
relatively low detection rates.
hCG, uE3, DIA, h-hCG, PAPP-A, and NT. Refer to Tables 17 and 18 for Down
syndrome detection and false-positive rates of various multiple marker
Diagnostic Tests
combinations. Multiple marker screening is currently not considered a
Amniotic fluid AFP is recommended for patients with an unexplained
substitute for amniocentesis and cytogenetic analysis in women 35 years
elevated AFP MoM and for women who have had children with NTDs. If
of age or older; however, the test may be offered to such women if genetic
the AFP is elevated, acetylcholinesterase and fetal hemoglobin assays
counseling is provided. Such counseling should include a thorough
are then performed (at an additional charge). Acetylcholinesterase
discussion of the relative sensitivities of screening and diagnostic tests.5
testing is preferably performed at or after 14 weeks’ gestation due to
the higher false-positive rate obtained prior to 14 weeks. Fetal
hemoglobin testing may be helpful when AFP is positive and
Table 16. Tests Offered for Screening and Diagnosis of NTD acetylcholinesterase is negative.
and Fetal Aneuploidy
Chromosome analysis is recommended for patients with an unexplained
Test Name Markers Included increase in Down syndrome or trisomy 18 risk. Chromosome analysis is
NTD screening also routinely performed on amniotic fluid cells from patients with an
Maternal Serum AFP* AFP elevated NTD risk, since chromosomal abnormalities may be detected in
these patients.
First trimester screening for Down
syndrome and trisomy 18 Figures 8 and 9 illustrate suggested algorithms for screening and
First Trimester Screen, h-hCG Maternal age, h-hCG, NT,† diagnosis.
PAPP-A
First Trimester Screen, hCG Maternal age, hCG, NT,†
PAPP-A
Second trimester screening for NTD, Table 18. Down Syndrome Screening Sensitivity and
Down syndrome, and trisomy 18 Specificity in the Second Trimester
Triple Screen Maternal age, AFP, hCG, uE3
Quad Screen Maternal Age, AFP, hCG, uE3, Detection Rate (%)*
DIA Palomaki, et al.† Wald, et al.‡
Penta Screen Maternal Age, AFP, hCG, uE3,
DIA, h-hCG Age, AFP NG 42
Diagnosis of NTD and fetal aneuploidy Age, AFP, hCG 67 66
Alpha-Fetoprotein, Amniotic Fluid AFP, with reflex to Age, AFP, hCG, uE3 72 74
with Reflex (x2)‡ acetylcholinesterase and
HbF when AFP is elevated Age, AFP, hCG, uE3, DIA 79 81
Chromosome Analysis & AFP Karyotype and AFP, with reflex Age, AFP, hCG, uE3, DIA, h-hCG 83 NG
w/Reflex to AchE & Fetal Hgb, to acetylcholinesterase and
Amniotic Fluid‡ HbF when AFP is elevated NG, not given.
*At a 5% false-positive rate.
Fetal Hemoglobin, Amniotic Fluid HbF †
Case control study based on 45 Down syndrome-affected and 238 unaffected
*This test is not needed when a second trimester screen is ordered. pregnancies.7
‡
†
Nuchal translucency measurement (mm), provided by the ordering physician. Prospective study based on 101 Down syndrome-affected and 47,053 unaffected
‡
There is an additional charge for the acetylcholinesterase and fetal hemoglobin tests. pregnancies (SURUSS).8
84

Section 5 Genetics

Patient Information & Informed Consent

Blood Test
(16-18 weeks’ gestation preferred;
14-22 weeks accepted)

Abnormal Screen (NTD) Negative Screen

Level II or III Ultrasound Routine Prenatal Care

& Genetic Counseling

Unexplained Explained
Corrected Gestational Age Negative Screen

AFP MoM >2.5 but <3.5

GA <19 weeks Neural Tube Defect Genetic Counseling

Repeat AFP Multiple Pregnancy High Risk/Special Prenatal Care

Abdominal Wall Defect Genetic Counseling

Negative Screen
Fetal Demise

Routine Prenatal Care Abnormal Screen

AFP MoM >2.5 but <3.5

and GA >19 weeks

Amniocentesis,
AFP MoM >3.5 Chromosome Analysis & AFP

Normal High Risk/Special Prenatal Care

Abnormal Chromosome Genetic Counseling

Elevated AFP

Acetylcholinesterase &
Fetal Hemoglobin

Abnormal Normal
(NTD, Fetal Blood, Other Fetal Anomalies)

High Risk/Special
High Resolution Ultrasound Prenatal Care

Genetic Counseling

Figure 8. Algorithm for maternal serum screening and diagnosis of neural tube defects.
 85

Section 5 Genetics

Patient Information & Informed Consent

Blood Test
(1st or 2nd trimester)*

Abnormal Screen (1st Trimester) Abnormal Screen (2nd Trimester) Negative Screen

Routine Prenatal Care

Genetic Counseling Level II or III Ultrasound
& Genetic Counseling

Chorionic Villus
Sampling or Amniocentisis† &
Chromosome Analysis Unexplained Explained

Corrected
Gestational Age Negative Screen
Amniocentesis †

Chromosome Analysis
Multiple Gestation Negative Screen

Abnormal Normal
Fetal Demise
Genetic Special
Counseling Prenatal Care Abnormal Normal

Genetic Special
Counseling Prenatal Care
*For 1st trimester testing, sample collection during 11 to 13 weeks’ gestation is preferred; sample collection during 9 to 13 weeks is accepted. For 2nd trimester, sample collection during 16
to 18 weeks’ gestation is preferred; sample collection during 14 to 21 weeks is accepted.
†
Transcervical or transabdominal chorionic villus sampling (CVS) is recommended for 9 1⁄2 to 12 weeks’ gestation; transabdominal CVS is recommended during 12 to 15 weeks’ gestation; and
amniocentesis is recommended from 15 weeks’ gestation to term.

Figure 9. Algorithm for maternal serum screening and diagnosis of Down syndrome.

Ordering Information
Screening Tests
Submit the patient’s date of birth (not chronological age), specimen
collection date, gestational age (see below), weight at time of sample
collection, race, insulin-dependent diabetes status prior to the
pregnancy, whether this is a repeat sample, number of fetuses, and
history of NTD. All information listed must be provided to obtain a
complete report.
For calculation of gestational age in the first trimester, provide an
ultrasound measurement of fetal crown-rump length in millimeters
(mm). LMP dating is not acceptable. For second trimester, gestational
age is expressed as the estimated date of delivery (EDD) and is most
accurately determined from ultrasound measurement of fetal biparietal
diameter. LMP dating may also be used. Gestational ages are expressed
in decimal weeks, which are based on the number of completed weeks
and the number of additional days.
The optimal specimen collection time for NTD screening is 16 to 18
weeks’ gestation. The NTD interpretation, which is based on AFP MoM,
is not provided for samples collected prior to 14.0 or after 22.9 weeks’
gestation (ie, 14 weeks, 0 days or 22 weeks, 6 days). The NTD risk,
which is usually provided along with the AFP MoM, is not provided for
samples collected prior to 15.0 weeks’ gestation. Down syndrome and
trisomy 18 risks are not provided in the second trimester screens for
86
Section 5
samples collected prior to 14.0 or after 22.9 weeks’ gestation. The
optimal time for first-trimester Down syndrome screening is 11.0 to 13.9
weeks’ gestation; NT will not be included in the risk calculation for
samples collected prior to 10.0 weeks’ gestation.
Diagnostic Tests
Submit the gestational age (specify method of determination; ie,
ultrasound or LMP date), specimen collection date, clinical indication,
serum AFP and MoM result (if available), and amniotic fluid AFP MoM if
already performed. Although rare, culture for cytogenetic analysis may
be unsuccessful (ie, absence of adequate metaphase cells) due to a low
volume of amniotic fluid, a bloody tap, advanced gestation (>24 weeks),
or fetal pathology (eg, fetal demise).
Inaccurate gestational age is one of the most frequent causes of
inaccurate screening and diagnostic test results. MoMs and screening
risks can be recalculated if subsequent ultrasound information suggests
more accurate gestational dating. For second trimester screening,
however, it is recommended that recalculation only be done if there is
more than a 7-day difference between the LMP and ultrasound dates.
Refer to the Quest Diagnostics Directory of Services for test codes,
specimen requirements, etc. Additional questions can be addressed to a
Quest Diagnostics Genetic Counselor by calling 866-GENE-INFO.
Test Interpretation
Screening
Maternal serum AFP elevation is associated with open NTDs, as fetal
AFP leaks from the open neural tube lesion into the amniotic fluid and
then diffuses passively into maternal blood. Elevated levels are also
associated with multiple gestation; placental anomalies; a variety of
fetal disorders including ventral abdominal wall defects, congenital
nephrosis, and oligohydramnios; fetal loss; and maternal disorders such
as liver cancer, hepatitis, and cirrhosis. Low levels of maternal serum
AFP have been associated with Down syndrome, fetal loss, hydatidiform
mole, and other fetal chromosomal abnormalities. Closed NTD-affected
pregnancies seldom have an abnormal AFP level.
Dimeric Inhibin A levels vary throughout the normal menstrual cycle;
the nadir occurs during early follicular phase and the peak in mid-luteal
phase. Levels are high during the first trimester of pregnancy, decrease
to lower levels during the second trimester, and increase again in the
third trimester. Abnormally elevated levels are associated with Down
syndrome, preeclampsia, and ovarian granulosa cell tumors. Abnormally
low levels are associated with polycystic ovarian syndrome and
postmenopausal status.
hCG elevation in maternal serum has been associated with Down
syndrome, hyperemesis of pregnancy, fetal demise, and Turner
syndrome with hydrops fetalis. Elevated levels are also observed in
association with choriocarcinoma, hydatidiform mole, and normal twin
pregnancy. Low levels of hCG have been associated with trisomy 18 and
fetal demise.
h-hCG is a hyperglycosylated form of hCG that is produced very early in
pregnancy, peaking at about 9 weeks’ gestation. Elevation in maternal
serum has been associated with Down syndrome: levels are more than
twice those of unaffected pregnancies in the first trimester and
approximately 3 times the levels in the second trimester.6,7 In normal
twin pregnancies, h-hCG levels are about 2 times the levels observed in
singleton pregnancies.9 Elevated levels are also associated with trisomy
22 (1 case)10 and invasive choriocarcinoma.11 Low levels of h-hCG have
Genetics
been associated with trisomy 18, trisomy 13 (1 case), triploidy, and
preeclampsia.10,12
PAPP-A levels are low in fetal aneuploidy (ie, trisomy 13, 18, and 21
[Down syndrome]) and fetal demise. Low levels may also be associated
with other adverse pregnancy outcomes such as intrauterine growth
restriction and proteinuric pregnancy-induced hypertension.13
uE3 levels are low in fetal anencephaly, Down syndrome, trisomy 18,
hydrops fetalis, Turner syndrome, Smith-Lemli-Opitz syndrome, steroid
sulfatase deficiency, and fetal demise.8 A maternal serum uE3 level is
the strongest individual predictor of risk for trisomy 18 and thus has the
greatest effect on trisomy 18 risk assessment.14
Since these tests are rarely used singly, it is important to interpret them
in context of one another (Tables 19 and 20). The following information
pertains to interpretation of various test groupings.
For NTD screening, AFP is considered elevated in a singleton pregnancy if
the MoM is 2.50 or more (1.90 or more for those with insulin-dependent
diabetes). For a twin pregnancy, AFP is considered elevated if the MoM is
4.0 or more. The singleton pregnancy detection rates are 88% for
anencephaly and 79% for open spina bifida (false-positive rate, 3%).25
In the event of an elevated maternal serum AFP screen (ie, an increased
risk for NTD), ultrasonography is recommended to confirm the
gestational age or detect the presence of twins, anencephaly, spina
bifida, encephalocele, or abdominal wall defect. When the gestational
age is <19 weeks and an increased AFP MoM of r 2.5 but <3.5 is still
unexplained, repeat blood sampling and testing are recommended to
confirm the elevation. Repeat blood sampling is not recommended when
1) the Down syndrome risk is elevated; 2) the AFP MoM is r2.5 and the
gestational age is advanced (r19 weeks); or 3) the AFP MoM is r3.5.
Follow-up for abnormal AFP results includes genetic counseling, level II
or III ultrasound examination, and consideration of amniocentesis for
chromosome and AFP analysis. Following these procedures, an
unexplained AFP elevation indicates an increased risk for obstetrical
complications, including rupture of membranes and premature labor,
intrauterine growth retardation, and stillbirth.
Normal levels do not ensure birth of a normal infant; AFP screening has
a false-negative rate of 12% for anencephaly and 21% for open spina
bifida. Closed NTD will not be detected in most cases. In addition, 2%
to 3% of newborns have some type of physical or mental defect, many
of which may be undetectable with current prenatal diagnostic
procedures.
For Down syndrome screening, test results are considered abnormal (ie,
at increased risk for Down syndrome) if the risk for Down syndrome is
Table 19. First-trimester Maternal Serum hCG, h-hCG,
PAPP-A, and NT Levels in Various Disorders6,8,10,15-18
hCG MoM h-hCG MoM PAPP-A MoM NT MoM
Down syndrome High High Low High
Trisomy 18 Low Low Low High
Trisomy 13 Unknowna Unknownb Low High
Fetal demise Unknown Unknown Low High
a
The median total hCG MoM was 0.379 in 42 cases of trisomy 13. Confirmatory studies
are not available.
b
The h-hCG level was low in 1 known case of trisomy 13.
 87

Section 5 Genetics

Table 20. Levels of Second-trimester Maternal Serum Markers in Various Disorders7,8,10,14,19-24

AFP MoM hCG MoM uE3 MoM Inhibin A MoM h-hCG MoM
Open spina bifida High Normal Normal Normal Unknown
Anencephaly High, very high Normal Very low Normal Unknown
Down syndrome Low High Low High High
Trisomy 18 Low Low Low Low Normal Low
Trisomy 18 & NTD High or low Low Low Unknown Unknown
Turner syndrome (45,X)
Without hydrops fetalis Low Low Very low Normal or low Unknown
With hydrops fetalis Low High Very low High Unknown
Fetal demise High or low High or low Very low High or low Unknown

r1:270 in the first trimester or the same as or greater than that of a 35- An amniotic fluid AFP MoM <2.0 is indicative of an unaffected fetus;
year-old woman (1:270) in the second trimester. The detection rate and however, a small percentage (<3%) of open NTDs may have levels in
false-positive rate will vary with the markers used (Tables 17 and 18). this range. A closed NTD (5% to 10% of all NTDs) will almost always
have levels in this range.
In the event first-trimester test results indicate an increased risk for
Down syndrome, genetic counseling and cytogenetic analysis of either a Chromosome analyses with 3 copies of either chromosome 21 or
chorionic villus sample (CVS) or amniotic fluid cells, depending on the chromosome 18 are indicative of Down syndrome or trisomy 18,
gestational age, are recommended. respectively. Other chromosomal aneuploidies are detected when
present. Results are >99% sensitive and specific for trisomy, assuming
In the event second-trimester test results indicate an increased risk for successful culture (metaphase cells obtained).
Down syndrome, obtaining a repeat blood specimen is contraindicated
unless the gestational age was earlier than 14 weeks at the time of the Fetal hemoglobin positive results indicate the presence of fetal blood in
first blood draw. Ultrasonography will provide a more accurate the amniotic fluid. This may be iatrogenic or caused by fetal anomalies
estimation of the gestational age and may resolve the increased risk. If such as NTD or ventral wall defects. High-resolution ultrasound is
not, genetic counseling and cytogenetic analysis of amniotic fluid cells recommended in such cases.
are recommended. Approximately 2% of pregnancies at increased risk
would be expected to have an affected fetus. For NTD diagnosis, interpretation varies for different combinations of
test results (Tables 21 and 22). In general, positive acetylcholinesterase
For trisomy 18 screening, test results are considered abnormal results confirm positive amniotic fluid AFP results, whereas negative
(increased risk for trisomy 18) if the risk for trisomy 18 is r1:100. In the acetylcholinesterase results indicate a high probability of a false-
first trimester, the detection rate is 75% at a 0.5% false-positive rate positive AFP. Both positive AFP and acetylcholinesterase may be caused
when based on maternal age, PAPP-A, and NT.15 In the second trimester, by fetal blood contamination. High-resolution ultrasound is
the detection rate is 60% with a 0.2% false-positive rate when based recommended as a follow-up to all positive AFP and
on maternal age, AFP, hCG, and uE3; 1 in 9 pregnancies at increased risk acetylcholinesterase results. Since NTD may be associated with
would be expected to have an affected fetus.14 In the event of increased chromosomal imbalances, chromosome analysis is also recommended.
risk for trisomy 18, genetic counseling and cytogenetic studies of CVS or
amniotic fluid cells are recommended; repeat screening is For Down syndrome and trisomy 18 diagnosis, a cytogenetic analysis
contraindicated. of amniotic fluid cells is sufficient for diagnosis. If results are positive
for these or any other chromosome abnormality, further genetic
Diagnostic Tests counseling is recommended.
Acetylcholinesterase results are positive in >99% of NTDs. Positive
results may also be associated with gastroschisis (96% sensitivity),26
other ventral wall defects (75% sensitivity), Turner syndrome (45,X),
teratoma, hypoplasia of heart and lung, fetal demise, fetal blood
contamination (16% false-positive rate), and unknown causes (2.5%
false-positive rate).27 False-positive results occur more frequently before Table 21. Diagnostic Sensitivity and Specificity for NTD29
14 weeks’ gestation.
DR (%) DR (%) DR (%) FPR
Amniotic fluid AFP results are considered elevated if the MoM is r2.0. (OSB) (Anencephaly) (CSB) (%)
Elevated levels are associated with open NTDs, abdominal wall defects,
Turner syndrome, esophageal and duodenal atresia, congenital Amniotic fluid AFP 90 98 22 0.46
hydronephrosis, and fetal blood contamination. In the UK Collaborative Acetylcholinesterase 99 98 26 0.34
Study, AFP was elevated in 99% of open NTD with 0.4% apparently
false-positive cases.28 Elevated levels are automatically reflexed to AFP & Acetylcholin- 96 97 26 0.14
acetylcholinesterase and fetal hemoglobin testing. Follow up with a esterase
high-resolution ultrasound (if not already performed) is recommended. DR, detection rate; FPR, false-positive rate; OSB, open spina bifida; CSB, closed spina
bifida.
88
Section 5
Table 22. Interpretation of NTD Diagnostic Test Results
Amniotic
Fluid AFP Acetylcholinesterase Interpretation
Elevated Negative Open NTD unlikely; explore
other causes
Elevated Weak positive Ventral wall defect likely; rule
out fetal blood contamination
Elevated Positive Open NTD or other defect very
likely
Elevated Inconclusive Pseudocholinesterase
deficiency or degraded
specimen; NTD not ruled out
References
1. Centers for Disease Control and Prevention (CDC). Spina bifida and
anencephaly before and after folic acid mandate—United States,
1995-1996 and 1999-2000. MMWR Morb Mortal Wkly Rep.
2004;53:362-365.
2. Gardner RJM, Sutherland, GR. In: Oxford Monographs on Medical
Genetics: Chromosome Abnormalities and Genetic Counseling. 3rd
ed. Oxford, England: Oxford University Press; 2004:363-372.
3. Miller OJ, Therman E. Human Chromosomes. 4th ed. New York:
Springer–Verlag; 2001:175-185.
4. ACOG Committee Opinion #296: first-trimester screening for fetal
aneuploidy. Obstet Gynecol. 2004;104:215-217.
5. American College of Obstetricians and Gynecologists Committee on
Practice Bulletins—Obstetrics. ACOG practice bulletin. Clinical
management guidelines for obstetrician-gynecologists. Prenatal
diagnosis of fetal chromosomal abnormalities. Obstet Gynecol.
2001;97(5 Pt 1):suppl 1-12.
6. Palomaki GE, Knight GJ, Neveux LM, et al. Maternal serum invasive
trophoblast antigen and first trimester Down syndrome screening.
Clin Chem. 2005;51:1499-1504.
7. Palomaki GE, Neveux LM, Knight GJ, et al. Maternal serum invasive
trophoblast antigen (hyperglycosylated hCG) as a screening marker
for Down syndrome during the second trimester. Clin Chem. 2004;
50:1804-1808.
8. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimester
antenatal screening for Down’s syndrome: the results of the Serum,
Urine and Ultrasound Screening Study (SURUSS). J Med Screen.
2003;10:56-104.
9. Lee JES, Petersen P, Carlton E, et al. Invasive trophoblast antigen
(ITA) levels in second trimester twin pregnancies [abstract]. In:
Abstracts of the American Society of Human Genetics 54th
Annual Meeting. October 27-29, 2004; Toronto, Canada. Abstract
2791.
10. Sancken U, Taube, D. Invasive trophoblast antigen (ITA) clinical
utility in first and second trimester prenatal screening for trisomy 21
and other chromosomal abnormalities: preliminary report of a
prospective study in Germany [abstract]. In: Abstracts of the
American Society of Human Genetics 54th Annual Meeting. October
27-29, 2004; Toronto, Canada. Abstract 2755.
11. Khanlian SA, Smith HO, Cole LA. Persistent low levels of human
chorionic gonadotropin: a premalignant gestational trophoblastic
disease. Am J Obstet Gynecol. 2003;188;1254-1259.
12. Bahado-Singh RO, Oz UA, Kingston JM, et al: The role of
hyperglycosylated hCG in trophoblast invasion and the prediction of
subsequent pre-eclampsia. Prenat Diagn. 2002;22:478-481.
13. Yaron Y, Heifetz S, Ochshorn Y, et al. Decreased first trimester PAPP-
Genetics
A is a predictor of adverse pregnancy outcome. Prenat Diagn.
2002;22:778-782.
14. Palomaki GE, Haddow JE, Knight GJ, et al. Risk-based prenatal
screening for trisomy 18 using alpha-fetoprotein, unconjugated
estriol and human chorionic gonadotropin. Prenat Diagn.
1995;15:713-723.
15. Tul N, Spencer K, Noble P, et al. Screening for trisomy 18 by fetal
nuchal translucency and maternal serum free C-hCG and PAPP-A at
10-14 weeks of gestation. Prenat Diagn. 1999;19:1035-1042.
16. Spencer K, Ong C, Skenton H, et al. Screening for trisomy 13 by
fetal nuchal translucency and maternal serum free C-hCG and PAPP-
A at 10-14 weeks of gestation. Prenat Diagn. 2000;20:411-416.
17. Goetzl L, Krantz D, Simpson JL, et al. Pregnancy-associated plasma
protein A, free beta-hCG, nuchal translucency, and risk of pregnancy
loss. Obstet Gynecol. 2004;104:30-36.
18. Spencer K, Heath V, Flack N, et al. First trimester maternal serum
AFP and total hCG in aneuploidies other than trisomy 21. Prenat
Diagn. 2000;20:635-639.
19. Milunsky A, Jick SS, Bruell CL, et al. Predictive values, relative
risks, and overall benefits of high and low maternal serum alpha-
fetoprotein screening in singleton pregnancies: new epidemiologic
data. Am J Obstet Gynecol. 1989;161:291-297.
20. Saller DN Jr, Canick JA, Schwartz S, et al. Multiple-marker
screening in pregnancies with hydropic and nonhydropic Turner
syndrome. Am J Obstet Gynecol. 1992;167(4Pt 1):1021-1024.
21. Lambert-Messerlian GM, Palomaki GE, Canick JA. Second trimester
levels of maternal serum inhibin A in pregnancies affected by fetal
neural tube defects. Prenat Diagn. 2000;20:680-682.
22. Cuckle HS, Sehmi IK, Jones RG. Inhibin A and non-Down syndrome
aneuploidy. Prenat Diagn. 1999;19:787-792.
23. Lambert-Messerlian GM, Saller DN Jr, Tumber MB, et al. Second-
trimester maternal serum inhibin A levels in fetal trisomy 18 and
Turner syndrome with and without hydrops. Prenat Diagn.
1998;18:1061-1067.
24. Wenstrom KD, Chu DC, Owen J, et al. Maternal serum B-fetoprotein
and dimeric inhibin A detect aneuploidies other than Down
syndrome. Am J Obstet Gynecol. 1998;179:966-970.
25. Wald NJ, Cuckle H, Brock JH, et al. Maternal serum alpha-
fetoprotein measurement in antenatal screening for anencephaly
and spina bifida in early pregnancy. Lancet. 1977;1(8026):1323-1332.
26. Goldfine C, Haddow JE, Knight GJ, et al. Amniotic fluid alpha-
fetoprotein and acetylcholinesterase measurements in pregnancies
associated with gastroschisis. Prenat Diagn. 1989;9:697-700.
27. Report of the Collaborative Acetylcholinesterase Study: Amniotic
fluid acetylcholinesterase electrophoresis as a secondary test in the
diagnosis of anencephaly and open spina bifida in early pregnancy.
Lancet. 1981;2(8242):321-324.
28. Second Report of the U.K. Collaborative Study on Alpha-fetoprotein
in Relation to Neural-tube Defects. Amniotic fluid alpha-fetoprotein
measurement in antenatal diagnosis of anencephaly and open spina
bifida in early pregnancy. Lancet. 1979;2(8144):651-652.
29. Second Report of the Collaborative Acetylcholinesterase Study:
Amniotic fluid acetylcholinesterase measurement in the prenatal
diagnosis of open neural tube defects. Prenat Diagn. 1989;9:813-
829.
5.2.7.1 First Trimester Screen, Hyperglycosylated hCG (h-hCG)
Clinical Use: This test is used for prenatal (first trimester) risk
assessment for Down syndrome and trisomy 18.
Clinical Background: Prenatal testing for Down syndrome and
trisomy 18 risk assessment is routinely offered to pregnant women in
the second trimester of pregnancy. First-trimester screening, however, is

Section 5
advantageous in that 1) positive results can lead to earlier diagnosis
and 2) when pregnancy termination is elected, there is more privacy and
reduced maternal morbidity. The first trimester screen detection rate, at
a constant false-positive rate, is higher than that of the triple screen
and the same as that of the quad screen performed in the second
trimester.1 The American College of Obstetricians and Gynecologists
(ACOG) now considers first-trimester screening a reasonable option.1
This first-trimester maternal serum screening test includes maternal
age, pregnancy-associated plasma protein-A (PAPP-A), hyperglycosyl-
ated hCG (h-hCG), and nuchal translucency (NT). PAPP-A is a placental
protein generally present in lower concentrations in Down syndrome-
affected pregnancies relative to unaffected pregnancies. h-hCG is a
hyperglycosylated form of human chorionic gonadotropin (hCG) that is
produced by cytotrophoblasts during embryonic implantation and
trophoblast invasion of the uterine wall. Levels tend to be increased in
Down syndrome-affected pregnancies. NT is an ultrasonographic
measurement of a fluid-filled space at the back of the fetal neck. NT
tends to be elevated in cases of fetal aneuploidy (eg, Down syndrome).
Palomaki and colleagues showed that this screening test is equivalent
to the PAPP-A, free-C hCG, and NT combination for Down syndrome
screening (see Table 23).2,3
Individuals Suitable for Testing include women in their first
trimester of pregnancy.
Method: The level of PAPP-A is measured using an enzyme
immunoassay (EIA), whereas h-hCG is measured using an
electrochemiluminescence assay. The multiple of the median (MoM) is
calculated for PAPP-A, h-hCG, and NT. PAPP-A and h-hCG MoM values
are adjusted for maternal weight. Down syndrome risk is based on all 3
MoM values, combined with the maternal age at time of delivery;
expected detection rate is 84% at a 5% false-positive rate.2 Trisomy 18
risk is based on maternal age, PAPP-A, and NT; expected detection rate
is 75% at a 0.5% false-positive rate.4 If the NT measurement is not
provided, the Down syndrome risk will be based on age, PAPP-A, and
h-hCG, and the trisomy 18 risk will be based on age and PAPP-A. Neural
tube defect (NTD) risk is not provided; for NTD risk, order a second-
trimester (16-18 weeks’ gestation) AFP test. Aliases include Maternal
Serum Screen, 1st Trimester and invasive trophoblast antigen (ITA,
h-hCG).
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
Interpretive Information: Women with a risk <1:270 (Down
syndrome) or <1:100 (trisomy 18) are considered at low risk of carrying
an affected fetus. Since this is a screening test, such risks cannot
guarantee the birth of an unaffected baby.
Table 23. Down Syndrome Detection Rates (DR)2
DR, %*
Age, PAPP-A, h-hCG, NT 84
Age, PAPP-A, free-C hCG, NT 84
Age, PAPP-A, h-hCG 67
Age, PAPP-A, free-C hCG 67
*At a 5% false-positive rate.
89
Genetics
Women with a risk r1:270 (Down syndrome) or r1:100 (trisomy 18) are
considered at increased risk of carrying an affected fetus. Genetic
counseling and possible diagnostic testing are recommended.
Inaccurate gestational age and NT measurements can substantially
impact risk assessment. Ultrasound confirmation of gestational age is
strongly suggested. Risks can be recalculated if necessary; call 1-800-
642-4657, ext. 4455.
References
1. ACOG Committee Opinion #296: first-trimester screening for fetal
aneuploidy. Obstet Gynecol. 2004;104:215-217.
2. Palomaki GE, Knight GJ, Neveux LM, et al. Maternal serum invasive
trophoblast antigen and first trimester Down syndrome screening.
Clin Chem. 2005;51:1499-1504.
3. Palomaki GE, Neveux LM, Haddow JE, et al. Hyperglycosolated-hCG
(h-hCG) and Down syndrome screening in the first and second
trimesters of pregnancy. Prenat Diagn. 2007;27:808-813.*
4. Tul N, Spencer K, Noble P, et al. Screening for trisomy 18 by fetal
nuchal translucency and maternal serum free C-hCG and PAPP-A at
10-14 weeks of gestation. Prenat Diagn. 1999;19:1035-1042.
Related References
5. Weinans MJN, Sancken U, Pandian R, et al. Invasive trophoblast
antigen (hyperglycosylated hCG) as a first-trimester serum marker
for Down’s syndrome. In: Weinans M, ed. Operationalization of First
Trimester Maternal Serum Screening for Down’s Syndrome.
Wageningen: Ponsen & Looyen BV; 2004:111-122.*
6. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimester
antenatal screening for Down’s syndrome: the results of the Serum,
Urine and Ultrasound Screening Study (SURUSS). J Med Screen.
2003;10:56-104.
7. Strom CM, Palomaki GE, Knight GJ, et al. Maternal urine invasive
trophoblastic antigen (ITA) is a useful marker for Down syndrome
detection in the 1st trimester [abstract]. Amer J Hum Genet.
2001;69:664.*
8. Weinans MJ, Butler SA, Mantingh A, Cole LA. Urinary
hyperglycosylated hCG in first trimester screening for chromosomal
abnormalities. Prenat Diagn. 2000;20:976-978.
9. Cuckle HS, Shahabi S, Sehmi IK, et al. Maternal urine
hyperglycosylated hCG in pregnancies with Down syndrome. Prenat
Diagn. 1999;19:918-920.
10. Cole LA, Shahabi S, Oz UA, et al. Urinary screening tests for fetal
Down syndrome: II. Hyperglycosylated hCG. Prenat Diagn.
1999;19:351-359.
11. Cole LA, Omrani A, Cermik D, et al. Hyperglycosylated hCG, a
potential alternative to hCG in Down syndrome screening. Prenat
Diagn. 1998;18:926-933.
*This study was funded in part by Quest Diagnostics; however, Quest Diagnostics did not
participate in the data analysis or influence the conclusions reached by the authors.
5.2.7.2 Quad Screen
Clinical Use: This test is used for prenatal risk assessment for neural
tube defects (NTDs), Down syndrome, and trisomy 18.
Clinical Background: Prenatal testing is routinely offered to pregnant
women for NTDs, Down syndrome, and trisomy 18 risk assessment.
Neural tube risk assessment is based on alpha-fetoprotein (AFP) alone,
whereas Down syndrome and trisomy 18 risk assessment are based on
multiple marker combinations such as the “triple screen” that includes
maternal age, AFP, human chorionic gonadotropin (hCG), and
unconjugated estriol (uE3).
90

Section 5 Genetics

Studies have demonstrated, however, that adding inhibin A to the triple
screen improves the detection rate by approximately 10%. During
pregnancy, inhibin is secreted from both the corpus luteum and the
placenta. In Down syndrome pregnancies, inhibin A levels are 2-fold
higher than in unaffected pregnancies, leading to detection of
approximately 40% of Down syndrome fetuses at a 5% false positive
rate. When combined with maternal age, AFP, hCG, and uE3, the
detection rate increases to approximately 75%.
Method: Concentrations of AFP, hCG, uE3, and inhibin A are
determined. The multiple of the median (MoM) is calculated for each.
Different medians are used for white, African American, Hispanic, and
Asian populations for AFP, hCG, uE3, and DIA. MoM values for all
analytes are adjusted for maternal weight; however, only the AFP MoM
is adjusted for insulin dependent diabetes status. All 4 MoM values are
combined with maternal age at time of delivery to determine the Down
syndrome risk. Trisomy 18 risk is based on maternal age and AFP, hCG,
and uE3 MoMs. NTD risk is based on the AFP MoM only.
Analytical methods used are as follows:
AFP Immunochemiluminescence assay
hCG Immunochemiluminescence assay
uE3 Immunochemiluminescence assay
Inhibin A Enzyme immunoassay
Interpretive Information: See Table 24.
5.2.7.3 Penta Screen
Clinical Use: This test is used for prenatal risk assessment for neural
tube defects (NTDs), Down syndrome, and trisomy 18.
Clinical Background: Prenatal screening is routinely offered for
neural tube defects (NTDs), Down syndrome, and trisomy 18 risk
assessment. NTD risk assessment is based on alpha-fetoprotein (AFP)
alone, whereas Down syndrome and trisomy 18 risk assessments are
based on multiple marker combinations that may include maternal age,
AFP, human chorionic gonadotropin (hCG), unconjugated estriol (uE3),
and dimeric inhibin A (DIA). In this screening test, we include an
additional marker: hyperglycosylated hCG (h-hCG).
Multiple studies have demonstrated the utility of h-hCG in Down
syndrome screening.1-12 h-hCG is a hyperglycosylated form of hCG that is
produced by cytotrophoblasts during embryonic implantation and
trophoblast invasion of the uterine wall. Levels tend to be increased in
Down syndrome-affected pregnancies. As shown in Table 25, the
addition of h-hCG improves screening sensitivity (ie, detection rate).
Based on previous experience,13 the improvement in sensitivity gained
from an additional marker should lead to a lower false-positive rate in
clinical practice. Thus, the number of amniocenteses required for follow-
up of “positive” test results may be reduced.
Table 24. Risk Cut-offs for NTD, Down Syndrome, and
Trisomy 18
Normal risk Increased risk
Open neural tube defect
Singleton pregnancy AFP MoM <2.5 AFP MoM r2.5
Twin pregnancy AFP MoM <4.0 AFP MoM r4.0
Insulin-dependent diabetic AFP MoM <1.9 AFP MoM r1.9
Down syndrome Risk <1:270 Risk r1:270
Trisomy 18 Risk <1:100 Risk r1:100
Individuals Suitable for Testing include women in their second
trimester of pregnancy (16 to 18 weeks’ gestation preferred; 14.0 to
22.9 weeks accepted, but risk of NTD not provided for samples collected
prior to 15.0 weeks).
Method: AFP, hCG, uE3, DIA, and h-hCG are measured using marker-
specific immunoassays. The multiple of the median (MoM) is calculated
for all markers. Race-specific medians are used for AFP, hCG, uE3, and
DIA. MoM adjustments are made for maternal weight (all markers) and
insulin dependent diabetes (AFP only). NTD risk is based on the
maternal age and AFP MoM; the singleton pregnancy detection rates
are 88% for anencephaly and 79% for open spina bifida (false-positive
rate, 3%).14 Down syndrome risk is based on MoM values of all 5
markers and maternal age at time of delivery. The singleton pregnancy
detection rate is 83% at a 5% false-positive rate.12 Trisomy 18 risk is
based on maternal age and AFP, hCG, and uE3 MoMs. The singleton
pregnancy detection rate is 60% at a 0.2% false-positive rate.15 Aliases
include Maternal Serum Screen 5 (MSS 5) and invasive trophoblast
antigen (ITA, h-hCG).
Reference Range: See Table 26.
Interpretive Information: Women with values above the cut-off listed
in Table 26 are considered at increased risk of carrying an affected
fetus. Inaccurate patient information can substantially affect risk
assessment. Risks can be recalculated using corrected patient
information; call 1-800-642-4657, ext. 4455.
Normal risk for NTD: Normal levels do not ensure birth of a normal
infant; AFP screening has a false-negative rate of 8% for anencephaly
and 38% for spina bifida.15 Closed NTD will not be detected in most
cases.
Increased risk for NTD: Ultrasonography is recommended to confirm
the gestational age or detect the presence of twins or anencephaly.
When the gestational age is <19 weeks and an increased AFP MoM of
>2.5 but <3.5 is still unexplained, repeat blood sampling and testing are
recommended to confirm the elevation. Repeat blood sampling is not
recommended when 1) the Down syndrome risk is elevated; 2) the AFP

Table 25. Sensitivity and Specificity of Prenatal Screening Tests for Down Syndrome12

Test Name Alias Markers Included DR, %*

Triple Screen Maternal Serum Screen 3 Age, AFP, hCG, uE3 72
Quad Screen Maternal Serum Screen 4 Age, AFP, hCG, uE3, DIA 79
Penta Screen Maternal Serum Screen 5 Age, AFP, hCG, uE3, DIA, h-hCG 83
*Detection rate at a 5% false-positive rate.

Section 5
Table 26. Cut-off Used to Define a Normal Screen
Disorder Normal Screen
Neural tube defect <2.50 AFP MoM (singleton pregnancy)
<1.90 AFP MoM (insulin-dependent diabetic)
<4.00 AFP MoM (twins)
<3.50 AFP MoM (insulin-dependent diabetic, twins)
<4.50 AFP MoM (triplets)
Down syndrome Risk <1:270
Trisomy 18 Risk <1:100
MoM is >2.5 and the gestational age is advanced (>19 weeks); or 3) the
AFP MoM is >3.5. Follow-up for abnormal AFP results includes genetic
counseling, level II or III ultrasound examination, and consideration of
amniocentesis for chromosome and AFP analysis. Following these
procedures, an unexplained maternal serum AFP elevation indicates an
increased risk for obstetrical complications, including rupture of
membranes and premature labor, intrauterine growth retardation, and
stillbirth.
Increased risk for Down syndrome: Obtaining a repeat blood
specimen is contraindicated unless the gestational age was <14 weeks
at the time of the first blood draw. Ultrasonography will provide a more
accurate estimation of the gestational age and may resolve the
increased risk. If not, genetic counseling and cytogenetic analysis of
amniotic fluid cells are recommended. Approximately 2% of pregnancies
at increased risk would be expected to have an affected fetus.
Increased risk for trisomy 18: One in 9 pregnancies at increased risk
would be expected to have an affected fetus.15 Genetic counseling and
cytogenetic studies of amniotic fluid cells are recommended.
References
1. Bahado-Singh R, Shahabi S, Karaca M, et al. The comprehensive
midtrimester test: high-sensitivity Down syndrome test. Am J
Obstet Gynecol. 2002;186:803-808.
2. Bahado-Singh RO, Oz U, Shahabi S, et al. Comparison of urinary
hyperglycosylated human chorionic gonadotropin concentration with
the serum triple screen for Down syndrome detection in high-risk
pregnancies. Am J Obstet Gynecol. 2000;183:1114-1118.
3. Bahado-Singh RO, Oz UA, Kovanci E, et al. A high sensitivity
alternative to “routine” genetic amniocentesis: Multiple urinary
analytes, nuchal thickness and age. Am J Obstet Gynecol.
1999;180:169-173.
4. Bahado-Singh RO, Oz UA, Shahabi S, et al. Urine hyperglycosylated
hCG plus ultrasound biometry for detection of Down syndrome in
the second trimester in a high-risk population. Obstet Gynecol.
2000;95:889-894.
5. Cole LA, Omrani A, Cermik D, et al. Hyperglycosylated hCG, a
potential alternative to hCG in Down syndrome screening. Prenat
Diagn. 1998;18:926-933.
6. Cole LA, Shahabi S, Oz UA, et al. Hyperglycosylated human
chorionic gonadotropin (invasive trophoblast antigen) immunoassay:
a new basis for gestational Down syndrome screening. Clin Chem.
1999;45:2109-2119.
7. Cole LA, Shahabi S, Oz UA, et al. Urinary screening tests for fetal
Down syndrome: II. Hyperglycosylated hCG. Prenat Diagn.
1999;19:351-359.
91
Genetics
8. Cuckle HS, Shahabi S, Sehmi IK, et al. Maternal urine
hyperglycosylated hCG in pregnancies with Down syndrome. Prenat
Diagn. 1999;19:918-920.
9. Palomaki GE, Knight GJ, Roberson MM, et al. Invasive trophoblast
antigen (hyperglycosylated human chorionic gonadotropin) in
second-trimester maternal urine as a marker for Down syndrome:
preliminary results of an observational study on fresh samples. Clin
Chem. 2004;50:182-189.*
10. Pandian R, Cole LA, Palomaki GE. Second-trimester maternal serum
invasive trophoblast antigen: a marker for Down syndrome
screening. Clin Chem. 2004;50:1433-1435.*
11. Wald NJ, Rodeck C, Hackshaw AK, et al. First and second trimester
antenatal screening for Down’s syndrome: the results of the Serum,
Urine and Ultrasound Screening Study (SURUSS). J Med Screen.
2003;10:56-104.
12. Palomaki GE, Neveux LM, Knight GJ, et al. Maternal serum invasive
trophoblast antigen (hyperglycosylated hCG) as a screening marker
for Down syndrome during the second trimester. Clin Chem. 2004;
50:1804-1808.*
13. Haddow JE, Palomaki GE, Knight GF, et al. Second trimester
screening for Down’s syndrome using maternal serum dimeric
inhibin A. J Med Screen. 1998;5:115-119.
14. Wald NJ, Cuckle H, Brock JH, et al. Maternal serum alpha-
fetoprotein measurement in antenatal screening for anencephaly
and spina bifida in early pregnancy. Lancet. 1977;1(8026):1323-1332.
15. Palomaki GE, Haddow JE, Knight GJ, et al. Risk-based prenatal
screening for trisomy 18 using alpha-fetoprotein, unconjugated
estriol and human chorionic gonadotropin. Prenat Diagn.
1995;15:713-723.
16. Norem CT, Schoen EJ, Walton DL, et al. Routine ultrasound
compared with maternal serum alpha-fetoprotein for neural tube
defect screening. Obstet Gynecol. 2005;106:747-752.
*This study was funded in part by Quest Diagnostics; however, Quest Diagnostics did not
participate in the data analysis or influence the conclusions reached by the authors.
5.2.8 Porphyrins, Delta Aminolevulinic Acid, and
Porphobilinogen
Deficiencies of enzymes in the heme synthetic pathway lead to 8
different genetic disorders called porphyrias. Five of the deficiencies are
autosomal dominant, 2 are autosomal recessive, and 1 is X-linked
recessive. Clinical symptoms include paroxysmal attacks of abdominal
pain, episodes of dementia or psychosis, and characteristic skin lesions.
See Table 27 for selected porphyrias matched with the associated
metabolite elevations and Table 28 for terminology and synthetic
pathways.
Porphyric attacks may be induced by exposure to a variety of drugs,
including barbiturates, chloral hydrate, diazepam, hydantoin,
griseofulvin, and others or by hormonal changes, such as during puberty,
menstruation, or pregnancy. Minor elevations of porphyrins (less than 2
to 3 times normal) may be due to an acute illness, liver disease, ethanol
intake, estrogens, iron overload, hemodialysis, and exposure to heavy
metals, pesticides, and herbicides. Care should be taken in specimen
collection and handling since exposure to light or increased temperature
leads to decreased levels of these metabolites.
Tests available to assist in diagnosis and management are listed in
Table 29.
92

Section 5 Genetics

Table 27. Selected Porphyria and Associated Metabolic Elevations

Elevated Metabolite*
Disorder Urine Feces Plasma
Acute intermittent porphyria Delta aminolevulinic acid
Porphobilinogen
ALA dehydratase deficiency porphyria Delta aminolevulinic acid
Congenital erythropoietic porphyria Uroporphyrin Coproporphyrin
Coproporphyrin
Delta aminolevulinic acid dehydratase Delta aminolevulinic acid
deficiency
Erythropoietic protoporphyria None Protoporphyrin Protoporphyrin
Hepatoerythropoietic porphyria Uroporphyrin Uroporphyrin
Heptacarboxyporphyrin Heptacarboxyporphyrin
Hereditary coproporphyria Coproporphyrin Coproporphyrin Coproporphyrin
Pentacarboxyporphyrin
Delta aminolevulinic acid
Porphobilinogen
Porphyria cutanea tarda Uroporphyrin Uroporphyrin Uroporphyrin
Heptacarboxyporphyrin Heptacarboxyporphyrin Heptacarboxyporphyrin
Variegate porphyria Coproporphyrin Coproporphyrin Coproporphyrin
Pentaporphyrin Protoporphyrin Protoporphyrin
Delta aminolevulinic acid
Porphobilinogen
*Patients with hereditary forms of porphyria usually present with profound elevations (>5-fold) during acute episodes. Moderate elevations (<3-fold) are often due to medications or
environmental factors.

5.3 Cytogenetics developmental delay, or abnormalities of sexual differentiation and

development. Neoplasia may result from acquired tissue-specific
5.3.1 Chromosome Analysis cytogenetic aberrations in otherwise normal individuals.

Clinical Use: This test is used to determine the genetic cause for
mental retardation, congenital anomalies, infertility, miscarriage,
stillbirth, and ambiguous genitalia; to confirm or exclude the diagnosis
of known chromosomal syndromes; and to diagnose and manage
neoplastic conditions such as hematologic malignancies and solid
tumors.
Clinical Background: Chromosome analysis is the microscopic
examination of chromosomes in dividing cells. Such analysis can detect
changes in chromosomal number and structure. Deletions (eg, partial
monosomy), duplications (eg, partial trisomy), and structural
abnormalities such as translocations, inversions, and rings can be
detected. These chromosomal changes may be associated with
infertility, miscarriage, stillbirth, birth defects, mental retardation,
Sensitivity of this conventional chromosome analysis method is best for
larger chromosomal aberrations. Methods that utilize molecular probes
(eg, fluorescence in situ hybridization [FISH]) may be required to detect
smaller, subtler, changes. The advantage of chromosome analysis,
however, is that specimens can be screened for multiple cytogenetic
abnormalities, whereas molecular methods require a suspicion or
knowledge of the specific abnormality at the time of testing so that the
appropriate probe(s) can be used.
Method: Cell culture is performed initially in order to stimulate mitosis.
The cells are then harvested and stained to produce G-bands.
Metaphase chromosomes are viewed microscopically at a resolution of
400-550 bands and aligned in a standard sequence based on size,
centromere location, and banding pattern. Special stains such as C-

Table 28. Report Name and Synthetic Pathway

Reporting Name Other name Comment

Uroporphyrin Octacarboxyporphyrin
Heptacarboxyporphyrin Intermediate in synthetic pathway of uroporphyrin to coproporphyrin
Hexacarboxyporphyrin Intermediate in synthetic pathway of uroporphyrin to coproporphyrin
Pentacarboxyporphyrin Intermediate in synthetic pathway of uroporphyrin to coproporphyrin
Coproporphyrin Tetracarboxyporphyrin

Section 5
Table 29. Porphyria-related Tests
Delta Aminolevulinic Acid, 24 Hour Urine
Delta Aminolevulinic Acid, Random Urine
Porphobilinogen, Quantitative, 24-Hour Urine
Porphobilinogen, Quantitative, Random Urine
Porphyrins, Fractionated, Plasma
Porphyrins, Fractionated, Quantitative, 24-Hour Urine
Porphyrins, Fractionated, Quantitative, Random Urine
Porphyrins, Fractionated, Quantitative & Porphobilinogen, 24-Hour Urine
Porphyrins, Total, Plasma
banding, Q-banding, and Ag-NOR (silver) staining are used as needed to
distinguish between normal variations and clinically significant
abnormalities. B- or T-cell mitogens are used in cultures when indicated
for diagnosing leukemia and lymphoma.
Interpretive Information: A patient-specific interpretive report is
provided. The report includes chromosomal findings, clinical
significance, and recommendations for additional studies (eg, FISH)
when indicated.
5.3.2 FISH, SKY®’ Marker Chromosome
Clinical Use: This test is used to further characterize a previously
detected chromosomal abnormality, assess patient prognosis, correlate
with similar, previously reported cases, and facilitate patient
management.
Clinical Background: Cytogenetic analysis is routinely used to detect
chromosomal abnormalities. Although adequate for the majority of
cases, conventional cytogenetic analysis is sometimes limited in its
ability to completely define the detected abnormality. For example,
chromosomal rearrangements such as translocations and markers, rings,
and derivative chromosomes may be of unidentified origin in congenital
anomalies and malignancies. Further characterization of these
unidentified chromosomal segments can facilitate prognosis, correlation
with similar, previously reported cases, and patient management
decisions.
Spectral karyotyping (SKY) provides a 24-color karyotype by displaying a
distinct color for each chromosome pair. This interferometer-based
optical method characterizes all 24 chromosomes (1-22, X and Y) with a
single hybridization. The distinct colors enable determination of the
origin of chromosomal segments in most cases.
Method: Metaphase chromosomal preparations are subjected to a
single hybridization with fluorochrome-labeled DNA libraries specific for
each of the 24 chromosomes. Results are visualized using a microscope
equipped with a SpectraCube, Sagnac interferometer, a CCD camera,
and specialized computer hardware and software for image production.
The SKY image is correlated with conventional chromosome banding
results and confirmed with chromosome-specific painting prior to
interpretation and reporting.
Note: Intra-chromosomal rearrangements (eg, inversions), some subtle
exchanges at the terminal regions of chromosome arms, deletions, and
duplications cannot be identified by this method.
93
Genetics
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A patient-specific interpretive report is
provided.
5.4 Molecular Genetics
5.4.1 Ashkenazi Jewish Panel
Clinical Use: This test is used to screen for carrier status or assess
risk of having a child with any of 8 genetic disorders commonly
occurring in the Ashkenazi Jewish (Eastern European descent)
population.
Clinical Background: This Ashkenazi Jewish Panel detects mutations
associated with 8 disorders that commonly occur in Ashkenazi Jewish
(Eastern European Jewish) individuals (see below). The panel simplifies
test ordering for Ashkenazi Jewish individuals who wish to know their
carrier status and/or their risk of having a child with any of these
disorders. It is most frequently used for Ashkenazi Jews and their
partners who are pregnant or contemplating pregnancy. Since all of
these disorders are autosomal recessive, both parents must be carriers
for the couple to have an affected child. If one partner is Ashkenazi
Jewish and the other is not, sequential screening, beginning with the
Ashkenazi Jew, is recommended.
The Disorders
Bloom syndrome: Children with Bloom syndrome are affected with
growth retardation, abnormalities in skin pigmentation,
immunodeficiency, a predisposition to cancer, and chromosomal
instability. Death usually occurs in the teens or twenties, most often
caused by cancer.
Canavan disease: Canavan disease (aspartoacylase deficiency) is a
progressive neurologic disease characterized by increased head
circumference, decreasing muscle tone and motor activity, progressive
loss of visual responsiveness, and mental retardation. Death usually
occurs by age 4.
Cystic fibrosis: Characteristic manifestations include recurrent lung
infections, malabsorption, malnutrition, and infertility (especially in
males). Median survival is 30 years.
Familial dysautonomia: Familial dysautonomia is characterized by
abnormal functioning of the sensory and autonomic nervous systems.
This causes decreased sensitivity to pain, abnormal regulation of body
temperature, paroxysmal hypertension, and gastrointestinal
abnormalities.
Fanconi anemia group C: Fanconi anemia is characterized by deficient
bone development and bone marrow function. This can lead to
pancytopenia, anemia, leukemia, and malformations of the limbs,
kidneys, and heart. The disorder may be mild or severe.
Gaucher disease: Gaucher disease is a lysosomal glycolipid storage
disorder caused by an enzymatic deficiency (acid beta galactosidase
deficiency). Individuals may have an enlarged liver and spleen,
thrombocytopenia, anemia, bone pain, bone lesions, and fractures. Life
expectancy depends on severity of the symptoms.
94

Section 5 Genetics

Niemann-Pick disease types A and B: This lysosomal storage disorder Table 30. Individual Tests Included in the Panel
is characterized by diminished acid sphingomyelinase activity. Type A is
usually fatal within 2 to 3 years. These children fail to thrive, have an Test Code Test Name
enlarged liver and spleen, and exhibit progressive mental and physical
degeneration. Individuals with type B also have hepatosplenomegaly 10224X Bloom Syndrome DNA Mutation Analysis
(along with cirrhosis, portal hypertension, ascites, and pancytopenia), 31650X Canavan Disease Mutation Analysis
but little to no neurologic involvement. They often survive into
adolescence and adulthood. 10458X Cystic Fibrosis Carrier Screen
16040X Familial Dysautonomia Mutation Analysis
Tay-Sachs disease: Tay-Sachs disease is a progressive,
neurodegenerative disorder caused by an enzymatic deficiency 10221X Fanconi Anemia DNA Mutation Analysis
(hexosaminidase A). The classic infantile form is characterized by 21503X Gaucher Disease DNA Mutation Analysis
developmental retardation followed by paralysis, dementia, seizures,
and blindness. Death usually occurs by age 4. 10222X Niemann-Pick Disease Mutation Analysis
21502X Tay-Sachs Disease Mutation Analysis
Methods: All tests, with the exception of CF carrier testing, are carried
out by multiplex-polymerase chain reaction (PCR) amplification, allele-
specific primer extension, and allelic discrimination using color-coded local Quest Diagnostics genetic counselor or call 1-866-GENE-INFO
microspheres. CF testing is carried out by PCR amplification with allelic (436-3463).
discrimination using an oligonucleotide ligation-based assay. The names
and codes of individual tests in this panel are provided in Table 30. The 5.4.2 CAH (21-Hydroxylase Deficiency) Common Mutations
number of mutations detected and the carrier detection rate for each See Genetics, “Congenital Adrenal Hyperplasia (CAH)” section 5.2.2.1.
disorder in the Ashkenazi Jewish Population are listed in Table 31. Table
32 lists the CF mutations detected in the CF Carrier Screen. 5.4.3 CAH (21-Hydroxylase Deficiency) Rare Mutations
See Genetics, “Congenital Adrenal Hyperplasia (CAH)” section 5.2.2.1.
All tests were developed and their performance characteristics determined by Quest
Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not 5.4.4 Central Diabetes Insipidus (CDI) Mutations
necessary. Performance characteristics refer to the analytical performance of the test. See The Quest Diagnostics Manual, Endocrinology Test Selection and
Niemann-Pick Disease and Canavan Disease testing are performed pursuant to a license
Interpretation at http://www.questdiagnostics.com/hcp/intguide/
agreement with Orchid Biosciences, Inc. EndoMetab/EndoManual.pdf.

Interpretive Information: The presence of a single mutation in an
asymptomatic individual identifies that person as a carrier. The absence
of a mutation(s) significantly reduces, but does not eliminate, the risk of
being a carrier. The residual risk of being a carrier (ie, of having a
mutation not screened for in this assay) is influenced by the individual’s
clinical and family history and ethnicity. Table 31 lists the residual risk
of being a carrier for each disorder following a negative test result in an
individual of full Ashkenazi-Jewish heritage.
Since genetic variation and other problems can affect the accuracy of
direct mutation testing, the results for all 8 tests should always be
interpreted in light of clinical and familial data. DNA-based testing is
highly accurate, but rare false negative/false positive results may occur.
For assistance with interpretation of these results, please contact your
5.4.5 Cystic Fibrosis (CF)
Table 33 provides a guide for selecting the appropriate test in various
clinical situations.
5.4.5.1 Cystic Fibrosis Screen
Clinical Use: This test is used to detect cystic fibrosis (CF) carriers,
determine a couple’s risk of having a child with CF, identify familial
mutations in affected individuals, and diagnose CF (pre- and
postnatally).
Clinical Background: CF is a common autosomal recessive disease
affecting primarily Caucasians of Northern European descent, with an
incidence of approximately 1 in 2500 births and a carrier rate of 1 in 25.

Table 31. Carrier Frequency, Detection Rate, and Residual Risk of Carrying a Mutation in the Ashkenazi Jewish Population

Disease Carrier Frequency Number of Mutations Detected Carrier Detection Rate, % Residual Risk
Bloom syndrome 1 in 100 1 97 1:3300
Canavan disease 1 in 40 4 99 1:3900
Cystic fibrosis 1 in 24 23 94 1:400
Familial dysautonomia 1 in 30 2 99.5 1:5800
Fanconi anemia group C 1 in 89 1 99 1:8800
Gaucher disease 1 in 13 6 96 1:301
Niemann-Pick disease 1 in 90 (type A) 4 95 1:1780
Unknown (type B)
Tay-Sachs disease 1 in 30 6 98 1:1450
 95

Section 5 Genetics

Table 32. CF Mutations and Polymorphisms Detected with psychosocial support; however, 90% of affected individuals die from
Carrier Screen lung damage. Median survival is approximately 30 years. Much milder
forms of the disease are well described with clinical onset being
Mutations Polymorphisms delayed until adult life.
621+1GmT 3120+1GmA G85E R347P 5T/7T/9T* CF has been attributed to mutations in the cystic fibrosis
711+1GmT 3659delC G542X R553X I506V transmembrane conductance regulator (CFTR) gene located on the long
arm of chromosome 7 (7q31.2). Over 1000 mutations have been
1717-1GmA 3849+10kbCmT G551D R560T I507V identified; however, the %F508 mutation accounts for about 70% of all
1898+1GmA A455E N1303K R1162X CFTR mutations in many, but not all, ethnic groups. Mutations result in a
defective CFTR protein that, in turn, results in defective cellular chloride
2184delA %I507 R117H W1282X transport.
2789+5GmA %F508 R334W
Individuals with CBAVD, chronic pancreatitis, or idiopathic pancreatitis
Additional mutations with clinical significance tested for in the CF Carrier Screen but not
included in the American College of Medical Genetics (ACMG) list of recommended
have a significantly increased risk of having 1 or more CF mutation(s).
mutations: 1078delT, 3876delA, 3905insT, 394delTT, A455E, R347H, S549R, V520F.
*May be of clinical significance in congenital bilateral absence of the vas deferens Individuals Suitable for Testing include those with a family history
(CBAVD) and other disorders. of CF or CFTR mutations, symptomatic children and adults, males with
CBAVD, patients with chronic or idiopathic pancreatitis, and
reproductively active individuals or couples.
The disorder may be characterized by chronic obstructive pulmonary
disease, pancreatic exocrine deficiency with malabsorption and Method: Selected regions of the CFTR gene are amplified, followed by
malnutrition, and congenital bilateral absence of the vas deferens detection of wild type and mutant sequences. This assay includes the
(CBAVD) leading to male infertility. Diagnosis of severe disease usually 23 most common CF mutations as recommended by the American
occurs within the first years of life and is based on chronic obstructive College of Medical Genetics, as well as 8 other mutations that occur at
lung disease, persistent pulmonary infection (primarily Pseudomonas low frequency (Table 32). The report specifies the mutations screened,
and Staphylococcus), meconium ileus, and pancreatic insufficiency with the mutations identified, and interpretive information.
failure to thrive. Diagnosis is confirmed by a positive sweat chloride test This test was developed and its performance characteristics determined by Quest
and/or detection of a CF-associated mutation on both chromosomes. Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Treatment is focused on improved airway clearance, antibiotic therapy Drug Administration. The FDA has determined that such clearance or approval is not
for infections, pancreatic enzyme replacement, proper nutrition, and necessary. Performance characteristics refer to the analytical performance of the test.

Table 33. Cystic Fibrosis Test Selection Guide*

Test Name Test Code Clinical Application

Cystic Fibrosis Screen 10458X General screen for carrier status and assessment of CF risk
CFTR Intron 8 Poly-T Analysis 15053X Differential diagnosis of CBAVD, idiopathic pancreatitis, bronchiectasis, etc. (used in
conjunction with the CF Carrier Screen)
Cystic Fibrosis DNA Analysis, Fetus 10226X Diagnose CF in a fetus
Cystic Fibrosis Complete (CF Complete™) Rare 10917X CF-affected individual, or an individual with atypical CF: identify rare mutation(s) when
Mutation Analysis, Entire Gene Sequence† only 1 or no mutations were detected in the general screening assay.
Individual with a family history of CF: identify a rare familial mutation when no
mutations were detected in the general screening assay.
Cystic Fibrosis CFTR Gene Deletion or 16080X Diagnose CF in symptomatic individuals when only 1 or no mutations were detected in
Duplication the general screening assay
Determine carrier status or diagnose CF when there is a known familial CF-causing gene
deletion or duplication
Cystic Fibrosis Rare Mutation Analysis, 10913X Screen for carrier status or diagnose CF when there is a known familial mutation that is
One Exon† not detectable in the general screening assay.
Cystic Fibrosis Rare Mutation Analysis, 10915X Screen for carrier status or diagnose CF when there are 2 known familial mutations that
Two Exon† are not detectable in the general screening assay.
Cystic Fibrosis D1152H Mutation Analysis 15335X Identify disease-causing mutation in individuals with CBAVD or mild CF symptoms and
no or only 1 other identified mutation
CBAVD, congenital bilateral absence of the vas deferens.
*These tests were developed and their performance characteristics determined by Quest Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the tests.
†
Contact your local Quest Diagnostics genetic counselor or call toll free 1-866-GENE-INFO (1-866-436-3463) before submission.
96
Section 5
Interpretive Information
The following information will help with interpretation of test results.
Additional assistance is available from our Genetic Counselors by
calling 1-866-GENE-INFO (1-866-436-3463).
Diagnosis
Detection of 2 mutant alleles in conjunction with positive clinical
findings or family history is consistent with CF. Failure to detect 1 or
more mutant alleles in a symptomatic patient, however, does not
exclude a diagnosis of CF. Approximately 18% of affected Caucasian
individuals have only 1 detectable mutation, and 1% have no detectable
mutations when using this screen. Sweat chloride testing should be
performed in all suspected CF cases.
Carrier Detection
The presence of a single CF mutation in an asymptomatic individual
identifies that person as a carrier. As shown in Table 34, absence of a
CF mutation significantly reduces, but does not eliminate, the risk of
being a carrier. The residual risk of being a carrier (ie, of having a CF
mutation not screened for in this assay) is influenced by the individual’s
clinical and family history and ethnicity. If clinically indicated, additional
testing is available.
IVS8 5T/7T/9T Polymorphism
• A single 5T variant with an R117H mutation on the same allele (in
cis) acts as a classic CF mutation. Thus, an individual with this
genotype is a CF carrier.
• A 7T or 9T variant with an R117H mutation in cis acts as a mild CF
mutation. Thus, an individual with this genotype is a CF carrier. When
coupled with a classic CF mutation, male patients may have CBAVD.
In summary, identification of an R117H mutation is followed by reflex
testing for the 5T/7T/9T polymorphism in intron 8. If a 5T variant is
identified, testing of family members is required to determine if the
variant is in cis or trans. Genetic counseling is recommended.
I506V and I507V variants
• I506V and I507V are not associated with CF or CBAVD.
• Individuals heterozygous for a %F507 or %F508 mutation who have an
I506V or I507V variant on the other chromosome appear to be
homozygotes on many CF detection assays (ie, carriers may be
misclassified as CF-affected individuals). Thus, when results indicate
a %F507 or %F508 homozygote, reflex testing for I506V and I507V is
performed to rule out a false-positive CF test.
Risk calculation for a CF-affected fetus
A couple’s risk of having a CF-affected fetus = [(mother’s carrier risk)
(father’s carrier risk)] w 4. This risk is the same for each pregnancy,
regardless of the outcomes of prior pregnancies.
Table 34. CF Carrier Risk Based on a Negative CF Mutation Analysis and Ethnicity*
Ethnicity Detection Rate, %
Ashkenazi Jewish 94
Non-Hispanic Caucasian 88
Hispanic American 72
African American 64
Asian American 49
Adapted from references 1, 2, and 4.
*Risks are based on the assumption that the relative’s specific CFTR mutation is unknown.
Genetics
References
1. American College Of Medical Genetics. Technical Standards and
Guidelines for CFTR Mutation Testing. 2006 Edition. Available at
http://www.acmg.net/Pages/ACMG_Activities/stds-
2002/cf.htm#Table1. Accessed October 23, 2006.
2. Committee on Genetics, American College of Obstetricians and
Gynecologists. ACOG Committee Opinion. Number 325, December
2005. Update on carrier screening for cystic fibrosis. Obstet
Gynecol. 2005;106:1465-1468.
3. Cystic Fibrosis; CF. In: Online Mendelian Inheritance in Man, OMIM‘.
John Hopkins University. Baltimore, MD: John Hopkins University.
MIM number 219700. Available at: http://www.ncbi.nlm.nih.gov/
omim/
4. Genetic testing for cystic fibrosis. NIH Consensus Statement
1997;15:1-37.
5. Grody WW, Cutting GR, Klinger KW, et al. Laboratory standards and
guidelines for population-based cystic fibrosis carrier screening.
Genet Med. 2001;3:149-154.
6. Kant JA, Mifflin TE, McGlennen R, et al. Molecular diagnosis of
cystic fibrosis. Clin Lab Med. 1995;15:877-898.
7. Richards CS, Bradley LA, Amos J, et al. Standards and guidelines
for CFTR mutation testing. Genet Med. 2002;4:379-391.
8. Stern RC. The diagnosis of cystic fibrosis. N Engl J Med.
1997;336:487-491.
9. Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet.
1992;8:392-398.
10. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population
carrier screening: 2004 revision of American College of Medical
Genetics mutation panel. Genet Med. 2004;6:387-391.
5.4.5.2 Cystic Fibrosis Complete (CF Complete“) Rare
Mutation Analysis, Entire Gene Sequence
Clinical Use: This test is used to identify rare familial mutations in
obligate CF carriers when they have tested negative in the routine
screening test. CF Complete should also be used to identify a rare
mutation in a CF-affected person when that person has tested negative
in the routine screen.
Clinical Background: Cystic fibrosis is an autosomal recessive
disease that affects approximately 1 in 2,500 Caucasians of Northern
European descent. It also occurs in other ethnic groups, but at a lower
incidence. To date, more than 1,300 disease-causing mutations have
been identified. Routine carrier screening detects 23 of these mutations,
identifying a maximum of 97% of carriers (Ashkenazi Jewish
population). In other populations, the detection rate can be substantially
lower (eg, only 57% in the Hispanic-American population). Use of the CF
Complete test, however, can dramatically increase the CF detection rate
because it detects >98% of all currently known mutations.
Prior Risk Risk After Negative CF Mutation Analysis
1/24 1/400
1/25 1/208
1/58 1/208
1/61 1/170
1/94 1/184

Section 5
Nevertheless, the CF Complete test should not replace the general
carrier screen test. Rather, the CF Complete test should be used to
identify rare familial mutations in obligate CF carriers when they have
tested negative in the routine screening test. CF Complete should also
be used to identify a rare mutation in a CF-affected person when that
person has tested negative in the routine screen.
Method: Genomic DNA is first isolated from the patient’s specimen.
Using this genomic DNA and polymerase chain reaction (PCR), the entire
coding region, as well as the splice junction sites, part of introns 11 and
19, and the promotor region of the CFTR gene, is amplified. The
resulting amplicons are then subjected to dye terminator cycle
sequencing reactions. The sequencing reaction products are analyzed on
an automated capillary DNA sequencer.
This test will theoretically identify >98% of disease-causing mutations.
It will not identify large deletions of the CFTR gene or potential intronic
mutations affecting mRNA splicing.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: CF mutation detection by sequencing will
detect more than 98% of known, as well as novel, CF mutations in all
different ethnic groups. However, large deletion or insertion mutations
and intronic mutations in regions not assayed will not be detected. An
individual positive for 1 copy of a known CF mutation is at least a CF
carrier. Individuals positive for 2 different CF mutations, or positive for 2
copies of the same CF mutation, will be reported as CF patients.
Individuals who test negative will still have a small risk of having
unidentified CF mutations.
5.4.5.3 Cystic Fibrosis CFTR Gene Deletion or Duplication
Clinical Use: This test is used to diagnose cystic fibrosis (CF),
diagnose atypical CF in individuals with congenital bilateral absence of
the vas deferens (CBAVD) or other conditions associated with the CF
transmembrane conductance regulator (CFTR) gene, and to assess the
risk of having a child with CF. It is used only when clinically indicated
after 1) standard mutation panel testing has demonstrated <2 CFTR
mutations; or 2) a large CFTR deletion or duplication has been detected
in a family member.
Clinical Background: CF is a common autosomal recessive disorder
characterized by chronic obstructive pulmonary disease, pancreatic
exocrine deficiency with malabsorption and malnutrition, and CBAVD
leading to male infertility. Atypical CF often affects only 1 organ system.
CF is caused by mutations in the CFTR gene, located on the long arm of
chromosome 7 (7q31.2). More than 1300 CFTR mutations have been
identified to date.1 Although most affect only 1 or a few nucleotides,
more than 25 large deletions or duplications have been described.
Because standard mutation panels and sequencing assays do not detect
such rearrangements, their actual frequency may be grossly
underestimated.
Routine carrier screening detects 23 of the most commonly identified
CFTR mutations.2 The sensitivity of the screening panel for identifying
mutant alleles is highest for Ashkenazi Jewish Caucasians (97%) and
can be much lower for other populations: 90% for non-Hispanic
Caucasians, 69% for African Americans, and 57% for Hispanic
Americans; the sensitivity among Asian Americans is unknown.3 Thus,
further testing is sometimes needed to identify both mutations in
97
Genetics
individuals with classic or atypical CF. Family members of CF patients
whose mutations are not known may also require additional testing.
Extensive sequence analysis (eg, CF Complete™ Rare Mutation
Analysis) can markedly increase the sensitivity of CFTR mutation
screening, but this approach will not identify large duplications or
deletions. The Cystic Fibrosis CFTR Gene Deletion or Duplication assay
detects deletions and duplications within the promotor region and all 27
exons of the CFTR gene. Recent studies suggest that such
rearrangements may account for 16% to 24% of mutant CFTR alleles
not identified after extensive sequencing.4-7 Because of the shorter
turnaround time of the CFTR deletion/duplication test, clinicians may
prefer to request it first and order extensive sequencing only if the
result is negative.
Individuals Suitable for Testing include individuals with symptoms
of classic or atypical CF who have <2 CFTR mutations detected with
standard mutation screening, those with a family history of a large
deletion or duplication in the CFTR gene, and family members of
individuals with CF whose mutations are unknown.
Method: This semi-quantitative fluorescent polymerase chain reaction
(SQF-PCR) method includes amplification of the CFTR promotor region
and all exons (1-27) in a single multiplex SQF-PCR, automated (capillary
electrophoresis) separation of fragments, and automated data analysis.
CFTR exon mutations are detected as an approximate 50% signal
decrease for deleted exon(s) and a 30% to 50% signal increase for
duplicated exon(s).
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The following information will help with
interpretation of test results. Additional assistance is available from our
Genetic Counselors by calling 1-866-GENE-INFO (1-866-436-3463).
Diagnosis:In the presence of positive clinical findings or family history,
detection of 2 known CFTR mutations—or 1 in addition to a previously
identified mutation—is consistent with a diagnosis of CF. The absence
of clinical correlation data for novel mutations precludes a firm
interpretation of their clinical effect. However, large deletions and
duplications would be expected to impair CFTR protein function and
thereby lead to disease in the presence of a second mutant allele.
Negative results do not rule out the presence of an undetected CFTR
mutation and therefore do not exclude a diagnosis of CF. This assay will
not detect translocations or mutations outside the regions tested and
may not detect small mutations such as single-nucleotide substitutions.
Testing with extensive CFTR sequencing may detect rare mutations not
identified by this assay.
Carrier Screening: The presence of a single known CF mutation in an
asymptomatic individual identifies that person as a carrier. The
relevance of a single novel mutation in this setting is not known.
However, as described above, large deletions or duplications would be
expected to negatively affect CFTR function and lead to disease in the
presence of a second mutant allele. Negative results do not eliminate
the risk of being a carrier. Testing with extensive CFTR sequencing may
detect rare mutations not identified by this assay.
References
1. Cystic Fibrosis Mutation Database. Cystic Fibrosis Consortium Web
site. Available at http://www.genet.sickkids.on.ca/cftr/. Accessed
May 12, 2005.
98
Section 5
2. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population
carrier screening: 2004 revision of American College of Medical
Genetics mutation panel. Genet Med. 2004;6:387-391.
3. Richards CS, Bradley LA, Amos J, et al. Standards and guidelines
for CFTR mutation testing. Genet Med. 2002:4:379-391.
4. Niel F, Martin J, Dastot-Le Moal F, et al. Rapid detection of CFTR
gene rearrangements impacts on genetic counselling in cystic
fibrosis. J Med Genet. 2004;41:e118.
5. Audrezet MP, Chen JM, Raguenes O, et al. Genomic rearrangements
in the CFTR gene: extensive allelic heterogeneity and diverse
mutational mechanisms. Hum Mutat. 2004;23:343-357.
6. Chevalier-Porst F, Souche G, Bozon D. Identification and
characterization of three large deletions and a
deletion/polymorphism in the CFTR gene. Hum Mutat. 2005;25:504.
7. Bombieri C, Bonizzato A, Castellani C, et al. Frequency of large CFTR
gene rearrangements in Italian CF patients. Eur J Hum Genet.
2005;13:687-689.
5.4.6 Factor V (Leiden) Mutation Analysis
See Coagulation, “Thrombophilia“ sections 3.5.1 and 3.5.4.
Genomic Alterations, Postnatal, ClariSure™ CGH
Clinical Use: This test is used to determine the genetic cause of
developmental delay (DD) or mental retardation (MR), confirm or exclude
the diagnosis of known chromosomal syndromes, and assist in clinical
management and genetic counseling.
Clinical Background: MR, defined as an intelligence quotient (IQ) of
<70, affects roughly 1% to 3% of the general population.1 The term
“developmental delay” (DD) is generally used for children younger than
5, as IQ is difficult to assess accurately before this age. Both
environmental and genetic factors—alone or in combination—may play
a causative role in DD/MR.
The initial investigation of unexplained DD/MR generally begins with
karyotype analysis, which detects large segmental aneusomies in about
3.7% of patients.2 This approach does not reliably detect smaller
duplications or deletions involving spans of less than 5 megabases
(Mb). Fluorescence in situ hybridization (FISH) with probes specific to
subtelomeric regions can detect chromosomal aberrations in another
4% of individuals with DD/MR3; additional specific probes are required
to detect aberrations outside of these regions (ie, microdeletion
syndromes). Thus, FISH is particularly useful when a known
microdeletion/duplication syndrome is suspected. A relatively new
approach to the detection of chromosomal abnormalities is microarray-
based comparative genomic hybridization (array-CGH), which can reveal
cytogenetic aberrations throughout the genome in a single assay.
The postnatal array-CGH assay uses bacterial artificial chromosomes
(BACs), containing known regions of the human genome, printed on
coated glass slides. This array includes nearly 1300 BACs per slide,
which equates to an average interval of 1 BAC every 3 Mb across the
genome and is comparable to classical G-banded chromosome analysis
with 650 bands/genome resolution. However, BAC coverage is enriched
at subtelomeric and pericentromeric regions and in close proximity to
regions associated with specific dosage abnormality syndromes.
Although the application of array-CGH to investigation of DD/MR is
relatively new, guidelines from the American College of Medical
Genetics include CGH as an option when no chromosomal or molecular
cytogenetic abnormalities are detected and the results of fragile X
testing are negative.1 Recent studies indicate that an array-CGH-based
assay targeting genomic regions associated with DD/MR can detect
Genetics
clinically relevant genomic alterations in roughly 5.6% to 11% of
patients with idiopathic developmental conditions.4,5
Individuals Suitable for Testing include those with idiopathic DD or
MR with or without dysmorphic features.
Method: Following extraction of DNA from whole blood, an array-CGH
is performed. This includes random priming amplification (performed in
duplicate) with differential labeling of patient and reference DNA,
hybridization of patient and reference DNA to a microarray containing
nearly 1300 individual BACS (each in triplicate), and comparison of
reference and patient DNA fluorescence signals to determine copy
number ratios. Duplications or deletions detected are verified with
specific FISH assays. Abnormal results are reported as ideograms of the
chromosome(s) involved, with a description of any genomic gains or
losses detected, along with a clinical interpretation. The analytical
sensitivity is >97%.
This test can identify large deletions and duplications associated with
the genetic disorders listed Table 35. The list is meant to designate the
regions targeted in the ClariSure array; however, it is not
comprehensive.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the test
Interpretive Information: Genomic gains and losses are reported
according to International System Cytogenetic Nomenclature (ISCN).6
The presence of specific chromosomal alterations previously associated
with DD/MR indicates that these rearrangements are the cause of
DD/MR. Parental testing and FISH verification of aberrations detected
with array-CGH should rule out false-positive results.
This assay does not detect genetically balanced translocations, low-
level mosaicism, or genomic alterations outside the regions represented
in the array. Thus, negative results do not rule out the possibility of
chromosomal abnormalities.
References
1. Shaffer LG; American College of Medical Genetics Professional
Practice and Guidelines Committee. American College of Medical
Genetics guideline on the cytogenetic evaluation of the individual
with developmental delay or mental retardation. Genet Med.
2005;7:650-654.
2. Shevell M, Ashwal S, Donley D, et al. Practice parameter:
evaluation of the child with global developmental delay: report of
the Quality Standards Subcommittee of the American Academy of
Neurology and the Practice Committee of the Child Neurology
Society. Neurology. 2003;60:367-380.
3. Anguiano A, Sulcova V, Morrone R, et al. Validation and
performance of computer-assisted fluorescence in situ hybridization
(FISH) analysis for subtelomeric abnormalities: experience with 2200
cases at a national reference laboratory [abstract]. 2006 Annual
Clinical Genetics Meeting; March 23–26, 2006; San Diego, CA.
Abstract 131.
4. Shaffer LG, Kashork CD, Saleki R, et al. Targeted genomic
microarray analysis for identification of chromosome abnormalities
in 1500 consecutive clinical cases. J Pediatr. 2006;149:98-102.
5. Poss AF, Goldenberg PC, Rehder CW, et al. Clinical experience with
array CGH: case presentations from nine months of practice. Am J
Med Genet A. 2006;140:2050-2056.
6. Shaffer LG, Tommerup N, eds. ISCN 2005: An International System
for Human Cytogenetic Nomenclature (Cytogenetic & Genome
Research S.). Basel: Karger, 2004.
 99

Section 5 Genetics

Table 35. Postnatal Genomic Alterations ClariSure Assay: Disorders and Associated Gene Targets

OMIM # Disorder Gene Target Gene Map Locus

607872 1p36 deletion syndrome Multiple 1p36
609425 3q29 microdeletion syndrome Multiple in 3q29, including PAK2 and DLG1 3q29
610443 17q21.31 microdeletion syndrome MAPT, CRHR1 17q21.31
608363 22q11.2 duplication syndrome Duplication of 3-Mb region in 22q11 22q11.2
610253 9q34.3 deletion syndrome EHMT1 9q34.3
300200 Adrenal hypoplasia, congenital NROB1 Xp21.3-p21.2
118450 Alagille syndrome JAG1 20p12
All unique subtelomeric regions Multiple 41 subtelomeres
All unique pericentromeric regions Multiple 43 pericentromeric regions
Aneuploidy Multiple All chromosomes
105830 Angelman syndrome UBE3A 15q11.2-q12
106210 Aniridia, type II PAX6 11p13
608636 Autism susceptibility Duplication of 15q11-q13 15q11
400003 Azoospermia factors (a, b, and c) DAZ 1-4 Yq11
400026
400027
109400 Basal cell nevus syndrome (Gorlin syndrome) PTCH1 9q22.3
130650 Beckwith-Wiedemann syndrome IGF2, CDKN1C 11p15.5
600430 Brachydactyly-mental retardation syndrome Deletion 2q37 2q37
113650 Branchiootorenal syndrome 1 EYA1 8q13.3
300300 Bruton agammaglobulinemia tyrosine kinase BTK Xq21.3-q22
114290 Campomelic dysplasia SOX9 17q24.3-q25.1
115470 Cat eye syndrome 22q11
118220 Charcot-Marie-Tooth disease, demyelinating, type 1A PMP22 17p11.2
214800 CHARGE CHD7 8q12.1
119600 Cleidocranial dysplasia RUNX2 6p21
122470 Cornelia de Lange syndrome NIPBL 5p13.1
123450 Cri-du-chat syndrome Multiple 5p15.2
220200 Dandy-Walker syndrome ZIC4, ZIC1 3q24
142340 Diaphragmatic hernia, congenital CHD2, NR2F2 15q26.1
188400 DiGeorge/Velocardiofacial syndrome 1 HIRA, TBX1 22q11.2
192430
601362 DiGeorge/Velocardiofacial syndrome 2 Unknown 10p14-p13
300018 Dosage-sensitive sex reversal NROB1 Xp21.3-p21.2
190685 Down syndrome critical region Multiple 21q22
175100 Familial adenomatous polyposis (FAP)/ APC 5q21-q22
Gardner syndrome
164280 Feingold syndrome MYCN 2p24.1
175700 Greig cephalopolysyndactyly syndrome GLI3 7p13
306955 Heterotaxy, visceral, X-linked ZIC3 Xq26.2
162500 Hereditary neuropathy with pressure palsies (HNPP) PMP22 17p11.2
236100 Holoprosencephaly 1 TMEM1 21q22.3
(continued)
100

Section 5 Genetics

Table 35. Postnatal Genomic Alterations ClariSure Assay: Disorders and Associated Gene Targets (Continued)

OMIM # Disorder Gene Target Gene Map Locus

157170 Holoprosencephaly 2 SIX3 2p21
142945 Holoprosencephaly 3 SHH 7q36
142946 Holoprosencephaly 4 TGIF 18p11.3
609637 Holoprosencephaly 5 ZIC2 13q32
307030 Hyperglycerolemia (glycerol kinase deficiency) GK Xp21.3-p21.2
300474
146255 Hypoparathyroidism, sensorineural deafness, GATA3 10p15
renal disease (HDR)
147791 Jacobsen Deletion 11q23; many genes 11q23
308700 Kallmann syndrome 1 KAL1 Xp22.3
Klinefelter (XXY male)
150230 Langer-Giedion syndrome TRPS1, EXT1 8q24.11-q24.13
(trichorhinophalangeal syndrome)
127300 Leri-Weill dyschondrosteosis SHOX Xpter-p22.32, Ypter-p11.2
300067 Lissencephaly, X-linked DCX Xq22.3-q23
300123 Mental retardation, X-linked, with isolated SOX3 Xq26.3
growth hormone deficiency
247200 Miller-Dieker lissencephaly syndrome LIS1 17p13.3
607432
161200 Nail-patella syndrome LMX1B 9q34.1
256100 Nephronophthisis 1 NPHP1 2q13
162200 Neurofibromatosis type I NF1 17q11.2
101000 Neurofibromatosis type II NF2 22q12.2
163950 Noonan syndrome 1 PTPN11 12q24.1
312080 Pelizaeus-Merzbacher disease PLP1 Xq22
601313 Polycystic kidney disease 1 PKD1 16p13.3-p13.12
610883 Potocki-Lupski syndrome RAI1 17p11.2
601224 Potocki-Shaffer syndrome EXT2, ALX4 11p11.2
176270 Prader-Willi SNRPN 15q11-q12
176270 Prader-Willi-like phenotype SIM1 6q16.3-q21
180200 Retinoblastoma RB1 13q14.1-q14.2
312750 Rett syndrome MECP2 Xq28
180500 Rieger syndrome, type 1 PITX2 4q25-q26
180849 Rubinstein-Taybi syndrome CREBBP 16p13.3
101400 Saethre-Chotzen syndrome TWIST1 7p21
182290 Smith-Magenis syndrome RAI1 17p11.2
117550 Sotos NSD1 5q35
183600 Split hand/foot malformation 1 SHFM1 7q21.2-q21.3
600095 Split hand/foot malformation 3 SHFM3 10q24
605289 Split hand/foot malformation 4 TP73L 3q27
606708 Split hand/foot malformation 5 DLX1, DLX2 2q31
308100 Steroid sulfatase deficiency (ichthyosis, X-linked) STS Xp22.32
(continued)
 101

Section 5 Genetics

Table 35. Postnatal Genomic Alterations ClariSure Assay: Disorders and Associated Gene Targets (Continued)

OMIM # Disorder Gene Target Gene Map Locus

186000 Synpolydactyly 1 HOXD gene cluster 2q31-q32
190350 Trichorhinophalangeal 1 TRPS1 8q24.12
605284 Tuberous sclerosis 1 TSC1 9q34
191092 Tuberous sclerosis 2 TSC2 16p13.3
194072 WAGR syndrome WT1 11p13
194050 Williams-Beuren syndrome ELN, LIMK1 7q11.23
194190 Wolf-Hirschhorn 165kb critical region contains FGFR3 4p16.3

5.4.8 Nephrogenic Diabetes Insipidus (Autosomal) Mutations disorders are frequently diagnosed in the 2nd or 3rd year of life.4 Affected
See The Quest Diagnostics Manual, Endocrinology Test Selection and females have a variable phenotype that can range from normal
Interpretation at http://www.questdiagnostics.com/hcp/intguide/ intelligence to severe mental retardation, with or without learning
EndoMetab/EndoManual.pdf. disabilities or personality disorders.

5.4.9 Nephrogenic Diabetes Insipidus (X-linked) Mutations
See The Quest Diagnostics Manual, Endocrinology Test Selection and
Interpretation at http://www.questdiagnostics.com/hcp/intguide/
EndoMetab/EndoManual.pdf
5.4.10 Prothrombin (Factor II) 20210GmA Mutation Analysis
See Coagulation, “Thrombophilia “sections 3.5.1, 3.5.7, 3.5.9, and
3.5.10.
5.4.11 XSense™, Fragile X with Reflex
In more than 99% of cases, FXS is caused by an expansion of a
polymorphic CGG trinucleotide repeat in the 5a untranslated region of
the FMR1 gene, located on the X chromosome, resulting in
hypermethylation of the FMR1 promoter.5 The extent of expansion and
hypermethylation correlates negatively with the amount of a protein
(absent in affected males and reduced in affected females) that plays a
role in brain synaptic development. The severity of the phenotype is
related to the extent of expansion (Table 36). Other rare mutations of
FMR1 associated with FXS include large deletions, point mutations, and
missense mutations.

Clinical Use: This test is used to detect fragile X syndrome (FXS)
carriers, determine an individual’s risk of having a child with FXS, and
diagnose FXS postnatally.
Clinical Background: FXS is the most common inherited cause of
developmental delay and mental retardation, occurring in approximately
1 in 4000 males and 1 in 6000 to 8000 females.1 The prevalence of
carriers in the Caucasian population is an estimated 1 per 259 females
and 1 per 813 males.2,3 Affected males usually have moderate to severe
mental retardation, pervasive speech delay, and behavioral problems
(eg, attention deficit hyperactivity disorder [ADHD]). Autism-spectrum
FMR1-related disorders are inherited in an X-linked dominant manner
with variable penetrance, and inheritance is affected by the number of
CGG repeats present (Table 37).7 Individuals with CGG repeats in the
intermediate and premutation range are carriers.
The molecular diagnosis of FXS is based on detecting the number of
CGG repeats and methylation status of the FMR1 gene. PCR can detect
and accurately measure repeat numbers in the normal and small
premutation ranges; Southern blot is required to detect larger CGG
repeats. Southern blot, however, is a time-consuming, laborious
process, which has limited the potential of carrier screening. Therefore,

Table 36. Number of CGG Repeats in FMR1 and Associated Phenotype

Approximate Number
of CGG Repeatsa Classification Gene Function Phenotype
5 to 44 Normal Normal Not affected
45 to 54 Intermediate Normal Not affected
(“gray zone”)
55 to 200 Premutation Larger premutations may have Males: ~38% incidence of FXTAS after
decreased gene expression age 50 years
Females: ~20% incidence of premature ovarian
failure
>200 Full mutation Loss of gene expression Fragile X syndrome
FXTAS, fragile X-associated tremor/ataxia syndrome (ie, progressive cerebellar ataxia and intention tremor).
a
Cut-offs are approximate and based on current research.6
102

Section 5 Genetics

Table 37. Inheritance Pattern of FMR1 CGG Repeat Mutations

Mutation in Parent Result in Offspring

Females with
Intermediatea (“gray zone”) Number of CGG repeats may increase to premutation size in offspring
Premutation Premutation may expand during meiosis in oocytes; thus, mother may give birth to a child with a full mutationb
Full Mutation Full mutation
c
Males with
Intermediatea (“gray zone”) Number of CGG repeats may increase to premutation size in daughters
Premutation Premutation passed to daughters
Full Mutation Full mutation shrinks to premutation size in daughters
Mutation in Parent Result in Offspring
a
Individuals with an intermediate mutation status are considered carriers due to the potential of offspring inheriting the premutation.
b
The greater number of repeats, the greater chance of expansion to a full mutation.
c
Sons are not affected because they only inherit the paternal Y chromosome. Males with full mutations are not likely to reproduce.

a new method called capillary Southern analysis (CSA) has been
developed. CSA decreases the number of Southern blot tests required
by ruling out false-negative PCR results. The combination of CSA with
PCR and Southern blot methods produces a high-throughput assay
capable of accurately determining the number of CGG repeats in FMR1
over the range of 5 to 2,000.8
Individuals Suitable for Testing include those with a family history
of FXS or undiagnosed mental retardation, including those seeking
reproductive counseling, as well as symptomatic children and adults.
Method: Polymerase chain reaction and gel electrophoresis is
performed to determine gender and the number of CGG repeats. CSA is
then performed as a reflex (with an additional charge) in females with
apparently homozygous CGG repeats. CSA includes restriction enzyme
digestion of genomic DNA, size-fractionation by capillary
electrophoresis, and PCR-based detection of fractions containing CGG
repeats. The next step is Southern blot confirmation of FMR1
expansions performed as reflex (at additional charge) if the patient is
1) male and has >44 CGG repeats or no FMR1 amplification product and
2) female and has a positive CSA result or >44 CGG repeats. The
number of CGG repeats and the methylation status are reported.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the test.
Reference Range: Negative (FMR1 containing 5 to 44 CGG repeats)
Interpetive Information: A negative result indicates 5 to 44 CGG
repeats, ie, a normal gene.
When >44 CGG repeats are identified, an individual’s mutation status
and phenotype are determined by the number of repeats present (see
Table 36). The associated risk of having a child with FXS is explained in
Table 37.
False-negative results may occur in individuals with cytogenetic
abnormalities involving the X chromosome (eg, males with Klinefelter
syndrome, females with XXX). This assay does not detect other
mutations (eg, deletions, point mutations, missense mutations) that
disrupt the function of the FMR1 gene and/or protein.
Results should be interpreted in conjunction with other laboratory and
clinical findings. Additional assistance in interpretation of results is
available from our Genetic Counselors by calling 1-866-GENE-INFO
(1-866-436-3463).
References
1. Pembrey ME, Barnicoat AJ, Carmichael B, et al. An assessment of
screening strategies for fragile X syndrome in the UK. Health Technol
Assess. 2001;5:1-95.
2. Rousseau F, Rouillard P, Morel ML, et al. Prevalence of carriers of
premutation-size alleles of the FMRI gene—and implications for the
population genetics of the fragile X syndrome. Am J Hum Genet.
1995;57:1006-1018.
3. Dombrowski C, Levesque S, Morel ML, et al. Premutation and
intermediate-size FMR1 alleles in 10572 males from the general
population: loss of an AGG interruption is a late event in the
generation of fragile X syndrome alleles. Hum Mol Genet.
2002;11:371-378.
4. Belmonte MK, Bourgeron T. Fragile X syndrome and autism at the
intersection of genetic and neural networks. Nat Neurosci.
2006;9:1221-1225.
5. Crawford DC, Acuna JM, Sherman SL. FMR1 and the fragile X
syndrome: human genome epidemiology review. Genet Med.
2001;5:359-371.
6. Technical standards and guidelines for fragile X testing: a revision to
the disease-specific supplements to the Standards and Guidelines for
Clinical Genetics Laboratories of the American College of Medical
Genetics. In: American College of Medical Genetics Standards and
Guidelines for Clinical Genetics Laboratories. 2006 ed. American
College of Medical Genetics Web site. http://www.acmg.net/Pages/
ACMG_Activities/stds-2002/fx.htm. Accessed: February 23, 2007.
7. Hagerman PJ, Hagerman RJ. The fragile-X premutation: a maturing
perspective. Am J Hum Genet. 2004;74:805-816.
8. Strom CM, Huang D, Li Y, et al. Development of a novel, accurate,
automated, rapid, high-throughput technique suitable for population-
based carrier screening for Fragile X syndrome. Genet Med.
2007;9:199-207.
5.5 Oncology-related Genetics
See Hematology/Oncology, section 6.
 103

Section 5 Genetics

5.6 Developmental Delay/Mental Canavan Disease Mutation Analysis*

Test Code: 31650X
Retardation Clinical Use: Diagnose Canavan disease; determine carrier status
Individuals Suitable for Testing: Those with seizures, spasticity,
Figure 10 provides a testing algorithm for the laboratory evaluation of
poor visual fixation, irritability, poor feeding, poor head control,
patients with developmental delay/mental retardation.
hypotonia, increased deep tendon reflexes, increased head
circumference, or a family history of Canavan disease
Individuals who present with DD/MR may benefit from the tests listed
below. Some patients not yet diagnosed with DD/MR may manifest
Carnitine, LC/MS/MS (Includes free and total carnitine and carnitine
other symptoms suggestive of a genetic or metabolic disorder. These
esters)
symptoms are presented herein to help identify patients who may
Test Code: 30299X
benefit from a specific test.
Clinical Use: Diagnose primary or secondary carnitine deficiency;
monitor patients with carnitine deficiency
Acylcarnitine, Plasma
Individuals Suitable for Testing: Those with tachycardia,
Test Code: 14531X
hepatomegaly, hypotonia, lethargy; those diagnosed with carnitine
Clinical Use: Diagnose organic aciduria or fatty acid disorder
deficiency
Individuals Suitable for Testing: Those with cardiac arrhythmia,
marked hypoglycemia, hepatomegaly, seizures, coma, muscle disease,
Carnitine, LC/MS/MS and Acylcarnitine
lethargy
Test Code: 15948X
Clinical Use: Diagnose organic aciduria, fatty acid disorder, primary or
Amino Acid Analysis for MSUD, LC/MS, Plasma
secondary carnitine deficiency
Test Code: 19779X
Individuals Suitable for Testing: Those with cardiac arrhythmia or
Clinical Use: Diagnose maple syrup urine disease (MSUD); monitor
tachycardia, hepatomegaly, seizures, hypotonia, coma, lethargy
branched chain amino acid levels in patients with MSUD
Individuals Suitable for Testing: Those with seizures, alternating
Chromosome Analysis, Blood
hypertonia/hypotonia, poor feeding, lethargy, failure to thrive; those
Test Code: 14596X
diagnosed with MSUD
Clinical Use: Determine genetic cause of DD/MR
Individuals Suitable for Testing: Those with dysmorphic features,
Amino Acid Analysis, LC/MS, CSF
birth defects, growth abnormalities, behavior problems
Test Code: 29881X
Clinical Use: Diagnose amino acid disorders in which CSF levels are
Chromosome Analysis, Follow-up
elevated relative to other sample types (eg, nonketotic hyperglycinemia)
Test Code: 10708X
Individuals Suitable for Testing: Those with convulsions, apnea,
Clinical Use: Diagnose a chromosomal abnormality
hypotonia, poor feeding, wandering eye movements, muscle spasms,
Individuals Suitable for Testing: Family members of individuals with
lethargy, hiccups
a known chromosomal abnormality
Amino Acid Analysis, LC/MS, Plasma (Includes 34 analytes)
Chromosome Analysis, High Resolution
Test Code: 767X
Test Code: 14595X
Clinical Use: Diagnose primary aminoacidopathies or screen for
Clinical Use: Diagnose DD/MR associated with small chromosomal
secondary aminoacidopathies; monitor response to therapy
deletions, duplications, or rearrangements
Individuals Suitable for Testing: Those with variable symptoms that
Individuals Suitable for Testing: Those with DD/MR being
may include coma, seizures, tachypnea, poor feeding, failure to thrive,
evaluated for subtle chromosomal abnormalities not identifiable by
vomiting, lethargy; those diagnosed with an aminoacidopathy
routine chromosome analysis
Amino Acid Analysis, Limited, LC/MS, Plasma (Includes 6 amino
FISH, Angelman*
acids associated with the most common aminoacidopathies)
Test Code: 14608X
Test Code: 1776X
Clinical Use: Diagnose Angelman syndrome
Clinical Use: Diagnose primary aminoacidopathies or screen for
Individuals Suitable for Testing: Those with seizures, wide mouth,
secondary aminoacidopathies; monitor response to therapy
protruding tongue, prominent jaw, thin upper lip, absent speech, ataxic
Individuals Suitable for Testing: Those with variable symptoms that
gate, paroxysmal laughter, light hair/skin pigmentation
may include coma, seizures, tachypnea, poor feeding, failure to thrive,
vomiting, lethargy; those diagnosed with an aminoacidopathy
FISH, Cri du chat*
Test Code: 14614X
Amino Acid Analysis, LC/MS, Urine (Includes creatinine)
Clinical Use: Diagnose Cri du chat syndrome
Test Code: 36183X
Individuals Suitable for Testing: Those with cat-like cry, severe
Clinical Use: Diagnose primary and secondary aminoacidopathies
psychomotor dysfunction, microencephaly, round face, hypertelorism,
Individuals Suitable for Testing: Those with variable symptoms that
small jaw, low-set ears, epicanthal folds, hypotonia
may include coma, seizures, tachypnea, poor feeding, failure to thrive,
vomiting, lethargy
FISH, DiGeorge, Velocardiofacial (VCFS)*
Test Code: 14610X
Plasma is preferred over urine except when abnormalities are more
Clinical Use: Diagnose DiGeorge/velocardiofacial syndrome
likely to manifest in urine (eg, arginosuccinic acid lyase deficiency,
Individuals Suitable for Testing: Those with cardiac malformations,
disorders of transsulfuration/amino acid transport)
seizures, hypocalcemia, high susceptibility to infections, hypertelorism,
cleft palate, bifid uvula, small jaw, low-set ears, speech delay, scoliosis,
hernia, schizophrenia/mental health disorders
104

Section 5 Genetics

Section 5
FISH, Miller-Dieker*
Test Code: 14612X
Clinical Use: Diagnose Miller-Dieker syndrome
Individuals Suitable for Testing: Those with central nervous system
disorders, microencephaly/lissencephaly, prominent forehead,
bitemporal hollowing, small nose with upturned nares, protuberant
upper lip, thin vermilion border of upper lip
FISH, Smith-Magenis*
Test Code: 14611X
Clinical Use: Diagnose Smith-Magenis syndrome
Individuals Suitable for Testing: Those with heart defect,
brachycephaly, hearing loss, speech delay with hoarse/deep voice, sleep
disturbance, hyperactivity, self-destructive behavior, broad face/nasal
bridge, Down syndrome-like facies
FISH, Microdeletion Syndromes Panel*
Test Code: 37559X
Clinical Use: Diagnose Angelman, DiGeorge, Kallmann, Prader-Willi,
Smith-Magenis, and Williams syndromes
Individuals Suitable for Testing: Those with symptoms associated
with microdeletion syndromes
FISH, Neonatal Screen
Test Code: 36053X
Clinical Use: Determine genetic cause of DD/MR (includes +13, +18,
+21, X, Y)
Individuals Suitable for Testing: Those with dysmorphic features,
birth defects, growth abnormalities, behavior problems
FISH, Subtelomere Screen*
Test Code: 10468X
Clinical Use: Determine genetic cause of DD/MR
Individuals Suitable for Testing: Those with negative chromosome
and fragile X analyses whose phenotype remains suspicious for a
genetic abnormality
FISH, Wolf-Hirschhorn*
Test Code: 14613X
Clinical Use: Diagnose or confirm the diagnosis of Wolf-Hirschhorn
syndrome
Individuals Suitable for Testing: Those with cardiac septal defects,
microencephaly, hypotonia, seizures, hypospadias/cryptorchidism
(males), absent uterus (females), prominent glabella, short philtrum,
hypertelorism, downturned corners of mouth
Fragile X with Reflex†,‡
Test Code: 19757X
Clinical Use: Diagnose fragile X syndrome; determine carrier status
Individuals Suitable for Testing: Those with dysmorphic features,
birth defects, growth abnormalities, and/or behavior problems, and
those at risk of carrying fragile X abnormality
Fragile X with Reflex†,‡ and Chromosome Analysis, Blood
Test Code: 19792X
Clinical Use: Determine genetic cause of DD/MR; diagnose fragile X
syndrome; determine fragile X carrier status
Individuals Suitable for Testing: Those with dysmorphic features,
birth defects, growth abnormalities, and/or behavior problems, and
those at risk of carrying fragile X abnormality
Gaucher Disease, DNA Mutation Analysis*
Test Code: 21503X
Clinical Use: Diagnose Gaucher disease; determine carrier status
105
Genetics
Individuals Suitable for Testing: Those with hepatosplenomegaly,
bone fractures and lesions, thrombocytopenia, or a family history of
Gaucher disease
Genomic Alterations, Postnatal, ClariSure™ CGH*
Test Code: 16135X
Clinical Use: Determine genetic cause of DD/MR
Individuals Suitable for Testing: Those with negative chromosome,
fragile X, and subtelomeric FISH analyses
Homocysteine (Nutritional and Congenital), Serum
Test Code: 36362X
Clinical Use: Diagnose homocystinuria; monitor homocysteine levels
in patients with homocystinuria
Individuals Suitable for Testing: Those with dislocated ocular lens,
osteoporosis, scoliosis, thinning/lengthening of long bones,
thromboembolism, psychiatric disorders, or diagnosed homocystinuria
Maple Syrup Disease (MSUD) Mutation Analysis (Ashkenazi
Jewish)*
Test Code: 16067X
Clinical Use: Diagnose MSUD; determine carrier status
Individuals Suitable for Testing: Those with seizures, alternating
hypertonia/hypotonia, poor feeding, lethargy, failure to thrive, or a
family history of MSUD
Methylmalonic Acid
Test Code: 34879X
Clinical Use: Diagnose methylmalonic acidemia; monitor
methylmalonic acid levels after diagnosis
Individuals Suitable for Testing: Those with respiratory distress,
hepatomegaly, lethargy, failure to thrive, vomiting, hypotonia, or with
diagnosed methylmalonic acidemia
Methylmalonic Acid, Urine (Includes creatinine)
Test Code: 34877X
Clinical Use: Diagnose methylmalonic acidemia; monitor
methylmalonic acid levels after diagnosis
Individuals Suitable for Testing: Those with respiratory distress,
hepatomegaly, lethargy, failure to thrive, vomiting, hypotonia, or with
diagnosed methylmalonic acidemia
Niemann-Pick Disease Mutation Analysis*
Test Code: 10222X
Clinical Use: Diagnose Niemann-Pick disease; determine carrier
status
Individuals Suitable for Testing: Those with hepatosplenomegaly,
hypotonia, muscle weakness, vomiting, constipation, and/or cherry-red
corneal spot and those with a family history of Niemann-Pick disease
Organic Acids, Qualitative, Urine
Test Code: 10049X
Clinical Use: Screen for organic aciduria
Individuals Suitable for Testing: Those with variable symptoms that
may include coma, liver disease, seizures, hypotonia, ataxia, failure to
thrive, lethargy
Organic Acids, Quantitative, Random Urine, Full Panel
Test Code: 38067X
Clinical Use: Diagnose organic aciduria
Individuals Suitable for Testing: Those with variable symptoms that
may include coma, liver disease, seizures, hypotonia, ataxia, failure to
thrive, lethargy
106

Section 5 Genetics

Phenylalanine References
Test Code: 37356X
1. Curry CJ, Stevenson RE, Aughton D, et al. Evaluation of mental
Clinical Use: Diagnose phenylketonuria; monitor phenylalanine levels
retardation: recommendations of a consensus conference. Am J Med
after diagnosis
Genetics.1997;72:468-477.
Individuals Suitable for Testing: Those with seizures, poor feeding,
2. Shaffer LG. American College of Medical Genetics guideline on the
vomiting, hyperactivity, eczema, hypopigmentation; those diagnosed
cytogenetic evaluation of the individual with developmental delay or
with phenylketonuria
mental retardation. Genet Med. 2005;7:650-654.
3. Sherman S, Pletcher BA, Driscoll DA. Fragile X syndrome: diagnostic
Phenylalanine and Tyrosine
and carrier testing. Genet Med. 2005;7:584-587.
Test Code: 26336X
Clinical Use: Monitor response to treatment of phenylketonuria
Individuals Suitable for Testing: Those with diagnosed
phenylketonuria

Prader-Willi/Angelman Syndrome*
Test Code: 11369Z
Clinical Use: Diagnose Prader-Willi or Angelman syndrome
Individuals Suitable for Testing: Those with severe hypotonia, poor
feeding leading to gavage feeding, cryptorchidism (males), hypoplastic
labia (females), strabismus, narrow bitemporal diameter, upslanting
fissures, short stature, small hands and feet, obesity, hyperphagia,
ataxic gate, paroxysmal laughter

Rett Syndrome Mutation Analysis*

Test Code: 15088X
Clinical Use: Diagnose Rett syndrome
Individuals Suitable for Testing: Males with unexplained neonatal
encephalopathy; those with Angelman-type symptoms with normal
chromosome 15q11.2-q13; or those with X-linked mental retardation and
a negative fragile X test

Tay-Sachs Disease Mutation Analysis*

Test Code: 21502X
Clinical Use: Diagnose Tay-Sachs disease; determine carrier status
Individuals Suitable for Testing: Those with macular pallor with
cherry red spot, blindness, dementia, apathy; family members at risk of
carrying a Tay-Sachs mutation

Tryptophan, LC/MS
Test Code: 959X
Clinical Use: Diagnose tryptophanuria; monitor tryptophan levels after
diagnosis
Individuals Suitable for Testing: Those with photosensitive skin
rash, short stature, cerebellar-like ataxia; those with diagnosed
tryptophanuria

Tyrosine
Test Code: 902X
Clinical Use: Diagnose tyrosinemia; monitor tyrosine levels after
diagnosis
Individuals Suitable for Testing: Those with liver disease, eye
symptoms of lacrimation, photophobia, redness, pain; nonpruritic,
painful hyperkeratotic skin lesions; those with diagnosed tyrosinemia
*This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
†
Reflex tests are performed at an additional charge and are associated with additional CPT
codes. For males, if PCR result suggests carrier status, an abnormal result, or no FMR1
amplification product, then Southern blot is performed. For females, if PCR result suggests
carrier status, normal homozygous FMR1 alleles, or gray zone, then capillary
electrophoresis Southern analysis (CSA) is performed; abnormal CSA results are followed-
up with Southern blot.
‡
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the test.
 107

Section 6 Hematology/Oncology

6.1 Available Tests HER2 (HercepTest®’), IHC with Reflex to FISH

HER2 (HercepTest®’), IHC, Paraffin Block
Adrenal Cortex Cancer HER2, ELISA
Aldosterone, 24-Hour Urine IGF Binding Protein-2 (IGFBP-2)1
Aldosterone, LC/MS/MS, Serum Immunohistochemistry (IHC) Marker, Stain Only (No Interpretation)1
Androstenedione, LC/MS/MS Ki-67, IHC with Interpretation1
Cancer of Unknown Primary (Identification of Origin)1 Lipid-Associated Sialic Acid (LSA, LASA)
Cortisol, Amniotic Fluid Micrometastasis Detection in Lymph Nodes, IHC1
Cortisol, Free and Total, LC/MS/MS p53 Gene Mutation Analysis, Cell-based1
Cortisol, Free, 24-Hour Urine p53 Gene Mutation Analysis, Plasma-based, Leumeta™1
Cortisol, Free, LC/MS/MS p53 Oncoprotein1
Cortisol, Saliva, LCMSMS Plasminogen Activator Inhibitor-1
Cortisol, Serum Progesterone Receptor (PR-IHC), Paraffin Block1
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Progesterone Receptor (PR-IHC), with Photomicrograph1
DHEA (Dehydroepiandrosterone), Amniotic Fluid Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, CSF1
DHEA (Dehydroepiandrosterone), LCMSMS Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, Serum1
DHEA (Dehydroepiandrosterone), Urine Vascular Endothelial Growth Factor (VEGF), ELISA2
DHEA Sulfate
Estradiol, 24-Hour Urine Eye and Ocular Adenxa Cancer (includes retinoblastoma,
Estradiol, Amniotic Fluid mesenchyme)
Estradiol, Bioavailable Cancer of Unknown Primary (Identification of Origin)1
Estradiol, Free Chromosome Analysis, Solid Tumor
Estradiol, Ultra Sensitive Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
Estrogens, Fractionated, LC/MS/MS IGF-I
IGF-I
Testosterone, Free and Total, LC/MS/MS Gastrointestinal Cancer (includes carcinoid, colon, esophagus,
Testosterone, Total (Males), ICMA omentum, rectum, stomach)
Testosterone, Total (Women and Children), LC/MS/MS 5-HIAA (5-Hydroxyindoleacetic Acid), 24-Hour Urine
5-HIAA (5-Hydroxyindoleacetic Acid), Random Urine
Breast Cancer CA 125
Breast Cancer Gene Expression Ratio (HOXB13:IL17BR)1 CA 125, CSF
CA 125 CA 125, Peritoneal Fluid
CA 125, CSF CA 125, Pleural Fluid
CA 125, Peritoneal Fluid CA 19-9 & CEA
CA 125, Pleural Fluid CA 19-9, CSF
CA 15-3 CA 19-9, Pericardial Fluid
CA 27.29 CA 19-9, Peritoneal Fluid
CA 27.29, CSF CA 19-9, Pleural Fluid
CA 72-4 CA 19-9, Serum
Cancer of Unknown Primary (Identification of Origin)1 CA 72-4
CEA with HAMA Treatment Cancer of Unknown Primary (Identification of Origin)1
CEA, CSF CD117, IHC1
CEA, Pericardial Fluid CEA with HAMA Treatment
CEA, Peritoneal Fluid CEA, CSF
CEA, Pleural Fluid CEA, Pericardial Fluid
CEA, Serum CEA, Peritoneal Fluid
CellSearch®’ Circulating Tumor Cells, Breast Cancer CEA, Pleural Fluid
Cyclin-D1 (BCL-1), IHC with Interpretation CEA, Serum
Cyclin-D1, IHC1 Chromogranin A1
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Chromosome Analysis, Solid Tumor
DNA Cell Cycle Analysis with Reflex to Ki-67 Circulating Endothelial Cell (CEC) Detection2
DNA Cell Cycle Analysis, Paraffin Block c-kit Mutation Analysis, Cell-based1
Epidermal Growth Factor Receptor (EGFR) Mutation Analysis (TK c-kit Mutation Analysis, Plasma-based, Leumeta™1
Domain)1 Colorectal Cancer (CRC) Pharmacogenomic Panel1
Epidermal Growth Factor Receptor (EGFR), ELISA2 Includes mutation analysis for UGT1A1, TS 6del, TS 2R/3R (28 bp repeat), XRCC1
Epidermal Growth Factor Receptor (EGFR), IHC (Arg399Gln), ERCC1 (C>T in codon 118), GSTP1 (lle 105Val), XPD (Lys751 Gln), and
ER/PR, Paraffin Block1 DPD (IVS14+1G>A).
ER/PR/DNA, Paraffin Block1 Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
ER/PR/DNA/HER2 w/Reflex to HER2 FISH, Paraffin Block1 DNA Cell Cycle Analysis, Paraffin Block
ER/PR/HER2 w/Reflex to HER2 FISH, Paraffin Block1 Epidermal Growth Factor Receptor (EGFR) Mutation Analysis (TK
ER/PR/MIB-1 (Ki-67), Paraffin Block1 Domain)1
Estrogen Receptor (ER-IHC), Paraffin Block1 Epidermal Growth Factor Receptor (EGFR), ELISA2
FISH, EGFR1 Epidermal Growth Factor Receptor (EGFR), IHC
FISH, HER-2/neu, Paraffin Block FISH, EGFR1
108
Section 6
Helicobacter pylori Antibodies (IgA, IgG)
Helicobacter pylori Antibodies (IgG, IgA, IgM)
Helicobacter pylori Antibodies (IgG,IgA), Western Blot1
Helicobacter pylori Antibody (IgA)
Helicobacter pylori Antibody (IgA), Western Blot1
Helicobacter pylori Antibody (IgG), Qualitative
Helicobacter pylori Antibody (IgG), Quantitative
Helicobacter pylori Antibody (IgG), Western Blot1
Helicobacter pylori Antibody (IgM)1
Helicobacter pylori Antigen Detection, EIA, Stool
Helicobacter pylori Urea Breath Test (UBiT®’)
Hemoquant®’, Feces
Lipid-Associated Sialic Acid (LSA, LASA)
Micrometastasis Detection in Lymph Nodes, IHC1
Microsatellite Instability (MSI), HNPCC1
MLH1 and MSH2 Mutations (Deletion and Duplication), HNPCC1
MLH1 and MSH2 Mutations, HNPCC1
MLH1 Mutation, One Exon, HNPCC1
MSH2 Mutation, One Exon, HNPCC1
MSH6 Mutation, HNPCC1
MSH6 Mutation, One Exon, HNPCC1
p53 Gene Mutation Analysis, Cell-based1
p53 Gene Mutation Analysis, Plasma-based, Leumeta™1
p53 Oncoprotein1
ras Mutation Analysis, Cell-based1
ras Mutation Analysis, Plasma-based Leumeta™1
Serotonin, Blood
Serotonin, Serum
UGT1A1 Gene Polymorphism (TA Repeat)1
Vascular Endothelial Growth Factor (VEGF), ELISA2
Gynecologic Cancer (includes cervix, choriocarcinoma,
endometrium, fallopian tube, germ cell, hydatidiform mole,
ovary, trophoblast, vagina, vulva)
Alpha Subunit1
CA 125
CA 125, CSF
CA 125, Peritoneal Fluid
CA 125, Pleural Fluid
CA 15-3
CA 27.29
CA 27.29, CSF
CA 72-4
Cancer of Unknown Primary (Identification of Origin)1
Chromosome Analysis, Solid Tumor
Cytology, Conventional Pap Smear
Cytology, Liquid-based Pap Test and HPV (SurePath®’)
Cytology, Liquid-based Pap Test and HPV (ThinPrep®’)
Cytology, Liquid-based Pap Test with Reflex to HPV (SurePath®’)
Cytology, Liquid-based Pap Test with Reflex to HPV (ThinPrep®’)
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
DNA Cell Cycle Analysis, Hydatidiform Mole, Paraffin Block
DNA Cell Cycle Analysis, Paraffin Block
Estradiol, 24-Hour Urine
Estradiol, Amniotic Fluid
Estradiol, Bioavailable
Estradiol, Free
Estradiol, Ultra Sensitive
Estrogens, Fractionated, LC/MS/MS
hCG, Total, CSF
hCG, Total, Pericardial Fluid
hCG, Total, Peritoneal Fluid
hCG, Total, Pleural Fluid
HPV (Human Papillomavirus) Genotypes 16 and 181
Hematology/Oncology
HPV (Human Papillomavirus), High Risk DNA, Hybrid Capture II
HPV (Human Papillomavirus), Hybrid Capture II
HPV (Papillomavirus) High Risk, Hybrid Capture II with Reflex to
Genotypes 16,181
HPV (Papillomavirus), Low/High Risk, Hybrid Capture II with Reflex to
Types 16,181
HPV Typing In Situ1
Inhibin A
Inhibin B, ELISA2
Lipid-Associated Sialic Acid (LSA, LASA)
Micrometastasis Detection in Lymph Nodes, IHC1
p53 Gene Mutation Analysis, Cell-based1
p53 Gene Mutation Analysis, Plasma-based, Leumeta™1
p53 Oncoprotein1
Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, CSF1
Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, Serum1
Testosterone, Free and Total, LC/MS/MS
Testosterone, Total (Women and Children), LC/MS/MS
Vascular Endothelial Growth Factor (VEGF), ELISA2
Head and Neck Cancer (includes brain, hypothalamus,
nasopharynx, parathyroid, salivary gland, thymus, thyroid)
Calcitonin
Calcium, 24-Hour Urine (with Creatinine)
Calcium, 24-Hour Urine (without Creatinine)
Calcium, Ionized
Calcium, Pediatric Urine
Calcium, Total & Ionized, Serum
Calcium, Total, Serum
Cancer of Unknown Primary (Identification of Origin)1
Chromogranin A1
Chromosome Analysis, Solid Tumor
Corticotropin Releasing Hormone
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
Epstein Barr Virus DNA, Qualitative Real-Time PCR3
Epstein Barr Virus DNA, Quantitative Real-Time PCR3
Epstein-Barr Virus Antibody (IgG) to Viral Capsid Antigen (VCA-IgG)
Epstein-Barr Virus Antibody (IgM) to Viral Capsid Antigen (VCA-IgM)
Epstein-Barr Virus, Antibody to Early Antigen D (IgG)
Epstein-Barr Virus Antibody to Nuclear Antigen (EBNA) (IgG)
Epstein-Barr Virus Panel
Includes EBV capsid IgG and IgM antibodies, EBNA IgG antibody, and strength of
signal for each.
Ferritin
FISH, Oligodendroglioma, 1p/19q1
Growth Hormone Releasing Hormone1
MEN2 and FMTC Mutations, Exons 10, 11, 13-161
Micrometastasis Detection in Lymph Nodes, IHC1
Neuron Specific Enolase (NSE)2
PTH, Intact (ICMA) & Ionized Calcium
PTH, Intact and Calcium
S-100, IHC1
T3, Total (Triiodothyronine)
T4, Free, Direct Dialysis
T4, Total (Thyroxine)
Thyroglobulin Panel
Includes thyroglobulin and thyroglobulin antibody.
Hepatobiliary Cancer (includes biliary ducts, gallbladder,
liver)
5’ Nucleotidase
5-HIAA (5-Hydroxyindoleacetic Acid), 24-Hour Urine
5-HIAA (5-Hydroxyindoleacetic Acid), Random Urine
Alpha-Fetoprotein (AFP) and AFP-L3
 109

Section 6 Hematology/Oncology

Alpha-Fetoprotein, CSF bcr/abl Gene Rearrangement, Quantitative PCR1

Alpha-Fetoprotein, Pericardial Fluid bcr/abl Gene Rearrangement, Quantitative PCR with Reflex to Subtype1
Alpha-Fetoprotein, Peritoneal Fluid bcr/abl Gene Rearrangement, Quantitative PCR, Plasma-based,
Alpha-Fetoprotein, Pleural Fluid Leumeta™1
Alpha-Fetoprotein, Tumor Marker bcr/abl Protein Quantitation (Total and Phosphorylated), Leumeta™1
CA 125 Bone Marrow Engraftment Study, STR Analysis2
CA 125, CSF Campath-1H Sensitivity (CD52)1
CA 125, Peritoneal Fluid CBFB/MYH11 inv(16), Quantitative Real-Time PCR1
CA 125, Pleural Fluid CD117, IHC1
CA 19-9 & CEA CD25, IHC with Interpretation1
CA 19-9, CSF CD57, CD3, CD8, Flow Cytometry1
CA 19-9, Pericardial Fluid CD57, Flow Cytometry1
CA 19-9, Peritoneal Fluid Cell Proliferation (BrdU Incorporation)2
CA 19-9, Pleural Fluid Cell Sorting, Specific Subpopulation2
CA 19-9, Serum Chromosome Analysis, CLL/LPD
Cancer of Unknown Primary (Identification of Origin)1 Chromosome Analysis, Hematologic Malignancy
CEA with HAMA Treatment Chromosome Analysis, Sister Chromatid Exchange
CEA, CSF Chromosomes, DEB Assay for Fanconi Anemia
CEA, Pericardial Fluid Chronic Lymphocytic Leukemia (CLL)/Lymphoma Diagnostic Panel1
CEA, Peritoneal Fluid Includes CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD19, CD20, CD23, CD38,
CEA, Pleural Fluid CD45, CD56, CD64, FMC7, kappa, lambda, and 3 additional markers.
CEA, Serum Chronic Lymphocytic Leukemia (CLL)/Lymphoma Follow-up Panel1
c-kit Mutation Analysis, Cell-based1 Includes CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD19, CD20, CD23, CD38, CD45,
c-kit Mutation Analysis, Plasma-based, Leumeta™1 DR, kappa, lambda, and 3 additional markers.
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Chronic Lymphocytic Leukemia, IgVH Mutation Status, Cell-based1
DNA Cell Cycle Analysis, Paraffin Block Chronic Lymphocytic Leukemia, IgVH Mutation Status, Leumeta™1
Ferritin CLL Prognostic Panel, Comprehensive1
Gamma-Glutamyltransferase (GGT) Includes beta2-microglobulin, CD38/CD19, CLL/LPD chromosome analysis, FISH B-
Glypican 32 cell CLL panel (11q22.3, 13q14.3, 13q34, 17p13, and chromosome 12), IGVH
Hepatitis B Surface Antigen mutation analysis, and ZAP-70.
Hepatitis B Surface Antigen with Reflex to Confirmation CLL Prognostic Panel, Comprehensive w/o Karyotype1
Hepatitis B Viral DNA, Quantitative, PCR1 Includes beta2-microglobulin, CD38/CD19, FISH B-cell CLL panel (11q22.3, 13q14.3,
Hepatitis B Virus DNA, Qualitative PCR1 13q34, 17p13, and chromosome 12), IGVH mutation analysis, and ZAP-70.
Hepatitis C Antibody Supplemental Testing CLL Prognostic Panel, Limited1
Hepatitis C Antibody with Reflex to Supplemental Testing Includes FISH B-cell CLL panel (11q22.3, 13q14.3, 13q34, 17p13, and chromosome
Hepatitis C Antibody, EIA 12), IGVH mutation analysis, and ZAP-70.
Hepatitis C Viral RNA, Qualitative PCR CLL Prognostic Panel, Monitoring1
Hepatitis C Viral RNA, Qualitative TMA Includes beta2-microglobulin, CD38/CD19, FISH B-cell CLL panel (11q22.3, 13q14.3,
Hepatitis C Viral RNA, Quantitative Real-Time PCR1 13q34, 17p13, and chromosome 12), and ZAP-70.
Hepatitis C Viral RNA, Quantitative, TMA1 Comprehensive Hematopathology Report
HEPTIMAX™ HCV RNA1 Includes hematopathologist interpretation of morphologic and ancillary studies.
Leukocyte Alkaline Phosphatase Stain Additional tests are performed, at an additional charge, when deemed medically
Micrometastasis Detection in Lymph Nodes, IHC1 necessary for the diagnosis. Such tests may include IHC and flow cytometric
Vascular Endothelial Growth Factor (VEGF), ELISA2 markers as well as chromosomal, FISH, or PCR genetic studies.
crkL Phosphorylation1
Leukemia crkL, Total1
ABL Kinase Domain Mutation in CML, Cell-based1 Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
ABL Kinase Domain Mutation in CML, Plasma-based, Leumeta™1 DNA Cell Cycle Analysis with Reflex to Ki-67
ABL T315I Mutation in CML, Cell-based1 DNA Cell Cycle Analysis, Blood
ABL T315I Mutation in CML, Plasma-based, Leumeta™1 FIP1L1-PDGFRA Gene Rearrangement [del(4q12)], Real-time PCR1
Acetyl Histone H3 and H4, IHC FISH, ALL, TEL/AML1 Translocation 12,211
Acid Hemolysis Test FISH, ALL/NHL, MYC-BA, 8q24 Rearrangement1
Acute Leukemia Follow-up Panel1 FISH, AML M3, PML/RARA,Translocation 15,171
Includes CD3, CD7, CD13, CD19, CD20, CD33, CD34, CD45, CD64, and 3 additional FISH, AML, AML1/ETO Translocation 8,211
markers. FISH, AML, CBFB/MYH11, Inversion 161
Alpha-1-Acid Glycoprotein FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel1
AML1/ETO t(8;21) Quantitative Real-Time PCR1 Includes 11q22.3, 13q14.3, 13q34, 17p13, and chromosome 12.
Apoptosis, Annexin V2 FISH, B-Cell Malignancy, IGH, 14q32 Rearrangement1
Apoptosis, Mitochondrial Membrane Potential2 FISH, Burkitt’s/NHL/ALL, IGH/MYC, t(8;14)1
B-Cell Gene Rearrangement, Minimal Residual Disease (MRD)1 FISH, Chromosome 20q Deletion1
B-Cell Gene Rearrangement, PCR1 FISH, Chromosome-Specific Probe (specify probe & chromosome
B-Cell Gene Rearrangement, Qualitative PCR, Plasma-based, Leumeta™1 number)1
B-Cell Gene Rearrangement, Quantitative PCR, Plasma-based, FISH, CML/ALL, bcr/abl Translocation 9,221
Leumeta™1 FISH, MLL (11q23) Gene Rearrangement1
110

Section 6 Hematology/Oncology

FISH, Myeloid Disorders Profile1 Epidermal Growth Factor Receptor (EGFR), ELISA2
FISH, SKY™ Marker Chromosome1 Epidermal Growth Factor Receptor (EGFR), IHC
FISH, X/Y, Post Bone Marrow Transplant FISH, EGFR1
FLT3 Mutations (ITD and D835)1 FISH, Lung Cancer1
Hairy Cell Leukemia (HCL)/Lymphoma Follow-up Panel1 IGF Binding Protein-2 (IGFBP-2)1
Includes CD11c, CD19, CD20, CD22, CD25, CD45, CD103, kappa, and lambda. Lipid-Associated Sialic Acid (LSA, LASA)
Hematopathology Consultation Micrometastasis Detection in Lymph Nodes, IHC1
Hematopathology Morphologic Evaluation Neuron Specific Enolase (NSE)2
Includes hematopathologist interpretation of bone marrow and/or peripheral blood Neuronal Nuclear (Hu) Antibody with Reflex to Titer & Western Blot1
smear morphology. Neuronal Nuclear (Hu) Antibody, IFA, with Reflex to Titer & Western
Histone Deacetylase-1 and -2, IHC Blot, CSF1
Histone H3, Phosphorylated, Quantitative Flow Cytometry1 p53 Gene Mutation Analysis, Cell-based1
Histone H3, Total, Quantitative Flow Cytometry1 p53 Gene Mutation Analysis, Plasma-based, Leumeta™1
HTLV I/II DNA, Qualitative Real-Time PCR1 p53 Oncoprotein1
HTLV I/II, Western Blot2 Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, CSF1
HTLV-I/II Antibody, EIA Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, Serum1
Immunohistochemistry (IHC) Marker, Stain Only (No Interpretation)1 Vascular Endothelial Growth Factor (VEGF), ELISA2
Intracellular Markers by Flow Cytometry1
Specify 1 or more of the following markers: CD3, CD22, CD79a, IgM, kappa, Lymphoma (includes Hodgkin’s disease)
lambda, MPO, TdT. B-Cell Gene Rearrangement, Minimal Residual Disease (MRD)1
JAK2 Mutation (V617F) Analysis, Cell-based1 B-Cell Gene Rearrangement, PCR1
JAK2 Mutation (V617F) Analysis, Plasma-based, Leumeta™1 B-Cell Gene Rearrangement, Qualitative PCR, Plasma-based, Leumeta™1
Ki-67, IHC with Interpretation1 B-Cell Gene Rearrangement, Quantitative PCR, Plasma-based,
Leukemia/Lymphoma Evaluation1 Leumeta™1
Includes CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD19, CD20, CD23, bcl-2, IHC1
CD33, CD34, CD38, CD45, CD56, CD64, CD117, HLA-DR, and coexpression of Beta-2-Microglobulin, CSF
CD19/kappa, CD19/lambda, and CD19/CD5. Beta-2-Microglobulin, Random Urine
Leukemia/Lymphoma Evaluation, Histogram Only (No Interpretation)1 Beta-2-Microglobulin, Serum
Includes CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD19, CD20, CD23, Bone Marrow Engraftment Study, STR Analysis2
CD33, CD34, CD38, CD45, CD56, CD64, CD117, HLA-DR, and coexpression of Cancer of Unknown Primary (Identification of Origin)1
CD19/kappa, CD19/lambda, and CD19/CD5. CD25, IHC with Interpretation1
Leukocyte Alkaline Phosphatase Stain Cell Proliferation (BrdU Incorporation)2
Lipid-Associated Sialic Acid (LSA, LASA) Chromosome Analysis, Hematologic Malignancy
MDR1 Activity2 Chromosome Analysis, Lymph Node
Myeloperoxidase (MPO) Antigen, Heparinized Plasma2 Chronic Lymphocytic Leukemia (CLL)/Lymphoma Diagnostic Panel1
Mylotarg Sensitivity (CD33)1 Includes CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD19, CD20, CD23, CD38,
NPM (Exon 12) Mutation Analysis, Cell-based1 CD45, CD56, CD64, FMC7, kappa, lambda, and 3 additional markers.
NPM (Exon 12) Mutation Analysis, Plasma-based, Leumeta™1 Chronic Lymphocytic Leukemia (CLL)/Lymphoma Follow-up Panel1
Ontak Sensitivity (CD25)1 Includes CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD19, CD20, CD23, CD38, CD45,
PML/RARA t(15;17), Quantitative PCR1 DR, kappa, lambda, and 3 additional markers.
ras Mutation Analysis, Cell-based1 Comprehensive Hematopathology Report
ras Mutation Analysis, Plasma-based Leumeta™1 Includes hematopathologist interpretation of morphologic and ancillary studies.
T & B Cells, Total Additional tests are performed, at an additional charge, when deemed medically
T-cell Receptor (TCR) Gene Rearrangement1 necessary for the diagnosis. Such tests may include IHC and flow cytometric
T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR, Leumeta™1 markers as well as chromosomal, FISH, or PCR genetic studies.
T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR, Cryoglobulin (% Cryocrit), Serum
Leumeta™1 Cryoglobulin Screen with Reflex to Cryoglobulin Profile, Serum
TCR-Gamma Gene Rearrangement, Quantitative PCR1 Cyclin-D1 (BCL-1), IHC with Interpretation
TPMT Genotype Cyclin-D1, IHC1
ZAP-701 Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
DNA Cell Cycle Analysis, Blood
Lung Cancer (includes bronchus, pleural fluid) Electrophoresis & Immunofixation, Serum with Tracing
Cancer of Unknown Primary (Identification of Origin)1 Electrophoresis, Protein and Kappa/Lambda, Serum
CEA with HAMA Treatment Electrophoresis, Protein and Total Protein and Tracing, CSF
CEA, CSF Electrophoresis, Protein and Total Protein, CSF
CEA, Pericardial Fluid Electrophoresis, Protein, 24-Hour Urine & Immunofixation Studies
CEA, Peritoneal Fluid Electrophoresis, Protein, 24-Hour Urine (with Total Protein)
CEA, Pleural Fluid Electrophoresis, Protein, CSF
CEA, Serum Electrophoresis, Protein, CSF, with Tracing
Chromogranin A1 Electrophoresis, Protein, Random Urine & Immunofixation Studies
Chromosome Analysis, Solid Tumor Electrophoresis, Protein, Random Urine (with Total Protein)
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Electrophoresis, Protein, Serum
Epidermal Growth Factor Receptor (EGFR) Mutation Analysis (TK Electrophoresis, Protein, Serum with Tracing
Domain)1 Electrophoresis, Protein, Urine
 111

Section 6 Hematology/Oncology

Epstein Barr Virus DNA, Qualitative Real-Time PCR3 Neural Crest Cancer (includes ganglioblastoma, ganglioma,
Epstein Barr Virus DNA, Quantitative Real-Time PCR3 neuroblastoma, paraganglioma, pheochromocytoma)
Epstein-Barr Virus Antibody (IgG) to Viral Capsid Antigen (VCA-IgG) Cancer of Unknown Primary (Identification of Origin)1
Epstein-Barr Virus Antibody (IgM) to Viral Capsid Antigen (VCA-IgM) Catecholamines, Fractionated, & VMA, 24-Hour Urine
Epstein-Barr Virus Antibody to Nuclear Antigen (EBNA) (IgG) Catecholamines, Fractionated, 24-Hour Urine
Epstein-Barr Virus Panel Catecholamines, Fractionated, Plasma
Includes EBV capsid IgG and IgM antibodies, EBNA IgG antibody, and strength of Catecholamines, Fractionated, Random Urine
signal for each. Chromogranin A1
Epstein-Barr Virus, Antibody to Early Antigen D (IgG) Chromosome Analysis, Solid Tumor
FISH, ALCL, ALK, 2p23 Rearrangements1 Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
FISH, ALL/NHL, MYC-BA, 8q24 Rearrangement1 FISH, N-myc Amplification, Neuroblastoma1
FISH, B-Cell Malignancy, IGH, 14q32 Rearrangement1 Homovanillic Acid, 24-Hour Urine
FISH, Burkitt’s/NHL/ALL, IGH/MYC, t(8;14)1 Homovanillic Acid, Random Urine
FISH, Follicular Lymphoma, IGH/BCL2, t(14;18)1 MEN2 and FMTC Mutations, Exons 10, 11, 13-161
FISH, Mantle Cell Lymphoma, IGH/CCND1, t(11;14)1 Metanephrines, Fractionated, LC/MS/MS, 24-Hour Urine
FISH, SKY™ Marker Chromosome1 Metanephrines, Fractionated, LC/MS/MS, Plasma
FISH, X/Y, Post Bone Marrow Transplant Metanephrines, Fractionated, LC/MS/MS, Random Urine
Follicular Lymphoma, bcl-2/JH t(14;18), Real-time PCR, Cell-based1 Neuron Specific Enolase (NSE)2
Follicular Lymphoma, bcl-2/JH t(14;18), Real-time PCR, Leumeta™1 Pheochromocytoma Evaluation
Hairy Cell Leukemia (HCL)/Lymphoma Follow-up Panel1 S-100, IHC1
Includes CD11c, CD19, CD20, CD22, CD25, CD45, CD103, kappa, and lambda. VMA (Vanillylmandelic Acid), Random Urine
Hematopathology Consultation VMA, 24-Hour Urine
Hematopathology Morphologic Evaluation
Histone Deacetylase-1 and -2, IHC Pancreatic Cancer
Histone H3, Phosphorylated, Quantitative Flow Cytometry1 CA 125
Histone H3, Total, Quantitative Flow Cytometry1 CA 125, CSF
Hodgkin’s Lymphoma Panel, IHC1 CA 125, Peritoneal Fluid
Includes Bob1, CD3, CD15, CD20, CD30, CD45RB (LCA), CD79a, EBV, and Oct-2. CA 125, Pleural Fluid
HTLV I/II DNA, Qualitative Real-Time PCR1 CA 19-9 & CEA
HTLV I/II, Western Blot2 CA 19-9, CSF
HTLV-I/II Antibody, EIA CA 19-9, Pericardial Fluid
Immunofixation, Serum CA 19-9, Peritoneal Fluid
Immunofixation, Urine CA 19-9, Pleural Fluid
Immunohistochemistry (IHC) Marker, Stain Only (No Interpretation)1 CA 19-9, Serum
Interleukin-2 Receptor, EIA2 CA 72-4
Intracellular Markers by Flow Cytometry1 Cancer of Unknown Primary (Identification of Origin)1
Kappa/Lambda Light Chain CEA with HAMA Treatment
Leukemia/Lymphoma Evaluation1 CEA, CSF
Leukemia/Lymphoma Evaluation, Histogram Only (No Interpretation)1 CEA, Pericardial Fluid
Lipid-Associated Sialic Acid (LSA, LASA) CEA, Peritoneal Fluid
Mantle Cell Lymphoma, bcl-1/JH t(11;14), Real-time PCR, Cell-based1 CEA, Pleural Fluid
Mantle Cell Lymphoma, bcl-1/JH t(11;14), Real-time PCR, Leumeta™1 CEA, Serum
MDR1 Activity2 Chromogranin A1
Ontak Sensitivity (CD25)1 C-Peptide
Rituxan Sensitivity (CD20)1 C-Peptide, 24-Hour Urine
T & B Cells, Total Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
T-cell Receptor (TCR) Gene Rearrangement1 DNA Cell Cycle Analysis, Paraffin Block
T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR, Leumeta™1 Gastrin
T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR, Glucagon1
Leumeta™1 IGF Binding Protein-1 (IGFBP-1)1
TCR-Gamma Gene Rearrangement, Quantitative PCR1 Insulin
Tumor Necrosis Factor-Alpha, Highly Sensitive2 Insulin, Free (Bioactive)1
Insulin, Total (Free and Antibody Bound)1
Malignant Melanoma Leukocyte Alkaline Phosphatase Stain
Cancer of Unknown Primary (Identification of Origin)1 Micrometastasis Detection in Lymph Nodes, IHC1
Chromosome Analysis, Solid Tumor Neuron Specific Enolase (NSE)2
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Pancreatic Elastase-1
Lipid-Associated Sialic Acid (LSA, LASA) Pancreatic Polypeptide1
Micrometastasis Detection in Lymph Nodes, IHC1 Proinsulin
S-100, IHC1 ras Mutation Analysis, Cell-based1
TA90 (Melanoma-associated Antigen)1 ras Mutation Analysis, Plasma-based Leumeta™1
Somatostatin1
Vasoactive Intestinal Polypeptide (VIP)1
112

Section 6 Hematology/Oncology

Paraneoplastic (Ectopic) Endocrine Syndromes (includes necessary for the diagnosis. Such tests may include IHC and flow cytometric
Cushing’s syndrome, hypercalcemia, hypoglycemia, SIADH, markers as well as chromosomal, FISH, or PCR genetic studies.
etc.) Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
ACTH, Plasma Electrophoresis & Immunofixation, Serum with Tracing
Alpha Subunit1 Electrophoresis, Protein and Kappa/Lambda, Serum
Arginine Vasopressin & Osmolality, Random Urine Electrophoresis, Protein and Total Protein and Tracing, CSF
Arginine Vasopressin (AVP, Antidiuretic Hormone, ADH)1 Electrophoresis, Protein and Total Protein, CSF
Calcitonin Electrophoresis, Protein, 24-Hour Urine & Immunofixation Studies
Calcium, 24-Hour Urine (with Creatinine) Electrophoresis, Protein, 24-Hour Urine (with Total Protein)
Calcium, 24-Hour Urine (without Creatinine) Electrophoresis, Protein, CSF
Calcium, Ionized Electrophoresis, Protein, CSF, with Tracing
Calcium, Pediatric Urine Electrophoresis, Protein, Random Urine & Immunofixation Studies
Calcium, Total & Ionized, Serum Electrophoresis, Protein, Random Urine (with Total Protein)
Calcium, Total, Serum Electrophoresis, Protein, Serum
Cancer of Unknown Primary (Identification of Origin)1 Electrophoresis, Protein, Serum with Tracing
Corticotropin Releasing Hormone2 Electrophoresis, Protein, Urine
Cortisol, Serum FISH, Multiple Myeloma, 13q-, 17p-, rea 14q321
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA Hematopathology Morphologic Evaluation
Erythropoietin (EPO) Plasma Cell Labeling Index2
Gastrin Plasma Cell Neoplasia Follow-up Panel1
Glucagon1 Includes CD20, CD38, CD45, CD56, CD138, kappa, lambda, and 3 additional
Growth Hormone (GH) markers.
Growth Hormone Releasing Hormone1
hCG, Total, CSF Soft Tissue Sarcoma (includes Kaposi’s)
hCG, Total, Pericardial Fluid Cancer of Unknown Primary (Identification of Origin)1
hCG, Total, Peritoneal Fluid Chromosome Analysis, Solid Tumor
hCG, Total, Pleural Fluid Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
IGF-I Desmoplastic Small Round Cell Tumor (DSRCT) Mutation Analysis1
Pancreatic Polypeptide1 DNA Cell Cycle Analysis, Paraffin Block
Plasma Renin Activity1 Ewing’s Sarcoma t(11;22) and t(21;22)1
PTH-Related Protein (PTH-RP)2 Factor VIII Related Antigen, IHC1
Somatostatin1 FISH, Ewing/PNET, EWSR1, 22q12 Rearrangements1
Vasoactive Intestinal Polypeptide (VIP)1 Neopterin2
p53 Gene Mutation Analysis, Cell-based1
Pituitary Cancer p53 Gene Mutation Analysis, Plasma-based, Leumeta™1
ACTH, Plasma p53 Oncoprotein1
Alpha Subunit1 Rhabdomyosarcoma Mutation Analysis (Alveolar, PAX3/FKHR,
Arginine Vasopressin & Osmolality, Random Urine PAX7/FKHR)1
Arginine Vasopressin (AVP, Antidiuretic Hormone, ADH)1 Sarcoma Mutation Analysis (Synovial, SYT-SSX1, SYT-SSX2)1
Cancer of Unknown Primary (Identification of Origin)1 Sarcoma Mutation Analysis Panel, Pediatric, Real-Time PCR3
Chromogranin A1 Includes detection of EWS-Wt, EWS-FLI1, EWS-ERG, PAX3/FKHR, PAX7/FKHR, SYT-
Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA SSX1, and SYT-SSX2 translocations.
FSH & LH
FSH (Follicle Stimulating Hormone) Urological and Male Genital Cancer (includes bladder, kidney,
FSH (Follicle Stimulating Hormone), Pediatrics prostate, testis, Wilm’s tumor)
Growth Disorders Panel 1 Acid Phosphatase, Total & Prostatic Acid Phosphatase
Includes hGH and IGFBP-3 Alpha-2-Macroglobulin
Growth Disorders Panel 2 Alpha-Fetoprotein, Tumor Marker
Includes hGH, IGF-1, and IGFBP-3 Cancer of Unknown Primary (Identification of Origin)1
Growth Hormone (GH) Chromosome Analysis, Solid Tumor
LH (Luteinizing Hormone) Cytology, Non-Gynecological, Fluid, Washings, Brushings, or FNA
LH (Luteinizing Hormone), Pediatrics DNA Cell Cycle Analysis with Reflex to Ki-67
Prolactin DNA Cell Cycle Analysis, Paraffin Block
Thyroid Stimulating Hormone (TSH) with HAMA Treatment E-cadherin, IHC1
TSH, 3rd Generation Erythropoietin (EPO)
Estrogens, Fractionated, LC/MS/MS
Plasma Cell Dyscrasia (includes multiple myeloma) Ferritin
Beta-2-Microglobulin, CSF FISH, Bladder Cancer, Bladder Washing
Beta-2-Microglobulin, Random Urine FISH, Prostate Cancer1
Beta-2-Microglobulin, Serum FISH, Vysis®’ UroVysion™’, Bladder Cancer
Cancer of Unknown Primary (Identification of Origin)1 hCG, Total, CSF
Comprehensive Hematopathology Report hCG, Total, Pericardial Fluid
Includes hematopathologist interpretation of morphologic and ancillary studies. hCG, Total, Peritoneal Fluid
Additional tests are performed, at an additional charge, when deemed medically hCG, Total, Pleural Fluid
 113

Section 6 Hematology/Oncology

IGF Binding Protein-2 (IGFBP-2)1 values were 27% and 95%, respectively (prevalence, 10.8%).2 Thus, the
IGF-II (Insulin Like Growth Factor II) Vysis UroVysion test appears useful for diagnosis of bladder cancer in
Ki-67, IHC with Interpretation1 patients with hematuria.
Leukocyte Alkaline Phosphatase Stain
Micrometastasis Detection in Lymph Nodes, IHC1 Researchers have found that increased chromosomal instability and
Neuron Specific Enolase (NSE)2 aneuploidy, such as that detected by the Vysis UroVysion test, are
Nuclear Matrix Proteins (NMP) characteristic of bladder tumor progression. Due to its high specificity
p53 Gene Mutation Analysis, Cell-based1 (~96%) and increased sensitivity (Table 1), the Vysis UroVysion test is
p53 Gene Mutation Analysis, Plasma-based, Leumeta™1 useful for early detection of bladder cancer recurrence when used in
p53 Oncoprotein1 conjunction with cystoscopy. When the Vysis UroVysion test is positive
Plasma Renin Activity1 and cystoscopy is negative, cancer recurs on average 4 months earlier
Prostate Specific Antigen (PSA) than when both tests are negative; thus, a positive Vysis UroVysion test
Prostate Specific Antigen (PSA), Free and Total may indicate a need for increased surveillance in these cases.
Prostate Specific Antigen (PSA), Post Prostatectomy
Prostate Specific Antigen (PSA), Post Prostatectomy & PAP Method: In this fluorescence in-situ hybridization (FISH) method, a
Prostate Specific Antigen (PSA), Post Prostatectomy with HAMA mixture of CEP 3, CEP 7, CEP 17, and LSI p16 probes, each labeled with
Treatment a different fluorochrome, is used to enumerate chromosomes 3, 7, and
Prostatic Acid Phosphatase (PAP) 17 and detect the 9p21 locus deletion on chromosome 9.
Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, CSF1
Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, Serum1 Interpretive Information: A positive result is consistent with a
Testosterone, Free and Total, LC/MS/MS diagnosis of bladder cancer or bladder cancer recurrence, either in the
Testosterone, Total (Males), ICMA bladder or in another site within the urinary system. A negative result is
Testosterone, Total (Women and Children), LC/MS/MS suggestive of the absence of bladder cancer but does not rule it out.
Vascular Endothelial Growth Factor (VEGF), ELISA2
1
This test was developed and its performance characteristics have been determined by References
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food 1. Leading sites of new cancer cases and deaths. American Cancer
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test. Society Web site. Available at: http://www.cancer.org/downloads/
2
This test is performed using a kit that has not been approved or cleared by the FDA. The stt/Leading_Sites_of_New_Cancer_Cases_and_Deaths___2005_Est
analytical performance characteristics of this test have been determined by Quest imates.pdf. Accessed 9-15-05.
Diagnostics Nichols Institute. This test should not be used for diagnosis without 2. UroVysion™ Bladder Cancer Kit - P030052. Food and Drug
confirmation by other medically established means.
3
This test was developed and its performance characteristics have been determined by Administration Web site. Available at:
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical http://www.fda.gov/cdrh/pdf3/p030052b.pdf. Accessed 9-15-05.
performance of the test. 3. Halling KC, King W, Sokolova IA, et al. A comparison of cytology and
Reflex tests are performed at an additional charge and are associated with an additional fluorescence in situ hybridization for the detection of urothelial
CPT code. carcinoma. J Urol. 2000;164;1768-1775.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
6.2.2 Nuclear Matrix Proteins (NMP)
6.2 Bladder Cancer Clinical Use: This test is used to diagnose bladder cancer and to
®’ monitor patients for bladder cancer recurrence.
6.2.1 FISH, Vysis UroVysion™’, Bladder Cancer
Clinical Background: Bladder cancer detection often begins with
Clinical Use: This test is used to diagnose bladder cancer in patients
voided urine cytology (VUC) testing of high-risk or symptomatic
with hematuria (in conjunction with standard diagnostic procedures) and
to detect bladder cancer recurrence.

Clinical Background: Bladder cancer is the fourth most common Table 1. Sensitivity of Urine Cytology and UroVysion
cancer in men and the ninth in women, with a combined estimate of According to Cancer Stage3
63,210 new cases in the U.S. in 2005.1 Non-invasive, superficial cancers
can usually be cured, especially when well differentiated. Many bladder Urine Cytology FISH
cancers, however, will recur: despite complete tumor resection, about
66% of patients will have a recurrence within 5 years and 88% within Stage
15 years. Frequent monitoring (up to four times per year) for early PTa 47 65
detection of recurrence is key to increased survival of these patients.
Traditionally, cystoscopy-guided biopsy, followed by histology, has been PTis 78 100
used for initial diagnosis and cystoscopy and voided urine cytology PT1-pT4 60 95
(VUC) have been used for monitoring.
Grade
Vysis UroVysion is a molecular cytology test that detects aneuploidy of 1 27 36
chromosomes 3, 7, and 17 and deletion of the 9p21 locus via
fluorescence in situ hybridization (FISH) in urine specimens. In a study of 2 54 76
497 patients with hematuria, the Vysis UroVysion test showed a 3 71 97
diagnostic sensitivity of 69% and specificity of 78% compared with
cystoscopy followed by histology. The positive and negative predictive Overall sensitivity 58 81
114

Section 6 Hematology/Oncology

individuals. VUC is highly specific but has relatively low sensitivity, Table 3. Diagnostic Sensitivity (%) of the NMP22 Test
especially for low-grade lesions; therefore, VUC cannot rule out a Compared to Cytology5
bladder cancer diagnosis. Ultimately, diagnosis is based on cystoscopy,
which is expensive and invasive. VUC continues to be used, however, Cytology NMP22
because it is non-invasive and can detect flat transitional cell
carcinomas not detected by cystoscopy. Because of the limitations of Stage
VUC and cystoscopy, additional non-invasive markers have been sought Ta 23 69
for diagnosis of primary and recurrent bladder cancer.
T1 64 81
Nuclear matrix proteins (NMPs) make up the internal structural T2-T4 73 91
framework of the nucleus and are associated with functions such as
DNA replication and RNA synthesis. Specific NMPs have been identified Grade
for various tumor types (eg, NMP22®’ for bladder cancer). Glas et al1 1 8 73
performed a meta-analysis that shows NMP22 has better sensitivity
than VUC (Table 2). Because this difference in sensitivity is greatest at 2 43 72
lower grades and stages (Table 3), use of NMP22 may facilitate earlier 3 77 81
detection of recurrence. Furthermore, a negative NMP22 result may
lengthen the interval between surveillance cystoscopies owing to the Tumor size (mm)
overall increase in sensitivity. Although NMP22 has less specificity than b10 32 76
VUC (Table 2), ruling out common causes of false-positive results (eg,
inflammation, infection, bladder or renal calculi) could increase the 11–20 37 67
specificity and thereby improve clinical usefulness.2 21–30 37 75
Neither NMP22 nor VUC can replace cystoscopy, but there may be value r31 85 93
in combining VUC and NMP22 results.3,4 Such a strategy would take
advantage of each marker’s strengths while diminishing the impact of
its limitations, potentially improving diagnostic sensitivity and 2. Lokeshwar VB, Soloway MS. Current bladder tumor tests: does their
specificity. These strategies have not yet been proven to be effective for projected utility fulfill clinical necessity? J Urol. 2001;165:1067-1077.
clinical use. NMP22 is currently FDA approved as an adjunct to, but not 3. Konety BR, Getzenberg RH. Urine based markers of urological
a replacement for, cytology. malignancy. J Urol. 2001;165:600-611.
4. Ross JS, Cohen MB. Ancillary methods for the detection of recurrent
Individuals Suitable for Testing include those at risk for or urothelial neoplasia. Cancer. 2000;90:75-86.
presenting with symptoms of bladder cancer and those being monitored 5. Boman H, Hedelin H, Jacobsson S, et al. Newly diagnosed bladder
for bladder cancer recurrence. cancer: the relationship of initial symptoms, degree of
microhematuria and tumor marker status. J Urol. 2002;168:1955-
Method: This enzyme immunoassay (EIA) uses 2 monoclonal antibodies 1959.
specific for nuclear matrix protein 22. The analytical sensitivity is 2.1 6. NMP22 Test Kit directional insert. Matritech, Inc. Oct. 1998.
U/mL. Results are reported in U/mL. Aliases for this test include
NMP22, Matritech NMP22, and nuclear mitotic apparatus protein 6.3 Breast Cancer
(NuMA).
6.3.1 Breast Cancer Gene Expression Ratio (HOXB13:IL17BR)
Interpretive Information: Increased (>10.0 U/mL) NMP
concentrations are associated with urinary tract transitional cell Clinical Use: This test is used to predict risk of breast cancer
carcinoma, renal cell carcinoma, bladder inflammation or infection, recurrence.
intestinal diversion, renal or bladder calculi, and recent history of a
foreign body in the urinary tract.2,3 Refer to Table 4 for additional Clinical Background: Breast cancer is the most frequently diagnosed
information about the levels of NMP in various disorders. cancer in women, with approximately 213,000 new cases and 41,000
deaths expected in the United States in 2006.1 Though often curable,
References breast cancer has a heterogeneous clinical course. Treatment options,
including hormone therapy and/or chemotherapy, are chosen on the
1. Glas AS, Roos D, Deutekom M, et al. Tumor markers in the diagnosis basis of endocrine responsiveness and prognostic risk factors.2
of primary bladder cancer. A systematic review. J Urol. Prognosis has historically been based on tumor size and grade, lymph
2003;169:1975-1982. node status, and patient age; this risk stratification combined with
menopausal status and hormone receptor status of the tumor is then
used to select therapy. However, breast cancer can recur even in treated
Table 2. Comparison of VUC and NMP22 for Diagnosis of patients in whom a favorable outcome is likely (ie, estrogen receptor
Primary Bladder Cancer1 [ER]-positive/lymph node-negative). The NSABP clinical trial noted
recurrence in up to 24% of such patients treated with tamoxifen alone
Sensitivity Specificity and up to 13% treated with both tamoxifen and chemotherapy over a 12
% (95% CI) % (95% CI) year follow-up period.3 This study suggests the need for additional
prognostic markers.
VUC 55 (48–62) 94 (90–96)
NMP22 67 (60–73) 78 (72–83) Ma and colleagues reported an association between tumor recurrence
and the expression of 2 genes, the homeobox gene (HOXB13) and the
CI, confidence interval.
 115

Section 6 Hematology/Oncology

Table 4. NMP22 Values in Health and Disease6

% with NMP in Indicated Range

No. of 0–10 10.1–20.0 20.1–50.0 50.1–100.0 >100.0
Patient Group Individuals U/mL U/mL U/mL U/mL U/mL
Healthy Individuals
Male >50 years 215 95 3 2 0 0
Female >50 years 151 87 7 5 1 1
Male & female <50 years 32 91 9 0 0 0
Total 398 92 5 3 0 0
Benign Disease
UTI & cystitis 26 85 12 4 0 0
Urinary calculi 16 94 0 0 6 0
BPH & prostatitis 52 92 8 0 0 0
Other 37 84 5 8 3 0
Total 117 88 8 3 1 0
Malignant Disease
Head and neck 6 83 0 17 0 0
GI Tract 12 83 0 8 0 8
Cardiovascular & pulmonary 12 58 8 17 17 0
Leukemia/lymphoma 11 64 18 9 9 0
Prostate 22 82 0 14 4 0
Kidney (non-TCC) 18 78 11 11 0 0
Other 17 65 18 6 0 12
UTI, urinary tract infection; BPH, benign prostatic hyperplasia; TCC, transitional cell carcinoma.

interleukin-17B receptor gene (IL-17BR).4 The expression ratio of
HOXB13:IL17BR (H:I expression ratio) predicted recurrence in a
multivariate analysis, whereas current clinicopathologic markers did not.
The clinical utility of the H:I expression index was further explored in a
cohort of 206 tamoxifen-treated postmenopausal women with curatively
resected ER-positive breast tumors.5 A high H:I ratio was predictive of
both early relapse and death in node-negative but not node-positive
patients. In a subsequent study of 225 tumor-bank samples from
patients with ER-positive/node-negative breast cancer, the H:I
expression ratio predicted relapse-free survival in a multivariate
analysis (hazard ratio = 3.9, 95% confidence interval [CI] 1.5–10.3, P =
0.007).6 An optimized cut-point (H:I value >1.0) was used, resulting in a
relapse-free survival prediction that was independent of tamoxifen
therapy and standard clinical and pathologic prognostic markers. In
addition, risk increased with increasing H:I expression ratio (Figure 1).
Individuals Suitable for Testing include treatment-naïve individuals
with ER-positive/lymph node-negative breast cancer.
Method: Sample analysis begins with laser capture microdissection if
the sample contains <30% cancer cells. Real-time PCR analysis of
HOXB13 and IL-17BR gene expression is then performed. Results are
reported as normalized H:I expression ratio with 5-year recurrence risk.
Aliases for this test include Homeobox 13 (HOXB13) and Interleukin 17B
Receptor (IL-17BR).
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: The H:I ratio serves as a continuous marker
of recurrence risk in untreated ER-positive/node-negative patients
(Figure 1).
The H:I ratio should not be used to predict response to therapy. The
results should be interpreted in light of other relevant clinical and
laboratory findings.
References
1. American Cancer Society: Cancer facts and figures 2006. Available
at: http://www.cancer.org/downloads/STT/CAFF2006PWSecured.pdf.
Accessed September 1, 2006.
2. Goldhirsch A, Glick JH, Gelber RD, et al. Meeting highlights:
International expert consensus on the primary therapy of early breast
cancer 2005. Ann Oncol. 2005;16:1569-1583.
3. Fisher B, Jeong J-H, Bryant J, et al. Treatment of lymph-node-
negative, oestrogen-receptor-positive breast cancer: long-term
findings from National Surgical Adjuvant Breast and Bowel Project
randomised clinical trials. Lancet. 2004;364:858-868.
4. Ma X-J, Wang Z, Ryan PD, et al. A two-gene expression ratio
116
Section 6
    





     






   
Figure 1. HOXB13:IL17BR gene expression ratio as a continuous predictor of recurrence at 5 years in patients (n=308) with early stage (ie, I or II)
primary, untreated, ER-positive/node-negative breast cancer.6 Vertical line, ratio = 1.0; solid line, predicted recurrence rate by H:I ratio; dashed lines,
95% CI. Reprinted with permission from the American Society of Clinical Oncology.6
predicts clinical outcome in breast cancer patients treated with
tamoxifen. Cancer Cell. 2004;5:607-616.
5. Goetz MP, Suman VJ, Ingle JN, et al. A two-gene expression ratio of
homeobox 13 and interleukin-17B receptor for prediction of
recurrence and survival in women receiving adjuvant tamoxifen. Clin
Cancer Res. 2006;12:2080-2087.
6. Ma X-J, Hilsenbeck SG, Wang W, et al. The HOXB13:IL17BR
expression ratio is a prognostic factor in early-stage breast cancer.
J Clin Oncol. 2006;24:4611-4619.
6.3.2 CA 15-3
Clinical Use: This test is used for therapeutic monitoring of patients
with metastatic breast cancer and for early detection of recurrent breast
cancer.
Clinical Background: Both CA 15-3 and CA 27.29 have received FDA
approval for monitoring patients with advanced breast cancer. The
American Society of Clinical Oncology (ASCO) guidelines, however, do
not encourage routine use of these markers for monitoring response to
treatment. Nevertheless, the ASCO guidelines do support using these
markers to suggest treatment failure in the absence of readily
measurable disease (J Clin Oncol. 2001;19:1865-1878 and 4185-4188).
Interpretive Information: Normal values for CA 15-3 are <32 U/mL.
CA15-3 levels are increased in less than 50% of patients with early-
stage breast cancer, substantially limiting the usefulness in that
Hematology/Oncology
      
 


population. However, in more advanced disease, a rising CA 15-3 level
may have clinical utility (Clin Chem. 2002;48:1151-1159).
6.3.3 CA 27.29
Clinical Use: This test is used for therapeutic monitoring of patients
with metastatic breast cancer and for early detection of recurrent breast
cancer.
Clinical Background: CA 27.29 is an epitope on the protein core of
the MUC-1 mucin glycoprotein (a breast cancer-associated antigen). This
epitope is molecularly similar to the one recognized by the DF3
monoclonal antibody used in the CA 15-3 assay;1 therefore, both
antibodies detect the same antigen. Elevated CA 27.29 levels are
primarily associated with metastatic breast cancer. In a prospective
study of 97 patients with metastatic breast cancer, 73% of patients with
disease progression had a r20% increase in CA 27.29 levels.
Conversely, 71% of those without progression did not have a 20%
increase in CA 27.29 levels.2 Additional studies further support the use
of CA 27.29 levels for therapeutic monitoring.3,4
In another prospective study of 162 stage II and stage III breast cancer
patients who were clinically free of disease at time of enrollment,
CA 27.29 was positive in 60% of the patients who subsequently
suffered a cancer recurrence (sensitivity = 60%). CA 27.29 was not
elevated in 93% of the patients free of recurrence (specificity = 93%).
Two consecutive positive CA 27.29 values were predictive of breast
 117

Section 6 Hematology/Oncology

cancer recurrence 80% of the time and, conversely, a negative CA 27.29 Table 5. Distribution of CA 27.292,6
value was predictive of the absence of a recurrence 91% of the time.2
Similar results had been obtained previously;5 consequently, CA 27.29 Number of Percent (%)
levels may be useful for predicting recurrent breast cancer. Subjects r39 U/mL

CA 27.29 levels are not useful for screening or diagnosis of malignant Apparently Healthy Women 314 1
disorders. Malignant Conditions

Individuals Suitable for Testing include patients with a metastatic Primary breast 37 5
breast cancer diagnosis. Metastatic breast 97 81

Method: This immunochemiluminescence assay (ICMA, ADVIA Stomach 29 7

Centaur®’ BR, Bayer Corporation) uses a mouse-monoclonal antibody Colon 43 26
(Mab B27.29) targeted toward the SAPDTRPA amino acid sequence on
the MUC-1 molecule.1 The analytical sensitivity is 3.5 U/mL. The assay Lung 47 43
is not affected by chemotherapeutic agents, therapeutic drugs, CEA, Pancreas 45 47
CA 125, CA 19-9, bilirubin, triglycerides, hemoglobin, or total protein.
Aliases include BR, cancer antigen 27.29, breast cancer marker, breast Ovarian 50 56
carcinoma associated mucin antigen, and cancer associated breast Liver 20 55
antigen (CABA).
Prostate 34 18
Values obtained from different methods are not interchangeable due to Uterus 30 13
variations in reagent specificity and other methodological differences.
Use only one method when monitoring patients. If the methodology is Nonmalignant Conditions
changed, re-baselining prior to making clinical decisions is highly Benign breast 129 3
recommended.
Pregnancy 49 2
Interpretive Information: Elevated CA 27.29 levels are associated Lactating women 37 11
with metastatic breast cancer and sometimes with primary breast
cancer.5,6 CA 27.29 is also elevated in ovarian, liver, pancreatic, and lung Cirrhosis 25 24
cancer, as well as in the nonmalignant conditions detailed in Table 5. Endometriosis 24 8
Rising levels suggest lack of therapeutic response and/or progressive
disease, whereas decreasing levels suggest response to treatment and Chronic hepatitis 48 10
disease regression. CA 27.29 elevations in patients with a history of Ovarian cyst 50 6
breast cancer and no clinical evidence of disease suggest recurrence.
Renal impairment 20 15
Falsely elevated or depressed values may occur in samples obtained
from patients who have received mouse monoclonal antibody
preparations during diagnosis or therapy. Such patients may have 6. Bon GG, von Mensdorff-Pouilly S, Kenemans P, et al. Clinical and
developed human anti-mouse antibodies (HAMA) that interfere with technical evaluation of ACS BR serum assay of MUC1 gene-derived
accurate analysis. Levels within the normal range do not preclude the glycoprotein in breast cancer, and comparison with CA 15-3 assays.
presence of cancer, nor are elevated results an absolute indication of Clin Chem. 1997;43:585-593.
malignancy. CA 27.29 test results should be interpreted in conjunction
with other clinical and laboratory findings. The serum half-life of 6.3.4 CellSearch®’ Circulating Tumor Cells, Breast Cancer
CA 27.29 has not been established.
Clinical Use: This test is used to predict progression-free survival
References (PFS) and overall survival (OS) in patients with metastatic breast cancer.
1. Reddish MA, Helbrecht N, Almeida A, et al. Epitope mapping of Mab Clinical Background: Circulating tumor cells (CTCs) are extremely
B27.29 within the peptide core of the malignant breast carcinoma- rare in healthy individuals and patients with nonmalignant diseases but
associated mucin antigen coded for by the human MUC 1 gene. J are present in patients with various metastatic carcinomas with a wide
Tumor Marker Oncol. 1992;7:19-27. range of frequencies.1-2 Some clinical studies indicate the assessment of
2. CA 27.29 assay directional insert. Bayer Corporation, 2000. CTCs can assist physicians in monitoring and predicting cancer
3. Abbate I, Correale M, Musci MD, et al. Monoclonal antibody B27.29 progression and in evaluating response to therapy in patients with
against mucinous breast cancer associated antigen CA 27.29 in metastatic cancer.3-5
breast cancer. Breast Cancer ResTreat. 1991;19:123.
4. Dnistrain AM, Schwartz MK, Greenberg EJ, et al. Evaluation of a In patients with metastatic breast cancer, the number of CTCs has been
breast cancer antigen assayed on the Ciba Corning ACS:180 shown to be a significant predictor of PFS and OS.6,7 In a study by
Automated Chemiluminescence System. J Tumor Marker Oncol. Cristofanilli et al,7 patients undergoing treatment for metastatic breast
1996;11:5-10. cancer with r5 CTCs/7.5 mL whole blood had shorter median PFS (2.7
5. Chan DW, Beveridge RA, Muss H, et al. Use of Truquant BR months vs 7.0 months, P0.001) and shorter OS (10.1 months vs 18
radioimmunoassay for early detection of breast cancer recurrence in months, P0.001) than patients having 5 CTCs/7.5 mL blood. In the
patients with stage II and stage III disease. J Clin Oncol. same study, a decrease in the number of CTCs to 5 from baseline to
1997;15:2322-2328. first follow-up (4-5 weeks after initiation of new therapy) was also
118
Section 6
found to be predictive of PFS and OS. These results suggest the number
of CTCs is a useful prognostic guide for patients with metastatic breast
cancer and can reliably estimate disease progression and survival
earlier (4-5 weeks vs 8-12 weeks, respectively) than traditional imaging
methods.
Individuals Suitable for Testing include patients with metastatic
breast cancer, prior to a new course of therapy and at follow-up.
Method: This method begins with an immunomagnetic sample
enrichment using antibodies targeting epithelial cell adhesion molecule
(EpCAM). Enrichment is followed by cell labeling with fluorescent
nucleic acid dye. Epithelial cells are distinguished from leukocytes by
using fluorescent labeled monoclonal antibodies specific for leukocytes
(CD-45) and epithelial cells (cytokeratins 8,18, and 19). Results are
reported as the number of CTCs/7.5 mL of whole blood. The analytical
sensitivity and specificity are 1 CTC/7.5 mL whole blood and 99.7%2,
respectively.
Interpretive Information: A CTC count r5/7.5 mL prior to therapy or
at first follow-up is predictive of shorter PFS and OS.7,8 A decrease in
the number of CTCs to 5/7.5 mL from baseline to first follow-up is
associated with longer PFS and OS (Table 6).
Antibodies used in the CellSearch assay are targeted at cell markers
(EpCAM and cytokeratins 8, 18, and 19) expressed by
adenocarcinomas.9-12 CTCs that do not express these markers will not be
detected by the CellSearch assay, whereas CTCs from non-breast
malignancies expressing these markers may be detected. CellSearch
test results should be interpreted in conjunction with other clinical and
laboratory findings.
References
1. Molnar B, Sipos F, Galamb O, et al. Molecular detection of
circulating cancer cells. Dig Dis. 2003;21:320-325.
2. Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the
peripheral blood of all major carcinomas but not in healthy subjects
or patients with nonmalignant diseases. Clin Cancer Res.
2004;10:6897-6904.
Table 6. Prediction of Survival Based on Circulating Tumor
Cell Count7
CTC Count
(# CTCs/7.5mL blood) PFS (months)
Prior to therapy
5 7
r5 2.7
First follow-up
5 6.1
r5 1.3
Baseline & first follow-up
Both 5 7 18
Baseline r5; follow-up 5 7.6
Both r5 2.1
CTC, circulating tumor cell; PFS, progression-free survival; OS, overall survival.
Hematology/Oncology
3. Aquino A, Prete SP, Balduzzi A, et al. A novel method for monitoring
response to chemotherapy based on the detection of circulating
tumor cells: a case report. J Chemother. 2002;14:412-416.
4. Katoh M, Neumaier M, Nezam R, et al. Correlation of circulating
tumor cells with tumor size and metastatic load in a spontaneous
lung metastasis model. Anticancer Res. 2004;24:1421-1425.
5. Berrepoot LV, Mehra N, Vermaat JS, et al. Increased levels of viable
circulating endothelial cells are an indicator of progressive disease
in cancer patients. Ann Oncol. 2004;15:139-145.
6. Weigelt B, Bosma AJ, Hart AA, Rodenhuis S, et al. Marker genes
for circulating tumour cells predict survival in metastasized breast
cancer patients. Br J Cancer. 2003;88:1091-1094.
7. Cristofanilli M, Budd T, Ellis M, et al. Circulating tumor cells,
disease progression, and survival in metastatic breast cancer.
N Engl J Med. 2004;351:781-791.
8. Cristofanilli M, Hayes DF, Budd GT, et al. Circulating tumor cells: a
novel prognostic factor for newly diagnosed metastatic breast
cancer. J Clin Oncol. 2005;23:1420-1430.
9. Osta W, Chen Y, Mikhitarian K, et al. EpCAM is overexpressed in
breast cancer and is a potential target for breast cancer gene
therapy. Cancer Research. 2004;64:5818-5824.
10. Armstrong A, Eck S. EpCAM: A new therapeutic target for an old
cancer antigen. Cancer Biology & Therapy. 2003;2:320-326.
11. Gaforio JJ, Serrano MJ, Sanchez-Rovira P, et al. Detection of breast
cancer cells in the peripheral blood is positively correlated with
estrogen-receptor status and predicts for poor prognosis. Int J
Cancer. 2003;107:984-990.
12. Abd El-Rehim DM, Pinder SE, Paish CE, et al. Expression of luminal
and basal cytokeratins in human breast carcinoma. J Pathol.
2004;203:661-671.
6.3.5 HER-2/neu Testing
Clinical Use: This test is used to predict 5-year disease-free and
overall survival in patients with breast cancer, assess eligibility for
trastuzumab (Herceptin®’) treatment, assist in dose selection for certain
drugs, and predict response to drug therapies.
Clinical Background: The HER-2/neu (human epidermal growth
factor receptor 2) proto-oncogene, 1 of a family of 4 closely related
growth factor receptor genes, encodes a 185-kd tyrosine kinase. HER-
2/neu gene amplification can lead to overproduction of the HER-2
protein and to tumor development through enhanced cell proliferation,
survival, motility, and adhesion. HER-2/neu amplification and
overexpression are observed in approximately 20% of invasive breast
cancers and are associated with an aggressive disease course and
decreased disease-free and overall survival.1,2
OS (months)
HER-2 status is most often used to determine patient eligibility for
trastuzumab immunotherapy. Trastuzumab, a humanized monoclonal
18 antibody directed against the extracellular domain of HER-2, inhibits
10.1 proliferation of human tumors cells that overexpress HER-2.
Documentation of HER-2 overexpression, either directly with immuno-
histochemistry (IHC) or indirectly with fluorescence in situ hybridization
18 (FISH), is therefore recommended before prescribing trastuzumab therapy.3
Patients whose breast tumors amplify the HER-2 gene and/or overexpress
7 the HER-2 protein are suitable candidates for this treatment.4-6
HER-2 amplification or overexpression status can also be helpful when
considering other types of therapy. HER-2-positive tumors have been
14.6 associated with increased sensitivity to anthracycline (eg, doxorubicin
[Adriamycin®’]) and cyclophosphamide/doxorubicin/5-fluorouracil (CAF)
8.2 chemotherapy.7-10 Conversely, such patients do not benefit as much from
cyclophosphamide/methotrexate/5-fluorouracil (CMF) regimens as do

Section 6
those with HER-2-negative tumors.11-14 HER-2-positive patients also tend
to have diminished sensitivity to endocrine therapy (eg, tamoxifen);
however, the data are somewhat conflicting, and decisions regarding
endocrine treatment should not be based on the results of HER-2
testing.15 Similarly, the data regarding HER-2 status and taxanes are
insufficient for clinical use, although the tendency is for HER-2-positive
tumors to respond well to treatment.15 For example, a recent article
reported benefit from the addition of paclitaxel after adjuvant
doxorubicin/cyclophosphamide therapy in patients with node-positive,
HER-2-positive breast cancer, while patients with node-positive, HER-2-
negative breast cancer did not benefit.16
Individuals Suitable for Testing include all patients with invasive
breast cancer.1
Test Selection: There are several approaches for detecting HER-2/neu
overexpression or amplification. Currently, the only FDA-cleared
methods for assessing HER-2 status are IHC and FISH. Quest
Diagnostics offers both methods and follows the testing and reporting
recommendations of the expert American Society of Clinical
Oncology/College of American Pathologists (ASCO/CAP) Panel.1 The
Panel expressed no preference of one method over the other. Figure 2
portrays an algorithm for use and interpretation of IHC and FISH
methods; the algorithm is based on the ASCO/CAP Panel
recommendations.
IHC Method: The IHC method determines protein overexpression
status of the invasive component of the cancer by using an anti-human
HER-2 antibody. The stained tissue is visually examined and graded
using a 4-point scale. Protein overexpression is indicated by a score of
3+. Staining variations between the in situ and invasive components
may occur.
FISH Method: The FISH assay determines gene amplification status of
the invasive component of the cancer by using probes specific for the
HER-2/neu locus and CEP 17; CEP 17 is included as an internal control
and to account for aneusomy of chromosome 17. Results are reported as
the ratio of HER-2/neu signal to CEP 17 signal. A ratio of >2.2 indicates
gene amplification. This cut-point is recommended by ASCO/CAP Panel
and the NCCN,1,17 even though many clinical trials used a ratio cut-point
of r2.0.6 The FISH assay has an analytical sensitivity (ratio) of 1.5 and
has no known cross-hybridization with other loci.
Comparative information for these 2 methods can be found in Table 7.
Aliases for these assays include c-erbB-2, HER-2/neu, human epidermal
growth factor receptor-2, and p185 (IHC only).
Test Interpretation: HER-2 IHC and FISH results are reported as
shown in Table 7. HER-2 overexpression or amplification suggests
patient eligibility for trastuzumab treatment (Figure 2). In addition, stage
II, node-positive breast cancer patients with amplification may benefit
from higher doses of CAF.8,9
Lack of HER-2 overexpression or amplification suggests the patient will
not respond to trastuzumab therapy; however, clinical trials are needed
to confirm this presumption. Similarly, trastuzumab response, or lack
thereof, in patients with confirmed equivocal HER-2 test results is
uncertain. Trastuzumab is clinically indicated only for patients in whom
HER-2 overexpression or amplification can be confirmed.
References Cited
1. Wolff AC, Hammond MEH, Schwartz JN, et al. American Society of
Clinical Oncology/College of American Pathologists guideline
119
Hematology/Oncology
recommendations for human epidermal growth factor receptor 2
testing in breast cancer. Arch Pathol Lab Med. 2007;131:18-43.
2. Nunes RA, Harris LN. The HER2 extracellular domain as a prognostic
and predictive factor in breast cancer. Clin Breast Cancer.
2002;3:125-135; discussion 136-137.
3. Herceptin® (trastuzumab) package insert. South San Francisco, CA:
Genentech, Inc. 2006. Available at: http://www.gene.com/gene/
products/information/oncology/herceptin/insert.jsp. Accessed
October 24, 2007.
4. Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly
intravenous recombinant humanized anti-p185HER2 monoclonal
antibody in patients with HER2/neu-overexpressing metastatic
breast cancer. J Clin Oncol. 1996;14:737-744.
5. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the
efficacy and safety of humanized anti-HER2 monoclonal antibody in
women who have HER2-overexpressing metastatic breast cancer
that has progressed after chemotherapy for metastatic disease.
J Clin Oncol. 1999:17:2639-2648.
6. Gonzales-Angulo AN, Hortobagyi GN, Esteva FJ. Adjuvant therapy
with trastuzumab for HER-2/neu–positive breast cancer. Oncologist.
2006;11:857-867.
7. Paik S, Bryant J, Park C, et al. erbB-2 and response to doxorubicin in
patients with axillary lymph node-positive, hormone receptor-
negative breast cancer. J Natl Cancer Inst. 1998;90:1361-1370.
8. Muss HB, Thor AD, Berry DA, et al. c-erbB-2 expression and
response to adjuvant therapy in women with node-positive early
breast cancer [erratum appears in N Engl J Med. 1994;331:211].
N Engl J Med. 1994;330:1260-1266.
9. Thor AD, Berry DA, Budman DR, et al. erbB-2, p53, and efficacy of
adjuvant therapy in lymph node-positive breast cancer. J Natl
Cancer Inst. 1998;90:1346-1360.
10. Dressler LG, Berry DA, Broadwater G, et al. Comparison of HER2
status by fluorescence in situ hybridization and
immunohistochemistry to predict benefit from dose escalation of
adjuvant doxorubicin-based therapy in node-positive breast cancer
patients. J Clin Oncol. 2005;23:4287-4297.
11. Allred DC, Clark GM, Tandon AK, et al. HER-2/neu in node-negative
breast cancer: prognostic significance of overexpression influenced
by the presence of in situ carcinoma. J Clin Oncol. 1992;10:599-605.
12. Gusterson BA, Gelber RD, Goldhirsch A, et al. Prognostic importance
of c-erbB-2 expression in breast cancer. International (Ludwig)
Breast Cancer Study Group. J Clin Oncol. 1992;10:1049-1056.
13. Menard S, Valagussa P, Pilotti S, et al. Response to
cyclophosphamide, methotrexate, and fluorouracil in lymph node-
positive breast cancer according to HER2 overexpression and other
tumor biologic variables. J Clin Oncol. 2001;19:329-335.
14. Kostopoulos I, Arapantoni-Dadioti P, Gogas H, et al. Evaluation of
the prognostic value of HER-2 and VEGF in breast cancer patients
participating in a randomized study with dose-dense sequential
adjuvant chemotherapy. Breast Cancer Res Treat. 2006;96:251-261.
15. Yamauchi H, Stearns V, Hayes DF. When is a tumor marker ready for
prime time? A case study of c-erbB-2 as a predictive factor in breast
cancer. J Clin Oncol. 2001;19:2334-2356.
16. Hayes DF, Thor AD, Dressler LG, et al for the Cancer and Leukemia
Group B (CALGB) Investigators. HER2 and response to paclitaxel in
node-positive breast cancer. N Engl J Med. 2007;357:1496-1506.
17. Carlson RW, Moench SJ, Hammond MEH, et al. HER2 testing in
breast cancer: NCCN Task Force report and recommendations.
J Natl Compr Canc Netw. 2006;4(Suppl 3):S1-S22. Also available at:
http://www.nccn.org/JNCCN/PDF/her22006.pdf. Accessed October
24, 2007.
120

Section 6 Hematology/Oncology
 121

Section 6 Hematology/Oncology

Table 7. Technology Guide to HER-2 Tests Used by Quest Diagnostics

Immunohistochemistry (IHC) Fluorescence In Situ Hybridization (FISH)

Test code 15547X 14620X
Clinical use (FDA-cleared) Evaluate HER-2/neu overexpression in breast tumor tissue Detect HER-2/neu oncogene amplification
Assess eligibility for trastuzumab (Herceptin) treatment Assess eligibility for trastuzumab treatment
Assess prognosis of stage II, node-positive breast cancer
patients
Predict disease-free and overall survival in patients with
stage II, node-positive breast cancer treated with adjuvant
cyclophosphamide, doxorubicin (Adriamycin), 5-fluorouracil
(CAF) chemotherapy
Assesses Protein overexpression Gene amplification
Method Immunohistochemistry (IHC) with reflex to FISH Fluorescence in situ hybridization (FISH) with reflex to IHC
(see below) (see below)
Primary reagent Polyclonal anti-human HER-2 antibody DNA probe specific for the HER-2/neu gene locus
(17q11.2-q12) plus an internal control
Test interpretation Cell membrane stain intensity: Ratio of HER-2/neu: CEP 17 control
0 = negative <1.8 = negative for HER-2/neu amplification
1+ = negative
2+ = equivocal; reflexed to FISH at additional 1.8 to 2.2 = equivocala; reflexed to IHC at additional charge
charge
3+ = strongly positive for presence of HER-2/neu >2.2 = positive for HER-2/neu amplification
overexpression
Sample requirements 1 paraffin block containing invasive malignant tumor 1 paraffin block containing invasive malignant tumor
preserved in 10% neutral buffered formalin; ship at preserved in 10% neutral buffered formalin; ship at room
room temperature. temperature.
Alternatively, five 4-micron unstained sections mounted
on treated slides (SuperFrost Plus, Poly-L-lysine coated,
or silanized). Submit at room temperature within 4 to 6
weeks of sectioning.
Analysis time 1 to 3 days 2 to 4 days
a
Verified by a second observer, counting additional cells, and repeating the FISH test.

Additional References
1. Press MF, Slamon DF, Klom KJ, et al. Evaluation of HER-2/neu gene
amplification and overexpression: comparison of frequently used
assay methods in a molecularly characterized cohort of breast cancer
specimens. J Clin Oncol. 2002;20:3095-3105.
2. Hayes DF, Thor AD. c-erbB2 in breast cancer: development of a
clinically useful marker. Semin Oncol. 2002;29:231-245.
6.3.6 MIB-1 (Ki-67), IHC with Interpretation
Clinical Use: This assay is used to assess tumor cell proliferation
(analogous to flow cytometric S-phase fraction) and to predict 5-year
disease-free and overall survival in patients with breast cancer. This
test is also used to assist in diagnosis of anaplastic large cell non-
Hodgkin’s lymphoma and may be used to determine prognosis in
patients with malignancies of the central nervous system, lung, ovary,
prostate, thyroid, renal pelvis, and ureter.
Clinical Background: MIB-1 is a monoclonal antibody raised against
recombinant segments of the Ki-67 antigen that is present in all stages
of the cell cycle except G0. Ki-67 increases during the latter half of the
S-phase and reaches a peak in the G2 and M phases of mitosis. MIB-1
reactivity correlates analytically with monoclonal Ki-67 antibody
reactivity1,2 and with the S-phase fraction.3 Unlike the Ki-67 monoclonal
antibody, which is reactive only in fresh frozen tissue, the MIB-1 antibody
is suitable for testing archival paraffin embedded tissues. This assay can
be used for tissue samples that cannot be analyzed by flow cytometry.
MIB-1 reactivity, ie, percent cells staining positive, correlates with 5-year
disease-free and overall survival in breast cancer patients,4,5 and is useful
in the diagnosis of anaplastic large cell non-Hodgkin’s lymphoma.6,7
Because MIB-1 staining is an indication of cellular proliferative activity, it
appears to be a useful indicator of poorer prognosis in individuals with
malignancies of the lung,8 ovary,9 prostate,10 renal pelvis and ureter,11 and
menigiomas.12 MIB-1 expression in cervical Papanicolaou tests correlates
with dysplasia in subsequent cervical biopsies13 and is useful in
distinguishing benign from malignant nodular thyroid lesions.14
Individuals Suitable for Testing include those with breast cancer,
non-Hodgkin’s lymphoma, and those with malignancies of the central
nervous system, lung, ovary, prostate, thyroid, renal pelvis, and ureter.
Method: This immunohistochemical assay utilizes standard antigen
unmasking procedures followed by incubation of 5-micron tissue
sections with MIB-1 antibody. Immunoreactivity is detected with a
labeled streptavidin-biotin and diaminobenzedine (DAB) system. A
122
Section 6
Table 8. Probability of Survival in Individuals with Breast Cancer
Tumor Cells Stained (%) Interpretation
<10 Negative
10–20 Borderline
>20 Positive
pathologist reports the estimated percent of malignant cells staining
positive with the MIB-1 antibody. This assay is suitable for small
samples (eg, sextant needle biopsies and cytology cell blocks) and
archival paraffin-embedded tissues.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Positive results are associated with more
aggressive breast cancer and shorter survival. Negative results are
associated with a prolonged survival and less aggressive disease (Table
8). An increased percentage of cells staining positive indicates a greater
degree of proliferative activity, and, thus, may be associated with a
more aggressive tumor type and poorer prognosis.
References
1. Kelleher L, Magee HM, and Dervan PA: Evaluation of cell-
proliferation antibodies reactive in paraffin sections. Appl
Immunohistochem. 1994;2:164-170.
2. Mauri FA, Girlando S, Palma PD, et al: Ki-67 antibodies (Ki-S5, MIB-
1, and Ki-67) in breast carcinomas: a brief quantitative comparison.
Appl Immunohistochem. 1994;2:171-176.
3. Ellis PA, Makris A, Burton SA, et al: Comparison of MIB-1
proliferation index with S-phase fraction in human breast
carcinomas. Br J Cancer. 1996;73:640-643.
4. Brown RW, Allred DC, Clark GM, et al: Prognostic value of Ki-67
compared to S-phase fraction in axillary node negative breast
cancer. Clin Cancer Res. 1996;2:585-592.
5. Veronese SM, Maissano C, and Scibilia J: Comparative prognostic
value of Ki-67 and MIB-1 proliferation indices in breast cancer.
Anticancer Res. 1995;15:2717-2722.
6. Frost M, Newell J, Lones MA, et al. Comparative
immunohistochemical analysis of pediatric Burkitt lymphoma and
diffuse large B-cell lymphoma. Am J Clin Pathol. 2004;121:384-392.
7. Colomo L, Lopez-Guillermo A, Perales M, et al. Clinical impact of the
differentiation profile assessed by immunophenotyping in patients
with diffuse large B-cell lymphoma. Blood. 2003;101:78-84.
8. Shiba M, Kohno H, Kakizawa K, et al. Ki-67 immumostaining and
other prognostic factors including tobacco smoking in patients with
resected nonsmall cell lung carcinoma. Cancer. 2000;89:1457-1465.
9. Munstedt K, von Georgi R, Franke FE. Correlation between MIB1-
determined tumor growth fraction and incidence of tumor
recurrence in early ovarian carcinomas. Cancer Invest. 2004;22:185-
194.
10. Pollack A, DeSilvio M, Khor LY, et al. Ki-67 staining is a strong
predictor of distant metastasis and mortality for men with prostate
cancer treated with radiotherapy and mortality for men with
prostate cancer treated with radiotherapy plus androgen
deprivation: Radiation Therapy Oncology Group Trial 92-02. J Clin
Oncol. 2004;22:2133-2140.
11. KojimaK, Narou S, Kanayma H, et al. Evaluation of Ki67 antigen
using MIB1 antibody as a prognostic factor in renal pelvis and
ureteral cancer. Nippon Hinyokika Gakkai Zasshi. 1996;87:822-830.
Hematology/Oncology
Proliferative Index Indicated Probability of Survival
Low Increased
Borderline Borderline
High Decreased
12. Torp SH, Lindboe CF, Gronberg BH, et al. Prognostic significance of
Ki-67/MIB-1 proliferation index in meningiomas. Clin Neuropathol.
2005;24:170-174.
13. Zeng Z, Del PG, Cohen JM, et al. MIB-1 expression in cervical
Papanicolaou tests correlates with dysplasia in subsequent cervical
biopsies. Appl Immunohistochem Mol Morphol. 2002;10:15-19.
14. Lewy-Trenda I, Janczukowicz J, Wierzchniewska-Lawska A. Pratical
application of proliferation markers’ (MIB-1, PCNA, AgNOR)
expression analysis for the differential diagnostics of nodular
thyroid lesions. Wiad Lek. 2006;59:32-37.
6.3.7 Micrometastasis Detection, IHC, Lymph Node or Bone
Marrow
Clinical Use: This assay is used to detect micrometastases of
epithelial cell origin (eg, breast cancer), determine the stage of
epithelial cancers, and to predict cancer recurrence/relapse and
diminished overall survival.
Clinical Background: Cytokeratins are expressed by both normal and
malignant epithelial cells, but not by lymph node cells. Thus, the
presence of cytokeratin-positive cells in lymph nodes is suggestive of
metastatic tumor. Studies detecting multiple chromosomal aberrations
in these suspected cytokeratin-positive micrometastases further
substantiate that these cells are tumor cells.
Studies have shown improved breast cancer staging when cytokeratin
immunocytochemical staining of the sentinel lymph node (SLN) biopsy
was combined with routine hematoxylin and eosin (H&E) stain. SLN
sensitivity for node-positive cancer was improved 10% to 25% by
identification of cytokeratin-positive cells. Other studies, however, show
no improvement in sensitivity due to cytokeratin staining when careful
histological examination of serial sections is employed. Variations in study
results may be due to the low number of subjects involved in the studies,
differences in the cytokeratin antibody specificity, as well as other factors.
A bone marrow study (N Engl J Med. 2000;342:525-533) showed that
the presence of cytokeratin-positive micrometastases increases the risk
of relapse in breast cancer stages I-III. Four-year overall survival was
93% for patients without versus 68% for patients with bone marrow
micrometastasis at the time of primary surgery (p <0.001). This
difference in survival data was irrespective of lymph node status and
was observed in the absence of adjuvant chemotherapy. Bone marrow
micrometastasis in lymph node-negative cases raised the risk to that of
node-positive cases. As reported in another study, finding
micrometastases after chemotherapy similarly increases risk of relapse.
Method: This immunohistochemical assay (IHC) uses the AE1/AE3
monoclonal antibody to detect the presence of cells expressing both low
and high molecular weight cytokeratins. The stained tissue sections are
reviewed by a pathologist who subsequently issues a detailed
interpretive report.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and

Section 6
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A positive result indicates the presence of
cytokeratin-positive cells suggestive of micrometastasis. In sentinel
lymph nodes, such a micrometastasis may more accurately define the
cancer stage. In bone marrow, such micrometastases may predict future
overt distant metastasis and diminished overall survival.
6.4 Cervical Cancer
6.4.1 Cervical Cancer Screening, SurePath™’ Liquid-Based
Pap Test
Clinical Use: This test is used to screen for cervical cancer.
Clinical Background: The Papanicolaou (Pap) test is the standard of
care for early detection and prevention of cervical cancer. The US
Preventive Services Task Force recommends routine Pap testing at least
every 3 years for all women with a cervix who are, or have been,
sexually active. The American College of Obstetricians and Gynecologists
(ACOG) recommends that the first screeing occur approximately 3 years
after first sexual intercourse or by age 21, whichever comes first.
Thereafter, ACOG recommends annual Pap testing for women under 30
years of age and for those at high risk.1 For women older than 30 years
who have had 3 consecutive, annual tests that are negative for
intraepithelial lesions and malignancy, ACOG recommends one of two
options.1 The first is that such women be re-screened by Pap alone every
2-3 years, and the second is that they be re-secreened by the combined
use of a Pap test and an HPV (Human Papillomavirus) test, as further
described in the section below (Section 6.4.2).
Conventional Pap testing is based on microscopic review of a cervical
specimen smeared on a slide and stained. Though successful in
reducing the incidence of invasive cervical cancer, conventional Pap
testing is limited, and there were no technological advancements for
about 50 years. In the late 1990s, however, new FDA-approved,
automated, liquid-based technologies were introduced. These
technologies substantially improve the number of satisfactory samples
and improve disease detection by identifying more low- and high-grade
squamous intraepithelial lesions (LSIL and HSIL, respectively).2-4
The SurePath Pap test employs this liquid-based technology. Cervical
samples are collected, and the entire sample is immediately placed in a
vial of liquid preservative, ensuring that 100% of cells collected are
received in the laboratory. During processing, non-diagnostic debris is
partially removed and the number of white blood cells is significantly
reduced. Fixed cells are then sedimented as a thin layer on a slide and
stained. Thus, epithelial cells, diagnostically relevant cells, and
infectious organisms are more clearly visible. The technology employed
by the SurePath Pap test results in a 39% reduction in unsatisfactory
slides and a 44% reduction in satisfactory, but limited, slides relative to
conventionally (non-liquid-based) prepared slides.5
Method: Cervical cytology is performed after liquid-based, density
gradient sedimentation of cervical cells. When a physician
interpretation is required, there will be an additional charge.
Interpretive Information: Quest Diagnostics follows the Bethesda
System of reporting for results and their interpretation.
References
1. American College of Obstetricians and Gynecologists. ACOG Practice
Bulletin. Clinical management guidelines for obstetrician-
123
Hematology/Oncology
gynecologists. Number 45, August 2003. Cervical Cytology Screening.
Obstet Gynecol. 2003; 102:417-427.
2. Vassilakos P, Saurel J, Rondez R. Direct-to-vial use of the AutoCyte
PREP liquid-based preparation for cervical-vaginal specimens in three
European laboratories. Acta Cytol. 1999;43:65-68.
3. Day SJ, Deszo EL, Freund GG. Dual sampling of the endocervix and
its impact on AutoCyte Prep endocervical adequacy. Am J Clin Pathol.
2002;118:41-46.
4. Vassilakos P, Carrel S, Petignat P, et al. Use of automated primary
screening on liquid-based, thin-layer preparations. Acta Cytol.
2002;46:291-295.
5. PrepStain™ Slide Processor Product Insert. Doc. No. 61CR000021,
Rev 4. Available at http://www.tripathimaging.com/us_ps_bpi_
pi.htm. Accessed 12-16-05.
6.4.2 HPV (Human Papillomavirus), Hybrid Capture II
Clinical Use: This test is used to determine the need for colposcopy in
individuals with ASCUS (atypical squamous cells of uncertain
significance) Pap test results, and as an adjunct to cervical cytology to
assist in guiding patient management.
Clinical Background: Human papillomavirus (HPV) infection is a very
common infection that is associated with condyloma, Bowenoid
papulosis, squamous intraepithelial lesions (SIL), and cancer. Although
HPV infection does not always progress to cancer, >93% of cervical
cancer cases are associated with HPV. Thirty of the more than 100 HPV
types infect the genital tract. This test detects 18 types: 5 low-risk types
that rarely develop into cancer and 13 types of intermediate to high risk
of developing into cancer. Both low-risk and intermediate/high-risk
types can cause cervical epithelial cell abnormalities.
Current guidelines suggest that the use of the combination of HPV DNA
testing and cervical cytology should be discontinued at the same age,
and under the same circumstances, as cervical cytology screening.1
Method: This nucleic acid hybridization method utilizes two DNA probe
cocktails, one specific for low-risk serotypes (types 6,11,42,43,44) and
one specific for intermediate/high-risk serotypes (types
16,18,31,33,35,39,45,51,52,56,58,59,68). Although the specific
serotype(s) cannot be reported, a result of positive or not detected is
reported for the low-risk serotypes and for the intermediate/high-risk
serotypes.
Interpretive Information: A “not detected” result is consistent with
the absence of HPV DNA, a level of HPV DNA below the detection limit
of the assay, or presence of a serotype other than those listed above.
The likelihood of HPV infection is low. The false-negative rate is
estimated to be b7.5% relative to HPV determination by polymerase
chain reaction (PCR). In individuals with ASCUS or LSIL Pap smear
results, absence of HPV DNA indicates a low probability of finding a
higher disease stage or severe disease at colposcopy.
A “positive” result indicates the presence of one or more of the HPV
serotypes listed above. The presence of low-risk serotypes indicates a
low probability of finding a higher disease stage at colposcopy and a
low probability of progression to cervical cancer. Conversely, the
presence of intermediate/high-risk HPV serotypes is associated with an
increased probability of finding high-grade disease at colposcopy and an
increased risk of progression to cervical cancer.
HPV DNA results must be interpreted in conjunction with other clinical
and laboratory data.
124

Section 6 Hematology/Oncology

Reference
1. Wright TC Jr, Schiffman M, Solomon D, et al. Interim guidance for
the use of human papillomavirus DNA testing as an adjunct to
cervical cytology for screening. Obstet Gynecol. 2004;103:304-309.
6.4.3 High-Risk HPV + Pap Test
Clinical Use: This test is used to screen for cervical cancer.
Clinical Background: The American Cancer Society (ACS)1 and the
American College of Obstetrics and Gynecology (ACOG)2 now
recommend a combination of cervical cytology and high-risk HPV testing
for routine cervical cancer screening in women age 30 and older. This
combination demonstrates high sensitivity for identifying high-grade
cervical intraepithelial neoplasia or cancer (CIN 2+). In numerous
studies, high-risk HPV was found in 85% to 100% of women with
biopsy-proven CIN 2+ and was a more sensitive indicator of CIN 2+ than
a single Pap test.1,3 Thus, the addition of high-risk HPV testing to
cervical cytology and other clinical information helps ensure appropriate
follow-up of women at increased risk of cervical cancer.
References
1. Saslow D, Runowicz C, Solomon D, et al. American Cancer Society
guideline for the early detection of cervical neoplasia and cancer.
CA Cancer J Clin. 2002;52:342-362.
2. ACOG Committee on Practice Bulletins. ACOG Practice Bulletin:
clinical management guidelines for obstetrician-gynecologists.
Number 45, August 2003. Cervical cytology screening (replaces
committee opinion 152, March 1995). Obstet Gynecol. 2003;102:417-
427.
3. Wright TC Jr, Schiffman M, Solomon D, et al. Interim guidance for
the use of human papillomavirus DNA testing as an adjunct to
cervical cytology for screening. Obstet Gynecol. 2004;103:304-309.
6.5 Gastrointestinal Cancer
6.5.1 CA 19-9
Clinical Use: This test is primarily used to monitor therapy in
individuals with pancreatic cancer. It is also useful for monitoring
therapy in selected individuals with gastric and colon cancer.

The negative predictive value (NPV) of this combination is very high: in Clinical Background: CA 19-9 is a mucin-glycoprotein derived from a
women with negative results on both tests, the risk of developing CIN human colorectal carcinoma cell line. It is related to the Lewis blood
2+ in the next 3 years is <2 in 1000 (summarized in reference 3). The group protein and is present in epithelial tissue of the stomach, gall
high NPV thus allows less-frequent screening for women with negative bladder, pancreas, and prostate. Blood levels may be increased
results on both tests. modestly in patients with pancreatitis, biliary tract disease,
inflammatory bowel disease, and cystic fibrosis. The distribution of CA
Although routine HPV testing is not recommended for women younger 19-9 in healthy individuals, benign disorders, and malignant disease is
than 30, HPV reflex testing is useful for directing follow-up in young reflected in Tables 9 and 10.
women with ASC-US cervical cytology results.
Interpretive Information: CA 19-9 concentrations are increased in
Method: This test uses microscopy for the Pap test and Hybrid patients with pancreatic, gastric, and colon cancer as well as in some
Capture®’ hc2 DNA detection for HPV testing. non-malignant conditions (Table 9). Increasing levels generally indicate
disease progression, whereas decreasing levels suggest therapeutic
Interpretive Information: Below is a summary of follow-up response.
recommendations for specific combinations of HPV and cervical cytology
results in women 30 years of age and older3:
• Women with negative cervical cytology and negative high-risk HPV
DNA results should not be re-screened in less than 3 years. Table 9. Distribution of CA 19-9
• Women with negative cytology findings but positive high-risk HPV
DNA results should be re-screened with both tests in 6 to 12 months. Number of % with CA 19-9
• If both tests show normal results at follow-up, routine screening Patient Group Patients >37.0 U/mL
at 3 years is appropriate.
• Women who are negative for high-risk HPV DNA but have cytology Healthy Subjects 1020 0.6
findings of ASCUS at follow-up should receive repeat cytology Benign Disease
and high-risk HPV DNA testing at 12 months.
• Women who remain positive for high-risk HPV DNA or have Pancreatic 21 0
cytology results >ASCUS (“cannot exclude high-grade squamous Inflammatory Bowel Disease 56 2
intraepithelial lesion,” low-grade squamous intraepithelial lesions,
or high-grade squamous intraepithelial lesions) at follow-up Polyps 27 0
should undergo colposcopy. Other Diseases 219 2
• In women with an ASCUS cervical cytology result, a concomitant
negative high-risk HPV result suggests a low probability of finding a Malignant Disease
higher disease stage at colposcopy; cytology should be repeated in Pancreatic Adenocarcinoma 80 79
12 months.1
• In women with an ASCUS cervical cytology result, a positive high-risk Gastric Adenocarcinoma 24 50
HPV DNA result suggests a low, but increased, probability of higher Colorectal Adenocarcinoma
stage disease; colposcopy should be performed.
• In women with cytology results >ASCUS, colposcopy should be Advanced 164 46
performed regardless of the HPV result. Localized 25 8
Inactive 21 0
Reference: DelVillano BC, et al. Clin Chem 1983;29:549-552.
 125

Section 6 Hematology/Oncology

Table 10. Distribution of CA 19-9 Table 11. Distribution of CA 72-4

% with CA 19-9 % with CA 72-4

Number of
in Indicated Range
Patient Group Patients >37 U/mL >75 U/mL Number of
Subject Group Subjects >6.0 U/mL >10.0 U/mL
Nonmalignant Disease
Disease Free 744 3.5 0.5
Abdominal Pain 34 3 3
Benign Disease
Jaundice 58 19 12
Stomach 75 5 1
Malabsorption 7 0 0
Colorectal 59 8 3
Pancreatic 48 19 6
Miscellaneous 17 0 0
Renal Failure 10 20 0
Malignant Disease
Malignant Disease
Gastric 199 42 34
Pancreas 37 89 86
Reference: Steinberg WM, et al. Gastroenterology. 1986;90:343-349. Stage IV 77 55 47
Colorectal 104 38 29

6.5.2 CA 72-4 Stage IV 41 54 39

Lung 165 16 12
Clinical Use: This test is used to monitor therapy and detect recurrence
in patients with CA 72-4-positive cancer (eg, gastrointestinal cancers). Adenocarcinoma 28 36 25
Squamous Cell 60 17 10
Clinical Background: CA 72-4 (TAG-72) is a high-molecular-weight
mucin glycoprotein derived from a tumor cell line. It is most useful as a Small Cell 61 11 7
marker for gastrointestinal cancer, but blood levels may be increased in Non-Small Cell 16 19 19
other malignancies (eg, lung cancer) and in non-malignant disorders
involving gastrointestinal tissues. Ovarian 92 24 20
Stage IV 15 53 40
Method: This enzyme immunoassay uses a capture antibody specific
for tumor associated glycoprotein 72 (TAG-72) and a biotin-conjugated Breast 66 12 11
detection antibody that binds to a different epitope of the TAG-72 Advanced prostatic 50 20 10
molecule. Addition of a streptavidin-labeled fluorescent tag initiates a
fluoroescent signal that is measured in a flow cytometer. Squamous 25 4 0
Sarcoma 29 3 3
Interpretive Information: Elevated levels (r4 U/mL) are associated Reference: Gero EJ, Colcher D, Ferroni P, et al. J Clin Lab Analysis. 1989;3:360-369.
with multiple tumor types and some non-malignant disorders (Table 11).
Increasing levels are associated with tumor progression, whereas
decreasing levels are associated with therapeutic response. This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
6.5.3 CD117, IHC necessary. Performance characteristics refer to the analytical performance of the test.

Clinical Use: This test is used to determine eligibility for Gleevec®’
(imatinib mesylate; STI571) treatment in patients with c-kit-positive
gastrointestinal stromal tumors (GISTs).
Clinical Background: The transmembrane glycoprotein c-kit (CD117),
a member of the receptor tyrosine kinase subclass III family, has been
implicated in a number of malignancies: GISTs, mast cell diseases,
acute myeloid leukemia, small cell lung carcinoma, and Ewing’s
sarcoma. Imatinib mesylate, a tyrosine kinase inhibitor, appears to be
effective in treating GISTs and other tumors that express c-kit.
Therefore, testing tumor tissue for c-kit immunoreactivity can help
predict sensitivity to tyrosine kinase inhibitor therapy.
Method: This immunohistochemical (IHC) assay uses a polyclonal
rabbit anti-human CD117/c-kit antibody, along with the Dako
EnVision®’+ Peroxidase detection kit (DakoCytomation), to detect
CD117/c-kit expression on tissue sections and cell preparations. The
EnVision+ detection system is a 2-step IHC staining technique
employing a horseradish peroxidase-labeled polymer conjugated with
secondary antibodies.
Interpretive Information: A positive result indicates tumor expression
of CD117/c-kit and eligibility for Gleevec therapy.
6.5.4 Colorectal Cancer
6.5.4.1 Laboratory Support of Diagnosis and Management
Clinical Background: Colorectal cancer (CRC) is the second leading
cause of cancer death in the United States, with projections of 55,000
deaths and 149,000 new cases diagnosed in 2006.1 About three-
quarters of CRC cases are sporadic, apparently resulting from
environmental factors, diet, and aging (Table 12). The remaining 25%
are familial or inherited. Familial CRC is not well understood and is
characterized by increased risk of CRC and an unclear pattern of
inheritance. Inherited CRC, on the other hand, exhibits a clear pattern of
inheritance and includes hereditary nonpolyposis colorectal cancer
(HNPCC) and familial adenomatous polyposis (FAP) as well as the more
rare MYH-associated neoplasia, Peutz-Jeghers, and juvenile polyposis
syndromes. Determining the type of CRC has important implications for
screening and follow-up of patients and family members.
126

Section 6 Hematology/Oncology

Table 12. Probability of Developing Colorectal Cancer2,3 Molecular Characteristics of CRC

The natural progression of CRC from benign adenoma to carcinoma to
Etiology % of New Cases Probability of Developing CRC metastatic disease is associated with 1of 2 distinct molecular
pathways. The most common pathway, chromosomal instability, occurs
Sporadic CRC 75 4% by age 79 in 80% to 85% of cases and is characterized by allelic loss (loss of
Familial CRC 15 to 20 1 first-degree relative with CRC: heterozygosity), chromosomal rearrangements, or loss of whole
9% by age 79 chromosomes. Such characteristics are associated with most sporadic
CRCs and FAP. The remaining 15% to 20% are associated with
More than 1 first-degree relative impairment of nucleotide mismatch repair (MMR) that normally occurs
with CRC: 16% by age 79 during DNA replication or recombination.
1 first-degree relative with CRC
before age 45: 15% by age 79 In tumor tissue, MMR defects are characterized by microsatellite
instability (MSI), defined as insertions or deletions of nucleotides within
1 first-degree relative with colo- repeated DNA nucleotide sequences known as microsatellites. The
rectal adenoma: 8% by age 79 MMR defect pathway is exemplified by HNPCC, an autosomal dominant
Hereditary CRC syndrome also known as Lynch syndrome. Individuals with HNPCC
HNPCC 5 80% by age 75 typically inherit 1 copy of a defective MMR allele; subsequent somatic
mutations may cause loss of the normal allele, leading to defective DNA
FAP 1 90% by age 45 repair. Approximately 51% of the mutations occur in MLH1, 38% in
Other Rare Attenuated FAP: 69% by age 80 MSH2, 10% in MSH6, and 2% in PMS2.4
MYH-associated neoplasia: Germline mutation of the adenomatous polyposis coli (APC) gene, a
unknown tumor suppressor gene, causes FAP, an autosomal dominant disorder
Peutz-Jeghers syndrome: 39% by with a prevalence of approximately 1 in 8000.
age 64
Screening for CRC
Juvenile polyposis syndrome: The American Cancer Society (ACS) recommends screening average-risk
17% to 68% by age 60 individuals with one of several options beginning at age 50 years (Table
CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; FAP, familial 13).5 Screening options should be chosen based on individual risk,
adenomatous polyposis. personal preference, and access. Of these screening tests, the fecal
occult blood test (FOBT) or fecal immunochemical test (FIT) are the only

Table 13. CRC Screening Recommendations

Population Screening Options Age to Begin Screening
Average-risk men and women* • Annual FOBT or FIT 50 years
• Flexible sigmoidoscopy every 5 years
• Annual FOBT and flexible sigmoidoscopy
every 5 years
• Double contrast barium enema every 5 years
• Colonoscopy every 10 years
1 second- or any third-degree Same as for those with average risk 50 years
relative† with CRC‡
First-degree relative of an individual with Same as for those with average risk 40 years
CRC or AP at age r60 years‡
2 second-degree relatives with CRC‡ Same as for those with average risk 40 years
r2 first-degree relatives with CRC, or Colonoscopy every 5 years 40 years or 10 years younger than the earliest
1 first-degree relative with CRC or AP at diagnosis in the family, whichever comes first
age <60 years‡
High risk for HNPCC (first-degree relative Colonoscopy every 1 to 2 years 20 to 25 years or 10 years younger than the
with HNPCC or carrier of known MMR gene earliest diagnosis in the family, whichever comes
mutation) ‡ first
High risk for FAP (first-degree relative with Annual sigmoidoscopy 10 to 12 years
FAP or carrier of known APC gene mutation) ‡
FOBT, fecal occult blood test; FIT, fecal immunochemical test; AP, adenomatous polyp; HNPCC, hereditary nonpolyposis colorectal cancer; FAP, familial adenomatous polyposis; APC,
adenomatous polyposis coli gene.
*Recommended by the American Cancer Society5
†
Third-degree relatives include great-grandparents and cousins.
‡
Recommended by the American Gastroenterology Association.7

Section 6
non-invasive tests; FOBT has led to the detection and surgical excision
of precancerous polyps, thereby significantly reducing the incidence of
CRC and related mortality.6 Flexible sigmoidoscopy is also associated
with reduced mortality for CRC,7 and combining FOBT with flexible
sigmoidoscopy is more effective than either test alone.5 Although less
commonly used, double contrast barium enema has the advantage of
examining the entire colon, but is less sensitive than colonoscopy in
detecting polyps.7
The American Gastroenterology Association recommends that high-risk
individuals be screened at a younger age and that screening be
performed more frequently (Table 13).7
Differential Diagnosis of CRC and Risk Assessment
Because HNPCC is the most common of the hereditary CRCs, criteria to
identify families with the disorder have been developed by the National
Cancer Institute (Bethesda guidelines)8 and the International
Collaborative Group on HNPCC (Amsterdam criteria).9
HNPCC is characterized by a high lifetime cancer risk and early age at
onset (mean z45 years). Because HNPCC progresses rapidly, early
detection is critical for affected individuals and their first-degree
relatives; close surveillance of affected family members can reduce
overall mortality by z65%.10 Additionally, women with HNPCC are at
high risk for endometrial cancer (40% to 70%) and ovarian cancer (10%
to 12%),4 and early detection of HNPCC or familial MMR mutations
allows close monitoring for these cancers.
FAP is characterized clinically by the presence of hundreds to thousands
of colorectal polyps identified at an early age (<30 years). FAP will
progress to colon cancer unless colectomy is performed. It is also
associated with an increased lifetime risk of other cancers, including
duodenal cancer (5% to 11% risk).11
Selection of Therapy for Patients with CRC
Pharmacogenomics, the study of genetic influences on drug response, is
becoming increasingly important in the selection of therapeutic agents.
Genetic polymorphisms are partially responsible for inter-patient
variability in drug efficacy and/or toxicity; detecting such polymorphisms
prior to treatment may help optimize drug selection and dosage.
Individuals Suitable for Testing
Screening, Differential Diagnosis, and Risk Assessment
Individuals suitable for testing include those recommended for CRC
screening (Table 13), those with CRC who meet any of the Bethesda
criteria (Table 14), those who meet the Amsterdam II criteria (Table 15),
and first-degree relatives of individuals with a known MMR gene
mutation.
Determine Prognosis, Select and Monitor Therapy
Individuals suitable for testing include those diagnosed with CRC.
Test Availability
Table 16 lists tests used to assess risk, screen, diagnose, determine
prognosis, and select and monitor therapy for CRC.
Test Selection and Interpretation
FOBT Screening
In 2003 the ACS guidelines for stool blood tests were expanded to
include fecal immunochemical tests (FITs).5 The traditional guaiac-based
FOBTs (eg, Hemoccult®’) suffer from false-positive results due to
ingestion of red meat, some raw fruits and uncooked vegetables, non-
127
Hematology/Oncology
Table 14. Bethesda Guidelines for Testing Colorectal Tumors
for MSI8
Colorectal tumors should be tested for MSI in individuals meeting any of
the following:
• CRC diagnosed at age <50 years
• Presence of synchronous CRC (multiple CRCs b6 months after initial
tumor removal), metachronous CRC (CRC recurrence >6 months after
initial tumor removal), or other HNPCC-associated tumors*
• CRC with the MSI-H histology† diagnosed at age <60 years
• CRC in r1 first-degree relative with an HNPCC-related tumor with 1
of the cancers diagnosed at age <50 years
• CRC diagnosed in r2 first- or second-degree relatives with HNPCC-
related tumors
*Endometrial, stomach, ovarian, pancreas, ureter and renal pelvis, biliary tract, and brain
tumors, sebaceous gland adenomas and keratoacanthomas in Muir-Torre syndrome, and
carcinoma of the small bowel.
†
Presence of tumor infiltrating lymphocytes, Crohn’s-like lymphocytic reaction, mucinous/
signet-ring differentiation, or medullary growth pattern.
steroidal anti-inflammatory drugs, and aspirin. Additionally, ingestion of
vitamin C (>250 mg/day) from supplements or citrus fruits may lead to
false-negative results. Thus, dietary and medication restrictions are
required prior to sample collection. Conversely, FITs such as InSure®’are
more specific and consequently do not require dietary and medication
restrictions.13 Furthermore, InSure is more specific for occult bleeding in
the colon and rectum thus making it less likely that bleeding is from the
upper gastrointestinal tract.13 In a recent clinical study comparing InSure
with a guaiac-based FOBT, InSure had a better true-positive rate for
early stage CRC (92.3% vs 30.8%, n = 13 stage I patients), all stages of
CRC (87.5% vs 54.2%, n = 24), and significant adenoma (42.6% vs
23.0%, n = 61).14 False-positive rates were z3%.
Positive FOBT or FIT results generally reflect the presence of blood in
the stool and may be associated with CRC. The ACS recommends
colonoscopy as follow-up to a positive test; flexible sigmoidoscopy or
repeat testing are not indicated.5 Negative results do not rule out CRC;
false-negative results can occur because of uneven distribution of blood
in the feces or intermittent bleeding.
Differential Diagnosis of CRC and Risk Assessment of Relatives
Since patients with CRC present with non-specific symptoms (eg,
change in bowel habit, unexplained weight loss, abdominal pain,
mucous discharge, or rectal bleeding) or with no symptoms, diagnosis is
based on colonoscopy and pathologic examination of the suspicious
tissue.
Table 15. Amsterdam II Criteria for Identifying Families with
HNPCC9
r3 relatives with an HNPCC-associated tumor*
• 1 is a first-degree relative of the other 2
• r2 successive generations affected
• r1 diagnosed at <50 years of age
• FAP should be excluded in the patients with CRC
• Tumor should be verified by pathologic examination
FAP, familial adenomatous polyposis.
*Defined in the first footnote of Table 14.
128

Section 6 Hematology/Oncology

Table 16. Tests Available for Diagnosis and Management of Colorectal Cancer
Test Code Assay Method Description Clinical Use
Screen
11290X Fecal Globin, Immunochemistry targeting globin portion of Screen for lower GI bleeding associated with CRC,
Immunochemistry hemoglobin; colorimetric detection adenomas, polyps, and other lower GI conditions
(InSure®’)
Not Hemoccult®’ Oxidation of guaiac by hemoglobin peroxidase; Screen for upper and lower GI bleeding
Applicable colorimetric detection
Diagnose or Assess Risk
14517X Tissue, Gastrointestinal Hematoxylin and eosin stain; microscopy Diagnose CRC
Pathology Report
14989X Microsatellite Instability Multiplex PCR amplification of 5 NCI-recommended Differential diagnosis of CRC; assess risk of HNPCC
(MSI), HNPCC* microsatellites; fluorescent fragment analysis in patients with CRC
14986X MLH1 and MSH2 PCR amplification of MMR gene regions;
Mutations, HNPCC* DNA sequencing

16051X MLH1 and MSH2 Multiplex PCR amplification of MMR gene regions; Confirmation of clinical diagnosis of HNPCC;
Mutations (Deletion and fluorescent fragment analysis identification of familial mutations in affected
Duplication), HNPCC* individuals

14982X MSH6 Mutation, PCR amplification of MMR gene regions;

HNPCC* DNA sequencing
14984X MLH1 Mutation,
One Exon, HNPCC*
14981X MSH2 Mutation, PCR amplification of MMR gene regions; Identify MMR gene mutation in family members
One Exon, HNPCC* DNA sequencing when the family mutation is known
14983X MSH6 Mutation,
One Exon, HNPCC*
Determine Prognosis
978X Carcinoembryonic Immunochemiluminometric assay Predict prognosis pretreatment
Antigen (CEA)
14603X Chromosome Analysis, Culture, microscopy, karyotype Assess prognosis in patients with CRC†
Solid Tumor
36158X DNA Cell Cycle Analysis, Flow cytometry Predict overall survival in patients with CRC
Paraffin Block
14989X Microsatellite Instability Multiplex PCR amplification of 5 NCI-recommended Predict overall survival in patients with CRC
(MSI), HNPCC* microsatellites; fluorescent fragment analysis
36162X p53 Oncoprotein* Immunohistochemical assay Predict recurrence-free survival in patients with
CRC
14517X Tissue, Gastrointestinal Hematoxylin and eosin stain; microscopy Predict survival in patients with CRC
Pathology Report
Select and Monitor Therapy
4698X CA 19-9 Immunochemiluminometric assay
15583X CA 72-4 EIA, fluorometric quantitation Monitor therapeutic response; detect residual
disease, detect recurrence
978X Carcinoembryonic Immunochemiluminometric assay
Antigen (CEA)
16109X Colorectal Cancer (CRC) PCR amplification followed by fluorescent fragment Guide chemotherapy selection (5-fluorouracil,
Pharmacogenomic Panel* analysis or single nucleotide primer extension oxaliplatin, and irinotecan) to minimize toxicity in
Includes UGT1A1, TYMS, advanced CRC; predict survival in patients
XRCC1, ERCC1, GSTP1, XPD, receiving these chemotherapeutic drugs
and DPYD gene analysis.

(continued)

Section 6
Table 16. Tests Available for Diagnosis and Management of Colorectal Cancer (continued)
Test Code Assay Method Description
15538X Dihydropyrimidine PCR amplification of target regions followed by
Dehydrogenase (DPYD) hybridization with mutant and wild-type
Gene Mutation Analysis*‡ oligonucleotides
10920X Epidermal Growth Factor Immunoassay
Receptor (EGFR), ELISA§
10479X Epidermal Growth Factor Immunohistochemical assay
Receptor (EGFR), IHC
19041X FISH, EGFR* Fluorescence in situ hybridization
17813X UGT1A1 Gene PCR amplification of promoter region of UGT1A1;
Polymorphism (TA fluorescent detection
Repeat)*
CRC, colorectal cancer; GI, gastrointestinal; HNPCC, hereditary nonpolyposis colorectal cancer; NCI, National Cancer Institute; MMR, mismatch repair.
*This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
†
See reference 12 for more details.
‡
This test is performed pursuant to a license agreement with Roche Molecular Systems Inc.
§
This test was performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been
determined by Quest Diagnostics Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means.
Once CRC is diagnosed, it is important to determine if the cancer is
hereditary. If HNPCC is diagnosed, or if family members have an
HNPCC-associated mutation, increased surveillance is required. Figure 3
depicts a suggested testing algorithm for the differential diagnosis of
CRC and risk assessment of family members. Details regarding these
tests follow.
Microsatellite Instability
The diagnosis of HNPCC begins with consideration of MSI testing.
Colorectal tumors should be tested for MSI when any of the Bethesda
criteria are met (Table 14). In a study of 1222 patients with CRC,
combining Bethesda criteria with MSI testing was more effective in
determining which patients should be tested for MMR mutations than
the use of either alone.15 The combination resulted in a sensitivity of
81.8%, a specificity of 98.0%, and an overall accuracy of 97.9% for
identifying patients with MLH1 or MSH2 mutations. Furthermore,
combining Bethesda criteria with MSI testing was more cost effective.
Results are reported as MSI-high (MSI-H), MSI-low (MSI-L), or negative
for MSI (microsatellite stable, MSS). A MSI-H result is reported if r2 of
the 5 National Cancer Institute-recommended markers show instability
and requires follow-up with MMR gene mutation testing (Figure 3).
Although an MSI-H result is the hallmark of HNPCC, it is also found in
15% to 20% of sporadic CRC cases.11 An MSI-L result, reflecting
instability in 1 marker, is found in <10% of HNPCC cases and in most
MSI-positive sporadic CRCs. Since MSI results do not rule out HNPCC,
MMR gene mutation testing should be considered regardless of MSI
status in families with a strong suspicion of HNPCC.8
MMR Gene Mutation Testing
For patients with suspected HNPCC and no known familial mutation,
begin MMR gene testing with MLH1 and MSH2. MLH1/MSH2 mutation
testing should include DNA sequence changes, which are more
commonly identified, and also deletion/duplication mutations, which are
found in z27% of families with HNPCC.16 If no MLH1/MSH2 mutations
are detected, MSH6 mutation testing should be performed (Figure 3).
Detection of an MMR gene mutation in a patient with CRC is diagnostic
of HNPCC. Failure to identify a relevant mutation makes HNPCC unlikely
but does not rule it out.
129
Hematology/Oncology
Clinical Use
Predict toxicity from pyrimidine-based
chemotherapeutic agents (5-fluorouracil,
capecitabine)
Determine suitability for EGFR-targeted drugs
Predict irinotecan toxicity; assist in selecting initial
dosage for patients with metastatic or recurrent
CRC
Once a diagnosis of HNPCC is established and an MMR gene mutation
is identified, first-degree relatives should be tested for the mutation
using a single-exon assay (Table 16; Figure 3). Relatives who carry the
family mutation are at high risk for HNPCC and should be monitored
closely (Table 13). For first-degree relatives of HNPCC patients who
meet the Amsterdam criteria but do not know the family mutation,
MLH1 and MSH2 mutation testing should be performed first; MSH6
mutation testing should then be considered for patients with negative
results (Figure 3). Because not all HNPCC families meet the Amsterdam
criteria, MMR mutation testing should also be considered when there is
a strong suspicion of HNPCC.3,16 Detection of an MMR gene mutation is
associated with high risk for HNPCC, whereas negative results are
consistent with average risk but do not rule out HNPCC.
Determining Prognosis
Tumor Staging
Tumor staging using either the American Joint Committee on Cancer
(AJCC) system17 or the Dukes system has historically been a powerful
tool for determining the prognosis of patients with CRC. Tumor staging
reflects the extent of CRC and provides prognostic information, as
demonstrated by the data in Table 17. The data were derived by
correlating AJCC stage with survival in more than 119,000 colon cancer
patients.18 Interestingly, stage IIIa CRC had a better prognosis than
stage IIb (P <0.001); this may be due to the current practice of initiating
chemotherapy for patients with stage III but not stage II disease.18 The
Dukes stage that corresponds to the AJCC stage is provided to give the
reader estimated survival times for the various Dukes stages.
Microsatellite Instability
Tissue testing for MSI may be used to assess prognosis independent of
CRC tumor stage. In a meta-analysis combining the results from 32
studies including 7,642 cases, overall survival was more favorable with
MSI than with MSS tumors (hazard ratio [HR] = 0.65, 95% confidence
interval [CI] 0.59–0.71).19 The survival advantage of patients with MSI
tumors was maintained when HRs were pooled for stage II and stage III
CRC patients (n = 2,935). However, the use of microsatellite status to
assess prognosis has not been investigated in a prospective clinical
trial.
130
Section 6
DNA Cell Cycle Analysis
DNA cell cycle analysis is used to determine ploidy, specifically
aneuploidy (an abnormal complement of chromosomes), in CRC tissue.
Because of conflicting data on the association of aneuploidy with a poor
outcome, the American Society of Clinical Oncology (ASCO) does not
recommend routine testing for ploidy.20
p53 Oncoprotein
Overproduction of p53 oncoprotein serves as a surrogate marker of p53
oncogene mutation, which is a common event in tumorigenesis.
Although some studies have found an association between p53-positive
tumor tissue and poor prognosis in patients with CRC, results have been
inconsistent.20
Hematology/Oncology
Carcinoembryonic Antigen (CEA)
Elevated pretreatment CEA levels (>5 ng/mL) are associated with poor
prognosis and predict eventual tumor recurrence.20
Selecting and Monitoring Therapy
Carcinoembryonic Antigen (CEA)
A CEA level, if elevated preoperatively, can be used to detect residual
disease following curative intent surgery in patients with primary CRC.
If tumor removal was complete, the CEA level should return to normal
within z6 weeks following surgery; persistently elevated levels suggest
residual or metastatic disease.21
Approximately 50% of patients who undergo surgery with curative intent
develop recurrent or metastatic disease.21 Serial CEA monitoring post

Section 6
Table 17. CRC Prognosis by Disease Stage18
AJCC Stage Dukes Stage TNM
I A T1 or T2, N0, M0
Ila B T3, N0, M0
IIb B T4, N0, M0
IIIa C T1 or T2, N1, M0
IIIb C T3 or T4, N1, M0
IIIc C Any T, N2, M0
IV D Any T, Any N, M1
AJCC, American Joint Committee on Cancer; TNM, tumor-node-metastasis.
*Colon cancer specific.
surgery is useful for detecting these conditions: the sensitivity and
specificity are z80% and z70%, respectively.21 Sensitivity is higher
(z100%) for detecting liver metastasis, which accounts for about 80% of
CRC recurrences, than for detecting locoregional recurrence (sensitivity
z60%).21 ASCO recommends testing every 3 months for at least 3 years
following diagnosis of stage II or III disease, providing the patient is a
candidate for further surgery or systemic therapy.20 While stable or
falling CEA levels suggest no disease progression, elevated levels, if
confirmed by retesting, are associated with disease progression and
warrant reevaluation for recurrent and metastatic disease.
Stage IV CRC (distant metastases) and locoregional recurrence may be
treated with surgical resection, chemotherapy, or radiation, depending
on the site and extent of the metastases or recurrence. In this setting,
CEA levels are useful for evaluating the success of surgical removal of
the metastasis and for monitoring chemotherapy. ASCO considers CEA
to be the marker of choice for monitoring chemotherapy and
recommends measurement before treatment and every 1 to 3 months
during treatment.20 While decreases in CEA levels during chemotherapy
suggest a favorable treatment response, persistently rising values above
pretreatment levels suggest disease progression. Rising values should
prompt reevaluation and consideration of alternative treatment.20,21
However, transient increases in CEA can occur with chemotherapy that
are not associated with disease progression (eg, within 2 weeks
following 5-FU-based treatment and within 4 to 6 weeks after
oxaliplatin therapy).20,22 Thus, timing of sample collection for CEA
determination should be considered in context with the therapy
prescribed.
Although there is no universally accepted definition of what constitutes
a clinically significant change in CEA levels, guidelines have been
proposed: 1) r30% increase over the previous value, confirmed by a
second sample collected within 1 month; or 2) >15% increase
maintained over r3 successive samples.21
Since z25% of patients with CRC do not have elevated levels of CEA,
monitoring treatment with alternative tumor markers such as CA 19-9 or
CA 72-4 may be of benefit.21 ASCO does not recommend the routine use
of these markers, however.20
Epidermal Growth Factor Receptor (EGFR)
Cetuximab (Erbitux®’) is a recombinant, human/mouse chimeric
monoclonal antibody that blocks signal transduction and cell growth
when bound to the extracellular domain of EGFR. Thus, EGFR expression
was assumed to be a prerequisite for response to cetuximab, and the
clinical trials conducted to demonstrate drug efficacy required a positive
EGFR immunohistochemical (IHC) stain for patient inclusion. Later
131
Hematology/Oncology
Pathologic Description 5-year Survival*
Cancer limited to submucosa or muscularis 93.2%
Cancer extends into serosa 84.7%
Cancer extends through serosa 72.2%
Cancer extends to regional lymph nodes 83.4%
Cancer extends beyond the muscularis 64.1%
Cancer extends to increased number of lymph nodes 44.3%
Distant metastases (eg, liver, lung, bone) 8.1%
studies, however, showed that patients with EGFR-negative tumors
might also respond. 23,24 Nevertheless, the Erbitux package insert states
that a positive EGFR IHC stain is a prerequisite for use of the drug.25
Clinical trials have shown no correlation between efficacy of treatment
and the intensity (ie, 1+ to 4+) of EGFR IHC staining.26
Determination of EGFR gene copy number by FISH or EGFR protein
expression by ELISA (using serum rather than tissue) have been
proposed as alternatives to IHC testing. Although preliminary results
suggest FISH results predict response to cetuximab-based therapy,27
confirmatory studies are required.
Pharmacogenomic Testing
The goal of pharmacogenomic testing is to improve patient outcome by
enabling optimal patient-specific drug selection and dosing.
Theoretically, genetic markers can identify patients who will 1) respond
to a specific medication using standard doses, 2) respond only with
increased doses, or 3) have a toxic reaction that either requires a
reduced dose or selection of an alternative therapy. To date, most
studies correlating genetic markers with CRC treatment outcomes are
retrospective, derived from small sample sizes, and often originate from
a single research group. The information presented below and in Table
18 summarizes the available literature and classifies the clinical utility
of each test.
5-Fluorouracil (5-FU)
DPYD Polymorphism
Deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting
enzyme in the catabolism of pyrimidine-based chemotherapeutic agents
(eg, 5-FU and capecitabine), has been linked to severe myelosuppression
and death in patients treated with standard doses.28 Approximately 50%
of DPD-deficient patients have 1 or 2 copies of a DPYD allele containing
the IVS14+1GmA mutation; roughly one-third of patients with grade 3
or 4 toxicity due to 5-FU treatment have this mutation.28-30
Consequently, presence of this mutation suggests an increased risk of
severe myelosuppression in patients treated with 5-FU; however, a
negative test result (lack of mutation) does not rule out this risk.
TYMS Polymorphism
5-FU exerts its effect primarily by inhibiting thymidine nucleotide
production catalyzed by thymidylate synthase (TS). Polymorphisms in the
5a- and 3a-untranslated regions (UTRs) of the gene that encodes TS
(TYMS) influence the activity of the enzyme and may, therefore, affect
patient response to 5-FU-based treatment as detailed in Table 18.31-33
132

Section 6 Hematology/Oncology

Table 18. Prediction of Chemotherapy Outcome in Patients Treated for Advanced CRC

Clinical Utility
Therapy Test Favorable Result Outcome Predicted of Test*
5-Fluorouracil DPYD IVS14+1GmA Negative for IVS14+1GmA Reduced toxicity28-30 2
31,32
TYMS 3’-UTR Negative for 6-bp deletion Reduced disease progression 3
TYMS 5’-UTR 2R (2 tandem repeats of a Improved response rate33 3
28-bp sequence) Improved survival31,32 3
Irinotecan UGT1A1 TA repeat Negative for TA repeat Reduced toxicity34-36 1
32,37
Oxaliplatin ERCC1-118 Negative for C118T Improved survival 2
32,38
GSTP Ile105Val Val105Val Improved survival 3
Reduced disease progression32 3
Reduced toxicity39 3
XPD-751 Negative for Lys751Gln Improved survival32,40 2
Reduced disease progression32,40 3
Improved response rate40 3
XRCC1 Arg399Gln Negative for Arg399Gln Improved response rate41 3
UTR, untranslated region; bp, base pair.
*1, currently defined; 2, potential for future use; 3, not currently defined.

Irinotecan
UGT1A1 Polymorphism
Irinotecan (Camptosar®’) therapy can result in dose-limiting toxicity
manifesting as neutropenia, diarrhea, and asthenia. The risk of toxicity
can be assessed by testing for an additional TA repeat in the promoter
region of the gene encoding uridine diphosphate glucuronosyltrans-
ferase 1A1 (UGT1A1).34,35 This hepatic enzyme metabolizes SN-38, the
active form of irinotecan and the cause of drug toxicity.
The presence of an additional TA repeat (ie, positive for TA repeat) is
consistent with reduced UGT1A1 enzyme activity and SN-38
metabolism, leading to increased likelihood of irinotecan toxicity.
Consequently, the irinotecan product insert suggests a reduced initial
dose for patients homozygous for the TA repeat.36 Heterozygous patients
have intermediate enzyme activity and may be at increased risk for
neutropenia; however, such patients have been shown to tolerate
normal initial doses.35 Patients negative for the TA repeat are the least
likely to suffer from dose-limiting toxicity. The UGT1A1 TA repeat assay
does not detect other mutations in the UGT1A1 gene that may affect
UGT1A1 enzyme activity.
Oxaliplatin
ERCC1-118 Polymorphism
Excision repair cross-complementation group 1 (ERCC1) is an excision
nuclease within the nucleotide excision repair (NER) pathway.
Polymorphisms in the ERCC1 gene may increase DNA repair activity via
the NER pathway, resulting in resistance to platinum-based
chemotherapeutic drugs such as oxaliplatin.37 A single-nucleotide
polymorphism (CmT) in codon 118 was shown to result in worse
survival when advanced CRC patients were treated with 5-FU/
oxaliplatin.32,37 Median survival was improved in patients homozygous
for the 118C genotype (C/C) relative to those with 1 or more thymine
alleles (Kaplan-Meier analysis, P = 0.021).37
GSTP1 Ile105Val Polymorphism
The activity of glutathione S-transferase P1 (GSTP1), an enzyme involved
in inactivation of platinum-based chemotherapeutic agents, is decreased
with GSTP1 polymorphisms that lead to an amino acid change at codon
105. Homozygosity for the 105Val allele has been associated with
improved survival, slower disease progression, and less cumulative
peripheral neuropathy in patients treated with oxaliplatin.32,38,39
Shorter median survival associated with a dose-dependent presence of
Ile genotype was significant in 1 study (Ile/Ile, 7.9 months; Ile/Val, 13.3
months; Val/Val >24.9 months, P<0.001),38 but not in another after
multivariate analysis (P=0.072).32 Whereas median survival data are
conflicting, the risk of disease progression was significantly higher (2
times) in patients with the Ile/Ile genotype than in those with the
Val/Val genotype (95% CI 0.85–4.71, P=0.018).32
Preliminary results indicate that risk of dose-limiting toxicity manifesting
as cumulative peripheral neuropathy is increased with the combined
Ile/Ile and Ile/Val genotypes compared to the Val/Val genotype (odds
ratio, 5.75; 95% CI 1.08–30.74; P=0.02).39
XPD-751 Polymorphism
Xeroderma pigmentosum group D (XPD), another enzymatic component
of the NER pathway, is also involved with DNA repair resulting in
resistance to platinum-based drugs. K751Q polymorphisms in the XPD
gene (Lys/Gln and Gln/Gln) have been associated with reduced response
to 5-FU/oxaliplatin treatment and reduced median survival.32,40 Among
patients (n = 70) with advanced CRC treated with 5-FU/oxaliplatin, the
Lys/Lys genotype was associated with longer median survival (17.4
months, 95% CI 7.9–26.5) than was the Lys/Gln (12.8 months, 95% CI
8.5–25.9) or Gln/Gln (3.3 months, 95% CI 1.4–6.5) genotype.40
XRCC1 Arg399Gln Polymorphism
The X-ray cross-complementation group 1 (XRCC1) enzyme is another
DNA repair enzyme associated with resistance to platinum-based
treatment.41 In a small study of XRCC1 polymorphisms, homozygosity for
the 399Arg allele (Arg/Arg) predicted successful 5-FU/oxaliplatin
treatment in patients with advanced CRC.41 Patients with at least 1 Gln
allele (Arg/Gln or Gln/Gln) were at a 5.2-fold increased risk (95% CI
1.21–22.07) of not responding to 5-FU/oxaliplatin chemotherapy.
 133

Section 6 Hematology/Oncology

References biochemical markers in colorectal cancer: European Group on

Tumour Markers (EGTM) guidelines. Eur J Cancer. 2003;39:718-727.
1. American Cancer Society: Cancer facts and figures 2006. Available
22. Sorbye H, Dahl O. Transient CEA increase at start of oxaliplatin
at: http://www.cancer.org/downloads/STT/CAFF2006PW
combination therapy for metastatic colorectal cancer. Acta
Secured.pdf. Accessed June 12, 2006.
Oncologica. 2004;43:495-498.
2. Engstrom PF. Colorectal Cancer. In: Lenhard RE, Osteen RT, Gansler
23. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in
T, ed. Clinical Oncology. Atlanta, GA: American Cancer Society Inc;
colorectal cancer patients with tumors that do not express the
2001:361-371.
epidermal growth factor receptor by immunohistochemistry. J Clin
3. Genetics of Colorectal Cancer (PDQ®). Available at:
Oncol. 2005;23:1803-1810.
http://www.cancer.gov/cancertopics/pdq/genetics/colorectal/Health
24. Hebbar M, Wacrenier A, Desauw C, et al. Lack of usefulness of
Professional/page1. Accessed August 11, 2006.
epidermal growth factor receptor expression determination for
4. Lynch HT, de la Chapelle A. Hereditary colorectal cancer. New Engl
cetuximab therapy in patients with colorectal cancer. Anticancer
J Med. 2003;348:919-932.
Drugs. 2006;17:855-857.
5. Smith RA, Cokkinides V, Eyre HJ. American Cancer Society
25. Erbitux [package insert]. Branchburg, NJ:ImClone Systems Inc;
guidelines for the early detection of cancer, 2006. CA Cancer J Clin.
March 2006.
2006;56:11-25.
26. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy
6. Mandel JS, Church TR, Bond JH, et al. The effect of fecal occult-
and cetuximab plus irinotecan in irinotecan-refractory metastatic
blood screening on the incidence of colorectal cancer. New Engl J
colorectal cancer. N Engl J Med. 2004;351:337-345.
Med. 2000;343:1603-1607.
27. Moroni M, Veronese S, Benvenuti, et al. Gene copy number for
7. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening
epidermal growth factor receptor (EGFR) and clinical response to
and surveillance: Clinical guidelines and rationale-update based on
antiEGRF treatment in colorectal cancer: a cohort study. Lancet
new evidence. Gastroenterology. 2003;124:544-560.
Oncol. 2005;6:279-286.
8. Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda guidelines
28. Raida M, Schwabe W, Hausler P, et al. Prevalence of a common
for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and
point mutation in the dihydropyrimidine dehydrogenase (DPD) gene
microsatellite instability. J Natl Cancer Inst. 2004;96:261-268.
within the 5’-splice donor site of intron 14 in patients with severe
9. Vasen HF, Watson P, Mecklin J-P, et al. New clinical criteria for
5-fluorouracil (5-FU)-related toxicity compared with controls. Clin
hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome)
Cancer Res. 2001;7:2832-2839.
proposed by the International Collaborative Group on HNPCC.
29. Van Kuilenburg AB, Meinsma R, Zoetekouw L, et al. High prevalence
Gastroenterology. 1999;116:1453-1456.
of the IVS14+1G>A mutation in the dihydropyrimidine
10. Jarvinen HJ, Aarnio M, Mustonen H, et al. Controlled 15-year trial
dehydrogenase gene of patients with severe 5-fluorouracil-
on screening for colorectal cancer in families with hereditary
associated toxicity. Pharmacogenetics. 2002;12:555-558.
nonpolyposis colorectal cancer. Gastroenterology. 2000;118:829-834.
30. Van Kuilenburg AB, Meinsma R, Zoetekouw L, et al. Increased risk
11. Cruz-Correa M, Giardiello FM. Diagnosis and management of
of grade IV neutropenia after administration of 5-fluorouracil due to
hereditary colon cancer. Hematol Oncol Clin N Am. 2003;17:539-551.
a dihydropyrimidine dehydrogenase deficiency: high prevalence of
12. Bardi G, Fenger C, Johansson B, et al. Tumor karyotype predicts
the IVS14+1G>A mutation. Int J Cancer. 2002;101:253-258.
clinical outcome in colorectal cancer patients. J Clin Oncol.
31. Hitre E, Budai B, Adleff V, et al. Influence of thymidylate synthase
2004;22:2623-2634.
gene polymorphisms on the survival of colorectal cancer patients
13. Levin B, Brooks D, Smith RA, et al. Emerging technologies in
receiving adjuvant 5-fluorouracil. Pharmacogenet Genomics.
screening for colorectal cancer: CT colonoscopy, immunochemical
2005;15:723-730.
fecal occult blood tests, and stool screening using molecular
32. Stoehlmacher J, Park DJ, Zhang W, et al. A multivariate analysis of
markers. CA Cancer J Clin. 2003;53:44-55.
genomic polymorphisms: prediction of clinical outcome to 5-FU/
14. Smith A, Young GP, Cole SR, et al. Comparison of a brush-sampling
oxaliplatin combination chemotherapy in refractory colorectal
fecal immunochemical test for hemoglobin with a sensitive guaiac-
cancer. Br J Cancer. 2004;91:344-354.
based fecal occult blood test in detection of colorectal neoplasia.
33. Marsh S, McKay JA, Cassidy J, et al. Polymorphism in the
Cancer. 2006;107:2152-2159.
thymidylate synthase promoter enhancer region in colorectal cancer.
15. Pinol V, Castells A, Andreu M, et al. Accuracy of revised Bethesda
Int J Oncology. 2001;19:383-386.
guidelines, microsatellite instability, and immunochemistry for the
34. Massacesi C, Terrazzino S, Marcucci F, et al. Uridine diphosphate
identification of patients with hereditary nonpolyposis colorectal
glucuronosyl transferase 1A1 promotor polymorphism predicts the
cancer. JAMA. 2005;293:1986-1994.
risk of gastrointestinal toxicity and fatigue induced by irinotecan-
16. Lynch HT, Riley BD, Weismann S, et al. Hereditary nonpolyposis
based chemotherapy. Cancer. 2006;106:1007-1016.
colorectal carcinoma (HNPCC) and HNPCC-like families: problems in
35. Rouits E, Boisdron-Celle M, Dumont A, et al. Relevance of different
diagnosis, surveillance, and management. Cancer. 2004;100:53-64.
UGT1A1 polymorphisms in irinotecan-induced toxicity: a molecular
17. AJCC Cancer Staging Manual, 6th ed. New York, NY: Springer;
and clinical study of 75 patients. Clin Cancer Res. 2004;10:5151-
2002.
5159.
18. O’Connell JB, Maggard MA, Ko CY. Colon cancer survival rates with
36. Camptosar [package insert]. New York, NY: Pfizer Inc; 2006.
the new American Joint Commission Committee on cancer sixth
37. Park DJ, Zhang W, Stoehlmacher J, et al. ERCC1 gene polymorphism
edition staging. J Natl Cancer Inst. 2004;96:1420-1425.
as a predictor for clinical outcome in advanced colorectal cancer
19. Popat S, Hubner R, Houlston RS. Systematic review of microsatellite
patients treated with platinum-based chemotherapy. Adv Hematol
instability and colorectal cancer prognosis. J Clin Oncol.
Oncol. 2003;1:162-166.
2005;23:609-618.
38. Stoehlmacher J, Park DJ, Zhang W, et al. Association between
20. Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of
glutathione S-transferase P1, T1, and M1 genetic polymorphism and
recommendations for the use of tumor markers in gastrointestinal
survival of patients with metastatic colorectal cancer. J Natl Cancer
cancer. J Clin Oncol. 2006;24:5313-5327.
Inst. 2002;94:936-942.
21. Duffy MJ, vanDalen A, Haglund C, et al. Clinical utility of
39. Lecomte T, Landi B, Beaune P, et al. Glutathione S-transferase P1
134
Section 6
polymorphism (Ile105Val) predicts cumulative neuropathy in patients
receiving oxaliplatin-based chemotherapy. Clin Cancer Res.
2006;12:3050-3056.
40. Park DJ, Stoehlmacher J, Zhang W, et al. A xeroderma
pigmentosum group D gene polymorphism predicts clinical outcome
to platinum-based chemotherapy in patients with advanced
colorectal cancer. Cancer Res. 2001;61:8654-8658.
41. Stoehlmacher J, Ghaderi V, Iobal S, et al. A polymorphism of the
XRCC1 gene predicts for response to platinum based treatment in
advanced colorectal cancer. Anticancer Res. 2001;21:3075-3079.
6.5.4.2 Carcinoembryonic Antigen (CEA)
Clinical Use: This test is used to monitor persistent, metastatic, or
recurrent adenocarcinoma of the colon following curative surgery.
Clinical Background: CEA is an oncofetal glycoprotein present in the
gastrointestinal tract and body fluids of the embryo and fetus. This 180-
kilodalton antigen is also present in certain adult gastrointestinal cells,
including the mucosal cells of the colorectum, and small amounts are
present in blood. Although its physiological role is not clear, CEA is a
useful tumor marker. Blood levels are usually not increased in localized
or primary disease, but are often elevated in patients with disseminated
cancers and in some patients with non-malignant disease.
CEA is useful for detecting recurrence of colon cancer; increased levels
may precede clinical evidence of recurrence by as much as 6 months.
The sensitivity for detecting recurrence is 97% in patients whose CEA
was elevated preoperatively, but only 66% in those with normal
preoperative levels.
CEA is not recommended for screening because of low sensitivity and
specificity, particularly in the early stages of neoplastic disease. The
American Society of Clinical Oncology (ASCO) has recommended the
use of CEA testing for staging/prognosis, detecting recurrence,
monitoring therapy, and screening for hepatic metastases in patients
with colon cancer (J Clin Oncol. 2001;19:1865-1878).
Interpretive Information: Normal CEA concentrations (<2.5 ng/mL)
are seen in about 97% of apparently healthy individuals. Elevations may
be seen in 19% of heavy smokers and 7% of former smokers,
individuals with inflammatory diseases of the gastrointestinal tract (eg,
peptic ulcer, diverticulitis, etc), liver diseases including cirrhosis and
chronic active hepatitis, advanced renal disease, and fibrocystic disease
of the breast. Levels can also be increased in as many as 30% of
patients with breast, lung, hepatocellular, and pancreatic carcinoma
(Table 19). About 30% of patients with metastatic colon cancer have
normal CEA levels.
6.5.4.3 Epidermal Growth Factor Receptor (EGFR), IHC
Clinical Use: This test is used to determine eligibility for cetuximab
(Erbitux™’) treatment in patients with advanced metastatic colorectal
cancer.
Clinical Background: EGFR is a 170-kd receptor tyrosine kinase
encoded by the c-erb-B (HER-1) proto-oncogene. It is expressed in
various solid tumors, including colorectal, prostate, head and neck, and
lung cancer, as well as in certain normal tissues. When bound by ligands
such as epidermal growth factor (EGF) and transforming growth factor-B,
EGFR undergoes conformational changes that activate its intracellular
tyrosine kinase activity, initiating autophosphorylation and downstream
signal transduction pathways. Activation can mediate a variety of cellular
responses, including gene expression, cell proliferation, and cell survival.
Hematology/Oncology
Dysregulation of the EGFR signaling pathway due to EGFR
overexpression, genetic aberrations, or other causes leads to malignant
transformation. Thus, EGFR has become an important target for anti-
cancer drug development; cetuximab is the first EGFR inhibitor to receive
FDA approval for treatment of colorectal cancer.
Cetuximab is a human–mouse chimeric monoclonal antibody directed
against the extracellular ligand-binding domain of EGFR. This drug
competitively inhibits binding of EGFR by EGF and transforming growth
factor-B, thereby blocking downstream signal transduction pathways
and arresting cell growth. Cetuximab is indicated for treatment of
advanced metastatic colorectal cancer resistant to irinotecan-based
therapy, and as a single agent in patients who cannot tolerate
irinotecan. Pre-treatment testing for EGFR expression in tumor tissue is
required, as only patients with EGFR-expressing tumors are eligible for
cetuximab therapy. However, levels of EGFR expression do not appear to
correlate with clinical response to cetuximab.
Method: This immunohistochemical assay uses a mouse monoclonal
anti-human antibody specific for the extracellular region of EGFR.
Results are reported as positive or negative for detection of EGFR;
values >0 (ie, 1+, 2+, or 3+) are considered positive. No cross-reaction
with HER-2, HER-3, or HER-4 has been noted. Aliases include EGF
Receptor, HER-1, cetuximab sensitivity, and Erbitux®’ sensitivity.
Interpretive Information: A positive result indicates the presence of
EGFR on cell membranes of tumor tissue and eligibility for treatment
with cetuximab. A negative result suggests that the patient is not
suitable for cetuximab treatment. Specimens from individuals with
hepatitis B virus infection with HBsAg may exhibit non-specific staining
with horseradish peroxidase.
6.5.4.4 Fecal Globin, Immunochemistry (InSure®)
Clinical Use: This test is used to screen for lower gastrointestinal
bleeding associated with colorectal cancer, adenomas, polyps, and other
lower gastrointestinal conditions.
Clinical Background: Colorectal cancer is the third most common form
of cancer and the third leading cause of cancer death in the United
States. According to American Cancer Society (ACS) estimates, 145,290
new cases of colorectal cancer were expected to be diagnosed and more
than 56,000 people were estimated to die of this disease in 2005,
accounting for 10% of cancer deaths in each sex.1 The most common risk
factor is age: >90% of colorectal cancers are diagnosed in people >50
years of age.1 Screening and early detection are crucial, as survival rates
decrease dramatically with increasing cancer stage: 5-year survival ranges
from >90% for Dukes stage A to <5% for Dukes stage D. Moreover,
detection and removal of precancerous polyps can reduce the incidence of
colorectal cancer by 76% to 90%.2 Although routine screening is
recommended for average-risk individuals r50 years of age,3-5 fewer than
half of age-eligible adults receive appropriate screening.6,7
Because cancerous and precancerous colorectal lesions tend to cause
low-level bleeding, assays for occult blood in feces have become an
important screening tool. Annual screening with a fecal occult blood
test (FOBT) can decrease colorectal cancer mortality by up to 33%.8 ACS
guidelines indicate that a yearly FOBT is an acceptable screening
method for average-risk individuals r50 years of age; combining an
annual FOBT with flexible sigmoidoscopy every 5 years is preferred over
the annual use of either test alone.3
One drawback to the most common currently used FOBTs is that they
are guaiac based; they detect heme peroxidase activity and are not
 135

Section 6 Hematology/Oncology

Table 19. Distribution of CEA

% with CEA in Indicated Range

Subject Group No. of Subjects 0–2.5 ng/mL 2.6–5.0 ng/mL 5.1–10.0 ng/mL 10.0–20.0 ng/mL >20.0 ng/mL
Healthy Subjects
Non-smokers 225 98 2 0 0 0
Smokers 150 87 8 5 0 0
Malignant Disease
Colorectal 250 38 11 7 6 38
Lung 158 46 16 11 7 20
Breast 221 68 15 8 2 7
Gastric 35 60 17 9 6 9
Ovarian 35 83 11 3 3 0
Pancreatic 43 44 21 16 7 12
Liver 6 50 17 0 33 0
Lymphoproliferative 15 80 20 0 0 0
Other* 40 75 10 10 5 0
Non-Malignant Disease
Cirrhosis 53 42 13 32 13 0
Other benign liver 5 40 20 0 40 0
Ulcerative colitis 11 91 9 0 0 0
Benign polyps 23 65 22 13 0 0
Colon and intestinal 15 67 20 0 13 0
Gastrointestinal 21 76 14 0 5 5
Breast 53 96 0 2 0 2
Kidney and bladder 12 83 8 0 8 0
Lung 29 69 24 7 0 0
†
Other 117 86 8 4 1 0
Source: Bayer Corporation.
*Other cancerous diseases include bladder, prostate, esophagus, head and neck, sarcoma, and kidney cancer.
†
Other non-malignant diseases include benign uterine, cervical and vaginal, benign ovary, and benign male genital as well as other benign conditions.

specific for human hemoglobin. Thus, hemoglobin from red meat,
peroxidase from fruits and vegetables, and certain medications can
cause false-positive reactions and need to be avoided for several days
before the test. In addition, vitamin C (in excess of 250 mg/day) from
supplements or citrus fruits and juices may cause a false-negative
guaiac test result. While these FOBTs are non-invasive and specimens
can be collected at home, strict dietary and medication restrictions may
decrease adherence.7,9 Newer immunochemical assays such as InSure
do not react with non-human hemoglobin or peroxidase, so food
restrictions are not necessary. Immunochemical FOBTs are also more
specific for lower gastrointestinal bleeding because they target the
globin portion of hemoglobin, which does not survive passage through
the upper gastrointestinal tract. Based on these features and published
performance characteristics of immunochemical FOBTs, ACS guidelines
suggest that immunochemical FOBTs such as InSure “are more patient-
friendly, and are likely to be equal or better in sensitivity and
specificity” relative to guaiac-based tests.3,10
Individuals Suitable for Testing include those undergoing routine
screening for colorectal lesions or other sources of bleeding in the lower
gastrointestinal tract.
Samples should not be collected 1) during, or 3 days before or after, a
menstrual period; 2) if bleeding hemorrhoids are present; 3) if there is
visible blood in the urine or toilet bowl; 4) if there are bleeding cuts on
the hands; or 5) if the toilet contains rust or saltwater. Toilet freshener
should be removed and the toilet flushed prior to sample collection.
Dietary roughage may increase test sensitivity, but no dietary changes
or restrictions are required.
Method: This immunochemistry assay uses monoclonal, mouse anti-
human hemoglobin-coated chromatography test strips with colorimetric
detection. The analytical sensitivity of the assay is 50 Ng Hb/g feces.11 It
is specific for colorectal bleeding and does not detect blood from the
upper gastrointestinal tract.9
136
Section 6
Interpretive Information: Positive results indicate occult blood in the
feces and should be followed up with physician consultation and
possible endoscopic evaluation. Negative results indicate the absence
of fecal blood; however, false-negatives can occur because of uneven
distribution of blood in the feces or intermittent bleeding.
References
1. American Cancer Society. Cancer facts and figures 2005. Available
at: http://www.cancer.org/docroot/STT/stt_0.asp. Accessed Sept.
20, 2005.
2. Winawer SJ, Zauber AG, Ho MN, et al. Prevention of colorectal
cancer by colonoscopic polypectomy. The National Polyp Study
Workgroup. N Engl J Med. 1993;329:1977-1981.
3. Smith RA, Cokkinides V, Eyre HJ. American Cancer Society
guidelines for the early detection of cancer, 2003. CA Cancer J Clin.
2003;53:27-43.
4. US Preventive Services Task Force. Screening for colorectal cancer:
recommendations and rationale. AHRQ Pub. No. 03-510A. July
2002. Available at: http://www.ahcpr.gov/clinic/3rduspstf/
colorectal/colorr.pdf. Accessed July 18, 2003.
5. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening
and surveillance: clinical guidelines and rationale—update based
on new evidence. Gastroenterology. 2003;124:544-560.
6. Centers for Disease Control and Prevention. Colorectal cancer test
use among persons aged r50 years—United States, 2001. MMWR.
2003; 52:193-196.
7. Vernon SW. Participation in colorectal cancer screening: a review.
J Natl Cancer Inst. 1997;89:1406-1422.
8. Mandel JS, Bond JH, Church TR, et al. Reducing mortality from
colorectal cancer by screening for fecal occult blood. Minnesota
Colon Cancer Control Study. N Engl J Med. 1993;328:1365-1371.
9. Robinson MH, Pye G, Thomas WM, et al. Haemoccult screening for
colorectal cancer: the effect of dietary restriction on compliance.
Eur J Surg Oncol. 1994;20:545-548.
10. Levin B, Brooks D, Smith RA, et al. Emerging technologies in
screening for colorectal cancer: CT colonography, immunochemical
fecal occult blood tests, and stool screening using molecular
markers. CA Cancer J Clin. 2003;53:44-55.
11. InSure Product Instructions. Falmouth, ME: Enterix Inc; 2000.
6.5.4.5 Microsatellite Instability (MSI), HNPCC
Clinical Use: This assay is used to assess the need to test individuals
or their family members for hereditary nonpolyposis colorectal cancer
(HNPCC)-associated mutations.
Clinical Background: HNPCC, an autosomal dominant condition
caused by mutations in mismatch repair (MMR) genes, accounts for
about 3% to 5% of colorectal cancers. In individuals with an MMR
mutation, the lifetime risk of developing colorectal cancer is about
80%;1 these mutations may also predispose to endometrial, ovarian,
stomach, and other forms of cancer. Although HNPCC may have a
somewhat better prognosis than other forms of colorectal cancer, it is
characterized by rapid progression from adenomatous polyps to
malignant lesions. Identifying HNPCC in affected individuals is
important, as close surveillance of at-risk family members has been
found to reduce the rate of colorectal cancer and overall mortality by
>60%.2 Family members with an MMR mutation require aggressive
follow-up (colonoscopy every 1–2 years), while those without the
familial mutation are at the same risk as the general population and do
not require intensive screening.
HNPCC can be diagnosed on the basis of clinical and family history,3,4 as
well as by direct detection of MMR mutations. In suspected HNPCC
Hematology/Oncology
patients without a known familial mutation, the first step in the genetic
diagnosis is identifying microsatellite instability (MSI) in tumor cells.
Microsatellites are sequences of repetitive DNA with repeating units of
1 to 7 base pairs. These regions are prone to replication errors, which
are normally repaired by MMR enzymes. If one of the MMR enzymes is
impaired, the microsatellites become unstable and will have varying
repeat numbers at specific loci. Although MSI does not seem to have
any clinical effect, it is a marker of faulty DNA repair; cancer arises
when faulty DNA repair leads to mutations in tumor suppressor or
oncogenes. MSI is found in most HNPCCs but only about 15% of
sporadic colorectal tumors,5,6 and therefore cannot be used alone for
diagnosis of HNPCC. However, a finding of MSI strongly indicates the
presence of MMR mutations and the need for further genetic testing.7
The National Cancer Institute has recommended a panel of 5
microsatellites for MSI screening: BAT 25 and BAT 26 (mononucleotide
repeats), D2S123, D5S346, and D17S250 (dinucleotide repeats).
Individuals Suitable for Testing include those who meet any of the
Bethesda criteria (Table 20) and in whom a familial mutation is not
known.
Method: This assay detects microsatellite instability in tumor tissue.
Paraffin sections containing r30% tumor cells are processed with
selective scraping of the tumor-rich area, whereas those containing
<30% tumor cells are processed with laser capture microdissection.
Whole blood is used as control (non-tumor) tissue. Microsatellite
instability is detected in a multiplex polymerase chain reaction (PCR)-
based assay in which 5 STR loci (BAT 25, BAT 26, D2S123, D5S346,
D17S250) are amplified from tumor and normal tissue. One primer from
each set of PCR primers is labeled with either 6-FAM (blue) or HEX
(green) fluorescein; the 5 markers are distinguished by peak size and
fluorescent labels. Amplification products are separated by capillary
electrophoresis, followed by data analysis with GeneMapper®’ software
to detect peaks and determine allele size. The tumor and normal peaks
are visually compared to detect MSI. Aliases for this test are HNPCC,
Table 20 Bethesda Guidelines for Testing Colorectal
Cancers for Microsatellite Instability Testing8
• Individuals with cancer in families that meet the Amsterdam criteria3
• Individuals with 2 HNPCC-related cancers, including synchronous and
metachronous colorectal cancers or associated extracolonic cancers*
• Individuals with colorectal cancer and a first-degree relative with
colorectal cancer and/or HNPCC-related extracolonic cancer and/or a
colorectal adenoma; one of the cancers diagnosed at age <45 years,
and the adenoma diagnosed at age <40 years
• Individuals with colorectal cancer or endometrial cancer diagnosed at
age <45 years
• Individuals with right-sided colorectal cancer with an undifferen-
tiated pattern (solid/cribriform) on histopathology diagnosed at age
<45 years†
• Individuals with signet-ring-cell-type colorectal cancer‡ diagnosed at
<45 years
• Individuals with adenomas diagnosed at age <40 years
*Endometrial, ovarian, gastric, hepatobiliary or small bowel cancer or transitional cell
carcinoma of the renal pelvis or ureter.
†
Solid/cribriform defined as poorly differentiated or undifferentiated carcinoma composed
of irregular, solid sheets of large eosinophilic cells and containing small gland-like spaces.
‡
Composed of 50% signet-ring cells.

Section 6
Lynch syndrome, and replication error (RER+).
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The presence of 2 or more unstable
microsatellite loci indicates MSI; instability at b1 locus indicates
absence of MSI. Individuals with MSI tumors should be screened for
germline mutations in MLH1 and MSH2, and in MSH6 if no MLH1 or
MSH2 mutations are found. Because most HNPCCs exhibit MSI,
germline MMR mutation analysis is generally not necessary in patients
without MSI-H. However, MSI is frequently absent in tumors associated
with MSH6 mutations.9
References
1. Vasen HF, Wijnen JT, Menko FH, et al. Cancer risk in families with
hereditary nonpolyposis colorectal cancer diagnosed by mutation
analysis. Gastroenterology. 1996;110:1020-1027.
2. Jarvinen HJ, Aarnio M, Mustonen H, et al. Controlled 15-year trial on
screening for colorectal cancer in families with hereditary
nonpolyposis colorectal cancer. Gastroenterology. 2000;118:829-834.
3. Vasen HF, Mecklin JP, Khan PM, et al. The International Collaborative
Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC).
Dis Colon Rectum. 1991;34:424-425.
4. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for
hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome)
proposed by the International Collaborative group on HNPCC.
Gastroenterology. 1999;116:1453-1456.
5. Giardiello FM, Brensinger JD, Petersen GM. AGA technical review on
hereditary colorectal cancer and genetic testing. Gastroenterology.
2001;121:198-213.
6. Terdiman JP, Gum JR Jr, Conrad PG, et al. Efficient detection of
hereditary nonpolyposis colorectal cancer gene carriers by screening
for tumor microsatellite instability before germline genetic testing.
Gastroenterology. 2001;120:21-30.
7. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening and
surveillance: clinical guidelines and rationale-update based on new
evidence. Gastroenterology. 2003;124:544-560.
8. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National
Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal
Cancer Syndrome: meeting highlights and Bethesda guidelines.
J Natl Cancer Inst. 1997;89:1758-1762.
9. Berends MJ, Wu Y, Sijmons RH, et al. Molecular and clinical
characteristics of MSH6 variants: an analysis of 25 index carriers of a
germline variant. Am J Hum Genet. 2002;70:26-37.
6.5.4.6 MLH1, MSH2, and MSH6 Mutations for HNPCC
Clinical Use: This test is used to differentiate hereditary nonpolyposis
colorectal cancer (HNPCC) from non-HNPCC colorectal cancer (CRC) and
to assess the risk of HNPCC in family members of individuals with
HNPCC.
Clinical Background: HNPCC, which accounts for about 3% to 5% of
CRCs, is caused by defects in mismatch repair (MMR) enzymes. These
defects may also increase the risk of endometrial, cervical, stomach,
ovarian, and other forms of cancer. About 90% of individuals with
identifiable HNPCC mutations have mutations in the MLH1 or MSH21
MMR gene. Mutations in MSH6, PMS1, and PMS2 have also been
implicated.
HNPCC is an autosomal dominant condition with variable penetrance.
Individuals with HNPCC typically inherit 1 copy of a defective MMR
137
Hematology/Oncology
allele; mutations in somatic tissue may cause loss of the normal allele,
leading to an increased rate of mutations in affected cells. Mutations in
oncogenes or tumor suppressor genes then lead to carcinogenesis. The
lifetime risk of CRC in individuals with an MMR gene mutation is about
80%.2
Diagnosis of HNPCC is based on clinical features and familial cancer
patterns,3,4 or on detection of mutations in MMR genes. Although
HNPCC appears to have a slightly more favorable prognosis than non-
HNPCC CRC, the often rapid progression from adenomatous polyps to
malignant lesions necessitates aggressive follow up for individuals with
HNPCC and their first-degree relatives; close surveillance of family
members can reduce the rate of CRC and overall mortality by >60%.5,6
Detection of MMR mutations is important for counseling individuals
with HNPCC and their families, as family members without a known
familial mutation are at the same risk as the general population and do
not require intensive screening.
If a familial mutation is not known, testing suspected HNPCC tumors for
microsatellite instability (MSI) can help determine the need for MMR
mutation analysis. Microsatellites are regions of repetitive DNA with
repeating units of 1 to 7 base pairs. These regions are prone to
replication errors, which can be detected as heterogeneous repeat
numbers at specific microsatellite regions. Because MMR enzymes
normally repair such errors, detection of MSI in tumor tissue suggests
the presence of MMR defects. MSI is found in most HNPCCs7,8 but only
about 15% of sporadic colorectal tumors.
Because of the relative frequencies of MMR mutations, MLH1 and
MSH2 should be examined first; if no mutations are found, mutations in
MSH6 should be sought. If the family mutation is known, only the
relevant exon in the affected gene should be tested.
Individuals Suitable for Testing include individuals with CRC who
meet the Amsterdam criteria for diagnosis of HNPCC3; individuals with
documented MSI in a colorectal tumor; first-degree relatives of
individuals who have a known MMR mutation; and, if the familial MMR
mutation is not known, first-degree relatives of individuals who
• Meet the Amsterdam criteria; or
• Have 2 HNPCC-related cancers, including synchronous and
metachronous CRCs or associated extracolonic cancers (endometrial,
ovarian, gastric, hepatobiliary or small bowel cancer or transitional
cell carcinoma of the renal pelvis or ureter); or
• Have CRC and have a first-degree relative with CRC and/or HNPCC-
related extracolonic cancer and/or a colorectal adenoma; one of the
cancers diagnosed at age <45 years, and the adenoma diagnosed at
age <40 years.9
Method: In this assay, relevant MMR gene coding regions and splice
junction sites are amplified by polymerase chain reaction (PCR). Both
strands of each amplified product are then sequenced, with automated
fluorescence detection. The test report lists nucleotide changes in
coding sequences and known splice sites. This assay detects
heterozygous point mutations, small deletions, and insertions in the
relevant MMR gene(s), with an analytical sensitivity of >98%. Aliases
include Lynch Syndrome Mutation and HNPCC Mismatch Repair Gene
Mutation.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
138
Section 6
Interpretive Information
Individuals with CRC: Detection of an MMR gene mutation in an
individual with colon cancer suggests the need for aggressive screening
for recurrent CRC and, possibly, other cancers associated with HNPCC
(eg, endometrial and ovarian cancer). American Gastroenterological
Association (AGA) guidelines recommend colonoscopy every 1 to 2 years
for individuals with a clinical or genetic diagnosis of HNPCC.6 Genetic
counseling should be offered to the patient and first-degree family
members, who may benefit from germline mutation testing.
If mutations are not found in MLH1 or MSH2, mutations in MSH6
should be sought. Mutations not identified in these assays may also be
associated with HNPCC.
At-risk family members: If the familial mutation is known, detection
of that mutation implies an increased risk of HNPCC and associated
cancers. AGA guidelines recommend colonoscopy every 1 to 2 years for
those at increased risk of HNPCC, beginning at age 20 to 25, or 10 years
before the earliest age of CRC diagnosis in the family.6 Individuals
without the mutation are not at increased risk and can be followed up
according to screening recommendations for the normal-risk population.
If the familial mutation is not known, negative MLH1 and MSH2
findings should be followed with tests for mutations in MSH6.
References
1. Lynch HT, de la Chapelle A. Genetic susceptibility to non-polyposis
colorectal cancer. J Med Genet. 1999;36:801-818.
2. Vasen HF, Wijnen JT, Menko FH, et al. Cancer risk in families with
hereditary nonpolyposis colorectal cancer diagnosed by mutation
analysis. Gastroenterology. 1996;110:1020-1027.
3. Vasen HF, Mecklin JP, Khan PM, et al. The International Collaborative
Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC).
Dis Colon Rectum. 1991;34:424-425.
4. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for
hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome)
proposed by the International Collaborative group on HNPCC.
Gastroenterology. 1999;116:1453-1456.
5. Jarvinen HJ, Aarnio M, Mustonen H, et al. Controlled 15-year trial on
screening for colorectal cancer in families with hereditary
nonpolyposis colorectal cancer. Gastroenterology. 2000;118:829-834.
6. Winawer S, Fletcher R, Rex D, et al. Colorectal cancer screening and
surveillance: clinical guidelines and rationale-update based on new
evidence. Gastroenterology. 2003;124:544-560.
7. Giardiello FM, Brensinger JD, Petersen GM. AGA technical review on
hereditary colorectal cancer and genetic testing. Gastroenterology.
2001;121:198-213.
8. Terdiman JP, Gum JR Jr, Conrad PG, et al. Efficient detection of
hereditary nonpolyposis colorectal cancer gene carriers by screening
for tumor microsatellite instability before germline genetic testing.
Gastroenterology. 2001;120:21-30.
9. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National
Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal
Cancer Syndrome: meeting highlights and Bethesda guidelines.
J Natl Cancer Inst. 1997;89:1758-1762.
6.5.4.7 UGT1A1 Gene Polymorphism (TA Repeat)
Clinical Use: This test is used to predict toxicity from irinotecan
therapy, assist in selection of initial irinotecan dosage, and support
diagnosis of Gilbert’s syndrome.
Clinical Background: Irinotecan (Campostar®’, Pfizer Inc, New York,
NY) is a topoisomerase-I inhibitor widely used for treatment of
Hematology/Oncology
metastatic and recurrent colorectal cancer. The most common dose-
limiting adverse effects of irinotecan are neutropenia, diarrhea, and
asthenia. Variations in the gene encoding uridine diphosphate
glucuronosyltransferase 1A1 (UGT1A1) may help predict which patients
are most likely to develop these adverse effects.
UGT1A1 is a hepatic enzyme primarily responsible for conjugation of
bilirubin. UGT1A1 also catalyzes the glucuronidation of SN-38, the
active metabolite of irinotecan and the main source of treatment-related
toxicity. Glucuronidation is thought to protect against the toxicity of
SN-38.1 However, the presence of an additional TA repeat in the TATA
region of the UGT1A1 promoter (ie, 7 TA repeats; UGT1A1*28) markedly
decreases UGT1A1 production, leading to reduced glucuronidation.2,3
Patients homozygous for the UGT1A1*28 allele therefore accumulate
higher levels of SN-38 and are more likely to experience severe adverse
effects during irinotecan chemotherapy.4-6 Thus, knowledge of the
UGT1A1 polymorphism status could help guide the selection of
appropriate starting dosages, reducing the risk of severe toxicity and
improving the chances that therapy could be maintained. Homozygosity
for this allele is also associated with Gilbert’s syndrome, a mild form of
unconjugated hyperbilirubinemia.
Roughly 10% of the US population is homozygous for UGT1A1*28. The
frequency of the UGT1A1*28 allele varies among ethnicities,7 being
highest in those of African (43%) or European (39%) descent and lowest
in those of Asian (16%) descent.8 Variants with 5 or 8 repeats occur at
much lower frequencies, primarily in individuals of African descent. The
presence of 8 TA repeats has been associated with Gilbert’s syndrome
and with decreased glucuronidation in vitro.8 Other variations in the
genes encoding UGT1A1 and other uridine diphosphate glucuronosyl-
transferases may also influence glucuronidation and have been reported
at varying frequencies across ethnicities.
Individuals Suitable for Testing include patients being considered
for treatment with irinotecan and individuals with suspected Gilbert’s
syndrome.
Method: This fluorescent polymerase chain reaction (PCR) method uses
primers specific for the 5a untranslated region of UGT1A1 and
automated detection of PCR products. Results reported as negative,
heterozygous, or homozygous for the UGT1A1*28 allele.
Interpretive Information: Absence of the UGT1A1*28 allele usually
indicates a wild-type genotype, although some individuals—especially
those of African descent—may have 5 or 8 TA repeats. Patients with a
wild-type UGT1A1 genotype have a low risk of severe toxicity from
standard starting dosages of irinotecan. Similarly, in patients with 1
copy of the UGT1A1*28 allele (heterozygous), the other copy is most
likely wild-type. These patients are at increased risk of irinotecan
toxicity but may still tolerate normal initial dosages. Individuals with 2
copies of the UGT1A1*28 allele (homozygous) are at increased risk of
severe toxicity from irinotecan. Irinotecan product information indicates
that a reduced initial dosage should be considered for such patients.9
Other chemotherapeutic options may also be considered.
This assay does not detect other polymorphisms or mutations in the
UGT1A1 gene that may impair irinotecan detoxification, nor does it
examine other modifiers of irinotecan metabolism such as CYP3A4
activity. Since genetic variation can affect the accuracy of direct
mutation testing, the results should be interpreted in light of other
clinical and laboratory findings.
In symptomatic patients, homozygosity for UGT1A1*28 is consistent
with a diagnosis of Gilbert’s syndrome. Because other UGT variants

Section 6
have been associated with Gilbert’s syndrome, absence of the
UGT1A1*28 allele does not rule out this condition.
References:
1. Gupta E, Lestingi TM, Mick R, et al. Metabolic fate of irinotecan in
humans: correlation of glucuronidation with diarrhea. Cancer Res.
1994;54:3723-3725.
2. Iyer L, King CD, Whitington PF, et al. Genetic predisposition to the
metabolism of irinotecan (CPT-11). Role of uridine diphosphate
glucuronosyltransferase isoform 1A1 in the glucuronidation of its
active metabolite (SN-38) in human liver microsomes. J Clin Invest.
1998;101:847-854.
3. Iyer L, Das S, Janisch L, et al. UGT1A1*28 polymorphism as a
determinant of irinotecan disposition and toxicity. Pharmacogenomics
J. 2002;2:43-47.
4. Massacesi C, Terrazzino S, Marcucci F, et al. Uridine diphosphate
glucuronosyl transferase 1A1 promoter polymorphism predicts the
risk of gastrointestinal toxicity and fatigue induced by irinotecan-
based chemotherapy. Cancer. 2006; [Epub ahead of print]
5. Rouits E, Boisdron-Celle M, Dumont A, et al. Relevance of different
UGT1A1 polymorphisms in irinotecan-induced toxicity: a molecular
and clinical study of 75 patients. Clin Cancer Res. 2004;10:5151-5159.
6. Innocenti F, Undevia SD, Iyer L, et al. Genetic variants in the UDP-
glucuronosyltransferase 1A1 gene predict the risk of severe
neutropenia of irinotecan. J Clin Oncol. 2004;22:1382-1388.
7. Innocenti F, Grimsley C, Das S, et al. Haplotype structure of the UDP-
glucuronosyltransferase 1A1 promoter in different ethnic groups.
Pharmacogenetics. 2002;12:725-733. Erratum in: Pharmacogenetics.
2003;13:183.
8. Beutler E, Gelbart T, Demina A. Racial variability in the UDP-
glucuronosyltransferase 1 (UGT1A1) promoter: a balanced
polymorphism for regulation of bilirubin metabolism? Proc Natl Acad
Sci U S A. 1998;95:8170-8174.
9. Campostar® [package insert]. New York, NY: Pfizer Inc; 2005.
6.6 Hematopoietic/Lymphoid Disorders
6.6.1 AML1/ETO t(8;21) Quantitative Real-time PCR
Clinical Use: This test is used to diagnose acute myeloid leukemia
(AML) with t(8;21) chromosome translocation; monitor treatment
response; monitor minimal residual disease (MRD); and predict early
relapse.
Clinical Background: The translocation t(8;21)(q22;q22) is one of the
most common structural chromosomal aberrations in patients with AML,
occurring in about 15% of adult AML cases and 40% of AML cases with
differentiation (AML-M2). AML with t(8;21) has a mean onset age of
about 30 years and is most common in children and younger adults; it is
relatively rare in elderly patients. The presence of t(8;21) is associated
with the highest complete remission rate (90%) and the highest
probability (50%-70%) of remaining in complete remission at 5 years.
However, the disease may become resistant to therapy upon relapse.
Translocation t(8;21)(q22;q22) fuses the 5’-portion of the AML1 gene on
chromosome 21 to the almost complete open reading frame of the ETO
gene on chromosome 8. Thus, the transactivation domain in the middle
of AML1 is replaced with part of the ETO. This results in constant
expression of AML1/ETO fusion mRNA, which can be detected by
reverse transcription-polymerase chain reaction (RT-PCR). The
quantitative t(8;21) assay can be used not only to diagnose AML, but
also to serially monitor patients for MRD, evaluate the effectiveness of
treatment, and predict early relapse.
139
Hematology/Oncology
Method: In this real-time quantitative RT-PCR assay, the AML1-ETO
fusion transcript and an internal control (ABL transcript) are amplified
from the same RNA sample. The internal control serves as a control for
real-time PCR performance and as a reference for relative quantitation;
the relative ratio of AML1-ETO to internal control is reported. The
current sample will be tested side-by-side with a previously stored
sample, if available, to monitor quantitative changes with time (trend).
This assay can detect 1 leukemic cell in a background of 105 normal
cells. There is no known cross-reaction with other AML translocations.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: If the AML1-ETO (t[8;21]) translocation is
detected, the result will be reported as positive and the ratio will be
provided. Higher ratios are associated with higher percentages of
leukemic cells. A negative result (a ratio of zero) indicates absence of
the AML1-ETO translocation.
For monitoring MRD, we recommend monitoring trends rather than the
absolute ratio from a single measurement.
6.6.2 B- and T-Cell Gene Rearrangements, PCR
Clinical Use: These tests are used to diagnose B-cell or T-cell
malignancies, to determine leukemia and lymphoma lineage, and to
detect minimal residual disease or recurrent disease.
Clinical Background: The evaluation of lymph nodes, bone marrow,
and other tissues for the presence of lymphoma usually involves a
multiparameter approach. The obligatory first step when evaluating a
tissue for suspected lymphoma is to examine the tissue microscopically
for morphology. In many cases, morphologic examination is sufficient to
establish a diagnosis of malignant lymphoma. There is, however, a
significant proportion of cases in which additional studies are needed in
order to establish a definitive diagnosis. Those additional studies
include immunoperoxidase staining of the tissue sections, flow
cytometric evaluation of fresh cells from the specimen, and molecular
analysis. Molecular analysis includes such modalities as cytogenetics
(including FISH) and polymerase chain reaction (PCR). All of these
special studies are intended to provide some evidence that can help to
distinguish between benign lymphadenopathy and malignant lymphoma.
In addition, the special tests can sometimes help to establish both the
lineage and the presence of prognostically significant subtypes of
malignant lymphoma.
Methods
B-Cell Gene Rearrangement, Qualitative PCR: Following
extraction, DNA is amplified by PCR, utilizing 3 primer sets [FRI, FRII,
and FRIII]. The amplification products are analyzed by capillary
electrophoresis. Results are reported as negative or positive for the
presence of a clonal B-cell expansion.
B-Cell Gene Rearrangement, Quantitative PCR: Following
extraction, DNA is amplified by PCR, utilizing a primer set (FR3/CDR3)
targeting the variable and joining regions of the immunoglobulin heavy
chain gene. The amplification products are analyzed by capillary
electrophoresis. Results are reported as the ratio of the clonal peak area
to that of the internal control (Ki-RAS). This test should be used to
monitor patients being treated for B-cell malignancies. The analytical
140
Section 6
sensitivity is 1 monoclonal cell/10,000 normal cells when the initial pre-
therapy sample is provided for comparison.
T-Cell Receptor (TCR) Gene Rearrangement, Qualitative PCR:
Following extraction, DNA is amplified by PCR, utilizing primers for the
V and J regions of the T-cell receptor gamma chain gene. The
amplification products are analyzed by capillary electrophoresis, and
results are reported as positive or negative for the presence of a TCR-
gamma clonal expansion.
T-Cell Receptor (TCR) Gene Rearrangement, Quantitative PCR:
Following extraction, DNA is amplified by semi-nested PCR, utilizing
primers for the V and J regions of the T-cell receptor gamma chain
gene. The amplification products are analyzed by capillary
electrophoresis, and results are reported as the ratio of the clonal peak
area to that of the internal control (Ki-RAS). This test should be used to
monitor patients being treated for T-cell malignancies. The analytical
sensitivity is 1 monoclonal cell/10,000 normal cells when the initial pre-
therapy sample is provided for comparison.
These tests were developed and their performance characteristics determined by Quest
Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Positive results are highly suggestive of
malignancy; however, a positive result should not be used as the sole
criterion for diagnosis. Both positive and negative results should be
interpreted in the context of all clinical and laboratory findings. Up to
20% of B- and T-cell neoplasms can yield a false-negative result in
these assays.
6.6.3 CBFB/MYH11 inv(16), Quantitative Real-Time PCR
Clinical Use: This test is used to diagnose acute myelomonocytic
leukemia (AML) with abnormal eosinophils, with inv(16) or t(16;16,) (ie,
CBFB/MYH11); monitor effectiveness of treatment; monitor minimal
residual disease (MRD); and predict early relapse.
Clinical Background: The pericentric inversion of chromosome
16(p13;q22), and less frequently the t(16;16) (p13;q22) translocation,
accounts for 16% of the chromosomal aberrations associated with AML.
This inversion results in fusion of the core binding factor C (CBFB) gene
on 16q22 with the smooth muscle myosin heavy chain gene (MYH11) on
16p13, leading to the formation of a chimeric CBFB/MYH11 fusion
protein. Clinically, the inv(16) or t(16;16) is associated with AML with
abnormal eosinophils (French–American–British classification M4E0
subtype), with abnormal eosinophils being part of the malignant clone.
Patients with inv(16) or t(16;16) generally have relatively good response
and long-term disease-free survival rates.
The quantitative real-time reverse transcription-polymerase chain
reaction (RT-PCR) assay for CBFB/MYH11 can be used as a sensitive
tool for diagnosis of AML subtype M4E0, monitoring of MRD, and
assessment of the potential for disease relapse during and after
chemotherapy.
Method: This quantitative real-time RT-PCR method utilizes primers to
amplify the CBFB/MYH11 fusion transcript. Amplification of the abl gene
transcript is performed as a control for sample RNA quality and as a
reference for relative quantification. Results are reported as positive or
negative; if positive, the ratio of CBFB/MYH11 to abl transcript
amplification is reported. The current specimen will be tested side-by-side
with a previous sample, if available, to monitor the quantitative change
with time (trend). This assay can detect 1 tumor cell in 100,000 cells.
Hematology/Oncology
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems Inc.
Interpretive Information: Positive results are indicative of AML
subtype M4E0. Results are reported as the ratio of amplified fusion
product from the patient sample to that of the internal control. For
monitoring MRD, we recommend monitoring changes with time (trend)
rather than absolute ratio from a single measurement.
6.6.4 CD25, IHC
See Hematology/Oncology, “ONTAK®’ Sensitivity (CD25)” section 6.6.13.
6.6.5 Chronic Myeloproliferative Diseases
6.6.5.1 Laboratory Support of Diagnosis and Management
Clinical Background
Chronic myeloproliferative diseases (cMPDs) are clonal stem cell
disorders characterized by proliferation of 1 or more of the granulocytic,
erythrocytic, myelomastocytic, or megakaryocytic cell lines. These
diseases collectively have an incidence of 6 to 9 per 100,000 population
annually.1 This Clinical Focus describes the various cMPDs and the use
of laboratory testing for diagnosis and management.
The cMPDs include chronic myelogenous leukemia (CML), essential
thrombocythemia (ET), polycythemia vera (PV), chronic idiopathic
myelofibrosis (CIMF), chronic eosinophilic leukemia (CEL)/hypereo-
sinophilic syndrome (HES), systemic mastocytosis (SM), chronic
neutrophilic leukemia (CNL), and 8p11 myeloproliferative syndrome
(MPS). All cMPDs exhibit at least 1 of the features listed in Table 21.
cMPDs typically occur in adults 40 to 60 years old and are uncommon in
people <20 years old. Frequently, the onset is insidious and the clinical
course indolent. Patient complaints may include fatigue and lethargy,
weight loss, abdominal discomfort, easy bruising, night sweats, and
swollen, painful joints. Physical examination may reveal pallor,
enlargement of the spleen or liver, and petechiae.
Distinguishing between the cMPDs is often difficult because of the
overlap of clinical and laboratory findings. For example, most cMPDs
result in organomegaly, leukocytosis, and excessive megakaryocyte
proliferation. Each cMPD begins with effective hematopoiesis resulting
in circulating mature blood cells, but eventually progresses to
ineffective hematopoiesis and bone marrow failure or, potentially, acute
leukemia. Table 22 details hematologic characteristics of the various
cMPDs, including those considered diagnostic by the World Health
Organization (WHO).
The finding that constitutive (unregulated) protein-tyrosine kinase (PTK)
activity is associated with cMPDs has led to the development of
molecularly targeted therapies. For example, imatinib mesylate
(Gleevec) is a potent PTK inhibitor and prevents cellular proliferation in
bcr/abl-positive patients. Imatinib is the treatment of choice for CML
with a reported 76% complete cytogenetic response (CCR) rate in
chronic-stage patients.5 Imatinib has also been used to treat other
cMPDs including PV,6 CEL/HES,7 and certain subtypes of SM.4
Chronic Myelogenous Leukemia
Patients may present with splenomegaly, but more commonly CML is
detected with a routine complete blood count (CBC) in asymptomatic
 141

Section 6 Hematology/Oncology

Table 21. Clinical and Laboratory Features of cMPDs1-4

Feature CML ET PV CIMF CEL/HES SM CNL 8p11 MPS

Overproduction of 1 or more blood cell lines
with dominance of a transformed clone + + + + + – + +
Increased fibrosis in bone marrow b40% – +/– + – – – –
Increased cellularity in bone marrow + +/- + +/– + + + +
Chromosomal abnormalities* 100% z5% z15% z35% ? ? z10% 100%
Thrombotic and/or hemorrhagic
complications – + + + – + z27% –
Extramedullary hematopoiesis (EMH)
by liver and/or spleen + +/– + + – – – –
Transformation to AML z70% <5% z10% z18% – <5% – +
Overlapping clinical features + + + + + + + +
CML, chronic myelogenous leukemia; ET, essential thrombocythemia; PV, polycythemia vera; CIMF, chronic idiopathic myelofibrosis; CEL, chronic eosinophilic leukemia; HES,
hypereosinophilic syndrome; SM, systemic mastocytosis; CNL chronic neutrophilic leukemia; AML, acute myeloid leukemia; 8p11 MPS, 8p11 myeloproliferative syndrome.
*Does not include somatic mutations.

patients. The hallmark of CML is the presence of the Philadelphia
chromosome (Ph) and/or the bcr/abl fusion gene.1 CML progresses
through 3 sequential phases (chronic, accelerated, and blast), each of
which is progressively more resistant to therapy. Thus, early diagnosis
and treatment is imperative.
experience constitutional symptoms including fever, fatigue, cough,
angioedema, muscle pains, pruritus, and diarrhea. Tissue infiltration by
eosinophils, especially in the heart, skin, nervous system, and lungs may
lead to more serious symptoms. Organ involvement, especially in the
heart, is the most severe complication of CEL and HES.

Essential Thrombocythemia Systemic Mastocytosis

Although some patients present with symptoms of vascular occlusion or
hemorrhage, ET is asymptomatic in more than 50% of patients and is
identified fortuitously with a routine CBC that reveals an elevated
platelet count.1 Thrombocytosis due to secondary causes such as
systemic infections, inflammatory conditions, bleeding, or malignancy
must be ruled out before clonal ET can be diagnosed. Thrombosis and
hemorrhage are the most frequent clinical complications in patients
with ET.
Polycythemia Vera
The most serious complications of PV are thrombosis, hemorrhage, and
hypertension. Approximately 25% of patients present with venous or
arterial thrombosis, myocardial ischemia, or stroke.1 Major complaints at
diagnosis include headache, dizziness, visual disturbances, and
numbness/tingling. Erythromelalgia (vasodilation with burning), pruritus,
and gout may also be present. About 70% of patients have plethora or
splenomegaly and 40% have hepatomegaly.
Chronic Idiopathic Myelofibrosis
CIMF, also known as agnogenic myeloid metaplasia, is characterized by
anemia, progressive splenomegaly and bone marrow fibrosis, and multi-
organ extramedullary hematopoiesis (EMH). Up to 30% of patients with
CIMF are asymptomatic at diagnosis and CBC findings or splenomegaly
seen during a routine physical examination trigger the diagnostic
workup.1 The remainder present with signs of EMH, which accounts for
many of the peripheral blood findings in fibrotic CIMF. Major causes of
morbidity and mortality include acute myeloid leukemia (AML), which
develops in 5% to 30% of CIMF patients; bone marrow failure due to
hemorrhage or infection; thromboembolic events; portal hypertension;
and cardiac failure.1
Clinical symptoms of SM can be grouped into 1) constitutional
symptoms; 2) skin conditions such as pruritus and urticaria; 3) mast cell
mediator-related features including abdominal pain, flushing, headache,
and respiratory symptoms; and 4) bone-related complaints including
fractures and bone and joint pain.3 Symptoms range from mild to life-
threatening depending on the degree of organ involvement. SM should
be distinguished from cutaneous mastocytosis, a childhood disorder
usually confined to the skin that frequently shows spontaneous
regression.4
Chronic Neutrophilic Leukemia
Splenomegaly, the most constant clinical feature, is caused by
neutrophilic infiltration and may be symptomatic.1 Most patients have
hepatomegaly and 25% to 30% report a history of bleeding.
8p11 Myeloproliferative Syndrome
8p11 MPS is a rare disorder (<50 cases) characterized by gene changes
involving chromosome 8.8 Presenting symptoms may include lethargy,
weight loss, fevers, night sweats, lymphadenopathy, and splenomegaly.
Bone marrow findings include marked eosinophilia, which is also seen
in peripheral blood. Other CBC results include marked leukocytosis with
mostly neutrophils, metamyelocytes, and myelocytes; basophilia and
thrombocytosis are typically absent. Unlike other cMPDs, 8p11 MPS
progresses to high-grade T-cell lymphoblastic lymphoma in 60% to 70%
of patients and rapidly transforms to AML.
Individuals Suitable for Testing
• Individuals with the hematologic abnormalities listed in Table 22,
with or without clinical symptoms of cMPDs
• Individuals being monitored following diagnosis of a cMPD

Chronic Eosinophilic Leukemia/Hypereosinophilic Syndrome Test Availability

In about 10% of CEL or HES patients, hypereosinophilia is detected Table 23 lists tests used for diagnosis and management of cMPDs.
incidentally and no symptoms are apparent.1 Other patients may
142

Section 6 Hematology/Oncology

Table 22. Chronic Myeloproliferative Disease Incidence and Hematologic Characteristics, Including WHO Diagnostic Criteria
(Bolded)1,3
Incidence
cMPD (x 100,000) M:F Peripheral Blood Characteristics Bone Marrow Characteristics
Chronic 1–1.5 z1.7:1 Positive for Ph and/or bcr/abl Positive for Ph and/or bcr/abl
Myelogenous Leukocytosis (median WBC count ~170 x 109/L) Hypercellularity due to l neutrophils at different
Leukemia Predominance of neutrophils in different stages stages of maturation
of maturation Pseudo-Gaucher cells and sea-blue histocytes ±
Absolute basophilia; eosinophilia p Blasts <5%; l eosinophils
Platelet count N or l Small megakaryocytes
Mild anemia ± Reticulin fibers l in up to 40% of patients
Essential 1–2.5 1:1 Platelet count r600 x 109/L for r2 months Predominantly megakaryocyte proliferation
Thrombocythemia with mostly mature, enlarged forms
Polycythemia Vera 0.8–1.0 z1.7:1 l hemoglobin (>18.5 g/dL in men; >16.5 g/dL l cellularity with trilineage proliferation
in women) and hematocrit OR Stainable iron ±
l RBC mass (>25% above mean normal
predicted value)
WBC count >12 x 109/L*
Platelet count >400 x 109/L
Chronic Idiopathic 0.5–1.5 1:1 Prefibrotic stage Prefibrotic stage
Myelofibrosis • Mild to marked thrombocytosis • l cellularity
• Mild to moderate leukocytosis • Neutrophilic proliferation
• Mild anemia • Megakaryocyte proliferation with
Fibrotic stage • abnormal forms
• Moderate to marked anemia • Minimal or absent reticulin fibrosis
• Leukoerythroblastosis Fibrotic stage
• Prominent RBC poikilocytosis with • n cellularity
• teardrop shapes • Presence of reticulin and/or collagen
fibrosis
• Dilated marrow sinuses with intraluminal
• hematopoiesis
• Atypical and prominent megakaryocyte
• proliferation
• New bone formation (ie, osteosclerosis)
Chronic Eosinophilic Unknown z9:1 Persistent eosinophilia r1.5 x 109/L for l eosinophils
Leukemia/Hyper- r6 months Myeloblasts <20%
eosinophilic Syndrome Myeloblasts <20%
Systemic Mastocytosis Unknown z1:2 Anemia ± Multi-focal clusters or aggregates of mast
Leukopenia or leukocytosis ± cells (>15/ aggregate)
Thrombocytopenia or thrombocytosis ± Staining for tryptase may confirm the diagnosis
Mast cells may be spindle-shaped and may have
reniform or indented nuclei
Chronic Neutrophilic Unknown, 1:1 WBC count r25 x 109/L l cellularity with granulocytic hyperplasia
Leukemia but rare Segmented neutrophils and bands >80% Myeloblasts <5% of nucleated marrow cells
Immature granulocytes <10% N neutrophilic maturation pattern
Myeloblasts <1%
M:F, male to female ratio; Ph, Philadelphia chromosome; l, increased; N, normal; n, decreased; p, may be present.
*In the absence of fever or infection.

Test Selection and Interpretation
Chromosome Abnormalities
Because of the clonal nature of the cMPDs, detecting chromosomal
abnormalities and somatic mutations is important for diagnosis,
treatment selection, and monitoring. In the case of CML, a specific
chromosomal abnormality (ie, Ph+) is considered diagnostic. No such
specific abnormality or molecular marker has been identified for the
other disorders. However, recurring abnormalities that are not disease-
specific have been associated with the non-CML disorders (Table 24).
Detection of these abnormalities meets the WHO requirement for
establishing clonality and thus diagnosing a cMPD.12
Baseline bone marrow karyotyping data are used to identify clonal
evolution, which is the appearance of a genetic abnormality not present
previously. Clonal evolution is associated with a poor prognosis and, in
CML, is indicative of passing from the chronic to the accelerated or
blast phase (Table 24). Additionally, identification of a chromosomal
abnormality rules out a non-malignant reactive disorder.
 143

Section 6 Hematology/Oncology

Table 23. Tests Available to Support Chronic Myeloproliferative Disease Diagnosis and Management*

Test Code Assay Clinical Use

16031X ABL Kinase Domain Mutation in CML, Predict imatinib drug resistance prior to clinical relapse in patients with CML
Plasma-based, Leumeta™ ‡ Identify individuals who may benefit from alternative therapy
17853X bcr/abl Gene Rearrangement, Quantitative PCR, Diagnose CML
Plasma-based, Leumeta†‡ Monitor CML patients for therapeutic response, MRD, and early relapse
15101X bcr/abl Gene Rearrangement, Quantitative PCR Diagnose CML
with Reflex to Subtype†§ Distinguish ALL (subtype e1a2) from CML (subtypes b2a2/b3a2)
4420X C-Reactive Protein Rule out inflammatory conditions as a cause of reactive thrombocytosis,
leukocytosis, or eosinophilia
16104X c-kit Mutation Analysis, Plasma-based, Leumeta†‡ Diagnose SM
Predict response to imatinib therapy
14600X Chromosome Analysis, Hematological Malignancy† Diagnose, assess prognosis, and select treatment for cMPDs
Detect clonal evolution in cMPD disease progression
17734X Comprehensive Hematopathology Report Diagnose hematologic and hematopoietic disorders involving the bone marrow
Graphic report integrates morphologic, flow cytometric, and genetic test results
427X Erythropoietin (EPO) Differentiate secondary from primary polycythemia
457X Ferritin Rule out iron deficiency as cause of anemia
Differentiate PV from secondary erythrocytosis or from ET
16099X FIP1L1-PDGFRA Gene Rearrangement [del (4q12)], Support the diagnosis of CEL and rule out HES
Real-time PCR† Predict response to imatinib therapy
Monitor therapeutic response in FIP1L1-PDGFRA-positive patients10,19
12070X FISH, CML/ALL, bcr/abl Translocation 9;22† Diagnose or rule out CML
Assess prognosis and effectiveness of therapy in bcr/abl-positive patients
10055X FISH, Chromosome 20q Deletion† Assess prognosis in patients with PV, CIMF, CNL, and CEL1,11
10709X FISH, Myeloid Disorders Profile† Diagnose myeloid malignancies
Monitor therapeutic response in myeloid marker-positive patients
36743X Hematopathology Consultation Differential diagnosis of hematologic disorders
15684X Immunohistochemistry (IHC) Marker† Differential diagnosis of SM: identify CD2 and/or CD25 positive mast cells 3
10248X Intracellular Markers by Flow Cytometry† Differentiate myeloid from lymphoid malignancy
16101X JAK2 Mutation (V617F) Analysis, Diagnose or confirm the diagnosis of PV, ET, or CIMF
Plasma-based, Leumeta†‡
35080X Leukemia/Lymphoma Evaluation† Diagnose leukemia or lymphoma
Monitor therapeutic response and detect relapse
233X Leukocyte Alkaline Phosphatase Stain Differentiate CML from other causes of leukocytosis including CNL and PV
17862X T-Cell Receptor (TCR) Gene Rearrangement, Differentiate HES due to T-cell clonality from CEL due to eosinophilic clonality
Qualitative PCR, Leumeta†‡ Determine leukemia and lymphoma lineage
17861X T-Cell Receptor (TCR) Gene Rearrangement, Differentiate HES due to T-cell clonality from CEL due to eosinophilic clonality
Quantitative PCR, Leumeta†‡ Determine leukemia and lymphoma lineage
Detect and monitor MRD in patients with T-cell neoplasm
34484X Tryptase]] Differentiate systemic from cutaneous mastocytosis In the absence of other
myeloid disease4
Monitor effectiveness of SM therapy
928X Vitamin B12 Binding Capacity, Unsaturated Differentiate PV from secondary polycythemia
(Transcobalamin)
CML, chronic myelogenous; MRD, minimal residual disease; ALL, acute lymphoblastic leukemia; SM, systemic mastocytosis; HES hypereosinophillic syndrome; PV, polycythemia vera.
*Refer to Directory of Services for CPT codes and specimen collection and handling requirements.
†
This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
‡
Cell-based tests are also available. Plasma-based (ie, Leumeta) tests may replace cell-based tests that require bone marrow sample or tissue biopsy. Plasma-based tests may be more
sensitive in some situations.
§
Reflex tests are performed at an additional charge and are associated with an additional CPT code(s).
]]
This test was performed using a kit that has not been approved or cleared by the FDA. The analytical performance characteristics of this test have been determined by Quest Diagnostics
Nichols Institute. This test should not be used for diagnosis without confirmation by other medically established means.
144

Section 6 Hematology/Oncology

Table 24. Recurring Chromosomal Abnormalities Associated with cMPDs1,4,8,11,13

Disease Abnormalities
CML-chronic, accelerated, and blast phase t(9;22)(q34:q11), bcr/abl
CML-accelerated or blast phase +8, +Ph, +19, inv(17q), t(3;21)(q26;q22)(evi1/aml1)
ET +8, del(13q), t(X;5), inv(3), t(13;14), t(2;3), del(11q21)
PV +8, +9, del(20q), del(13q), del(1p11), +19, t(Y;1), t(3;17)
CIMF +8, del(20q), del(12p), -7/del(7q), del(11q), del(13q), del(3q), t(1;20), t(1;7), 5q-, 7q-, t(4;13), der(1q9p),
t(5;17)
CEL +8, t(5;12)(q33;p13)(tel/pdgfrb), dic(1;7), 8p11(fgfr1), del(20q), -7
SM +9, del 20(q12), t(8;21)
CNL +8, +9, del(20q), del(11q14), t(2;2)(q32:p24)
8p11 MPS t(8;13), t(8;9), t(6;8), t(8;22), t(8;19), t(8;17), t(8;11), t(8;12), ins(12;8)
CML, chronic myelogenous leukemia; ET, essential thrombocythemia; PV, polycythemia vera; CIMF, chronic idiopathic myelofibrosis; CEL, chronic eosinophilic leukemia; SM, systemic
mastocytosis; CNL, chronic neutrophilic leukemia; 8p11 MPS, 8p11 myeloproliferative syndrome.

cMPD-associated somatic mutations have been identified using Studies using the V617F mutation to determine prognosis have yielded
polymerase chain reaction (PCR) techniques (Table 25). Identification of inconsistent results. In one study of 244 patients with cMPDs (128 with
such mutations meets the WHO diagnostic criterion for establishing PV, 93 with ET, and 23 with CIMF), the V617F mutation was associated
clonality and may be useful in selecting therapy and evaluating with a longer duration of disease and a higher frequency of
prognosis. complications including secondary fibrosis, hemorrhage, and
thrombosis.15 In another study of 110 patients with CIMF, V617F-positive
JAK2 V617F Mutation patients had significantly worse survival than V617F-negative patients
JAK2 V617F is the first acquired somatic mutation found to be (hazard ratio [HR] = 3.30, 95% confidence interval [CI] 1.26–8.68,
associated with PV, CIMF, and ET; its detection supports the diagnosis of P=0.01).18 However, the presence of the V617F mutation was not
these cMPDs but does not distinguish between them. A negative result predictive of inferior survival in150 patients with ET followed for a
does not rule out the diagnosis, however, because the mutation is not median of 11.4 years.19
always present (Table 25). Several clinical studies have indicated that
the JAK2 V617F mutation is not present in patients with CML, Determining the zygosity of patients who are positive for the JAK2
secondary erythrocytosis, SM, or normal control subjects,15,16 but is V617F mutation improves the clinical use of the mutation as a
present in up to 2% of de novo AML cases.17 prognostic indicator. Quest Diagnostics Nichols Institute reports positive
results as heterozygous or homozygous/hemizygous (hemizygosity is
defined as 1 mutant and 1 deleted wild-type gene). In a preliminary
study of patients with cMPD (n=84), heterozygous patients had a
Table 25. Somatic Gene Mutations and Their Frequencies in survival advantage when compared to patients who were
homozygous/hemizygous or negative for the V617F mutation.20 When the
cMPDs4,7,14
frequency of thrombosis was examined in patients with ET (n=639),
homozygosity was associated with more frequent events than in
Disease Genetic Mutation Frequency (%)
patients who were heterozygous or negative for V617F; however, no
ET JAK2 V617F 31 such differences were apparent in patients with PV.21 Furthermore, in
multivariate analysis, homozygous patients with ET had increased risk of
PV JAK2 V617F 76
a cardiovascular event when compared with patients who were V617F-
CIMF JAK2 V617F 50 negative (HR = 3.97, 95% CI 1.34–11.7, P = 0.013).21
CEL FIP1L1/PDGFRA 26
FIP1L1-PDGFRA Fusion Gene
SM c-kit D816V >80 The presence of the FIP1L1/PDGFRA somatic mutation confirms the
diagnosis of CEL and predicts a favorable response to imatinib
c-kit D816Y <5
treatment.7,9 This mutation produces a PTK, which is the cause of CEL,
c-kit D816H <5 and inhibition of the PTK activity with imatinib accounts for the
effectiveness of this treatment.22 Such responses are also seen in a
c-kit D820G <5
small subset of SM patients with eosinophilia who are positive for
c-kit V560G <5 FIP1L1/PDGFRA (Table 25). FIP1L1/PDGFRA mutation testing is used to
monitor therapy; molecular remission is documented when positive
c-kit F522C <5
results become negative.10
c-kit V530I <5
Bone Marrow Histology
SM with eosinophilia FIP1L1/PDGFRA <5
WHO diagnostic guidelines include bone marrow findings as criteria for
ET, essential thrombocythemia; PV, polycythemia vera; CIMF, chronic idiopathic identifying and distinguishing between the cMPDs (Table 22). The WHO
myelofibrosis; CEL, chronic eosinophilic leukemia; SM, systemic mastocytosis.

Section 6
recommends that bone marrow biopsy and peripheral blood specimens
be evaluated together to reach a diagnosis.1 Quest Diagnostics uses
various stains and immunohistochemical markers to evaluate bone
marrow. Cellularity, collagen and reticulin fibrosis, and proliferation of
granulocytes, megakaryocytes, mast cells, and erythrocytes are routinely
evaluated to aid in the diagnosis of a cMPD.
cMPD progression is associated with increased bone marrow fibrosis
and transformation to AML. Thus, once a cMPD has been diagnosed,
disease progression may be monitored with periodic evaluation of bone
marrow cellularity, degree of fibrosis, and cytogenetic changes.
Immunophenotyping
Flow cytometric analysis for cell surface markers (immunophenotyping) is
used for differential diagnosis of leukemia and lymphoma, to assess
response to therapy, and to detect relapse. Lineage-specific markers
distinguish myeloid from lymphoid disorders and include myeloperoxidase
found in myeloid disorders, cytoplasmic CD3 in T-lymphocytes, and
cytoplasmic CD22 and IgM in B-lymphocytes.23 Immunophenotyping helps
guide therapeutic decisions when a cMPD progresses as reflected by
increased aberrant cells. On the other hand, decreases in abnormal
marker-positive cells are associated with therapeutic success.
Immunophenotype testing (ie, the Leukemia/Lymphoma Evaluation test) is
particularly important when blast cells increase to>10% in blood or
marrow for defining cell lineage associated with disease progression.
Chronic Myelogenous Leukemia Testing
Differential diagnosis begins with the diagnosis or exclusion of CML in
symptomatic patients or in those who have cMPD-related abnormalities
in their CBC (Figure 4). Fluorescence in situ hybridization (FISH) testing
provides qualitative results for bcr/abl, whereas reverse transcription-
PCR (RT-PCR) results are quantitative. Positive results are diagnostic of
CML, while negative results eliminate CML. Other cMPDs (bcr/abl-
negative) should then be considered.
Approximately 95% of patients with CML are Ph+, whereas all are
positive for bcr/abl by FISH and RT-PCR. This seeming discrepancy can
be explained by the presence of a masked Ph, which is not observed by
conventional karyotyping but is detected by the molecular tests.1
Following interferon or imatinib therapies, conventional bone marrow
cytogenetic testing best predicts disease-free and overall survival. Once
a complete cytogenetic response (CCR) is achieved (ie, the absence of
Ph+ metaphase cells), molecular monitoring should be used.5,24 RT-PCR
is favored over FISH because bcr/abl mRNA correlates with CCR, can be
used to monitor the kinetics of leukemia disease burden, and can be
performed on peripheral blood.24 Most studies have found a good
correlation between blood and marrow PCR values.24 At a recent
consensus conference held by the National Institutes of Health (NIH),
testing intervals of 3 to 6 months were recommended, although rising
bcr/abl levels warrant more frequent testing.25 In the International
Randomized Study of Interferon versus STI571 (IRIS), a r3-log reduction
of bcr/abl transcript levels in peripheral blood was associated with a
negligible risk of disease progression.26
Treatment with imatinib can result in the acquired resistance that is
most commonly associated with point mutation(s) in the PTK domain of
bcr/abl.27 Such mutations may precede or accompany progression to a
more aggressive disease.27,28 ABL kinase domain mutation testing is
appropriate for patients presenting with advanced disease and for
chronic-phase patients with inadequate initial response (failure to
achieve a complete hematologic response at 3 months, minimal
cytogenetic response at 6 months, or CCR at 12 months).25
145
Hematology/Oncology
Although risk of early mortality and chronic morbidity is present,
allogeneic hematopoietic stem cell transplantation (AHSCT) provides
another approach to CML treatment. As with drug treatments, AHSCT is
most effective in the chronic phase (60% long-term remission) compared
to either accelerated (z30%) or blast phase (z14%).29 Following such
treatment, detection of bcr/abl by RT-PCR is strongly associated with an
increased risk of relapse. In a study of 346 patients, positive results at 6
to 12 months after transplant were associated with a 42% risk of
relapse at a median of 200 days; negative results were associated with
a 3% risk.30 Transplant centers typically test for peripheral blood bcr/abl
mRNA every 3 to 6 months for the first 2 years and yearly thereafter.24
Table 26 summarizes testing used in selecting and monitoring therapy
and assessing prognosis for CML and other cMPDs.
Essential Thrombocythemia
Although ET is diagnosed mainly by exclusion, positive criteria for the
diagnosis are a sustained platelet count of r600 x 109/L and increased
megakaryocyte proliferation in bone marrow studies (Table 22). Normal
C-reactive protein levels rule out reactive causes of thrombocytosis,
which are more common than clonal ET. However, elevated C-reactive
protein levels are not helpful for diagnosis in this setting.31
Additional exclusion criteria include absence of underlying neoplasm
and other cMPDs associated with increased megakaryocyte
proliferation.1 A positive result for the JAK2 V617F mutation is likely to
rule out CML, myelodysplastic syndrome (V617F-positive in 5% of
patients), and non-hematologic cancer.15-17 A negative result requires
chromosome testing to rule out malignancies such as CML, 5q-
syndrome, AML with inv(3), and myelodysplastic syndrome.1 PV is ruled
out with normal hemoglobin and/or RBC mass, and fibrotic stage CIMF
is ruled out by the absence of reticulin and/or collagen fibrosis in bone
marrow.1
A laboratory test guide for the differential diagnosis of ET is presented
in Figure 5. Laboratory tests used to select and monitor therapy and to
assess prognosis for ET are presented in Table 26. The use of
cytoreductive therapy to reduce elevated platelet counts has been
suggested for high-risk patients (ie, age r60 years or a history of
thrombosis).32
Polycythemia Vera
Increased RBC mass, measured using radioactive chromium, is the
hallmark of PV diagnosis. This direct measurement is not necessary
when the hemoglobin is >18.5 g/dL in men or >16.5g/dL in women.
The WHO criteria for diagnosing PV include ruling out the more common
inherited and secondary, acquired erythrocytosis.1 Acquired
erythrocytosis can be due to chronic hypoxia, treatment with
erythropoietin (EPO) or androgens, or EPO-secreting tumors. Once
elevated RBC mass or hemoglobin levels are observed (Table 22), a
subnormal serum EPO level and a positive JAK2 V617F mutation
excludes secondary erythrocytosis and inherited polycythemia (see
Figure 6). An elevated EPO level rules out PV, while a normal level is
inconclusive. Negative JAK2 V617F mutation makes the diagnosis of PV
less likely but does not rule it out.
The WHO provides a second alternative to diagnose PV: hemoglobin or
RBC mass is elevated, inherited and secondary erythrocytosis are ruled
out, and 2 other criteria are met. These latter criteria include >400 x
109/L platelet count, >12 x 109/L WBC count, characteristic bone marrow
cytology (Table 22), and low serum EPO level.
146

Section 6 Hematology/Oncology

Complete Blood Count

cMPD-associated abnormalities
(Table 2)

bcr/abl (FISH [12070]*

or
Quantitative PCR) [17853]

Negative Positive

JAK2 Mutation (V617F) [16101] CML diagnosis likely

Positive† Negative†

Suspect CEL
Suspect ET Platelet count 600 x 10 9 /L or HES Eosinophil count
See Fig 2 for 2 months See Fig 5 1.5 x 10 9 /L for 6 months

Suspect PV Elevated hemoglobin/ Symptoms including

See Fig 3 hematocrit or RBC mass Suspect SM flushing, pruritis, urticaria,
See Fig 6 abdominal cramping,
diarrhea, bronchoconstriction,
Anemia, thrombocytosis, and headache
leukocytosis, splenomegaly,
Suspect CIMF and/or leukoerythroblastosis
See Fig 4 with RBC poikilocytosis WBC 2 5x10 9 /L,
(teardrop shapes) Suspect CNL segmented neutrophils and
See Fig 7 bands >80%, and immature
granulocytes <10%
Suspect Overlapping clinical and
unclassifiable laboratory features of
cMPD cMPDs present Suspect 8p11 Marked eosinophilia,
See text MPS leukocytosis with
See Fig 8 neutrophilia and left shift
*Test codes are included in brackets for all figures.
†
See text for interpretation of JAK2 mutation results.

Figure 4. Differential diagnosis of cMPDs.1, 3, 4, 8

Laboratory tests to select and monitor therapy and to assess prognosis decreased numbers of erythroid precursors, and marked atypical forms
for PV are presented in Table 26. Phlebotomy targeting reduction in of megakaryocytes in bone marrow confirms a diagnosis of prefibrotic
hematocrit is the cornerstone of PV therapy. CIMF.12

Chronic Idiopathic Myelofibrosis
The classical presentation of CIMF is the appearance of a
leukoerythroblastic blood smear with teardrop poikilocytosis, anemia,
splenomegaly and possibly hepatomegaly due to EMH, and bone
marrow fibrosis.1 This picture is characteristic of the fibrotic, advanced
stage of the disease. Diagnosis is more complicated in the 20% to 30%
of patients who are at the prefibrotic stage, which can resemble PV or
ET. The differential diagnosis is important since CIMF has worse survival
than ET or PV.12 Observation of prominent neutrophil proliferation,
Detection of the JAK2 V617F mutation supports the diagnosis of CIMF
but does not distinguish between PV, ET, and CIMF. Absence of the
mutation does not rule out the diagnosis. Detection of a clonal
aberration, which is relatively common in CIMF (z35%), also supports
the diagnosis. A laboratory test guide for the differential diagnosis of
CIMF is presented in Figure 7.
Table 26 summarizes tests used to monitor therapy and assess
prognosis of CIMF. A combination of age >60 years, hemoglobin levels
 147

Section 6 Hematology/Oncology

Table 26. Tests Used in the Management of Patients with cMPD

Assay Select Therapy Monitor Therapy Assess Prognosis
CML
ABL Kinase Domain Mutation in CML X X X
bcr/abl Gene Rearrangement. Quantitative PCR X X X
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X X X
% Blast Cells X X X
ET
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X
Platelet Count X X X
PV
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X X
Hematocrit X X
CIMF
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X X
Platelet Count, Hemoglobin, and % Blast Cells X X
CEL/HES
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X
Eosinophil Count X
FIP1L1/PDGFRA Gene Rearrangement X X X
% Blast Cells X
SM
Bone Marrow Histology X
c-kit Mutation Analysis X X
FIP1L1/PDGFRA Gene Rearrangement X X X
Tryptase X
CNL
Bone Marrow Histology X
Bone Marrow Chromosome Analysis X
% Blast Cells X X
8p11 MPS
Bone Marrow Histology X X
Bone Marrow Chromosome Analysis X
CML, chronic myelogenous leukemia; ET, essential thrombocythemia; PV, polycythemia vera; CIMF, chronic idiopathic myelofibrosis; CEL, chronic eosinophilic leukemia; HES,
hypereosinophilic syndrome; SM, systemic mastocytosis; CNL, chronic neutrophilic leukemia; 8p11 MPS, 8p11 myeloproliferative syndrome.

<10 g/dL, platelet counts <100 x 109/L, and >3% circulating blast cells
predict a median survival of <5 years as compared to z15 years in their
absence.33 The detection of certain chromosomal abnormalities also
appears to be prognostic. In one study of 165 patients with CIMF,
trisomy 8 or 12p deletion was associated with a poor prognosis while
deletions of 13q or 20q were not.13
Chronic Eosinophilic Leukemia/Hypereosinophilic Syndrome
Hypereosinophilia (r1.5 x 109/L) may be due to reactive eosinophilia,
idiopathic HES, or CEL. The differential diagnosis of CEL and HES begins
with the exclusion of all causes of reactive eosinophilia, including
parasitic infection, infectious disease, allergic reaction, pulmonary
diseases such as hypersensitivity pneumonitis, collagen vascular
diseases, and underlying neoplastic disease.1 Neoplastic diseases to be
excluded include T-cell lymphomas, Hodgkin lymphoma, acute
lymphoblastic leukemia/lymphoma, other cMPDs, AML, and
myelodysplastic syndromes. Once these diagnoses have been excluded,
CEL is diagnosed if there is evidence of a clonal myeloid abnormality
(eg, the presence of the FIP1L1/PDGFRA fusion gene); HES is diagnosed
if no such evidence is found.1
Molecular testing can help distinguish the causes of hypereosinophilia.
In a series of patients with persistent hypereosinophilia, T-cell receptor
gene rearrangements were present in 32% leading to the diagnosis of
T-cell associated HES and the FIP1L1/PDGFRA fusion gene was detected
in 17%, confirming the diagnosis of CEL.10 Thus, detection of these 2
genetic mutations led to a specific diagnosis in nearly 50% of the
patients and was useful in ruling out T-cell-associated HES in the
remainder. Because the FIP1L1/PDGFRA fusion gene is not present in all
patients with CEL, its absence does not rule out CEL. A laboratory test
guide for CEL and HES diagnosis is presented in Figure 8.
Detection of the FIP1L1/PDGFRA fusion gene can also be used to predict
response to imatinib.7,9 Although the number of patients studied was
148

Section 6 Hematology/Oncology

Systemic Mastocytosis
Bone Marrow Histology [36743] and The major diagnostic criterion for SM is the presence of dense
Chromosomal Analysis [14600] multifocal clusters or aggregates of mast cells (>15 per aggregate) in a
bone marrow biopsy specimen.3 Minor criteria include: 1) abnormal
morphology in >25% of mast cells; 2) c-kit mutation at codon 816;
3) mast cells coexpressing CD117 and CD2 and/or CD25; and 4) serum
Megakaryocyte proliferation with clusters; tryptase levels persistently >20 ng/mL in the absence of an associated
mostly mature forms hematologic clonal non-mast cell lineage disease. SM is diagnosed if at
least the major criterion plus 1 minor criterion, or at least 3 minor
Minimal or absent reticulin fibrosis; absent criteria, are met. A laboratory test guide for SM diagnosis is presented
collagen fibrosis in Figure 9.
Stainable iron present
An elevated tryptase level is an important marker of SM, but may also
No chromosomal evidence or granulocytic dysplasia be seen in acute and chronic myeloid leukemias, other cMPDs,
associated with myelodysplastic syndrome myelodysplastic syndromes, and myelomastocytic leukemia.4

In addition to being a criterion for diagnosis, the c-kit D816V mutation

ET diagnosis likely
Figure 5. Differential diagnosis of ET.1 (Continued from Figure 4)
limited, 57 of 57 (100%) positive for FIP1L1/PDGFRA achieved complete
hematological response with imatinib.7 Negative FIP1L1/PDGFRA
results, however, did not rule out response to therapy; 12 of 53 (23%)
achieved either a partial or a complete hematologic response.
In preliminary studies, MRD detection using FIP1L1/PDGFRA testing was
useful for monitoring response to imatinib therapy.9,10 Table 26
summarizes tests used to select and monitor therapy and assess
prognosis in patients with CEL or HES.
confers resistance to imatinib by interfering with the binding of the drug
to the catalytic site of the KIT PTK. Conversely, the presence of wild-
type or F522C c-kit or the FIP1L1/PDGFRA fusion gene is associated with
sensitivity to imatinib.4
Table 26 summarizes tests used to select and monitor therapy and
assess prognosis in patients with SM.
Chronic Neutrophilic Leukemia
CNL is diagnosed when the hematologic criteria are met (Table 22),
hepatosplenomegaly is present, no evidence of a physiologic
neutrophilia is found, and other cMPDs and myelodysplastic disorders
are ruled out.1 A normal C-reactive protein level rules out inflammation
and infection that could produce neutrophilia; however, an elevated

Erythropoietin [427] /
JAK2 Mutation (V617F) [16101]

Low/Positive Low/Negative Normal/Positive or Negative High/Negative

Rules out secondary PV diagnosis less likely PV diagnosis unlikely

erythrocytosis and
inherited polycythemia

PV diagnosis likely

Bone Marrow Histology [36743] and

Chromosomal Analysis [14600]

Hypercellularity with trilineage proliferation

Stainable iron may be absent
Chromosomal abnormality, if present,
documents clonality

PV diagnosis likely, especially with splenomegaly

Figure 6. Differential diagnosis of PV.1 (Continued from Figure 4)
 149

Section 6 Hematology/Oncology

8p11 Myeloproliferative Syndrome

Bone Marrow Histology [36743] and
Chromosomal Analysis [14600]
Neutrophililc proliferation, megakaryocyte
proliferation with maturation defects, and
presence of reticulin and collagen fibrosis
Chromosomal abnormality, if present,
documents clonality
CIMF diagnosis likely
Figure 7. Differential diagnosis of CIMF.1 (Continued from Figure 4)
level does not necessarily rule out CNL. Negative results for Ph or
bcr/abl rule out CML, which also presents with neutrophilia. Other
cMPDs are ruled out by the absence of their usual hematologic
characteristics (Table 22). Furthermore, myelodysplastic disorders are
ruled out by the absence of granulocytic dysplasia and myelodysplastic
changes in other myeloid lineages. A laboratory test guide for CNL
diagnosis is presented in Figure 10.
A summary of tests used to monitor therapy and assess prognosis of
CNL is presented in Table 26.
Suspicion of the 8p11 MPS is triggered by eosinophil counts ranging
from 1.2 x 109/L to 40 x 109/L (median 4 x109/L) as reported in a series
of 14 patients.8 Bone marrow studies show myeloid hyperplasia, and
chromosome analysis identifies abnormalities associated with
chromosome 8 (see Figure 11). Transformation to AML or lymphoma has
been associated with +21 and this clonal evolution may be identified
with karyotyping (see Table 26).8
Unclassifiable cMPD
In approximately 15% of cMPD cases, clinical and laboratory features
characteristic of a myeloproliferative disease are present but fail to meet
the diagnostic criteria of any one cMPD.1 Such patients are either in the
early stages of the disease and characteristic features of a particular
cMPD will develop with time or are in the advanced stages of the
disease with marked marrow fibrosis or blastic infiltration. In the former
case, reevaluation at intervals of 4 to 6 months is recommended.1
References
1. Jaffe ES, Harris NL, Stein H, et al. eds. World Health Organization
Classification of Tumours: Pathology and Genetics of Tumours of
Hematopoietic and Lymphoid Tissues. Lyon, France: IARC Press;
2001.
2. Chronic Myeloproliferative Disorders (PDQ®’): Treatment. Available
at: http://www.cancer.gov/cancertopics/pdq/treatment/
myeloproliferative/healthprofessional. Accessed August 2, 2006.
3. Valent P, Horny H-P, Escribano L, et al. Diagnostic criteria and
classification of mastocytosis: a consensus proposal. Leuk Res.
2001;25:603-625.

FIP1L1-PDGFRA Gene Rearrangement [16099] /

T-Cell Receptor Gene Rearrangement [17861]

Negative/Negative Positive/Negative Negative/Positive

CEL diagnosis likely Diagnose T-cell

associated HES

Bone Marrow Histology [36743] and

Chromosomal Analysis [14600]

Increased numbers of eosinophils Normocellular, normal

<20% myeloblasts trilineage hematopoiesis,
and normal karyotype
Chromosomal abnormality, if
present, documents clonality

Possible early stage HES or

reactive eosinophilia
CEL diagnosis likely with clonality;
otherwise, diagnose HES Retest if eosinophilia persists

Figure 8. Differential diagnosis of CEL/HES.1, 10 (Continued from Figure 4)

150

Section 6 Hematology/Oncology

c-kit Mutation [16104]

Positive Negative

SM diagnosis likely SM diagnosis unlikely

Bone Marrow Histology [36743] and

Chromosomal Analysis [14600]

Mast cell aggregates ( >15 per aggregate)

CD2+ and/or CD25+ mast cells
>25% of mast cells are spindle-shaped or atypical

SM diagnosis likely
Figure 9. Differential diagnosis of SM.3 (Continued from Figure 4)

4. Valent P, Akin C, Sperr WR, et al. Mastocytosis: pathology, genetics,
and current options for therapy. Leuk Lymphoma. 2005;46:35-48.
5. O’Brien SG, Guilhot F, Larson RA, et al. Imatinib compared with
interferon and low-dose cytarabine for newly diagnosed chronic-
phase chronic myeloid leukemia. N Engl J Med. 2003;348:994-1004.
6. Silver RT. Treatment of polycythemia vera with recombinant
interferon B (rIFN B) or imatinib mesylate. Curr Hematol Rep.
2005;4:235-237.
7. Pardanani A, Ketterling RP, Li C-Y, et al. FIP1L1-PDGFRA in
eosinophilic disorders: prevalence in routine clinical practice, long-
term experience with imatinib therapy, and a critical review of the
literature. Leuk Res. 2006;30:965-970.
8. Macdonald D, Reiter A, Cross NCP. The 8p11 myeloproliferative
syndrome: A distinct clinical entity caused by a constitutive
activation of FGFR1. Acta Haematol. 2002;107:101-107.
Bone Marrow Histology [36743] and
Chromosomal Analysis [14600]
9. Gotlib J, Cools J, Malone JM, et al. The FIP1L1-PDGFRB fusion
tyrosine kinase in hypereosinophilic syndrome and chronic
eosinophilic leukemia: implications for diagnosis, classification, and
management. Blood. 2004;103:2879-2891.
10. Roche-Lestienne C, Lepers S, Soenen-Cornu V, et al. Molecular
characterization of the idiopathic hypereosinophilic syndrome (HES)
in 35 French patients with normal conventional cytogenetics.
Leukemia. 2005;19:792-798.
11. Albitar M. Myeloproliferative diseases: molecular genetics. In:
Encyclopedia of Life Sciences. New York, NY: John Wiley & Sons,
Ltd; 2005:1-6.
12. Vardiman JW, Harris NL, Brunning RD. The World Health
Organization (WHO) classification of the myeloid neoplasms. Blood.
2002;100:2292-2302.
13. Tefferi A, Mesa RA, Schroeder G, et al. Cytogenetic findings and
their clinical relevance in myelofibrosis with myeloid metaplasia.
Br J Haematol. 2001;113:763-771.
14. Skoda R, Prchal JT. Chronic myeloproliferative disorders-
introduction. Semin Hematol. 2005;42:181-183.
15. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function

Hypercellularity, neutrophilic proliferation with a normal Bone Marrow Histology [36743] and
maturation pattern Chromosomal Analysis [14600]

Myeloblasts <5% of nucleated cells

No evidence of other cMPDs Myeloid hyperplasia

Marked eosinophilia
No chromosomal evidence of myelodysplastic syndrome
or myeloid dysplasia Presence of t(8;13), t(8;9), t(6;8), t(8;22), t(8;19),
t(8;17), t(8;11), t(8;12), or ins(12;8)
Chromosomal abnormality, if present, documents clonality

Diagnose 8p11 MPS

CNL diagnosis likely, especially with hepatosplenomegaly
Figure 11. Differential diagnosis of 8p11 myeloproliferative syndrome.8
Figure 10. Differential diagnosis of CNL.1 (Continued from Figure 4) (Continued from Figure 4)

Section 6
mutation of JAK2 in myeloproliferative disorders. N Engl J Med.
2005;352:1779-1790.
16. Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2
V617F mutation in chronic myeloproliferative disorders. Blood.
2005;106:2162-2168.
17. Lee JW, Kim YG, Soung JH, et al. The JAK2 V617F mutation in de
novo acute myelogenous leukemias. Oncogene. 2006;25:1434-1436.
18. Campbell PJ, Griesshammer M, Dohner K, et al. The V617F mutation
in JAK2 is associated with poorer survival in idiopathic
myelofibrosis. Blood. 2006;107:2098-2100.
19. Wolanskyj AP, Lasho TL, Schwager SM, et al. JAK2 mutation in
essential thrombocythaemia: clinical associations and long-term
prognostic relevance. Br J Haematol. 2005;131:208-213.
20. Ma W, Kantarjian H, Jilani I, et al. Hemizygous/homozygous and
heterozygous JAK2 mutation detected in plasma of patients with
myeloproliferative diseases: correlation with clinical behavior. Br J
Haematol. 2006;134:341-343.
21. Vannucchi AM, Antonioli E, Gugliemelli P, et al. Clinical profile of
homozygous JAK2 V617F mutation in patients with polycythemia
vera or essential thrombocythemia. Blood. 2007;110:840-846.
22. Cools J, DeAngelo DJ, Gotlib J, et al. A tyrosine kinase created by
fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of
imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med.
2003;348:1201-1214.
23. Todd WM. Acute myeloid leukemia and related conditions. Hematol
Oncol Clin N Am. 2002;16:301-319.
24. Oehler VG, Radich JP. Monitoring bcr/abl by polymerase chain
reaction in the treatment of chronic myeloid leukemia. Current
Oncology Reports. 2003;5:426-435.
25. Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients
responding to treatment with tyrosine kinase inhibitors-review and
recommendations for “harmonizing” current methodology for
detecting BCR-ABL transcripts and kinase domain mutations and for
expressing results. Blood. 2006;108:28-37.
26. Hughes TP, Kaeda J, Branford S, et al. Frequency of major molecular
responses to imatinib or interferon alfa plus cytarabine in newly
diagnosed chronic myeloid leukemia. N Engl J Med. 2003;349:1423-
1432.
27. Branford S, Rudzki Z, Parkinson I, et al. Real-time quantitative PCR
analysis can be used as a primary screen to identify patients with
CML treated with imatinib who have BCR-ABL kinase domain
mutations. Blood. 2004;104:2926-2932.
28. Soverini S, Martinelli G, Rosti G, et al. ABL mutations in late chronic
myeloid leukemia patients with up-front cytogenetic resistance to
imatinib are associated with a greater likelihood of progression to
blast crisis and shorter survival: a study by the GIMEMA Working
Party on Chronic Myeloid Leukemia. J Clin Oncol. 2005;23:4100-4109.
29. Tefferi A, Dewald GW, Litzow ML, et al. Chronic myeloid leukemia:
Current application of cytogenetics and molecular testing for
diagnosis and treatment. Mayo Clin Proc. 2005;80:390-402.
30. Radich JP, Gehly G, Gooley T, et al. PCR detection of the bcr/abl
fusion transcript after allogeneic marrow transplantation for chronic
myeloid leukemia: results and implications in 346 patients. Blood.
1995;85:2632-2638.
31. Anastasi J, Vardiman JW. Chronic myelogenous leukemia and the
chronic myeloproliferative diseases. In: Knowles DM, ed. Neoplastic
Hematopathology. 2nd ed. Philadelphia, PA: Lippincott Williams &
Wilkins; 2001:1745-1790.
32. Tefferi A, Barbui T. bcr/abl-negative, classic myeloproliferative
disorders: diagnosis and treatment. Mayo Clin Proc. 2005;80:1220-
1232.
33. Okamura T, Kinukawa N, Niho Y, et al. Primary chronic
myelofibrosis: clinical and prognostic evaluation in 336 Japanese
patients. Int J Hematol. 2001;73:194-198.
151
Hematology/Oncology
6.6.5.2 bcr/abl Gene Rearrangement, FISH and Quantitative PCR
Clinical Use: These tests are used to diagnose chronic myelogenous
leukemia (CML) in the presence or absence of Philadelphia chromosome,
determine prognosis, detect relapse, and identify acute lymphocytic
leukemia (ALL) patients who have the bcr/abl rearrangement. The
quantitative polymerase chain reaction (PCR) test is used to monitor
CML patients for therapeutic response, minimal residual disease (MRD),
and early relapse.
Clinical Background: The bcr/abl rearrangement results in
production of an abnormal tyrosine kinase molecule with increased
tyrosine kinase activity, postulated to be responsible for development of
leukemia. The bcr/abl fusion gene, formed by rearrangement of the
breakpoint cluster region (bcr) on chromosome 22 with the c-abl proto-
oncogene on chromosome 9, is present in virtually all CML patients. It is
also identified in some cases of ALL, in which it is associated with poor
prognosis.
The t(9;22)(q34;q11) translocation associated with bcr/abl leads to a
cytogenetic aberration known as Philadelphia chromosome, although
this rearrangement may also be detected in the absence of
cytogenetically defined Philadelphia chromosome. In CML without
Philadelphia chromosome, the diagnosis is usually CML in blast crisis,
which differentiates along several pathways with varying prognosis.
Four fusion transcripts can result from the bcr/abl rearrangement,
depending on the breakpoint on chromosome 22: b2a2, b3a2, e1a2, and
e19a2. b2a2 and b3a2 are detected mainly in CML and e1a2 is detected
mainly in ALL. e19a2 results from a rare rearrangement detected in
CML. With the quantitative PCR, a reflex test code is available that
allows specimens with positive bcr/abl results to be reflexed to an
additional PCR assay (at additional charge) to distinguish e1a2 from
b2a2 and b3a2.
FISH Method: [FISH, CML/ALL, bcr/abl Translocation 9;22] This
fluorescence in situ hybridization (FISH) method employs probes
representative of the chimeric bcr/abl junction. The assay detects both
major and minor chimeric fusions but does not discriminate between
them.
PCR Method: [bcr/abl Gene Rearrangement, Quantitative PCR] In this
quantitative assay, extracted RNA is subjected to real-time reverse
transcription-polymerase chain reaction (RT-PCR) to amplify 3 types of
bcr/abl fusion transcripts: b2a2, b3a2, and e1a2. An additional
amplification of the abl gene is performed as a control for sample RNA
quality and as a reference for relative quantification. For follow-up and
monitoring the effectiveness of treatment, a previously stored sample, if
available, will be analyzed along with the current sample to assess
quantitative changes with time (trend). If there is no amplification of
fused mRNA (BCR:ABL ratio = 0), the result is reported as negative. For
samples with positive results, the ratio between the quantities of the
fused mRNA (BCR/ABL) and control mRNA (ABL) is reported. The
analytical sensitivity of this test is 1 tumor cell in 100,000 normal cells.
This assay does not detect the e19a2 transcript.
A reflex test can be ordered to determine the type of translocation.
With this test, specimens with positive bcr/abl results are reflexed to an
additional PCR assay (at additional charge) to distinguish e1a2
(expressed in ALL) from b2a2 and b3a2 (expressed in CML). Results are
reported as positive or negative for e1a2 and b2a2/b3a2.
These tests were developed and their performance characteristics determined by Quest
Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
152
Section 6
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: The bcr/abl gene rearrangement is
observed in CML, ALL, and, rarely, AML. A positive result indicates the
presence of Philadelphia chromosome, but the diagnosis of CML, ALL, or
AML should be based on the presence of characteristic cellular
abnormalities in bone marrow. In patients with ALL, the bcr/abl
rearrangement is associated with poor prognosis.
Serial monitoring of assay values may provide a quantitative measure of
tumor burden and response to therapy. Increasing levels of BCR/ABL
detected with quantitative PCR are associated with clinical progression.
To monitor MRD, we recommend evaluating changes with time (trend)
rather than the absolute ratio from a single time point.
6.6.5.3 Campath®’ Sensitivity (CD52)
Clinical Use: This test is used to determine eligibility for Campath
(alemtuzumab; anti-CD52) treatment in patients with chronic
lymphocytic leukemia (CLL).
Clinical Background: Campath is a humanized antibody targeted
against CD52, an antigen that can be expressed at high density on the
surface of malignant CLL cells. Binding of Campath to CD52 on the
target cells is necessary for cell death and therapeutic response.
Because this drug can cause significant morbidity, including neutropenia
and thrombocytopenia, drug sensitivity should be established before
treatment is initiated.
Method: In this 4-color, multiparametric flow cytometry assay, white
blood cells are stained with fluorescently labeled CD52, CD19, CD5, and
CD45 monoclonal antibodies. The stained cells are run on a flow
cytometer, lymphocytes are selected by CD45 vs side scatter gating, and
a subsequent CD19+CD5+ population is selected. The presence or
absence of CD52 on the surface of the selected lymphocytes is
determined by mean fluorescence intensity.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A positive result indicates the presence of
CD52 on the patient’s lymphocytes and eligibility for Campath therapy.
6.6.5.4 Chronic Lymphocytic Leukemia, IgVH Mutation Status
Clinical Use: This test is used to assess prognosis for patients with
chronic lymphocytic leukemia (CLL).
Clinical Background: CLL is the most common leukemia in the
Western world. Patients with CLL follow heterogeneous clinical courses.
Some survive for prolonged periods without definitive therapy, while
others die rapidly, despite aggressive treatment. CLL patients can be
divided into 2 basic groups on the basis of the mutational status of the
immunoglobulin heavy-chain variable-region (IgVH) gene in leukemic
cells: patients with IgVH gene mutations have longer survival than those
without. Thus, mutation analysis may be useful for planning
management strategies.
Two potential surrogate markers, CD38 and ZAP-70, have been
investigated because of their association with lack of IgVH mutation, but
the correlation is not 100%.
Hematology/Oncology
Method: Following extraction of total RNA from blood or bone marrow
samples, the IgVH gene is amplified by reverse transcription-polymerase
chain reaction (RT-PCR) and sequenced by dideoxy chain termination
cycle sequencing with automated base calling. The nucleotide sequences
are then aligned to NCBI IgBlast. A mutated status is assigned when
there is r2% deviation from germline IgVH sequence. Sequencing may
not be possible for specimens with <10% clonal B-cells.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A “mutated” result indicates r2% deviation
from the germline sequence. Patients with IgVH mutations tend to have
a more favorable prognosis, with longer overall survival.
6.6.5.5 ZAP-70
Clinical Use: This test is used to assess prognosis and the need for
aggressive therapy in patients with chronic lymphocytic leukemia (CLL).
Clinical Background: CLL has a highly variable course. Some
patients survive for decades without needing treatment, whereas others
progress rapidly and require aggressive therapy within several years
after diagnosis. Predicting which patients will progress rapidly can help
determine the need for close follow-up and may hold potential for risk-
adapted treatment strategies.
The most established predictor of disease progression is lack of mutation
in the immunoglobulin heavy chain variable region (IgVH) in neoplastic
cells. However, because IgVH mutation testing is not widely available,
several surrogate markers have been investigated. To date, the most
effective such marker is expression of zeta-associated protein 70 (ZAP-70),
a 70-kD member of the Syk family of protein tyrosine kinases. ZAP-70 is
expressed primarily in T-cells and natural killer (NK) cells and is critical for
signal transduction following T-cell receptor engagement. In CLL B-cells,
elevated ZAP-70 expression appears to predict the need for therapy as
effectively as IgVH mutation status. Although ZAP-70 expression is
strongly correlated with IgVH mutation status, the combination of the 2
markers may provide greater prognostic value than either marker alone.
Method: This 4-color flow cytometry assay utilizes ZAP-70, CD3, CD19,
and CD45 monoclonal antibodies. The fluorescently labeled lymphocyte
population is selected by CD45 versus side scatter gating. The
percentages of CD3 (B-cell)-positive and CD19 (T-cell)-positive
lymphocytes expressing ZAP-70 are reported; samples in which >10% of
the B-cell population expresses ZAP-70 are considered positive.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Positive ZAP-70 results predict an
aggressive disease course. Patients with positive results require close
follow-up to detect changes in clinical status. Negative results predict a
more indolent disease course.
Testing for IgVH mutation status, available through Quest Diagnostics,
may provide additional prognostic information.
6.6.6 FISH, ALL, TEL/AML1 Translocation 12;21
Clinical Use: This test is used for differential diagnosis of acute
lymphoblastic leukemia (ALL), to determine prognosis of patients with
ALL, and to monitor patients with ALL.

Section 6
Clinical Background: The TEL/AML1 is a gene fusion resulting from
a t(12;21)(p13;q22) chromosomal translocation and is usually
undetectable by conventional chromosome analysis. Although it occurs
in only about 3% of adult acute lymphoblastic leukemia (ALL) cases, it is
the most common genetic rearrangement in B-lineage pediatric ALL
(frequency ~25%). Unlike other ALL-associated translocations [eg,
t(9;22) and t(4;11)], the TEL/AML1 gene fusion is associated with a more
favorable prognosis as evidenced by a significantly lower relapse rate.
Evaluation of this and other prognostic markers aids in selecting low-
toxicity versus high-toxicity therapies. Successful therapy occurs in 90%
of pediatric cases and usually results in the loss of the TEL/AML1 fusion
transcript. Relapse is associated with reappearance of the transcript;
thus FISH testing is useful for monitoring patients for current disease
status.
Method: The FISH method employs dual color probes to detect the TEL
and AML1 loci. A TEL/AML1 fusion signal is observed in cells with the
translocation. In cases with negative cytogenetic results, a FISH assay
may be performed on the same sample.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The presence of the TEL/AML1 gene fusion
is indicative of acute lymphoblastic leukemia (ALL) with a favorable
prognosis. In patients who are currently being treated or who have just
completed the treatment regimen, presence of the TEL/AML1 gene
fusion indicates residual disease. In patients thought to be in remission,
presence of the TEL/AML1 gene fusion indicates recurrent disease.
6.6.7 Follicular Lymphoma, bcl-2/JH t(14;18), Real-time PCR
Clinical Use: This test is used to diagnose B-cell lymphomas,
including follicular lymphoma and other B-cell lymphomas harboring
bcl-2, t(14;18); determine prognosis for patients with diffuse large B-cell
lymphomas; monitor minimal residual disease (MRD); and predict early
relapse.
Clinical Background: The bcl-2 gene translocation t(14;18), a
rearrangement of the bcl-2 proto-oncogene on chromosome 18 with the
immunoglobulin heavy chain region on chromosome 14, leads to
deregulated BCL-2 production and interferes with normal apoptosis. The
majority of the breakpoints on chromosome 18 are tightly clustered in
the major breakpoint cluster region (mbr) and minor cluster region (mcr),
although some fall outside of these areas.
The t(14;18) translocation is characteristic of B-cell lymphomas,
occurring in up to 90% of follicular lymphomas. It is also found in 20%
to 30% of diffuse large B-cell lymphomas, where it is an indicator of
poor prognosis. The bcl-2/JH assay is useful for establishing diagnosis,
predicting prognosis, monitoring therapeutic response, and detecting
MRD or recurrent disease.
Method: In this quantitative real-time polymerase chain reaction (PCR)-
based assay, DNA is amplified using primers specific for the mbr and
the mcr within the bcl-2 proto-oncogene on chromosome 18, and the
immunoglobulin heavy chain region on chromosome 14 (JH). An
additional amplification for the Ki-Ras gene is performed as a control for
sample DNA quality and as a reference for relative quantification.
Results are reported as the ratio of amplified bcl-2 product to amplified
Ki-Ras product. The current specimen will be tested side-by-side with a
previously stored sample, if available, to assess quantitative changes
over time (trend).
153
Hematology/Oncology
This assay detects both major (mbr) and minor (mcr) fusion sequences
and has an analytical sensitivity of 1 tumor cell in 100,000 normal cells.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems Inc.
Interpretive Information: Positive results indicate that the cells carry
the t(14;18) translocation and are reported as a ratio between the
quantities of the amplified target gene bcl-2 and the control Ki-Ras
gene. A positive result indicates the presence of a follicular B-cell
lymphoma, a large diffuse B-cell lymphoma, or an undifferentiated B-cell
lymphoma. If there is no bcl-2 amplification (ratio = 0), the result is
reported as negative. Because translocation breakpoints sometimes
occur outside the areas covered in this assay, a negative result does not
rule out absence of this translocation.
For monitoring MRD, we recommend monitoring changes with time
(trend) rather than the absolute ratio from a single measurement.
6.6.8 Hematopathology Consultation
Clinical Use: This procedure is used to diagnose and classify benign
and malignant hematological disorders (listed below); monitor the
effectiveness of therapy; predict relapse; and detect progression.
Clinical Background: The diagnosis and classification of hematologic
disorders, including leukemias and lymphomas, depends on a
multiparameter approach integrating morphologic evaluation with
ancillary studies such as cytochemistry, immunohistochemistry, flow
cytometry, cytogenetic, and molecular analyses. The hematopathology
group at Nichols Institute offers a comprehensive panel of
immunohistological and chemical stains, flow cytometry assays, and
cytogenetic and molecular studies that may help resolve difficult cases.
Thorough evaluation of samples (bone marrow, lymph nodes, peripheral
blood, other lymphoid tissues) is carried out by highly qualified
hematopathologists and clinical cytogeneticists.
Consultation Process: Along with the clinical history and laboratory
data, bone marrow aspirate smears and H&E-stained bone marrow
biopsy and clot sections are reviewed by a highly qualified
hematopathologist. A preliminary diagnostic report is then issued, and
the results are often discussed with the referring physician or
pathologist by telephone. If additional studies (such as
immunohistochemical stains) are felt necessary, but have not been
requested by the referring physician or pathologist, we will consult with
the referring physician/pathologist before performing such studies.
(Additional tests are performed at an additional charge.) The case will
be reviewed again when the ancillary studies are completed, and a final
report will then be issued.
6.6.9 Interleukin-2 Receptor, EIA
Clinical Use: This test is used to assess prognosis in patients with
lymphoma, monitor therapeutic response in patients with hairy cell
leukemia, and assess T-cell activation following transplantation.
Clinical Background: Elevated levels of soluble IL-2 receptor are
detected in AIDS, autoimmune diseases, sarcoidosis, and a variety of
leukemias and lymphomas. In HIV-positive individuals, the IL-2 receptor
level is elevated during the asymptomatic phase, as well as during
persistent generalized lymphadenopathy and symptomatic phases. IL-2
154
Section 6
receptor detection may be useful in monitoring HIV and in assessing
T-cell activation following transplantation. Elevated IL-2 receptor levels
may also have clinical and prognostic significance in patients with
malignant lymphoma, non-Hodgkin’s lymphoma, B-cell, and
undifferentiated lymphomas.
Studies have suggested that IL-2 receptor levels in a broad spectrum of
conditions associated with T- or B-cell immune activation offer a rapid
and reliable measure of disease activity, response to therapy, and, in
some cases, prognosis. Measurement of the soluble IL-2 receptor level
is also helpful in assessing therapeutic response in patients with hairy
cell leukemia.
Method: Enzyme immunoassay (EIA)
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
6.6.10 Leukemia/Lymphoma Evaluation, Flow Cytometry
Clinical Use: This test is used for differential diagnosis, therapeutic
monitoring, and detection of relapse.
Clinical Background: Neoplasms of hematopoietic and lymphoid
origin can be characterized by the expression of various cell surface
(membrane) markers. Such expression, coupled with morphology,
cytochemical stains, chromosome analysis, and gene rearrangement
studies, is integral to the differential diagnosis that serves as the basis
for treatment selection. Following initiation of therapy, significant
decreases in the targeted cell populations, as determined by
phenotyping, are indicative of therapeutic success. A return or rise in
such cell populations indicates relapse of the disorder.
Method: Based on a preliminary review of the available clinical and
morphologic information and initial flow cytometric features of the case,
the laboratory will select an appropriate panel of 22 cell surface
markers. Gating on the proper cell population is based on thorough
evaluation of available data. Results of flow cytometric analysis will be
correlated with morphology prior to preparation of the interpretive
report. Following consultation with the referring physician, additional
markers may be included for difficult and unusual cases.
This test was developed and its performance characteristics determined by Quest
Diagnostics. It has not been cleared or approved by the U.S. Food and Drug Administration.
The FDA has determined that such clearance or approval is not necessary. Performance
characteristics refer to the analytical performance of the test.
Interpretive Information: A patient-specific, detailed interpretation is
issued by an experienced hematopathologist.
6.6.11 Mantle Cell Lymphoma, bcl-1/JH t(11;14), Real-time PCR
Clinical Use: This test is used to diagnose mantle cell lymphoma,
assess the effectiveness of therapy, monitor minimal residual disease
(MRD), and predict early relapse.
Clinical Background: Mantle cell lymphoma is an aggressive non-
follicular small B-cell lymphoma that is associated with significantly
shorter survival (median survival is 3 years) despite a low-grade
histology. Patients typically present at 50 to 70 years of age with
widespread disease that may involve the bone marrow, peripheral
blood, spleen, liver, gastrointestinal tract, and various lymphoid tissues.
Because of the aggressive nature of mantle cell lymphoma, accurate
diagnosis is important for prognosis and management.
Hematology/Oncology
The t(11;14)(q13;q32) translocation causes deregulation of the bcl-1
gene and over-expression of cyclin D1, which may in turn lead to
lymphoma genesis. The bcl-1 translocation is specific for mantle cell
lymphoma and occurs in the majority of cases. Detection of the bcl-1/JH
gene rearrangement [t(11;14)(q13;q32)] can be helpful in the differential
diagnosis of mantle cell lymphoma and in following up patients during
and after treatment.
Method: This quantitative real-time polymerase chain reaction (PCR)
assay utilizes 2 primers from the bcl-1 gene and 1 primer from the
immunoglobulin heavy chain (IgH) gene specific for the t(11;14)
translocation. An additional amplification for the Ki-Ras gene is
performed as a control for sample DNA quality and as a reference for
relative quantification. Positive results are reported as the ratio of
amplified bcl-1 product to that of Ki-Ras. The current specimen will be
tested side-by-side with a previously stored sample, if available, to
assess quantitative changes with time (trend). The analytical sensitivity
of this test is 1 tumor cell in 100,000 normal cells.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: If there is no bcl-1 amplification (bcl-1:Ki-
Ras ratio = 0), the result is reported as negative. If there is amplification
of bcl-1, the result is positive and the ratio is reported.
A positive result indicates that the cells carry the bcl-1 t(11;14)
translocation characteristic of mantle cell lymphoma. Because this
translocation occurs in only about two-thirds of cases, a negative result
does not preclude the diagnosis of mantle cell lymphoma. The results
should be interpreted in the context of other clinical and pathologic
features.
To monitor MRD, we recommend evaluating changes with time (trend)
rather than the absolute ratio from a single measurement.
6.6.12 Mylotarg®’ Sensitivity (CD33)
Clinical Use: This test is used to determine eligibility for Mylotarg
(semtuzumab ozogamicin; anti-CD33) treatment in patients with acute
myeloid leukemia (AML).
Clinical Background: Mylotarg is a chemotherapy agent that
consists of a recombinant, humanized IgG kappa antibody conjugated to
a cytotoxic anti-tumor antibiotic, calicheamicin, which binds specifically
to the CD33 antigen. This antigen is found on the surface of leukemic
blasts and immature normal cells of myelomonocytic lineage, but not in
normal hematopoietic stem cells. Mylotarg is indicated for treatment of
patients with CD33-positive AML. Its safety and efficacy in patients
with poor performance status has not been established, and
pretreatment testing to establish CD33 sensitivity is recommended.
Method: In this 4-color, multiparametric flow cytometry assay, white
blood cells are stained with fluorescently labeled CD33, CD45, CD13,
and HLA DR monoclonal antibodies. The stained cells are run on a flow
cytometer, and blast cells are selected by CD45 vs side scatter gating
and expression of CD13 and HLA DR. The presence or absence of CD33
on the surface of the blast cells is determined by mean fluorescence
intensity.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and

Section 6
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A positive result indicates the presence of
CD33 antigen on the patient’s blast cells and eligibility for Mylotarg
treatment.
6.6.13 ONTAK®’ Sensitivity (CD25)
Clinical Use: This test is used to determine eligibility for ONTAK
(denileukin diftitox) treatment in patients with persistent or recurrent
cutaneous T-cell lymphoma (CTCL).
Clinical Background: CTCL is a slowly progressing form of non-
Hodgkin’s lymphoma (NHL) that initially manifests in the skin but may
eventually involve other organs. Prognosis depends on disease stage at
the time of diagnosis; median survival is >10 years for early-stage
patients but <3 years for late-stage patients with visceral involvement.
Treatment options include interferon, chemotherapy, and electron-beam
radiation therapy.
ONTAK is a CTCL therapy that targets the high-affinity interleukin-2
(IL-2) receptor. The IL-2 receptor may exist in a low-affinity form (CD25),
an intermediate-affinity form (CD122/CD132), and a high-affinity form
(CD25/CD122/CD132). Patients whose malignant cells express the CD25
component of the IL-2 receptor may respond to ONTAK therapy. Thus,
CD25 expression is one of the selection criteria used for evaluating a
patient’s eligibility for this therapy.
Flow Cytometry Method: In this 4-color, multiparametric flow
cytometry assay, white blood cells are stained with fluorescently
labeled CD25, CD45, CD3, and CD7 monoclonal antibodies. The stained
cells are run on a flow cytometer and T-cells are selected by sequential
gating (CD45 vs side scatter; CD7/CD3 coexpression). The presence or
absence of CD25 on the surface of the selected T-cells is determined by
mean fluorescence intensity.
IHC Method: This immunohistochemistry assay detects CD25
expression on the cell surface and is equivalent to the assay used in the
ONTAK phase III clinical trial.
Aliases for CD25 include interleukin-2 receptor, IL-2 receptor, low
affinity IL-2 receptor, and p55.
These tests were developed and their performance characteristics determined by Quest
Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: A positive result indicates the presence of
CD25 antigen on the patient’s T-cells and eligibility for ONTAK therapy.
6.6.14 PML/RARA t(15;17) Translocation, FISH and Quantitative
PCR
Clinical Use: Both FISH and PCR methods may be used to diagnose
acute promyelocytic leukemia (APL), monitor patients for APL
recurrence, and predict therapeutic response to all-trans-retinoic acid
and arsenic trioxide therapy. For suspected low-level recurrent or
minimal residual disease (MRD) detection and monitoring, PCR is
recommended.
Clinical Background: Acute promyelocytic leukemia (APL or AML-
M3) is a subtype of acute myeloid leukemia with distinct clinical and
histopathologic features. Historically one of the most lethal forms of
acute myeloid leukemia, APL leads to disseminated intravascular
155
Hematology/Oncology
coagulation and death when not diagnosed and treated. Treatment with
all-trans-retinoic acid substantially improves survival in patients who
have failed anthracycline chemotherapy or for whom anthracycline is
contraindicated. Similarly, arsenic trioxide is beneficial in APL patients
who have failed or have contraindications for treatment with
anthracycline or retinoid-based therapy.
Genetically, APL is characterized by a unique chromosomal anomaly:
More than 99% of APL patients harbor a translocation between
chromosomes 15 and 17, which results in fusion of the retinoic acid
receptor alpha (RARA) gene on chromosome 17 with the PML gene on
chromosome 15. Either a short or a long transcript isoform is produced,
depending on the PML breakpoint. The short isoform has been
associated with poor outcome, although this remains controversial.
Thus, the PML/RARA, t(15;17) assays are useful for establishing the
diagnosis of APL and predicting therapeutic response to all-trans-
retinoic acid and arsenic trioxide. Detection of the PML/RARA fusion
transcript by reverse transcription–PCR (RT–PCR) is more sensitive than
conventional cytogenetic detection of the t(15;17) translocation and best
predicts therapeutic response to all-trans-retinoic acid. The quantitative
PCR assay is also useful for monitoring therapeutic response and
detecting minimal residual or recurrent disease. Although APL can be
diagnosed on the basis of morphology, presence of the t(15;17)
translocation or PML/RARA gene expression is required for response to
all-trans-retinoic acid as well as arsenic trioxide.
PCR Method: [PML/RARA t(15;17), Quantitative PCR] In this assay,
extracted RNA is subjected to 2 separate quantitative real-time reverse
transcription-polymerase chain reaction (RT-PCR) procedures to detect
the 2 types of PML/RAR-alpha fusion transcripts (long and short
isoforms). An additional amplification for the abl gene is performed as a
control for sample RNA quality and as a reference for relative
quantification. If the PML/RARA t(15;17) translocation is detected, the
result is considered positive and the ratio from amplified PML/RARA
fusion transcript to the control ABL transcript is reported. If there is no
PML/RARA amplification (ratio = 0), the result is reported as negative.
The isoform (short or long) is also reported. If available, a previously
stored sample will be tested alongside the current specimen to assess
quantitative changes with time (trend). The analytical sensitivity of this
test is 1 tumor cell in 100,000 normal cells.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
FISH Method: [FISH, AML M3, PML/RARA, Translocation 15;17] In this
fluorescence in situ hybridization (FISH) method, dual color probes are
used to detect the PML and RARA loci. A PML/RARA fusion signal is
observed in cells with the translocation. In cases with negative
cytogenetic results, a FISH assay may be performed on the same
sample.
These tests were developed and their performance characteristics determined by Quest
Diagnostics Nichols Institute. They have not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Positive results with either assay indicate
that the cells carry the PML/RARA t(15;17) translocation and are
diagnostic for APL. Because the efficacy of all-trans-retinoic acid and
arsenic trioxide has not been demonstrated in patients who lack the
PML/RARA t(15;17) translocation, other forms of treatment should be
considered for patients without evidence of this rearrangement.
In the PCR assay, the short isoform may be associated with shorter
disease-free and overall survival. To monitor MRD with the PCR assay,
156

Section 6 Hematology/Oncology

we recommend evaluating changes with time (trend) rather than the 6.7.2 Alpha-fetoprotein (AFP) and AFP-L3
absolute ratio from a single measurement.
Clinical Use: This test is used to determine risk of hepatocellular
Suggested Reading carcinoma (HCC), diagnose HCC, determine prognosis for individuals
with HCC, and monitor HCC therapy.
1. Dyck JA, Warrell RP Jr, Evans RM, et al. Rapid diagnosis of acute
promyelocytic leukemia by immunohistochemical localization of
Clinical Background: HCC is the fifth most common cancer in the
PML/RAR-B protein. Blood. 1995;86:862-867.
world, with an incidence of >20 per 100,000 in China and eastern Asia
2. Flenghi L, Fagiolo M, et al. Characterization of a new monoclonal
and 8 to 10 per 100,000 in the United States.1,2 The most significant risk
antibody (PG-M3), directed against the amino-terminal portion of the
factors for HCC are chronic infection with hepatitis B or C virus (HBV
PML gene product. Blood. 1995;85:1871-1880.
and HCV, respectively) and hepatic cirrhosis. Individuals at risk for HCC
3. Gallagher RE, Yeap BY, Bi W, et al. Quantitative real-time RT-PCR
are typically monitored with serial hepatobiliary ultrasounds, as well as
analysis of PML-RARB mRNA in acute promyelocytic leukemia:
other imaging techniques (eg, CT scans), and serial measurements of
assessment of prognostic significance in adult patients from
alpha-fetoprotein (AFP).
intergroup protocol 0129. Blood. 2003;101:2521-2528.
4. Grimwade D, Gorman P, Duprez E, et al. Characterization of cryptic
AFP is a glycoprotein synthesized by the fetal yolk sac, fetal liver,
rearrangements and variant translocations in acute promyelocytic
testicular non-seminomatous germ cell cancers, and malignant hepatic
leukemia. Blood. 1997;90:4876-4885.
cells. It is the most established marker of HCC. Reliance on AFP levels to
5. Miller WH Jr, Kakizuka A, Frankel SR, et al. Reverse transcription
detect HCC, however, is confounded by the fact that AFP may be elevated
polymerase chain reaction for the re-arranged retinoic acid receptor
in individuals with chronic HBV or HCV infection and hepatic cirrhosis. The
B clarifies diagnosis and detects minimal residual disease in acute
sensitivity and specificity of AFP for diagnosing HCC vary with the
promyelocytic leukemia. Proc Natl Acad Sci USA. 1992;89:2694-2698.
population studied and the cut-off value above which AFP is considered
6. Slack JL, Bi W, Livak KJ, et al. Pre-clinical validation of a novel,
positive. They range from 52% to 80% and 90% to 98%, respectively.3
highly sensitive assay to detect PML-RARB mRNA using real-time
reverse-transcription polymerase chain reaction. J Mol Diagn. 2001;3:
Measurement of an AFP glycoform may prove to be clinically superior to
141-149.
measuring AFP. Three glycoforms, determined by the degree of
fucosylation of the N-acetylglucosamine-linked sugar chain, have been
6.6.15 Rituxan®’ Sensitivity (CD20)
identified. The glycoforms are separated in vitro by their ability to bind
the lectin Lens culinaris agglutinin (LCA), a carbohydrate binding protein
Clinical Use: This test is used to determine eligibility for Rituxan
isolated from lentil seeds. AFP-L1 is non-LCA binding and the major
(rituximab; anti-CD20) treatment in patients with B-cell non-Hodgkin’s
glycoform found in individuals with nonmalignant hepatopathy (eg,
lymphomas (NHL).
cirrhosis or chronic HBV infection). AFP-L2 has an intermediate LCA
binding capacity and is primarily produced by yolk sac tumors. AFP-L3 is
Clinical Background: Rituxan is a genetically engineered, chimeric
produced by malignant liver cells, binds to LCA with high affinity, and is
murine/human monoclonal antibody directed against the CD20 antigen
the major glycoform found in individuals with HCC.4,5
found on the surface of normal and malignant B-cell lymphocytes. This
antigen is expressed on the surface of >90% of B-cell NHL, and binding
AFP-L3 levels r10% are associated with a 7-fold increased risk of
to it is a prerequisite for Rituxan’s anti-tumor effect. Because NHL
developing HCC within the next 21 months and can be elevated 3 to 21
subtypes may differ in their response to Rituxan, determination of drug
months before HCC is detected by standard imaging techniques.6,7 The
sensitivity is important for choosing therapy.
diagnostic sensitivity and specificity ranges from 36% to 66% and 77%
to 95%, respectively.8,9 Because AFP-L3 is produced by malignant
Method: In this 4-color, multiparametric flow cytometry assay, white
hepatocytes, its measurement helps distinguish non-malignant hepatic
blood cells are stained with fluorescently labeled CD20, CD45, CD5, and
disease from HCC.6,8-12 Malignant liver cells that produce AFP-L3 have an
CD19 monoclonal antibodies. The stained cells are run on a flow
increased tendency for rapid growth, early invasion, and intra-hepatic
cytometer, B-cell lymphocytes are selected by sequential gating (CD45
metastasis, thus making AFP-L3 an indicator of poor prognosis in
vs side scatter; CD19/CD5 coexpression), and the presence or absence
affected individuals.6,8-12
of CD20 on the surface of the selected B-lymphocytes is determined by
mean fluorescence intensity.
Individuals Suitable for Testing include those at risk for HCC and
This test was developed and its performance characteristics determined by Quest those with primary or recurrent HCC.
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test. Method: This immunofluorescent liquid-phase binding assay
simultaneously measures AFP-L3 and non-AFP-L3 forms of AFP. Ion
Interpretive Information: A positive result indicates the presence of exchange chromatography is used to separate AFP-L3 and the non-AFP-
CD20 antigen on the patient’s B-cells and eligibility for Rituxan therapy. L3 glycoforms, followed by fluorescence detection of the 2
chromatography fractions. Total AFP and the percentage of AFP-L3 are
6.6.16 TPMT Genotype reported. The analytical sensitivity is 0.8 ng/mL for AFP and 0.5% for
See Immunology, “Gastrointestinal Diseases” section 7.4.4. AFP-L3. There is no interference from vitamins B1, B6, and B12 or from
alpha, beta, and gamma interferon; ibuprofen; acetaminophen; and
6.7 Hepatocellular Cancer acetylsalicylic acid. 6

6.7.1 Alpha-fetoprotein (AFP) Reference Range

See Hematology/Oncology, “Tumor Markers” section 6.12.1. AFP: 1.6 – 4.5 ng/mL
AFP-L3: 0.5 – 9.9%

These ranges apply to non-pregnant adults only.


Section 6
Interpretive Information: Individuals with an increased percentage of
AFP-L3 (AFP-L3%), relative to total AFP present in serum, are at
increased risk of developing HCC. AFP-L3-secreting hepatocytes have an
increased tendency for rapid growth, early invasion, and intra-hepatic
metastasis, thus leading to a poorer prognosis in affected individuals.
AFP and AFP-L3 levels decline to normal levels with effective therapy;
rising levels suggest disease progression or recurrence.5
Not all hepatic cancers secrete AFP or AFP-L3. AFP and AFP-L3% may
be elevated in individuals with germ cell tumors and other cancers
including those of the gastric and biliary tract, lung, pancreas, and
testes.8 AFP may also be elevated in individuals with viral infections,
liver diseases other than HCC, women who are pregnant, and children
less than 18 months of age. Decreased AFP-L3% may occur if free and
conjugated bilirubin are >20 mg/dL.6
Results should be interpreted in conjunction with other laboratory and
clinical findings.
References
1. Bosch FX, Ribes J, Borras J. Epidemiology of primary liver cancer.
Semin Liver Dis. 1999;19:271-285.
2. American Cancer Society: Cancer Facts and Figures 2006. Atlanta,
Ga: American Cancer Society, 2006. Available at
http://www.cancer.org/downloads/STT/CAFF2006PWSecured.pdf.
Accessed April 19, 2006.
3. Lopez JB, Thambyrajah V, Balasegaram, et al. Appropriate cut-off
levels for serum alpha-fetoprotein in hepatocellular carcinoma.
Diagnostic Oncology. 1994-1995;4:287-291.
4. Sato Y, Nakata K, Kato Y, et al. Early recognition of hepatocellular
carcinoma based on altered profiles of alpha-fetoprotein. N Engl J
Med. 1993;328:1802-1806.
5. Li D, Mallory T, Satomura S. AFP-L3: a new generation of tumor
marker for hepatocellular carcinoma. Clin Chim Acta. 2001;313:15-
19.
6. LBA AFP-L3 Manufacturer’s Package Insert, Rev. #1/0505DDD00K,
Wako Pure Chemical Industries, Ltd., (1/05/05)
7. Kumada T, Nakano S, Takeda I, et al. Clinical utility of Lens culinaris
agglutinin-reactive alpha-fetoprotein in small hepatocellular
carcinoma: special reference to imaging diagnosis. J Hepatol.
1999;30:125-130.
8. Taketa K, Endo Y, Sekiya C, et al. A collaborative study for the
evaluation of lectin-reactive B-fetoprotein in early detection of
hepatocellular carcinoma. Cancer Res. 1993;53:5419-5423.
9. Oka H, Saito A, Ito K, et al. Multicenter prospective analysis of
newly diagnosed hepatocellular carcinoma with respect to the
percentage of Lens culinaris agglutinin-reactive B-fetoprotein. J
Gastroen Hepatol. 2001;16:1378-1383.
10. Tada T, Kumada T, Toyoda H, et al. Relationship between Lens
culinaris agglutinin-reactive B-fetoprotein and pathological features
of hepatocellular carcinoma. Liver Int. 2005;25:848-853.
11. Yoshida S, Kurokohchi K, Arima Keiji, et al. Clinical significance of
Lens culinaris agglutinin-reactive fraction of serum B-fetoprotein in
patients with hepatocellular carcinoma. Int J Oncol. 2002;20:305-
309.
12. Toyoda H, Kumada T, Kiriyama S, et al. Prognostic significance of
simultaneous measurement of three tumor markers in patients with
hepatocellular carcinoma. Clin Gastroenterol Hepatol. 2006;4:111-
117.
157
Hematology/Oncology
6.8 Melanoma
6.8.1 TA90 (Melanoma-associated Antigen)
Clinical Use: This test is used to assess prognosis after curative
resection of malignant melanoma and to monitor patients for melanoma
recurrence after curative resection.
Clinical Background: Existing prognostic factors such as lymph node
status and tumor thickness and depth are helpful for predicting which
melanomas will metastasize after curative resection. However, even
patients whose tumors are considered relatively low-risk, based on
standard prognostic factors, can develop metastasis and locoregional
recurrence. Markers that accurately detect subclinical metastasis and
predict recurrence may help determine which patients should receive
adjuvant therapy.
TA90 is a 90-kd tumor-associated antigen that is expressed by >70% of
melanomas. After curative resection of malignant melanoma, patients
with occult metastasis may exhibit elevated levels of a TA90-IgG
immune complex (TA90-IC).1 Several reports have indicated that TA90-IC
is a sensitive and specific marker of recurrence in patients with
malignant melanoma and is associated with shortened survival.1-4
Patients with TA90-IC detected early after curative resection of
American Joint Committee on Cancer (AJCC) Stage I to III melanoma
were found to have significantly lower 5-year overall survival (36% vs
84%, P <.001) and disease-free survival (24% vs 74%, P <.001) than
TA90-IC-negative patients.2 In that study, TA90-IC status was
independent of standard prognostic factors, including lymph-node
status; Breslow depth was the only other significant predictor of
outcome in multivariate analysis. Similar results were found for patients
with thick (r4 mm) melanomas.3
Serial monitoring of TA90-IC levels after curative resection of Stage I to
III melanoma may also help predict recurrence, even in patients who are
initially TA90-IC negative. Kelley et al found that 58 of 74 (78%)
patients who developed distant metastasis had at least 2 consecutive
positive TA90-IC results, first elevated an average of 19 months before
clinical evidence of recurrence.2 Only 20 of 88 (23%) patients without
metastasis had at least 2 positive TA90-IC results; when patients who
received a polyvalent melanoma vaccine (known to elicit TA90-IC
formation) were excluded, the sensitivity (92%) and specificity (86%) of
the assay increased. Thus, detection of TA90-IC might be helpful in
selecting patients for early intervention, when adjuvant therapy may be
most effective.
Individuals Suitable for Testing include patients who have recently
undergone curative resection of malignant melanoma and those being
followed up for recurrence of malignant melanoma.
Method: This enzyme-linked immunosorbent assay (ELISA) uses a
murine anti-TA90 monoclonal capture antibody with a goat antihuman
immunoglobulin detection antibody; the latter is Fab-conjugated with
alkaline phosphatase. There is no known cross-reactivity with other
tumor markers.
This test was developed and its performance characteristics determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: TA90-IC positivity soon after curative
resection of Stage I to III or thick (r4 mm) melanoma is associated with
lower rates of overall and disease-free survival (Table 27) and a greater
risk of recurrence (Table 28). During serial monitoring, positivity is
158

Section 6 Hematology/Oncology

Table 27. TA90-IC Status and Survival

5-Year Overall Survival (OS) 5-Year Disease-Free Survival (DFS)

TA90-IC- TA90-IC- TA90-IC- TA90-IC-
positive negative P value positive negative P value
Stage I-III melanoma* 36% 84% <.001 24% 74% <.001
†
Thick melanoma (r4 mm)* 26% 84% <.001 10% 73% <.001
*TA90-IC measured within 6 months after tumor resection.
†
Estimated 5-year OS and DFS (median follow-up period was 25 months).

associated with recurrence and appears, on average, 19 months before
clinical or radiologic evidence of recurrence.2 The results of this assay
should be interpreted in light of other relevant clinical and laboratory
findings.
References
1. Kelley MC, Jones RC, Gupta RK, et al. Tumor-associated antigen TA-
90 immune complex assay predicts subclinical metastasis and
survival for patients with early stage melanoma. Cancer.
1998;83:1355-1361.
2. Kelley MC, Gupta RK, Hsueh EC, et al. Tumor-associated antigen
TA90 immune complex assay predicts recurrence and survival after
surgical treatment of stage I-III melanoma. J Clin Oncol.
2001;19:1176-1182.
3. Chung MH, Gupta RK, Essner R, et al. Serum TA90 immune complex
assay can predict outcome after resection of thick (r4 mm) primary
melanoma and sentinel lymphadenectomy. Ann Surg Oncol.
2002;9:120-126.
4. Litvak DA, Gupta RK, Yee R, et al. Endogenous immune response to
early- and intermediate-stage melanoma is correlated with outcomes
and is independent of locoregional relapse and standard prognostic
factors. J Am Coll Surg. 2004;198:27-35.
6.9 Ovarian Cancer
6.9.1 CA 125, ICMA
Clinical Use: This test is used for therapeutic monitoring, residual
disease detection, and early detection of ovarian cancer recurrence.
Clinical Background: CA 125 is a high molecular weight glycoprotein
consisting of both protein and carbohydrate moieties. Elevated levels
are associated with more than 50% of ovarian cancers and 24% to 45%
of breast, cervical, colorectal, endometrial, lung, and pancreatic
malignancies.1 Since CA 125 can be produced by mesothelial cells,2
elevated levels may also be observed in certain non-malignant disorders
such as pericarditis and pelvic inflammatory disease, as well as in 1%
to 2% of apparently healthy individuals.1,3 CA 125 is not useful for
screening or diagnosis of malignant disorders.4
Serum studies, nevertheless, have correlated increasing CA 125 levels
with malignant disease and poor therapeutic response. Decreasing
levels, on the other hand, are associated with a favorable therapeutic
response. Overall, CA 125 levels correlate with disease in 87% of
individuals.3 Elevated post-treatment CA 125 levels (r21 U/mL) were
found to be indicative of residual ovarian tumors, possibly negating the
need for a second-look, exploratory laparotomy.5-7 Low levels (<21 U/mL)
do not rule out the presence of residual disease; patients should still
receive regular general physical and pelvic examinations.4-6,8
Individuals Suitable for Testing include patients diagnosed with
ovarian carcinoma.
Method: This immunochemiluminometric assay ([ICMA], Immulite®’
2000 OM-MA, Diagnostic Products Corporation) utilizes a murine
monoclonal antibody as the capture antibody. This antibody’s specificity
is similar to that of the M11 antibody that is commonly used in CA 125
assays. A rabbit polyclonal antibody is used as the detection antibody.
The analytical sensitivity is 1 U/mL; the assay is not affected by
cisplatin, cyclophosphamide, doxorubicin hydrochloride, 5-fluorouracil,
mitomycin C, or vincristine. Commonly used aliases include cancer
antigen 125 and OC125.
Values obtained from different methods are not interchangeable due to
variations in reagent specificity and other methodological differences.
Use only one method when monitoring patients. If the methodology is
changed, re-baselining prior to making clinical decisions is highly
recommended.

Interpretive Information: Elevated CA 125 serum levels may be seen

in patients with ovarian, lung, pancreatic, and uterine malignancies, as
Table 28. Sensitivity and Specificity of TA90-IC Assay for well as certain non-malignant disorders such as pericarditis, cirrhosis,
Predicting Recurrence After Curative Resection of hepatitis and other hepatic disorders, endometriosis, and ovarian cysts
(Table 29). Elevated levels have also been reported to occur during
Melanoma menstruation and the first trimester of pregnancy.
Sensitivity Specificity In patients who are, or have been, treated for cancer, a doubling of
(%) (%) CA 125 levels suggests disease progression or recurrence, whereas a
Stage I-III melanoma halving of levels suggests a therapeutic response.3,8 Detection of
elevated levels in previously treated patients suggests the presence of
Soon after curative resection1 77 76 residual tumor. Levels are likely to be within the normal range in
Serial monitoring during follow-up 2
78 77 patients with no evidence of disease. Following complete remission, a
rise in CA 125 levels suggests tumor recurrence.3
Thick melanoma (r4 mm)
Soon after curative resection3 70 85 Falsely elevated or depressed values may occur in samples obtained
from patients who have received mouse monoclonal antibody
 159

Section 6 Hematology/Oncology

Table 29. Distribution of CA 1251

Percent of Subjects
Number of 0–20 21–49 50–99 r100
Subjects U/mL U/mL U/mL U/mL
Apparently Healthy Females
<50 years 50 100 0 0 0
r50 years 14 100 0 0 0
Malignant Conditions
Ovarian 96 38 10 7 45
Breast 14 79 14 7 0
Gastrointestinal 6 50 33 17 0
Genitourinary 11 36 9 18 36
Other 18 50 17 0 33
Nonmalignant Conditions
Gastrointestinal 6 83 0 17 0
Genitourinary 8 88 0 0 12
Other 44 91 9 0 0

preparations during diagnosis or therapy. Such patients may have
developed human anti-mouse antibodies (HAMA) that interfere with
accurate analysis.
Levels within the normal range do not preclude the presence of cancer,
nor are elevated results an absolute indication of malignancy. CA 125
test results should be interpreted in conjunction with other clinical and
laboratory findings.
References
1. CA 125 assay directional insert. Diagnostic Products Corporation.
July, 2005.
2. Ho-dac-Pannekeet MM, Hiralall JK, Struijk DG, et al. Longitudinal
follow-up of CA 125 in peritoneal effluent. Kidney Int. 1997;51:888-
893.
3. Kenemans P, Yedema CA, Bon GG, et al. CA 125 in gynecologic
pathology—a review. Eur J Obstet Gynecol Reprod Biol. 1993;49:115-
124.
4. NIH Consensus Conference. Ovarian cancer. Screening, treatment,
and follow-up. NIH Consensus Development Panel on Ovarian Cancer.
JAMA. 1995;273:491-497.
5. Niloff JM, Bast RC Jr, Schaetzl EM, et al. Predictive value of CA 125
antigen levels in second-look procedures for ovarian cancer. Am J
Obstet Gynecol. 1985;151:981-986.
6. Berek JS, Knapp RC, Malkasian GD, et al. CA 125 serum levels
correlated with second-look operations among ovarian cancer
patients. Obstet Gynecol. 1986;67:685-689.
7. Hording U, Toftager-Larsen K, Lund B, et al. The value of CA 125
measurement before second-look laparotomy in patients with ovarian
carcinoma. Eur J Gynaecol Oncol. 1994;15:217-221.
8. Atack DB, Nisker JA, Allen HH, et al. CA 125 surveillance and
second-look laparotomy in ovarian carcinoma. Am J Obstet Gynecol.
1986;154:287-289.
6.10 Paroxysmal Nocturnal
Hemoglobinuria (PNH)
6.10.1 Red Cell CD55 and CD59 Expression
Clinical Use: This test is used to diagnose paroxysmal nocturnal
hemoglobinuria (PNH).
Clinical Background: PNH is a rare hematopoietic stem cell defect in
which a somatic mutation of the X-linked PIG-A (phosphatidyl inositol
glycan-class A) gene results in a partial or absolute deficiency of
glycosyl phosphatidyl inositol-linked proteins (GPI): for example, CD55
and CD59 red cell membrane antigens. A deficiency in both antigens
leads to the characteristic manifestations of PNH, namely increased
sensitivity of red blood cells to complement-mediated lysis. Hemolytic
bouts occur irregularly and may be initiated by infection, surgery, or
strenuous exercise. Patients with PNH are also susceptible to
thrombotic complications due to platelet abnormalities. In addition,
aplastic anemia or myelodysplasia may develop in 25% of patients with
PNH and practically all patients have evidence of hemosiderinuria.
Although PNH can be diagnosed with the Ham (acidified serum) and
sucrose hemolysis tests, flow cytometry is considered the diagnostic
test of choice because of its greater sensitivity and specificity. Absence
of CD55 and CD59 on red blood cells documented by flow cytometry is
diagnostic for PNH.
Method: This single-color flow cytometry assay utilizes anti-CD55 and
anti-CD59 monoclonal antibodies to detect the relative amounts of
CD55- and CD59-deficient red blood cells. Threshold gating
differentiates normal red blood cells from deficient red blood cells.
Results are reported for each marker as normal or abnormal.
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
160

Section 6 Hematology/Oncology

Interpretive Information: Abnormal expression of both CD55 and
CD59 is necessary for the diagnosis of PNH. If only 1 of the markers is
abnormal, the test result is considered to be equivocal. In these cases,
consider submitting another sample for re-testing or analyzing for the
presence of PIG-A gene mutations.
6.11 Prostate Cancer
6.11.1 Laboratory Support of Diagnosis and Management
Clinical Background: Prostate cancer is the most commonly
detected cancer in men in the United States, affecting approximately
1 out of every 6 men.1 It is the second leading cause of cancer death
among men in the United States.1 Although prostate cancer is thought
to begin when men are in their thirties and forties, it is most often
diagnosed in men over 65 years of age. Prevalence increases with
increasing age.1
prostate as well as in the breast and salivary glands. When secreted
into seminal fluid, PSA liquifies the gel-forming proteins in semen.
The normal function of PSA in breast and salivary glands is not
known.
Individuals Suitable for Testing
Screening
The following men are suitable for prostate cancer screening: 1) men
r50 years of age who have a life expectancy of r10 years; 2) African-
American men and men who have a first-degree relative diagnosed with
prostate cancer before age 65 (begin screening at 45 years of age); 3)
men who have 2 or more first-degree relatives diagnosed with prostate
cancer before age 65 (begin screening at age 40); and 4) men who
would be candidates for active treatment if a potentially curable
prostate cancer diagnosis was established (candidacy based on age,
comorbidity, and an interest in being treated).

Prostate cancer typically progresses slowly over the course of 15 or
more years. When organ-confined, it is potentially curable by radical
prostatectomy or radiation therapy (ie, external-beam radiation or
brachytherapy). Such therapy, however, may be associated with
significant morbidity, including urinary incontinence and impotence. For
patients with a good prognosis, watchful waiting may be a better
alternative. For metastatic disease, therapeutic options include
androgen ablation, adjuvant hormone-radiotherapy, or chemotherapy.
Prognosis for patients with metastatic disease is poor, nonetheless.
Patients should be actively involved in the decision to undergo prostate
cancer screening and should be fully informed of the potential benefits,
limitations, and risks associated with screening. Screening is not
recommended for the general population.
Other Applications
The following men are suitable for testing in the non-screening setting:
1) symptomatic men; 2) men with a prostate cancer diagnosis; and 3)
men who are undergoing or have completed prostate cancer therapy.

Laboratory testing can assist with screening, diagnosis, staging,
prognosis, detection of residual or recurrent disease, and therapeutic
monitoring. The primary test used for these purposes is prostate-
specific antigen (PSA). PSA is an androgen-regulated, kallikrein-like
serine protease produced by normal and malignant epithelium in the
Test Availability
Laboratory tests that can be used to support screening, diagnosis, and
management of prostate cancer are listed in Table 30.

Table 30. Laboratory Tests for Screening, Diagnosis, and Management of Prostate Cancer

Available Tests Method Description

Primary Tests
Free PSA ICMA Includes free, total, and % free/total PSA
Total PSA ICMA Analytical sensitivity: 0.1 ng/mL
PSA Post-prostatectomy ICMA Analytical sensitivity: 0.01 ng/mL
PSA Post-prostatectomy ICMA Analytical sensitivity: 0.01 ng/mL; eliminates interference from human anti-mouse antibodies
with HAMA Treatment (HAMA) that may be present in patients who have received mouse monoclonal antibody
preparations
Secondary Tests
bcl-2 IHC Detects bcl-2 expression in tissue
DNA Cell Cycle Analysis Flow cytometry Includes ploidy status and % S-phase in tissue
E-cadherin IHC Detects E-cadherin protein in tissue
FISH, Prostate Cancer FISH Detects 8p22 (lipoprotein lipase [LPL]) deletion, chromosome 8 aneusomy, 8q24 (C-MYC gene)
gain, 7q31 deletion, and chromosome 7 aneusomy
Ki-67 (MIB-1) IHC Detects MIB-1 protein in tissue
p53 Oncoprotein IHC Detects mutated p53 oncoprotein in tissue
Testosterone, Total LC/MS/MS Suitable for measuring the low levels of testosterone associated with gonadotropin-releasing
hormone analogs and antiandrogen therapies
ICMA, immunochemiluminometric assay; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; LC/MS/MS, liquid chromatography, tandem mass spectrometry.

Section 6
Test Application
Screening
Prostate cancer screening remains controversial. Proponents point to the
high incidence, absence of preventive agents, ease of screening,
potential curability of organ-confined disease, and lack of effective
therapy for advanced disease. Opponents tout the slow progression of
many tumors, high number of false positives, overdiagnosis of indolent
disease, and treatment side effects that lead to diminished quality of
life. Since screening commenced in the late 1980s, incidence of
metastatic disease has decreased, while the incidence of organ-
confined, moderately differentiated disease has increased.2This
suggests screening has been effective in identifying clinically
significant, potentially curable disease while reducing the incidence of
incurable disease. Mortality has decreased as well,3 but this reduction
may be due to factors other than screening and early detection.4 Results
from the first randomized controlled trial to show direct evidence that
screening reduces prostate cancer mortality have been controversial.5
Two more studies addressing this issue are ongoing; their expected
completion dates are between 2006 and 2010.6,7
Pending additional data, the American Cancer Society8 and the American
Urological Association9 have recommended that digital rectal exam
(DRE) and total PSA be offered annually to men r50 years of age who
have a minimum life expectancy of 10 years. Screening is recommended
earlier (eg, age 40 to 45 years) for African-American men and those
with an affected first-degree relative. These organizations also
encourage patient education to facilitate informed decision-making, as
do the American College of Physicians10 and the American College of
Preventive Medicine.11 The latter groups do not support routine
screening, nor does the U.S. Preventive Services Task Force.12
High risk individual*
Suspicious DRE
Any total PSA level
Non-suspicious DRE
Total PSA 4.0 ng/mL
Total PSA
and DRE
Non-suspicious DRE
Total PSA 4.1-10.0 ng/mL
Non-suspicious DRE
Total PSA >10.0 ng/mL
*Age, race, family history; note that patient informed consent is encouraged (see text for details).
†
Alternatively, consider biopsy if PSA is 2.6–4.0 ng/mL or PSA velocity (PSAV) is r0.75 ng/mL/y.
Adapted, with changes, from NCCN guidelines.13
Figure 12. Indications for prostate biopsy following DRE and total PSA screen. Options shown for cases with non-suspicious DRE and total PSA in the
4.1–10.0 ng/mL range can be selected at the discretion of the urologist.
161
Hematology/Oncology
Contrary to those of other organizations, the new National Comprehensive
Cancer Network (NCCN) Clinical Practice Guidelines recommend offering
PSA testing (baseline) to all men beginning at age 40 years. Follow-up
recommendations (PSA or PSA and DRE at specified frequency) then
depend on the PSA concentration and patient age, life expectancy, and
risk of prostate cancer. Furthermore, the NCCN strategy incorporates use
of free PSA and PSA velocity (see next section, Diagnosis) when making
decisions to biopsy men with a total PSA level <10.0 ng/mL.13
Diagnosis
Prostate cancer diagnosis begins with DRE and total PSA. If either is
suggestive of cancer, a transrectal ultrasound (TRUS)-guided biopsy is
generally the next step (Figure 12). Investigators have attempted to
reduce the number of unnecessary biopsies (ie, enhance the specificity
of total PSA testing) without reducing the cancer detection rate. To this
end, age-specific PSA reference ranges have been proposed as well as
calculations of the prostate-specific antigen density (PSAD), PSA
velocity, and the percentage of free to total PSA (% free PSA). Although
conflicting studies have been published regarding the benefit of these
strategies, guidelines for use and interpretation are provided herein for
physicians who want to use them.
Age-related Reference Ranges
Serum PSA levels increase with increasing age and prostate size in
healthy men. Use of age-related reference ranges (Table 31)
theoretically would increase sensitivity in younger men and increase
specificity (reducing unnecessary biopsies) in older men. Use of age-
related reference ranges has not been widely adopted due to studies
demonstrating diminished sensitivity in older men.18 Some investigators
support the use of age-specific ranges only in men younger than 60
years of age.19
TRUS-guided biopsy
Retest in 1-2 years †
TRUS-guided biopsy
or
% Free PSA 10%
and/or TRUS-guided biopsy
PSAV 0.75 ng/mL/y
TRUS-guided biopsy
162

Section 6 Hematology/Oncology

Table 31. Age- and Race-Specific Reference Ranges

Total PSA (ng/mL)

Age, Free PSA14 Complexed PSA14
15
(years) (ng/mL) (ng/mL) Caucasians African Americans16 Asians17
40–49 <0.5 <1.0 <2.5 <2.0 <2.0
50–59 <0.7 <1.5 <3.5 <4.0 <3.0
60–69 <1.0 <2.0 <4.5 <4.5 <4.0
70–79 <1.2 <3.0 <6.5 <5.5 <5.0

PSA Density
Use of PSAD combines total PSA level with prostate gland volume to
assess the probability of a positive biopsy. It is defined as the total PSA
divided by the prostate gland volume as determined by TRUS. Levels
b0.15 ng/mL/cm3 have been associated with a low likelihood of
prostate cancer. PSAD has not been widely used owing to lack of
consistent evidence regarding clinical benefit.
PSA Velocity
In individuals with pre-clinical prostate cancer, there is an accelerated
increase in serum PSA levels, beginning 7 to 9 years prior to diagnosis.
Thus, evaluation of the rate of change in PSA levels (PSA velocity,
PSAV) may assist in early detection of cancer. Carter et al found that
PSA velocity more accurately detected prostate cancer (sensitivity 72%,
specificity 90%) than total PSA (sensitivity 78%, specificity 60%).20 As
mentioned above, NCCN guidelines incorporate use of PSAV in the early
detection of prostate cancer. The guidelines suggest prostate biopsy be
performed when the PSAV is r0.75 ng/mL/y in men with PSA <10.0
ng/mL. Furthermore, the guidelines recommend using PSAV to monitor
men who have had a negative biopsy.13 Instructions for calculating PSAV
are provided in Appendix 1.
% Free PSA
Since the overlap in total PSA levels is substantial among men with
benign prostatic hyperplasia (BPH) and prostate cancer, use of % free
PSA may help distinguish BPH from cancer. PSA circulates in both free
and bound, or complexed, forms. Immunoreactive PSA is primarily bound
to the B1-antichymotrypsin (ACT) protease inhibitor; thus, PSA levels in
healthy individuals mainly reflect complexed PSA. Individuals with
benign prostatic hypertrophy (BPH) tend to have lower proportions of
complexed PSA (ie, higher % free PSA) relative to those with prostate
cancer. Studies using % free PSA (Table 32) have shown improved
specificity in men with borderline total PSA levels (4.1-10.0 ng/mL),
resulting in a 13% to 37% reduction in negative biopsies. Furthermore,
in men with “normal” PSA levels (2.6 to 4.0 ng/mL) use of % free PSA
may increase the sensitivity of PSA by detecting cancers that would
have been missed by total PSA alone.22,25 NCCN guidelines now approve
use of % free PSA to determine need for biopsy in selected men with
PSA levels in the 4.1–10.0 ng/mL or 2.6–4.0 ng/mL range.13
Staging
Tumor staging provides prognostic information and assists in selection
of the therapeutic modality. In general, total PSA levels increase with
increasing stage, although there is significant overlap (Table 33). A
combination of markers has proved to be more useful than any single
marker. Nomograms using patient-specific clinical (TNM) stage, biopsy
data (eg, Gleason score), and pretreatment total PSA level are
commonly used.32 Data from various imaging techniques (eg, bone scan)
can also be used.
Prognosis
The College of American Pathologists (CAP) and the World Health
Organization (WHO) both recommend routine use of PSA as a
prognostic factor in conjunction with TNM stage, Gleason score, and
surgical margin status.33 The NCCN also recommends incorporating
PSA levels when determining prognosis.34 The American Joint
Committee on Cancer (AJCC), however, excludes inclusion of PSA as a
prognostic factor pending evaluation of survival data from multiple
institutions.

Table 32. Improved Specificity Using % Free PSA

Reference Free PSA Cut Point (%) Sensitivity (%) Negative Biopsies Avoided (%)
22
Catalona WJ, et al 27* 90 18
23 †
Catalona WJ, et al 25 95 20
Luderer AA, et al24 25† 100 31
Roehl KA, et al25 25‡ 85 19
26 †
Van Iersel MP, et al 25 90 25
27 †
Vashi AR, et al 24 95 13
28 †
Catalona WJ, et al 23 90 30
29 †
Catalona WJ, et al 23 90 31
30 †
Bangma CH, et al 20 89 37
*Total PSA 2.6–4.0 ng/mL.
†
Total PSA 4.1–10.0 ng/mL.
‡
Total PSA 2.6–4.0 ng/mL; using a cut point of 30%, sensitivity was 93% but only 9% of negative biopsies could be avoided.
 163

Section 6 Hematology/Oncology

Table 33. Relation Between Total PSA Concentration and before diagnosis can be used to predict survival. PSADT calculated
Localized Disease31 using PSA levels obtained after primary therapy can be used to predict
both survival and the extent of the recurrence (Table 34).
PSA ng/mL Likelihood of Organ-confined Cancer %
The time to biochemical recurrence, following primary therapy (eg,
<4.0 83 radical prostatectomy or radiation therapy), provides prognostic
4.0–10.0 67 information regarding the extent of the recurrence (local vs distant)
(Table 34). Following radical prostatectomy, biochemical recurrence is
10.1–20.0 56 defined as a persistent rise in total PSA r0.2 ng/mL.35 Following
>20.0 30 radiation therapy, biochemical recurrence is defined as 3 consecutive
rises in PSA levels at least 3 months apart.36 The date of biochemical
recurrence is defined as the midpoint between the post radiation nadir
PSA Kinetics PSA and the first of the 3 consecutive rises.
PSA kinetics are becoming more accepted for predicting disease-free
survival, prostate cancer-related mortality, and local vs distant disease Other laboratory tests that may assist with prognostic assessments are
at time of biochemical relapse. These predictions are then used to help listed in Table 35.
select appropriate therapeutic regimens.
Initial Treatment Selection
The same PSAV used in the diagnosis of prostate cancer (see previous PSA levels can be helpful in making initial treatment decisions when
discussion) can be used to predict disease-free and cancer-related used in combination with TNM-stage, Gleason score, and life
survival, irrespective of treatment. The cut points for interpretation, expectancy. Cut points most frequently used for such decision-making
however are different; see Table 34 for the cut point used when are 10 ng/mL and 20 ng/mL.9,34 PSA levels <20 ng/mL are associated
determining prognosis. PSAV calculated from PSA levels obtained after with negative bone scans, and PSA levels <10 ng/mL are associated
primary therapy (eg, radical prostatectomy or radiotherapy) can help with local disease.
predict the extent of the recurrence (ie, local vs distant). The guidelines
for interpretation are listed in Table 34. Therapeutic Monitoring, Residual and Recurrent Disease
Detection
PSA doubling time (PSADT) is the time required for total PSA levels to For patients being “treated” with expectant management or watchful
double. Instructions for calculating PSADT are provided in Appendix 2. waiting, total PSA should be performed every 6 months if life
Similar to PSAV, PSADT calculated using PSA concentrations obtained expectancy is r10 years.34 If life expectancy is <10 years, PSA testing
should be performed every 6 to 12 months. Following definitive therapy,
ultrasensitive PSA levels help detect residual disease or document
eradication of the tumor (Figure 13). Absence of rising PSA levels is the
Table 34. Guidelines for Use of PSA Kinetics in Making best indicator of total tumor eradication.43 A 6-month testing interval for
Treatment Decisions21, 37-41 the first 5 years followed by annual testing thereafter is suggested for
recurrent disease detection following curative treatment.34 For patients
PSA Kinetic Result Clinical Significance with metastatic disease, PSA testing should be performed every 3 to 6
months.34
PSADT
Pre diagnosis <18 mo Decreased cancer- Sample Collection Considerations
related survival Serum PSA levels are affected by the relatively long half-life (2-3 days)
of total PSA and various other factors. The adverse effects of many of
After primary therapy <3 mo Decreased cancer- these factors can be reduced or eliminated with proper timing of sample
related survival
r10 mo Recurrence likely to be
local
<10 mo Recurrence likely to be Table 35. Test Results Associated with Unfavorable Prognosis
distant
Test Result
PSAV
7q31 deletion Present
Pre diagnosis >2.0 ng/mL/y Decreased disease-free
survival 8p22 deletion Present
Decreased cancer- 8q24 gain Present
related survival
bcl-2 Overexpressed
After primary therapy <0.75 ng/mL/y Recurrence likely to be
local Chromosome 7 aneusomy Present
>0.75 ng/mL/y Recurrence likely to be Chromosome 8 aneusomy Present
distant
DNA ploidy Aneuploid, tetraploid
Time to biochemical >2 y Recurrence likely to be
(PSA) recurrence after local E-cadherin Underexpressed
primary therapy b2 y Recurrence likely to be Ki-67 (MIB-1) Overexpressed
distant
PSADT, PSA doubling time; PSAV, PSA velocity.
p53 mutation Present
164

Section 6 Hematology/Oncology

Radical prostatectomy

<0.2 ng/mL No residual cancer; retest every 6 months

for 5 years and annually thereafter

PSA
3 months later One-time elevation; no residual cancer;
<0.2 ng/mL retest every 6 months for 5 years and
annually thereafter
Retest in
0.2 ng/mL
1 month
Repeat at 3- to 6-month intervals to
PSADT, PSA doubling time. 0.2 ng/mL determine PSADT; consider radiation
Adapted, with changes, from NCCN guidelines.34 therapy while PSA is <1.5 ng/mL

Figure 13. Patient follow-up after radical prostatectomy.

collection (Table 36). Furthermore, 3 analyses, each obtained from a pathologically organ-confined (64%) and thus potentially curable.54
separate collection, are recommended prior to biopsy to rule out effects Approximately 90% are clinically significant tumors, being 1 cc or
of physiologic and assay variation.48 greater in size.22

Test Interpretation
Total PSA levels >4.0 ng/mL are generally considered elevated, although
lower cut points are sometimes used in younger men13 (Table 31).
Elevated levels are associated with BPH, acute urinary retention, urinary
tract infections (including acute prostatitis), prostatic intraepithelial
neoplasia, and prostate cancer. Transient elevations may be observed
following DRE, ejaculation, prostate biopsy, or surgery (Table 36).
Total PSA levels b4.0 ng/mL do not guarantee the absence of prostate
cancer in asymptomatic individuals; 25% of men with cancer have a
PSA level in this range. Indeed, there is no level of PSA at which
clinically significant cancer does not occur (Table 38). Consequently,
some proponents of screening recommend lowering the PSA cut point to
2.6 ng/mL56 and using % free PSA to reduce unnecessary biopsies.
NCCN has recently adopted such a strategy.13

A 50% or greater change in serial PSA levels is considered clinically Among patients with borderline elevated PSA levels (4.1-10.0 ng/mL),
significant.46 Decreases in PSA not related to prostate cancer can be approximately 25% will have cancer. Levels >10.0 ng/mL are associated
caused by medication and herbal supplementation (Table 37). with extracapsular cancer that is less likely to be curable.

Screening and Diagnosis
Similar to other screening programs (eg, mammography), two-thirds of
screened individuals with an elevated total PSA (>4.0 ng/mL) do not
have prostate cancer (66% false-positive rate); however, most of the
cancers that are detected are clinically localized (92%) and
Since free PSA is usually eliminated by the kidney, % free PSA can be
increased in men with chronic renal failure and in those receiving
dialysis. It can also be increased by DRE, prostate biopsy, and
prostatectomy. False elevations can occur if the sample is collected
prior to 48 to 72 hours after prostate manipulation.

Table 36. Recommended Timing of Sample Collection44-47

Factor Effect on PSA Level Recommended Timing of Sample Collection*

Cystoscopy None or small l Prior to, or 24–48 hours after, procedure

DRE None or small l Prior to the procedure
Ejaculation l r48 hours after ejaculation
Prostate biopsy l Prior to, or 4–6 weeks after, biopsy
Prostate massage 2-fold l Prior to the procedure
Prostatitis l 8 weeks after end of treatment
TRUS None or small l Prior to, or 24–48 hours after procedure
†
TURP l Prior to, or r6 weeks after, surgery
Urethral catheterization None Anytime
Urinary retention l r4 days after relief
DRE, digital rectal exam; TRUS, transrectal ultrasonography; TURP, transurethral resection of prostate.
*To reduce or eliminate effect on PSA level.
†
Includes electrovaporization techniques.
 165

Section 6 Hematology/Oncology

Table 37. Pharmacologic Effects that Confound Interpretation of PSA Test Results9,49-53

Medication/Supplement Clinical Indication Effect on Total PSA

®’
Finasteride Male pattern hair loss (Propecia ) 50% n after 6–12 months*†
BPH (Proscar®’)
Dutasteride (Avodart®’) BPH ~50% n†
Terazosin BPH None
Antiandrogens (eg, flutamide, Prostate cancer r50% n for 3–14 months after withdrawal of
bicalutamide, megestrol acetate) medication
Saw palmetto (Serenoa Herbal supplement for BPH and Possible n, similar to finasteride
repens) lower urinary tract symptoms
*May vary widely in individual men.
†
% Free PSA not affected.

Staging and Prognosis Appendix 1. PSA Velocity (PSAV) Calculations

Table 33 provides the risk of organ-confined prostate cancer associated
with various concentrations of total PSA. Refer to Table 34 for
suggested PSAV and PSADT cut points used to assign risk of local vs
distant recurrence and disease-free and cancer-related survival. Table
34 also includes a cut point based on time to PSA recurrence that
predicts local vs distant disease.
Therapeutic Monitoring, Residual and Recurrent Disease
Detection
Following successful radical prostatectomy, total PSA levels should be
undetectable. A persistent rise in total PSA (r0.2 ng/mL35) is suggestive
of biochemical recurrence, which can precede clinical recurrence by r5
years.
Following successful radiation therapy, PSA does not always reach
undetectable levels; however, a nadir of 0.5 to 1.0 ng/mL is considered
favorable. Biochemical failure after radiotherapy is defined as 3
consecutive rises in PSA levels at least 3 months apart.36
Following successful antiandrogen therapy, there is an initial drop in
PSA to normal or even undetectable levels. Relapse caused by
androgen-independent tumor cells is characterized by castration levels
of testosterone (<50 ng/mL) and 3 consecutive rises in PSA levels 2
weeks apart. Two of these increases should be 50% over the nadir
level. Use of PSA for monitoring therapy in individuals with androgen-
independent cancer is limited; however, a r50% decline from
pretreatment levels is considered a sign of a better outcome, relative to
a <50% decline, when maintained for 8 weeks.57
Additional interpretive information is provided in Table 39.
Table 38. Prevalence of Prostate Cancer in Men with PSA
Levels <4.0 ng/mL55
PSA ng/mL Prevalence (%) of Prostate Cancer
0–0.5 7
0.6–1.0 10
1.1–2.0 17
2.1–3.0 24
3.1–4.0 27
PSAV can be calculated using linear regression.21 Alternatively, calculate
PSAV using 3 consecutive PSA measurements20:
1. PSAV = [PSAt – PSAt-1] w [yeart – yeart-1] where t is the most recent
PSA level and t-1 is the previous, consecutive measurement.
2. Average PSAV = [PSAVt + PSAVt-1] w 2 where t is the most recent
PSAV and t-1 is the previous, consecutive PSAV.
Always use PSA measurements from a minimum of 3 samples collected
over at least 18 months. Furthermore, all PSA test results should be
generated using reagents and method from the same manufacturer.
Appendix 2. PSA Doubling Time (PSADT) Calculations
PSADT can be calculated using one of the following 2 methods42:
1. PSADT = natural log of 2 (ie, 0.693) w slope of the line derived from a
plot of log[PSA] vs time of PSA measurement
2. PSADT = (natural log of 2)(time interval) w (log[final PSA] – log[initial
PSA]) where the time interval is the period between initial and final
PSA measurements.
References
1. American Cancer Society. Cancer Facts and Figures 2005. Atlanta:
American Cancer Society; 2005. Available at: http://www.cancer.
org/docroot/STT/stt_0.asp. Accessed February 6, 2006.
2. Farkas A, Schneider D, Perrotti M, et al. National trends in the
epidemiology of prostate cancer, 1973 to 1994; evidence for the
effectiveness of prostate-specific antigen screening. Urology.
1998;52:444-449.
3. Brenner H, Arndt V. Long-term survival rates of patients with
prostate cancer in the prostate-specific antigen screening era:
population-based estimates for the year 2000 by period analysis.
J Clin Oncol. 2005;23:441-447.
4. Etzioni R, Legler JM, Feuer EJ, et al. Cancer surveillance series:
interpreting trends in prostate cancer – Part III: quantifying the link
between population prostate-specific antigen testing and recent
declines in prostate cancer mortality. J Natl Cancer Inst.
1999;91:1033-1039.
5. Boer R, Schroder FH. Quebec randomized controlled trial on prostate
cancer screening shows no evidence for mortality reduction.
Prostate. 1999;40:130-134.
6. Gohagan JK, Prorok PC, Kramer BS, et al. Prostate cancer screening
in the prostate, lung, colorectal and ovarian cancer screening trial of
the National Cancer Institute. J Urol. 1994;152:1905-1909.
7. Schroder FH. Detection of prostate cancer: the impact of the
European Randomized Study of Screening for Prostate Cancer
(ERSPC). Can J Urol. 2005;12(Suppl 1):2-6.
166

Section 6 Hematology/Oncology

Table 39. Interpretation of Test Results13,20,23,25,35,43,55,58,59

Test Result Clinical Significance

Free PSA* When total PSA is 2.6 to 4.0 ng/mL, probability for prostate cancer is:
>30% of total PSA 20%
21–30% 19%
11–20% 25%
1–10% 43%
When total PSA is 4.1–10.0 ng/mL, probability of prostate cancer is:
r26% of total PSA 8% Biopsy not recommended
21–25% 16%
16–20% 20% Indeterminant; consider other factors before deciding to biopsy
11–15% 28%
0–10% 56% Biopsy recommended
PSA velocity† <0.75 ng/mL/year Low likelihood of prostate cancer
r0.75 ng/mL/year Suspicious for cancer; consider biopsy
Total PSA‡ Likelihood of prostate cancer:
b4.0 ng/mL 15%
4.1–10.0 ng/mL 27%
>10.0 ng/mL 59%
20.0–29.9 ng/mL 74%
r30 ng/mL 96%
PSA, Post Prostatectomy <0.01 ng/mL Undetectable level of PSA; absence of recurrence
<0.10 ng/mL Low likelihood of residual or recurrent cancer post radical prostatectomy
r0.2 ng/mL§ Residual or recurrent cancer likely after radical prostatectomy
0.5–1.5 ng/mL Consider 2nd line treatment with radiotherapyII
*Not helpful in 2/3 of all men, ie, those who have % free PSA between 10% and 25%.
†
See Table 34 for cut points associated with survival and local vs distant disease prognosis.
‡See Table 38 for prostate cancer prevalence associated with PSA b4.0 ng/mL.
§NCCN guidelines recommend >0.3 ng/mL and rising on 2 or more measurements.34
II
Initiate radiotherapy in patients with post prostatectomy recurrence while PSA is b1.5 ng/mL for best therapeutic response.

8. Smith RA, Cokkinides V, Eyre HJ. American Cancer Society
guidelines for the early detection of cancer, 2006. CA Cancer J Clin.
2006;56:11-25. Also available at: http://caonline.amcancersoc.org/
cgi/content/full/56/1/11. Accessed February 6, 2006.
9. Prostate-specific antigen (PSA) best practice policy. American
Urological Association (AUA). Oncology. 2000;14:267-286. Also
available at: http://www.cancernetwork.com/journals/oncology/
o0002e.htm?_requestid=165766. Accessed February 6, 2006.
10. Coley CM, Barry MJ, Mulley AG. Screening for prostate cancer.
American College of Physicians. Ann Intern Med. 1997;126:480-484.
Also available at: www.annals.org/cgi/content/full/126/6/480.
Accessed February 6, 2006.
11. Ferrini R, Woolf SH. American College of Preventive Medicine
practice policy: screening for prostate cancer in American men. Am
J Prev Med. 1998;15:81-84. Also available at:
http://www.acpm.org/prostate.htm. Accessed March 8, 2006.
12. U.S. Preventive Services Task Force. Screening for Prostate Cancer:
Recommendations and Rationale. Ann Intern Med. 2002;137:915-
916. Also available at: http://www.ahrq.gov/clinic/3rduspstf/
prostatescr/prostaterr.htm. Accessed February 6, 2006.
13. Babaian R and Catalona WJ. National Comprehensive Cancer
Network clinical practice guidelines in oncology – v.1.2005: prostate
cancer early detection. Available at: http://www.nccn.org/
professionals/physician_gls/PDF/prostate_detection.pdf. Accessed
February 16, 2006.
14. Oesterling JE, Jacobsen SJ, Klee GG, et al. Free, complexed and
total serum prostate specific antigen: the establishment of
appropriate reference ranges for their concentrations and ratios.
J Urol. 1995;154:1090-1095.
15. Oesterling JE, Jacobsen SJ, Chute CG, et al. Serum prostate-
specific antigen in a community-based population of healthy men.
Establishment of age-specific reference ranges. JAMA.
1993;270:860-864.
16. Morgan TO, Jacobsen SJ, McCarthy WF, et al. Age-specific
reference ranges for serum prostate-specific antigen in black men.
N Engl J Med. 1996;335:304-310.
17. Oesterling JE, Kumamoto Y, Tsukamoto T, et al. Serum prostate-
specific antigen in a community-based population of healthy
Japanese men: lower values than for similarly aged white men.
Br J Urol. 1995;75:347-353.
18. Catalona WJ, Hudson MA, Scardino PT, et al. Selection of optimal
prostate specific antigen cutoffs for early detection of prostate
cancer: receiver operating characteristic curves. J Urol.
1994;152:2037-2042.
19. Partin AW, Criley SR, Subong EN, et al. Standard versus age-
specific prostate specific antigen reference ranges among men with
clinically localized prostate cancer: a pathological analysis. J Urol.
1996;155:1336-1339.
20. Carter HB, Pearson JD, Metter EJ, et al. Longitudinal evaluation of
prostate-specific antigen levels in men with and without prostate
disease. JAMA. 1992;267:2215-2220.
21. D’Amico AV, Chen MH, Roehl KA, et al. Preoperative PSA velocity
and the risk of death from prostate cancer after radical
prostatectomy. N Engl J Med. 2004;351:125-135.
22. Catalona WJ, Smith DS, Ornstein DK. Prostate cancer detection in
men with serum PSA concentrations of 2.6 to 4.0 ng/mL and benign
prostate examination. Enhancement of specificity with free PSA
measurements. JAMA. 1997; 277:1452-1455.

Section 6
23. Catalona WJ, Partin AW, Slawin KM, et al. Use of the percentage
of free prostate-specific antigen to enhance differentiation of
prostate cancer from benign prostatic disease. A prospective
multicenter clinical trial. JAMA. 1998;279:1542-1547.
24. Luderer AA, Chen YT, Soriano TF, et al. Measurement of the
proportion of free to total prostate-specific antigen improves
diagnostic performance of prostate-specific antigen in the
diagnostic gray zone of total prostate-specific antigen. Urology.
1995;46:187-194.
25. Roehl KA, Antenor JOV, Catalona WJ. Robustness of free prostate
specific antigen measurements to reduce unnecessary biopsies in
the 2.6 to 4.0 ng/mL range. J Urol. 2002;168:922-925.
26. Van Iersel MP, Witjes WPJ, Thomas CMG, et al. Review on the
simultaneous detection of total prostate-specific antigen and free
prostate-specific antigen. Prostate Suppl. 1996;7:48-57.
27. Vashi AR, Wojno KJ, Henricks W, et al. Determination of the “reflex
range” and appropriate cutpoints for percent free prostate-specific
antigen in 413 men referred for prostatic evaluation using the
AxSYM system. Urology. 1997;49:19-27.
28. Catalona WJ. Clinical utility of measurement of free and total
prostate-specific antigen (PSA): a review. Prostate Suppl. 1996;7:64-
69.
29. Catalona WJ, Smith DS, Wolfert RL, et al. Evaluation of percentage
of free serum prostate-specific antigen to improve specificity of
prostate cancer screening. JAMA. 1995;274:1214-1220.
30. Bangma CH, Kranse R, Blijenberg BG, et al. The value of screening
tests in the detection of prostate cancer. Part II: Retrospective
analysis of free/total prostate-specific analysis ratio, age-specific
reference ranges, and PSA density. Urology. 1995; 46:779-784.
31. Narayan P, Gajendran V, Taylor S, et al. The role of transrectal
ultrasound-guided biopsy-based staging, preoperative serum prostate-
specific antigen, and biopsy Gleason score in prediction of final
pathologic diagnosis in prostate cancer. Urology. 1995; 46:205-212.
32. Partin AW, Mangold LA, Lamm DM, et al. Contemporary update of
prostate cancer staging nomograms (Partin tables) for the new
millennium. Urology. 2001;58:843-848.
33. Bostwick DG, Foster CS. Predictive factors in prostate cancer:
current concepts from the 1999 College of American Pathologists
Conference on Solid Tumor Prognostic Factors and the 1999 World
Health Organization Second International Consultation on Prostate
Cancer. Semin Urol Oncol. 1999;17:222-272.
34. National Comprehensive Cancer Network clinical practice guidelines
in oncology – v.2.2005: prostate cancer. Available at:
http://www.nccn.org/professionals/physician_gls/PDF/prostate.pdf.
Accessed February 16, 2006.
35. Freedland SJ, Sutter ME, Dorey F, et al. Defining the ideal cutpoint
for determining PSA recurrence after radical prostatectomy.
Prostate-specific antigen. Urology. 2003;61:365-369.
36. Consensus statement: guidelines for PSA following radiation
therapy. American Society for Therapeutic Radiology and Oncology
Consensus Panel. Int J Radiat Oncol Biol Phys. 1997;37:1035-1041.
37. Sengupta S, Myers RP, Slezak JM, et al. Preoperative prostate
specific antigen doubling time and velocity are strong and
independent predictors of outcomes following radical
prostatectomy. J Urol. 2005;174:2191-2196.
38. D’Amico AV, Moul J, Carroll PR, et al. Prostate specific antigen
doubling time as a surrogate end point for prostate cancer specific
mortality following radical prostatectomy or radiation therapy.
J Urol. 2004;172:S42-S47.
39. Pound CR, Partin AW, Eisenberger MA, et al. Natural history of
progression after PSA elevation following radical prostatectomy.
JAMA. 1999;281:1591-1597.
40. Patel DA, Presti JC Jr, McNeal JE, et al. Preoperative PSA velocity
167
Hematology/Oncology
is an independent prognostic factor for relapse after radical
prostatectomy. J Clin Oncol. 2005;23:6157-6162.
41. Partin AW, Pearson JD, Landis PK, et al. Evaluation of serum
prostate-specific antigen velocity after radical prostatectomy to
distinguish local recurrence from distant metastases. Urology.
1994;43;649-659.
42. Cannon GM Jr, Walsh PC, Partin AW, et al. Prostate-specific antigen
doubling time in the identification of patients at risk for progression
after treatment and biochemical recurrence for prostate cancer.
Urology. 2003;62(Suppl 6B):2-8.
43. Cox JD, Gallagher MJ, Hammond EH, et al. Consensus statements
on radiation therapy of prostate cancer: guidelines for prostate re-
biopsy after radiation and for radiation therapy with rising prostate-
specific antigen levels after radical prostatectomy. American Society
for Therapeutic Radiology and Oncology Consensus Panel. J Clin
Oncol. 1999;17:1155-1163.
44. Caplan A, Kratz A. Prostate-specific antigen and the early diagnosis
of prostate cancer. Am J Clin Pathol. 2002;117(Suppl 1):S104-S108.
45. Talic RF, El-Tiraifi AM, Altaf S, et al. Changes of serum prostate-
specific antigen following high energy thick loop prostatectomy. Int
Urol Nephrol. 2000;32:271-274.
46. Oesterling JE, Rice DC, Glenski WJ, et al. Effect of cystoscopy,
prostate biopsy, and transurethral resection of prostate on serum
prostate-specific antigen concentration. Urology. 1993;42:276-282.
47. Dew T, Coker C, Saadeh F, et al. Influence of investigative and
operative procedures on serum prostate-specific antigen
concentration. Ann Clin Biochem. 1999;36(Pt3):340-346.
48. Soletormos G, Semjonow A, Sibley PEC, et al. Biological variation of
total prostate-specific antigen: a survey of published estimates and
consequences for clinical practice. Clin Chem. 2005;51:1342-1351.
49. Gormley GJ, Stoner E, Bruskewitz RC, et al. The effect of finasteride
in men with benign prostatic hyperplasia. The Finasteride Study
Group. N Engl J Med. 1992;327:1185-1191.
50. Pannek J, Marks LS, Pearson JD, et al. Influence of finasteride on
free and total serum prostate specific antigen levels in men with
benign prostatic hyperplasia. J Urol. 1998;159:449-453.
51. Andriole GL, Kirby R. Safety and tolerability of the dual 5alpha-
reductase inhibitor dutasteride in the treatment of benign prostatic
hyperplasia. Eur Urol. 2003;44:82-88.
52. Brawer MK, Lin DW, Williford WO, et al. Effect of finasteride and/or
terazosin on serum PSA: results of VA Cooperative Study #359.
Prostate. 1999;39:234-239.
53. Paul R, Breul J. Antiandrogen withdrawal syndrome associated with
prostate cancer therapies: incidence and clinical significance. Drug
Saf. 2000;23:381-390.
54. Mettlin C. The American Cancer Society National Prostate Cancer
Detection Project and national patterns of prostate cancer detection
and treatment. CA Cancer J Clin. 1997;47:265-272.
55. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate
cancer among men with a prostate-specific antigen level b4.0 ng
per milliliter. N Engl J Med. 2004;350:2239-2246.
56. Krumholtz JS, Carvalhal GF, Ramos CG, et al. Prostate-specific
antigen cutoff of 2.6 ng/mL for prostate cancer screening is
associated with favorable pathologic tumor features. Urology.
2002;60:469-474.
57. Smith DC, Dunn RL, Strawderman MS, et al. Change in serum
prostate-specific antigen as a marker of response to cytotoxic
therapy for hormone-refractory prostate cancer. J Clin Oncol.
1998;16:1835-1843.
58. Catalona WJ, Smith DS, Ratliff TL, et al. Detection of organ-
confined prostate cancer is increased through prostate-specific
antigen-based screening. JAMA. 1993;270:948-954.
59. Gerstenbluth RE, Seftel AD, Hampel N, et al. The accuracy of the
increased prostate specific antigen level (greater than or equal to 20
168

Section 6 Hematology/Oncology

ng/mL) in predicting prostate cancer: is biopsy always required? AFP levels are increased in patients with primary hepatocellular
J Urol. 2002;168:1990-1993. carcinoma; increased concentrations may precede clinical evidence of
liver cancer. Thus, serial measurements have been recommended in
6.12 Tumor Markers hepatitis B carriers, who have an increased risk of liver cancer.

Tumor markers are membrane protein or secretory derivatives released
into circulation from tissue cells. Blood levels are generally low in
normal individuals but increased in patients with selected tumors. These
markers are not specific for malignancy or for a particular tumor type.
Because of false-positive and false-negative results, they are not
recommended for diagnosis and are rarely recommended for widespread
general population screening; however, screening may be useful in
selected high-risk populations. Tumor markers may be of prognostic
significance when combined with other clinical and pathologic findings;
however, these markers are most useful for patient management during
or after therapy.
6.12.1 Alpha-fetoprotein (AFP)
Clinical Use: This test is used to distinguish between seminomatous
and nonseminomatous testicular germ cell cancer, monitor therapy and
detect recurrence in individuals with nonseminomatous testicular germ
cell cancer, determine prognosis in individuals with fulminant hepatitis,
monitor therapy in individuals with hepatocellular carcinoma, and
monitor hepatitis B carriers for evidence of liver cancer.
Clinical Background: AFP is an oncofetal protein produced by the
fetal yolk sac and liver. Levels in maternal blood are relatively elevated,
with peak concentrations early in the second trimester of pregnancy.
After birth, blood concentrations diminish rapidly to adult levels. AFP
serves as a marker for testicular nonseminomatous germ cell cancers
(embryonal carcinoma, choriocarcinoma, teratomas). The presence of
increased AFP levels in blood indicates yolk sac tumor elements. Thus,
AFP measurements are useful to distinguish between seminomatous
and nonseminomatous testicular germ cell cancer. AFP, as well as lactic
dehydrogenase (LDH) and beta-hCG, is recognized by the International
Germ Cell Consensus Classification as an independent prognostic factor
that can categorize nonseminomatous germ cell tumors into prognostic
groups (J Clin Oncol. 1997;15:594-603).
Interpretive Information: AFP concentrations are increased in
patients with liver cancer, hepatitis, and liver cirrhosis (Table 40).
Increased levels in severe hepatitis indicate liver cell regeneration.
Thus, the absence of AFP in patients with fulminant hepatitis is
considered a poor prognostic sign.
AFP concentrations decline to normal levels with effective therapy.
Persistently elevated levels suggest residual disease; rising levels
suggest disease progression or recurrence.
6.12.2 CA 125
See Hematology/Oncology, “Ovarian Cancer” section 6.9.1.
6.12.3 CA 15-3
See Hematology/Oncology, “Breast Cancer” section 6.3.2.
6.12.4 CA 19-9
See Hematology/Oncology, “Gastrointestinal Cancer” section 6.5.1.
6.12.5 CA 27.29
See Hematology/Oncology, “Breast Cancer” section 6.3.3.
6.12.6 CA 72-4
See Hematology/Oncology, “Gastrointestinal Cancer” section 6.5.2
6.12.7 Carcinoembryonic Antigen (CEA)
See Hematology/Oncology, “Gastrointestinal Cancer” section 6.5.4.2
6.12.8 Lipid Associated Sialic Acid (LASA, LSA)
Clinical Use: This test is used to monitor tumor burden in patients
with various malignant conditions, including Hodgkin’s disease,
leukemia, and melanoma.

Table 40. Distribution of AFP

Number with AFP Levels

Number of
Subjects <5 IU/mL 5–15 IU/mL 15–100 IU/mL >100 IU/mL
Healthy Subjects
Men 119 118 1 0 0
Women 29 0 0 0 0
Malignant Disease
Testicular
Seminomatous 6 6 0 0 0
Nonseminomatous 60 14 8 15 23
Liver 10 3 0 2 5
Other 40 36 1 0 3
Other (Women) 20 18 0 1 1
Nonmalignant Disease
Cirrhosis 4 3 1 0 0
Hepatitis 24 19 4 1 0
Other 6 5 0 0 1
Other (Women) 16 15 0 1 0
All data were derived from men except where noted.
Source: IMMULITE 2000 AFP package insert (DPC).
 169

Section 6 Hematology/Oncology

Clinical Background: Sialic acid (N-acetylneuraminic acid) is an Because of lack of clinical specificity and sensitivity, this test is not
important component of the glycoproteins and glycolipids that compose recommended for diagnosis of malignancy or detection of recurrence.
the cellular membranes. It mediates cell-cell interactions such as
substance recognition, antigenicity, and adhesion and is involved in Interpretive Information: Increased levels are associated with breast
hormone action, enzyme properties, transport mechanisms, and cancer, colorectal cancer, gynecologic cancer, lung cancer, hematologic
neurotransmission. Sialic acid containing glycoproteins and glycolipids malignancies, melanoma, and benign, inflammatory, and chronic
are significantly altered during neoplastic transformation and are diseases. Decreased levels are associated with therapeutic response.
released by malignant cells into the blood.
References
The LASA test measures the sialoglycoproteins and sialoglycolipids
1. Dnistrian AM, Schwartz MK, Katopodis N, et al. Serum lipid-bound
released by malignant cells. Elevated serum levels have been observed
sialic acid as a marker in breast cancer. Cancer. 1982;50:1815-1819.
in patients with a wide variety of malignancies: gynecologic, breast,
2. Erbil KM, Jones JD, Klee GG. Use and limitations of serum total and
colon, gastroenteric, lung, melanoma, leukemia, lymphoma, Hodgkin’s
lipid-bound sialic acid concentrations as markers for colorectal
disease, and others. Levels are also frequently elevated in benign,
cancer. Cancer. 1985;55:404-409.
inflammatory, and chronic disease. Table 41 summarizes the variable
3. Schwartz PE, Chambers SK, Chambers JT, et al. Circulating tumor
sensitivity and specificity.
markers in the monitoring of gynecologic malignancies. Cancer.
1987;60:353-361.
Method: In this colorimetric assay, the lipid fraction is extracted from
4. Dnistrian AM, Schwartz MK. Plasma lipid-bound sialic acid and
serum samples via chloroform/methanol. Sialolipids are then
carcinoembryonic antigen in cancer patients. Clin Chem.
precipitated with phosphotungstic acid. The precipitate is treated with
1981;27:1737-1739.
resorcinol and butyl acetate/n-butyl alcohol. The intensity of the
resultant color is directly proportional to the concentration of LASA. The
6.12.9 Neuron Specific Enolase (NSE)
analytical sensitivity is 2 mg/dL. Results are reported in mg/dL.
Clinical Use: This test is used to monitor disease progression and
therapy in individuals with small cell lung cancer (SCLC). It is also used
to monitor therapy in various other cancers (Table 42).
Table 41. Distribution of Lipid-Associated Sialic Acid Clinical Background: NSE is a glycolytic enzyme that catalyzes the
conversion of phosphoglycerate to phosphoenol pyruvate. It is present in
Patient Group Number of Subjects % Elevated neurons, neuroendocrine cells, and amine precursor uptake and
Breast 1 decarboxylation (APUD) cells. Elevated NSE concentrations are observed
in patients with neuroblastoma, pancreatic islet cell carcinoma,
Normal 78 0 medullary thyroid carcinoma, pheochromocytoma, and other
Benign 106 13 neuroendocrine tumors as well as in certain benign conditions. NSE
levels are frequently increased in patients with SCLC and infrequently in
Primary cancer 64 47 patients with non-SCLC. NSE has therefore been used to monitor
Recurrent metastatic cancer 61 62 disease progression and management in SCLC.
Colorectal2 Interpretive Information: Distribution of NSE concentrations in
Benign colorectal polyps 17 18 healthy subjects and various benign and malignant disorders is provided
in Table 42.
Local and regional cancer 43 40
This test is performed using a kit that has not been approved or cleared by the FDA. The
Distant hepatic and nonhepatic 54 69 analytical performance characteristics of this test have been determined by Quest
metastases Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
Gynecologic malignancies3
Ovarian (NED) 189 10 6.12.10 Nuclear Matrix Proteins (NMP)
See Hematology/Oncology, “Bladder Cancer” section 6.2.2.
Ovarian (ED) 100 71
Endometrial (NED) 70 3 6.12.11 Prostate Specific Antigen (PSA)
See Hematology/Oncology, “Prostate Cancer” section 6.11.1.
Endometrial (ED) 45 43
Cervical (NED) 36 12 6.12.12 Vysis UroVysion
See Hematology/Oncology, “Bladder Cancer” section 6.2.1.
Cervical (ED) 35 62
4
Lung cancer 18 72
Leukemia4 21 90
4
Lymphoma 18 78
4
Hodgkin’s disease 18 94
4
Melanoma 12 83
NED, no evidence of disease; ED, evidence of disease.
170

Section 6 Hematology/Oncology

Table 42. Distribution of NSE

% with NSE in Indicated Range

No. of Patients
Patient Groups Studied <13 ng/mL 13–25 ng/mL 25–50 ng/mL >50 ng/mL
Blood Donors 33 94 6 0 0
Benign Disorders
Bronchopneumonia 20 65 30 5 0
Lung lesions 11 91 9 0 0
Gastrointestinal & liver 45 84 16 0 0
Malignant Disorders
SCLC pre-treatment:
Limited disease 38 34 24 29 13
Extensive disease 39 12 10 31 46
Non SCLC 94 83 10 3 4
Lung metastases 10 80 20 0 0
Breast & gastrointestinal with 20 85 15 0 0
hepatic metastasis
Prostate 42 74 19 7 0
Reference: Cooper et al. Br J Cancer. 1985;52:333-338.
 171

Section 7 Immunology

7.1 Available Tests Reticulin IgG Antibody

Tissue Transglutaminase Antibody (IgA)
Allergic Diseases Tissue Transglutaminase Antibody (IgG)
Aspergillus Antibody, Immunodiffusion Tissue Transglutaminase Antibody (IgG, IgA)
Eosinophil Cationic Protein (ECP)1
Eosinophil Count, Blood Inflammatory Bowel Disease
Eosinophil Count, Urine Inflammatory Bowel Disease Differentiation Panel
Histamine, 24-Hour Urine1 Includes Saccharomyces cerevisiae Antibodies (IgA, IgG), and ANCA screen with
Histamine, Plasma1 reflex to titer.
IgE, Total, Serum Lactoferrin, Qualitative, Stool
Theophylline Lactoferrin, Quantitative, Stool
Tryptase1 Pancreatic Exocrine Cell Antibody2
Quest Diagnostics offers allergy testing for food, pollens (trees, grasses, weeds), drugs,
Saccharomyces cerevisiae Antibodies (ASCA) (IgA)
molds, insect venoms, and occupational-related allergens. Please refer to the Quest Saccharomyces cerevisiae Antibodies (ASCA) (IgG)
Diagnostics’ Directory of Services for a complete list of specific allergen tests. TPMT Genotype

Cytokines Other Gastrointestinal Diseases (including autoimmune

Interleukin-1 Beta1 hepatitis)
Interleukin-2 (IL-2)1 Actin (Smooth Muscle) Antibody (IgG)
Interleukin-2 Receptor, EIA1 Autoimmune Hepatitis Diagnostic Panel
Interleukin-61 Autoimmune Hepatitis Extended Panel
Tumor Necrosis Factor-Alpha, Highly Sensitive1 Autoimmune Hepatitis Panel, Primary
Vascular Endothelial Growth Factor (VEGF), ELISA1 Gastric Parietal Cell Antibody, ELISA
HLA-B27 Antigen
Dermatologic Diseases HLA-B27, DNA Typing
Bullous Pemphigoid Antigen (BP 180) Antibody1 Intrinsic Factor Blocking Antibody
Bullous Pemphigoid BP230 IgG1 Liver Kidney Microsome (LKM-1) Antibody (IgG)
Desmoglein Antibodies (1 and 3) Mitochondria M2 Antibody (IgG), EIA
Epidermal Antibodies with Reflex to Titers Mitochondrial Antibody, IFA
RNA Polymerase III Antibody Primary Biliary Cirrhosis Diagnostic Panel, Comprehensive
Soluble Liver Antigen (SLA) Autoantibody
Endocrine Diseases
Adrenal Antibody Screen with Reflex to Titer Hematologic Diseases
Glutamic Acid Decarboxylase-65 Autoantibodies CD55 and CD59 Expression, Red Cells and Granulocytes1
Growth Hormone Antibody2 Coombs, Indirect (Antibody Screen)
HLA-DR4, Flow Cytometry2 Neutrophil Antibody, Flow Cytometry2
21-Hydroxylase Antibody1 Platelet Antibody, Direct, Flow Cytometry2
IA-2 Antibody2 Platelet Antibody, Indirect (IgG)
Insulin Autoantibody2
Islet Cell Antibody Screen with Reflex to Titer2 HLA and Transplantation
Ovarian Antibody Screen with Reflex to Titer, IFA Cyclosporine, LC/MS/MS, Whole Blood
PTH Antibody Cyclosporine A, Trough, Blood
Sperm Antibody (IgA, IgG)2 FISH, X/Y, Post Bone Marrow Transplant
T3 (Triiodothyronine) Antibody HLA Typing, Celiac Disease
T4 (Thyroxine) Antibody HLA Typing, Narcolepsy
TBII (Thyrotropin-Binding Inhibitory Immunoglobulin) HLA-A Class I DNA Typing
Thyroglobulin Antibody HLA-A High Resolution SBT Typing2
Thyroid Peroxidase Antibody (Anti-TPO) HLA-A2 Typing w/Reflex A2 High Res. SBT Subtyping2
Thyroid Stimulating Hormone (TSH) Antibody HLA-A,B Class I DNA Typing
TSI (Thyroid Stimulating Immunoglobulin)2 HLA-A,B,C Class I DNA Typing
HLA-A,B,C Phenotype
Gastrointestinal Diseases HLA-B Class I DNA Typing
Celiac Disease HLA-B High Resolution SBT Typing2
Celiac Disease Comprehensive Panel HLA-B27 Antigen
Celiac Disease Panel HLA-B27, DNA Typing
Celiac Disease Panel without Gliadin HLA-C Class I DNA Typing
Endomysial Antibody Screen (IgA) with Reflex to Titer HLA-C High Resolution SBT Typing2
Gliadin Antibody (IgA) HLA-DQA1 DNA Typing
Gliadin Antibody (IgG) HLA-DQB1 Class II DNA Typing
Gliadin Antibody (IgA, IgG) HLA-DQB1 High Resolution SBT Typing2
HLA Typing, Celiac Disease HLA-DR, DQ Class II DNA Typing
Includes DQB1, DQ2, and DQ8. HLA-DR4, Flow Cytometry2
IgA, Serum HLA-DRB1 Class II DNA Typing
Reticulin Antibody (IgA) with Reflex to Titer2 HLA-DRB1 High Resolution SBT Typing2
172

Section 7 Immunology

HLA-DRB3*4*5 DNA Typing Complement Component C4, Pleural Fluid

Immune Cell Function Complement Component C62
Mycophenolic Acid Complement Component C82
Neopterin1 Complement Profile
Tacrolimus (FK506), Trough Includes C4 and C3 complement, properdin factor B, and C1q complement.
TPMT Genotype Complement, Total (CH50)
Immune Complex Detection by C1q Binding
Immunodeficiencies Mannose Binding Lectin (MBL)
Cellular Immunodeficiencies Properdin Factor B (C3 Proactivator)
Adenosine Deaminase, CSF1
Adenosine Deaminase, Peritoneal Fluid1 Humoral Immunity
Adenosine Deaminase, Pleural Fluid1 Humoral Immunity Evaluation Panel
Lymphocyte Subset Panel 1 Includes diphtheria and tetanus toxoid antibodies, Haemophilus influenza IgG
Includes absolute lymphocyte count, CD19, CD3, CD4, CD8, CD16/CD56, and antibody, and Streptococcus pneumoniae serotypes 1, 3, 14, 19 (19F), 23 (23F), and
CD4/CD8 ratio. 51 (7F).
Lymphocyte Subset Panel 2 IgA, Serum
Includes absolute lymphocyte count, CD19, CD3, CD4, CD8, and CD4/CD8 ratio. IgA Subclasses
Lymphocyte Subset Panel 3 IgD, Serum
Includes absolute lymphocyte count, CD3, CD4, CD8, and CD4/CD8 ratio. IgE, Total, Serum
Lymphocyte Subset Panel 4 IgG Subclass IgG1
Includes absolute lymphocyte count, CD4, CD8, and CD4/CD8 ratio. IgG Subclass IgG2
Lymphocyte Subset Panel 5 IgG Subclass IgG3
Includes absolute lymphocyte count and CD4. IgG Subclass IgG4
Natural Killer Cells2 IgG Subclasses and Total IgG
Includes absolute lymphocyte count and CD16/CD56. IgG, CSF
T & B Cells, Total IgG, Serum
IgG/Albumin Ratio, CSF
Complement Deficiencies IgM, Serum
Angioedema Panel Immunoglobulin Profile, CSF
Angioedema Panel, Acquired Includes IgA, IgG, and IgM.
Angioedema Panel, Hereditary Immunoglobulin Profile, Serum
Includes C1 inhibitor protein and functional C1 inhibitor. Includes IgA, IgG, and IgM.
Angioedema Panel, Hereditary, Comprehensive2
Angioedema Profile 1 Infection- and Vaccine-related Humoral Immunity
Includes C1 inhibitor protein, functional C1 inhibitor, C1q complement component, Adenosine Deaminase, CSF1
and C4 complement component. Adenosine Deaminase, Peritoneal Fluid1
C1 Inhibitor, Functional Adenosine Deaminase, Pleural Fluid1
C1 Inhibitor, Protein2 Diphtheria Toxoid & Tetanus Toxoid Antibodies
C1q Complement Component Diphtheria Toxoid Antibody2
C2 Complement Component2 Diphtheria Toxoid, Pre and Post2
C3 Complement Component Diphtheria Toxoid, Titer to Endpoint2
C3, C4, and Total (CH50) Complement Haemophilus influenzae Serotyping (A-F), SA
C3a desArg Fragment1 Haemophilus influenzae Type B Antibody (IgG)
C3d Circulating Immune Complexes Hepatitis B Surface Antibody
C4 & C3 Complement Group Hepatitis B Surface Antigen
C4 Activation Panel Hepatitis C Antibody, EIA
Includes C4 complement component and C4d fragment. Tetanus Toxoid Antibody (EIA)2
C4 Binding Protein, Plasma2
C4 Binding Protein2 Lymphoproliferative Disorders
C4 Complement Component Electrophoresis & Immunofixation, Serum with Tracing
C4d Fragment, EIA2 Electrophoresis, Protein and Kappa/Lambda, Serum
C5 Complement Component Electrophoresis, Protein and Total Protein and Tracing, CSF
C7 Complement Component1 Electrophoresis, Protein and Total Protein, CSF
C9 Complement Component1 Electrophoresis, Protein, 24-Hour Urine & Immunofixation Studies
Circulating Immune Complex Electrophoresis, Protein, 24-Hour Urine (with Total Protein)
Includes C4 and C3 complement, C3d circulating immune complexes, and immune Electrophoresis, Protein, CSF
complex detection via C1q binding. Electrophoresis, Protein, CSF, with Tracing
Complement Activation Panel2 Electrophoresis, Protein, Random Urine & Immunofixation Studies
Includes C3 complement component, properdin factor B, and C4d fragment. Electrophoresis, Protein, Random Urine (with Total Protein)
Complement Component C3, Pericardial Fluid Electrophoresis, Protein, Serum
Complement Component C3, Peritoneal Fluid Electrophoresis, Protein, Serum with Tracing
Complement Component C3, Pleural Fluid Electrophoresis, Protein, Urine
Complement Component C4, Pericardial Fluid Immunofixation with Quantitation of IgA, IgG, and IgM
Complement Component C4, Peritoneal Fluid Immunofixation, Serum
 173

Section 7 Immunology

Immunofixation, Urine MAG (Western Blot) and MAG-SGPG Antibodies w/Reflex to MAG, EIA2
Kappa Lambda Light Chains w/Calculation, 24-Hour Urine Motor & Sensory Neuropathy Evaluation with GQ1B Ab & IFE2
Kappa Lambda Light Chains, Total, Random Urine Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1a (IgG, IgM),
Kappa Light Chain, Free GD1b (IgG, IgM), GQ1b (IgG), MAG-SGPG (IgM), and MAG (IgM) antibodies; Hu
Kappa Light Chain, Total, Random Urine antibodies with reflex to titer
Kappa/Lambda Light Chain Motor Neuropathy Antibody Panel 1
Kappa/Lambda Light Chains, Free with Ratio Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM), and
Kappa/Lambda Light Chains, Free with Ratio and Reflex to MAG-SGPG (IgM) antibodies.
Immunofixation Motor Neuropathy Antibody Panel 2
Lambda Light Chain, Free Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM),
Lambda Light Chain, Total, Random Urine MAG-SGPG (IgM), and MAG (IgM) antibodies and Hu antibodies with reflex to titer
Surface Light Chains2 and Western blot.
Viscosity, Serum Motor Neuropathy Antibody Panel, Serum2
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM),
Neurologic Diseases GD1a (IgG, IgM), MAG-SGPG (IgM), and MAG (IgM) antibodies.
Multiple Sclerosis Myelin-Associated Glycoprotein (MAG)-SGPG Antibody (IgM)2
Albumin & IgG, CSF Myelin-Associated Glycoprotein (MAG) Antibody (IgM), EIA2
IgG Synthesis Rate/Index, CSF Neuronal Nuclear (Anti-Ri) Ab, IFA with Reflex to Titer and Western
Interferon-beta (IFNb) IgG, ELISA with Reflex to Neutralization Blot2
Multiple Sclerosis Panel 1 Neuronal Nuclear (Hu and Ri) Antibodies with Reflex to Titer & Western
Includes oligoclonal bands (IgG) and IgG synthesis rate/index on CSF. Blot2
Multiple Sclerosis Panel 2 Neuronal Nuclear (Hu) Antibody with Reflex to Titer & Western Blot2
Includes oligoclonal bands (IgG), IgG synthesis rate/index, and myelin basic protein Neuronal Nuclear (Hu) Antibody, IFA, w/Rfx to Titer & Western Blot,
on CSF. CSF2
Myelin Basic Protein2 Paraneoplastic Syndrome Antibody Panel, Serum2
Oligoclonal Bands (IgG), CSF Includes Hu antibody screen with reflexes to titer and Western blot, Yo antibody
Tysabri (Natalizumab) Antibodies, ELISA screen with reflex to titer, and Ri antibody screen with reflexes to titer and
Western blot.
Myasthenia Gravis Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, CSF2
Acetylcholine Receptor Binding Antibody Purkinje Cell (Yo) Antibody Screen with Reflex to Titer, IFA, Serum2
Acetylcholine Receptor Blocking Antibody2 Sensory & Motor Neuropathy Antibody Panel
Acetylcholine Receptor Modulating Antibody2 Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM),
Myasthenia Gravis Panel 1 GQ1b (IgG), MAG-SGPG (IgM), and MAG (IgM) antibodies.
Includes anti-striated muscle antibody screen with reflex to titer and acetylcholine Sensory Neuropathy Antibody Panel 12
receptor binding antibody. Includes Hu antibody screen with reflex to titer and Western blot, MAG-SGPG
Striated Muscle Antibody with Reflex to Titer2 (IgM), and MAG (IgM) antibodies.
Sensory Neuropathy Antibody Panel 22
Neuropathies Includes Hu antibody screen with reflex to titer and Western blot, Yo antibody
Anti-Yo & Anti-Hu Panel screen with reflex to titer, Ri antibody screen with reflex to titer and Western blot,
Includes Yo antibody screen with reflex to titer and Hu antibody screen with and MAG-SGPG (IgM) and MAG (IgM) antibodies.
reflexes to titer and Western blot.
Ganglioside Antibody Panel 1 Pulmonary Diseases
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM), and Alpha-1 Antitrypsin (AAT) Mutation Analysis2
GQ1b (IgG) antibodies. Alpha-1-Antitrypsin (AAT) Quantitation and Mutation Analysis2
Ganglioside Antibody Panel 2 Alpha-1-Antitrypsin Quantitation
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), and GD1b (IgM) C3a desArg Fragment1
antibodies. Hypersensitivity Pneumonitis Screen2
Ganglioside Antibody Panel 3
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgM), and GD1b (IgM) Rheumatic and Related Systemic Diseases
antibodies. Inflammatory Myopathies
Ganglioside Antibody Panel 4 Jo-1 Antibody
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), GD1b (IgG, IgM), and PM-Scl Antibody2
GD1a (IgG, IgM) antibodies. Polymyositis/Dermatomyositis Antibody Panel
Ganglioside Antibody Panel 5 Includes Jo-1 and Pm-Scl antibodies.
Includes ganglioside GM-1 (IgG, IgM), asialo-GM-1 (IgG, IgM), and GD1b (IgG, IgM) TPMT Genotype
antibodies.
Ganglioside Asialo-GM-1 Antibody (IgG), EIA Mixed Connective Tissue Disease
Ganglioside Asialo-GM-1 Antibody (IgM), EIA ANAchoice™ with Reflex to Mixed Connective Tissue Disease Antibody
Ganglioside GD1a Antibody (IgG), EIA ENA Screen with Reflex to ENA Antibodies
Ganglioside GD1a Antibody (IgM), EIA Mixed Connective Tissue Disease Syndrome Panel
Ganglioside GD1b Antibody (IgG), EIA RNP Antibody
Ganglioside GD1b Antibody (IgM), EIA Sm and Sm/RNP Antibodies
Ganglioside GM-1 Antibodies (IgG and IgM), EIA Sm Antibodies
Ganglioside GQ1b Antibody (IgG), EIA Sm/RNP Antibody
174

Section 7 Immunology

Rheumatic Fever and Other Cardiovascular Diseases Spondylarthropathies

Intercalated Disk Antibodies2 HLA-B27 Antigen
Myocardial Antibody Screen with Reflex to Titer2 HLA-B27, DNA Typing

Rheumatoid Arthritis and Other Arthropathies Systemic Lupus Erythematosus

ANAchoice™ Rheumatoid Arthritis Diagnostic Panel ANAchoice™ Screen, with Reflex to dsDNA
ANA Comprehensive Panel 1 ANAchoice™ Screen, with Reflex to Titer, IFA
Includes ANA screen with reflex to titer and pattern, rheumatoid factor, DNA ANAchoice™ with Reflex to Lupus/SLE Antibodies
antibody with reflex to titer, and Scl-70, Sjögren’s (SS-A, SS-B), Sm, and Sm/RNP ANA Comprehensive Panel 1
antibodies. Includes ANA screen with reflex to titer and pattern, rheumatoid factor, DNA
C-Reactive Protein (CRP) antibody with reflex to titer, and Scl-70, Sjögren’s (SS-A, SS-B), Sm, and Sm/RNP
C-Reactive Protein (CRP), Highly Sensitive, CSF antibodies.
Cyclic Citrullinated Peptide (CCP) IgG ANA Comprehensive Panel 3 with Reflex to ANA Titer and Pattern
HLA-B27 Antigen Includes ANA screen with reflex to titer and pattern and dsDNA, scleroderma (Scl-
HLA-B27, DNA Typing 70), Sm and Sm/RNP, and Sjögren’s (SS-A, SS-B) antibodies.
Lupus Erythematosus (SLE)/Rheumatoid Arthritis Panel w/Reflex to ANA ANA Screen (IFA) with Reflex to Titer
Titer Autoimmune Disorders Panel with Reflex to ANA Titer and Pattern
Includes ANA screen with reflex to titer and pattern, cyclic citrullinated peptide C2-Glycoprotein I Antibody (IgA)
(CCP) IgG antibody, and rheumatoid factor antibody. C2-Glycoprotein I Antibody (IgG)
Rheumatoid Arthritis Diagnostic Panel C2-Glycoprotein I Antibody (IgM)
Includes rheumatoid factor and cyclic citrullinated peptide antibody (IgG). C2-Glycoprotein I Antibodies (IgA, IgG, IgM)
Rheumatoid Arthritis Diagnostic Panel, Comprehensive C3a desArg Fragment1
Rheumatoid Factor Cardiolipin Antibody (IgA)
Rheumatoid Factor, CSF Cardiolipin Antibody (IgG)
Rheumatoid Factor (IgA) Cardiolipin Antibody (IgM)
Rheumatoid Factor (IgG) Cardiolipin Antibody Screen w/ Reflex to IgG and IgM
Rheumatoid Factor (IgA, IgG, IgM) Cardiolipin Antibody Screen w/ Reflex to IgG, IgA, IgM
Rheumatoid Factor Screen with Reflex to Titer, Synovial Fluid Chromatin Antibodies (IgG)
DNA (DS) Antibodies
Scleroderma and CREST Syndrome DNA (DS) Antibody, Crithidia IFA w/Reflex to Titer
ANAchoice™ with Reflex to Scleroderma Antibodies DNA (DS) Antibody, High Avidity ELISA
ANA Comprehensive Panel 1 Histone Antibodies
Includes ANA screen with reflex to titer and pattern, rheumatoid factor, DNA Lupus Activity Panel 21
antibody with reflex to titer, and Scl-70, Sjögren’s (SS-A, SS-B), Sm, and Sm/RNP Includes C3 and C4 complement and high avidity dsDNA antibody.
antibodies. Lupus (SLE) Panel
ANA Comprehensive Panel 3 with Reflex to ANA Titer and Pattern Includes C4 and C3 complement; TPO, rheumatoid factor, SS-A, SS-B, Sm, Sm/RNP,
Includes ANA screen with reflex to titer and pattern and dsDNA, scleroderma (Scl- Scl-70, ribosomal P, actin (IgG), and gastric parietal cell antibodies; and reticulin,
70), Sm and Sm/RNP, and Sjögren’s (SS-A, SS-B) antibodies. mitochondrial, dsDNA, ANA, striated muscle, and myocardial antibody screens with
Centromere Antibody, EIA reflex to titers.
Centromere Antibody Screen with Reflex to Titer Lupus Erythematosus Panel
PM-Scl Antibody2 Includes ANA screen with reflex to titer; dsDNA, histone, Sm, and Sm/RNP
Polymyositis/Dermatomyositis Antibody Panel antibodies; and CRP.
Includes Jo-1 and Pm-Scl antibodies. PCNA Antibody, IFA
Scleroderma (Scl-70) and Jo-1 Antibodies Phosphatidylserine Antibody (IgA)
Scleroderma Antibody (Scl-70) Phosphatidylserine Antibodies (IgG, IgM)
Scleroderma Panel 1 Phosphatidylserine Antibodies (IgA, IgG, IgM)
Includes Scl-70 antibody and centromere antibody screen with reflex to titer. Ribosomal P Antibody
Scleroderma Panel 2 Sm Antibodies
Includes ANA screen with reflex to titer and pattern and PM-Scl and Scl-70 Systemic Lupus Erythematosus (SLE) Comprehensive Diagnostic Panel
antibodies. Systemic Lupus Erythematosus (SLE) Diagnostic Panel, Drug Induced
Systemic Lupus Erythematosus (SLE) Disease Activity Panel
Sjögren’s Syndrome
ANAchoice™,Sjögren’s Panel Vasculitis
ANA Comprehensive Panel 1 ANCA Screen with Reflex to C- & P-ANCA Titers and MPO or PR-3
Includes ANA screen with reflex to titer and pattern, rheumatoid factor, DNA Antibody
antibody with reflex to titer, and Scl-70, Sjögren’s (SS-A, SS-B), Sm, and Sm/RNP ANCA Vasculitides
antibodies. Includes ANCA, proteinase-3, and myeloperoxidase antibodies.
ANA Comprehensive Panel 3 with Reflex to ANA Titer and Pattern C3d Circulating Immune Complexes
Includes ANA screen with reflex to titer and pattern and dsDNA, scleroderma (Scl- Cryoglobulin (% Cryocrit), Serum
70), Sm and Sm/RNP, and Sjögren’s (SS-A, SS-B) antibodies. Cryoglobulin Screen with Reflex to Cryoglobulin Profile, Serum
Sjögren’s Antibodies (SS-A, [Ro]) Profile includes % cryocrit, cryocrit immunofixation and immunodiffusion, and
Sjögren’s Antibodies (SS-A, SS-B, [Ro, La]) rheumatoid factor.
Sjögren’s Antibodies (SS-B [La]) Glomerular Basement Membrane Antibody (IgG)
Sjögren’s Syndrome Diagnostic Panel, Comprehensive Immune Complex Detection by C1q Binding

Section 7
Immune Complex Detection Panel 1
Includes C3d circulating immune complexes and immune complex detection by C1q
binding.
Immune Complex Detection Panel 2
Includes C3d circulating immune complexes, immune complex detection by C1q
binding, and total complement (CH50).
Lung Hemorrhage and Nephritis Panel
Myeloperoxidase Antibody
Neutrophil Cytoplasmic Antibody (ANCA) Screen with Reflex to Titer
Proteinase-3 Antibody
Vasculitis Diagnostic Panel
1
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
2
This test was developed and its performance characteristics were determined by Quest
Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food and
Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Reflex tests are performed at an additional charge.
7.2 Allergic Diseases
7.2.1 Childhood Allergy (Food and Environmental) Profile
Clinical Use: This test is used to diagnose suspected IgE antibody-
mediated allergic reactions.
Clinical Background: The prevalence of allergic disease in children
has increased markedly in recent decades.1,2 Symptoms include primarily
gastrointestinal distress, recurrent otitis media, wheezing, and eczema
at very young ages, with rhinitis, conjunctivitis, and asthma
predominating in later years. Limiting exposure to allergens during early
childhood can reduce symptoms3 and even the prevalence of asthma
later in childhood.4 Avoidance of allergenic food substances may also
lead to future tolerance.5 In contrast, inappropriate elimination of food
from the diet may negatively affect quality of life, nutritional status, and
growth in children.5 Thus, accurate diagnosis of childhood allergy is
critical for appropriate management.
In addition to patient history and physical evaluation, specific laboratory
tests may be useful in evaluating the role of allergy in the patient’s
illness.1,5 Use of specific IgE testing combined with clinical judgment
can improve the ability of clinicians to identify atopy, reducing
misclassification and influencing advice for allergen avoidance.1
The ImmunoCAP®’ Childhood Allergy Profile measures IgE antibodies to
common food and inhalant allergens, providing an accurate and
convenient approach for confirming or ruling out many common allergies
in symptomatic children.
Method: This test includes 12 ImmunoCAP specific IgE allergens: cat
dander, cockroach, codfish, dog dander, egg white, house dust mite
(Dermatophagoides farinae), milk, mold (Alternaria alternata), peanut,
soybean, wheat, and total IgE.
References
1. Duran-Tauleria E, Vignati G, Guedan MJ, et al. The utility of specific
immunoglobulin E measurements in primary care. Allergy. 2004;59
Suppl 78:35-41.
2. Sly RM. Changing prevalence of allergic rhinitis and asthma. Ann
Allergy Asthma Immunol. 1999;82:233-248.
3. Lever R, MacDonald C, Waugh P, et al. Randomised controlled trial of
175
Immunology
advice on an egg exclusion diet in young children with atopic eczema
and sensitivity to eggs. Pediatr Allergy Immunol. 1998;9:13-9.
4. Chan-Yeung M, Ferguson A, Watson W, et al. The Canadian
Childhood Asthma Primary Prevention Study: outcomes at 7 years of
age. J Allergy Clin Immunol. 2005;116:49-55.
5. American College of Allergy, Asthma, and Immunology. Food allergy:
a practice parameter. Ann Allergy Asthma Immunol. 2006;96:s1-s68.
7.2.2 Food Allergy: Joint Task Force Guidelines for Diagnosis
and Management
Introduction: Guidelines for diagnosis and management of food allergy
have been developed by the Joint Task Force on Practice Parameters
representing the American Academy of Allergy, Asthma, and
Immunology; the American College of Allergy, Asthma, and Immunology;
and the Joint Council of Allergy, Asthma and Immunology.1 Here we
provide a summary of these guidelines with a particular focus on the
use of laboratory testing for the diagnosis and management of food
allergy (IgE-mediated). It is important to bear in mind, however, that the
majority of adverse reactions to food are not caused by IgE-mediated
allergies. Refer to the complete Practice Parameter1 for more
information on the differential diagnosis of non-IgE-mediated adverse
food reactions.
Clinical Background: It is important to diagnose food allergies to
avoid unnecessary dietary restrictions that could negatively affect
quality of life, nutritional status, and, in children, growth. The diagnostic
algorithm developed by the Joint Task Force (Figure 1) begins with a
relevant clinical history provided by the patient. This includes a list of
suspected foods and the amount consumed, the time between ingestion
and reaction, the type and duration of symptoms, repeatability of the
reaction to the suspect food, requirement for cofactors (eg, exercise),
and the time from last reaction. The second step is performance of a
relevant physical examination including inspection of the upper and
lower respiratory tract and the cutaneous and gastrointestinal (GI)
systems. Third, determine if clinical and physical findings are consistent
with an IgE-mediated reaction, which is characterized by a short time
(ie, usually within minutes, rarely up to a few hours) from ingestion of
the food to symptom onset, a severe reaction resulting from small
amounts of food, an adverse reaction occurring with reexposure, and
symptoms of pruritus, urticaria or angioedema, GI disturbance,
rhinoconjunctivitis, bronchospasm, and anaphylaxis. Fourth, perform
food-specific IgE testing when the history is consistent with an IgE-
mediated food reaction. In vivo or in vitro tests can be used. Finally,
perform an oral food challenge or trial elimination diet as indicated by
food-specific IgE test results and clinical symptoms (see Figure 1).
Individuals Suitable for Testing include individuals who have a
history consistent with an IgE-mediated food reaction as described
above.
Test Availability: Available tests include food-specific IgE tests such
as in vivo skin prick or puncture tests (intracutaneous or percutaneous)
and in vitro (serum) tests (eg, ImmunoCAP®’, AlaSTAT®’, HY-TEC™’) as
well as oral food challenge tests and a trial elimination diet.
In vivo food-specific IgE testing has a high negative predictive value
(r95%) but a low positive predictive value (b50%). These tests are
useful, therefore, as an initial test to rule out an IgE-mediated reaction
to a highly suspected food. Percutaneous skin tests (PSTs) are
recommended over intracutaneous tests because intracutaneous tests
are overly sensitive, and thus have a high rate of false-positive results,
and are associated with risk of anaphylaxis and death.
176

Section 7 Immunology
 177

Section 7 Immunology

In vitro IgE tests have roughly the same sensitivity and specificity as in
vivo IgE tests and consequently have the same clinical application. In
vitro tests are especially useful when the patient has a history of a life-
threatening reaction or a medical condition that prevents accurate skin
testing (eg, extensive atopic dermatitis or dermatographism) and when
the patient is known to have a nonreactive histamine control or is
known to be pregnant. Radioimmunoassay procedures
(radioallergosorbent test [RAST]) are no longer used. Current methods
use a food-specific allergen bound to a solid phase to detect IgE in the
patient’s serum. World Health Organization-based standards are used to
quantify results, which are reported in kIU/L.
Since a positive test alone does not necessarily indicate ingestion of the
food will result in a clinical response, further testing is required (Figure
1) in patients without an anaphylactic response to rule out cross-
reacting proteins or other causes of a false-positive result (eg,
eosinophilic gastroenteropathy). A larger PST response size or greater
concentration of IgE correlates with a greater likelihood of a clinical
reaction to the food, but does not correlate with the type or severity of
that reaction.
Interpretation of these and other available diagnostic tests is
summarized in Table 2.

Oral food challenge tests and trial elimination diets (Table 1) may lead
to or confirm a diagnosis of food allergy and may be useful for patient
management.
Interpretive Information: A negative food-specific IgE test result
generally indicates lack of an IgE-mediated food reaction and probable
tolerance of the food. However, results may be affected by the patient’s
age, the reagents used, and the testing techniques used. For example,
tests that use extracts from foods with stable proteins (eg, peanut, milk,
egg, tree nuts, fish, and shellfish) are more reliable than those that use
extracts from foods with labile proteins (eg, some fruits and
vegetables). In vitro tests and PSTs use purified antigen that may differ
from food challenge antigen that is raw, cooked, or otherwise processed
resulting in seemingly inconsistent results. Furthermore, in vivo skin
tests are affected by the skin test devices used, the location of test
placement, and the mode of measurement. In the event of a severe
reaction and a negative test result, an open food challenge may be
appropriate to rule out a food allergy. Preparation for a possible
anaphylactic response should be made prior to the food challenge.
Patient Management: The following steps may be taken when
managing a patient with a food allergy. First, educate and advise the
patient to avoid the specific food allergen. Educational topics include
how to read food labels, including an awareness of risk imposed by
unfamiliar terms and cross-contamination during food processing;
available dietary alternatives, including supplements; and how to avoid
unintentional food allergen exposure (eg, in schools or restaurants.
Second, educate and advise the patient on treating an anaphylactic
response (eg, use of injectable epinephrine with or without
antihistamine and seeking medical care for a systemic reaction). Third,
perform periodic testing to determine newly-developed tolerance.
Avoidance of the food allergen may lead to future tolerance.
Additionally, children are likely to outgrow some types of food allergies
(eg, cow’s milk, wheat, and egg). A subsequent negative food-specific
IgE test is likely to indicate tolerance to the food; however, a positive
test is inconclusive since these tests may remain positive even when
the patient no longer has clinical symptoms. An oral food challenge may
be helpful in determining current tolerance status.

A positive food-specific IgE test result in a patient with a history of Reference

anaphylactic response to that food is considered diagnostic. There is no
1. American College of Allergy, Asthma, and Immunology. Food allergy:
need for further testing.
a practice parameter. Ann Allergy Asthma Immunol. 2006;96:S1-S68.

Table 1. Non-cutaneous In Vivo Tests Used in Diagnosis and Management of Food Allergy*

Food Challenge Test Definition Advantages Disadvantages

Open challenge Uses either suspect food in its natural
form or a placebo; not blinded to
patient or physician
Oral challenge Uses either suspect food (given in
Single-blind gradually increasing amounts) or a
placebo disguised and blinded to the
patient; reactions are judged by
patient and physician
Double-blind Uses either suspect food (given in
gradually increasing amounts) or a
placebo disguised and blinded to the
patient and the physician; reactions
are judged independently by patient
and physician
Trial elimination diet Eliminates suspected foods from the
diet for several weeks and then
gradually reintroduces them one at a
time with reactions observed
*Anaphylaxis is a risk and thus food challenges should be performed where emergency treatment is readily available.
Simplest to perform; best when
several foods are suspect
Eliminates patient bias and need for
test interpreter to be blinded; offers
protocol flexibility
Gold standard for diagnosis of food
allergy
Best when symptoms are chronic;
replaces individual food challenges
when several suspect foods have been
identified
Subject to patient and physician bias;
positive result may require blinded
confirmation testing
Test interpreter potentially biased
Requires a third person to prepare the
blinded food
Positive result requires compelling
supportive evidence for a definitive
diagnosis
178

Section 7 Immunology

Table 2. Interpretation of Tests Used for Diagnosis of Food Allergy

Tests Positive Test Results Negative Test Results

Food-specific IgE testing
Open oral food challenge
Single blind oral food challenge
Double blind oral food challenge
Trial elimination diet
With history of anaphylaxis: diagnostic;
no further testing needed
Without history of anaphylaxis: suggestive, but not
diagnostic; further testing needed
Reaction is suggestive of food allergy, but results
are prone to bias
Reaction is suggestive of food allergy, but results
are prone to bias
Reaction is highly suggestive of food allergy
Food allergy is probable when chronic
symptoms resolve
Tested foods are likely to be tolerated (negative
predictive value z95%)
Consider open challenge in some cases with severe
reaction
Tested foods are likely to be tolerated
Tested foods are likely to be tolerated irrespective of
food-specific IgE test results
Tested foods are likely to be tolerated irrespective of
food-specific IgE test results
Perform oral challenge when chronic symptoms persist

7.2.3 Food Allergy Profile
Clinical Use: This test is used to diagnose suspected IgE antibody-
mediated food reactions.
Clinical Background: Accurate diagnosis of food allergy is important
not only for avoidance of foods that may cause a reaction, but also to
avoid unnecessary dietary restrictions that may adversely affect quality
of life.
Diagnosis of food allergies begins with assessment of clinical history
and physical examination to evaluate respiratory and gastrointestinal
symptoms. When findings are consistent with an IgE antibody-mediated
food reaction, food-specific IgE testing is performed with in vitro or in
vivo tests. In vitro testing is particularly useful in patients at risk of
severe anaphylactic reactions from skin-prick testing and others with
medical conditions that contraindicate in vivo testing.1
Clinical Background: The ImmunoCAP®’ Respiratory Allergy Profile
provides an accurate and convenient method of confirming or excluding
atopy in patients with symptoms that might be attributed to allergy.1 In
the United States, allergic rhinitis is the most common cause of rhinitis,
affecting up to 40% of children and 10% to 30% of adults.2 These cases
require different treatment than that used for non-allergic rhinitis.
Furthermore, allergy testing helps identify allergy-triggered asthma that
is present in 60% to 90% of children and 50% of adults with asthma.3
Treating the allergy is often beneficial in these cases. Thus, allergy
testing results can be helpful when making treatment decisions.
Because different types of allergens are found in different parts of the
country, Quest Diagnostics offers Regional Profiles that focus on
allergens specific to that region; for example, region XIV covers the
California central valley area. By using the appropriate Regional Profile,
your patients’ test results reflect the allergen exposure from a particular
geographic location.

The Food Allergy Profile provides rapid and convenient measurement of
food-specific IgE antibodies. The profile includes allergens associated
with common food allergies and is useful in confirming or excluding
suspected food allergies. Results can help direct subsequent testing and
management options, including trial elimination diet, oral food
challenge, and further evaluation of non-IgE-related reactions.
Method: This test includes total IgE and 21 ImmunoCap specific IgE
allergens: Bermuda grass; cat dander; cockroach; common pigweed;
cultivated oat; dog dander; grey alder; house dust mite (D farinae and D
pteronyssinus); Japanese cedar; lamb’s-quarters (goosefoot); maple box-
elder; mold (Alternaria alternata, Aspergillus fumigatus, and Cladosporium
herbarum); oak; olive; redtop bentgrass; scale, lenscale; and walnut.

Method: This test includes 11 ImmunoCAP®’ specific IgE allergens: References

clam, cod fish, corn (maize), egg white, milk, peanut, scallop, shrimp,
soybean, walnut, and wheat.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics. It has not been cleared or approved by the U.S. Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary.
Performance characteristics refer to the analytical performance of the test.
Reference
1. American College of Allergy, Asthma, and Immunology. Food allergy:
a practice parameter. Ann Allergy Asthma Immunol. 2006;96:s1-s68.
7.2.4 Respiratory Allergy Profile, Region XIV
Clinical Use: This test is used to diagnose suspected IgE antibody-
mediated respiratory reactions.
1. American Academy of Allergy, Asthma, and Immunology. AAAAI
Work Group Report: allergy diagnosis in clinical practice. November
2006. Available at: www.aaaai.org. Accessed June 6, 2007.
2. Dykewicz MS, Fineman S. Executive summary of joint task force
practice parameters on diagnosis and management of rhinitis. Am
Allergy Asthma Immunol. 1998;81:463-468.
3. Li JT, Pearlman DS, Nicklas RA, et al. Algorithm for the diagnosis and
management of asthma: a practice parameter update. Am Allergy
Asthma Immunol. 1998;81:415-420.
7.3 Cytokines
7.3.1 Interleukin-2 (IL-2) Receptor
Clinical Use: This test is used to assess T-cell activation following
transplantation, assess prognosis in patients with lymphoma, and
monitor therapeutic response in patients with hairy cell leukemia.

Section 7
Clinical Background: Elevated levels of soluble IL-2 receptor are
detected in AIDS, autoimmune diseases, sarcoidosis, and a variety of
leukemias and lymphomas. In HIV-positive individuals, the IL-2 receptor
level is elevated during the asymptomatic phase, as well as during
persistent generalized lymphadenopathy and symptomatic phases. IL-2
receptor detection may be useful in monitoring HIV and in assessing T-
cell activation following transplantation. Elevated IL-2 receptor levels
may also have clinical and prognostic significance in patients with
malignant lymphoma, non-Hodgkin’s lymphoma, B-cell, and
undifferentiated lymphomas.
Studies have suggested that IL-2 receptor levels in a broad spectrum of
conditions associated with T- or B-cell immune activation offer a rapid
and reliable measure of disease activity, response to therapy, and, in
some cases, prognosis. Measurement of the soluble IL-2 receptor level
is also helpful in assessing therapeutic response in patients with hairy
cell leukemia.
This test was performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
7.4 Gastrointestinal Diseases
7.4.1 Celiac Disease Comprehensive Panel
Clinical Use: This test is used to diagnose celiac disease, differentiate
celiac disease from irritable bowel syndrome, and monitor adherence to
a gluten-free diet in patients with celiac disease.
Clinical Background: Celiac disease is caused by an immune
response to gluten in genetically susceptible individuals. Patients may
develop partial to complete villous atrophy of the small intestine, crypt
hyperplasia, and lymphocytic infiltration of the epithelium and lamina
propria. This disease is more common than once thought, affecting as
many as 1 in 133 “not-at-risk” Americans (ie, those without family
history or gastrointestinal symptoms); rates are even higher among first-
and second-degree relatives of patients.1 Untreated, celiac disease may
be accompanied by progression of villous atrophy and development of
other autoimmune diseases (eg, thyroid disease and insulin-dependent
diabetes mellitus), osteoporosis, and neoplasia, including T-cell
lymphoma and adenocarcinoma of the small intestine.
Diagnosis is based on biopsy of the small intestine, but serologic assays
help identify patients who require this invasive procedure. Tissue
transglutaminase (tTG; IgA) antibody is an excellent first-line marker,
with high sensitivity and specificity in untreated individuals.2 The
endomysial antibody (EMA; IgA) assay has high specificity for celiac
disease and is used to confirm positive IgA anti-tTG results. Although
this panel tests for EMA only when IgA anti-tTG results are positive,
EMA testing can be ordered separately if the anti-tTG result is negative
but clinical suspicion remains high. Some patients with limited villous
atrophy have been reported to lack EMA and tTG antibodies; testing for
IgA antigliadin antibody (AGA) may help detect celiac disease in such
patients.3 Total serum IgA is measured to identify selective IgA
deficiency, present in about 2% to 10% of celiac disease patients. Such
patients would have negative results on IgA anti-tTG and EMA assays
but may have positive IgG anti-tTG results.
Because levels of anti-tTG and EMA tend to wane in the absence of
gluten ingestion, these markers are useful to monitor adherence to a
gluten-free diet.
179
Immunology
Method: This panel includes tests for tTG IgA, gliadin IgA, and total
IgA. Additional tests are performed, at an additional charge, as follows:
An EMA screen is performed when the tTG IgA is positive, and, if
positive, an endomysial antibody titer is performed. Further, a tTG IgG
test is performed when the total IgA is low.
References
1. Fasano A, Berti I, Gerarduzzi T, et al. Prevalence of celiac disease in
at-risk and not-at-risk groups in the United States: a large multicenter
study. Arch Intern Med. 2003;163:286-292.
2. Farrell RJ, Kelly CP. Diagnosis of celiac sprue. Am J Gastroenterol.
2001;96:3237-3246.
3. Green PH, Jabri B. Coeliac disease. Lancet. 2003;362:383-391.
7.4.2 Inflammatory Bowel Disease (IBD) Differentiation Panel
Clinical Use: This test is used to differentiate Crohn’s disease from
ulcerative colitis, to stratify Crohn’s disease subtypes,1 and to assess
need for further invasive testing in children with IBD symptoms.2
Clinical Background: Crohn’s disease (CD) and ulcerative colitis (UC)
are the most common forms of IBD. Although UC and CD are typically
differentiated on the basis of clinical, radiographic, and endoscopic
findings, distinguishing between these conditions can be difficult in
about 10% to 15% of patients, especially when disease is confined to
the colon. Because the treatment and prognosis of UC and CD differ,
accurate diagnosis is critical for management.
Numerous studies have investigated the utility of 2 serologic markers,
perinuclear anti-neutrophil cytoplasmic antibody (pANCA) and anti-
Saccharomyces cerevisiae antibody (ASCA), in differentiating between
UC and CD. The pANCA associated with IBD differs from that found in
the vasculitides, having an “atypical” perinuclear staining pattern that
can be identified by differential staining patterns with ethanol–formalin
fixation. This atypical pANCA is found in about 50% to 80% of UC
patients but only 10% to 30% of those with CD. ASCA, on the other
hand, is more common in CD (46% to 70%) than in UC (6%-12%).3,4 The
combination of these markers has high specificity for UC (94%-97%;
pANCA+/ASCA-) and CD (81%-98%; ASCA+/pANCA-).5 Serologic results
can also assist in stratification of CD: pANCA-positive CD is associated
with a clinical phenotype similar to that of UC (UC-like CD),4 while
positivity for ASCA IgG and IgA is associated with non-UC-like disease.6
Several reports have noted the potential utility of serologic testing,
combined with other clinical and laboratory information, to identify
children with suspected IBD who may not require invasive testing.2,7
Method: This panel includes a pANCA screen, with reflex to titer at an
additional charge, and an ASCA IgG and IgA test.
References
1. Klebl FH, Bataille F, Bertea CR, et al. Association of perinuclear
antineutrophil cytoplasmic antibodies and anti-Saccharomyces
cerevisiae antibodies with Vienna classification subtypes of Crohn’s
disease. Inflamm Bowel Dis. 2003;9:302-307.
2. Dubinsky MC, Ofman JJ, Urman M, et al. Clinical utility of
serodiagnostic testing in suspected pediatric inflammatory bowel
disease. Am J Gastroenterol. 2001;96:758-765.
3. Savige J, Dimech W, Fritzler M, et al. Addendum to the International
Consensus Statement on testing and reporting of antineutrophil
cytoplasmic antibodies. Quality control guidelines, comments, and
recommendations for testing in other autoimmune diseases. Am J
Clin Pathol. 2003;120:312-318.
180
Section 7
4. Abreu MT, Vasiliauskas EA, Kam LY, et al. Use of serologic tests in
Crohn’s disease. Clinical Perspectives in Gastroenterology.
2001;4:155-164.
5. Reumaux D, Sendid B, Poulain D, et al. Serological markers in
inflammatory bowel diseases. Best Pract Res Clin Gastroenterol.
2003;17:19-35.
6. Walker LJ, Aldhous MC, Drummond HE, et al. Anti-Saccharomyces
cerevisiae antibodies (ASCA) in Crohn’s disease are associated with
disease severity but not NOD2/CARD15 mutations. Clin Exp Immunol.
2004;135:490-496.
7. Bartunkova Kolarova I, Sediva A, et al. Antineutrophil cytoplasmic
antibodies, anti-Saccharomyces cerevisiae antibodies, and specific
IgE to food allergens in children with inflammatory bowel diseases.
Clin Immunol. 2002;102:162-168.
7.4.3 Lactoferrin
Clinical Use: This test is used to rule out irritable bowel syndrome
(IBS) in patients presenting with IBD symptoms.
Clinical Background: An estimated 30 million Americans suffer from
irritable bowel syndrome (IBS), a disorder characterized by crampy
abdominal pain, bloating, constipation, and/or diarrhea. The same
clinical picture may be seen in individuals with ulcerative colitis (UC)
and Crohn’s disease (CD). Collectively known as inflammatory bowel
disease (IBD), UC and CD affect more than 1 million Americans.
Although individuals with IBS may experience severe discomfort and
require symptomatic treatment, patients with IBD may develop rectal
bleeding and permanent intestinal damage. Furthermore, patients with
IBD frequently require long-term steroid therapy and
immunosuppressive agents. Consequently, distinguishing IBS from IBD
is critical for patient management.
In patients with active IBD, lactoferrin, a proven marker of inflammation,
is released from leukocytes infiltrating the intestinal mucosa. Whereas
fecal lactoferrin tends to be elevated in patients with active IBD, it is
minimally present in patients with IBS. In the stool of patients
presenting with symptoms of IBD, lactoferrin is 86% sensitive and
100% specific in distinguishing IBD from IBS, thus making fecal
lactoferrin an important diagnostic tool (Am J Gastroenterol.
2003;98:1309-1314).
The evaluation of fecal lactoferrin offers a safe, non-invasive, accurate
method to quickly differentiate IBD from IBS once infectious causes of
intestinal inflammation and colorectal cancer are ruled out. A positive
fecal lactoferrin result can be complemented by the Quest Diagnostics
Inflammatory Bowel Disease Differentiation Panel (separate order code)
to assist in distinguishing UC from CD.
7.4.4 TPMT Genotype
Clinical Use: This test is used to identify patients at risk for toxicity
from thiopurine drugs and to determine the need to adjust drug dosage
or select alternative therapy.
Clinical Background: Thiopurine drugs (azathioprine, 6-
mercaptopurine, and 6-thioguanine) are used to treat patients with
leukemia, rheumatic disease, inflammatory bowel disease, or solid
organ transplantation. These drugs require conversion to thioguanine
nucleotides to exert their therapeutic (cytotoxic) effect; however, that
conversion can be blocked by methylation or oxidation.1 The methylation
pathway depends on thiopurine methyltransferase (TPMT) activity,
which varies among individuals: approximately 90% have normal
activity, 10% have intermediate activity, and 0.3% have low or no
Immunology
detectable activity.1 Thioguanine nucleotides can accumulate in patients
who have reduced TPMT activity and who are receiving standard
thiopurine doses, resulting in hematopoietic toxicity (eg,
myelosuppression).2,3 Dosage reduction can minimize toxicity in such
patients.4
Reduced TPMT activity can be caused by polymorphisms in the TPMT
gene.1 Molecular studies have identified 4 variant alleles that together
account for >95% of reduced TPMT activity: TPMT*2 (238GmC),
TPMT*3A (460GmA and 719AmG), TPMT*3B (460GmA), and
TPMT*3C (719AmG).3,5 Individuals with 2 variant alleles have low or no
TPMT activity, while those with 1 variant allele have intermediate TPMT
activity. Wild-type (TPMT*1) homozygotes, on the other hand, have
normal enzyme activity.
Methods for measuring red blood cell (RBC) TPMT activity are available,
but results may be falsely elevated by recent blood transfusions and
falsely lowered by RBC aging.5,6 TPMT genotype testing can predict
reduced TPMT activity5,7 and is not affected by these variables. The Quest
Diagnostics Nichols Institute TPMT genotype assay uses polymerase
chain reaction (PCR) amplification followed by single nucleotide primer
extension (SNPE) to detect the 4 common TPMT variants.
TPMT genotyping results have predicted thiopurine drug toxicity in a
variety of disorders, including rheumatic disease,8 acute lymphoblastic
leukemia,7 renal transplantion,9 and Crohn’s disease.2 Genotype
analysis can thus help identify patients at increased risk of hematologic
toxicity, although prospective clinical studies are needed to determine
appropriate starting dosage for such patients.1
Individuals Suitable for Testing include patients being considered
for thiopurine therapy.
Method: This assay uses PCR to amplify target regions of the TPMT
gene, followed by multiplex SNPE targeting nucleotides 238, 460, and
719. Following hybridization through linker oligonucleotides to
microspheres, detection of reporter fluorescence on a specific
microsphere indicates the presence of an allele. Results are reported as
genotype detected or not detected. This assay is specific for wild-type
TPMT*1 and variants TPMT*2, *3A, *3B, and *3C; other variants are
not detected.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the test.
Interpretive Information: The wild-type TPMT*1/TPMT*1 genotype is
consistent with normal TPMT enzyme activity. Standard doses of
thiopurine drugs are less likely to be toxic in individuals with this
genotype. Heterozygotes with 1 wild type and 1 variant allele are
predicted to have intermediate TPMT activity, are at increased risk of
hematologic toxicity, and may require a lower dosage.1,4
Patients who lack a wild-type allele are predicted to have low or no
detectable enzyme activity and are at high risk for life-threatening
hematologic toxicity if given full doses of thiopurine medication.4
Alternative therapy or reduced dosage should be considered for these
patients.1,4
This assay does not detect rare alleles. In addition, because the
TPMT*3A allele contains the polymorphisms found in the TPMT*3B and
TPMT*3C alleles, this assay cannot distinguish the TPMT*1/TPMT*3A
(intermediate enzyme activity) from the TPMT*3B/TPMT*3C genotype
(no or low enzyme activity).3 However, the TPMT*3B/TPMT*3C genotype
is extremely rare in the United States.

Section 7
Results should be interpreted in conjunction with other laboratory and
clinical findings.
References
1. McLeod HL, Siva C. The thiopurine S-methyltransferase gene locus–
implications for clinical pharmacogenomics. Pharmacogenomics.
2002;3:89-98.
2. Colombel JF, Ferrari N, Debuysere H, et al. Genotypic analysis of
thiopurine S-methyltransferase in patients with Crohn’s disease and
severe myelosuppression during azathioprine therapy.
Gastroenterology. 2000;118:1025-1030.
3. Evans WE. Pharmacogenetics of thiopurine S-methyltransferase and
thiopurine therapy. Ther Drug Monit. 2004;26:186-191.
4. Evans WE, Hon YY, Bomgaars L, et al. Preponderance of thiopurine S-
methyltransferase deficiency and heterozygosity among patients
intolerant to mercaptopurine or azathioprine. J Clin Oncol.
2001;19:2293-2301.
5. Yates CR, Krynetski EY, Loennechen T, et al. Molecular diagnosis of
thiopurine S-methyltransferase deficiency: genetic basis for
azathioprine and mercaptopurine intolerance. Ann Intern
Med.1997;26:608-614.
6. Lennard L, Chew TS, Lilleyman JS. Human thiopurine
methyltransferase activity varies with red blood cell age. Br J Clin
Pharmacol. 2001;52:539-546.
7. Relling MV, Hancock ML, Rivera GK, et al. Mercaptopurine therapy
intolerance and heterozygosity at the thiopurine S-methyltransferase
gene locus. J Natl Cancer Inst. 1999; 91:2001-2008.
8. Black AJ, McLeod HL, Capell HA, et al. Thiopurine methyltransferase
genotype predicts therapy-limiting severe toxicity from azathioprine.
Ann Intern Med. 1998;129:716-718.
9. Kurzawski M, Dziewanowski K, Gawronska-Szklarz B, et al. The
impact of thiopurine S-methyltransferase polymorphism on
azathioprine-induced myelotoxicity in renal transplant recipients.
Ther Drug Monitor. 2005;27:435-441.
7.5 Hematologic Diseases
7.5.1 Platelet Antibody, Direct
See Coagulation, “Platelet Immune Disorders” section 3.3.1.
7.5.2 Platelet Antibody, Indirect (IgG)
See Coagulation, “Platelet Immune Disorders” section 3.3.2.
7.5.3 Platelet Glycoprotein Antibody
See Coagulation, “Platelet Immune Disorders” section 3.3.3.
7.5.4 Red Cell CD55 and CD59 Expression
See Hematology/Oncology, “Paroxysmal Nocturnal Hemoglobinuria
(PNH)” section 6.10.1.
7.5.5 TPMT Genotype
See Immunology, “Gastrointestinal Diseases” section 7.4.4.
Table 3. Correlation of ATP Level with Immune Response5-9
ATP Range
(ng/mL) Immune Response
b225 Low
226-524 Moderate
r525 Strong
181
Immunology
7.6 HLA and Transplantation
7.6.1 Immune Cell Function
Clinical Use: This test is used to monitor cell-mediated immunity in
immunosuppressed individuals.
Clinical Background: Cell-mediated immunity is expressed by
T-lymphocytes through direct cytotoxicity and the release of
lymphokines. The functions of cell-mediated immunity include the
destruction of fungi and tumor cells and the elimination of viral
infections. In addition, cell-mediated immunity is responsible for graft-
versus-host disease in allograft recipients.
Transplant recipients generally require prolonged treatment with
immunosuppressive agents to prevent rejection of the allograft. Correct
dosing of immunosuppressives is critical. Over-medicating may leave the
individual susceptible to infections or lead to drug toxicity, while under-
dosing can lead to shortened graft survival due to the immune response
to the transplanted tissue.1,2 Though immunosuppressive drug levels are
routinely monitored, they do not always correlate with the degree of
immunosuppression.1,2 Additionally, many current in vitro methods used
to study cell-mediated immunity (eg, cytokine production and
lymphoproliferation) are unsuitable for clinical practice because they
require long turnaround times or are unreliable at predicting graft
rejection.2
This assay measures the increase in intracellular ATP production that
occurs in T-lymphocytes within 24 hours of stimulation by antigens or
mitogens.3 Recent studies indicate that this ATP level correlates with
T-lymphocyte activity and, consequently, cell-mediated immune function,
thus making ATP a useful clinical indicator of cell-mediated immune
function.4-9
Individuals Suitable for Testing include those receiving
immunosuppressive therapy.
Method: This assay begins with phytohemagglutinin stimulation of
lymphocyte ATP production. CD4 positive T-lymphocytes are then
separated magnetically, and the level of ATP is measured via
chemiluminescence. The analytical sensitivity is 1 ng/mL. The assay is
specific for ATP produced by CD4 positive T-lymphocytes. Aliases
include Immuknow™’ and Lymphocyte Stimulation.
Interpretive Information: The ATP ranges shown in Table 3 are
correlated with cell-mediated immune function.5-9 An ATP level between
226-524 ng/mL indicates a level of cell-mediated immunity at which the
risk for both infection and rejection is at a minimum. It is the desired
ATP range for transplant recipients on immunosuppressive therapy.5-9
Results may be unreliable in some patients immediately following
transplantation because of immune system instability caused by surgical
Interpretation
Risk of Infection Risk of Rejection
Increased Decreased
Decreased Decreased
Decreased Increased
182

Section 7 Immunology

trauma, anesthesia, transfusion, immunosuppressive therapy, or very asymptomatic, while others may develop allergic disease, repeated
low CD4 count.5 Results should be interpreted in conjunction with other sinopulmonary or gastroenterologic infections, and/or autoimmune
laboratory and clinical findings. disease. Individuals with complete absence of IgA (<5 mg/dL) may
develop autoantibodies to IgA after blood or intravenous
References immunoglobulin infusions and may experience anaphylaxis on repeat
exposure.
1. Kowalski R, Post D, Schneider M, et al. Immune cell function testing:
an adjunct to therapeutic drug monitoring in transplant patient
Method: In this nephelometry method, human anti-IgA binds to IgA in
management. Clin Transplant. 2003;17:77-88.
the patient sample, forming an insoluble complex. The amount of light
2. Schulick RD, Weir MB, Miller MW, et al. Longitudinal study of in vitro
scattered by this insoluble complex is proportional to the concentration
CD4+ helper cell function in recently transplanted renal allograft
of IgA present in the sample.
patients undergoing tapering of their immunosuppressive drugs.
Transplantation. 1993;56:590-596.
Interpretive Information: Age-related IgA reference ranges are listed
3. Buttgereit F, Burmester GR, Brand MD. Bioenergetics of immune
in Table 4.
functions: fundamental and therapeutic aspects. Immunology Today.
2000;21:192-199.
7.7.2 IgA Subclasses
4. Sottong PR, Rosebrock JA, Britz JA, et al. Measurement of T-
lymphocyte response in whole-blood cultures using newly
Clinical Use: This test is used to diagnose IgA subclass deficiencies
synthesized DNA and ATP. Clin Diagn Lab Immunol. 2002;7:307-311.
and to determine the etiology of recurrent infections.
5. Kowalski RJ, Post DR, Burdick J, et al. Assessing relative risks of
infection and rejection in renal transplant recipients: a meta-analysis
Clinical Background: IgA is the first line of defense for the majority
using the Cylex® ImmuKnow™ Assay. Paper presented at: 3rd
of infections and consists of 2 subclasses. IgA1 is the dominant
International Congress on Immunosuppression; December 8, 2004
subclass, accounting for 80% to 90% of total serum IgA and greater
San Diego, CA. Available at: http://www.cylex.net/abstracts.html.
than half of the IgA in secretions such as milk, saliva, and tears. IgA2 is
Accessed February 25, 2005.
more concentrated in secretions than in blood. IgA2 is more resistant to
6. Zeevi A, Harris C, Zak, et al. The impact of immunosuppression
proteolytic cleavage and may be more functionally active than IgA1.
tapering on T cell function in intestinal transplant recipients as
assessed by the Cylex immune cell function assay. Paper presented
A deficiency in an IgA subclass may result in a compensatory increase
at: The British Transplantation Society 6th annual congress 2003;
in the other IgA subclass. Also, IgA deficiencies are often associated
April 8, 2003; London, England. Available at:
with a deficiency in one or more IgG subclasses leading to an additive
http://www.cylex.net/abstracts.html. Accessed February 25, 2005.
effect in causing recurrent infections. The majority of published studies
7. Kowalski RJ, Post DR, Bentlejewski C, et al. Retrospective analysis of
address total IgA deficiency, not selective deficiency of either subclass.
monitoring small bowel transplant recipients with the Cylex®
Diseases associated with IgA deficiency include ataxia telangiectasia,
ImmuKnow™ assay. Paper presented at: American Society for
common variable immunodeficiency, and recurrent respiratory tract
Histocompatibility and Immunogenetics; October 2, 2004; San
infections. Immunoglobulin replacement therapy may be of benefit.
Antonio, TX. Available at: http://www.cylex.net/abstracts.html.
Accessed February 25, 2005.
Method: Three antibodies—1 anti-IgA, 1 monospecific for IgA1, and 1
8. Rai R, Jing T, Dunbar K, et al. Assessment of cell-mediated immunity
monospecific for IgA2—are utilized. These antibodies bind to the IgA,
in patients with chronic hepatitis C: initial experience with 103
IgA1, and IgA2 antigens, respectively, forming insoluble immune
patients at Johns Hopkins Hospital. Paper presented at: The Liver
complexes that scatter light. The light-scatter is directly related to the
Meeting 55th Annual Meeting of the AASLD; October 29, 2004;
Boston, MA. Available at: http://www.cylex.net/abstracts.html.
Accessed February 25, 2005.
9. ImmuKnow™ [package insert]. Columbia, MD: Cylex Table 4. IgA Reference Ranges in Children and Adults
Incorporated;2003. Available at: http://www.cylex.net/pdf/Immu
KnowPkgInsert50157006.pdf. Accessed February 18, 2005. Age mg/dL
7.7 Immunodeficiencies Cord blood 1-3
1 mo 2-43
7.7.1 IgA, Serum
2-5 mo 3-66
Clinical Use: This test is used to diagnose IgA deficiencies and to 6-9 mo 7-66
determine the etiology of recurrent infections.
10-12 mo 12-75
Clinical Background: IgA is the first line of defense for the majority 1-3 y 24-121
of infections at mucosal surfaces and consists of 2 subclasses. IgA1 is
the dominant subclass, accounting for 80% to 90% of total serum IgA 4-6 y 33-235
and greater than half of the IgA in secretions such as milk, saliva, and 7-9 y 41-368
tears. IgA2, on the other hand, is more concentrated in secretions than
in blood. IgA2 is more resistant to proteolytic cleavage and may be 10-11 y 64-246
more functionally active than IgA1. 12-13 y 70-432
IgA deficiency is the most prevalent isotype deficiency, occurring in 14-15 y 57-300
1/400 to 1/700 individuals. Many patients with IgA deficiency are r16 y 81-463
 183

Section 7 Immunology

Table 5. IgA Subclass Reference Ranges in Children Table 7. Reference Ranges for IgG Subclasses
and Adults
Age, y IgG 1, mg/dL IgG 2, mg/dL IgG 3, mg/dL IgG 4, mg/dL
Age IgA1, mg/dL IgA2, mg/dL Total IgA, mg/dL
0–1 194-842 23-300 19-85 0.5-78
6–11 mo 1–115 0–19 3–101
2–3 315-945 38-225 17-68 1.0-54
1y 3–120 0–23 6–112
4–5 308-945 61-345 10-122 2.0-112
2y 7–132 1–23 11–134
6–7 288-918 44-375 16-85 0.4-98
3y 11–143 1–25 16–155
8–9 432-1020 72-430 13-85 2.0-95
4–7 y 23–175 2–33 31–214
10–11 423-1080 78-355 17-173 2.0-115
8–11 y 33–204 2–37 43–268
12–13 342-1150 100-455 28-125 4.0-136
12–17 y 47–249 4–50 65–356
14–17 315-855 64-495 23-198 11-157
r18 y 46–378 13–91 81–463
Adults 382-929 241-700 22-178 4-86
Source: Quest Diagnostics Nichols Institute Clinical Correlations Department.
antigen concentration and is determined in a nephelometer. Results are
reported as mg/dL.

Interpretive Information: Age-related IgA reference ranges are
provided on the patient report; adult and pediatric reference ranges are
provided for each IgA subclass (Table 5). Very high levels of either IgA
subclass are associated with IgA myeloma. IgA2 deficiencies may be
associated with recurrent sinopulmonary infections.
7.7.3 IgG Subclasses and Total IgG
See Tables 6 and 7 for age-related reference ranges for total IgG and
IgG subclasses, respectively.
cells, and natural killer (NK) cells. T-cells are involved in combating
intracellular infections, cancer cells, and foreign tissue. B-cells give rise
to the humoral immune system that is targeted against bacterial and
viral infections. NK cells play a role in defense against viral infections
and tumors. These lymphocyte subsets can be discerned by the
antigenic properties of cell surface (membrane) markers. For example,
T-cells are CD3 positive, B-cells are CD19 positive, and NK cells are CD3
negative and CD16 and CD56 positive. T-cells are further classified as
helper cells (CD4 positive) or cytotoxic cells (CD8 positive).

7.7.4 Lymphocyte Subset Panels
Clinical Use: These tests are used to determine the immune status of
patients with HIV infection, to monitor antiretroviral and
immunosuppressive therapy, and for the differential diagnosis of
congenital and acquired immune deficiencies.
Clinical Background: The cellular and humoral immune systems are
mediated by distinct lymphocyte classes or subsets including T-cells, B-
Although there is a relatively fixed number and proportion of these
lymphocyte subsets in normal individuals, the absolute number and
proportion is altered in various diseases. For example, in individuals
with human immunodeficiency virus (HIV) infection, the number of CD4
cells and the proportion of CD4 cells relative to CD8 cells vary based on
the stage of disease and therapeutic response. Thus, lymphocyte subset
analysis can provide information regarding the immune status of the
patient, can assist in monitoring therapy, and can help characterize
congenital and acquired immune deficiencies.

Method: In this 4-color multiparametric flow cytometry method, whole

Table 6. Total IgG Reference Ranges blood is first incubated with fluorochrome-conjugated monoclonal
antibodies targeted against the various cell surface antigens. Following
Age Total IgG, mg/dL red cell lysis, the sample is passed through a flow cytometer. Using a
CD45 vs side scatter gating technique, lymphocytes are selected and
Cord blood 553–1360 percentages of the various cell surface markers (CD3, CD4, CD8, CD19,
1 month 213–765 and/or CD16/CD56) are determined. The absolute cell count of each
marker (ie, the number of cells bearing each marker) is calculated using
2–5 months 170–595 the absolute lymphocyte count (obtained from standard hematology
6–9 months 187–765 instrumentation) and the marker percentage. Both the absolute cell
count and the percentage are reported for each marker.
10–12 months 247–910
1–3 years 533–1078 Quest Diagnostics offers the lymphocyte subset panels listed in Table 8.
4–6 years 592–1723 Interpretive Information: In patients with HIV infection, the CD4+
7–9 years 673–1734 T-cell level and the CD4/CD8 ratio typically decline shortly after
seroconversion. CD4+ levels above 500 cells/mm3 are usually associated
10–11 years 821–1835 with asymptomatic infection, whereas levels <200 cells/mm3 are
12–13 years 893–1823 consistent with a transition to AIDS. CD4 levels rise in response to
effective anti-retroviral therapy. Aside from a history of AIDS-defining
14–15 years 842–2013 illness or severe HIV-related symptoms, the CD4 cell count is generally
r16 years 694–1618 the most important factor in determining when to initiate therapy.
Source: Quest Diagnostics Nichols Institute Clinical Correlations Department.
184

Section 7 Immunology

Table 8. Components of the Lympocyte Subset Panels

WBC Total Lymph Total B Total T T Lymph Subsets Helper/Cytotoxic Natural Killer
Lymphocyte Subset Panel 1 • • • • • • •
Lymphocyte Subset Panel 2 • • • • • •
Lymphocyte Subset Panel 3 • • • • •
Lymphocyte Subset Panel 4 • • • •
Lymphocyte Subset Panel 5 • • •
Natural Killer Cells1 •
T & B Cells, Total • • • •

7.8 Neurologic Diseases
7.8.1 Myasthenia Gravis and Acetylcholine Receptor
Antibodies
Clinical Use: Acetylcholine receptor antibody tests are useful for the
differential diagnosis of myasthenia gravis (MG)-like muscle weakness
and for monitoring therapeutic response in patients with MG.
Clinical Background: MG is an autoimmune disease characterized by
skeletal muscle weakness due to defective neuromuscular transmission.
Onset may be gradual or acute following viral infection or pregnancy.
Treatment options include anticholinesterase drugs, thymectomy,
corticosteroids, plasmapheresis, and immunosuppressive therapy.
Approximately 90% of MG patients demonstrate elevated serum levels
of acetylcholine receptor antibodies. These antibodies cause a loss of
receptors, complement-mediated focal lysis of the postsynaptic
membrane, and partial or complete inhibition of receptor function. Three
types of antibodies may be involved: binding, blocking, and modulating.
Binding antibodies are detected in approximately 90% of MG patients
with generalized disease and approximately 70% of MG patients with
only ocular muscle involvement. Modulating antibodies are found in
about the same frequency as binding antibodies; however,
approximately 8% of patients have only 1 of the 2 tests positive.
Blocking antibodies are detectable in about 52% of patients with
generalized disease and about 30% of patients with ocular MG.
Blocking antibodies are found in the absence of binding antibodies in
approximately 1% of MG patients. Guidelines for ordering binding,
blocking, and modulating tests can be found in Table 9.
Modulating antibody (test code 26474X): This radiobinding assay
employs muscle-type acetylcholine receptors in live human sarcoma
cells (TE671). Modulating antibodies in the patient serum bind to the
receptors followed by binding of any remaining non-modulated
acetylcholine receptor binding sites to radiolabeled bungarotoxin. The
bound fraction is separated from the free radiolabeled bungarotoxin by
repeated washing and quantified. Binding specificity is determined by
pre-addition of the carbamylcholine agonist in parallel cultures. Note
that this test detects both blocking and modulating antibodies.
The blocking and modulating antibody tests were developed and their performance
characteristics have been determined by Quest Diagnostics Nichols Institute. They have
not been cleared or approved by the U.S. Food and Drug Administration. The FDA has
determined that such clearance or approval is not necessary. Performance characteristics
refer to the analytical performance of the test.
Interpretive Information: Elevated levels of receptor antibodies are
found in 80% to 86% of patients with MG; levels are higher in more
severe cases. Husain et al found that binding antibodies were present in
82% of patients with moderate/severe disease, 69% of patients with
mild, generalized disease, and 59% of patients with ocular myasthenia.
Seventy-nine percent of all the patients had binding antibodies; 57%
had modulating antibodies; and 23% had blocking antibodies.
References
1. Conroy WG, Saedis MS, Lindstrom J. TE671 cells express an
abundance of a partially mature acetylcholine receptor a subunit
which has characteristics of an assembly intermediate. J Biol Chem.
1990;265:21642-21651.
2. Drachman DB, Adams RN, Josifek LF, et al. Functional activities of

Methods
Binding antibody (test code 206X): Patient specimens and
Table 9. Myasthenia Gravis Test Selection Guide
calibrators are incubated with detergent-solubilized, fetal and adult
Binding Blocking Modulating
acetylcholine receptors (AChR) that are labeled with 125I-alpha-
Applications Antibody Antibody Antibody
bungarotoxin. The resulting labeled AChR/autoantibody complex is then
precipitated with anti-human IgG. The amount of radioactivity in the Initial screening x
precipitate is directly proportional to the amount of antibody present.
Confirmation of diagnosis x x1
Blocking antibody (test code 34459X): Patient specimens and Monitoring therapeutic x
calibrators are incubated with detergent-solubilized, fetal and adult response and disease
acetylcholine receptors (AChR), and 125I-alpha-bungarotoxin. progression
Acetylcholine blocking antibody in the patient sample competes with
Recent onset of MG (< 1 year) x
the 125I-alpha-bungarotoxin for a limited number of binding sites on the
receptor. The resulting AChR/blocking antibody complexes are then Mild muscle weakness only x
precipitated with concanavalin-A Sepharose®´. The amount of
Ocular muscle weakness only x
radioactivity in the precipitate is inversely proportional to the amount of
1
antibody present. When binding antibody is negative.

Section 7
autoantibodies to acetylcholine receptors and the clinical severity of
myasthenia gravis. N Engl J Med. 1982;307:769-775.
3. Howard FM Jr, Lennon VA, Finley J, et al. Clinical correlations of
antibodies that bind, block, or modulate human acetylcholine
receptors in myasthenia gravis. Ann NY Acad Sci. 1987;505:526-
538.
4. Husain AM, Massey JM, Howard JF, et al. Acetylcholine receptor
antibody measurements in acquired myasthenia gravis. Diagnostic
sensitivity and predictive value for thymoma. Ann NY Acad Sci.
1998;841:471-474.
5. Lennon VA, Jones G, Howard F, at al. Autoantibodies to
acetylcholine receptors in myasthenia gravis (letter). N Engl J Med.
1983;308:402-403.
6. Levinson AI, Wheatley LM. Myasthenia gravis. In: Rich R, ed.
Clinical Immunology Principles and Practice. St. Louis, MO: Mosby
Publishers; 1996.
7. Mittag TW. Antibodies to receptor proteins: the significance of
nicotinic acetylcholine receptor antibodies. J Clin Immunoassay.
1983;6:S25-S32.
8. Pachner AR. Anti-acetylcholine receptor antibodies block
bungarotoxin binding to native human acetylcholine receptor on the
surface of TE671 cells. Neurology. 1989;39:1057-1061.
9. Penn AS, Richman DP, Ruff RL, et al, eds. Myasthenia Gravis and
Related Disorders: Experimental and Clinical Aspects. Conference
proceedings. Washington, DC, April 12-15, 1992. Ann N Y Acad Sci.
1993;681:1-622.
10. Vincent A. Acetylcholine receptor autoantibodies. In:
Autoantibodies. Peter JB, Shoenfeld Y, eds. Amsterdam: Elsevier
Science;1996:1-9.
7.8.2 Peripheral Neuropathy
Introduction: Peripheral neuropathy usually presents with weakness
and sensory loss or pain in the arms and legs. It is estimated that over 10
million people in the United States suffer from neuropathy, the incidence
of which increases with age. Neuropathies are classified according to
cause (endocrine, metabolic, nutritional, toxic, etc) or clinical
presentation (sensory, motor, autonomic, mixed sensory and motor,
mononeuritis, mononeuritis multiplex, etc). Due to the diverse causes of
neuropathy, laboratory testing is invariably required for diagnosis or
etiologic identification. The following is a brief review of the known
causes of acquired peripheral neuropathies and the laboratory tests
available for their evaluation and diagnosis (see also Table 10).
Endocrine and Metabolic Causes: Endocrine causes of neuropathy
include diabetes mellitus and hypothyroidism. The most common cause
of neuropathy is diabetes mellitus, which accounts for approximately
30% of cases. Approximately 10% of individuals with diabetes manifest
overt clinical symptoms of neuropathy, and in some cases neuropathy is
the presenting complaint. The most frequent presentation is distal
sensory polyneuropathy, but patients may also present with small fiber
neuropathy (commonly caused by glucose intolerance), sensorimotor
neuropathy, amyotrophy, mononeuritis, or mononeuritis multiplex.
Diagnostic tests for diabetes mellitus include blood glucose and glucose
tolerance assays. Glycated hemoglobin (ie, hemoglobin A1c) is also
useful for monitoring diabetic control. Hypothyroidism presents
predominantly as a sensory neuropathy and can be diagnosed with
thyroid function tests including TSH and T4.
Metabolic causes of neuropathy include renal failure and porphyria.
Renal failure, indicated by elevated serum creatinine and BUN, is
associated with a predominantly sensory axonal neuropathy. Porphyria
is associated with an acute, predominantly motor, neuropathy and is
detected by urine porphyrin analysis.
185
Immunology
Nutritional and Toxic Causes: Vitamin deficiency (B1, B6, B12, and E)
and folate deficiency, as well as excessive intake of vitamin B6, can
cause peripheral neuropathy. B12 deficiency is associated with
achlorhydria or pernicious anemia and sometimes with anti-parietal cell
or intrinsic factor antibodies. Vitamin E deficiency is often associated
with ataxia. Measurement of serum vitamin levels is useful in making
the diagnosis.
Peripheral neuropathy may also be caused by several heavy metals.
Lead toxicity is associated with motor neuropathy, whereas arsenic and
mercury cause sensory neuropathy. The 24-hour urine heavy metal test
is the most useful test for diagnosis of heavy metal toxicity.
Immune-Mediated Peripheral Neuropathies: The immune system
mediates peripheral neuropathies in autoimmune diseases, in systemic
diseases such as vasculitis and primary amyloidosis, and in
paraneoplastic syndromes. Autoimmune neuropathies are usually
divided into Guillain-Barre syndrome, variants that are of acute onset
and self-limiting, and variants that are chronic and follow a progressive
or relapsing course. In recent years, several novel glycoconjugate
antigens, both glycoproteins and glycolipids, have been identified as
putative targets for many of these autoimmune polyneuropathies. In
general, IgG anti-glycoconjugate autoantibodies have been associated
with acute neuropathies, whereas IgM autoantibodies are associated
with the chronic neuropathic syndromes.
Acute immune-mediated neuropathies include the Guillain-Barre
syndrome (GBS; acute inflammatory demyelinating polyneuropathy),
acute motor axonal polyneuropathy, acute sensory polyneuropathy,
acute autonomic polyneuropathy, and the Miller-Fisher syndrome in
which the extra-ocular muscles are affected. Increased titers of IgG
anti-GM1 or GD1a ganglioside antibodies have been associated with
GBS and acute motor axonal neuropathy, whereas increased IgG anti-
GQ1b ganglioside antibodies are closely associated with the Miller-
Fisher syndrome. Tests for these autoantibodies are useful aids in the
evaluation of patients suspected of having these syndromes.
Chronic immune-mediated polyneuropathies in which the peripheral
nerves are selectively affected include chronic inflammatory
demyelinating polyneuropathy (CIDP), demyelinating polyneuropathy
associated with IgM anti-MAG (myelin-associated glycoprotein)
antibodies or anti-SGPG (sulfoglucuronyl paragloboside) antibodies,
multifocal motor neuropathy associated with IgM anti-GM1 or GD1a
antibodies, and sensory polyneuropathy associated with IgM anti-
sulfatide antibodies or anti-GD1b or disialosyl ganglioside antibodies.
Other neuropathies may be associated with anti-GM2 antibodies. The
presence of increased titers of these autoantibodies helps diagnose an
immune-mediated polyneuropathy that may respond to specific
immunotherapy. Some of these autoantibodies also occur as IgM
monoclonal gammopathies in patients with non-malignant monoclonal
gammopathies or in association with B-cell lymphoproliferative
disorders (see paraneoplastic syndromes below).
Peripheral neuropathy can also occur in patients with rheumatologic
diseases or systemic vasculitis, including systemic lupus erythematosus,
Sjögren’s syndrome, rheumatoid arthritis, and Wegener’s
granulomatosis. These can be diagnosed with the aid of tests for anti-
nuclear antibodies (ANA), extractable nuclear antigen antibodies (ENA)
or anti-SSA-Ro/SSB-La antibodies, rheumatoid factor and cyclic
citrullinated peptide, and anti-neutrophilic cytoplasm antibodies (ANCA),
respectively. Polyarteritis nodosa is another disease that is associated
with vasculitis of the peripheral nerves, sometimes associated with
hepatitis B. Chronic hepatitis C virus infection is often associated with
cryoglobulinemia, in which deposition of cryoglobulin-containing
186

Section 7 Immunology

Table 10. Acquired Peripheral Neuropathies and Associated Laboratory Tests

Cause of Neuropathy Laboratory Test

Endocrine and Metabolic Diseases
Diabetes Blood glucose, glycated hemoglobin
Hypothyroidism Thyroid function
Renal failure BUN, serum creatinine
Porphyria Urine porphyrins
Nutritional Diseases
Vitamin B12 deficiency CBC, serum B12
Vitamin B6 deficiency Serum B6
Vitamin B6 toxicity Serum B6
Vitamin B1 deficiency Serum B1
Vitamin E deficiency Serum vitamin E
Folate deficiency Serum folate
Heavy Metal Toxicity
Lead Urine heavy metals
Arsenic Urine heavy metals
Mercury Urine heavy metals
Autoantibodies to Peripheral Nerve Antigens
Anti-Asialo-GM-1 ganglioside neuropathy Asialo-GM-1 antibody
Anti-GD1a ganglioside neuropathy GD1a antibody
Anti-GD1b ganglioside neuropathy GD1b antibody
Anti-GM-1 ganglioside neuropathy GM-1 antibody
Anti-GM-2 ganglioside neuropathy GM-2 antibody
Anti-GQ1b ganglioside neuropathy GQ1b antibody
Anti-MAG/SGPG neuropathy MAG/SGPG antibody
Anti-sulfatide neuropathy Sulfatide antibody
Rheumatological & Vasculitic Disease
Polyarteritis Cryoglobulins, immune complexes, CH50, hepatitis B and C serology, parvovirus serology,
HIV-1 serology
Systemic lupus erythematosus ANA, dsDNA antibodies
Rheumatoid arthritis Cyclic citrullinated peptide antibody, rheumatoid factor
Wegener’s granulomatosis Anti-neutrophil cytoplasmic antibody (ANCA)
Sjögren’s syndrome SS-A/Ro antibody, SS-B/La antibody
Celiac disease Gliadin, transglutaminase, and endomysial antibodies
Paraneoplastic Disease
Lung cancer Hu, Ri, and Yo antibodies
Monoclonal gammopathy Immunoglobulin profile, IFE, serum & urine
Myeloma Immunoglobulin profile, IFE, serum & urine
Macroglobulinemia Immunoglobulin profile, IFE, serum & urine
Chronic lymphocytic leukemia Immunoglobulin profile, IFE, serum & urine
Primary Amyloidosis Immunoglobulin profile, IFE, serum & urine
Kappa/lambda light chains, free with ratio, serum
Infectious and Inflammatory Disease
AIDS HIV-1 antibody
Lyme disease Borrelia burgdorferi antibodies (total, IgG, IgM)
Herpes zoster Varicella zoster antigen and antibodies (IgG, IgM)
Cytomegalovirus CMV antigen and antibodies (IgG, IgM)
Hepatitis B HBs antigen, HBc antibodies (IgG, IgM)
Hepatitis C Hepatitis C antibody
Sarcoidosis Angiotensin converting enzyme

Section 7
immune complexes causes small- and medium-size vessel disease. In
immunosuppressed patients, vasculitis can also result from viral
infections such as parvovirus. In viral infections, circulating immune
complexes, cryoglobulins, or decreased complement levels (CH50) may
be present. Vasculitic neuropathies typically present as mononeuritis,
mononeuritis multiplex, or polyneuritis, although Sjögren’s syndrome,
which is associated with anti-SSA-Ro/SSB-La antibodies, sometimes
presents with sensory neuropathy or ganglioneuritis. Celiac disease, an
inflammatory disease of the gut that results from gluten intolerance,
may be associated with a sensory neuropathy, sometimes with anti-
ganglioside antibodies. It can be recognized by the presence of gliadin,
transglutaminase, or endomysial antibodies. Endomysial antibodies
commonly, but not always, react with transglutaminase.
Other neuropathies that are, in part, mediated by the immune system
are those associated with neoplasia, monoclonal gammopathies, and
primary amyloidosis (see below).
Paraneoplastic Neuropathies, Monoclonal Gammopathies, and
Primary Amyloidosis: Paraneoplastic syndromes are thought to be
caused by indirect effects of tumors, usually via immune or metabolic
mechanisms. Several paraneoplastic neuropathic syndromes have been
recognized. One of these is a predominantly sensory neuropathy that
occurs in patients with carcinoma of the lung in association with anti-
Hu antibodies, which serve as a marker for the disease. Neuropathy is
also associated with IgM monoclonal gammopathies in patients with
Waldenstrom’s macroglobulinemia or B-cell leukemia or lymphoma and
with IgG or IgA monoclonal gammopathies in myeloma. The monoclonal
IgMs in patients with neuropathy frequently exhibit reactivity to one of
the glycoconjugate antigens in peripheral nerve (see above). In
myeloma, the monoclonal IgG or IgA antibodies do not have
demonstrable autoantibody activity, but in osteosclerotic myeloma, in
which 50% of the patients have neuropathy, there are highly elevated
levels of serum vascular endothelial growth factor (VEGF). Monoclonal
gammopathy of any isotype, or light chain disease, can also be
associated with primary amyloidosis in which the amyloid deposits
contain fragments of the monoclonal light chains. The same neuropathic
syndromes can also be associated with non-malignant IgM, IgG, or IgA
monoclonal gammopathies, or monoclonal gammopathies of unknown
significance (MGUS). Laboratory tests that are useful for detecting
monoclonal gammopathies include an immunoglobulin profile (IgA, IgG,
IgM) and immunofixation electrophoresis of serum and urine.
Measurement of free kappa and lambda light chain is useful for
detecting light chain disease.
Polyneuropathies Caused by Infections or Inflammatory
Diseases: Several infectious diseases also cause peripheral
neuropathy. Human immunodeficiency virus-1 (HIV-1) infection is
typically associated with a distal sensory neuropathy. Lyme disease can
cause mononeuritis multiplex or diffuse polyneuropathy.
Cytomegalovirus (CMV) infection of nerves causes an ascending
polyradiculopathy. Herpes zoster infection can cause radiculopathy
(shingles). Hepatitis B or C infections, or parvovirus infection in
immunocompromised patients, can be associated with polyarteritis
nodosa and vasculitic neuropathy. Sarcoidosis can also cause a
multifocal or diffuse neuropathy. Serologic testing for suspected
infections or for angiotensin converting enzyme (ACE) levels in
sarcoidosis is helpful in the evaluation and diagnosis of patients with
neuropathy.
References
1. Asbury AK, Thomas PK. Peripheral nerve disorders 2. Butterworth
Heinemann Ltd, 1995.
187
Immunology
2. Bollensen E, Schipper HI, Steck AJ. Motor neuropathy with activity
of monoclonal IgM antibody to GD1a ganglioside. J Neurol.
1989;236:353-355.
3. Chiba A, Kusunoki S, Obata H, et al. Serum anti-GQ1b antibodies
are associated with ophthalmoplegia in Miller-Fisher syndrome and
Guillaine-Barre syndrome. Neurology. 1993;43:1911-1917.
4. Duane GC, Farrer RG, Dalakas MC, et al. Sensory neuropathy
associated with immunoglobulin M to GD1b ganglioside. Ann
Neurol. 1992;31:683-685.
5. Dyck PJ, Thomas PK, Griffin JW, et al. Peripheral Neuropathy, 3rd
Ed. Philadelphia: WB Saunders; 1993.
6. Kelly JJ Jr, Kyle RA, Miles JM, et al. The spectrum of peripheral
neuropathy in myeloma. Neurology. 1981;31:24-31.
7. Kelly JJ Jr, Kyle RA, Obrien PC, et al. The prevalence of monoclonal
gammopathy in peripheral neuropathy. Neurology. 1981;31:1480-
1483.
8. Kinsella LJ, Lange DJ, Trojaborg W, et al. The clinical and
electrophysiological correlates of elevated anti-GM1 antibody titers.
Neurology. 1994;44:1278-1282.
9. Kyle RA, Greip PR. Amyoloidosis (AL): clinical and laboratory
features of 229 cases. Mayo Clin Proc. 1983;58:665-683.
10. Latov N. Pathogenesis and therapy of neuropathies associated with
monoclonal gammopathies. Ann Neurol. 1995;37(S1):S32-42.
11. Latov N, Steck AJ. Neuropathies associated with glycoconjugate
antibodies and IgM monoclonal gammopathies. In: Asbury A,
Thomas PK (eds). Peripheral Nerve Disorders II. Boston:
Butterworth-Heinemann;1995:153-173.
12. Ogino M, Orazio N, Latov N. IgG anti-GM1 antibodies from patients
with acute motor neuropathy are predominantly of the IgG1 and
IgG3 subclasses. J Neuroimmunol. 1995; 58:77-80.
13. Pestronk A, Li F, Griffin J, et al. Polyneuropathy syndromes
associated with serum antibodies to sulfatide and myelin
associated glycoprotein. Neurology. 1991; 41:357-362.
14. van den Berg LH, Hays AP, Nobile-Orazio E, et al. Anti-MAG and
anti-SGPG antibodies in neuropathy. Muscle Nerve. 1996;19:637-
643.
7.9 Rheumatic and Related Systemic
Diseases
7.9.1 Rheumatic and Related Disease Screening
Laboratory work-up for patients with suspected rheumatic disease often
begins with an antinuclear antibody (ANA) screen (Figure 2). A positive
screen can be followed by various antibody tests as shown in the
Figure. Primary disease associations for these second-line antibody
tests are shown in Tables 11-13.
7.9.2 Rheumatoid Arthritis: Laboratory Markers for Diagnosis
and Prognosis
Clinical Background: Rheumatoid arthritis (RA) is an autoimmune
disease that affects mainly the synovial membranes and articular
structures and is characterized by chronic, systemic inflammation
involving multiple joints. Small joints in the hands and feet are usually
affected first, followed by the larger joints. Patients with RA have
periodic flare-ups that can lead to irreversible joint destruction.
Systemic effects may include damage to organs such as the heart and
lungs.
About 1% of the US population has RA, with prevalence being 2 to 3
times higher in women than men. Although the cause of RA remains
unknown, the increased risk in family members of patients with RA
suggests a genetic component. Environmental or hormonal factors may
188
Section 7
be involved in perpetuating the inflammatory process and joint
destruction.
Treatment with disease-modifying anti-rheumatic drugs (DMARDs) can
often ameliorate the disease. Because bone destruction can occur early,
with many RA patients showing radiographic evidence of bone
destruction in the first 2 years of disease,1 therapy should be initiated
promptly to minimize irreversible joint damage. American College of
Rheumatology (ACR) criteria for classification of RA require that 4 of the
following 7 conditions be present2:
• Morning stiffness in and around joints lasting r1 hour (present r6
weeks)
Immunology
• Physician-documented arthritis involving r3 joint areas
simultaneously (present r6 weeks)
• Arthritis of the proximal interphalangeal, metacarpophalangeal, or
wrist joints (present r6 weeks)
• Symmetric involvement of joint areas (present r6 weeks)
• Rheumatoid nodules
• Positive serum rheumatoid factor (RF)
• Radiographic evidence of erosions or periarticular osteopenia in hand
or wrist joints
Although these criteria demonstrate good sensitivity (91%-94%) and
specificity (89%) in differentiating patients with established RA from
 189

Section 7 Immunology

Table 11. Systemic Lupus Erythematosus (SLE) and Mixed Table 13. CREST Syndrome and Neurologic SLE
Connective Tissue Disease
Test CREST Syndrome Neurologic SLE
Systemic Lupus Mixed Connective Centromere antibody + –
Test Erythematosus Tissue Disease
Ribosomal P antibody – +
dsDNA antibodya + –
SLE, systemic lupus erythematosus.
Chromatin antibodya + –
b
Sm antibody + – primarily detects IgM RF. IgM RF, as well as IgA and IgG RF, can also be
Sm/RNP antibody + + (high titer) measured individually with specific immunoassays.
RNP antibody + + (high titer) Until the introduction of the anti-CCP assay (see below), RF was
a
Highly sensitive for SLE. considered the most useful laboratory indicator of RA. The reported
b
Highly specific for SLE. sensitivity of RF for RA generally ranges from 60% to 90%, but
specificity is relatively low (~70%-80%). Patients with other rheumatic
control subjects, they are less robust for classifying patients with early diseases or conditions that present with polyarthritis often have positive
inflammatory polyarthritis who may have mild symptoms and signs.3 RF results. The presence of IgG or IgA RF, or both, in patients with IgM
RF and joint disease markedly increases the likelihood that the patient
The recently developed anti-cyclic citrullinated peptide (anti-CCP) assay, has RA; these combinations are not typically found in patients with
in combination with RF, may help establish a diagnosis in the early other rheumatic diseases that may be accompanied by IgM RF. However,
stages of RA when treatment is most effective in preserving function. IgA and IgG RF are not highly sensitive and are not widely used in the
diagnosis of RA.
This Clinical Focus discusses laboratory tests available to assist
physicians in differentiating between RA and other conditions that Anti-CCP has emerged as a sensitive (~55%-80%) and highly specific
present with polyarticular arthritis, and in monitoring the disease (~90%-98%) marker of RA (Table 15).4,5,6 The data presented in this
course. clinical focus are based on studies using the second-generation anti-
CCP immunoassay, which has improved sensitivity and equivalent
Individuals Suitable for Testing include those with symptoms of specificity relative to first-generation assays.7 In most side-by-side
arthritis not attributed to other conditions and those with established comparisons, anti-CCP is as sensitive as and more specific than RF in
RA. various clinical situations (Table 15).

Test Availability: Table 14 provides a listing of tests that may be
useful in the diagnosis, assessment of prognosis, and follow-up of RA.
Test Selection: Because of the low overall prevalence of RA, these
assays are not suitable for routine screening of asymptomatic
individuals.
The combination of anti-CCP and RF appears to provide greater
sensitivity than either assay alone (Table 15)8,9 and is therefore useful
in the diagnostic work-up of suspected RA (Figure 3). Notably, in a
study of 561 patients with suspected RA, anti-CCP was detected in 30
of 87 (34%) RA patients who had negative results for all 3 RF isotypes
(IgM, IgA, and IgG by ELISA).8

Diagnosis RF and especially anti-CCP can be detected years before the onset of
Diagnosis of RA relies primarily on patient history and radiographic symptoms. In studies of blood donors, the sensitivity of anti-CCP
evidence of joint damage. Laboratory testing can help differentiate RA detection for future development of RA ranged from 29% to 37%, with
from other conditions that manifest with polyarthritis and can be a specificity of r98%.10,11,12 Sensitivity increased as the time to
especially useful early in the disease course for establishing diagnosis disease onset decreased. Anti-CCP testing may also predict a future
and prognosis. diagnosis of RA in patients with a diagnosis of undifferentiated
arthritis (UA).13
RF is the most widely used laboratory marker of RA and is the only one
included in the current (1988) ACR classification criteria.2 RF titer is Prognosis
most often assessed with latex fixation/immunoturbidimetry, which RA has a variable clinical course. Some patients have self-limiting
disease whereas others develop progressive joint damage. Predicting
which patients will experience progressive disease may help direct
aggressive treatment with DMARDs to patients who need it most, and
Table 12. Sjögren’s Syndrome, Scleroderma, and spare others from unnecessary exposure to the potential adverse effects
of these drugs.
Polymyositis
The presence of RF is generally associated with more severe disease
Sjögren’s and greater risk of progressive joint erosion. Very high titers may be
Test Syndrome Scleroderma Polymyositis associated with more severe joint disease, Felty’s syndrome, rheumatoid
SS-A antibody + – – nodules, peripheral neuropathy, and skin ulcers. IgA and IgG RF
positivity early in the course of RA is also predictive of more severe
SS-B antibody + – – disease and likelihood of radiographic progression.20,19
Scl-70 antibody – + –
Anti-CCP testing is also useful for determining the prognosis of RA,
Jo-1 antibody – – + being predictive of disease progression at 3 to 10 years after disease
190

Section 7 Immunology

Table 14. Tests Available to Support Diagnosis, Prognosis, and Follow-up of Rheumatoid Arthritis

Assay Method Clinical Use

Rheumatoid Factor (RF) Latex fixation/immunoturbidimetry Most widely used test to assist in diagnosis and determining
prognosis of RA; detects primarily IgM RF
Rheumatoid Factor (IgA) ELISA Provides added specificity when used in combination with other
RF or anti-CCP assays
Rheumatoid Factor (IgG) ELISA Provides added specificity when used in combination with other
RF or anti-CCP assays
Anti-Cyclic Citrullinated Peptide (anti-CCP) ELISA Assist in diagnosis and determining prognosis of RA—more
specific than RF
Rheumatoid Arthritis, Diagnostic Panel
RF Latex fixation/immunoturbidimetry Provides additional diagnostic and prognostic value relative to
Anti-CCP ELISA either assay alone
Erythrocyte Sedimentation Rate (ESR) Modified Westergren Assess disease activity
C-Reactive Protein (CRP) Immunoturbidimetry Assess disease activity
ELISA, enzyme-linked immunosorbent assay

onset. In most studies, anti-CCP positivity at baseline correlates with
poor prognosis in terms of radiographic and functional outcome.20,21 In a
study of patients with early arthritis, baseline values of anti-CCP (67%)
and RF (69%) showed similar sensitivity for prediction of radiographic
progression at 5 years, but anti-CCP showed greater specificity (56% vs
24%).20 Combining RF with anti-CCP results appeared to help in
prediction: RF+/anti-CCP+ patients had greater progression than
RF+/anti-CCP– and RF–/CCP– patients at 5 years.
Monitoring Disease Course
CRP and ESR are both acute-phase markers of inflammation sometimes
used to monitor RA disease activity. CRP is produced by the liver in

Table 15. Performance of the Second-generation Anti-CCP Assay and RF for Diagnosis of Rheumatoid Arthritis

Sensitivity (%) Specificity (%)

Anti- Anti- Anti- Anti-
Anti- CCP CCP Anti- CCP CCP
Reference Patients, N CCP RF and RF or RF CCP RF and RF or RF
Blood Donors (Before Clinical Manifestations)
Berglin et al12* 59 37 22 – – 98 94 – –
Rantapaa-Dahlqvist et al10* 83 34 20 15 – 98 95 99 –
Suspected Rheumatic Disease
Sauerland et al5 700 74 70 – – 94 70 – –
Vallbracht et al8* 561 64 66 – 79 97 82 – 81
Greiner et al14 333 81 86 – 90 98 82 – 81
De Rycke et al15 315 75 78 – – 97 81 – –
Variety of Rheumatic Diseases
Lee et al6 249 66 72 57 81 90 80 91 80
RA at Various Stages
Dubucquoi16* 140 64 60 – 75 96 ~70 – –
Suzuki et al17 549 88 70 – – 89 82 – –
Early (< 1 year) RA
Forslind et al18 379 55 61 – – – – – –
Suzuki et al17 91 84 78 – – – – – –
Long-standing (r4 yr) RA
De Rycke et al.15† 180 66 25 – – – – – –
CCP, cyclic citrullinated peptide; RF, rheumatoid factor; RA, rheumatoid arthritis.
RF analysis by latex fixation/immunoturbidimetry unless otherwise indicated; –, data not available.
*IgM RF by ELISA.
†
Cut-points were adapted to yield r98.5% specificity.

Section 7
Rheumatoid Arthritis, Diagnostic Panel
(RF and IgG anti-CCP)
Anti-CCP+ Anti-CCP+
RF+ RF-
Consistent with RA; Frequently present
predictive of progressive in early RA; predictive
joint destruction of progressive joint
destruction
Likelihood of RA
Figure 3. Flowchart for initial laboratory testing in patients with polyarthritis. Diagnosis should be based on clinical, radiographic, and laboratory
findings. See Interpretive Information section for detailed discussion. The arrow indicates the relative likelihood of RA with different combinations of
results; however, even patients with negative results on both tests may have RA.
response to tissue injury, infection, and inflammation. Levels increase
during periods of heightened RA disease activity, but elevations may
also reflect inflammation due to other causes, such as infection or
injury. The ESR typically rises 24 to 48 hours after an inflammatory
stimulus and returns to normal levels gradually thereafter. ESR
measurement may help assess disease activity when other clinical and
laboratory studies yield equivocal results.22
Supportive Testing
A complete blood count with white blood cell differential can help
document the mild anemia, leukocytosis, and other hematologic
abnormalities sometimes associated with RA. More severe anemia may
reflect gastrointestinal bleeding resulting from steroidal and non-
steroidal anti-inflammatory drugs. Urinalysis typically yields normal
results. Liver and kidney function should be assessed before starting
therapy with DMARDs, to establish baseline values, and at intervals
thereafter.
Test Interpretation: The result of each assay should be evaluated in
conjunction with clinical and radiographic findings and other serological
test results.
RF
Positive RF results are suggestive of RA, but the low specificity
precludes a definitive diagnosis. Positive results are also common in
patients with other rheumatic diseases and conditions that can mimic
RA (Table 16). Negative results are consistent with conditions other than
RA but do not rule out RA. Because RF-negative patients may
seroconvert, follow-up testing at intervals during the first year of
disease may be useful if the initial result is negative. IgA and IgG are
both highly specific for RA and are associated with greater disease
severity and likelihood of progression. Because of the relatively low
sensitivity of IgA and IgG RF, negative results do not indicate absence of
RA.
Anti-CCP
Positive anti-CCP results are highly specific for RA and are associated
with an aggressive disease course. Although this assay is generally
more specific than RF, patients with other rheumatic diseases may
191
Immunology
Anti-CCP- Anti-CCP-
RF+ RF-
More suggestive RA unlikely; observed
of other rheumatic in only 10% to 20%
diseases or infection, of patients with RA
but consistent with RA
rarely have elevated titers (Table 16). Negative results do not rule out a
diagnosis of RA but suggest that other rheumatic diseases may be
responsible for the patient’s symptoms.
Anti-CCP and RF (Figure 3)
The combination of a positive anti-CCP and IgM RF result is highly
specific for RA (~90%-100%) and is associated with an aggressive
disease course. However, this profile may be found in some patients
with other rheumatic diseases, such as SLE, scleroderma, and psoriatic
arthritis. Patients with positive anti-CCP and negative RF results are
also likely to have RA. RA is less likely in patients with a positive RF
and negative anti-CCP result, but cannot be ruled out. Negative results
on both assays indicate a very low likelihood of RA, but do not exclude
the diagnosis. RF is also associated with extra-articular manifestations
such as rheumatoid nodules, whereas anti-CCP is not. In RF-positive
patients with chronic HCV or other infections associated with
polyarticular arthritis, a positive anti-CCP result suggests a likely
diagnosis of RA; HCV patients with cryoglobulinemia typically have
negative anti-CCP results.23
CRP and ESR
Elevated levels of ESR and CRP in patients with RA suggest heightened
disease activity. However, elevations may also be due to other
inflammatory conditions. Normal ESR and CRP results indicate relatively
low disease activity. In patients with discordant ESR and CRP results,
CRP levels may be the more reliable marker of RA disease activity.24
Recent evidence suggests that CRP levels during early RA may be
predictive of long-term (10-year) disease progression.20
References
1. Fuchs HA, Kaye JJ, Callahan LF, et al. Evidence of significant
radiographic damage in rheumatoid arthritis within the first 2 years
of disease. J Rheumatol. 1989;16:585-591.
2. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism
Association 1987 revised criteria for the classification of rheumatoid
arthritis. Arthritis Rheum. 1988;31:315-324.
3. Harrison BJ, Symmons DP, Barrett EM, Silman AJ. The performance
of the 1987 ARA classification criteria for rheumatoid arthritis in a
population based cohort of patients with early inflammatory
192

Section 7 Immunology

Table 16. Reactivity of Rheumatoid Factor (RF) and Anti-Cyclic Citrullinated Antibody (Anti-CCP) Assays in Various Disorders

Percent Positive*
Anti-CCP
Population Sample Size, n RF (r20 Units)
RA5 231 70 74
8†
Healthy individuals/blood donors 154 7 1
25
SLE 201 13 6
26
Scleroderma 86 NA 12
27†
Primary Sjögren’s syndrome 134 59 8
28
Juvenile RA
Polyarticular onset 77 18 13
Pauciarticular onset 139 7 2
Polymyalgia rheumatica29 49 7 0
Mixed connective tissue disease/vasculitis8† 103 43 7
Psoriatic arthritis30 160 11 7
5
Non-inflammatory myalgia 52 19 8
5
Osteoarthritis 40 13 8
4‡
Lyme disease 20 NA 15
23
Hepatitis C infection (no cryoglobulinemia) 50 44 0
23
HCV-related cryoglobulinemia 29 76 7
NA, not available.
*The percentages shown are based on the specific references cited.
†
RF tested by IgM RF ELISA.
‡
First-generation anti-CCP assay.

polyarthritis. American Rheumatism Association. J Rheumatol.
1998;25:2324-2330.
4. Bizzaro N, Mazzanti G, Tonutti E, et al. Diagnostic accuracy of the
anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem.
2001;47:1089-1093. Erratum in: Clin Chem. 2001;47:1748.
5. Sauerland U, Becker H, Seidel M, et al. Clinical utility of the anti-
CCP assay: experiences with 700 patients. Ann N Y Acad Sci.
2005;1050:314-318.
6. Lee DM, Schur PH. Clinical utility of the anti-CCP assay in patients
with rheumatic diseases. Ann Rheum Dis. 2003;62:870-874.
7. van Gaalen FA, Visser H, Huizinga TW. A comparison of the
diagnostic accuracy and prognostic value of the first- and second
anti-cyclic citrullinated peptides autoantibody (CCP1 and CCP2) tests
for rheumatoid arthritis. Ann Rheum Dis. 2005 Mar 30; [Epub ahead
of print].
8. Vallbracht I, Rieber J, Oppermann M, et al. Diagnostic and clinical
value of anti-cyclic citrullinated peptide antibodies compared with
rheumatoid factor isotypes in rheumatoid arthritis. Ann Rheum Dis.
2004;63:1079-1084.
9. Visser H, le Cessie S, Vos K, et al. How to diagnose rheumatoid
arthritis early: a prediction model for persistent (erosive) arthritis.
Arthritis Rheum. 2002;46:357-365.
10. Rantapaa-Dahlqvist S, de Jong BA, Berglin E, et al. Antibodies
against cyclic citrullinated peptide and IgA rheumatoid factor
predict the development of rheumatoid arthritis. Arthritis Rheum.
2003;48:2741-2749.
11. Nielen MM, van Schaardenburg D, Reesink HW, et al. Specific
autoantibodies precede the symptoms of rheumatoid arthritis: a
study of serial measurements in blood donors. Arthritis Rheum.
2004;50:380-386.
12. Berglin E, Padyukov L, Sundin U, et al. A combination of
autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1
locus antigens is strongly associated with future onset of
rheumatoid arthritis. Arthritis Res Ther. 2004;6:R303-8.
13. van Gaalen FA, Linn-Rasker SP, van Venrooij WJ, et al.
Autoantibodies to cyclic citrullinated peptides predict progression to
rheumatoid arthritis in patients with undifferentiated arthritis: a
prospective cohort study. Arthritis Rheum. 2004;50:709-715.
14. Greiner A, Plischke H, Kellner H, et al. Association of anti-cyclic
citrullinated peptide antibodies, anti-citrullin antibodies, and IgM
and IgA rheumatoid factors with serological parameters of disease
activity in rheumatoid arthritis. Ann N Y Acad Sci. 2005;1050:295-
303.
15. De Rycke L, Peene I, Hoffman IE, et al. Rheumatoid factor and
anticitrullinated protein antibodies in rheumatoid arthritis:
diagnostic value, associations with radiological progression rate,
and extra-articular manifestations. Ann Rheum Dis. 2004;63:1587-
1593.
16. Dubucquoi S, Solau-Gervais E, Lefranc D, et al. Evaluation of anti-
citrullinated filaggrin antibodies as hallmarks for the diagnosis of
rheumatic diseases. Ann Rheum Dis. 2004;63:415-419.
17. Suzuki K, Sawada T, Murakami A, et al. High diagnostic
performance of ELISA detection of antibodies to citrullinated
antigens in rheumatoid arthritis. Scand J Rheumatol. 2003;32:197-
204.
18. Forslind K, Ahlmen M, Eberhardt K, et al; BARFOT Study Group.
Prediction of radiological outcome in early rheumatoid arthritis in
clinical practice: role of antibodies to citrullinated peptides (anti-
CCP). Ann Rheum Dis. 2004;63:1090-1095.
19. Vencovsky J, Machacek S, Sedova L, et al. Autoantibodies can be

Section 7
prognostic markers of an erosive disease in early rheumatoid
arthritis. Ann Rheum Dis. 2003;62:427-430.
20. Lindqvist E, Eberhardt K, Bendtzen K, at al. Prognostic laboratory
markers of joint damage in rheumatoid arthritis. Ann Rheum Dis.
2005;64:196-201.
21. Kastbom A, Strandberg G, Lindroos A, et al. Anti-CCP antibody test
predicts the disease course during 3 years in early rheumatoid
arthritis (the Swedish TIRA project). Ann Rheum Dis. 2004;63:1085-
1089.
22. Sox HC Jr, Liang MH. The erythrocyte sedimentation rate.
Guidelines for rational use. Ann Intern Med. 1986;104:515-523.
23. Wener MH, Hutchinson K, Morishima C, et al. Absence of
antibodies to cyclic citrullinated peptide in sera of patients with
hepatitis C virus infection and cryoglobulinemia. Arthritis Rheum.
2004;50:2305-2308.
24. Wolfe F. Comparative usefulness of C-reactive protein and
erythrocyte sedimentation rate in patients with rheumatoid arthritis.
J Rheumatol. 1997;24:1477-1485.
25. Hoffman IE, Peene I, Cebecauer L, et al. Presence of rheumatoid
factor and antibodies to citrullinated peptides in systemic lupus
erythematosus. Ann Rheum Dis. 2005;64:330–332.
26. QUANTA Lite™ CCP IgG ELISA [Package Insert]. Inova Diagnostics,
San Diego, CA; 2004.
27. Gottenberg JE, Mignot S, Nicaise-Rolland P, et al. Prevalence of
anti-cyclic citrullinated peptide and anti-keratin antibodies in
patients with primary Sjögren’s syndrome. Ann Rheum Dis.
2005;64:114-117.
28. Ferucci ED, Majka DS, Parrish LA, et al. Antibodies against cyclic
citrullinated peptide are associated with HLA-DR4 in simplex and
multiplex polyarticular-onset juvenile rheumatoid arthritis. Arthritis
Rheum. 2005;52:239-246.
29. Lopez-Hoyos M, Ruiz de Alegria C, Blanco R, et al. Clinical utility of
anti-CCP antibodies in the differential diagnosis of elderly-onset
rheumatoid arthritis and polymyalgia rheumatica. Rheumatology
(Oxford). 2004;43:655-657.
30. Alenius GM, Berglin E, Rantapaa Dahlqvist S. Antibodies against
cyclic citrullinated peptide (CCP) in psoriatic patients with or
without manifestation of joint inflammation. Ann Rheum Dis.
2006;65:398-400.
7.9.3 Cardiolipin Antibodies
See Coagulation, “Thrombophilia” sections 3.5.1 and 3.5.2.
7.9.4 Histone Antibody
Clinical Use: This test is used to rule out drug-induced systemic lupus
erythematosus (SLE).
Clinical Background: The diagnosis of drug-induced lupus is unlikely
in the absence of histone antibodies.
Method: Enzyme immunoassay (EIA)
Interpretive Information: Histone antibodies are present in 95% of
patients with drug-induced lupus and 50% of patients with idiopathic
SLE. They are occasionally detected in other connective tissue diseases.
7.9.5 Myocardial Antibody
Clinical Use: This test is used to determine autoimmune etiology in
patients with cardiomyopathy.
Clinical Background: Antibodies to cardiac antigens are commonly
found in patients after vascular damage to cardiac or pericardial tissue.
193
Immunology
Such antibodies are found in up to 20% to 40% of patients after cardiac
surgery. The presence of myocardial antibodies suggests an
autoimmune etiology in patients with cardiomyopathy, eg, idiopathic
dilated cardiomyopathy, myocarditis, rheumatic fever, and Dressler’s
syndrome. However, because of low sensitivity and specificity, this test
is of limited value in understanding cardiac autoimmunity.
Method: Indirect immunofluorescence assay; the antibody titer is
determined (at an additional charge, associated with an additional CPT
code) for samples with positive results.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The presence of a myocardial antibody
suggests an autoimmune etiology for the cardiomyopathy.
7.9.6 Neutrophil Cytoplasmic Antibodies
Clinical use: This test is used for differential diagnosis of systemic
necrotizing vasculitides.
Clinical Background: Neutrophil cytoplasmic antibodies are
associated with inflammatory diseases such as systemic necrotizing
vasculities and are therefore useful in the differential diagnosis. Once
an ANCA screen positive result is obtained, tests for more specific
autoantibodies can be ordered, for example, myeloperoxidase (MPO)
antibodies for perinuclear ANCA and proteinase-3 (PR3) antibodies for
cytoplasmic ANCA.
Anti-MPO and anti-PR3 are useful in the classification of systemic
vasculitides, including Wegener’s granulomatosis (WG), microscopic
polyarteritis (MPA), Churg-Strauss syndrome, syndrome of lung
hemorrhage and nephritis, and primary pauci-immune necrotizing and
crescentic glomerulonephritis. In addition, these autoantibodies are
helpful in distinguishing active versus inactive disease states,
monitoring the effect of therapy, and evaluating the possibility of
relapse.
Anti-PR3 antibodies are found in 70% to 95% of patients with WG.
Anti-MPO antibodies are commonly associated with MPA, Churg-
Strauss syndrome, and idiopathic crescentic glomerulonephritis (ICGN).
Ninety percent of patients with inflammatory bowel disease,
autoimmune hepatitis, or cholangitic liver disease are negative for MPO
antibodies even though perinuclear staining of neutrophils may be
observed (as seen in MPO antibody-positive individuals with vasculitis).
Close follow-up of MPO or PR3 antibody-positive patients is imperative
because these patients may have, or be at risk for, serious progressive
disease.
Method: ANCA immunofluorescence assay; the antibody titer is
determined (at an additional charge, associated with an additional CPT
code) for samples with positive results. This test does not specify the
type of ANCA present (eg, MPO or PR3 antibody). Separate tests codes
are available for that purpose.
Interpretive Information: A positive ANCA screen suggests a
systemic necrotizing vasculitides. Correlation with clinical findings aids
in diagnosis.
194

Section 7 Immunology

References
1. Kallenberg CG, Mulder AH, Tervaert JW. Antineutrophil cytoplasmic
antibodies: a still-growing class of autoantibodies in inflammatory
disorders. Am J Med. 1992;93:675-682.
2. Goekin JA, Bonsib SM. Anti-neutrophil cytoplasmic antibody and the
histopathologic diagnosis of vasculitis. ASCP National Meeting,
1992.
3. Hardarson S, Labrecque DR, Mitros FA, et al. Antineutrophil
cytoplasmic antibody in inflammatory bowel and hepatobiliary
diseases. High prevalence in ulcerative colitis, primary sclerosing
cholangitis, and autoimmune hepatitis. Am J Clin Pathol.
1993;99:277-281.
4. Jennette JC, Falk RJ. Antineutrophil cytoplasmic autoantibodies in
inflammatory bowel disease. Am J Clin Pathol. 1993;99:221-223.
 195

Section 8 Infectious Disease

8.1 Available Tests West Nile Virus Antibodies (IgG, IgM), CSF
West Nile Virus Antibodies (IgG, IgM), Serum
Central Nervous System/Neurologic West Nile Virus RNA, Qualitative Real-Time PCR3
Acid-Fast Stain
Adenosine Deaminase, CSF1 Diarrhea/Gastrointestinal
AFB Susceptibility, Custom Drug Panel Adenovirus Antigen Detection, Gastroenteritis, EIA
Specify 1 drug for testing. Campylobacter Culture
AFB Susceptibility, M tuberculosis Complex, Secondary Panel, Agar Campylobacter jejuni Antibody, ELISA
Method Clostridium difficile Culture with Reflex to Toxin Assay
Includes amikacin 6, amikacin 12, capreomycin 10, ciprofloxacin 2, cycloserine 30, Clostridium difficile Cytotoxin Antibody, Neutralization
ethionamide 5, and p-aminosalicylic acid 2. Clostridium difficile Toxin B Screen (Cytotoxin)
Bacterial Meningitis Antigen Panel Clostridium difficile Toxins A & B Detection, EIA
Includes Streptococcus Gp B, Streptococcus pneumoniae, and Haemophilus Cryptosporidium Antigen, DFA
influenzae type B antigens as well as Neisseria meningitis Gp A/Y, C/W135, and Culture, Stool (Salmonella and Shigella)
B/E coli K1 antigens. Culture, Stool (Salmonella, Shigella, and Campylobacter)
Coccidioides Antibody, Complement Fixation, CSF Cyclospora and Isospora Exam
Coxsackie B (1-6) Antibodies, Serum E coli O157 Culture
Cryptococcus Antibody E coli, Enteroinvasive (EIEC), CC
Cryptococcus Antigen Screen with Reflex to Titer E coli Shiga Toxins, EIA, Stool
Culture, Enterovirus Entamoeba histolytica Antibody (IgG), ELISA
Culture, Fungus, Miscellaneous Source Entamoeba histolytica Antigen, EIA
Culture, Herpes Simplex Virus with Reflex to Typing Gastroenteritis Pediatric Panel
Culture, Herpes Simplex Virus, Rapid Method Includes adenovirus and rotavirus antigens.
Culture, Mycobacterium Giardia Antibody, IFA
Culture, Virus, Body Fluids, Tissues Giardia and Cryptosporidium Antigen Panel
Cysticercus IgG Ab, Western Blot1 Includes Giardia and Cryptosporidium antigens.
Echovirus Antibodies, CSF Giardia Antigen, EIA
Echovirus Antibodies, Serum Giardia Antigen with Reflex to Ova & Parasites
Enterovirus Panel I, CF (Serum) Helicobacter pylori Antibodies (IgA, IgG)
Includes Coxsackie B (1-6), echovirus (4, 7, 9, 11, 30), and poliovirus (1, 2, 3) Helicobacter pylori Antibodies (IgG, IgA, IgM)2
antibodies. Helicobacter pylori Antibodies (IgG, IgA), Western Blot2
Enterovirus RNA, Qualitative Real-Time PCR2 Helicobacter pylori Antibody (IgG), Qualitative
Fungus Panel, CSF Helicobacter pylori Antibody (IgG), Quantitative
Includes Aspergillus, Blastomyces, Coccidioides (complement fixation and Helicobacter pylori Antibody (IgG), Western Blot2
immundiffusion), Cryptococcus, and Histoplasma antibodies and Cryptococcus Helicobacter pylori Antigen Detection, EIA, Stool
antigen screen with reflex to titer. Helicobacter pylori Urea Breath Test (UBiT®’)
Haemophilus influenzae Type B Antibody (IgG) Microsporidia Spore Detection
Herpes Simplex Virus, Type 1 & 2 DNA, Real-Time PCR3 Ova & Parasites and Giardia Antigen
HSV 1 IgG, HerpeSelect™ Type-specific Antibody Ova & Parasites, Stool Concentrate and Permanent Smear
HSV 1/2 IgG, HerpeSelect™ Type-specific Antibody Parasite Identification
JC Polyoma Virus DNA, Qualitative Real-Time PCR3 Pinworms, Direct Examination
Listeria Antibody, CF (Serum) Rotavirus Antigen Detection, EIA
Lyme Disease Antibody, Total, EIA with Reflex to CSF Ratio Salmonella Serotyping (Groups A-Z, 51-61 and VI)
Lyme Disease DNA, Real-Time PCR, CSF or Synovial Fluid2 Salmonella, Total Antibody, EIA
Lymphocytic Choriomeningitis Virus Antibody, IFA (Serum)2 Strongyloides IgG Antibody, ELISA2
Measles Antibodies (IgG, IgM) Trichinella IgG Antibody, ELISA
Measles Antibody (IgG) Tropheryma whipplei (Whipple’s) DNA, Qualitative, PCR3
Meningoencephalitis Comprehensive Panel (Serum) Vibrio cholerae Culture with Reflex to Susceptibility
Includes California, Eastern equine IgG and IgM, St. Louis IgG and IgM, Western Yersinia Culture
equine encephalitis IgG and IgM, LCM IgG and IgM, HSV 1 and 2 IgG indexes with Yersinia enterocolitica Antibody, MAT
interpretation, HSV 1/2 IgM index with confirmation and interpretation, adenovirus,
influenza types A and B, measles (rubeola) IgG and IgM with interpretation, mumps Hepatitis
IgG and IgM with interpretation, varicella-zoster, Coxsackie virus (A2, A4, A7, A9, Hepatitis A (IgM), Acute Status
A10, A16, B1-6), echovirus (4, 7, 9, 11, 30), and cytomegalovirus IgG and IgM Hepatitis A Total Antibody, EIA with Reflex to IgM
antibodies. Hepatitis A Total Antibody, Immune Status
Mumps Virus Antibodies (IgG, IgM) Hepatitis B Chronic Panel
Mumps Virus Antibody (IgG) Includes hepatitis B surface antigen and Be antibody and antigen.
Mumps Virus Direct Detection, DFA1 Hepatitis B Core Antibody (IgM)
Mycobacterium tuberculosis Complex, PCR, Non-respiratory2 Hepatitis B Core Total Antibody
Neisseria meningitidis IgG Vaccine Response, MAID Hepatitis B Core Total Antibody, EIA, with Reflex to IgM
Neisseria meningitidis Serotyping, SA Hepatitis B Diagnostic Panel
Rabies Antibody, ELISA Includes hepatitis B core IgM and total antibodies, B surface antibody and antigen,
Varicella Zoster Virus (VZV) DNA, Qualitative Real-Time PCR3 and Be antibody and antigen.
VDRL, CSF Hepatitis B Surface Antibody
196

Section 8 Infectious Disease

Hepatitis B Surface Antibody Quantitation HIV-1 RNA Quantitative PCR, Ultrasensitive

Hepatitis B Surface Antibody Quantitation, Liver Transplant HIV-1 RNA, Quantitative bDNA (v3.0)
Hepatitis B Surface Antigen HIV-1 RNA, Quantitative bDNA with Reflex to HIV-1 Genotype2
Hepatitis B Surface Antigen with Reflex to Confirmation HIV-1 RNA, Quantitative bDNA with Reflex to VirtualPhenotype™2
Hepatitis B Viral DNA, Quantitative, PCR2 HIV-1 RNA, Quantitative PCR
Hepatitis B Virus DNA, Qualitative PCR2 HIV-1 RNA, Quantitative PCR with Reflex to VirtualPhenotype™2
Hepatitis B Virus Drug Resistance, Genotype, and BCP/Precore HIV-1 RNA, Quantitative PCR, Expanded Range
Mutations2 HIV-1 VirtualPhenotype™ for Drug Resistance to PRI and RTI2
Hepatitis Be Antibody HIV-1/HIV-2 Antibodies, Western Blot/Immunoblot1
Hepatitis Be Antigen HIV-1/HIV-2 Antibody Screen with Progressive Reflex (WB, EIA,
Hepatitis Be Panel Immunoblot)
Includes hepatitis Be antigen and antibody. HIV 1/2 EIA Antibody Screen with Reflex to HIV-1 Western Blot
Hepatitis C Antibody Supplemental Testing HIV-2 Antibody, EIA, with Positives Reflexed to Immunoblot
Hepatitis C Antibody with Reflex to RIBA HIV-2 Antibody, Immunoblot1
Hepatitis C Antibody, EIA HIV-2 DNA/RNA Qualitative Real-Time PCR3
Hepatitis C Viral RNA Genotype, LiPA2 HLA-B*5701 Typing
Hepatitis C Viral RNA, Qualitative PCR
Hepatitis C Viral RNA, Qualitative PCR with Reflex to Genotype, LiPA2 Immunization/Immunity
Hepatitis C Viral RNA, Qualitative PCR, with Reflex to Quantitative Real- Diphtheria Antitoxoid & Tetanus Antitoxoid Antibodies
Time PCR2 Diphtheria Antitoxoid Antibody2
Hepatitis C Viral RNA, Qualitative TMA Diphtheria Antitoxoid, Pre and Post2
Hepatitis C Viral RNA, Quantitative bDNA Diphtheria Antitoxoid, Titer to Endpoint2
Hepatitis C Viral RNA, Quantitative bDNA with Reflex to Qualitative Haemophilus influenzae Type B Antibody (IgG)
TMA Hepatitis A Total Antibody, Immune Status
Hepatitis C Viral RNA, Quantitative bDNA/TMA2 Measles Antibody (IgG)
Hepatitis C Viral RNA, Quantitative PCR with Reflex to Genotype, LiPA2 Measles, Mumps, and Rubella Antibodies (IgG)
Hepatitis C Viral RNA, Quantitative Real-Time PCR2 Mumps Virus Antibody (IgG)
Hepatitis C Viral RNA, Quantitative Real-Time PCR with Reflex to Neisseria meningitidis IgG Vaccine Response, MAID
Qualitative TMA2 Rubella Antibodies (IgG, IgM)
Hepatitis C Viral RNA, Quantitative, TMA2 Rubella Immune Status
Hepatitis Comprehensive Panel 2 Streptococcus pneumoniae IgG Antibody Panel (14 Serotypes) MAID
Includes hepatitis A IgM and total antibodies, B IgM core and core total antibodies, Includes serotypes 1, 3, 4, 5, 8, 9 (9N), 12 (12F), 14, 19 (19F), 23 (23F), 26 (6B), 51
B surface antigen and antibody, and C antibody. (7F), 56 (18C), and 68 (9V).
Hepatitis D Antibody, Total1 Streptococcus pneumoniae IgG Antibody (6 Serotypes), MAID
Hepatitis D Antigen1 Streptococcus pneumoniae IgG Antibody (7 Serotypes), MAID
Hepatitis D Virus (HDV) IgM Antibody, EIA1 Streptococcus pneumoniae IgG, Pre- and Postvaccination (6 Serotypes)
Hepatitis E Antibodies (IgG, IgM)1 Streptococcus pneumoniae IgG, Pre- and Postvaccination (7 Serotypes)
Hepatitis E Antibody (IgG)1 Streptococcus pneumoniae IgG, Pre- and Postvaccination (14 Serotypes)
Hepatitis E Antibody (IgM)1 Tetanus Antitoxoid Antibody (EIA)2
Hepatitis G Virus RNA, Qualitative PCR3 Varicella-Zoster Virus Antibody (IgG)
Hepatitis Virus Panel, Acute Varicella-Zoster Virus Antibody, Serum
Includes hepatitis A IgM antibody, hepatitis B core (IgM) and surface antibodies,
and hepatitis C antibody. Immunocompromised
HEPTIMAX® HCV RNA2 5-Fluorocytosine Level, BA
HEPTIMAX® HCV RNA with Reflex to HCV Genotype, LiPA2 Antiviral Level, Ganciclovir, HPLC
Hyaluronic Acid2 Antiviral Susceptibility, Acyclovir
Liver Fibrosis Panel (HepaScore™)2 Antiviral Susceptibility, Foscarnet
Includes alpha2-macroglobulin, gammaglutamyltransferase (GGT), hyaluronic acid, Antiviral Susceptibility, Ganciclovir
total bilirubin, and the HepaScore (calculated). Adenovirus Antigen Detection, Gastroenteritis, EIA
Adenovirus Antigen Detection, Respiratory, DFA
HIV Adenovirus DNA, Qualitative Real-Time PCR2
Antiretroviral Level, Amprenavir, HPLC Aspergillus Antibody Screen
Antiretroviral Level, Delavirdine, HPLC Aspergillus Antibody, Immunodiffusion
HIV-1 Antibody Confirmation, Western Blot Aspergillus Antigen
HIV-1 Antibody, Western Blot with Reflex to HIV-2, EIA with Reflex to BK Virus DNA, PCR3
Immunoblot BK Virus DNA, Quantitative Real-Time PCR3
HIV-1 DNA, Qualitative PCR4 BK and JC Virus DNA, Qualitative Real-Time PCR3
HIV-1 Genotype and Phenotype, PhenoSense GT®’3 Blastomyces dermatitidis Identification, DNA Probe
HIV-1 Genotype with Graphical Report2 Candida albicans Panel
HIV-1 Genotype2 Includes Candida antigen and IgA, IgG, and IgM antibodies.
HIV-1 gp41 Envelope Genotype2 Candida Antibody
HIV-1 Phenotype, PhenoSense™’ Candida Antibody (IgG)2
HIV-1 RNA, Qualitative PCR Candida Antibodies (IgG, IgM, IgA)2
HIV-1 RNA Quantitative PCR with Reflex to Genotype2 Candida Antigen Detection
 197

Section 8 Infectious Disease

Coccidioides Antibody (IgG), EIA with Reflex to Complement Fixation Pregnancy

Coccidioides Antibody, Complement Fixation, CSF Bacterial Vaginosis/Vaginitis Panel
Coccidioides Antibody, Immunodiffusion Includes Candida species, Trichomonas vaginalis, and Gardnerella vaginalis.
Coccidioides Antibody, Immunodiffusion and Complement Fixation Cytomegalovirus (CMV) Genotype2
Coccidioides immitis Identification, DNA Probe Cytomegalovirus Antibodies (IgG, IgM)
Cryptococcus Antibody Cytomegalovirus Antibody (IgG) with Reflex to IgM
Cryptococcus Antigen Screen with Reflex to Titer Cytomegalovirus Culture, Rapid and Conventional
Cryptosporidium Antigen, DFA Cytomegalovirus DNA, Qualitative Real-Time PCR3
Culture, Fungus, Blood Cytomegalovirus DNA, Quantitative Real-Time PCR3
Culture, Fungus, Miscellaneous Source Cytomegalovirus IgG Antibody
Culture, Mycobacterium, Blood Cytomegalovirus IgG Avidity, ELISA
Culture, Yeast Herpes Simplex Virus Antigen Detection, DFA
Cyclospora and Isospora Exam Herpes Simplex Virus, Type 1 & 2 DNA, Real-Time PCR3
Cytomegalovirus (CMV) Genotype2 Herpesvirus 6 Antibodies (IgG, IgM)2
Cytomegalovirus Antibodies (IgG, IgM) Herpes Virus 6 DNA, Qualitative Real-Time PCR2
Cytomegalovirus Antibody (IgG) with Reflex to IgM HTLV I/II DNA, Qualitative Real-Time PCR2
Cytomegalovirus Culture, Rapid and Conventional Parvovirus B-19 Antibodies (IgG, IgM)
Cytomegalovirus DNA, Qualitative Real-Time PCR3 Parvovirus B-19 Antibody (IgG)
Cytomegalovirus DNA, Quantitative Real-Time PCR3 Parvovirus B19 DNA, Qualitative Real-Time PCR3
Cytomegalovirus IgG Antibody Parvovirus B-19 DNA, Real-Time PCR and Parvovirus B-19 Antibodies
Cytomegalovirus IgG Avidity, ELISA (IgG, IgM)2
Epstein Barr Virus DNA, Qualitative Real-Time PCR3 Rubella Antibodies (IgG, IgM)
Epstein Barr Virus DNA, Quantitative Real-Time PCR3 Rubella Immune Status
Fungal Identification, Molds Torch Panel (IgG & IgM)
Fungus Panel, Serum Includes cytomegalovirus (IgG, IgM), rubella (IgG, IgM), and Toxoplasma (IgG, IgM)
Includes Aspergillus, Blastomyces, Coccidioides (complement fixation, antibodies, HSV antibody screen with reflex to titer, and HSV 1/2 type-specific IgG
immundiffusion, and EIA), Cryptococcus, and Histoplasma (complement fixation and antibodies.
immunodiffusion) antibodies and Cryptococcus antigen screen with reflex to titer. Torch Panel, Convalescent
Hepatitis B Surface Antibody Quantitation, Liver Transplant Includes cytomegalovirus IgG, HSV 1/2 type-specific IgG, rubella IgG, and
Herpes Simplex Virus Antigen Detection, DFA Toxoplasma IgG and IgM antibodies.
Herpes Simplex Virus, Type 1 & 2 DNA, Real-Time PCR3 Toxoplasma Antibodies (IgG, IgM)
Herpes Simplex Virus/Varicella Zoster Virus Rapid Culture Toxoplasma gondii, DNA, Qualitative Real-Time PCR2
Herpes Virus 6 DNA, Qualitative Real-Time PCR2 Toxoplasma IgG Antibody with Reflex to Toxoplasma IgM Antibody
Herpes Virus 8 DNA, Qualitative Real-Time PCR3 Trichomonas vaginalis RNA, Qualitative TMA2
Herpesvirus 8 IgG Antibody, IFA1
Histoplasma Antibody Panel Respiratory
Includes complement fixation and immunodiffusion. AFB Susceptibility, Custom Drug Panel
Histoplasma Antibody, Complement Fixation Specify 1 drug for testing.
Histoplasma Antibody, Immunodiffusion Acid-Fast Stain
Histoplasma capsulatum Identification, DNA Probe Adenovirus Antibody, Serum
JC Polyoma Virus DNA, Qualitative Real-Time PCR3 Adenovirus Antigen Detection, Respiratory, DFA
Legionella Antigen Detection, DFA Adenovirus DNA, Qualitative Real-Time PCR2
Legionella Antigen, EIA, Urine Antistreptolysin-O
Legionella Culture Aspergillus Antibody Screen
Legionella DNA, Qualitative Real-Time PCR2 Aspergillus Antibody, Immunodiffusion
Lymphogranuloma Venereum (LGV) Differentiation Antibody Panel, MIF Aspergillus Antigen
Includes C trachomatis (L2) IgG, IgM, and IgA; C trachomatis (D-K) IgG, IgM, and Atypical Pneumonia DNA Panel, Qualitative Real-Time PCR2
IgA; C pneumoniae IgG, IgM, and IgA; C psittaci IgG, IgM, and IgA; and Includes C pneumoniae, L pneumophila, Legionella species, and M pneumoniae
interpretation. DNA.
Microsporidia Spore Detection Avian Influenza H5 Gene, Qualitative Real-Time PCR2
Modified Acid Fast Stain for Aerobic Actinomycetes Avian Influenza H5 Gene, QL Real-Time PCR with Reflex to A and B
Mycobacterium avium-intracellulare DNA, Qualitative PCR2 Culture, Rapid2
Parainfluenza Virus (Types 1, 2, and 3) RNA, Qualitative Real-Time PCR3 Avian Influenza H5 Gene, QL Real-Time PCR with Reflex to Influenza A
Parainfluenza Virus Antigen Detection, DFA and B RNA2
Parvovirus B19 DNA, Qualitative Real-Time PCR3 Blastomyces Antibody, Immunodiffusion
Pneumocystis carinii Detection, DFA Blastomyces dermatitidis Identification, DNA Probe
Susceptibility, Aerobic Actinomycetes (Nocardia and Rhodococcus), MIC1 Bordetella parapertussis/pertussis, DFA
Susceptibility, MAI Complex, MIC1 Bordetella pertussis Culture and DFA
Toxoplasma Antibodies (IgG, IgM) Bordetella pertussis/parapertussis Culture
Toxoplasma gondii DNA, Qualitative Real-Time PCR3 Bordetella pertussis/parapertussis DNA, Qualitative Real-Time PCR3
Toxoplasma IgG Antibody with Reflex to Toxoplasma IgM Antibody Burkholderia pseudomallei Antibody Panel, IFA
Varicella Zoster Virus (VZV) DNA, Qualitative Real-Time PCR3 Includes B pseudomallei IgG and IgM antibodies.
Varicella/Herpes Zoster Virus Antigen Detection, DFA Chlamydia/Chlamydophila Group Antibody
198

Section 8 Infectious Disease

Chlamydia and Chlamydophila Antibody Panel 1 (IgG)2 Mycoplasma pneumoniae Antibodies (IgG, IgM), EIA
Includes C trachomatis, C pneumoniae, and C psittaci IgG antibodies. Mycoplasma pneumoniae Antibody (IgG), EIA
Chlamydia and Chlamydophila Antibody Panel 2 (IgM)2 Mycoplasma pneumoniae Antibody (IgM)
Includes C trachomatis, C pneumoniae, and C psittaci IgM antibodies. Mycoplasma pneumoniae Culture
Chlamydia and Chlamydophila Antibody Panel 3 ( IgG, IgA, IgM)2 Mycoplasma pneumoniae DNA, Qualitative Real-Time PCR2
Includes C trachomatis, C pneumoniae, and C psittaci IgG, IgM, and IgA antibodies Mycoplasma/Ureaplasma Culture
with interpretation. Ova & Parasites, Sputum
Chlamydophila pneumoniae, Antibodies (IgG, IgA, IgM)2 Parainfluenza Virus (Types 1, 2, and 3) Antibodies, Serum
Chlamydophila pneumoniae DNA, Qualitative Real-Time PCR2 Parainfluenza Virus (Types 1, 2, and 3) RNA, Qualitative Real-Time PCR3
Chlamydophila psittaci Antibodies (IgG, IgA, IgM)2 Parainfluenza Virus Antigen Detection, DFA
Coccidioides Antibody (IgG), EIA with Reflex to Complement Fixation Pneumocystis carinii Detection, DFA
Coccidioides Antibody, Complement Fixation, CSF Rapid Respiratory DFA Viral Screen with Reflex
Coccidioides Antibody, Immunodiffusion Rapid Viral Respiratory Culture Screen with Reflex
Coccidioides Antibody, Immunodiffusion and Complement Fixation Respiratory Syncytial Virus Antibody, Serum
Coccidioides immitis Identification, DNA Probe Respiratory Virus Panel, Qualitative PCR3
Cryptococcus Antibody Includes adenovirus DNA; influenza A and B RNA; parainfluenza 1, 2, and 3 RNA;
Cryptococcus Antigen Screen with Reflex to Titer and RSV RNA.
Culture, Ear RSV (Respiratory Syncytial Virus) RNA, Qualitative Real-Time PCR2
Culture, Mycobacterium RSV (Respiratory Syncytial Virus) Antigen Detection, DFA
Culture, Mycobacterium, Blood SARS Coronavirus RNA, Qualitative RT-PCR3
Culture, Sputum/Lower Respiratory and Gram Stain with reflex to Streptococcus Group A, DNA Probe
Susceptibility Streptococcus pneumoniae IgG Antibody Panel (14 Serotypes) MAID
Culture, Streptococcus, Group A Includes serotypes 1, 3, 4, 5, 8, 9 (9N), 12 (12F), 14, 19 (19F), 23 (23F), 26 (6B), 51
Culture, Throat (7F), 56 (18C), and 68 (9V).
DNase-B Antibody Streptococcus pneumoniae IgG Antibody (6 Serotypes), MAID
Echovirus Antibodies, Serum Streptococcus pneumoniae IgG Antibody (7 Serotypes), MAID
Epstein-Barr Virus Antibody (IgM) to Viral Capsid Antigen (VCA-IgM) Streptococcus pneumoniae IgG, Pre- and Postvaccination (6 Serotypes)
Fungal Identification, Molds Streptococcus pneumoniae IgG, Pre- and Postvaccination (7 Serotypes)
Fungus Panel, Serum Streptococcus pneumoniae IgG, Pre- and Postvaccination (14 Serotypes)
Includes Aspergillus, Blastomyces, Coccidioides (complement fixation, Streptococcus pneumoniae (Pneumococcal) Serotyping
immundiffusion, and EIA), Cryptococcus, and Histoplasma (complement fixation and Streptozyme Screen with Reflex to Titer
immunodiffusion) antibodies and Cryptococcus antigen screen with reflex to titer. Susceptibility, Aerobic Actinomycetes (Nocardia and Rhodococcus), MIC1
Fungus, Direct Examination, Sputum, Tissue or Body Fluid Susceptibility, MAI Complex, MIC1
Haemophilus influenzae Serotyping (A-F), SA Susceptibility, Rapid Growing Bacteria, MIC1
Hantavirus Antibodies, ELISA1
Histoplasma Antibody Panel STI/Genitourinary Infections
Includes complement fixation and immunodiffusion. Antiviral Level, Ganciclovir, HPLC
Histoplasma Antibody, Complement Fixation Antiviral Susceptibility, Acyclovir
Histoplasma Antibody, Immunodiffusion Antiviral Susceptibility, Foscarnet
Histoplasma capsulatum Identification, DNA Probe Bacterial Vaginosis/Vaginitis Panel
Influenza A and B Culture, Rapid Method Includes Candida species, Trichomonas vaginalis, and Gardnerella vaginalis.
Influenza Type A and B Antibodies, Serum Chlamydia trachomatis & Neisseria gonorrhoeae, DNA Probe
Influenza Type A and B RNA, Qualitative Real-time RT-PCR3 Chlamydia trachomatis Antibodies (IgG, IgA, IgM)
Influenza Type A Antibody, Serum Chlamydia trachomatis Culture
Influenza Type B Antibody, Serum Chlamydia trachomatis, DFA
Influenza Virus Type A & B Antigen Detection, DFA Chlamydia trachomatis DNA, PCR
Legionella Antigen Detection, DFA Chlamydia trachomatis, DNA Probe, Endocervical, Male Urethral,
Legionella Culture Conjunctival
Legionella DNA, Qualitative Real-Time PCR2 Chlamydia trachomatis DNA, SDA
Legionella pneumophila Antibody (IgG), IFA Chlamydia trachomatis DNA, SDA, Pap Vial
Legionella pneumophila Antibody (IgM), IFA2 Chlamydia trachomatis RNA, TMA
Legionella Antigen, EIA, Urine Chlamydia trachomatis, TMA (Alternate Target)
Measles Antibodies (IgG, IgM) Chlamydia trachomatis/Neisseria gonorrhoeae RNA, TMA
Measles Antibody (IgG) Chlamydia/N gonorrhoeae DNA, PCR
Measles Antibody (IgM) Chlamydia/N gonorrhoeae DNA, SDA
Methicillin Resistant Staphylococcus aureus, PCR Chlamydia/N gonorrhoeae DNA, SDA, Pap Vial
Modified Acid Fast Stain for Aerobic Actinomycetes Culture, Herpes Simplex Virus with Reflex to Typing
Mycobacteria Identification Culture, Herpes Simplex Virus, Rapid Method
Mycobacteria PCR, Culture and Smear, Respiratory Culture, Mycobacterium
Mycobacterium avium-intracellulare DNA, Qualitative PCR2 Culture, Yeast
Mycobacterium tuberculosis Complex, PCR, Respiratory2 FTA-ABS (Fluorescent Treponemal Antibody-Absorption)
Mycobacterium tuberculosis Susceptibility Panel with PZA Herpes Simplex Virus Antigen Detection, DFA
Includes susceptibility to streptomycin, isoniazid, rifampin, ethambutol, and Herpes Simplex Virus Typing (Monoclonal)
pyrazinamide. Herpes Simplex Virus, Type 1 & 2 DNA, Real-Time PCR3
 199

Section 8 Infectious Disease

HPV (Human Papillomavirus) DNA, High and Low Risk, Anal-Rectal Burkholderia pseudomallei Antibody Panel, IFA
HPV (Human Papillomavirus) DNA, High Risk, Anal-Rectal Includes B pseudomallei IgG and IgM antibodies.
HPV (Human Papillomavirus) Genotypes 16 and 182 Colorado Tick Fever Antibody, IFA
HPV (Human Papillomavirus), High Risk DNA, Hybrid Capture II Cysticercus Antibody, ELISA (CSF)
HPV (Human Papillomavirus), Hybrid Capture II Dengue Fever Antibody (IgG)2
HPV (Papillomavirus) High Risk, Hybrid Capture II with Reflex to Dengue Fever Antibodies (IgG, IgM)2
Genotypes 16,182 Dengue Fever Antibody (IgM)2
HPV (Papillomavirus), Low/High Risk, Hybrid Capture II with Reflex to Echinococcus granulosus Antibody (IgG)1
Types 16,182 Ehrlichia chaffeensis Antibodies (IgG, IgM)2
HPV Typing In Situ2 Filaria IgG4 Antibody, ELISA
HSV 1 IgG, HerpeSelect™ Type-specific Antibody Francisella tularensis Antibody, DA
HSV 1/2 IgG, HerpeSelect™ Type-specific Antibody Francisella tularensis Screen
HSV 2 IgG, HerpeSelect™ Type-specific Antibody Leishmania Antibody, IFA
HSV 2 IgG, HerpeSelect™ Type-specific Antibody with Reflex to HSV-2 Leptospira Antibody
Inhibition Lyme Disease Antibodies (IgG, IgM), Western Blot
HSV-2 Inhibition, ELISA Lyme Disease Antibody (IgG), Western Blot
HSV1/2 IgG,HerpeSelect™ Type-specific Antibody with Reflex to HSV-2 Lyme Disease Antibody, Total, EIA with Reflex to Western Blot (IgG,
Inhibition IgM)
HTLV-I/II Antibody, EIA Lyme Disease Antibody, Total, EIA with Reflex to CSF Ratio
HTLV I/II DNA, Qualitative Real-Time PCR2 Lyme Disease C6 Antibodies, Total with Reflex to IgG and IgM, Western
HTLV I/II, Western Blot1 Blot
Lymphogranuloma Venereum (LGV) Differentiation Antibody Panel, MIF Lyme Disease DNA, Real-time PCR and Tick Identification
Includes C trachomatis (L2) IgG, IgM, and IgA; C trachomatis (D-K) IgG, IgM, and Lyme Disease DNA, Real-Time PCR, Blood2
IgA; C pneumoniae IgG, IgM, and IgA; C psittaci IgG, IgM, and IgA; and Lyme Disease DNA, Real-Time PCR, CSF or Synovial Fluid2
interpretation. Lyme Disease DNA, Real-Time PCR, Tick2
Mycobacteria PCR, Culture and Smear, Non-Respiratory Lyme Disease DNA, Real-Time PCR, Urine2
Mycobacterium tuberculosis Complex, PCR, Non-respiratory2 Malaria/Babesia Examination by Giemsa Stain
Mycobacterium tuberculosis Susceptibility Panel with PZA Q Fever (Coxiella burnetii) Antibodies (IgG, IgM) with Reflex to Titers
Includes susceptibility to streptomycin, isoniazid, rifampin, ethambutol, and Rickettsial Antibody Panel with Reflex to Titers
pyrazinamide. Includes Rocky Mountain spotted fever IgG and IgM antibody screens with reflex to
Mycoplasma/Ureaplasma Culture titers and typhus fever IgG and IgM antibody screens with reflex to titers.
Neisseria gonorrhoeae DNA, PCR Rickettsial Disease Panel
Neisseria gonorrhoeae DNA, SDA Includes Rocky Mountain spotted fever IgG and IgM antibody screens with reflex to
Neisseria gonorrhoeae DNA, SDA, Pap Vial titers, typhus fever IgG and IgM antibody screens with reflex to titers, and Q fever
Neisseria gonorrhoeae Probe, Endocervical or Male Urethral IgG and IgM phase I and II antibody screens with reflex to titers.
Neisseria gonorrhoeae RNA, TMA Rocky Mountain Spotted Fever Antibodies (IgG, IgM) with Reflex to
Neisseria gonorrhoeae, TMA (Alternate Target) Titers
RPR (Diagnosis) with Reflex to Titer Tick (and Other Arthropods) Identification
RPR (Diagnosis) with Reflex to Titer and Confirmatory Testing Tick/Arthropods ID with Reflex to Lyme Disease DNA, Tick
Treponema pallidum Ab, Particle Agglutination Trypanosoma cruzi Antibody Panel, IFA
Trichomonas vaginalis RNA, Qualitative TMA2 Includes T cruzi IgG and IgM antibodies.
VDRL, CSF Typhus (Murine) Antibody (IgG, IgM) with Reflex to Titers
VDRL, Serum West Nile Virus Antibodies (IgG, IgM), CSF
VDRL, Serum with Reflex to FTA-ABS West Nile Virus Antibodies (IgG, IgM), Serum
West Nile Virus RNA, Qualitative Real-Time PCR3
Vector-borne and Travel Medicine Yersinia Culture
Anaplasma phagocytophilum and Ehrlichia chafeensis Antibody Panel2 Yersinia enterocolitica Antibody, MAT
Includes A phagocytophilum IgG and IgM antibodies and E chafeensis IgG and IgM Yersinia pestis Screen
antibodies.
Anaplasma phagocytophilum (IgG/IgM)2 Wound and Skin
Arbovirus Antibody Panel, IFA (CSF) Aerobic Bacteria Culture with Reflex Susceptibility
Includes California, Eastern equine, Western equine, and St. Louis IgG and IgM Aerobic Bacteria Identification
antibodies with interpretation. Aerobic Bacteria Identification and Susceptibility, MIC
Arbovirus Antibody Panel, IFA (Serum) Anaerobic Bacteria Culture and Gram Stain with Reflex Susceptibility
Includes California, Eastern equine, Western equine encephalitits, and St. Louis IgG Anaerobic Bacteria Identification
and IgM with interpretation. Clindamycin Resistance (D Test)
Babesia microti Antibodies (IgG, IgM)2 Culture, Aerobic and Anaerobic with Gram Stain
Babesia microti DNA, PCR3 Culture, Fungus, Skin, Hair or Nails
Bartonella DNA, PCR3 Culture, Mycobacterium
Bartonella henselae Antibodies (IgG, IgM) with Reflex(es) to Titer2 Culture, Varicella-Zoster, Rapid Method
Bartonella Species Antibody (IgG, IgM) with Reflex(es) to Titer2 DNase-B Antibody
Borrelia hermsii Antibody Panel, IFA Francisella tularensis Antibody, DA
Includes B hermsii IgG and IgM antibodies. Francisella tularensis Screen
Brucella Antibody (IgG, IgM), EIA2 Fungus, Direct Examination, Skin, Hair or Nails
200

Section 8 Infectious Disease

Fungus, Direct Examination, Sputum, Tissue or Body Fluid Culture, Virus, Body Fluids, Tissues
Herpes Simplex Virus/Varicella Zoster Virus Rapid Culture Culture, Yeast
Methicillin Resistant Staphylococcus aureus, PCR Cycloserine Level, SP
Mycobacteria Identification Cysticercus IgG Ab, Western Blot1
Mycobacteria PCR, Culture and Smear, Non-Respiratory Dicloxacillin Level, BA
Mycobacterium avium-intracellulare DNA, Qualitative PCR2 Donor, Chagas Disease (T cruzi), RIPA2
Mycobacterium tuberculosis Complex, PCR, Non-respiratory2 Donor, Chagas Disease (T cruzi) Screen with Reflex to Chagas Disease,
Mycobacterium tuberculosis Susceptibility Panel with PZA RIPA
Includes susceptibility to streptomycin, isoniazid, rifampin, ethambutol, and Donor, Chlamydia trachomatis/Neisseria gonorrhoeae, RNA, TMA
pyrazinamide. Donor, Cytomegalovirus (CMV) Total Antibodies
Scabies Examination Donor, Hepatitis B Surface Antigen Confirmation
Streptozyme Screen with Reflex to Titer Donor, Hepatitis B Surface Antigen with Reflex to Confirmation
Susceptibility, Aerobic Bacterium, Custom MIC Donor, Hepatitis B Virus Core Total Antibody
Susceptibility, Anaerobic Bacteria, MIC Panel Donor, Hepatitis C Antibody (Anti-HCV) with Reflex to RIBA
Includes susceptibility to 13 antibiotics. Donor, HIV-1 Antibody, Western Blot
Susceptibility, MAI Complex, MIC1 Donor, HIV-1/HBV/HCV NAT Procleix®’ with Reflex to HIV-1/HBV/HCV
Susceptibility, Rapid Growing Bacteria, MIC1 Discriminatory
Varicella/Herpes Zoster Virus Antigen Detection, DFA Donor, HIV-1/HCV NAT Procleix®’ with Reflex(es)
Varicella Zoster Virus (VZV) DNA, Qualitative Real-Time PCR3 Donor, HIV-1/HIV-2 Antibody Screen with Reflex(es)
Donor, HIV-2 Antibody Type 2 (Anti-HIV-2)
Other Disorders and Miscellaneous Tests Donor, HTLV-I/II Antibody Screen
Adenosine Deaminase, Peritoneal Fluid1 Donor, Rapid Plasma Reagin (RPR)
Adenosine Deaminase, Pleural Fluid1 Donor, Rapid Plasma Reagin (RPR) with Reflex to Syphilis IgG
Aerobic Bacteria Identification Donor, Syphilis IgG Antibody
Aerobic Bacteria Identification and Susceptibility, MIC Donor, West Nile Virus, NAT
Amoxicillin Level, BA Doxycycline Level, BA
Ampicillin Level, BA Epstein-Barr Virus Antibody (IgG) to Viral Capsid Antigen (VCA-IgG)
Anaerobic Bacteria Identification Epstein-Barr Virus Antibody to Nuclear Antigen (EBNA) (IgG)
Antifungal Serum Level, Amphotericin B, HPLC Epstein Barr Virus DNA, Qualitative Real-Time PCR3
Antifungal Serum Level, Fluconazole, HPLC Epstein Barr Virus DNA, Quantitative Real-Time PCR3
Antifungal Serum Level, Itraconazole, HPLC Epstein-Barr Virus Panel
Antifungal Susceptibility, Custom MIC (1) Includes EBV IgG and IgM capsid antibodies and EBV IgG antibody to nuclear
Antifungal Susceptibility, Custom MIC (2) antigen.
Antifungal Susceptibility, Custom MIC (3) Epstein-Barr Virus, Antibody to Early Antigen D (IgG)
Antimicrobial Serum Level, Rifampin, HPLC Erythromycin Level, BA
Antimicrobial Serum Level, Streptomycin, HPLC Extended-Spectrum Beta-Lactamase Confirmation
Antimicrobial Serum Level, Sulfamethoxazole, SP Fungal Identification, Molds
Antimicrob Susceptibility, Aerobic Bacteria, Custom MIC (2) Gram Stain
AFB Susceptibility, M.tuberculosis Complex, Secondary Panel, Agar HTLV-I/II Antibody, EIA
Method HTLV I/II DNA, Qualitative Real-Time PCR2
Includes amikacin 6, amikacin 12, capreomycin 10, ciprofloxacin 2, cycloserine 30, HTLV I/II, Western Blot1
ethionamide 5, and p-aminosalicylic acid 2. Imipenem Level, BA
Antimicrobial Susceptibility, Serum Bactericidal Test (Schlichter) Kanamycin Level, BA
Antimicrobial Synergy Studies Ketoconazole Level, BA
Aztreonam Level, BA Levofloxacin Level, BA
Bacterial 16S rDNA Sequencing1 Lysozyme, Serum
Blastomyces dermatitidis Identification, DNA Probe Lysozyme, Urine
Burkholderia pseudomallei Antibody Panel, IFA Methicillin Resistant Staphylococcus aureus, PCR
Includes B pseudomallei IgG and IgM antibodies. Minocycline Level, BA
C3a desArg Fragment1 Mycobacteria Identification
Cefoxitin Level, Serum, BA Mycobacterium avium-intracellulare DNA, Qualitative PCR2
Ceftazidime Level, BA Mycobacterium tuberculosis Susceptibility Panel with PZA
Ceftriaxone Level, BA Includes susceptibility to streptomycin, isoniazid, rifampin, ethambutol, and
Cefuroxime Level, BA pyrazinamide.
Cephalexin Level, BA Myocarditis/Pericarditis Panel
Cephazolin Level, BA Includes Cocksackie B (1-6,) echovirus, influenza (type A and B), and Chlamydophila
Ciprofloxacin Level, BA pneumoniae (IgG, IgA, and IgM) antibodies.
Clindamycin Level, BA Nafcillin Level, BA
Clindamycin Resistance (D Test) Neomycin Level, BA
Coxsackie A Antibodies, Serum Oxacillin Level, BA
Coxsackie B (1-6) Antibodies, Serum Parvovirus B-19 Antibodies (IgG, IgM)
Culture, Fungus, Miscellaneous Source Parvovirus B-19 Antibody (IgG)
Culture, Urine, Routine with Reflex to Susceptibility Parvovirus B19 DNA, Qualitative Real-Time PCR1
Culture, Urine, Special with Reflex to Susceptibility Parvovirus B-19 DNA, PCR and Parvovirus B-19 Antibodies (IgG, IgM)2

Section 8
Penicillin Level, BA
Piperacillin Level, BA
Poliovirus Antibody, Neutralization
Pyrazinamide Level, SP
Susceptibility, Aerobic Actinomycetes (Nocardia and Rhodococcus), MIC1
Susceptibility, Aerobic Bacteria, MIC
Susceptibility, Anaerobic Bacteria, MIC Panel
Includes susceptibility to 13 antibiotics.
Susceptibility, MAI Complex, MIC1
Susceptibility, Rapid Growing Bacteria, MIC1
Susceptibility, Yeast, Comprehensive Panel1
Includes amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole,
voriconazole, and caspofungin.
Tetracycline Level, BA
Ticarcillin/Clavulanate Level, BA
Ticarcillin Level, BA
Trimethoprim/Sulfamethoxazole Serum Level, BA
Varicella Zoster Virus Antibodies (IgG, IgM)
Voriconazole Level, HPLC
Yeast D2 LSU rDNA Sequencing1
Yeast Identification
1
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
2
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
3
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the test.
4
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. Performance characteristics refer to the analytical performance
of the test. This test should not be used for diagnosis without confirmation by other
medically established means.
Reflex tests are performed at an additional charge.
8.2 Diarrhea/Gastrointestinal
8.2.1 E coli Shiga Toxins, EIA, Stool
Clinical Use: This test is used to diagnose Shiga toxin-producing
Escherichia coli infection.
Clinical Background: Escherichia coli Shiga toxins are in the same
toxin family as that produced by Shigella dysenteriae type 1.1 The main
types of Shiga toxins produced by E coli are Stx1 and Stx2. The bacteria
that produce these toxins are transmitted to humans via E coli-
contaminated food (undercooked meat, raw milk, vegetables, or fruit)
and water, as well as by person-to-person and animal contact. Shiga
toxin production in vivo may lead to asymptomatic infection or mild to
severe gastrointestinal disease characterized by abdominal pain, watery
or bloody diarrhea, and hemorrhagic colitis. Five to ten percent of cases
progress to hemolytic uremic syndrome (HUS),2 a life-threatening
complication that includes acute renal failure, microangiopathic
hemolytic anemia, and severe thrombocytopenia and purpura. Although
HUS mortality is <10%, 30% of HUS survivors have significant
sequelae, including chronic renal insufficiency, hypertension, diabetes,
and neurologic defects.2 Children and the elderly are the most often
affected. Treatment is primarily supportive, but early diagnosis may
prevent unnecessary invasive diagnostic procedures (eg, appendectomy
for severe abdominal pain) or inappropriate antibiotic therapy.3-5 Infected
patients may be monitored for complications, enabling early
intervention. Early diagnosis also helps limit further spread of the
organism.
201
Infectious Disease
Individuals Suitable for Testing include patients with diarrhea.
Testing is strongly recommended for patients with bloody diarrhea or a
history of bloody diarrhea; those with severe watery diarrhea and no
evidence of other intestinal pathogens; those with HUS; and individuals
epidemiologically linked to cases of Shiga toxin-producing E coli
infection.2,6
Method: This test utilizes an enzyme immunoassay (EIA) that detects
Stx1 and Stx2. The stool specimen is preincubated in GN broth for
optimal sensitivity. The EIA uses monoclonal Stx1 and Stx2 capture
antibodies, a polyclonal Shiga-like toxin antibody, and an enzyme-
conjugated polyclonal IgG antibody for toxin detection. The analytical
sensitivity is 7 pg/well for Stx1 and 15 pg/well for Stx2. The clinical
sensitivity is 89%8 to 100%.9 The specificity for O157:H7 serotype is
99.7%, while the specificity for non-O157 serotype has not been
evaluated.9 Synonyms for this test include Shiga-like toxin (SLT),
verotoxin (VT), and Premier EHEC.
Interpretive Information: A positive result (toxin detected) indicates
the presence of Shiga toxin produced by E coli or Shigella dysenteriae
type 1. Since Aeromonas hydrophila, A caviae, and Enterobacter
cloacae are known to produce Stx1 and since Citrobacter freundii may
produce Stx2, presence of these organisms has the potential to result in
a positive test (studies not yet performed).2 Positive results have been
obtained from several Pseudomonas aeruginosa strains.10 A negative
result (not detected) indicates the absence of Shiga toxins or a level of
toxin below that which can be detected by the test. Neither a positive
nor a negative result precludes the presence of other infectious agents.
References
1. McBrien AD, Karmali MA, Scotland SM. A proposal for rationalizing
the nomenclature of the Escherichia coli cytotoxins. In: Karmali MA,
Goglio AG (eds). Recent Advances in Verocytotoxin-Producing
Escherichia coli Infections. Amsterdam: Elsevier Science BV. 1994;
pp. 353-356.
2. Paton JC, Paton AW. Pathogenesis and diagnosis of Shiga toxin-
producing Escherichia coli infections. Clin Microbiol Rev.
1998;11:450-479.
3. Walterspiel JN, Ashkenazi S, Morrow AL, et al. Effect of
subinhibitory concentrations of antibiotics on extracellular Shiga-
like toxin I. Infection. 1992;20:25-29.
4. Carter AO, Borczyk AA, Carlson JA, et al. A severe outbreak of
Escherichia coli O157:H7—associated hemorrhagic colitis in a
nursing home. N Engl J Med. 1987;317:1496-1500.
5. Pavia AT, Nichols CR, Green DP, et al. Hemolytic-uremic syndrome
during an outbreak of Escherichia coli O157:H7 infections in
institutions for mentally retarded persons: clinical and epidemiologic
observations. J Pediatr. 1990;116:544-551.
6. Karch H, Bielaszewska M, Bitzan M, et al. Epidemiology and
diagnosis of Shiga toxin-producing Escherichia coli infections. Diagn
Microbiol Infect Dis. 1999;34:229-243.
7. Tarr PI, Neill MA, Clausen CR, et al. Escherichia coli O157:H7 and
the hemolytic uremic syndrome: importance of early cultures in
establishing the etiology. J Infect Dis. 1990;162:553-556.
8. MacKenzie AM, Lebel P, Orrbine E, et al: Sensitivities and
specificities of Premier E coli O157 and Premier EHEC enzyme
immunoassays for diagnosis of infection with verotoxin (Shiga-like
toxin)-producing Escherichia coli. J Clin Microbiol. 1998;36:1608-
1611.
9. Kehl KS, Havens P, Behnke CE, et al. Evaluation of the premier EHEC
assay for detection of Shiga toxin-producing Escherichia coli. J Clin
Microbiol. 1997;35:2051-2054.
10. Beutin L, Zimmermann S, Gleier K. Pseudomonas aeruginosa can
202

Section 8 Infectious Disease

cause false-positive identification of verotoxin (Shiga-like toxin) Test Availability

production by a commercial enzyme immune assay system for
detection of Shiga-like toxins (SLTs). Infection. 1996;24:267-268.
8.2.2 Helicobacter pylori Infection
8.2.2.1 Laboratory Support of Diagnosis and Management
Clinical Background: Helicobacter pylori has been associated with
duodenal and gastric ulcers and chronic active, chronic persistent, and
atrophic gastritis in adults and children. Infected persons have a 2- to 6-
fold increased risk of developing gastric cancer and mucosal-associated-
lymphoid-type (MALT) lymphoma. About 70% to 90% of infected
individuals respond to a multiple-drug regimen over a 1- to 2-week
period.
Individuals Suitable for Testing: Testing is appropriate for
symptomatic individuals with active peptic ulcer disease, a history of
documented peptic ulcer, or MALT lymphoma and to confirm eradication
post-therapy.1 Testing should be performed only when treatment is
intended, or to document H pylori eradication.
Several methods can be used to diagnose H pylori infection: 1) upper
gastrointestinal tract biopsy with histologic exam, rapid urease testing
(RUT), or culture; 2) urea breath test (UBT) employing 14C- or 13C-urea; 3)
detection of antigen in stool; and 4) antibody detection. The UBT has
not been FDA-cleared for use in individuals <18 years of age.
Biopsy-Based Assays
When endoscopy is indicated, RUT or histologic examination is useful to
determine the presence of H pylori infection.1 Culture may be useful for
antibiotic resistance testing in patients unresponsive to therapy, but is
not very sensitive and not widely available.2
Non-Invasive Assays
The urea breath test (UBT) and stool antigen test have been
recommended by both the American Gastroenterological Association
(AGA) and the American College of Gastroenterologists (ACG) as the
most accurate non-invasive tests for diagnosis and for confirmation of
eradication.1,3 Both methods are highly sensitive and specific and are
useful for diagnosis, therapeutic monitoring, and test of cure (Table 1).
Serology-based methods cannot distinguish between active and
resolved infection. However, its low cost, wide availability, and

Table 1. Characteristics of Non-invasive Tests for H pylori

Urea Breath Test (UBT)5 Antigen Detection6

Clinical Utility Diagnose current infection Diagnose current infection
Assess treatment response Assess treatment response
Document cure Document cure
13
Measures CO2 release H pylori antigen
Method Infrared spectrometry Enzyme immunoassay
13
Primary reagent C-urea Polyclonal anti-H pylori antibody
Sensitivity, %
For diagnosis 95 (95% CI, 93%–97%) 96 (95% CI, 90–99)
For eradicationa 97% (95% CI, 93%–99%) 95 (95% CI, 74–99.9)
Specificity, %
For diagnosis 90 (95% CI, 73%–98%) 96 (95% CI, 90–99)
For eradicationa 95% (95% CI, 89%–98%) 96 (95% CI, 90–99)
False positives Achlorhydria; other urease-producing None known
organisms (eg, H heilmannii)
False negatives Proton pump inhibitors Very low antigenic levels
Antibiotics Proton pump inhibitors
Bismuth-containing compounds Antibiotics
Bismuth-containing compounds
FDA status Clearedb Cleared
Sample type Breath Stool
Sample stability
Ambient 1 week 24 hoursc
Refrigerated Not applicable 3 days
Frozen Not applicable 3 days
Sample handling Room temperature Frozen
CI, confidence interval.
a
Testing performed at least 4 weeks after end of therapy for H pylori eradication.
b
The performance characteristics of this test have not been established for persons under 18 years of age; Quest Diagnostics does not accept pediatric samples for this assay.
c
Quest Diagnostic does not accept room-temperature samples for this assay.

Section 8
sensitivity (roughly 76%-85%) may make serology a viable screening
option in high-prevalence settings (ie, prevalence above ~20%).2
Test Selection and Interpretation
Diagnosis
Patients >55 Years of Age or with Alarm Features
Prompt esophagogastroduodenoscopy is recommended for symptomatic
patients >55 years of age and all patients with alarm features (eg,
anemia, gastrointestinal bleeding, family history of gastrointestinal
cancer, early satiety, unintended weight loss of >10%, history of
esophagogastric malignancy, previous documented peptic ulcer,
progressive dysphagia, odynophagia, persistent vomiting, palpable mass
or lymphadenopathy, or jaundice).1,3,4 Biopsy samples obtained during
endoscopy may be used for diagnosis of H pylori infection, generally
with RUT or histology. Positive results on biopsy indicate infection, and
guidelines indicate that patients with positive results should be offered
H pylori eradication therapy.3 Especially in the presence of acute upper
gastrointestinal bleeding, negative results do not rule out infection and
should be confirmed with additional testing.2
Patients b55 Years of Age Without Alarm Features
Although current guidelines differ somewhat, 2 main approaches are
used for diagnosis of H pylori infection in patients b55 years of age
without alarm features1,3:
1) Test and treat: First, test with a noninvasive assay for H pylori
infection. Positive results with UBT or stool antigen indicate the need
for treatment to eradicate H pylori, followed by a trial of acid
suppression if symptoms persist after successful eradication. Positive
serology results should generally be confirmed with UBT or stool
antigen testing, especially in low-prevalence populations. Negative
results on non-invasive assays are followed by a 4- to 8-week empiric
trial of acid suppression therapy with a PPI.
2) Empiric treatment: A 4- to 8-week trial of empiric acid-reducing
therapy with a PPI. If the PPI trial fails or the patient relapses after the
end of treatment, the test-and-treat strategy should be pursued before
contemplating endoscopy.1
Post-therapeutic Testing
Follow-up testing with a non-invasive assay (UBT or stool antigen), or an
endoscopy-based assay if endoscopy is indicated, has been
recommended to confirm eradication of H pylori infection after therapy.2
If endoscopy is performed, culture may be useful to assess H pylori
antibiotic sensitivity after failed eradication therapy.1 UBT and antigen-
based testing should be performed no sooner than 4 weeks post-
therapy.
References
1. Talley NJ; American Gastroenterological Association. American
Gastroenterological Association medical position statement:
evaluation of dyspepsia. Gastroenterology. 2005;129:1753-1755.
2. Chey WD, Wong BC; Practice Parameters Committee of the American
College of Gastroenterology. American College of Gastroenterology
guideline on the management of Helicobacter pylori infection. Am J
Gastroenterol. 2007;102:1808-1825. Epub 2007 Jun 29.
3. Talley NJ, Vakil N; Practice Parameters Committee of the American
College of Gastroenterology. Guidelines for the management of
dyspepsia. Am J Gastroenterol. 2005;100:2324-2337.
4. Talley NJ, Vakil NB, Moayyedi P. American Gastroenterological
Association technical review on the evaluation of dyspepsia.
Gastroenterology. 2005;129:1756-1780.
203
Infectious Disease
5. BreathTek UBT™’ for H pylori [package insert]. Lafayette, Colorado:
Meretek Diagnostics Inc; 2005.
6. Premier Platinum HpSA™’ [package insert]. Cincinnati, Ohio: Meridian
Bioscience Inc; 2001.
8.2.2.2 Helicobacter pylori Antigen
Clinical Use: This test is used for differential diagnosis of peptic ulcer
disease and chronic active gastritis, and for therapeutic monitoring and
documentation of cure in patients with H pylori infection.
Method: This enzyme immunoassay employs a polyclonal anti-H pylori
capture antibody adsorbed to microwells and a peroxidase-conjugated
polyclonal detection antibody. Based on the intensity of color developed,
results are reported as H pylori antigen not detected, equivocal, or
detected.
Performance characteristics have not been established for watery
diarrheal stools or for asymptomatic individuals.
Interpretive Information: A positive result (antigen detected) is
indicative of H pylori presence (96% sensitivity); however some
individuals may have H pylori antigen but no disease. A negative result
(antigen not detected) indicates absence of H pylori or an antigenic level
below the assay limit of detection (184 ng H pylori protein/mL of stool)
(96% specificity). False-negative results may be obtained on specimens
from patients who have ingested selected compounds (antimicrobials,
proton pump inhibitors, bismuth preparations) within the 2 weeks prior
to specimen collection. In populations with disease prevalence ranging
from 34% to 69% (average 52%), the positive and negative predictive
values were 96%.
A positive result r7 days post-therapy is indicative of treatment failure.
A negative result r4 weeks post-therapy indicates eradication of the
infection.
8.2.2.3 Helicobacter pylori Urea Breath Test (UBiT)
Clinical Use: This test is used for differential diagnosis of patients
with peptic ulcer disease and chronic active gastritis and for therapeutic
monitoring in patients with Helicobacter pylori infection.
Method: This urea breath test indirectly detects the presence of H
pylori-associated urease by measuring CO2 in the patient’s breath. A
baseline breath sample is collected before the patient ingests 13C-urea,
ie, urea labeled with a naturally occurring, non-radioactive carbon
isotope. A second sample is collected shortly after the ingestion. H
pylori-associated urease degrades the urea, producing ammonia and
CO2. The resultant CO2 is absorbed in the blood and then exhaled. An
increase in the ratio of 13CO2 to 12CO2 between the pre- and post-
ingestion samples indicates presence of H pylori-associated urease.
Interpretive Information: A negative result indicates the absence of
H pylori-associated urease but does not rule out the possibility of H
pylori infection. False-negative results may be due to antimicrobials,
proton pump inhibitors (PPIs), and bismuth preparations ingested by the
patient within 2 weeks prior to testing or due to collection of a test
sample before the recommended interval following 13C-urea ingestion.
When clinical signs warrant, a repeat test should be considered with
this, or an alternative, test method.
A positive result is associated with the presence of H pylori infection,
other gastric spiral organisms such as H heilmannii, and achlorhydria.
204

Section 8 Infectious Disease

The performance characteristics of this assay for individuals <18 years (7) hemodialysis patients; (8) recent visitors or immigrants from endemic
of age have not been established. areas; and (9) health care workers.

8.3 Hepatitis
8.3.1 Viral Hepatitis: Laboratory Support of Diagnosis and
Management
Clinical Background: Viral hepatitis is a relatively common disease
(25 per 100,000 individuals in the United States) caused by a diverse
group of hepatotropic agents that lead to liver inflammation and cell
death. Five hepatitis viruses have been well characterized (A, B, C, D,
and E; Table 2).
Available Tests: Tests for antibodies and antigens are available for
hepatitis A-E, including those specific for hepatitis B core, surface, and
e proteins. Techniques for antibody detection include enzyme
immunoassay (EIA) and recombinant immunoblot assay (RIBA®'). EIA
technology is also used for antigen testing. Branched DNA (bDNA),
solution hybridization-hybrid capture (Digene), transcription-mediated
amplification (TMA), and polymerase chain reaction (PCR) are used for
DNA and RNA testing to determine the presence of viremia and
measure the viral load. HCV and HBV genotypes can also be determined
with hybridization or sequencing-based assays.

Hepatitis A and E viruses (HAV, HEV) are transmitted through the fecal-
oral route and manifest as acute or asymptomatic disease. There is no
chronic carrier state and serious sequelae are rare, although both HAV
and HEV can cause acute fulminant liver failure and HEV can cause
fulminant disease in pregnant women. Hepatitis B, C, and D viruses
(HBV, HCV, HDV) may establish persistent infections with significant
morbidity and mortality. All 3 are transmitted parenterally. HBV and HCV
are also transmitted through sexual contact and perinatally. HDV is
unique in that it is a “defective” virus that can replicate only in the
presence of HBV. HDV coinfection (HDV and HBV) significantly increases
the severity of disease. Acute HCV infection may be asymptomatic, but
most infections are chronic; chronic infection with HBV or HCV may lead
to cirrhosis and hepatocellular carcinoma.
In the United States, viral hepatitis is generally caused by HAV, HBV, or
HCV. Other causative viruses include cytomegalovirus (CMV), Epstein-
Barr virus (EBV), and human immunodeficiency virus (HIV). Hepatitis may
also be due to other diseases or medications.
A variety of immunologic and molecular assays are available for
diagnosing viral hepatitis and monitoring treatment response. This guide
provides an overview of available tests and indications for their use.
Individuals Suitable for Testing include (1) individuals with clinical
symptoms or abnormal liver enzyme levels; (2) children born to infected
women; (3) household or sexual contacts of infected persons or carriers;
(4) individuals participating in high-risk sexual activities (multiple sexual
partners, men who have sex with men); (5) intravenous drug users;
(6) recipients of organ transplants or multiple blood transfusions;
Test Selection
Diagnose Acute Infection
Four initial tests are generally recommended to diagnose acute hepatitis
(Figure 1): HAV immunoglobulin M (IgM) antibody, HBV core IgM
antibody (HBcAb, IgM), HBV surface antigen (HBsAg), and HCV antibody
by EIA. HAV IgM antibody is the preferred test for diagnosis of acute
hepatitis A infection because it rises early and persists only 3 to 12
months. HBcAb IgM is detectable during acute but not chronic HBV
infection. HBsAg, however, is detectable in both stages. Simultaneous
use of these 2 tests therefore not only detects both acute and chronic
HBV infection, but also helps to differentiate between them. The EIA
(antibody test) is used as the initial assay for diagnosing HCV infection
because of its high sensitivity, wide availability, and low cost. However,
antibody is not detected for many months after infection. RNA tests can
detect virus prior to seroconversion and serve to differentiate between
active and resolved infection. RIBA may be used in patients with
positive EIA results and negative HCV RNA results, although a repeat
HCV RNA assay may also be used in this setting. A negative RIBA result
is required for re-entry into the blood donor pool.
In cases of fulminant hepatitis, the possibility of coinfection or
superinfection (HBV with HCV or HDV) should be explored (Figure 1).
Diagnose Chronic Infection
When screening for chronic hepatitis, 2 laboratory tests are
recommended (Figure 2): HBsAg and HCV antibody by EIA. Positive
HBsAg results obtained 6 months or more after the initial diagnosis
indicate chronic infection. Either HBV DNA or the HBeAg assay can be

Table 2. Clinical Spectrum of Viral Hepatitis

Incubation Association with

Hepatitis Period Mortality Likelihood of Likelihood of Hepatocellular
Virus Transmission (weeks) (%) Carrier State Chronic Disease Carcinoma
A Fecal-oral 2-6 1 None None No
B Parenteral, 4-26 1-2 10% (adults) 1%-10% Yes
perinatal, sexual 90% (infants)
C Parenteral, perinatal
& possibly sexual 2-23 1-5 50%-80% 80%-90% Yes
D Parenteral, sexual,
perinatal 6-26 2-20 Variable 80% in superinfection* Yes*
†
E Fecal-oral 2-9 1-2 None None No
*Requires coinfection with HBV. Simultaneous infection with HBV is associated with severe acute disease and low likelihood of chronic infection (<5%); superinfection of HBV carries high
likelihood of fulminant disease (2%-20%), chronic HDV infection (up to 80%), and cirrhosis (60-70%), and may progress to hepatocellular carcinoma.
†
15%-20% in pregnant women.
 205

Section 8 Infectious Disease

Symptomatic or
high-risk individual

HAV infection
HAV IgM Ab–
unlikely

HAV IgM Ab+ HAV infection

Test for:
HBc IgM Ab– HBV infection
HAV IgM Ab HBsAg– unlikely HDV infection
HDV Ab– unlikely
HBc IgM Ab
HBs Ag Consider
HCV Ab (EIA) HBc IgM Ab+ or – test for HBc IgM Ab+ HBV with HDV
HBsAg+ HBV infection
HDV Ab HDV Ab+ coinfection

HCV infection HBc IgM Ab– HBV with HDV

HCV Ab (EIA)– unlikely HDV Ab+ superinfection

HCV Ab (EIA)+, Active or resolved

s/co >8* HCV infection

HCV Ab (EIA)+,
s/co 1 but <8.0*

Confirm with HCV

RNA tes t or RIB A ® ‘

HCV RNA+ HCV RNA– RIBA+ RIBA – RIBA indeterminate

HCV infection Active HCV infection unlikely; Active or resolved HCV infection unlikely Test for HCV RNA,
rule out active or resolved HCV infection or repeat RIBA
infection with RIBA 1 month later

Note: If infection is not diagnosed in a symptomatic individual, consider testing for HEV.
*s/co (signal-to-cutoff ratio) is specific to the chemiluminescent EIA used by Quest Diagnostics.

Figure 1. Diagnosis of acute hepatitis.

used for confirmation. HCV RNA testing is used to confirm positive HCV
antibody results, but when clinical suspicion is high and results from the
2 tests are discrepant, RIBA can be helpful.
HDV infection should be considered in chronic HBV carriers who
experience a further acute attack and/or rapidly progressive liver
disease. The HDV antibody assay is recommended for the initial test;
positive results may be followed by the HDV antigen test for
confirmation. HDV antigen testing may also be useful in HBsAg-
positive/HDVAb-negative individuals if coinfection or superinfection is
strongly suspected.
Demonstrate Carrier Status
Following disappearance of clinical symptoms, carrier status can be
demonstrated by testing for persistence of HBV antigen, HBV DNA, or
HCV RNA. For HBV, the presence of the following conditions indicates
inactive carrier state: HBsAg positive for r6 months, HBeAg-negative,
HBeAb-positive, serum HBV DNA <105 copies/mL, and persistently
206

Section 8 Infectious Disease

HBsAg–
with no clinical HBV infection unlikely
symptoms

– HBV infection unlikely

HBsAg–
with clinical Test for
symptoms HB V DNA
+ HBV infection

Test for: HBV infection Consider test

HBsAg+
for HDV Ab
HBsAg
HCV Ab

HDV infection
HCV Ab (EIA)– HCV infection unlikely HDV Ab– unlikely

HDV Ab+ HBV/HDV

HCV Ab (EIA)+, Active or resolved infection
s/co >8* HCV infection

HCV Ab (EIA)+,
s/co 1 but <8*

Confirm with HCV

RIB ® ‘
RNA test or RIBA

HCV RNA+ HCV RNA– RIBA+ RIBA – RIBA indeterminate

HCV infection Active HCV infection unlikely; Active or resolved HCV infection unlikely Test for HCV RNA,
rule out active or resolved HCV infection or repeat RIBA
infection with RIBA 1 month later
*s/co (signal-to-cutoff ratio) is specific to the chemiluminescent EIA used by Quest Diagnostics.

Figure 2. Diagnosis of chronic hepatitis.

normal ALT/AST levels. For HCV, use a quantitative or qualitative RNA
test. For HDV, use the HDV antigen test.
Demonstrate Recovery or Differentiate Between Active and
Resolved Infection
Differentiating current vs past infection is possible using HBV DNA,
HBsAg, and HBeAg testing for HBV; highly sensitive quantitative or
qualitative HCV RNA testing for HCV; and antigen testing for HDV.
Recovery from hepatitis B (in patients with known history of acute or
chronic HBV or the presence of HBcAb and/or HBsAb) is signaled by the
disappearance of HBsAg and HBV DNA along with persistently normal
ALT levels. Some individuals have detectable DNA after disappearance
of HBsAg. While this may not be associated with active disease in
immunocompetent individuals, it may represent treatment failure or
failure of natural immunity when HBsAb is absent. Additionally, HBV
DNA-positive individuals may be at risk for recurrent HBV disease and
HBV DNA-positive organ donors could potentially transmit the infection
to organ transplant recipients. HCV recovery is indicated by repeatedly
negative HCV RNA test results. HDVAg disappears within months after
recovery.
Assist with Treatment Decision-making and Therapeutic
Monitoring (Figure 3)
The alanine aminotransferase (ALT) assay is important when assessing
liver function. In chronic HBV infection, the baseline ALT level is
associated with the likelihood of treatment response; elevated ALT in
the presence of a positive HBV DNA assay is an indication for treatment
initiation. Quantitative HBV DNA assays can help assess the likelihood
of response to treatment, monitor response to therapy, and predict the
emergence of resistance to antiviral agents. The HBV genotyping assay
is used to determine the HBV genotype, which is important for
epidemiologic studies and may be associated with the clinical course
 207

Section 8 Infectious Disease

3A

Diagnosed ALT levels every

1 to 2 weeks until
HAV infection normal

3B
HBsAg– Resolved
ALT levels every HBsAg HBsAb+ infection
Acute 1 to 2 weeks until HBsAb
normal after 6 months HBsAg+ Chronic
Diagnosed HBsAb– infection
HBV infection

Chronic Monitor therapy

with HBV DNA

3C

Diagnosed Monitor therapy

chronic with HBV DNA and
HBV/ HDV HDV Ag
infection

3D
Probable
Negative resolved
Monitor ALT infection
Acute levels periodically HCV RNA assay
for 6 months after 6 months
Positive Chronic
infection
Diagnosed
HCV infection
Perform HCV genotyping
and measure viral load at Monitor therapy
Chronic baseline to guide duration with HCV RNA
of treatment assay*

*Refer to Molecular Testing in the Management of Hepatitis C Virus Infection, section 8.3.3.1, for more detailed information.

Figure 3. Laboratory management of patients with hepatitis. 3A, hepatitis A; 3B, hepatitis B; 3C, hepatitis B with D coinfection or superinfection; 3D,
hepatitis C.

and response to therapy. The HBV genotype assay can also detect the
emergence of mutations associated with resistance to antiviral drugs.
In HCV infection, elevated ALT in the presence of a positive HCV RNA
assay is an indication for treatment initiation. Highly sensitive
qualitative and quantitative HCV RNA tests are useful to monitor
response to therapy and document resolved infection. Quantitative RNA
tests can also help predict the likelihood of response and select the
appropriate duration of therapy. The HCV genotype assay is important
for selecting the appropriate duration of therapy and evaluating the
likelihood of treatment response.
Screen for Immunity or Successful Vaccination
HAV total antibody, HBsAb, or HBc total antibody assays are generally
recommended to determine immune status following infection, or pre-
or post-vaccination.
208

Section 8 Infectious Disease

Table 3. Clinical Application of Laboratory Tests for Hepatitis

Test Clinical Application

Alanine Aminotransferase (ALT) Indicate hepatic disease
Monitor response to antiviral therapy
Indicate resolution of HAV or HEV infection
HAV Antibody (IgM) First-line diagnostic test for acute hepatitis A
HAV Total Antibody Screen for immunity prior to vaccination
HBV Core Antibody (IgM) First-line diagnostic test for acute hepatitis B
Indicate recent infection (within preceding 4-6 months)
HBV Core Total Antibody (IgG + IgM) Indicate current or prior infection
HBV DNA, Qualitative Determine need to treat chronic HBV infection
Indicator of chronic hepatitis when still positive 6 months after diagnosis of acute HBV infection
Monitor response to therapy
Demonstrate viral replication in patients with mutant HBV (eg, HBeAg- and HBeAb+ individuals)
HBV DNA, Quantitative All indications listed for qualitative HBV DNA assay above
Predict likelihood of response to therapy
Indicate emergence of resistant variants during antiviral therapy
HBV e Antibody Indicate treatment response
HBV e Antigen Indicate active viral replication and high infectivity
HBV Drug Resistance, Genotype, Detect HBV mutations associated with resistance to antiviral agents
and BCP/Precore Mutations Predict and monitor response to therapy
Identify HBV genotype (A–G) for epidemiologic and prognostic purposes
Detect mutations in precore and basal core promoter regions, which may influence immune response and outcome
HBV Surface Antibody, Qualitative Indicate immunity post-infection, vaccination, or HBIG
HBV Surface Antibody, Quantitative Indicate immunity post-infection, vaccination, or HBIG; high levels are suggestive of a protective response
HBV Surface Antigen First-line diagnostic test for acute hepatitis B
Indicates chronic hepatitis when still positive 6 months after diagnosis of acute HBV infection
HBV Surface Antigen Confirmation Routinely used to confirm positive HBV surface antigen tests
HCV Antibody, EIA First-line screening test for detection of acute and chronic hepatitis C
HCV Antibody, RIBA Confirm infection when the antibody and RNA tests are discrepant and clinical suspicion is high; negative
result required for re-entry to blood donor pool
HCV Genotyping Predict likelihood of therapeutic response
Determine the duration of treatment
HCV RNA, Qualitative Detect acute infection prior to seroconversion (ie, within 1-2 weeks post-exposure)
Confirm EIA diagnosis of acute or chronic infection (LOD <50 IU/mL)
Differentiate between resolved and active infection
Demonstrate resolution of infection
HCV RNA, Quantitative Highly sensitive quantitative assays (at least as sensitive as relevant qualitative assay) only: all indications
listed for qualitative HCV RNA assay above
Predict response to antiviral therapy
Differentiate lack of therapeutic response from partial therapeutic response
HDV Antibody Diagnose HDV infection in patients with fulminant hepatic failure or known previous HBV infection
HDV Antigen Diagnose HDV infection in symptomatic, HBsAg-positive, HDV antibody-negative individuals
HEV Antibody (IgG) Demonstrate recent or past hepatitis E infection
HEV Antibody (IgM) Diagnose acute HEV infection
HBIG, hepatitis B immune globulin; LOD, limit of detection.

Multiple tests may be required to completely characterize an individual Table 3. Figures 1–3 provide algorithms for the use of diagnostic assays
patient’s infection. To further clarify the role of the available viral in the diagnosis and management of viral hepatitis.
hepatitis tests, specific clinical applications for each are addressed in
 209

Section 8 Infectious Disease

Table 4. Diagnosis of Acute Hepatitis

HAV HBV HBc HBc HCV HCV HDV HDV HEV HEV
Hepatitis Virus IgM HBsAg DNA IgM Total Ab RNA Ag Ab IgM IgG
A + – – – – – – – – – –
B – + + + – – – – – – –
B with D coinfection – + + + – – – + + – –
B with D superinfection – + + – – – – + + – –
C – – – – – + + – – – –
E – – – – – – – – – + –/+

Test Interpretation: A negative antibody test result indicates lack of
immunologic response to that type of hepatitis virus. False-negative
results may occur in early-stage acute disease (prior to seroconversion)
or in patients with a suppressed or non-functioning immune system. If
clinical suspicion is high, negative results can be verified by testing for
type-specific antigen, DNA, or RNA, as appropriate.
If an antibody test is positive, the patient has generated an immune
response to that type of hepatitis virus and should be evaluated further
with type-specific supplemental testing (eg, antigen, DNA, or RNA
testing). In an infant less than 18 months of age, a positive antibody
test result may indicate passive transfer of maternal antibody. Testing
with a type-specific antigen or nucleic acid-based assay may reveal
active infection. Additional antibody-specific interpretive information
follows:
• Presence of total HAV antibody, in the absence of HAV IgM antibody,
indicates immunity against HAV infection.
• HBc IgM antibody positivity usually indicates HBV infection within
the preceding 4 to 6 months.
• HBc IgG antibody positivity indicates prior HBV infection and may be
associated with chronic or resolved infection.
• HBeAb presence indicates resolving infection or response to therapy.
• HBsAb presence indicates immunity against HBV infection.
• A positive HDVAb test, coincident with the presence of HBsAg,
indicates HBV/HDV coinfection.
• False-positive HCV antibody results may be generated by the EIA in
cases of alcoholic liver disease, autoimmune chronic active hepatitis,
or improper sample storage and testing conditions. A positive HCV
RNA test result confirms a positive EIA result. In the presence of a
positive EIA result, a negative HCV RNA result should be confirmed
by repeat RNA assay or RIBA.
• A negative RIBA excludes HCV as the cause of a positive EIA result
and is required for re-entry into the blood donor pool.
A negative HBsAg, HBV DNA, or HCV RNA test result indicates lack of
current infection. In rare cases, a negative result reflects a viral load
below the assay’s limit of detection. Thus, a negative result does not
exclude the possibility of infection. Repeatedly positive antigen, DNA, or
RNA test results, on the other hand, indicate active infection. A return to a
negative test result following therapy indicates resolution of the infection.
After resolution, reappearance of a marker may indicate a relapse.
HBsAg presence does not reflect the level of active virus, nor does it
differentiate between acute and chronic infection or between mild and
severe disease. False-positive results are uncommon, but when they
occur they are generally due to technical limitations of the test or may
occur in young infants post-immunization. In low-risk populations, the
HBsAg confirmation test can help verify a repeatedly reactive result as a
true positive.
Relatively low levels of HBV DNA or HCV RNA are associated with
acute or resolving infection and with the probability of a sustained
therapeutic response; high levels predict lack of therapeutic response. In
general, a stable or rising viral load is indicative of lack of therapeutic
response, while falling levels indicate that the patient is responding to
treatment. Failure to achieve a r2 log10 IU/mL decline in HCV viral load
from baseline by week 12 indicates a low likelihood of sustained
response. Persistent detection of HCV RNA at the end of
interferon/ribavirin combination therapy (3 months for genotypes 1 or 3
or 6 months for genotype 1) indicates that sustained response to
therapy is unlikely. DNA and RNA levels need to be interpreted in
combination with all available clinical, biochemical, and liver biopsy
information.
Even after apparent recovery from HBV infection, viable virus may
remain in the liver where it can be detected in biopsy material, can
infect a transplant recipient who is HBcAb negative, and may even
cause recurrent HBV disease in individuals with profound
immunosuppression.
Tables 4-6 detail test result patterns and their associated clinical
significance.
References
1. Buti M, Sanchez F, Cotrina M, et al. Quantitative hepatitis B virus
DNA testing for the early prediction of the maintenance of response

Table 5. Diagnosis of Chronic Hepatitis

HBc HBc HBe HBV HCV HCV
Hepatitis Virus HBsAg HBsAb IgM Total HBeAg Ab DNA Ab RNA
B + – – + + – + -– –
C – – – – – – – + +
210
Section 8
Table 6. Interpretation of Hepatitis B Markers
Chronic
Acute Acute (Low
Marker (Early) (Resolving) Infectivity)
HBc IgM Ab ++ + –
HBc total Ab + + ++
HBsAb – – – –
HBsAg + + + +
HBV DNA, quantitative + –c – +
HBeAb – p p –
HBeAg + – – p
NA, not applicable.
a
Present for at least 6 months; should be undetectable 1 year after acute infection.
b
< 105 copies/mL.
c
Very low levels may be detected with highly sensitive assays.
Adapted from references 6, 7, and 9.
during lamivudine therapy in patients with chronic hepatitis B. J
Infect Dis. 2001;183:1277-1280.
2. EASL International Consensus Conference on Hepatitis C. Paris, 26-
28 February, 1999, Consensus Statement. European Association for
the Study of the Liver. J Hepatol. 1999;30:956-961.
3. Hepatitis Resource Network. Hepatitis C treatment algorithm.
Available at http://www.h-r-n.org. Accessed January 9, 2002.
4. Hoofnagle JH, di Bisceglie AM. Serologic diagnosis of acute and
chronic viral hepatitis. Semin Liver Dis. 1991;11:73-83.
5. Hu KQ, Vierling JM. Molecular diagnostic techniques for viral
hepatitis. Gastroenterol Clin North Am. 1994;23:479-498.
6. Kao JH, Chen PJ, Lai MY, et al. Genotypes and clinical phenotypes
of hepatitis B virus in patients with chronic hepatitis B virus
infection. J Clin Microbiol. 2002;40:1207-1209.
7. Kao JH, Wu NH, Chen PJ, et al. Hepatitis B genotypes and the
response to interferon therapy. J Hepatol. 2000;33:998-1002.
8. Lindh M, Hannoun C, Dhillon AP, et al. Core promoter mutations and
genotypes in relation to viral replication and liver damage in East
Asian hepatitis B virus carriers. J Infect Dis. 1999;179:775-782.
9. Lok AS, McMahon BJ; Practice Guidelines Committee, American
Association for the Study of Liver Diseases. Chronic hepatitis B.
Hepatology. 2001;34:1225-1241.
10. National Guideline for the Management of the Viral Hepatitides A,
B, and C. Clinical Effectiveness Group (Association of Genitourinary
Medicine and the Medical Society for the Study of Venereal
Disease). Sex Transm Inf. 1999;75(Suppl 1):S57-S64.
11. National Institutes of Health Consensus Development Conference
Statement: Management of Hepatitis C: 2002. Preliminary Draft
Statement, June 12, 2002. Available at
http://odp.od.nih.gov/consensus/cons/116/116cdc_intro.htm.
Accessed June 24, 2002.
12. Noskin GA with the AMA Advisory Group on Prevention, Diagnosis,
and Management of Viral Hepatitis. Prevention, diagnosis, and
management of viral hepatitis. Arch Fam Med. 1995;4:923-934.
13. Ohkubo K, Kato Y, Ichikawa T, et al. Viral load is a significant
prognostic factor for hepatitis B virus-associated hepatocellular
carcinoma. Cancer. 2002;94:2663-2668.
14. Puchhammer-Stokl E, Mandl C, Kletzmayr J, et al. Monitoring the
virus load can predict the emergence of drug-resistant hepatitis B
virus strains in renal transplantation patients during lamivudine
therapy. J Infect Dis. 2000;181:2063-2066.
15. Recommendations for prevention and control of hepatitis C virus
Infectious Disease
Chronic Inactive
(High HBsAg Successful
Infectivity) Carrier State Resolved Vaccination
– NA – –
++ NA + –
NA p +
pa – –
pb –c –
+ p –
– – –
(HCV) infection and HCV-related chronic disease. Centers for
Disease Control and Prevention. MMWR. 1998;47(RR-19):1-39.
16. San Francisco and Miami Centers of Excellence in Hepatitis C
Research and Education and the Hepatitis C Technical Advisory
Group, Department of Veterans Affairs. Treatment recommendations
for patients with chronic hepatitis C: 2001 Version 1.0. Available at
http://www.va.gov/hepatitisc/pved/treatmntgdlnes_00.htm.
Accessed December 6, 2001.
17. Sarrazin C, Teuber G, Kokka R, et al. Detection of residual hepatitis
C virus RNA by transcription-mediated amplification in patients with
complete virologic response according to polymerase chain reaction-
based assays. Hepatology. 2000;32:818-823.
18. World Health Organization, Department of Communicable Disease
Surveillance and Response. Hepatitis delta [WHO Web site, 2001].
Available at http://www.who.int/csr/disease/hepatitis/
HepatitisD_whocdscsrncs2001_1.pdf. Accessed June 24, 2002.
19. Yang G, Vyas GN. Immunodiagnosis of viral hepatitides A to E and
non-A to -E. Clin Diagn Lab Immunol. 1996;3:247-256.
8.3.2 Hepatitis B
8.3.2.1 Hepatitis B Virus Drug Resistance, Genotype, and
BCP/Precore Mutations
Clinical Use: This test is used to confirm resistance-associated
mutation(s) as a potential cause of virologic breakthrough; assist with
selection of patient-specific therapy after virologic breakthrough; and
assess prognosis.
Clinical Background: Treatment of chronic hepatitis B (CHB) is
geared to achieving sustained suppression of hepatitis B virus (HBV)
replication and remission of liver disease, with the aim of preventing
liver failure, cirrhosis, and hepatocellular carcinoma.1 Several drugs
have been approved for treatment, including interferon-alfa,
peginterferon alfa-2, and the nucleotide/nucleoside analogs (NAs)
lamivudine (3TC), adefovir dipivoxil (ADV), entecavir (ETV), and
telbivudine (LdT). Mutations in the HBV genome, and the HBV genotype
itself, may affect treatment response and disease course.
Treatment with NAs can yield sustained response in patients with HBV
infection. However, prolonged therapy may select for mutations in the
HBV DNA polymerase gene (including M204V in the YMDD motif),

Section 8
leading to drug resistance (Table 7). The rate of acquired resistance
varies with the NA used and baseline viral load, among other factors.
During treatment with NAs, viral load should be assessed every 12 to 24
weeks with a quantitative HBV DNA assay.1 In patients with confirmed
treatment adherence who exhibit virologic breakthrough or rebound,
genotypic resistance testing can be used to confirm the presence of
mutations1 and therefore guide selection of subsequent therapy.
In addition to detecting resistance-associated mutations, this assay also
assesses HBV genotype (A–H) and mutations in the precore (pre-C) and
basal core promoter (BCP) regions. HBV genotypes A–H are classified
according to sequence variations in the HBV “S” gene and have distinct
geographic distributions. Genotype analysis may have prognostic value.
Genotype C has been associated with more severe liver disease than
genotype B,2,3 although contradictory findings have been reported.
Genotype has also been associated with response to interferon-based
therapy.4-6
Mutation at nucleotide 1896 of the pre-C region (TAG mutation)
abolishes production of the HBeAg, leading to HBeAg-negative CHB,
while mutations at nucleotides 1762 and 1764 of the BCP region may
play a role in HBeAg clearance. The pre-C and BCP mutations also
appear to influence response to interferon treatment.7 Testing for these
mutations may therefore have prognostic value.
Individuals Suitable for Testing include patients with diagnosed
CHB.
Method: This assay uses polymerase chain reaction with HBV-specific
primers to amplify relevant portions of the HBV genome. Sequence data
are analyzed for mutations at codons 169, 173, 180, 181, 184, 194, 202,
204, 207, 236, and 250 in the reverse transcriptase region of the
polymerase gene; nucleotide 1896 of the pre-C region; and nucleotides
1762 and 1764 of the BCP region. The HBV genotype is determined by
computer-aided alignment and phylogenetic analysis of the amplified
portion of the S gene. The analytical sensitivity of this assay is 1,000 HBV
copies/mL (567 IU/mL) plasma; 500 HBV copies/mL (298 IU/mL) serum.
There is no known cross-reaction with other blood-borne pathogens.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: Mutations associated with resistance to
available NAs are listed in Table 7. The presence of such mutations in
patients with virologic breakthrough or rebound suggests the need to add
or replace a drug in the regimen. Practice guidelines from the American
Association for the Study of Liver Diseases provide detailed information
on treatment options for patients with confirmed resistance to an NA.1
At present, the effects of HBV genotype (A–H) on disease course and
treatment response are not understood well enough to influence
treatment decisions.
Detection of BCP mutations at nucleotides 1762 and 1764 is associated
with low levels of HBeAg and may predict a more favorable response to
Table 7. HBV DNA Polymerase Gene Mutations Associated with Resistance to Nucleos(t)ide Analogs
Lamivudine (3TC) Adefovir (ADV)
Mutation(s) L180M+M204V/I; A181T N236T; A181V
a
Associated with reduced susceptibility to ETV when combined with lamivudine-associated mutations.
211
Infectious Disease
interferon treatment. The G1896A pre-C variant, which abrogates
HBeAg formation, is associated with lower rates of interferon response.
Patients with mutations in either region can return to high levels of HBV
viremia after loss of HBeAg.7
References
1. Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology. 2007;45:507-
539.
2. Kao JH, Chen PJ, Lai MY, et al. Genotypes and clinical phenotypes of
hepatitis B virus in patients with chronic hepatitis B virus infection. J
Clin Microbiol. 2002;40:1207-1209.
3. Lindh M, Hannoun C, Dhillon AP, et al. Core promoter mutations and
genotypes in relation to viral replication and liver damage in East
Asian hepatitis B virus carriers. J Infect Dis. 1999;179:775-782.
4. Bonino F, Marcellin P, Lau GK, et al. Predicting response to
peginterferon alfa-2a, lamivudine and the two combined for HBeAg-
negative chronic hepatitis B. Gut. 2007;56:699-705. Epub 2006 Nov
24.
5. Janssen HL, van Zonneveld M, Senturk H, et al. Pegylated interferon
alfa-2b alone or in combination with lamivudine for HBeAg-positive
chronic hepatitis B: a randomised trial. Lancet. 2005;365:123-129.
6. Kao JH, Wu NH, Chen PJ, et al. Hepatitis B genotypes and the
response to interferon therapy. J Hepatol. 2000;33:998-1002.
7. Keeffe EB, Dieterich DT, Han SH, et al. A treatment algorithm for the
management of chronic hepatitis B virus infection in the United
States: an update. Clin Gastroenterol Hepatol. 2006;4:936-962.
8.3.3 Hepatitis C
8.3.3.1 Molecular Testing in the Management of Hepatitis C
Virus Infection
A flowchart showing molecular assays used to assist with diagnosis
and management of HCV infection is shown in Figure 4. The reportable
ranges and clinical applications of relevant molecular assays are
summarized in Tables 8–10.
References
1. San Francisco and Miami Centers of Excellence in Hepatitis C
Research and Education and the Hepatitis C Technical Advisory
Group, Department of Veterans Affairs. Treatment recommendations
for patients with chronic hepatitis C: 2001 Version 1.0. Available at
http://www.va.gov/hepatitisc/pved/treatmntgdlnes_00.htm.
Accessed December 6, 2001.
2. EASL International Consensus Conference on Hepatitis C. Paris, 26-28
February, 1999, Consensus Statement. European Association for the
Study of the Liver. J Hepatol. 1999;30:956-961.
3. Hepatitis Resource Network. Hepatitis C treatment algorithm.
Available at http://www.h-r-n.org. Accessed January 9, 2002.
4. Sarrazin C, Teuber G, Kokka R, et al. Detection of residual hepatitis C
virus RNA by transcription-mediated amplification in patients with
complete virologic response according to polymerase chain reaction-
based assays. Hepatology. 2000;32:818-823.
5. Fried MW, Shiffman ML, Reddy RK, et al. Pegylated (40 kDa)
interferon alpha-2a (PEGASYS) in combination with ribavirin: efficacy
and safety results from a phase III randomized, controlled, actively
Entecavir (ETV) Telbivudine (LdT)
M250V; T184G; S202Ia M204I; L180M+M204V
212

Section 8 Infectious Disease

 213

Section 8 Infectious Disease

Table 8. Quantitative HCV RNA Assays

Test Code Reportable Range, Specimen

(CPT Codea) Test Name HCV RNA IU/mL Requirements Clinical Applications
10565X HEPTIMAX®b HCV RNA 5–50,000,000 3 mL (min. 1.0 mL) plasma in Confirm infection; establish viral load at
(87522) PPT-potassium EDTA (white-top baseline; monitor viral load during
tube). Ship frozen. therapy; determine duration of
treatment; assess likelihood of non-
response during treatment; assess
likelihood of non-sustained response at
end of treatment.
35645X Hepatitis C Viral RNA, 50-50,000,000 2 mL (min. 0.5 mL) plasma in Establish viral load at baseline; monitor
(87522) Quantitative Real-Time PPT-potassium EDTA (white- viral load during therapy; determine
PCRb top tube). Ship frozen. duration of treatment; assess likelihood
of non-response during treatment.
10073X Hepatitis C Viral RNA, 5–7,500 2 mL (min. 0.6 mL) plasma in Confirm infection; assess likelihood of
(87522) Quantitative TMAb PPT-potassium-EDTA (white- non-sustained response at end of
top tube). Ship frozen. treatment.
29271X Hepatitis C Viral RNA, 615–7,700,000 1 mL (min. 0.2 mL) plasma in Establish viral load at baseline; monitor
(87522) Quantitative bDNA potassium EDTA (white-top tube) viral load during therapy; determine
or no-additive serum-separator duration of treatment; assess likelihood
tube. Ship frozen. of non-response during treatment.
11348X Hepatitis C Viral RNA, 50-50,000,000 3 mL (min. 1.1 mL) plasma in Establish viral load and genotype at
(87522) Quantitative PCRb with (Reflex to genotype if PPT-potassium EDTA (white- baseline; determine duration of
Reflex to Genotype, >300 IU/mL) top tube). Ship frozen. treatment and likelihood of response.
LiPAb, c
19702X HEPTIMAX® HCV RNA 5–50,000,000 3.2 mL (min. 1.6 mL) plasma in Establish viral load and genotype at
(87522) with Reflex to Genotype, (Reflex to genotype if PPT-potassium EDTA (white- baseline; determine duration of
LiPAb, c >300 IU/mL) top tube). Ship frozen. treatment and likelihood of response.
a
The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions
regarding coding to the payor being billed.
bThis test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the US Food and Drug

Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
cReflex tests are performed at additional charge and are associated with an additional CPT code(s).

Table 9. Qualitative HCV RNA Assays

Test Code Limit of Detection, Specimen

(CPT Codea) Test Name (LOD), IU/mL Requirements Clinical Applications
37273X Hepatitis C Viral RNA, 10 2 mL (min. 0.6 mL) plasma in Confirm presence of chronic HCV
(87521) Qualitative TMA PPT-potassium EDTA (white-top infection; differentiate between resolved
tube). Ship frozen. and active infection.
34024X Hepatitis C Viral RNA, 50 2 mL (min. 0.6 mL) plasma in Confirm presence of chronic HCV
(87521) Qualitative PCR PPT-potassium EDTA (white-top infection; differentiate between resolved
tube). Ship frozen. and active infection.
a
The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions
regarding coding to the payor being billed.

Table 10. HCV Genotype Assay

Test Code Specimen

(CPT Codea) Test Name Limit of Detection Requirements Clinical Applications
37811X Hepatitis C Viral RNA Minimum viral load: 2 mL (min. 0.6 mL) plasma in Assess length of treatment required and
(87902) Genotype, LiPAb 300 IU/mL PPT potassium EDTA (white-top likelihood of response (used at
tube). Ship frozen. baseline).
a
The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions
regarding coding to the payor being billed.
b
This test was developed and its performance characteristics determined by Quest Diagnostics Nichols Institute. It has not been cleared or approved by the US Food and Drug
Administration. The FDA has determined that such clearance or approval is not necessary. Performance characteristics refer to the analytical performance of the test.
214
Section 8
controlled, multicenter study. Gastroenterology. 2001;120 (Suppl 1):A-
55. Abstract 289.
6. National Institutes of Health. Consensus Development Conference
Statement. Management of Hepatitis C: 2002. Available at
http://consensus.nih.gov/cons/116/116cdc_intro.htm. Accessed
January 8, 2003.
7. Alter MJ, Kuhnert WL, Finelli L; Centers for Disease Control and
Prevention. Guidelines for laboratory testing and result reporting of
antibody to hepatitis C virus. Centers for Disease Control and
Prevention. MMWR Recomm Rep. 2003;52(RR-3):1-16.
8.3.3.2 Hepatitis C Viral RNA Genotype, LiPA
Clinical Use: This test is used to determine whether to begin antiviral
therapy in patients with chronic hepatitis C virus (HCV) infection; predict
response to therapy; assess need for liver biopsy; and determine
duration and dosage of treatment.
Clinical Background: Hepatitis C virus (HCV) is a major cause of
hepatic disease and the leading reason for liver transplantation in the
United States. Acute HCV infection is often asymptomatic and usually
goes undiagnosed. However, 60% to 85% of patients develop chronic
infection, which is associated with increased risk of cirrhosis, end-stage
liver disease, and hepatocellular carcinoma.1,2
Antiviral therapy with pegylated interferon and ribavirin is aimed at
slowing disease progression and limiting hepatic complications,1,2 but
can also cause adverse effects. Thus, appropriate patient selection is
necessary to balance the potential benefits against the potential risk of
treatment. HCV genotype is the strongest predictor of sustained
virologic response to current therapy3,4 and may therefore play a role in
deciding whom to treat.
Of the 6 major genotypes and more than 50 subtypes of HCV,5
genotypes 1a/1b, 2b, and 3a are the most common in the United
States.6,7 Patients with genotype 1 infection have markedly lower
response rates than those with genotype 2 or 3, require a higher dosage
of ribavirin, and may benefit from a longer course of therapy.1,2 Given
their lower response rates, biopsy may be especially relevant for
patients with genotype 1 infection.
Previous versions of the HCV line probe assay (LiPA) were based solely
on variations in the 5’ untranslated region (UTR) of the HCV genome.
However, the current version analyzes the core region as well, which
improves accuracy by providing more precise distinction between
subtypes 6c-6l and 1a/1b.8
Individuals Suitable for Testing include patients with detectable
hepatitis C viral RNA (>300 IU/mL) who are being evaluated for
treatment.
Method: This assay uses reverse transcription and polymerase chain
reaction (RT-PCR) of specific regions of the HCV genome, followed by
line probe assay (LiPA) with genotype-specific probes from the HCV core
region and 7 regions of the HCV 5’ UTR. It distinguishes among major
types and most common subtypes of HCV: 1, 1a, 1b, 1a/b; 2, 2a/c, 2b; 3,
3a, 3b, 3c, 3k; 4, 4a/c/d, 4b, 4e, 4f, 4h; 5, 5a; and 6, 6a/b, 6c-l. This
assay does not distinguish between 2a and 2c; 4c and 4d; or 6a and 6b.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the tests.
Infectious Disease
Interpretive Information: Genotype 1 is associated with a relatively
low (42% to 46%) response rate to peginterferon alfa and ribavirin.3,4 A
higher ribavirin dosage is recommended for these patients, and they
typically require 48 weeks of therapy for a sustained response even if
the virus is undetectable at 24 weeks.1,2 However, those who show
rapid virologic response (<50 IU/mL HCV RNA at 4 weeks) and tolerate
therapy poorly may be considered for discontinuation at 24 weeks.2
Failure to achieve a r2 log10 drop in viral load by week 12 indicates
that further treatment is not likely to be effective and may be
discontinued.1 However, the decision to stop treatment should also take
into account the tolerability of therapy and other clinical factors.
Genotypes 2 and 3 are associated with a relatively high response rate
(76% to 82%). Twenty-four weeks of treatment is generally sufficient.1,2,9
Treatment decisions should also take into account additional factors
that may influence therapeutic response, such as pretreatment HCV viral
load and cirrhosis status, sex and age of the patient, and other clinical
and laboratory findings.
References
1. Strader DB, Wright T, Thomas DL, Seeff LB; American Association for
the Study of Liver Diseases. Diagnosis, management, and treatment
of hepatitis C. Hepatology. 2004;39:1147-1171. Erratum in:
Hepatology. 2004;40:269.
2. Department of Veterans Affairs Hepatitis C Resource Center; Yee HS,
Currie SL, Darling JM, et al. Management and treatment of hepatitis
C viral infection: recommendations from the Department of Veterans
Affairs Hepatitis C Resource Center program and the National
Hepatitis C Program office. Am J Gastroenterol. 2006;101:2360-2378.
3. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus
ribavirin for chronic hepatitis C virus infection. N Engl J Med.
2002;347:975-982.
4. Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b
plus ribavirin compared with interferon alfa-2b plus ribavirin for initial
treatment of chronic hepatitis C: a randomised trial. Lancet.
2001;358:958-965.
5. Simmonds P, Holmes EC, Cha T-A, et al. Classification of hepatitis C
virus into six major genotypes and a series of subtypes by
phylogenetic analysis of the NS-5 region. J Gen Virol. 1993;74:2391-
2399.
6. Zein NN, Rakela J, Krawitt EL, et al. Hepatitis C virus genotypes in
the United States: epidemiology, pathogenicity, and response to
interferon therapy. Collaborative Study Group. Ann Intern Med.
1996;125:634-639.
7. Zein NN. Clinical significance of hepatitis C virus genotypes. Clin
Microbiol Rev. 2000;13:223-235.
8. Noppornpanth S, Sablon E, De Nys K, et al. Genotyping hepatitis C
viruses from Southeast Asia by a novel line probe assay that
simultaneously detects core and 5’ untranslated regions. J Clin
Microbiol. 2006;44:3969-3974.
9. Dienstag JL, McHutchison JG. American Gastroenterological
Association technical review on the management of hepatitis C.
Gastroenterology. 2006;130:231-264.
8.3.3.3 Hepatitis C Viral RNA, Qualitative
Clinical Use: These tests are used to confirm hepatitis C virus (HCV)
infection and demonstrate resolution of infection.
Clinical Background: These qualitative HCV RNA tests detect the
presence of HCV circulating in the blood. They are used to confirm HCV

Section 8
diagnosis following a positive or indeterminate antibody test result.
These tests differentiate between resolved and active infection and may
be useful for detecting acute infection prior to seroconversion. They are
especially useful for confirming diagnosis in people with indeterminate
HCV immunoblot (RIBA) results, as well as in immunosuppressed or
immunoincompetent individuals.
Methods
PCR: In this polymerase chain reaction (PCR) method, viral RNA is
reverse-transcribed to cDNA and amplified with biotin-labeled HCV-
specific primers. Amplification products are then hybridized to HCV-
specific capture probes and detected with an avidin-HRP conjugate in a
colorimetric assay. This test detects HCV genotypes 1a and 1b; the
ability to detect other genotypes is currently unknown. Analytical
sensitivity is 50 IU/mL.
TMA: In this transcription-mediated amplification (TMA) method, RNA
is extracted from the patient sample and amplified by utilizing 2
enzymes (reverse transcriptase and T7 RNA polymerase) to cycle
between RNA and DNA intermediates, resulting in several billion RNA
amplicons. These amplicons are detected via hybridization protection
assay (HPA) in which only hybridized probes remain chemiluminescent
and are detected in a luminometer. Analytical sensitivity is 10 IU/mL.
Interpretive Information: A “detected” result with either method
indicates the presence of HCV RNA and is consistent with active
infection (acute or chronic). A “not detected” result, on the other hand,
is suggestive of the absence of detectable HCV RNA. False-negative
results might occur due to heparin therapy or HCV RNA levels below the
detectable limit of the assay. Because of the sensitivity of the TMA
method, some false-positive results may occur, particularly in specimens
that have been contaminated through aliquotting or some other
procedure. Six months following cessation of antiviral therapy, a
repeatedly negative test suggests clearance of the virus and recovery
from the infection. HCV RNA tests sensitive to 10 IU/mL or less are
better indicators of resolved infection than are tests with lesser
sensitivity.
8.3.3.4 Hepatitis C Viral RNA, Quantitative
Clinical Use: These tests are used to confirm active hepatitis C virus
(HCV) infection; monitor response to antiviral therapy; and confirm
resolution of infection (sustained virologic response, SVR). Note that,
because of its narrow linear range, the quantitative TMA is generally
not appropriate for establishing baseline viral load or early virologic
response (EVR) at week 12 of treatment. Conversely, the sensitivity of
the quantitative bDNA assay may not be adequate to establish rapid
virologic response (RVR) at 4 weeks, resolution of infection, or SVR.
Clinical Background: HCV infection is a major cause of hepatic
disease and the leading reason for liver transplantation in the United
States. Acute HCV infection is often asymptomatic and usually goes
undiagnosed. However, 60% to 85% of patients develop chronic
infection, which is associated with increased risk of cirrhosis, end-stage
liver disease, and hepatocellular carcinoma.1,2 Detection and
quantitation of HCV RNA is an important component of diagnosis and
treatment monitoring.
Individuals with suspected HCV infection are typically first tested for
HCV antibodies using an enzyme immunoassay (EIA); a sensitive HCV
RNA assay may be performed to document active infection in patients
with positive EIA results. Although qualitative HCV RNA tests are often
used at this step, a quantitative assay is necessary to establish the
215
Infectious Disease
baseline viral load (concentration of HCV in plasma) before beginning
treatment.
After treatment is initiated, measurement of HCV viral load at specified
times helps predict the likelihood of SVR and guide treatment
decisions.1,2 The same quantitative test should be used each time to
avoid technology-related variability.1,2 Rapid virologic response (RVR;
undetectable HCV RNA 4 weeks after treatment initiation) and EVR (r2
log10 IU/mL decline from baseline by week 12) are predictors of SVR and
help determine the duration of therapy. Viral load measurement at 24
weeks (genotypes 2 and 3) or 48 weeks (genotype 1) documents end-of-
treatment response (ETR).3 Twenty-four weeks after the end of
treatment, HCV RNA should be monitored to assess SVR. An HCV RNA
assay sensitive to at least 50 IU/mL is recommended to document both
ETR and SVR.2
Methods: Quest Diagnostics offers 3 technologies for quantitative HCV
RNA testing: real-time polymerase chain reaction (PCR), branched DNA
(bDNA) signal amplification, and transcription-mediated amplification
(TMA). Several combinations of these platforms are available, each with
different sensitivities and linear ranges. HEPTIMAX offers the greatest
sensitivity and broadest linear range of the available options (See
Molecular Testing in the Management of Hepatitis C Virus Infection,
section 8.3.3.1, for more information).
bDNA: In this branched DNA (bDNA) signal amplification method, viral
RNA is captured on the surface of a microtiter well by synthetic
oligonucleotides coating the well. The solid phase bound viral RNA is
hybridized to multiple branched DNA molecules. Alkaline phosphatase
labeled probes are in turn hybridized to the branched DNA and reacted
with a chemiluminescent substrate to produce a greatly amplified light
signal. The amount of light emitted is directly proportional to the
quantity of HCV RNA.
This method demonstrates equal quantification of HCV genotypes 1-6.
The linear range is 615 to 7,700,000 IU/mL.
Real-time PCR: Viral RNA is reverse-transcribed to cDNA and
amplified with HCV-specific primers and a fluorescent dye-labeled
probe. Amplification products are detected by measuring fluorescent
signals generated during the PCR. Viral copy numbers are quantified by
comparing fluorescent signals generated in the specimen to those
produced by calibration standards. The linear range of this assay is 50
to 50,000,000 IU/mL.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
TMA: Viral RNA is extracted from the patient sample and amplified by
utilizing 2 enzymes (reverse transcriptase and T7 RNA polymerase) to
cycle between RNA and DNA intermediates, resulting in several billion
RNA amplicons. These amplicons are detected via hybridization
protection assay (HPA) in which only hybridized probes remain
chemiluminescent and are detected in a luminometer. The linear range
is 5 to 7500 IU/mL. This assay is generally not appropriate for
establishing baseline viral load or EVR at week 12 of treatment
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
216
Section 8
HEPTIMAX: The HEPTIMAX test begins with the HCV RNA quantitative
real-time PCR method. If the HCV RNA level is below 50 IU/mL, then the
sample is assayed again using the quantitative TMA method. The
reportable range is 5 to 50,000,000 IU/mL.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
The following conversion factors are used when converting from
copies/mL to IU/mL:
PCR: Copies/mL = IU/mL x 2.7
TMA: Copies/mL = IU/mL x 5.2
bDNA: Copies/mL = IU/mL x 5.2
Individuals suitable for testing include those with reactive HCV
antibody EIA results and those who are being considered for treatment,
are receiving treatment, or have completed treatment for HCV infection.1
Interpretive Information
In patients with reactive EIA results, a negative HCV RNA result
typically indicates resolved infection but does not rule out carrier status;
negative results may also indicate a false-positive EIA result, false-
negative HCV RNA result, or intermittent viremia for a carrier. Results
r5 IU/mL for HEPTIMAX or quantitative TMA, r50 IU/mL for
quantitative PCR, or 615 IU/mL for bDNA suggest active HCV infection.
Results r7,500 IU/mL for TMA, r50,000,000 for HEPTIMAX or real-time
PCR, or r7,700,000 IU/mL for bDNA indicate that the HCV viral load is
above the linear range of the assay.
Monitoring Virologic Response
Treatment Week 4: An undetectable viral load at 4 weeks (ie, RVR; viral
load <50 IU/mL) indicates a high likelihood of SVR;2-5 some guidelines
have discussed shortening therapy in such cases.2,4
Treatment Week 12: Lack of EVR suggests a low (b3%) likelihood of
SVR; the decision to continue therapy is influenced by the goal of
treatment, stage of liver disease, viral genotype, and tolerability of drug
therapy, among other factors. Genotype 1 patients who have a slow
virologic response (HCV RNA detectable at 12 weeks, undetectable at
24 weeks) but tolerate therapy well may benefit from an additional 24
weeks of treatment beyond the standard 48 weeks (72 weeks total).2
Treatment Week 24: Detectable HCV RNA at week 24 indicates
treatment failure; further treatment is unlikely to yield SVR. Absence of
detectable HCV RNA at 24 weeks (<50 IU/mL) indicates resolved
infection in patients with genotype 2 or 3 infection, whereas patients
with genotype 1 infection generally require an additional 24 weeks of
treatment.
Follow-up: At 24 weeks after end of treatment, detectable HCV RNA
indicates relapse whereas undetectable levels (<50 IU/mL) indicate SVR.
Note that current guidelines are based largely on studies that used HCV
RNA tests sensitive to 50 IU/mL. The relevance of using results
between 5 IU/mL and 50 IU/mL (eg, quantitative TMA results) to
establish RVR, ETR, and SVR is not well established, although several
studies have indicated that detection of low-level viremia in this range
improves prediction of relapse or breakthrough viremia.6,7,8
Infectious Disease
References
1. Strader DB, Wright T, Thomas DL, et al; American Association for the
Study of Liver Diseases. Diagnosis, management, and treatment of
hepatitis C. Hepatology. 2004;39:1147-1171. Erratum in: Hepatology.
2004;40:269.
2. Department of Veterans Affairs Hepatitis C Resource Center; Yee HS,
Currie SL, Darling JM, et al. Management and treatment of hepatitis
C viral infection: recommendations from the Department of Veterans
Affairs Hepatitis C Resource Center program and the National
Hepatitis C Program office. Am J Gastroenterol. 2006;101:2360-2378.
3. Scott JD, Gretch DR. Molecular diagnostics of hepatitis C virus
infection: a systematic review. JAMA. 2007;297:724-732.
4. Dienstag JL, McHutchison JG. American Gastroenterological
Association technical review on the management of hepatitis C.
Gastroenterology. 2006;130:231-264.
5. Mangia A, Santoro R, Minerva N, et al. Peginterferon alfa-2b and
ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med.
2005;352:2609-2617.
6. Sarrazin C, Hendricks DA, Sedarati F, et al. Assessment, by
transcription-mediated amplification, of virologic response in patients
with chronic hepatitis C virus treated with peginterferon alpha-2a. J
Clin Microbiol. 2001;39:2850-2855.
7. Kadam JS, Gonzalez SA, Ahmed F,et al. Prognostic significance of
hepatitis C virus RNA detection by transcription-mediated
amplification with negative polymerase chain reaction during therapy
with peginterferon alpha and ribavirin. Dig Dis Sci. 2007 Apr 4; [Epub
ahead of print].
8. Gerotto M, Dal Pero F, Bortoletto G, et al. Hepatitis C minimal
residual viremia (MRV) detected by TMA at the end of Peg-IFN plus
ribavirin therapy predicts post-treatment relapse. J Hepatol.
2006;44:83-87.
8.3.3.4 Liver Fibrosis Panel, HepaScore™
Clinical Use: This assay is used to predict extent of liver fibrosis in
individuals infected with hepatitis C virus (HCV).
Clinical Background: Treatment of chronic HCV infection can prevent
cirrhosis and reduce the likelihood of hepatocellular carcinoma (HCC).1
Not all patients are treated with antiviral therapy in the early days
following onset since only 20% to 30% of untreated people will develop
cirrhosis.1 Treatment decisions are based in part on the stage of liver
fibrosis, which marks the progression to cirrhosis. Individuals with
minimal fibrosis (ie, F0 or F1 on the METAVIR scoring system) are not
likely to develop advanced fibrosis in the short-term, even in light of
long-standing disease, and are typically monitored every 3 to 5 years.1
Individuals with significant fibrosis, ie, METAVIR score rF2, are at
increased risk of developing cirrhosis and are usually treated.1
The gold standard for determining the extent of fibrosis is liver biopsy.2
However, the procedure carries a moderate risk of complications,
including bleeding and a small risk of death.3,4 Moreover, because
fibrosis is not uniformly distributed in the liver and a biopsy only
samples 1/50,000th to 1/30,000th of the liver mass,4,5 cirrhosis is missed
in an estimated 15% to 30% of liver biopsies.5-7 The difficulties
associated with liver biopsy have led to interest in the development of
non-invasive testing to determine the degree of hepatic fibrosis.
Progressive degrees of fibrosis, and ultimately cirrhosis, are reflected in
alterations in blood levels of various biomarkers (eg, as fibrosis
increases, serum levels of B2-macroglobulin increase and those of
apolipoprotein A1 decrease). The knowledge of such alterations has led
to the development of predictive models based on clinically determined

Section 8
algorithms that utilize selected markers. One such model, the
HepaScore, is based on serum levels of B2-macroglobulin, hyaluronic
acid, gamma glutamyl transferase (GGT), and total bilirubin, along with
age and sex. In one study, a HepaScore r0.5 (possible range, 0-1.0) was
63% sensitive and 89% specific for the presence of significant fibrosis
(METAVIR rF2), while a score <0.5 was 88% sensitive and 74% specific
for excluding advanced fibrosis (METAVIR rF3).8 In Quest Diagnostics’
internal validation using paired liver biopsy and serum samples from
patients with HCV infection, the optimum cutoff was 0.55. Based on
data from the entire population, a score of r0.55 was 83% sensitive
and 65% specific for the presence of hepatic fibrosis METAVIR score
rF2.9 Using the same cutoff in a subset of subjects with more than 1
elevated analyte, the sensitivity and specificity increased to 88% and
69%, respectively.
Individuals Suitable for Testing include patients with chronic HCV
infection.
Method: This assay measures B2-macroglobulin by fixed-time
nephelometry; total bilirubin by a colorimetric method; GGT in an
enzymatic/colorimetric method; and hyaluronic acid with an enzyme
immunoassay. The report includes individual analyte concentrations, the
HepaScore, patient-specific probability of having each METAVIR score
(see Table 11 for examples), the PPV and probability of a false-positive
result when positive, and the NPV and probability of a false-negative
result when negative (see Interpretive Information).
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information:
• A HepaScore <0.55 is considered “negative” and indicates a
METAVIR score of F0 or F1.
• A HepaScore r0.55 is considered “positive” and indicates a
METAVIR score of F2 to F4.
• The patient-specific probability (P) of having a particular METAVIR
score is based on the HepaScore sensitivity and specificity and the
prevalence of liver fibrosis in either the entire population or the
subset thereof (population described in the Clinical Background
section).
• The negative predictive value (NPV, probability that a negative test
correctly indicates the absence of significant fibrosis) = P F0-F1.
• The probability of a false-negative result = 4(P F2 + P F3 + P F4).
• The positive predictive value (ie, PPV, probability that a positive test
correctly indicates significant fibrosis) = 4(P F2 + P F3 + P F4).
• The probability of a false-positive result = P F0-F1.
Table 11. Probability of Having a Specific METAVIR Score
METAVIR Score Example 1a Example 2a
Probability (%) Probability (%)
F0-F1 16.2 86.4
F2 12.0 11.9
F3 22.2 1.7
F4 49.6 0.0
a
See text for details of the example.
217
Infectious Disease
Example 1: Patient has a HepaScore of 0.9 (positive result) and more
than 1 elevated analyte level; prevalence of significant fibrosis
(METAVIR score r2) is 53%. The probability of having a specific
METAVIR score is presented in Table 11. The PPV is 83.8% and the
probability of a false positive is 16.2%.
Example 2: Patient has a HepaScore of 0.2 (negative result) and no
elevated analyte concentrations; prevalence of significant fibrosis is
50.1%. The probability of having a specific METAVIR score is presented
in Table 11. The negative predictive value is 86.4% and the probability
of a false negative is 13.6%.
This test is meant as a screening tool to avoid unnecessary biopsies.
The lowest and highest scores may obviate the need for a biopsy, while
intermediary scores should be interpreted in the overall clinical context
of the individual patient.
References
1. Strader D, Wright T, Thomas D, et al. Diagnosis, management, and
treatment of hepatitis C. Hepatology. 2004;39:1147-1171.
2. Dienstag J. The role of liver biopsy in chronic hepatitis C. Hepatology.
2002;36:S152-160.
3. McGill DB, Rakela J, Zinsmeister AR, et al. A 21-year experience with
major hemorrhage after percutaneous liver biopsy. Gastroenterology.
1990;99:1396-1400.
4. Cadranel JF, Rufat P, Degos F. Practices of liver biopsy in France:
results of a prospective nationwide survey. For the Group of
Epidemiology of the French Association for the Study of the Liver
(AFEF). Hepatology. 2000;32:477-481.
5. Poynard T, Ratziu V, Bedossa P. Appropriateness of liver biopsy. Can J
Gastroenterol. 2000;14:543-548.
6. Regev A, Berho M, Jeffers LJ, et al. Sampling error and intraobserver
variation in liver biopsy patients with chronic HCV infection. Am J
Gastroenterol. 2002;97:2614-2618.
7. Colloredo G, Guido M, Sonzogni A, et al. Impact of liver biopsy size
on histological evaluation of chronic viral hepatitis: the smaller the
sample, the milder the disease. J Hepatol. 2003;39:239-244.
8. Adams LA, Bulsara M, Rossi E, et al. Hepascore: an accurate
validated predictor of liver fibrosis in chronic hepatitis C infection.
Clin Chem. 2005;51:1876-1873.
9. Data on file at Quest Diagnostics Nichols Institute, San Juan
Capistrano, CA.
8.3.4 Hepatitis E
8.3.4.1 Hepatitis E Antibody (IgG)
Clinical Use: This assay is used in the diagnosis of acute hepatitis E
virus (HEV) infection and in the differential diagnosis of enteric hepatitis.
Clinical Background: Hepatitis E virus (HEV) is the major etiologic
agent of enterically transmitted non-A, non-B hepatitis in developing
countries. In the United States, it is usually diagnosed in recent
travelers to endemic areas (India, Asia, Africa, and Central America).
Like hepatitis A, HEV occurs in both sporadic and epidemic forms and
causes an acute, moderately severe but not chronic hepatitis that is
frequently cholestatic. Unlike hepatitis A virus, HEV progresses to fatal
fulminant hepatitis in at least 15% to 30% of pregnant patients,
especially in the third trimester. The infection may also manifest
without jaundice or be present subclinically.
Hepatitis E IgG antibodies have been detected in up to 93% of patients
during the acute phase. In most patients, IgG antibodies are short lived,
218

Section 8 Infectious Disease

often being undetectable 6 to 12 months after onset; however, duration
up to 4.5 years has been observed.
Method: In this enzyme immunoassay (EIA), anti-HEV antibodies in
patient serum bind to 2 recombinant HEV antigens coating the
polystyrene well. Following a wash to remove unbound material, HRP-
conjugated goat anti-human IgG is added to bind to the antigen-
antibody complexes. After a second wash, o-phenylenediamine (OPD)
substrate is added, producing a yellow-orange color. The amount of
color produced is directly proportional to the amount of antibody present
in the patient specimen. Results are reported as non-reactive or
repeatedly reactive. Aliases include anti-HEV and HEV antibody.
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.
Interpretive Information: A “reactive” result is associated with HEV
infection. Non-reactive results are associated with absence of HEV
infection, early HEV infection (prior to seroconversion), and past,
resolved HEV infection.
References
1. Anonymous. Hepatitis E among US travelers, 1989-1992. Morbid
Mortal Weekly Rep. 1992;42:1-4.
2. Bradley DW. Enterically-transmitted non-A, non-B hepatitis. Br Med
Bull. 1990;46:442-461.
3. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived
from a blood-borne non-A, non-B hepatitis genome. Science.
1990;244:359-361.
4. Dawson G, Gutierrez PR, Pilot-Matias T, et al. Recombinant antigens
and synthetic peptides for serodiagnosis of hepatitis E infection. In:
Nishioka K, Suzuki H, Mishiro S, et al, eds. Viral Hepatitis and Liver
Disease. Tokyo: Springer-Verlag; 1994:371-374.
5. Fields HA, Favorov MO, Margolis HS. The hepatitis E virus: a review.
Journal of Clinical Immunoassays. 1993;16:215-222.
6. Purdy MA, Krawczynski K. Hepatitis E. Gastroenterol Clin North Am.
1994;23:537-546.
7. Reyes GR, Purdy MA, Kim JP, et al. Isolation of a cDNA from the
virus responsible for enterically-transmitted non-A, non-B hepatitis.
Science.1990; 247:1335-1339.
8. Ticehurst J. Hepatits E virus. In: PR Murray, ed. Manual of Clinical
Microbiology. 6th ed. Washington, DC: ASM Press;1995:1056-1067.
9. Yang G, Vyas GN. Immunodiagnosis of viral hepatitides A to E and
non-A to -E. Clin Diagn Lab Immunol. 1996;3:247-256.
10. Yarbough PO, Tam AW, Fry KE, et al. Hepatitis E virus: identification
of type-common epitopes. J Virol. 1991;65:5790.
8.4 HIV
8.4.1 HIV-1 Infection: Markers for Diagnosing and Monitoring
Therapy
Several laboratory markers are available to provide diagnostic and
prognostic information and guide therapy for patients with HIV infection
(Table 12).
HIV Antibody: Detection of HIV antibodies is the most efficient, and
the only FDA cleared, method for determining whether an individual has
HIV infection. Currently available enzyme immunoassays (EIAs) have
analytical sensitivities and specificities that exceed 99% and 98%,
respectively. Because of the possibility of false-positive results and their
implications, all reactive EIA or other screening HIV tests must be
confirmed with the Western blot assay.

Table 12. Laboratory Tests for HIV Infection

Test
HIV-1/2 EIA
HIV-1 antibody Western blot;
HIV-2 antibody Western blot
Absolute CD4 lymphocyte count
CD4 lymphocyte percentage
HIV-1 RNA, quantitative
HIV-1 genotype
HIV-1 phenotype
HIV-1 virtual phenotype
HIV-1 DNA PCR, Qualitative
C2-Microglobulin
HIV-1 culture
Clinical Use
Screening tests for HIV antibodies. Sensitivity >99.9%. Repeatedly reactive results must be confirmed with
Western blot to diagnose HIV infection.
Confirmatory test for HIV infection. Specificity is >99% when combined with a reactive EIA result. Western blot
must be performed on same specimen as EIA (or other screening test); otherwise, specificity and sensitivity are not
predictable. Indeterminate Western blot results may occur with early HIV infection or because of non-specific
cross-reacting antibodies.
Most widely used predictor of HIV-1 progression and indicator of when to start treatment. Risk of progression is
high with CD4 count <200 cells/mm3. Best short-term predictor for development of opportunistic infections.
Useful in conjunction with CD4 count. Risk of progression is high with percentage <20%.
Most useful molecular test for determining prognosis and monitoring therapy.
Useful for detection and assessment of resistance to antiretroviral therapy; can assist in drug selection prior to
initial treatment or following therapeutic failure.
Useful for prediction of resistance to antiretroviral therapy; can assist in drug selection for acute infection prior to
initial treatment or following therapeutic failure.
Useful for detection and assessment of resistance to antiretroviral therapy; can assist in drug selection prior to
initial treatment or following therapeutic failure.
Useful for detection of HIV-1 in select circumstances (see text).
Cell surface protein indicative of macrophage-monocyte stimulation. Levels >3.5 mg/dL associated with rapid
progression of disease. Limited usefulness.
Clinical research tool.

Section 8
The Western blot is interpreted as positive, indeterminate, or negative
on the basis of the banding pattern (number and type of bands) on the
assay strip. The Western blot is considered positive if it exhibits
antibody reactivity with at least 2 of the following bands: p24 (gag
region core protein), gp41, gp120/160 (env region envelope
glycoproteins).
A negative HIV Western blot does not exclude the possibility of
infection, since the time frame for seroconversion is variable. Repeat
testing of a new specimen is recommended for persons with recent HIV
exposure. False-negative results can occur when the tests are
performed before seroconversion, when the patient is
immunosuppressed, and, rarely, late in the course of AIDS.
An indeterminate Western blot may represent incomplete HIV antibody
response or nonspecific reactivity. Most HIV-infected individuals with an
initial indeterminate Western blot result seroconvert within 1 month.
Thus, persons with an initial indeterminate Western blot result should
be retested for HIV infection >1 month later. Those with continued
indeterminate Western blot results after 1 month are not likely to have
HIV infection. In certain settings, and in collaboration with clinical and
laboratory specialists, nucleic acid testing (eg, viral RNA or proviral
DNA amplification method) may be helpful in resolving an initial
indeterminate Western blot3; however, nucleic acids assays are not
FDA-cleared for diagnosis of infection.
CD4: The CD4+ T-cell (CD4) count is the most valuable indicator of
immune status in HIV-infected patients and is the best available
predictor of the short-term risk of developing a new opportunistic
infection. In general, the risk of opportunistic infections and HIV-
associated malignancies increases as the CD4 count decreases. The
trend in counts is more important than any single value; a 30% or
greater change in the absolute CD4 count between tests, or a 3%
change in the CD4 percentage, is considered clinically significant.4
The CD4 count is also generally the most important factor (aside from a
history of an AIDS-defining illness or severe HIV infection-related
symptoms) in determining whether to start therapy. Prior to the start of
therapy, CD4 counts should be measured every 3 to 6 months. Treatment
is indicated if the count is <200 cells/mm3, regardless of the viral load.
Because of the importance of starting therapy before the CD4 count
falls below 200 cells/mm3, treatment is generally offered to patients
with CD4 values of 200 to 350 cells/mm3; however, other factors such
as readiness of the patient to begin therapy and potential drug toxicity
should also be taken into account. Treatment should generally be
deferred if the CD4 count remains >350 cells/mm3. In these patients, a
viral load >100,000 indicates the need for frequent CD4 testing (at least
every 3 months), but treatment would not generally be offered unless
the physician is following an “early treatment” approach.4
After treatment is initiated, the CD4 count should be measured every 3
to 6 months to help assess the immunologic response and the need to
initiate chemoprophylaxis.
CD4 counts exhibit substantial diurnal variation (counts are generally
lower in the morning) and may be transiently depressed by an
intercurrent illness. Because of the potentially wide variation, obtaining
2 measurements may be advisable when deciding to initiate therapy; a
third measurement would be required if the results are discordant.
HIV-1 RNA, Quantitative: Three HIV-1 viral load assays are FDA-
cleared for quantifying HIV-1 RNA in plasma (viral load): an HIV-1
reverse transcription-polymerase chain reaction (RT-PCR) assay
(Amplicor HIV-1 Monitor®’ test, version 1.5, Roche Diagnostics); a
219
Infectious Disease
nucleic acid sequence-based amplification (NASBA) assay (NucliSens®’
HIV-1 QT, bioMérieux); and a signal amplification nucleic acid probe—or
branched DNA (bDNA)—assay (VERSANT®’ HIV-1 RNA 3.0, Bayer
Corporation).
Viral load is an important predictor of outcome and indicator of
treatment response. Prior to treatment initiation, the viral load provides
information on disease progression and may be used to supplement CD4
data in determining whether to start therapy. After treatment is
initiated, a primary goal is to decrease the viral load below the limits of
detection of the available assays within 16 to 24 weeks: <50 copies/mL
for the Amplicor assay, <75 copies/mL for the VERSANT assay, and <80
copies/mL for the NucliSens assay. Thereafter, the viral load is useful in
assessing the continuing effectiveness of therapy.
In patients with a clinical syndrome and recent high-risk activity
consistent with acute HIV-1 infection who have negative or
indeterminate antibody test results, RNA detection may be used for
preliminary diagnosis of infection. Positive results must be confirmed
with antibody testing (EIA and Western blot) 2 to 4 months after the
initial negative or indeterminate antibody results.4
The recommended frequency of HIV-1 RNA measurement depends on
the stage of disease management4:
• Prior to treatment initiation: At the time of diagnosis and every 3 to 4
months thereafter.
• Start of treatment: Immediately prior to initiation of therapy and
again 2 to 8 weeks later; viral load should decrease by at least 1.0
log10 copies/mL.
• Change in regimen because of suboptimal viral suppression: 2 to 8
weeks after change; viral load should decrease by at least 1.0 log10
copies/mL.
• Change in regimen because of treatment toxicity or regimen
simplification: Some experts recommend that viral load be measured
at 2 to 8 weeks in this setting as well, to assess the effectiveness of
the new regimen.
• Continuing therapy: Every 3 to 4 months or when there is a clinical
event or significant change in CD4 count. A 3-fold (0.5 log10) change
in HIV-1 RNA viral load is considered clinically significant.
In the event of a clinically unexplained viral load increase, Quest
Diagnostics offers retesting of the same specimen. If the rise in viral
load is not confirmed by retesting the original specimen, or if the latter
is not available, we will test a newly collected sample at no additional
charge for quality control purposes.
HIV-1 Resistance Testing: The development of drug resistant HIV
variants is an important cause of virologic failure—that is, persistent
viremia in the presence of drug treatment. Resistance assays are useful
for selecting active drugs when changing regimens because of virologic
failure and, possibly, in cases of suboptimal reduction in viral load.4
Testing should be performed on samples obtained while the patient is
still receiving the failing regimen. If samples are taken after a drug is
withdrawn, resistant variants may not be detected but may re-emerge if
the drug is reinstated.
Because drug-resistant HIV-1 variants can be transmitted and may
affect response to the initial drug regimen, resistance testing (preferably
genotypic) is also recommended prior to therapy in individuals with
chronic or acute infection.4 Genotypic testing may also be useful before
therapy is required.
Quest Diagnostics offers 3 types of HIV-1 resistance tests.
220

Section 8 Infectious Disease

HIV-1 Genotype: Certain mutations in the genetic sequence of HIV-1 Table 13. Abbreviations of Antiretroviral Drug
are associated with resistance to antiretroviral drugs and can cause
therapeutic failure. HIV-1 genotyping identifies such mutations in Abbreviation Name of Drug
individual patient viral populations. Quest Diagnostics employs a rules-
based algorithm developed by experts to interpret the results. Thus, Nucleoside Reverse Transcriptase
predicted drug resistance patterns are reported in addition to the actual Inhibitors (NRTI)
mutations. The HIV-1 subtype is reported as well. ABC Abacavir (Ziagen®')

The absence of mutations does not necessarily imply drug susceptibility; ddI Didanosine (Videx®')
mutations in minor viral populations may not be detected but may FTC Emtricitabine (Emtriva®')
become predominant in the future.
3TC Lamivudine (Epivir®')
HIV-1 Phenotype: Resistance to antiretroviral agents can also be d4T Stavudine (Zerit®')
predicted with the phenotype assay, which assesses the ability of a
patient’s HIV-1 to replicate in vitro in the presence of available TDF Tenofovir disoproxil fumarate
antiretroviral drugs. (Viread®')
ZDV (AZT) Zidovudine (Retrovir®')
HIV-1 Virtual Phenotype: A third alternative, the virtual phenotype,
uses sequence data to predict phenotypic resistance to drugs. Nonnucleoside Reverse Transcriptase
Phenotypic susceptibility is derived from the sequence data of the Inhibitors (NNRTI)
patient’s HIV-1 protease and reverse-transcriptase genes using a linear
model correlating matched genotype and phenotype data from tens of DLV Delavirdine (Rescriptor®')
thousands of patient samples. The predicted, or virtual, phenotype is EFV Efavirenz (Sustiva®')
expressed as the average fold change in IC50 for each available drug.
Clinical cutoffs for the fold change that correlate the fold change to NVP Nevirapine (Viramune®')
virologic response are now available for most reported drugs.
Protease Inhibitors (PI)
HIV-1 DNA, Qualitative PCR: The qualitative HIV-1 DNA test detects APV Amprenavir (Agenerase®')
the presence of HIV-1 proviral DNA, a form of the viral genome
produced by the integration of viral DNA into host chromosomes. During ATV Atazanavir (Reyataz®')
testing, proviral DNA is extracted from circulating infected lymphocytes DRV Darunavir (Prezista™')
and amplified. This assay can detect HIV-1 DNA prior to seroconversion
(antibody formation). HIV DNA analysis has been used to aid the early FPV Fosamprenavir (Lexiva®')
management of infants born to HIV-1 infected mothers. Maternal IDV Indinavir (Crixivan®')
antibodies may persist in the infant for many months, confounding
diagnosis. However, maternal antibodies do not interfere with the HIV-1 LPV/r Lopinavir/ritonavir (Kaletra®')
DNA test. Presumptive infection may be considered if 2 or more NFV Nelfinavir (Viracept®')
separate blood samples are positive for HIV-1 DNA. HIV-1 RNA appears
to be equally sensitive and specific for this purpose.5 Repeatedly SQV Saquinavir (Invirase®')
reactive HIV-1 EIA results with confirmatory Western blot results are TPV Tipranavir (Aptivus®')
always required to confirm the diagnosis HIV-1 infection.
Fusion Inhibitor
C2-Microglobulin, a marker of macrophage and monocyte
stimulation, is a cell-surface protein whose concentration increases at ENF (T-20) Enfuvirtide (Fuzeon®')
the time of HIV-1 seroconversion and continues to rise with progression
of disease. The test is not very useful; CD4 cell counts and viral load References
assays are stronger prognostic and therapeutic markers. 1. Makuwa M, Souquiere S, Niangui MT, et al. Reliability of rapid
diagnostic tests for HIV variant infection. J Virol Methods.
Culture: The definitive biologic test for active HIV-1 infection is 2002;103:183-190.
recovery and growth of virus from a patient’s cells. Blood is the 2. Celum CL, Coombs RW, Lafferty W, et al. Indeterminate human
specimen most often taken for viral culture. Peripheral blood immunodeficiency virus type 1 Western blots: seroconversion risk,
mononuclear cells from a patient’s specimen are incubated with virus- specificity of supplemental tests, and an algorithm for evaluation.
naive cells and medium in tissue culture for 3 weeks. Isolation of virus J Infect Dis. 1991;164:656-664.
is confirmed by the detection of p24 antigen in the culture media. While 3. Centers for Disease Control and Prevention. Revised guidelines for
the sensitivity of culture varies from 65% to 100%, the specificity HIV counseling, testing, and referral. MMWR Recomm Rep.
approaches 100%. However, a single negative culture may not be 2001;50(RR-19):1-57.
reliable. HIV-1 culture is cumbersome, time-consuming, requires 4. Panel on Clinical Practices for Treatment of HIV Infection convened by
biosafety level 3 laboratory procedures, and is very expensive. It is only the Department of Health and Human Services (DHHS). Guidelines for
appropriate as a clinical research tool. the use of antiretroviral agents in HIV-1-infected adults and
adolescents. May 4, 2006. Available at: http://aidsinfo.nih.gov/.
Drug Abbreviations used for antiretroviral drugs are defined in Table Accessed August 16, 2006.
13. 5. Lambert JS, Harris DR, Stiehm ER, et al. Performance characteristics
of HIV-1 culture and HIV-1 DNA and RNA amplification assays for

Section 8
early diagnosis of perinatal HIV-1 infection. J Acquir Immune Defic
Syndr. 2003;34:512-519.
8.4.2 HIV-1 DNA, Qualitative PCR
Clinical Use: This test is used to detect HIV-1 infection in infants up
to 18 months of age.
Clinical Background: The qualitative HIV-1 DNA test detects the
presence of the human immune deficiency proviral DNA, a form of the
HIV-1 genome produced by the integration of viral DNA into host cell
DNA. Qualitative HIV-1 DNA analysis can aid in early detection of HIV-1
in infants born to HIV-1-infected mothers. Maternal antibodies may
persist for the first 18 months of life, confounding diagnosis in the
infant; however, maternal antibodies do not interfere in the HIV-1 DNA
test. This assay may also detect HIV-1 in patients with acute infection
prior to seroconversion (antibody formation), as well as in patients with
agammaglobulinemia. It is recommended that positive results be
confirmed on two separate blood samples with one or a combination of
virus-specific tests.
ELISA and Western blot remain the primary tools for HIV-1 diagnosis.
The qualitative DNA PCR assay is advisable only for the situations
described above.
Individuals Suitable for Testing include infants 18 months of age or
less born to HIV-1 infected mothers and children and adults (see
“Clinical Use”).
Method: In this real-time PCR assay, proviral HIV-1 DNA extracted from
infected circulating lymphocytes is amplified with HIV-1-specific primers
and a fluorescent dye-labeled probe, followed by detection of the
resulting amplicons. The analytical sensitivity is 80 copies/mL.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. Performance characteristics refer to the analytical performance
of the test. This test should not be used for diagnosis without confirmation by other
medically established means.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: “Not detected” HIV-1 DNA results suggest
that HIV-1 proviral DNA was not found in the specimen, but do not
exclude the possibility of HIV-1 infection. Low lymphocyte counts may
lead to a false-negative result.
An “indeterminate” result indicates that the presence or absence of
HIV-1 DNA cannot be established for the current specimen. Repeat
testing of another specimen by the same or a different method is
usually indicated.
An HIV-1 DNA “detected” result is consistent with presence of HIV-1
infection. Presumptive infection may be considered if 2 or more
separate blood samples are positive for HIV-1 DNA. HIV-1 RNA testing
has been confirmed as equally sensitive and specific for early diagnosis
of perinatal HIV-1 infection (see reference 4). Positive results on ELISA
and Western blot assays for HIV-1 are required to confirm the diagnosis
of HIV-1 infection.
References
1. Centers for Disease Control and Prevention. Revised
recommendations for HIV screening of pregnant women. MMWR
Recomm Rep. 2001;50(RR-19):63-85.
2. Report of a consensus workshop, Siena, Italy, January 17-18, 1992.
221
Infectious Disease
Early diagnosis of HIV infection in infants. J Acquir Immune Defic
Syndr. 1992;5:1169-1178.
3. Jackson JB. Detection and quantitation of human immunodeficiency
virus type 1 with molecular DNA/RNA technology. Arch Path Lab
Med. 1993;17:473-477.
4. Lambert JS, Harris DR, Stiehm ER, et al. Performance characteristics
of HIV-1 culture and HIV-1 DNA and RNA amplification assays for
early diagnosis of perinatal HIV-1 infection. J Acquir Immune Defic
Syndr. 2003;34:512-519.
5. Mylonakis E, Paliou M, Lally M, Flanigan TP, Rich JD. Laboratory
testing for infection with the human immunodeficiency virus:
established and novel approaches. Am J Med. 2000;109:568-576.
6. Owens DK, Holodniy M, Garber AM, et al. Polymerase chain reaction
for the diagnosis of HIV infection in adults. Ann Intern Med.
1996;124:803-815.
7. Peckham C, Gibb D. Mother-to-child transmission of the human
immunodeficiency virus. N Engl J Med. 1995;333:298-302.
8. Proffitt MR, Yen-Lieberman B. Laboratory diagnosis of human
immunodeficiency virus infection. Infect Dis Clin North Am.
1993;7:203-220.
9. Whetsell AJ, Drew JB, Milman G, et al. Comparison of three
nonradioisotopic polymerase chain reaction-based methods for
detection of human immunodeficiency virus type 1. J Clin Microbiol.
1992;30:845-853.
8.4.3 HIV Viral Load Testing
8.4.3.1 HIV-1 RNA, Quantitative
Clinical Use: This test is used to assess prognosis, monitor
progression of HIV-1 infection, determine when to initiate therapy, and
monitor effect of antiretroviral drug therapy.
Clinical Background: Measurement of HIV-1 RNA plasma levels
(viral load) provides a direct assessment of viremia and should be used
in conjunction with CD4+ T-cell counts. The baseline (pre-treatment)
HIV-1 RNA level, combined with the baseline number of CD4+ T-cells,
predicts progression to AIDS and death.1,2 Periodic viral load assessment
can be used to track the actual progression of the infection and is an
essential parameter for determining when to initiate therapy. Once
therapy has begun, HIV-1 RNA levels provide important information
regarding therapeutic response. Following initiation or change in
antiviral regimen, responders show a rapid decline in viral load by 2 to 8
weeks and a maximum antiviral effect on HIV-1 RNA in 4 to 5 months.3
The goal is an approximate 1.0 log10 reduction within 2 to 8 weeks and
a decline to below detectable levels (<75 copies/mL by bDNA or <50
copies/mL by PCR) by 16 to 24 weeks.3 Optimally, undetectable levels
will be sustained 6 or more months. In multiple studies, decreases in
viral load have been correlated with improved clinical outcome (ie,
survival). Such correlation was independent of pretreatment HIV-1 RNA
levels, baseline CD4+ T-cells, and prior drug experience. Thus,
measurement of HIV-1 viral load is essential for treatment optimization
and should be used to assess therapeutic response and when making
changes in therapy.
Measurement of the HIV-1 RNA level and the CD4+ T-cell count is
recommended at the following times: 1) at time of diagnosis; 2) every 3
to 4 months (HIV-1 RNA) or every 3 to 6 months (CD4+ T-cell count) to
monitor progression in untreated patients; 3) on 2 occasions
immediately prior to initiation of therapy; 4) 2 to 8 weeks and again at 3
to 4 months after initiation of therapy; and 5) every 3 to 4 months
thereafter to monitor continuing effectiveness of therapy.3 If HIV-1 RNA
levels are still detectable after 16 to 24 weeks of therapy, the
measurement should be repeated for confirmation, using a second
222

Section 8 Infectious Disease

sample, prior to changing therapy.3 Do not perform HIV-1 RNA testing changed, re-baselining is highly recommended prior to making clinical
within 4 weeks of immunization or resolution of intercurrent infections.3 decisions.

In patients with a clinical syndrome and recent high-risk activity
consistent with acute HIV-1 infection who have negative or
indeterminate antibody test results, RNA detection may be used for
preliminary diagnosis of infection. Positive results must be confirmed
with antibody testing (EIA and Western blot) 2 to 4 months after the
initial negative or indeterminate antibody results.3
Individuals Suitable for Testing include patients with recently
diagnosed with HIV-1 infection, those in the clinically latent period of
the infection, and those undergoing antiretroviral therapy.
Method: Quest Diagnostics offers 2 technologies for quantitative HIV-1
RNA measurement: polymerase chain reaction (PCR) and branched DNA
(bDNA) signal amplification. (See Table 14 for more information). Values
obtained from different methods are not interchangeable, because of
variations in reagent specificity and other methodological differences.
Use only one method when monitoring patients. If the methodology is
PCR: This test uses reverse transcription of RNA, followed by PCR
amplification of cDNA and hybridization of amplicons to HIV-1-specific
capture probes. The minimum detectable change in viral load is reported
in Table 15. Reportable ranges for the quantitative, ultrasensitive, and
expanded range assays are 400 to 750,000 copies/mL, 50 to 100,000
copies/mL, and 50 to 7,500,000 copies/mL, respectively. This assay
(Amplicor HIV-1 Monitor, version 1.5) is specific for subtypes (clades) A
through G and provides a more reliable quantitation of non-B clades
than previous versions.
bDNA: This assay utilizes capture and hybridization of RNA to HIV-1
specific probes and multiple branched DNA (bDNA) molecules. bDNA
molecules are hybridized to alkaline phosphatase-labeled probes,
followed by chemiluminescent detection. The reportable range is 75 to
500,000 copies/mL. This assay is specific for HIV-1 group M subtypes
(clades) A through G.5

Table 14. Characteristics of Quantitative HIV Viral Load Tests

bDNA v3.0 Standard PCR Ultrasensitive PCR Expanded Range PCR

Sample Preparation
Sample matrix Plasma, Frozen Plasma, Frozen Plasma, Frozen Plasma, Frozen
Requested volume 3.0 mL 2.0 mL 2.0 mL 2.0 mL
Minimum volume 1.5 mL 0.5 mL 0.6 mL 1.0 mL
Collection container EDTA (lavender-top) EDTA (lavender-top) EDTA (lavender-top) EDTA (lavender-top)

Assay Description
Format Microwell Microwell Microwell Microwell
Method Direct hybridization of RNA Reverse-transcription & Reverse-transcription & Reverse-transcription &
to target probes and polymerase chain reaction polymerase chain reaction polymerase chain reaction
multiple branched DNA
molecules
Amplification Signal amplification Target amplification Target amplification Target amplification
Label Chemiluminescent Colorimetric Colorimetric Colorimetric
Subtypes detected A–G A–G* A–G* A–G*
Manufacturer Bayer Diagnostics Roche Molecular Systems Roche Molecular Systems Roche Molecular Systems
‘a ‘a
Kit name VERSANT HIV-1 AMPLICOR HIV-1 MONITOR AMPLICOR HIV-1 MONITOR AMPLICOR HIV-1 MONITOR
RNA 3.0 Assay (bDNA) Test, version 1.5 Test, version 1.5 Test, version 1.5
FDA cleared or approved Yes Yes Yes Yes

Reportable Range 75–500,000 copies/mL 400–750,000 copies/mL 50–100,000 copies/mL 50–7,500,000 copies/mL

Minimum Change 0.51 log10 (across range of 0.78 log10 (400–1,000 0.39 log10 (75– 100,000 Not determined for levels
Detectable assay)† copies/mL) copies/mL >750,000; for lower levels
0.5 log10 (1,000–750,000 0.44 log10 (~75 copies/mL) refer to Standard and
copies/mL)‡ 0.68 log10 (~50 copies/mL)‡ Ultrasensitive assays

CPT Code§ 87536 87536 87536 87536

*Linearity, reproducibility, and limit of detection have not been evaluated for non-B subtypes.
†
Versant HIV-1 RNA 3.0 assay (bDNA) [Package Insert]. Tarrytown, NY: Bayer Corporation; 2002.
‡
Amplicor HIV-1 Monitor test, version 1.5 [Package Insert]. Branchburg, NJ: Roche Molecular Systems; 2002.
§
The CPT code provided is based on AMA guidelines and is for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions regarding
coding to the payor being billed.

Section 8
Table 15. Minimum Detectable Change in Viral Load4
Minimum Change
Assay Detectable
Standard
400–1,000 HIV-1 RNA copies/mL 0.78 log10
1,000–750,000 HIV-1 RNA copies/mL 0.50 log10
Ultrasensitive
~50 HIV-1 RNA copies/mL 0.68 log10
~75 HIV-1 RNA copies/mL 0.44 log10
75–100,000 HIV-1 RNA copies/mL 0.39 log10
Expanded Range Not Determined
Interpretive Information: Viral load data should be interpreted in the
context of the patient’s immunodeficiency status, among other factors.
Patients with a history of an AIDS-defining illness or severe HIV-related
symptoms should receive antiretroviral therapy, regardless of CD4+ T-
cell count and viral load. In general, therapy is recommended for
asymptomatic individuals when the CD4+ T-cell count is <200/mm3,
regardless of viral load; therapy should be offered to those with 201 to
350 CD4+ T-cells/mm3. For asymptomatic patients with a plasma viral
load of >100,000 HIV-1 RNA copies/mL (by bDNA or RT-PCR), therapy is
generally deferred if the CD4+ T-cell count is >350 cells/mm3; some
physicians may consider initiating treatment in this setting.3 When the
viral load is <100,000 copies/mL and the CD4+ T-cell count is >350/mm3,
treatment should be deferred.
A 3-fold (0.5 log10) change in HIV-1 RNA viral load is considered
clinically significant.3 Increasing levels may be due to disease
progression, failed antiretroviral therapy, other active infections (eg, TB,
pneumococcal pneumonia), or immunization. Decreasing levels indicate
therapeutic response and improved outcome.
References
1. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+
lymphocytes as prognostic markers of HIV-1 infection. Ann Intern
Med. 1997;126:946-954.
2. Mofenson LM, Korelitz J, Meyer WA III, et al, for the National
Institute of Child Health and Human Development Intravenous
Immunoglobulin Clinical Trial Study Group. The relationship between
serum human immunodeficiency virus type 1 (HIV-1) RNA level, CD4
lymphocyte percent, and long-term mortality risk in HIV-1-infected
children. J Infect Dis. 1997;175:1029-1038.
3. Panel on Clinical Practices for Treatment of HIV Infection convened by
the Department of Health and Human Services (DHHS). Guidelines for
the use of antiretroviral agents in HIV-1-infected adults and
adolescents. May 4, 2006. Available at: http://aidsinfo.nih.gov/.
Accessed August 16, 2006.
4. Amplicor HIV-1 Monitor® test, version 1.5 [package insert].
Branchburg, NJ: Roche Molecular Systems; 2002.
5. Versant® HIV-1 RNA 3.0 assay (bDNA) [package insert]. Tarrytown,
NY: Bayer Corporation; 2002.
8.4.4 HIV Drug Resistance Testing
8.4.4.1 HIV-1 Genotype
Clinical Use: This test is used to detect HIV-1 mutations in the reverse
transcriptase (RT) and protease (Pr) genes.
223
Infectious Disease
Clinical Background: HIV-1 genotyping identifies mutations in the
HIV-1 reverse transcriptase (RT) and protease (Pr) genes. Therefore,
genotyping can be used to identify mutations associated with current or
evolving resistance and to monitor transmission of drug-resistant HIV-
1.2,3 Clinical evidence strongly supports the use of genotypic resistance
testing to help guide therapy decisions in patients who have
experienced virologic failure. Such guidance yields greater viral
suppression than when therapy selection is based on standard of care,
particularly when expert consultation is available.4,5
Individuals Suitable for Testing: Testing is recommended following
first antiretroviral regimen failure; following multiple regimen failure; in
pregnant women with HIV-1 infection; and in individuals with acute or
chronic HIV-1 infection prior to initiation of therapy. Testing should be
considered before therapy is required in individuals with a diagnosis of
acute or chronic HIV-1 infection.2,6
Method: This assay utilizes reverse transcription and polymerase chain
reaction (PCR) amplification of plasma HIV-1 RNA, followed by
automated DNA sequencing of the entire Pr gene (to codon 99) and RT
gene (to codon 400). Sequencing is performed with an Applied
Biosystems Model 3700 capillary automated sequencer. Multiple control
samples with known mutations are included in each assay run; because
these are placed at different positions on each run, they also serve as
plate identifiers. Each sequence is compared with sequences obtained
in the past 3 years in the Quest Diagnostics database to help ensure
detection of rare cross-contamination events or sample mix-ups.
Associated drug resistance is identified using the Quest
Diagnostics–Stanford rules-based algorithm. The report includes
detected mutations and predicted drug resistance.
An HIV-1 viral load of 600 copies/mL or more is required for detection of
mutations. Furthermore, mutations will be detected only in viral
populations accounting for >25% of the individual’s HIV-1 population.
This assay is specific for point, insertion, and deletion mutations in the
entire Pr gene and the RT gene up to codon 400. The coefficient of
variation is 5.5%.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: The report form lists individual mutations in
the RT and Pr genes, along with associated drug resistance for each
antiretroviral drug. The HIV-1 subtype, or clade, is also reported. The
interpretation algorithm used to associate mutations with drug
resistance is updated regularly by a panel of HIV experts but is not
reviewed by the FDA. Resistance may also be affected by as yet
uncharacterized mutations and interactions among mutations. In
addition, mutations in minor viral populations (b25% of the viral
population) may not be detected. Because wild-type virus tends to out-
compete resistant virus in the absence of selective pressure, resistance
testing is most reliable for drugs that the patient is taking at the time of
testing.
Therapeutic failure may be due to factors other than resistance,
including poor adherence to the drug regimen, suboptimal therapy or
drug bioavailability, and immunologic decline. Thus, in clinical practice,
physicians select therapeutic regimens on the basis of the patient’s
antiretroviral treatment history, viral load, clinical status, and potential
metabolic toxicity as well as resistance information.
224
Section 8
References
1. Coffin JM. HIV population dynamics in vivo: implications for genetic
variation, pathogenesis, and therapy. Science. 1995;267:483-489.
2. Hirsch MS, Brun-Vezinet F, Clotet B, et al. Antiretroviral drug
resistance testing in adults infected with human immunodeficiency
virus type 1: 2003 recommendations of an International AIDS Society-
USA Panel. Clin Infect Dis. 2003;37:113-128.
3. Duwe S, Brunn M, Altmann D, et al. Frequency of genotypic and
phenotypic drug-resistant HIV-1 among therapy-naive patients of the
German Seroconverter Study. J Acquir Immune Defic Syndr.
2001;26:266-273.
4. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of
antiretroviral management based on plasma genotypic antiretroviral
resistance testing in patients failing therapy. CPCRA 046 Study Team
for the Terry Beirn Community Programs for Clinical Research on
AIDS. AIDS. 2000;14:F83-93.
5. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping
in HIV-1 therapy: the VIRADAPT randomized controlled trial. Lancet.
1999;353:2195-2199.
6. Panel on Clinical Practices for Treatment of HIV Infection convened by
the Department of Health and Human Services (DHHS). Guidelines for
the use of antiretroviral agents in HIV-1-infected adults and
adolescents. May 4, 2006. Available at: http://aidsinfo.nih.gov/.
Accessed August 16, 2006.
8.4.4.2 HIV-1 gp41 Envelope Genotype
Clinical Use: This test is used to guide selection of antiretroviral
drugs; predict HIV-1 resistance to the fusion inhibitor Fuzeon; and
monitor transmission of drug-resistant HIV-1.
Clinical Background: Fuzeon (enfuvirtide; T-20) is the first FDA-
approved HIV-1 fusion inhibitor, a class of antiretrovirals that block entry
of HIV-1 into host cells. Enfuvirtide is a short (36-amino acid) peptide
modeled after the heptad repeat 2 (HR-2) region of the HIV-1 envelope
glycoprotein gp41; it inhibits fusion by binding the first helical region
(HR-1) of gp41. Variations in cellular and viral sequences affect the
sensitivity of HIV-1 to enfuvirtide.1,2 Sequence variations in certain
regions of gp41, most notably codons 36 to 45 of HR-1, enhance in vitro
resistance to enfuvirtide3 and have been identified in patients receiving
this drug.2
Individuals Suitable for Testing include HIV-1-infected individuals
receiving enfuvirtide who show evidence of virologic failure and those
HIV-1-infected individuals for whom enfuvirtide treatment is being
considered. This assay is not appropriate for HIV-2 infected individuals.
Method: A 600-bp region of the HIV-1 RNA gp41 gene (including HR-1
and HR-2) is amplified by reverse transcription-polymerase chain
reaction (RT-PCR) and then subjected to automated DNA sequencing in
an Applied Biosystems capillary automated DNA sequencer. A control
sample with known mutations is included each time the assay is run.
The report includes detected mutations and predicted drug resistance
for enfuvirtide. This assay exhibits >95% detection at 400 HIV-1 RNA
copies/mL and detects subpopulations accounting for r20% of the viral
population. It is specific for point, insertion, and deletion mutations in
the gp41 gene in HIV-1 group M subtypes A-D, G, H, and K. It does not
detect gp41 mutations in HIV-2. There is no cross-reaction with HCV,
HBV, enterovirus, CMV, or human RNA. The inter- and intra-assay
precision at the nucleotide level is 99.88%.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Infectious Disease
Interpretive Information: The report form lists individual mutations in
the gp41 region, along with associated drug resistance for enfuvirtide.
The interpretation algorithm used to associate mutations with drug
resistance is updated regularly by a panel of HIV experts but is not
reviewed by the FDA. Resistance may also be affected by as yet
uncharacterized mutations and interactions among mutations. In
addition, mutations in minor viral populations (<20% of the viral
population) may not be detected.
Therapeutic failure may be due to factors other than resistance,
including poor adherence to the drug regimen, suboptimal therapy or
drug bioavailability, and immunologic decline. Thus, in clinical practice,
physicians select therapeutic regimens on the basis of the patient’s
antiretroviral treatment history, viral load, clinical status, and potential
metabolic toxicity as well as resistance information.
References
1. Reeves JD, Gallo SA, Ahmad N, et al. Sensitivity of HIV-1 to entry
inhibitors correlates with envelope/coreceptor affinity, receptor
density, and fusion kinetics. Proc Natl Acad Sci U S A.
2002;99:16249-16254.
2. Wei X, Decker JM, Liu H, et al. Emergence of resistant human
immunodeficiency virus type 1 in patients receiving fusion inhibitor
(T-20) monotherapy. Antimicrob Agents Chemother. 2002;46:1896-
1905.
3. Mink M, Greenberg ML, Mosier S, et al. Impact of HIV-1 gp41 amino
acid substitutions (positions 36-45) on susceptibility to T-20
(enfuvirtide) in vitro. Analysis of primary virus isolates recovered from
patients during chronic enfuvirtide treatment and site-directed
mutants in NL4-3 [abstract]. Antivir Ther. 2002;7:S17.
8.4.4.3 HIV-1 Phenotype, PhenoSense®’
Clinical Use: This test is used to guide selection of antiretroviral
drugs, predict HIV drug resistance, and monitor transmission of drug-
resistant HIV.
Clinical Background: Despite continuing advances in treatment for
HIV-1 infection, highly active antiretroviral therapy (HAART) often fails.
The high replication rate of HIV-1 coupled with its rapid mutation rate
promotes the accumulation of mutations,1 some of which confer reduced
susceptibility to antiretroviral agents. If viral replication is not
adequately suppressed during HAART, HIV-1 variants containing
resistance-associated mutations will emerge, increasing the likelihood
of treatment failure. Transmission of mutant strains may also lead to
drug resistance in treatment-naive patients.
HIV-1 phenotyping is a quantitative measure of drug resistance that has
been shown to predict viral load response to new antiretroviral
treatment2,3 and to facilitate selection of active drugs (ie, drugs to which
the virus is susceptible).4 This test examines the ability of a patient’s
HIV-1 to replicate in vitro in the presence of different concentrations of
antiretroviral drugs and compares the results with a “wild-type” virus.
Thus, the combined effects of resistance factors, replication, and
infectivity of the patient’s various HIV-1 subpopulations are evaluated.
Phenotyping is technically more complex and has a longer turnaround
time than does genotyping. The technique can be beneficial, however,
when patients with a complex treatment history experience treatment
failure, when determining the potential for newly introduced
antiretroviral drugs in an individual patient, and when there are few
VirtualPhenotype®’ matches for an individual’s genotype. The
PhenoSense assay also assesses drug hypersusceptibility and viral
replication capacity (capacity of virus to replicate in the absence of

Section 8
drugs), which may be associated with immunologic and virologic
response to therapy.5,6
Individuals Suitable for Testing7,8: Testing is recommended
following first antiretroviral regimen failure; following multiple regimen
failure; in pregnant women with HIV-1 infection; and in individuals with
acute or chronic HIV-1 infection prior to initiation of therapy. Testing
should be considered before therapy is required in individuals with a
diagnosis of acute or chronic HIV-1 infection. However, genotypic
resistance testing is generally preferred for treatment-naive patients.8
Methods: RNA is extracted and the Pr and RT regions of the HIV-1 viral
genome are reverse-transcribed to cDNA. These regions are then
amplified via PCR and inserted into a test vector containing a luciferase
indicator gene. In vitro replication of resulting recombinant test vector
(in the presence of varying drug concentrations) is measured by
luciferase production; drug sensitivity is quantified as IC50. The graphical
report includes, for each drug, patient viral drug susceptibility expressed
as IC50 fold change relative to a drug-sensitive control virus; the IC50
change cut-off that corresponds to reduced drug susceptibility; and
susceptibility curves. The report also includes the patient viral
replication capacity (relative to a drug-sensitive control virus). This
assay is specific for all HIV-1 group M subtypes, requires a minimum
viral load of 500 HIV-1 RNA copies/mL, and detects viral subpopulations
accounting for as little as 10% of the patient’s viral population. Lab
testing time is about 14 days.
Interpretive Information: IC50 fold changes above the assay-defined
cutoffs indicate reduced drug susceptibility (ie, increased resistance).
Therapeutic failure may be due to factors other than resistance,
including poor adherence to the drug regimen, suboptimal therapy or
drug bioavailability, and immunologic decline. Thus, in clinical practice,
physicians select therapeutic regimens on the basis of the patient’s
antiretroviral treatment history, viral load, clinical status, and potential
metabolic toxicity as well as resistance information.
References
1. Coffin JM. HIV population dynamics in vivo: Implications for genetic
variation, pathogenesis, and therapy. Science. 1995;267:483-489.
2. Call SA, Saag MS, Westfall AO, et al. Phenotypic drug susceptibility
testing predicts long-term virologic suppression better than treatment
history in patients with human immunodeficiency virus infection. J
Infect Dis. 2001;183:401-408.
3. Piketty C, Race E, Castiel P, et al. Efficacy of a five-drug combination
including ritonavir, saquinavir and efavirenz in patients who failed on
a conventional triple-drug regimen: phenotypic resistance to protease
inhibitors predicts outcome of therapy. AIDS. 1999;13:F71-77.
4. Cohen CJ, Hunt S, Sension M, et al. A randomized trial assessing the
impact of phenotypic resistance testing on antiretroviral therapy.
AIDS. 2002;16:579-588.
5. Haubrich RH, Kemper CA, Hellmann NS, et al; California Collaborative
Treatment Group. The clinical relevance of non-nucleoside reverse
transcriptase inhibitor hypersusceptibility: a prospective cohort
analysis. AIDS. 2002;16:F33-40.
6. Sufka SA, Ferrari G, Gryszowka VE, et al. Prolonged CD4+ cell/virus
load discordance during treatment with protease inhibitor-based
highly active antiretroviral therapy: immune response and viral
control. J Infect Dis. 2003;187:1027-1037.
7. Hirsch MS, Brun-Vezinet F, Clotet B, et al. Antiretroviral drug
resistance testing in adults infected with human immunodeficiency
virus type 1: 2003 recommendations of an International AIDS Society-
USA Panel. Clin Infect Dis. 2003;37:113-128.
8. Panel on Clinical Practices for Treatment of HIV Infection convened by
225
Infectious Disease
the Department of Health and Human Services (DHHS). Guidelines for
the use of antiretroviral agents in HIV-1-infected adults and
adolescents. May 4, 2006. Available at: http://aidsinfo.nih.gov/.
Accessed August 16, 2006.
8.4.4.4 HIV-1 VirtualPhenotype for Drug Resistance to PRI
and RTI
Clinical Use: This test is used to guide selection of antiretroviral
drugs, predict HIV-1 drug resistance, and monitor transmission of drug-
resistant HIV-1.
Clinical Background: Despite continuing advances in treatment for
HIV-1 infection, highly active antiretroviral therapy (HAART) often fails.
The high replication rate of HIV-1 coupled with its rapid mutation rate
promotes the accumulation of mutations,1 some of which confer reduced
susceptibility to antiretroviral agents. If viral replication is not
adequately suppressed during HAART, HIV-1 variants containing
resistance-associated mutations will emerge, increasing the likelihood
of treatment failure. Transmission of mutant strains may also lead to
drug resistance in treatment-naive patients.
Genotyping of the reverse transcriptase (RT) and protease (Pr) genes has
proven effective in identifying mutations associated with resistance to
RT and Pr inhibitors.2 Genotyping improves selection of antiretroviral
drug regimens relative to standard of care, particularly if expert
consultation is available.3,4 Another approach to assessing drug
susceptibility is phenotyping, which examines the ability of a patient’s
HIV-1 to replicate in vitro in the presence of different concentrations of
antiretroviral drugs. Like genotyping, phenotyping helps predict viral
load response to new antiretroviral regimens5,6 and facilitates selection
of active drugs.7 Phenotyping can be especially beneficial in patients
with a complex treatment history, but is more complex and has a longer
turnaround time than genotyping.
A third alternative, the virtual phenotype, uses sequence data to predict
phenotypic resistance to drugs. Phenotypic susceptibility is derived from
the sequence data of the patient’s HIV-1 protease and reverse-
transcriptase genes using a linear model correlating matched genotype
and phenotype data from tens of thousands of patient samples. The
predicted, or virtual, phenotype is expressed as the average fold change
in IC50 for each available drug. Clinical cutoffs (CCOs) for the fold change
that correlate the fold change to virologic response are now available
for most reported drugs.
Individuals Suitable for Testing2,8: Testing is recommended
following first antiretroviral regimen failure; following multiple regimen
failure; in pregnant women with HIV-1 infection; and in individuals with
acute or chronic HIV-1 infection prior to initiation of therapy. Testing
should be considered before therapy is required in individuals with a
diagnosis of acute or chronic HIV-1 infection. Note that genotypic
resistance testing is generally preferred for treatment-naive patients.8
Method: Nucleic acid sequencing is performed on PCR-amplified cDNA.
The entire protease gene (codons 1-99) and the reverse transcriptase
gene (out to codon 400) are sequenced, covering all known mutations
involved in drug resistance. Linear modeling equations derived from
Virco’s matched genotype-phenotype database are used to correlate the
genotype to phenotypic susceptibility changes for each drug. The
average increase in resistance (fold change in IC50) is calculated for
each drug. The report displays the identified mutations, the subtype,
and—for each drug—the average fold change in IC50, and the lower
(CCO20) and upper (CCO80) CCOs for predicting virologic response. This
assay is specific for HIV-1 group M (including subtypes A–D, F–H, J, K
226
Section 8
and recombinant subtypes such as AE and AG), has an analytical
sensitivity of 600 HIV-1 RNA copies/mL, and detects sequences in viral
populations accounting for >25% of the individual’s HIV-1 population.
The lab testing time is about 1 week.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Interpretive Information: IC50 fold changes below a drug’s lower
clinical cutoff (CCO20) suggest maximal virologic response. Fold change
values between the CCO20 and upper cutoff (CCO80) suggest partial or
reduced virologic response. Fold change values above the CCO80 suggest
minimal virologic response. Therapeutic failure may be due to factors
other than resistance, including poor adherence to the drug regimen,
suboptimal therapy or drug bioavailability, and immunologic decline.
Thus, in clinical practice, physicians select therapeutic regimens on the
basis of the patient’s antiretroviral treatment history, viral load, clinical
status, and potential metabolic toxicity as well as resistance
information.8
References
1. Coffin JM. HIV population dynamics in vivo: implications for genetic
variation, pathogenesis, and therapy. Science. 1995;267:483-489.
2. Hirsch MS, Brun-Vezinet F, Clotet B, et al. Antiretroviral drug
resistance testing in adults infected with human immunodeficiency
virus type 1: 2003 recommendations of an International AIDS Society-
USA Panel. Clin Infect Dis. 2003;37:113-128.
3. Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of
antiretroviral management based on plasma genotypic antiretroviral
resistance testing in patients failing therapy. CPCRA 046 Study Team
for the Terry Beirn Community Programs for Clinical Research on
AIDS. AIDS. 2000;14:F83-93.
4. Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping
in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet.
1999;353:2195-2199.
5. Call SA, Saag MS, Westfall AO, et al. Phenotypic drug susceptibility
testing predicts long-term virologic suppression better than treatment
history in patients with human immunodeficiency virus infection. J
Infect Dis. 2001;183:401-408.
6. Piketty C, Race E, Castiel P, et al. Efficacy of a five-drug combination
including ritonavir, saquinavir and efavirenz in patients who failed on
a conventional triple-drug regimen: phenotypic resistance to protease
inhibitors predicts outcome of therapy. AIDS. 1999;13:F71-77.
7. Cohen CJ, Hunt S, Sension M, et al. A randomized trial assessing the
impact of phenotypic resistance testing on antiretroviral therapy.
AIDS. 2002;16:579-588.
8. Panel on Clinical Practices for Treatment of HIV Infection convened by
the Department of Health and Human Services (DHHS). Guidelines for
the use of antiretroviral agents in HIV-1-infected adults and
adolescents. May 4, 2006. Available at: http://aidsinfo.nih.gov/.
Accessed August 16, 2006.
8.4.5 Lymphocyte Subset Panels
See Immunology, section 7.7.4.
8.5 Immunocompromised
8.5.1 BK and JC Virus DNA, Real-Time PCR
Clinical Use: These tests are used to detect and monitor infection by
BK virus and JC virus.
Infectious Disease
Clinical Background: BK virus (BKV) and JC virus (JCV) are double-
stranded DNA, human polyomaviruses. More than 70% of the adult
population has antibodies to BKV and JCV, with primary infections
typically occurring in childhood.1 In the US, 50% of children develop
antibodies to BKV by 3 to 4 years of age and to JCV by 10 to 14 years of
age.1 In immunocompetent individuals, primary BKV infections usually
cause a mild respiratory illness and, rarely, cystitis, whereas primary
JCV infections are typically asymptomatic. After initial infection,
polyomaviruses establish latency in various tissues. The primary sites of
latency are uroepithelial cells for BK virus and B-lymphocytes and renal
tissue for JCV. Additional sites of latency for both viruses include the
ureters, brain, and spleen. BKV and JCV viruria is found in up to 3% of
pregnant women but is not associated with disease.1
Reactivation of latent as well as primary BKV and JCV infections may
occur in immunocompromised individuals. BKV infections can lead to
interstitial nephritis, hemorrhagic cystitis, and kidney allograft
rejection.2-4 BKV nephropathy is associated with BK viremia of >5,000
copies/mL (plasma) and BK viruria >107 copies/mL and is seen in
approximately 8% of kidney transplant recipients.2 Though latency is
typically associated with the absence of viremia, low levels (<2,000
copies/mL, plasma) are seen in some asymptomatic individuals.2 JCV is
responsible for progressive multifocal leukoencephalopathy, a fatal
demyelinating disease of the central nervous system seen in up to 70%
of AIDS patients.5 Additionally, BKV and JCV viruria are found in
approximately 40% of bone marrow transplant patients.2-4
PCR detects the presence of the virus, not antibodies to the virus; thus,
the detection of viral DNA may be indicative of an active infection. The
identification of viral DNA may warrant the institution of antiviral
therapies and/or a decrease of immunosuppressive therapies.2-4
Determination of viral DNA presence or concentration is also useful in
establishing the cause of allograft rejection.2-4
Individuals Suitable for Testing include transplant recipients
receiving immunosuppressive therapies and individuals with
immunosuppressive diseases such AIDS.
Method: Viral DNA is amplified by real-time polymerase chain reaction
using DNA primers with fluorogenic probes directed at highly conserved
sequences of the BKV and JCV genomes. These assays exhibit no cross-
reactivity with 20 viral and non-viral pathogens tested. The reportable
range for the quantitative BKV assay is 500 to 39,000,000 copies/mL.
These tests were developed and their performance characteristics have been determined
by Quest Diagnostics Nichols Institute. Performance characteristics refer to the analytical
performance of the tests.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: A not detected result in the BKV and JCV
qualitative assays indicates viral DNA either is not present in the
specimen or is present in a concentration below the assay’s limit of
detection. A detected result indicates viral DNA is present. Similarly, in
the BKV quantitative assay, a result <500 copies/mL indicates viral DNA
is not present in the sample or the concentration is less than the
detectable limit.
Inhibitors present indicates the presence of non-specific PCR inhibitors
in the sample. Inhibitors will slow the PCR reaction and may result in
falsely decreased values.
An increase or decrease in viral DNA concentration may indicate a
worsening or resolution of an active infection, respectively. While the
presence of viral DNA may indicate an active infection, DNA results
 227

Section 8 Infectious Disease

should not be the sole basis for diagnosis of active infection, as low
viral concentrations are sometimes seen in asymptomatic individuals.2,3
Results should be interpreted in conjunction with other laboratory and
clinical findings.
References
caspofungin, and posaconazole. Correlation with the NCCLS
microdilution method (reference method) is 90% to 97%.
This test is performed using a kit that has not been approved or cleared by the FDA. The
analytical performance characteristics of this test have been determined by Quest
Diagnostics Nichols Institute. This test should not be used for diagnosis without
confirmation by other medically established means.

1. Kazory A, Ducloux D. Renal transplantation and polyomavirus
infection: recent clinical facts and controversies. Transpl Infect Dis.
2003;5:65-71.
2. Randhawa P, Ho A, Shapiro R, et al. Correlates of quantitative
measurement of BK polyomavirus (BKV) DNA with clinical course of
BKV infection in renal transplant patients. J Clin Microbiol.
2004;42:1176-1180.
3. Ramos E, Drachenberg CB, Papadimitriou JC, et al. Clinical course of
polyoma virus nephropathy in 67 renal transplant patients. J Am Soc
Nephrol. 2002;13:2145-2151.
4. Randhawa P, Shapiro R, Vatas A. Quantitation of DNA of
polyomaviruses BK and JC in Human Kidneys. J Infect Dis.
2005;192:504-509.
5. Seth P, Diaz F, Major E. Advances in the biology of JC virus and
induction of progressive multifocal leukoencephalopathy.
J Neurovirol. 2003;9:236-246.
8.5.2 Susceptibility, Yeast, Comprehensive Panel
Clinical Use: This test is used to determine antifungal susceptibility of
non-fastidious yeasts and to aid in treatment selection for infected
patients.
Clinical Background: Non-fastidious, or rapid growing, yeasts
include those classified as Candida and Cryptococcus as well as other
yeasts such as Trichosporon. These normally occurring, opportunistic
fungi are most often of medical importance in patients debilitated by
hormone imbalance or an immunosuppressive state.
Method: In this minimum inhibitory concentration (MIC) assay, serial
dilutions of various antifungal agents are incubated with standardized
yeast suspensions. Utilizing a colorimetric growth indicator
(AlamarBlue®’), the lowest antifungal concentration that inhibits growth
(MIC) is determined. An interpretation is reported along with the MIC
whenever possible (see below).
Antifungal agents tested include amphotericin B, fluconazole,
itraconazole, ketoconazole, flucytosine (5-fluorocytosine), voriconazole,
Interpretive Information: Interpretive guidelines for Candida species
are provided in Table 16. Candida krusei (I orientalis) is assumed to be
resistant to fluconazole irrespective of the MIC. The MIC interpretation
guidelines for fluconazole and itraconazole activity against Candida
species are based on experience with mucosal infections and may not
apply to invasive infections. When results are interpreted as
susceptible-dose dependent to a specific antifungal agent, it is assumed
that maximum blood levels can be achieved. Interpretive guidelines are
not available for Cryptococcus and other non-fastidious yeasts.
8.6 Pregnancy
8.6.1 Parvovirus B-19 DNA, Qualitative Real-time PCR
Clinical Use: This test is used to determine the etiology of acute and
chronic anemias, erythema infectiosum, and polyarthropathy; diagnose
parvovirus infection as the causative agent for fetal hydrops; diagnose
parvovirus infection in immunosuppressed individuals (HIV-infected
individuals, transplant patients); and diagnose parvovirus infection prior
to seroconversion.
Clinical Physiology: Parvovirus B-19 is an unenveloped single-
stranded DNA virus that infects, replicates in, and lyses red cell
progenitors. Clinical symptoms of infection result from subsequent
erythroid aplasia or from the host immune response. In children, the
infection manifests as a nonspecific illness or with erythema
infectiosum (fifth disease) with a characteristic “slapped-cheek” rash.
Parvovirus B-19 occasionally causes polyarthritis in adults and transient
aplastic crisis may occur in patients with chronic hemolytic anemia
(sickle cell, hereditary spherocytosis, beta thalassemia, etc). Although
usually self-limiting, parvovirus infection may cause acute or chronic
anemia in the immunocompromised host and is the leading cause of red
cell aplasia in AIDS patients. In pregnant women, the infection may be
transmitted to the fetus. Fetal anemia, hydrops fetalis, or fetal demise
may occur in a small percentage of intrauterine infections.
An intense viremia develops 7 to14 days after parvovirus infection.
Clinical symptoms occur with the onset of specific IgM production,

Table 16. Candida Species Interpretive Guidelines

Antifungal Susceptible Susceptible–Dose Intermediate Resistant

Agent (μg/mL) Dependent (μg/mL) (μg/mL) (μg/mL)
Fluconazole* b8 16–32 — r64
Itraconazole b0.125 0.25–0.5 — r1
Flucytosine b4 — 8–16 r32
Voriconazole b1 2 — r4
Ketoconazole Interpretive guidelines unavailable from NCCLS
Amphotericin B Interpretive guidelines unavailable from NCCLS
Caspofungin Interpretive guidelines unavailable from NCCLS
Posaconazole Interpretive guidelines unavailable from NCCLS
*Candida krusei excluded.
228
Section 8
followed shortly by IgG production. Detection of parvovirus DNA is an
earlier and more sensitive marker of viral infection than anti-viral
antibodies and should be used in conjunction with anti-parvovirus IgM
detection for optimal diagnostic sensitivity. Parvovirus DNA is especially
useful for immunosuppressed patients in whom antibody levels may be
undetectable.
Method: In this real-time polymerase chain reaction (PCR) method, viral
DNA is amplified with primers specific for parvovirus B-19 virion
structural protein (VP1) and a fluorescent dye-labeled probe.
Amplification products are detected by measuring fluorescent signals
generated during the PCR. The analytical sensitivity of this assay is 400
parvovirus DNA copies/mL.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics Nichols Institute. It has not been cleared or approved by the U.S. Food
and Drug Administration. The FDA has determined that such clearance or approval is not
necessary. Performance characteristics refer to the analytical performance of the test.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems Inc.
Interpretive Information: A “detected” result is associated with
parvovirus B-19 infection. A “not detected” result is consistent with the
absence of parvovirus B-19 infection, but does not exclude the
diagnosis. Diagnosis of parvovirus infection should not rely solely on the
result of a PCR test.
References
1. Alger LS. Toxoplasmosis and parvovirus B19. Infect Dis Clin North
Am. 1997;11:55-75.
2. Cassinotti P, Bas S, Siegl G, et al. Association between human
parvovirus B19 infection and arthritis. Ann Rheum Dis. 1995;54:498-
500.
3. Clewley JP. Polymerase chain reaction assay of parvovirus B19 DNA
in clinical specimens. J Clin Microbiol. 1989;27:2647-2651.
4. Clewley JP. PCR detection of parvovirus B19. In: Persing DH, Smith
TF, Tevover FC, et al, eds. Diagnostic Molecular Microbiology:
Principles and Applications. Washington, DC: American Society for
Microbiology; 1993:367-373.
5. Mori J, Beattie P, Melton W, et al. Structure and mapping of the
DNA of human parvovirus B19. J Gen Virol. 1987;68:2797-2806.
6. Musiani M, Zerbini M, Gentilomi G, et al. Parvovirus B19 clearance
from peripheral blood after acute infection. J Infect Dis.
1995;172:1360-1363.
7. Portmore AC. Parvovirus (erythema infectiosum, aplastic crisis). In:
Mandell GL, Bennett JE, Dolan R, eds. Principles and Practice of
Infectious Diseases. 4th ed. New York: Churchill-Livingston;
1995:1439-1446.
8. Torok TJ, Wang Q, Gary GW Jr, et al. Prenatal diagnosis of
intrauterine infection with parvovirus B19 by the polymerase chain
reaction technique. Clin Infect Dis. 1992;14:149-155.
8.7 Respiratory Disease
8.7.1 Atypical Pneumonia DNA Panel, Qualitative Real-Time
PCR
Clinical Use: These tests are used for differential diagnosis of atypical
pneumonia (ie, pneumonia caused by the atypical respiratory pathogens
Chlamydophila pneumoniae (Chlamydia pneumoniae), Legionella
pneumophila, or Mycoplasma pneumoniae).
Clinical Background: Atypical pneumonia is a vague term used
historically to describe community-acquired pneumonia (CAP) that was
Infectious Disease
initially thought to be unique in clinical and radiographic presentation.
Subsequent studies revealed that the syndrome is diverse, and
diagnosis cannot always be made on clinical grounds or from chest x-
ray.1 The primary causes of atypical pneumonia are infections with C
pneumoniae, L pneumophila, and M pneumoniae, which together
account for 10% to 40% of CAP cases.2
Because mortality is reduced when antimicrobial therapy is
administered within 4 or 8 hours of presentation,3,4 antibiotics are
typically selected empirically. Guidelines for empiric therapy have been
developed for outpatients as well as for inpatients on a medical ward or
in an intensive care unit (ICU).1,5 However, identification of the causative
agent is important for selection of pathogen-specific antimicrobial
therapy and to ensure adequate treatment duration. Pathogen-specific
therapy reduces ineffective treatment, fosters the use of narrow-
spectrum antibiotics, and minimizes adverse drug reactions. In addition,
the atypical pathogens may require a longer course of treatment.1,5
Identification of these atypical pathogens has been based on direct
antigen detection, serology, or culture. Culture, however, is often
compromised when performed after initiation of antibiotic therapy and
requires at least 3 days to complete. Serology requires paired acute and
convalescent samples, usually collected 2 weeks apart, to diagnose
current infection. Thus, the time required for these tests makes them
useful only retrospectively. The Legionella urinary antigen test or
respiratory sample culture has been recommended for patients
hospitalized in the ICU with enigmatic pneumonia as well as during a
Legionella epidemic or when C-lactam antibiotic therapy has been
ineffective.5 The antigen test detects L pneumophila serogroup 1, which
accounts for 80% of legionellosis cases, but does not detect all 64
subgroups that are detected by culture and DNA testing.2
Real-time polymerase chain reaction (PCR) technology used to detect
the DNA of these organisms does not require the organism to be viable,
is not affected by previously administered antibiotic therapy, and does
not require paired acute and convalescent samples.6 PCR methods for
these atypical pathogens are at least as sensitive as culture and, in
most studies, more sensitive.6 In many cases, the organisms may be
detected by PCR prior to detection by immunological methods.
Individuals Suitable for Testing include patients presenting with
symptoms of pneumonia who 1) are suspected of having atypical
pneumonia; or 2) are not responding to antibiotic therapy.
Method: Real-time PCR-based tests with specific target primers and
probes are used to amplify and detect the DNA of each organism. These
assays exhibit no known cross-reactivity with other organisms or with
human DNA.
These tests were developed and their performance characteristics have been determined
by Quest Diagnostics Nichols Institute. They have not been cleared or approved by the U.S.
Food and Drug Administration. The FDA has determined that such clearance or approval is
not necessary. Performance characteristics refer to the analytical performance of the tests.
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Interpretive Information: A positive result is consistent with
infection by the organism detected. Diagnosis of pneumonia, however,
should rely on clinical and chest radiographic findings.
A negative test result is consistent with the absence of infection but
may also be due to DNA concentrations below the detection limit of the
assay. When there is epidemiologic evidence of legionellosis, treatment
of symptomatic patients is recommended even when Legionella test
results are negative.5
 229

Section 8 Infectious Disease

References 8.7.3 Respiratory Virus Panel, Qualitative Real-time PCR

1. Niederman MS, Mandell LA, Anzueto A, et al. Guidelines for the
Clinical Use: This panel is used for differential diagnosis of lower
management of adults with community-acquired pneumonia. Am J
respiratory infections (LRIs) caused by influenza virus A & B, respiratory
Respir Crit Care Med. 2001;163:1730-1754.
syncytial virus (RSV), parainfluenza virus 1, 2, and 3, or adenovirus; for
2. Bartlett JG. Diagnostic test for etiological agents of community-
differential diagnosis of upper as well as lower respiratory tract
acquired pneumonia. Infect Dis Clin N Am. 2004;18:809-827.
infections in immunocompromised patients; and to determine
3. Houck PM, Bratzler DW, Nsa W, et al. Timing of antibiotic
appropriate virus-specific treatments and avoid inappropriate antibiotic
administration and outcomes for Medicare patients hospitalized with
therapy.
community-acquired pneumonia. Arch Intern Med. 2004;164:637-644.
4. Meehan TP, Fine MJ, Krumholz HM, et al. Quality of care, process,
Clinical Background: Among the most common viruses causing LRIs
and outcomes in elderly patients with pneumonia. JAMA.
such as tracheobronchitis, bronchiolitis, and pneumonia are influenza
1997;278:2080-2084.
virus, RSV, parainfluenza virus, and adenovirus. Young children, the
5. Mandell LA, Bartlett JG, Dowell SF, et al. Update of practice
elderly, and patients with compromised cardiac, pulmonary, or immune
guidelines for the management of community-acquired pneumonia in
systems are at greatest risk for serious disease. In children, 15% to
immunocompetent adults. Clin Infect Dis. 2003;37:1405-1433.
25% of pneumonias are caused by RSV, 15% by parainfluenza virus, and
6. Murdoch DR. Nucleic acid amplification tests for the diagnosis of
7% to 9% by adenovirus.1 RSV infection is the most frequent cause of
pneumonia. Clin Infect Dis. 2003;36:1162-1170.
hospitalization in children <5 years of age. In the elderly, respiratory
viral infections cause up to 26% of hospital admissions for community-
8.7.2 Influenza A and B Culture, Rapid Method
acquired pneumonia.2
Clinical Use: This test is used for diagnosis of influenza virus A and B
Immunocompromised patients, including those with cancer or
infection.
transplants, are susceptible to the same seasonal viruses that cause
respiratory illness in the community at large.3 These viral pathogens can
Clinical Background: Influenza is caused by different influenza
lead to serious morbidity and mortality in such patients.3-6
viruses, most commonly type A but also type B. The infection usually
manifests as an acute upper respiratory illness lasting 7 to 10 days, but
Viral causes of LRI should be distinguished from bacterial causes to
it may also progress to viral pneumonia, or be complicated by a
avoid the unnecessary use of antibiotics and to help select specific
secondary bacterial infection. Effective anti-influenza drugs are
antiviral agents, when available, to treat or prevent infection. Laboratory
available, increasing the clinical importance of rapid diagnosis (ie,
identification of the virus responsible for a community epidemic or
within 2 days of the onset of symptoms).
seasonal disease is helpful for selecting prophylactic treatments, which
are available for influenza virus and RSV.7
In the past, laboratory tests have been primarily limited to
epidemiological purposes because such tests were lengthy and
Identification of viral respiratory pathogens has typically been based on
relatively insensitive. For example, direct fluorescent antibody (DFA)
direct antigen detection or culture; however, polymerase chain reaction
tests are rapid (1 day turnaround time) but insensitive, while culture
(PCR) is more sensitive for most respiratory viruses.5,7 This panel uses
tests are more sensitive but slow (5-14 days). Antibody detection tests
real-time PCR technology to identify these respiratory viral pathogens.
are impractical for diagnosis of acute disease. Direct antigen tests,
however, can differentiate type A and B influenza in a timely manner,
Individuals Suitable for Testing include immunocompetent patients
thereby assisting with treatment decisions.
who have symptoms of LRI and immunocompromised patients who have
symptoms of either upper or lower respiratory tract infection.
This rapid culture and antigen detection method appears to overcome
both limitations referred to above. Results are available within 8 to 24
Method: Real-time PCR-based testing with specific primers and probes
hours of sample arrival in the lab and the sensitivity is greater than
is used to amplify and detect target sequences of the respective viral
95%, significantly better than conventional culture, the current gold
genomes; for the RNA viruses, a reverse-transcription step is
standard. Specificity is 100%.
incorporated into the real-time PCR. These assays exhibit no known
cross-reactivity with other organisms or with human DNA.
Method: In this culture method, a mixed monolayer of mink lung cells
(strain Mv1Lu) and human adenocarcinoma cells (strain A549) is These tests were developed and their performance characteristics have been determined
inoculated with patient specimen. After 6 hours of incubation, one by Quest Diagnostics Nichols Institute. They have not been cleared or approved by the U.S.
Food and Drug Administration. The FDA has determined that such clearance or approval is
duplicate culture well is fixed and incubated with both influenza A and not necessary. Performance characteristics refer to the analytical performance of the tests.
B monoclonal antibodies and then stained with FITC-conjugated goat
anti-mouse antibody. If positive on fluorescent microscopic examination, Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
results are reported as positive for influenza A or B virus. If negative, a
second duplicate culture well is processed after 20 hours of incubation Interpretive Information: A positive result is consistent with
and examined microscopically. Results are reported as negative or infection by the virus(es) detected. Diagnosis of LRI, however, should
positive for influenza A or B virus. rely on clinical and chest radiographic findings.
Interpretive Information: A negative result suggests the absence of A negative test result is consistent with the absence of infection but
influenza A and B virus, while a positive result indicates the presence of may also be due to RNA or DNA concentrations below the detection
influenza A or B virus as specified. limit of the assay.
230
Section 8
References
1. Latham-Sadler BA, Morell VW. Viral and atypical pneumonias. Prim
Care. 1996;23:837-848.
2. Janssens J-P. Pneumonia in the elderly (geriatric) population. Curr
Opin Pulm Med. 2005;11:226-230.
3. Whimbey E, Englund JA, Couch RB. Community respiratory virus
infections in immunocompromised patients with cancer. Am J Med.
1997;102:10-18.
4. Ison MG, Hayden FG. Viral infections in immunocompromised
patients: what’s new with respiratory viruses? Curr Opin Pulm Med.
2002;15:355-367.
5. Roghmann M, Ball K, Erdman D et al. Active surveillance for
respiratory virus infections in adults who have undergone bone
marrow and peripheral blood stem cell transplantation. Bone Marrow
Transplantation. 2003;32:1085-1088.
6. Dykewicz CA, National Center for Infectious Diseases, Centers for
Disease Control and Prevention, Infectious Diseases Society of
America, American Society for Blood and Marrow Transplantation.
Guidelines for preventing opportunistic infections among
hematopoietic stem cell transplant recipients: focus on community
respiratory virus infections. Biol Blood Marrow Transplant. 2001;7
Suppl:19s-22s.
7. Henrickson KJ. Advances in the laboratory diagnosis of viral
respiratory disease. Pediatr Infect Dis J. 2004;23:s6-s10.
8.8 STI/Genitourinary Infections
8.8.1 Chlamydia and Gonorrhea Infections
8.8.1.1 Prevention of Pelvic Inflammatory Disease (PID):
Screening for Chlamydia trachomatis and Neisseria
gonorrhoeae
Clinical Background: Pelvic inflammatory disease (PID) is one of the
most common complications of sexually transmitted infections in
women. It affects various structures of the upper female genital tract,
leading to disorders such as endometritis, salpingitis, tubo-ovarian
abscess, and pelvic peritonitis.1 In the United States, PID affects about 1
million women each year, causing infertility in about 20% of affected
women, ectopic pregnancy in 9%, and chronic pelvic pain in 18%.1,2 PID
presents with a wide clinical spectrum and often goes undetected
because signs and symptoms are non-specific; therefore, a low
threshold for diagnosis should be maintained.
PID is often caused by sexually transmitted organisms, most commonly
Chlamydia trachomatis and Neisseria gonorrhoeae. Other causative
organisms include constituents of vaginal flora, such as anaerobic
bacteria, Gardnerella vaginalis, Haemophilus influenzae, enteric gram-
negative rods, and Streptococcus agalactiae. Cytomegalovirus,
Mycoplasma hominis, and Ureaplasma urealyticum have also been
implicated as causative agents.2,3
Chlamydia and gonorrhea are the 2 most common reportable infectious
diseases in the United States. Each year about 3 million new C
trachomatis infections occur among adolescents and adults in the
United States,4 associated with a total cost of $2.4 billion.5 N
gonorrhoeae is responsible for an estimated 650,000 new infections
annually.6 These diseases affect both men and women and are often
spread unknowingly by asymptomatic individuals. Infection during
pregnancy may result in neonatal transmission, leading to complications
such as ophthalmia neonatorum and pneumonia. Cure rates for
chlamydial infection are about 97% to 98% with a 7-day, multidose
regimen of doxycycline or a single dose of azithromycin.7 N gonorrhoeae
Infectious Disease
also responds well to treatment, although resistance to beta-lactams
and fluoroquinolones can complicate therapy, and fluoroquinolones are
no longer recommended for empiric therapy. Because chlamydia is
common in patients with gonorrhea, cotreatment should be considered
in individuals with diagnosed gonorrheal infection.2 Recommended
regimens for gonorrhea include cefixime or ceftriaxone and, if
chlamydial infection is not ruled out, azithromycin or doxycycline.
Subclinical PID has been reported in >25% of women with gonorrhea or
chlamydia;8 some studies have indicated that up to 40% of women with
untreated chlamydia develop PID.9,10 Because most people with C
trachomatis infection are asymptomatic and are not aware of their
infection, screening is important if these infections are to be adequately
diagnosed and treated. Chlamydia screening among asymptomatic,
sexually active women can decrease the incidence of PID11 and,
possibly, the prevalence of chlamydial infection. Guidelines from the
Centers for Disease Control and Prevention (CDC) now recommend
routine annual screening for all sexually active women under 25 years
of age and for all women at increased risk. Moreover, because repeat
infection is common in women with chlamydia, rescreening should be
considered 3 to 4 months after treatment.2 Although gonorrhea is a
common cause of PID, no studies have suggested that screening for
gonorrhea can reduce the incidence of PID.
Screening for cervical C trachomatis infection in asymptomatic women
can be cost effective. Screening with a DNA amplification assay,
coupled with a single dose of azithromycin in positive patients, is a
cost-effective strategy when the prevalence is 6% or greater.12
Screening high-risk women and treating those infected was found to
significantly lower the incidence of PID relative to a control population
(9/1009 [0.9%] vs 33/1598 [2.1%], respectively).11 Similarly, an economic
evaluation of a school-based sexually transmitted infection program in
New Orleans revealed a savings of $1524 for each case of PID
prevented.13 Thus, timely detection followed by effective therapy
appears to be a cost-effective way of preventing complications and
further spread of C trachomatis infection.
Individuals Suitable for Testing
Table 17 lists the patient populations that are appropriate for screening
and diagnosis.1,14,15
Test Availability
Nucleic Acid Amplification Tests
• Chlamydia trachomatis DNA, SDA: This test directly detects the
presence of C trachomatis DNA. It is based on strand displacement
amplification (SDA) and is highly sensitive and specific. The test can
be performed on either swab or urine specimens from men and
women. The test can also be performed on endocervical specimens
submitted in liquid-based Pap test vials.
• Chlamydia trachomatis RNA, TMA: This test directly detects the
presence of C trachomatis ribosomal RNA (rRNA). It is based on
transcription-mediated amplification (TMA) and is highly sensitive
and specific. The test can be performed on either swab or urine
specimens from men and women.
• Neisseria gonorrhoeae DNA, SDA: This test directly detects the
presence of N gonorrhoeae DNA. It is based on SDA and is both
sensitive and specific. The test can be performed on either swab or
urine specimens from men and women. The test can also be
performed on endocervical specimens submitted in liquid-based Pap
test vials.
 231

Section 8 Infectious Disease

Table 17. Individuals Suitable for C trachomatis and N gonorrhoeae Testing

Chlamydia Gonorrhea
Symptomatic individuals Symptomatic individuals
Sex partners of infected individuals (symptomatic and asymptomatic) Sex partners of infected individuals (symptomatic and asymptomatic)
All sexually active women b25 years of age; women >25 years of age at Women at high risk (see below)
high risk (see below)
Pregnant women at high risk (see below)
Pregnant women b25 years of age; other pregnant women at high risk
Women at high risk include
(see below)
• Sexually active women b25 years of age with 2 or more sex partners in
Women at high risk include the past year
•
• Women and teens attending STI, family-planning, or prenatal clinics Commercial sex workers
• Women with a previously diagnosed STI • Women with a history of repeated episodes of gonorrhea
• Women in high-prevalence settings
• Women undergoing elective abortion
• Women residing in detention facilities
• Women with new or multiple sex partners
• Women with inconsistent or incorrect use of barrier protection
• Women being evaluated for infertility
STI, sexually transmitted infection.
Note: Screening strategy will vary depending on prevalence in the patient population.

• Neisseria gonorrhoeae RNA, TMA: This test directly detects the tested for oxidase production and subcultured on chocolate agar for
presence of N gonorrhoeae rRNA. It is based on TMA and is highly confirmation.
sensitive and specific. The test can be performed on either swab or
urine specimens from men and women. Test Selection

Non-amplification Nucleic Acid Tests Chlamydia Screening

• Chlamydia trachomatis, DNA Probe: This hybridization method
directly detects the presence of C trachomatis by using a DNA probe
specific for the organism’s nucleic acid. Positive results are
automatically confirmed using organism-specific probes. The test can
be performed on swab specimens from men and women, but not on
urine specimens.
• Neisseria gonorrhoeae, DNA Probe: This hybridization method
directly detects the presence of N gonorrhoeae by using a DNA probe
specific for the organism’s nucleic acid. Positive results are confirmed
with organism-specific probes. The test can be performed on swab
specimens from men and women, but not on urine specimens.
Non-nucleic Acid Tests
• Chlamydia trachomatis, DFA: The direct fluorescence antibody assay
(DFA) directly detects the presence of C trachomatis. Specimens are
incubated with fluorescein-labeled monoclonal antibody and
examined microscopically for fluorescence of chlamydial elementary
bodies. Specimen quality is assessed by checking for the presence of
epithelial columnar cells.
• Chlamydia trachomatis Antibodies: This immunofluorescence assay
detects antibodies (IgM, IgG, and IgA) produced as an immune
response to C trachomatis infection.
• Chlamydia trachomatis Culture: This test detects the presence of C
trachomatis. The organism is grown in cell culture and then identified
via an immunofluorescence assay with monoclonal antibodies
specific for the major outer membrane protein (MOMP), present in all
15 known serovars of C trachomatis but not C pneumoniae or
C psittaci.
• Neisseria gonorrhoeae Culture: This test detects the presence of N
gonorrhoeae. Suspicious colonies of gram-negative diplococci are
CDC recommendations list the following options for chlamydia
screening in women, in order of preference: 1) a nucleic acid
amplification test performed on an endocervical swab, if a pelvic exam
is possible, or on urine if not; 2) a non-amplification-based nucleic acid
test, an enzyme immunoassay (ie, antibody test), or DFA performed on
an endocervical swab; and 3) culture performed on an endocervical
swab specimen.3
Chlamydia Diagnosis
Culture is no longer the standard for diagnosis of C trachomatis
infection. With the advent of nucleic acid amplification and detection
methods, culture is used infrequently because of low sensitivity, higher
cost, specimen viability requirements during collection and transport,
and slow turnaround time (3–7 days). Culture continues to be the most
specific method (~100%), followed closely by nucleic acid amplification
assays. The remaining methods range from 97% to 99% specific,
depending on the study. Thus, culture remains the test of choice for
medicolegal cases such as child abuse and sexual assault; only viable
infectious chlamydial elementary bodies are detected and there is
minimal risk of contamination.
Non-culture tests for C trachomatis are more rapid and standardized and
have less stringent specimen handling requirements. Nucleic acid
amplification methods such as SDA and TMA are the most sensitive
methods for detecting C trachomatis.3 Compared with amplification
methods, culture is only 70% to 85% sensitive.16,17 The remaining tests
(DNA probe and DFA) are all less sensitive than culture (sensitivity
roughly 70%–97% that of culture, depending on reagent manufacturer
and specimen type).
Positive test results are considered presumptive evidence of infection,
and confirmation testing should be considered when a false-positive
result could adversely affect the patient.3 Theoretically, the best
232
Section 8
approach is to use a second specimen with a different test that has a
different target, antigen, or phenotype and a different format. The least
desirable approach is to repeat the original test on the original
specimen.3 The CDC recommends that positive nucleic acid
amplification test results such as SDA and TMA be verified only with
another amplification test. For other non-culture tests, positive results
can be confirmed with culture, a non-culture test with a different target
than the initial test, or by use of a blocking antibody for EIA tests or a
competitive probe for DNA probe tests.3 DFA is frequently used to
confirm positive EIA results, as it can simultaneously assess specimen
adequacy through visualization of columnar epithelial cells and
examination for excessive cervical mucus and squamous epithelial cells.
The DFA test requires highly trained personnel for interpretation.
Chlamydia Test-of-Cure
Routine test-of-cure is not recommended for chlamydia when first-line
regimens are used.3 When test-of-cure is indicated, as in the case of
chlamydial infection during pregnancy, culture can be performed 3
weeks post-therapy.1 Non-culture detection methods should not be used
<3 weeks after treatment is completed.
Gonorrhea
As with chlamydia testing, nucleic acid amplification assays for
gonorrhea offer convenience and high sensitivity. The difference in
sensitivity between culture and nucleic acid amplification methods is
less pronounced than with chlamydia, although inappropriate storage
and transport conditions can lessen the sensitivity of culture. SDA has
shown specificity similar to that of culture and greater sensitivity.18
TMA also exhibits excellent specificity and sensitivity.19
Culture is required for medicolegal purposes and allows for subsequent
antibiotic susceptibility testing. CDC recommendations indicate that
while culture might be preferred because of the potential need for
antibiotic susceptibility testing, a nucleic acid amplification test can be
performed with endocervical swab specimens when problems in
controlling storage or transport conditions could compromise culture
sensitivity. Furthermore, some nucleic acid amplification tests can be
used for testing urine specimens (although with slightly less sensitivity),
extending test availability to venues where pelvic exams are not
performed.3
The theoretical considerations outlined above for confirming a positive
chlamydia result also apply for confirming a positive gonorrhea result.
Positive cultures can be confirmed with nucleic acid probe assays. For
positive non-culture results, culture is the preferred method for
confirmation if appropriate storage and transport conditions are
ensured. However, as storage and transport conditions are not always
optimal, a second non-culture method can be performed. Routine test-
of-cure is not recommended after treatment with a first-line antibiotic.
Test Interpretation
Positive results are considered presumptive evidence of infection. If a
false-positive result is expected to have adverse medical, social, or
psychological consequences, confirmatory testing should be
considered.3 Confirmatory testing should also be considered in low-
prevalence settings, where the positive predictive value (likelihood that
a positive result is a true positive) of assays is reduced. See the “Test
Selection” section for approaches to confirmatory testing. Treatment
probably should not be withheld while awaiting the results of
confirmatory testing.
Chlamydia
Antibody tests: C trachomatis infection confers little immunity against
reinfection, although secretory IgA may provide some protection. A
Infectious Disease
positive IgM antibody result may indicate recent infection with C
trachomatis; a negative result, however, does not indicate absence of C
trachomatis since IgM antibodies are frequently absent in infected
individuals. The presence of IgG indicates active or resolved infection.
High IgG titers often point to recent infection, whereas intermediate or
low titers may be due to early infection, resolved infection, or cross-
reaction with other Chlamydia species. When comparing antibody tests,
a 4-fold rise in titer best indicates active infection. Serologic tests are
not recommended for chlamydia screening.3
Culture: A positive culture result is highly specific for C trachomatis. A
negative result may indicate absence of C trachomatis infection or a
false-negative due to lack of organism viability or improper specimen
collection.
DFA: A positive DFA result indicates the presence of C trachomatis
infection, whereas a negative result implies absence of infection or a
false-negative due to lack of assay sensitivity.
DNA, SDA: A positive DNA result is highly indicative of C trachomatis
infection. False-positive results may be obtained due to laboratory
contamination (rare in an experienced lab) or sampling too soon after
cessation of therapy (ie, <3 weeks post-therapy). Negative results are
highly specific for absence of C trachomatis infection but may be caused
by interfering substances.
DNA Probe: A positive DNA probe test is indicative of C trachomatis in
high-prevalence patient populations; confirmation should be considered
in low-prevalence settings. A grossly bloody specimen may confound
the result, although confirmation of positive results minimizes problems
with blood-contaminated specimens. Because of its lesser sensitivity,
this technique is more likely to produce false-negative results than
nucleic acid amplification tests.
RNA, TMA: A positive TMA result is highly indicative of C trachomatis
infection, while a negative result is highly indicative of lack of infection.
False-positive results may be obtained due to laboratory contamination
(rare in an experienced lab) or sampling too soon after cessation of
therapy (ie, <3 weeks post-therapy).
Gonorrhea
Culture: A positive culture result is highly specific for N gonorrhoeae. A
negative result may indicate absence of infection or a false-negative
due to lack of organism viability or improper specimen collection.
DNA, SDA: A positive test result strongly suggests N gonorrhoeae
infection. False-positive results may be obtained due to laboratory
contamination (rare in an experienced lab) or sampling too soon after
cessation of therapy (ie, <3 weeks post-therapy). Negative results are
highly specific for absence of N gonorrhoeae infection but may be
caused by interfering substances.
DNA Probe: Positive results are automatically confirmed with organism-
specific probes and are indicative of infection. False-negative results
may be caused by an insufficient number of organisms in the specimen,
grossly bloody specimens, wood collection swabs, or unknown
endogenous substances.
RNA, TMA: A positive TMA result is highly indicative of N gonorrhoeae
infection, while a negative result is highly indicative of lack of infection.
False-positive results may be obtained due to laboratory contamination
(rare in an experienced lab) or sampling too soon after cessation of
therapy (ie, <3 weeks post-therapy).
 233

Section 8 Infectious Disease

References Method: In this assay, target DNA is amplified by isothermal strand

displacement amplification (SDA) using 2 enzymes (DNA polymerase
1. Centers for Disease Control and Prevention. Recommendations and
and a restriction endonuclease). Amplification products are detected
Reports: Sexually transmitted diseases treatment guidelines 2002.
simultaneously via fluorescence energy transfer from labeled probes.
MMWR. 2002:51(RR-6).
2. Westrom L, Joesoef R, Reynolds G, et al. Pelvic inflammatory
Interpretive Information: The clinical sensitivity and specificity of
disease and fertility. A cohort study of 1,844 women with
this assay depend on the presence or absence of clinical symptoms, the
laparoscopically verified disease and 657 control women with
patient’s sex, and the type of specimen tested. Seminal fluid, mucus,
normal laparoscopic results. Sex Transm Dis. 1992;19:185-192.
lubricants, and common ointments do not appear to interfere with the
3. Centers for Disease Control and Prevention. Recommendations and
assay. Samples containing leukocytes or blood (any amount of blood in
Reports: Screening tests to detect Chlamydia trachomatis and
urine or >5% in swabs) may give false-negative results. Additional
Neisseria gonorrhoeae infections—2002. MMWR. 2002;51(RR-
testing is recommended if false-positive or false-negative results could
15):1-38.
lead to adverse medical, social, or psychological consequences.
4. Groseclose SL, Zaidi AA, DeLisle SJ, et al. Estimated incidence and
prevalence of genital Chlamydia trachomatis infections in the
8.8.1.3 Neisseria gonorrhoeae DNA, SDA
United States, 1996. Sex Transm Dis. 1999;26:339-344.
5. Centers for Disease Control and Prevention. Special focus:
Clinical Use: This test is used to screen for and diagnose Neisseria
Surveillance for reproductive health. MMWR. 1993;42(SS-6):59.
gonorrhoeae infection.
6. American Social Health Association. Sexually Transmitted Disease
in America: How Many Cases and at What Cost? Menlo Park,
Method: In this assay, target DNA is amplified by isothermal strand
California: Kaiser Family Foundation; 1998.
displacement amplification (SDA) using 2 enzymes (DNA polymerase
7. Lau CY, Qureshi AK. Azithromycin versus doxycycline for genital
and a restriction endonuclease). Amplification products are detected
chlamydial infections: a meta-analysis of randomized clinical trials.
simultaneously via fluorescence energy transfer from labeled probes.
Sex Transm Dis. 2002;29:497-502.
8. Wiesenfeld HC, Hillier SL, Krohn MA, et al. Lower genital tract
Interpretive Information: The clinical sensitivity and specificity of
infection and endometritis: insight into subclinical pelvic
this assay vary based on the presence or absence of clinical symptoms,
inflammatory disease. Obstet Gynecol. 2002;100:456-463.
the patient’s sex, and the type of specimen tested. Seminal fluid,
9. Rees E. Treatment of pelvic inflammatory disease. Am J Obstet
mucus, lubricants, and common ointments do not appear to interfere
Gynecol. 1980;138:1042-1047.
with the assay. Samples containing leukocytes or blood (any amount of
10. Stamm WE, Guinan ME, Johnson C, et al. Effect of treatment
blood in urine or >5% in swabs) may give false-negative results.
regimens for Neisseria gonorrhoeae on simultaneous infection with
Additional testing is recommended if false-positive or false-negative
Chlamydia trachomatis. N Engl J Med. 1984;310:545-549.
results could lead to adverse medical, social, or psychological
11. Scholes D, Stergaghis A, Heidrich F, et al. Prevention of pelvic
consequences.
inflammatory disease by screening for cervical chlamydial infection.
N Engl J Med. 1996;334:1362-1366.
8.8.2 Herpes Simplex Virus (HSV) 1 and 2 IgG, HerpeSelect®
12. Genc M, Mardh P. A cost-effectiveness analysis of screening and
Type-Specific Antibodies
treatment for Chlamydia trachomatis infection in asymptomatic
women. Ann Intern Med. 1996;124:1-7.
Clinical Use: This test is used to detect and differentiate between
13. Wang LY, Burstein GR, Cohen DA. An economic evaluation of a
type-1 and type 2 herpes simplex virus (HSV) and to assist in diagnosis
school-based sexually transmitted disease screening program. Sex
and patient counseling.
Transm Dis. 2002;29:737-745.
14. US Preventive Services Task Force. Guide to Clinical Preventive
Clinical Background: Herpes simplex virus infection is extremely
Services, 2nd Edition, 1996. Available at: http://www.ahcpr.gov/
common in the United States: about 68% of individuals over age 12 are
clinic/cpsix.htm. Accessed November 18, 2002.
seropositive for HSV-1, and more than 20% are seropositive for HSV-2.
15. Nelson HD, Helfand M. Screening for chlamydial infection. Am J
Type-specific diagnosis has important implications for prognosis and
Prev Med. 2001;20(Suppl 3):95-107.
patient counseling. Although HSV-1 accounts for an increasing
16. Black CM. Current methods of laboratory diagnosis of Chlamydia
proportion of primary genital herpes, it is far less likely than HSV-2 to
trachomatis infections. Clin Microbiol Rev. 1997;10:160-184.
cause recurrent genital lesions. HSV-2 seropositivity, on the other hand,
17. Puolakkainen M, Hiltunen-Back E, Reunala T, et al. Comparison of
suggests a stronger likelihood of recurrent genital outbreaks and viral
performances of two commercially available tests, a PCR assay and
shedding, even when asymptomatic. Individuals who are seronegative
a ligase chain reaction test, in detection of urogenital Chlamydia
for HSV-1 and/or HSV-2 are at risk for acquiring infection from
trachomatis infection. J Clin Microbiol. 1998;36:1489-1493.
seropositive partners; seronegative women who become infected with
18. Van Dyck E, Ieven M, Pattyn S, et al. Detection of Chlamydia
either HSV-1 or HSV-2 during pregnancy are the most likely group of
trachomatis and Neisseria gonorrhoeae by enzyme immunoassay,
HSV-infected patients to pass on the virus to their neonate. Women
culture, and three nucleic acid amplification tests. J Clin Microbiol.
who are seropositive for both types early in pregnancy have a lower
2001;39:1751-1756.
likelihood of neonatal transmission than do women who have a first-
19. Gaydos CA, Quinn TC, Willis D, et al. Performance of the APTIMA
episode outbreak later in pregnancy, but a greater likelihood of
Combo 2 assay for detection of Chlamydia trachomatis and
transmission than women who remain uninfected throughout pregnancy.
Neisseria gonorrhoeae in female urine and endocervical swab
specimens. J Clin Microbiol. 2003;41:304-309.
Culture is the preferred virologic method for diagnosis, but has low
sensitivity when performed on samples from healing or recurrent
8.8.1.2 Chlamydia trachomatis DNA, SDA
lesions. Because IgG antibodies to HSV persist for life, serologic assays
can detect infection even in the absence of lesions. Most HSV serology
Clinical Use: This test is used to screen for and diagnose Chlamydia
assays, however, are not type-specific. The HerpeSelect IgG assays
trachomatis infection.
234

Section 8 Infectious Disease

distinguish between HSV-1 and HSV-2 on the basis of differences in the
patient’s immune response to HSV glycoprotein G (gG).
Interpretive Information: These assays are highly sensitive
(91%–100%) and specific (93%–100%) for HSV-1 and HSV-2 infection,
even in the absence of symptoms. Because antibodies may take several
weeks to reach detectable levels after primary infection, negative
results should be confirmed by repeat testing 4 to 6 weeks later in
cases of suspected early infection. Thus, a negative result suggests
absence of infection. A positive result strongly suggests infection.
8.8.3 HPV (Human Papillomavirus), High Risk DNA, Hybrid
Capture II
See Hematology/Oncology, section 6.4.2.
8.9 Vector-Borne Diseases and Travel
Medicine
8.9.1 Tick-borne Disease: Laboratory Support of Diagnosis
Babesiosis, Ehrlichiosis, Lyme Disease, Q Fever, Rocky
Mountain Spotted Fever, Tularemia
Clinical Background: Tick-borne diseases are those that are
transmitted to humans via a tick vector such as the deer tick, dog tick,
wood tick, and Lone Star tick. Transmission occurs primarily in the
spring and summer months throughout the United States. Causative
agents include bacteria, rickettsia, viruses, and protozoa. The incidence
varies based on geographic location and the causative agent; Lyme
disease is the most prevalent, especially in the Northeast (Table 18).
Clinical manifestations also vary depending on the disorder, but
frequently include fever, chills, sweats, headaches, myalgia, arthralgia,
nausea, and vomiting. A skin lesion at the site of the bite or a skin rash
may or may not be present. More severe disease may result in
hemolytic, respiratory, cardiac, and neurologic complications as well as
kidney or liver failure and arthritis. Although approximately 2% to 5% of
the cases end in death, antimicrobial therapy with doxycycline,
chloramphenicol, amoxicillin, etc, is usually effective. Coinfection with
more than 1 causative agent may occur (eg, Borrelia burgdorferi and
Babesia microti).
Diagnosis is primarily based on a history of exposure and clinical
presentation. Laboratory tests help confirm or support the clinical
diagnosis. Microscopic visualization of the causative organism from
blood or skin lesions, various serologic techniques, and polymerase
chain reaction (PCR) are employed.
Individuals Suitable for Testing include individuals with a history of
exposure in an endemic area and clinical presentation consistent with
tick-borne disease.
Test Availability
Non-specific Laboratory Tests
• Alanine Aminotransferase (ALT): This spectrophotometric (kinetic)
test measures the ALT level in serum.
• Aspartate Aminotransferase (AST): This spectrophotometric (kinetic)
test measures AST levels in the serum.
• Complete Blood Count (CBC): This test includes a white and red blood
cell count, a hemoglobin and hematocrit, and associated indices
(MCV, MCH, MCHC, RDW). In addition, a platelet count, mean
platelet volume, and automated differential cell count are provided.
• Erythrocyte Sedimentation Rate (ESR): This test is based on a
modified Westergren method.
Laboratory Tests for Support of the Clinical Diagnosis
• Babesia microti: This test detects the presence of B microti by
microscopic examination of Giemsa stained thick and thin peripheral
blood smears and an acridine orange stained buffy coat.
• Babesia Antibodies (IgG, IgM): This indirect immunofluorescence
antibody assay detects IgG and IgM antibodies produced as a
babesiosis immune response.
• Babesia microti DNA, PCR: This real-time PCR assay directly detects
the presence of B microti DNA.
• Ehrlichia chaffeensis Antibodies (IgG, IgM): Using E chaffeensis
antigen, this indirect immunofluorescence antibody assay detects IgG

Table 18. Tick-borne Disease Incidence

Disorder Causative Agent US Incidence US Geographic Distribution
Babesiosis Babesia microti Unknown Northeastern coast, WI
Colorado tick fever Coltivirus species 200–300/y Western US
Ehrlichiosis
Human monocyte (HME) Ehrlichia chaffeensis 50/y 30 states: South-central, Southeast, mid-Atlantic
Human granulocyte (HGE) E equi-like organism 25/y 11 states: upper Midwest and Northeast
Encephalitis Powassan virus 20 cases Unrestricted
Lyme disease Borrelia burgdorferi 10,000+/y 47 states, primarily Northeastern US, WI, MN, CA, OR
Q fever Coxiella burnetii Unknown Unrestricted
Rocky Mountain spotted Rickettsia rickettsii 600–1200/y 48 states, primarily Southeast, West, and South-Central US
fever (RMSF)
Tick paralysis Tick neurotoxin Unknown Northwest, Southern US
Tick-borne relapsing Borrelia species Unknown West, South, and Southwest US
fever (TBRF)
Tularemia Francisella tularensis 150–300/y AR, MO, OK, Southeast, Rocky Mountain states, UT, NV, CA

Section 8
and IgM antibodies produced as a human monocytic ehrlichiosis
immune response.
• Anaplasma phagocytophilum (IgG/IgM) (Human Granulocyte
Ehrlichiosis [HGE] Antibody): This immunofluorescence assay detects
IgG and IgM antibodies produced by the E equi-like organism.
• Lyme Disease Antibodies, EIA: This enzyme immunoassay detects the
non-immunoglobulin specific total antibodies (ie, polyvalent antibodies)
produced as an immune response to B burgdorferi infection.
• Lyme Disease Antibodies, Western Blot: Western blot assays can
detect IgG and IgM specific antibodies produced as an immune
response to B burgdorferi infection. During early disease, both IgG
and IgM antibody tests are performed, but only the IgG antibody test
is performed during late disease.
• Lyme Disease C6 Antibodies, Total with Reflex to IgG and IgM,
Western Blot*: This ELISA detects IgG and IgM antibodies to the C6
peptide, the immunodominant antigen of the VlsE surface protein of
Borrelia species. If the ELISA is positive or equivocal, Lyme disease
IgG and IgM confirmation by Western blot will be performed.
• Lyme Disease Antibody, Total, EIA with Reflex to CSF Ratio*: This
enzyme immunoassay detects antibodies produced as an immune
response to B burgdorferi infection in both CSF and serum. If the
immunoassay is positive, a CSF index is calculated based on the ratio
of CSF results to serum results.
• Lyme Disease DNA, Real-Time PCR: This test directly detects the
presence of B burgdorferi DNA based on polymerase chain reaction
(PCR) amplification of a specific flagellin gene sequence. The test
may be performed on whole blood, CSF, synovial fluid, or the tick
vector.
• Q Fever (Coxiella burnetii) Antibodies (IgG, IgM) with Reflex to
Titers*: This indirect immunofluorescence antibody assay detects IgG
and IgM antibodies produced as an immune response to C burnetii
infection. IgG and IgM results are reported for both phase I and
phase II antigens. Titers are performed if screens are positive.
• Rocky Mountain Spotted Fever (Rickettsia rickettsii) Antibodies (IgG,
IgM) with Reflex to Titers*: This indirect immunofluorescence
antibody assay detects IgG and IgM antibodies produced as an
immune response to R rickettsii infection. Titers are performed if
screens are positive.
• Tick (and Other Arthropods) Identification: This test provides a
species specific identification of an alcohol-preserved tick based on
direct microscopic examination.
• Francisella Tularensis Antibody, DA: This direct agglutination test
detects antibodies produced as an immune response to F tularensis
infection.
*Reflex tests are performed at an additional charge and are associated with additional
CPT codes.
Test Interpretation and Selection
Laboratory tests are primarily used to support the clinical diagnosis and
are not to be used for screening purposes.
Non-specific Laboratory Tests
Complete blood count (CBC) results may be abnormal in various tick-
borne diseases. For example, leukopenia may be observed in ehrlichiosis
and RMSF while leukocytosis may be observed in tick-borne relapsing
fever (TBRF) and tularemia. Thrombocytopenia may be observed in
ehrlichiosis as well as TBRF. Spirochetes (Lyme disease) and morulae
(ehrlichial inclusion bodies) may be observed in blood smears.
235
Infectious Disease
Erythrocyte sedimentation rate (ESR) may be increased in Lyme disease
and TBRF. As with all ESR results, normal results may not accurately
reflect the clinical state if the specimen is not tested within 8 hours
from the time of draw; abnormal results, however, are reliable.
Increased levels of the liver enzymes alanine aminotransferase (ALT)
and aspartate aminotransferase (AST) are commonly observed in
ehrlichiosis, tularemia, and RMSF.
Abnormal non-specific test results are by definition associated with a
multitude of other disorders. The laboratory tests listed below are much
more specific and therefore more useful in support of clinical diagnosis.
Laboratory Tests for Support of the Clinical Diagnosis
Babesiosis: Microscopic examination of Giemsa-stained thick and thin
peripheral blood smears and an acridine orange stained buffy coat can
assist in babesiosis diagnosis. Small intra-erythrocyte ring forms are
characteristic of both Babesia species and Plasmodium falciparum;
however, pigment deposits, schizonts, and gametocytes are absent in
the presence of Babesia species. A tetrad (“Maltese cross”)
configuration of budding trophozoites is diagnostic for B microti
infection.
Specific B microti antibodies are usually present by the time the patient
exhibits parasitemia and invariably within 4 weeks of onset unless the
patient is immunocompromised. Studies have indicated that
immunofluorescent antibody tests have a sensitivity of 88% to 96% and
a specificity of 90% to 100%. This method is known to cross-react with
other Babesia species (at lower titers) as well as with other blood and
tissue parasites and various tick-borne organisms; however, cross-
reactivity with malaria antibodies and Colorado tick fever is uncommon.
Although most patients with acute illness have titers r1024, testing of
acute and convalescent sera is recommended for more definitive
diagnosis. Elevated titers can persist for months following resolution of
symptoms.
Ehrlichiosis (HME and HGE): A single HME IgG titer of 64 or greater
indicates exposure at an unknown point in time to E chaffeensis. A 4-
fold or greater rise in either HME or HGE IgG titer between acute and
convalescent sera and/or an IgM titer of 20 or more is suggestive of
recent or current ehrlichiosis infection.
Lyme disease: Diagnosis of Lyme disease (B burgdorferi infection) is
best made on the basis of a history of exposure to the tick vector or tick
environment and clinical findings. Although biopsy with isolation of B
burgdorferi in culture is definitive, culture is often impractical and rarely
available. Since the sensitivity and specificity of anti-B burgdorferi
antibody tests are less than desired, a 2-step algorithm has been
recommended by the CDC/ASTPHLD Second National Conference on
Serologic Diagnosis of Lyme Disease: the first step is performance of
either total or immunoglobulin-specific (IgG and IgM) antibody assays
using EIA technology; the second step involves measurement of
antibodies with the Western blot technology on specimens having a
positive or equivocal EIA result. For early stage disease (<1 month
duration), IgM Western blot testing is recommended, whereas both IgG
and IgM testing are recommended for late-stage disease.
IgM antibodies may be present within a few weeks of disease onset
(early stage); however, IgG antibodies are produced later in the disease.
Both IgM and IgG antibodies may persist for many months or years. IgM
antibody testing is most sensitive between 1 and 2 months following
onset, whereas IgG antibody testing is most sensitive 3 months after
onset. Patients who are on antibiotic therapy and who have early stage
disease may have low or negative antibody titers. Thus, negative results
236
Section 8
may indicate lack of infection or lack of seroconversion (due to the time
of specimen collection relative to disease onset or to suppression of
antibody production subsequent to therapy, etc). If Lyme disease is still
suspected following a negative antibody result, testing a second
specimen collected 2 to 4 weeks after the first specimen is
recommended.
Positive results with polyvalent antibody assays may be due to current
or previous B burgdorferi infection, vaccination, or other spirochete-
caused diseases such as syphilis, yaws, pinta, leptospirosis, and
relapsing fever. Syphilis and Lyme disease may be differentiated by
VDRL and RPR tests, both of which are negative in Lyme disease and
positive in syphilis. Patients with autoimmune disorders (eg, lupus
erythematosus and rheumatoid arthritis), mononucleosis, rickettsia,
Ehrlichia, and bacterial infections (eg, Helicobacter pylori) may also have
a positive antibody test. Because the C6 antigen is highly specific for B
burgdorferi, the C6 assay does not typically yield false-positive results
in the above conditions. Additionally, the C6 assay does not react with
antibodies to the OspA vaccine antigen; thus, individuals who have
been vaccinated but not infected will test negative.
A positive IgM result in conjunction with a negative IgG result is
presumptive evidence of early infection, unless obtained on a specimen
collected more than 1 month following onset. A positive IgM result on a
specimen collected more than 1 month following onset is likely to be a
false-positive when the IgG result is negative. A positive IgG result with
a positive or negative IgM result is presumptive evidence of late
infection.
Expression of cerebrospinal fluid (CSF) and serum EIA antibody results
as a ratio may help correct for passive diffusion of antibodies across the
blood-brain barrier and thus can be used to further support a clinical
diagnosis of Lyme neuroborreliosis. Red cell contamination of CSF
specimens as well as xanthochromia and turbidity interfere with test
results. Collection of the serum and CSF specimen should be within 24
hours of each other; both specimens should be assayed simultaneously.
An index less than 0.76 is deemed negative and indicative of passive
blood-brain antibody diffusion, while an index greater than 1.13 is
deemed positive and is supportive of Lyme neuroborreliosis.
The interpretation of Western blot antibody assays is based on the
number and pattern of band positivity: 2 of 3 bands (23, 39, 41kd) for
IgM positivity and 5 of 10 bands (18, 23, 28, 30, 39, 41, 45, 58, 66, or 93
kd) for IgG positivity. The Western blot is to be used only following
initial EIA testing.
A positive PCR test result may be obtained prior to or following
seroconversion, and, although it is associated with active disease, it
does not necessarily prove the presence of active disease because PCR
cannot distinguish between live and dead organisms. A negative PCR
test is suggestive of the absence of B burgdorferi infection; however, a
negative PCR result does not exclude the diagnosis due to transient
spirochetemia, inadequate spirochete numbers in the sample, or DNA
sequence variances in different B burgdorferi strains. Although whole
blood specimens are clinically indicated under rare circumstances during
acute or early infection, such specimens are generally not recommended
since transient spirochetemia renders negative results useless. PCR
tests are most useful in patients with Lyme arthritis and Lyme
neuroborreliosis; the specimens of choice are synovial fluid and CSF,
respectively. Test results should be interpreted in context with clinical
findings.
Continued presence or absence of antibodies during treatment does not
imply therapeutic failure or success; however, PCR-detected B
Infectious Disease
burgdorferi DNA may be of use. Following adequate therapy, PCR
results are generally negative, while in cases of therapeutic failure,
evidenced by ongoing or worsening symptoms, PCR results are usually
positive.
Q fever: Indirect immunofluorescence is the method of choice for
detection of C burnetii antibodies; sensitivity and specificity are better
than with complement fixation (CF) or EIA. The ratio of phase II antibody
titer to phase I antibody titer is indicative of the stage of disease: in
acute disease, the ratio is greater than 1; in chronic disease, the ratio is
almost always much lower than 1. When the organism is in phase I, it is
extremely infectious, whereas in phase II it is avirulent. Although phase
II IgG titers of 256 or greater are suggestive of acute disease, a 4-fold
rise in titer between acute and convalescent samples is more definitive.
Rocky Mountain spotted fever (RMSF): Direct immunofluorescent
examination of a skin biopsy specimen is the most definitive test (70%
sensitivity, 100% specificity) for R rickettsii; however, it is impractical
and not widely used. Culture, too, is rarely performed due to biohazard
and stringent technologic requirements. The Weil-Felix assay, which
measures the agglutination of Proteus vulgaris OX-19 and OX-2
antigens, has been replaced by the more sensitive and specific indirect
immunofluorescence assay (IFA), which has 94% to 100% sensitivity
and 100% specificity.
A negative IFA IgM and IgG result (titer <64) does not exclude rickettsial
infection; a negative result obtained from a second specimen collected
7 to 14 days later may be necessary to exclude infection if early acute
stage infection is suspected. An IgM titer >64 is suggestive of current or
recent infection, but should be interpreted in conjunction with results
from a second specimen collected 7 to 14 days later, especially if the
IgG results are negative. An IgG titer r64 but <256 is suggestive of past
or early stage infection, and a second specimen is again needed. A 4-
fold or greater increase in titer between first and second specimens
strengthens the evidence for recent infection. An IgG titer 256 or greater
is presumptive evidence of recent or current infection. Cross-reactivity
among spotted fever rickettsiae precludes speciation of rickettsiae;
thus, this test is not specific for RMSF.
Tularemia: Culture of the F tularensis bacillus is not sensitive and is
rarely performed due to biohazard from airborne transmission; thus, the
agglutination test is most frequently used to support the clinical
diagnosis. A titer of 160 or greater is presumptive evidence of infection
and a 4-fold or greater increase in titer on a second specimen collected
2 to 3 weeks later is considered diagnostic. False-negative results may
be obtained in early stage disease since 30% of patients infected for 3
weeks have negative results. Because Brucella antibodies cross-react
with this test, positive specimens should also be tested for Brucella
antibody.
Tick identification: Identification of the tick vector may assist the
differential diagnosis by ruling in or out the feasibility of the suspected
tick-borne disease. Since most patients are unaware of having been
bitten by a tick, however, this test is rarely practical.
References
1. Association of State and Territorial Public Health Laboratory
Directors and the Centers for Disease Control and Prevention:
Recommendations. In Proceedings of the Second National
Conference on Serologic Diagnosis of Lyme Disease (Dearborn,
Michigan). Washington, DC: Association of State and Territorial
Public Health Laboratory Directors. 1995;173:5.
2. Centers for Disease Control and Prevention: Recommendations for

Section 8
test performance and interpretation from the second national
conference on serologic diagnosis of Lyme disease. MMWR.
1995;44:590-591.
3. Dressler F, Whalen JA, Reinhardt BN, et al. Western blotting in the
serodiagnosis of Lyme disease. J Infect Dis. 1993;167:392-400.
4. Fauci AS, Braunwald E, Isselbacher KJ, et al, eds. Harrison’s
Principles of Internal Medicine. 14th ed. New York: McGraw-Hill;
1998.
5. Krause PJ, Telford SR III, Ryan R, et al. Diagnosis of babesiosis:
evaluation of a serologic test for the detection of Babesia microti
antibody. J Infect Dis. 1994;169:923-926.
6. Krause PJ, Telford SR III, Spielman A, et al. Concurrent Lyme
disease and babesiosis: evidence for increased severity and
duration of illness. JAMA. 1996;275:1657-1660.
7. Lyme Disease—United States, 1995. MMWR. 1996;45:481-484.
8. MarDx Diagnostics, Inc. B. burgdorferi Marblot strip test system,
1997.
9. MarDx Diagnostics, Inc. Lyme disease EIA (IgG, IgM) test system,
1996.
10. MRL Diagnostics. Rickettsia IFA IgG. August, 1997.
11. Schmidt BL. PCR in laboratory diagnosis of human Borrelia
burgdorferi infections. Clin Microbiol Rev. 1997;10:185-201.
12. Spach DH, Liles WC, Campbell GL, et al. Tick-borne diseases in the
United States. N Engl J Med. 1993;329:936-947.
8.9.2 West Nile Virus: Detection with Immunologic and Real-
time PCR Assays
Clinical Background: The West Nile virus (WNV) is a single-stranded
RNA virus of the Flaviviridae family. Like other arboviruses (eg, St. Louis
encephalitis, dengue fever, and yellow fever), its main route of
transmission to humans is through mosquitoes (primarily Culex species)
that have acquired the virus from infected birds. Direct human-to-human
transmission is not common, although people have become infected
through blood transfusions, solid organ transplants, and percutaneous
exposure. Transmission during pregnancy and through breast-feeding
has also been reported.
The first cases of human WNV infection in the United States were
reported in New York City in 1999.1 The virus has since spread rapidly.
In 2002, infected birds or insects were found in 44 states;2 there were
4156 cases of human WNV infection resulting in 284 deaths.
Although WNV infection is usually asymptomatic, about 20% of those
infected develop West Nile fever lasting 3 to 6 days. The incubation
period from infection to the onset of symptoms, when present, is
typically 2 to 14 days. Symptoms are generally mild and may include
malaise, anorexia, nausea and vomiting, eye pain, headache, muscle
pain, rash, and lymphadenopathy. Only about 1 in 140 people infected
with WNV develop severe neurologic West Nile disease,3 which may
include meningitis, encephalitis, flaccid paralysis similar to that found in
polio, and other neurologic manifestations. Neurologic West Nile
disease is about 20 times as common in infected individuals >50 years
of age as in younger people; other potential risk factors for neurologic
involvement include compromised immunity and coexisting illness.
Hospitalized individuals have a case fatality rate of 4% to 18%,4 with
advanced age being the greatest risk factor.
Diagnosis of clinically suspected WNV infection is confirmed by a r4-
fold change in WNV-specific antibody titer on sequential specimens;
isolation of WNV or detection of WNV antigen or nucleic acid
sequences in clinical samples; or detection of WNV-specific IgM in
blood or CSF confirmed with detection of WNV-specific neutralizing
antibodies in the same or a subsequent sample.5
237
Infectious Disease
Test Availability: The IgM antibody capture enzyme-linked
immunosorbent assay (MAC–ELISA) is the most conclusive laboratory
method for diagnosing WNV infection of the CNS.6 Quest Diagnostics
currently offers an IgG ELISA/IgM MAC–ELISA as well as a real-time
polymerase chain reaction (PCR) assay for WNV; both are available for
serum and CSF (Table). A nucleic acid amplification test is also available
for donor testing.
Test Selection
West Nile Virus Antibodies (IgG, IgM)
These tests detect the host immune response to WNV infection.
Although viremia is detectable earlier than the immune response,
immunologic (IgG and IgM) assays are typically more sensitive for
detecting active and convalescent WNV infection. IgM is typically
detectable at the time of initial presentation; IgG can be detected as
early as 7 days after illness onset and within 3 weeks of exposure in
most infected individuals.7 Both assays (IgG and IgM) must be
performed on the same specimen to help establish whether or not the
infection is recent. For suspected neurologic WNV disease, CSF
specimens are ideal and should be collected at initial presentation. If
only serum samples are to be used for diagnosis, paired specimens
should be collected during acute illness and again 7 to 14 days later.
West Nile Virus RNA, Qualitative Real-time PCR
This test directly detects WNV RNA in serum and CSF. WNV viremia
peaks at about the time of symptom onset and rapidly fades to
undetectable levels. Thus, real-time PCR assays can detect WNV RNA
in clinical samples as early as several days before symptom onset, prior
to seroconversion. Although these assays have excellent analytical
sensitivity, they lack the clinical sensitivity of antibody tests and are
typically not used alone for screening or diagnosis. However, such tests
can be used for confirmation of suspected WNV isolates. They also may
be useful in patients with delayed or absent antibody response due to
immunosuppression or other factors.
This test was developed and its performance characteristics have been determined by
Quest Diagnostics. Performance characteristics refer to the analytical performance of the
test
Polymerase chain reaction (PCR) is performed pursuant to a license agreement with Roche
Molecular Systems, Inc.
Test Interpretation
West Nile Virus Antibodies (IgG, IgM)
Unlike the relatively brief IgM response to many viral infections, WNV
IgM in serum generally remains detectable for at least 1 to 2 months
after infection and often persists beyond 12 months.8 Thus, serum IgM
results must be interpreted in light of the patient’s clinical condition (eg,
presence of encephalitis or meningitis) and travel history, as well as
regional WNV activity.8
A negative IgM result on an acute-phase specimen strongly indicates
absence of WNV infection. A positive IgM result on an acute-phase
specimen, accompanied by clinical symptoms consistent with WNV
infection, suggests recent infection. In cases of WNV central nervous
system (CNS) infection, IgM is almost always detectable on the first day
of clinical illness. Detection of WNV IgM in CSF strongly suggests acute
CNS infection, as IgM does not cross the blood–brain barrier.9 Because
the MAC–ELISA may be cross-reactive with St. Louis encephalitis (SLE)
virus, SLE infection should be ruled out.
WNV IgG is often detectable as early as 7 days after illness onset and
persists indefinitely. Thus, a positive IgG result is consistent with
convalescence or immunity. A negative IgG result combined with a
238

Section 8 Infectious Disease

positive IgM result in acute-phase specimens suggests recent infection,

as does seroconversion from IgG-negative to -positive status from the
acute- to convalescent-phase sample.

False-positive WNV antibody results may occur in individuals infected

with or recently vaccinated against flaviviruses such as yellow fever,
dengue fever, and Japanese encephalitis, as well as those with
previous WNV infection or current SLE infection.

West Nile Virus RNA, Qualitative Real-time PCR

Although a positive real-time PCR result is diagnostic of WNV infection,
a negative result does not rule out infection.

References
1. Nash D, Mostashari F, Fine A, et al. The outbreak of West Nile virus
infection in the New York City area in 1999. N Engl J Med.
2001;344:1807-1814.
2. Centers for Disease Control and Prevention Web site. West Nile Virus
2002 Case Count. Available at http://www.cdc.gov/ncidod/dvbid/
westnile/surv&controlCaseCount02.htm. Accessed September 8,
2003.
3. Mostashari F, Bunning ML, Kitsutani PT, et al. Epidemic West Nile
encephalitis, New York, 1999: results of a household-based
seroepidemiological survey. Lancet. 2001;358:261-264.
4. Petersen LR, Marfin AA, Gubler DJ. West Nile virus. JAMA.
2003;290:524-528.
5. Centers for Disease Control and Prevention. Epidemic/epizootic West
Nile virus in the United States: guidelines for surveillance,
prevention, and control. Available at: http://www.cdc.gov/ncidod/
dvbid/westnile/resources/wnv-guidelines-apr-2001.pdf. Accessed
September 5, 2003.
6. Centers for Disease Control and Prevention. Fact sheet: West Nile
virus (WNV) infection: information for clinicians. Centers for Disease
Control and Prevention Web site. Available at http://www.cdc.gov/
ncidod/dvbid/westnile/resources/fact_sheet_clinician.htm. Accessed
October 1, 2003.
7. Lanciotti R. Diagnostic testing in humans—lessons learned in the
past three years. Fourth National Conference on West Nile Virus in
the United States. New Orleans, Louisiana. February 9-11, 2003.
8. Roehrig JT, Nash D, Maldin B, et al. Persistence of virus-reactive
serum immunoglobulin M antibody in confirmed West Nile virus
encephalitis cases. Emerg Infect Dis. 2003;9:376-379.
9. Petersen LR, Marfin AA. West Nile virus: a primer for the clinician.
Ann Intern Med.

Section 9
9.1 Drug Half-Life, Steady State, and
Recommended Sample Collection Time
A drug may accumulate in the body if the dosage is kept constant and
the concentration exceeds the total capacity for elimination (excretory
as well as nonexcretory). As a general rule, under conditions of first
order kinetics, 5.5 half-lives are required to achieve a steady state for
orally administered drugs on a fixed dose. Similarly, it will take 5.5 half-
lives to almost completely eliminate a discontinued orally administered
drug that had reached steady state. For example, a short half-life drug
such as primidone (6-8 hours) will reach steady state rapidly and be
eliminated rapidly (33-44 hours). A long half-life drug such as
phenobarbital (4 days) will take longer to reach steady state and will
persist long after being discontinued. Moreover, when a dose is
changed, it will take 5.5 half-lives to reach a new steady state. Thus,
following a dosage increase, it may not be appropriate to quantitate the
blood level of the drug after administration of the first higher dose.
Moreover, it is imperative to obtain a sample in the beta (elimination)
phase. A sample representative of the alpha (distribution) phase is not
helpful. An example of a commonly encountered drug for which this is a
problem is digoxin. Samples for digoxin should be obtained no sooner
than 6 hours after an oral dose.
The half-life also influences the interval at which a drug should be
administered. A drug should generally be given at an interval of 1/2 its
half-life. Such a drug administration schedule minimizes wide
fluctuations (peaks and valleys). The drug administration schedule will,
in turn, influence the time for specimen collection. Again, the proper
time to obtain a specimen for therapeutic monitoring is in the post-
absorptive (elimination phase) steady state. Remember, there are
exceptions to these generalities.
Table 1 contains elimination half-life and time to steady state guidelines
for therapeutic drug administration and monitoring. Elimination half-life
is defined as the time required for serum drug concentration (or amount
of drug in the body) to decrease by 50%. Steady state is the condition of
equilibrium achieved during chronic dosing in which the rate of drug
input (dose rate) equals the rate of drug elimination. Unless otherwise
noted, the half-life refers to oral administration.
References
1. Baselt RC. Disposition of Toxic Drugs and Chemicals in Man (7th Ed.).
Foster City, CA: Biomedical Publications, 2004.
2. Physicians’ Desk Reference. Montvale, NJ: Medical Economics Co.,
2006.
3. Gerson B. Essentials of Therapeutic Drug Monitoring. New York, NY:
Igaku-Shoin, 1983:389-416.
9.2 Sirolimus (Rapamycin)
Clinical Use: This test is used to monitor immunosuppressive therapy
following organ transplant.
Clinical Background: Sirolimus (rapamycin, Rapamune®’) is an
immunosuppressant that inhibits cytokine-stimulated T-cell proliferation.
Sirolimus acts by forming a complex with FK-binding protein-12 that in
turn binds to mTOR kinase, a specific cell cycle regulatory protein,
thereby inhibiting mTOR action. mTOR inhibition prevents cell cycle
progression from G1 to S phase in T-cells and, thus, T-cell proliferation.
mTOR inhibition is a different mechanism of action from that of
calcineurin-inhibiting agents such as cyclosporine (CsA), a fact that may
account for the synergistic effect of sirolimus/CsA combined therapy
following renal transplant. Sirolimus is currently recommended for use
239
Toxicology
in conjunction with cyclosporine (and corticosteroids) to reduce or
prevent graft rejection by the host. Sirolimus dose-related side effects
include increased serum levels of cholesterol, triglycerides, and
creatinine and decreased glomerular filtration rate. Hypertension, rash,
anemia, arthralgia, diarrhea, hypokalemia, leukopenia, and
thrombocytopenia also may occur.
Sirolimus bioavailability and clearance are dependent on intestinal and
hepatic metabolism by cytochrome P-450 (CYP) 3A4 enzyme and on
countertransport by the multidrug efflux pump p-glycoprotein in the
intestine. Pharmacokinetic studies reveal an approximate 4.5-fold range
in interindividual behavior (Table 2) and a correlation between trough
blood concentrations and both efficacy and toxicity. The pharmaco-
kinetics are altered during drug coadministration. For example, when
sirolimus is administered concomitantly with the microemulsion
formulation of CsA rather than administered separately 4 hours apart,
sirolimus trough levels increase. Furthermore, diltiazem and
ketoconazole increase sirolimus Cmax while rifampin decreases the Cmax.
Hence, therapeutic drug monitoring (TDM) may be needed to achieve
the best clinical outcome in selected cases (see below), even though
routine TDM is not an absolute requirement.
Individuals Suitable for Testing include patients who have had an
organ transplant and 1) have hepatic impairment; 2) are receiving
concurrent doses of strong CYP3A and p-glycoprotein inhibitors or
inducers; 3) in whom CsA doses are markedly reduced or discontinued;
4) have had marked changes in the relative timing of administration of
sirolimus and CsA; 5) are at high risk for rejection; or, 6) need to be
monitored for adherence to the drug regimen.
Method: This liquid chromatography/tandem mass spectrometry
(LC/MS/MS) method includes extraction of sirolimus from whole blood
via protein precipitation, followed by high-turbulence liquid chromatog-
raphy. Sirolimus in the eluate is further purified by on-line high-
performance liquid chromatography and subsequently detected by
tandem mass spectrometry. The limit of quantitation is 1.0 ng/mL and
there is no interference from the 33 analytes studied.
Therapeutic Range: 3.0 to 18.0 ng/mL (trough levels)
Interpretive Information: Steady-state trough sirolimus levels <3.0
ng/mL may place the patient at risk for host-graft rejection. Conversely,
levels >18.0 ng/mL are more likely to be associated with adverse
events. As mentioned above, levels may be affected by drug
coadministration and pharmacokinetics. Steady state is usually reached
5 to 7 days after a dose adjustment. Evaluation of multiple trough levels
7 days after the last dose change is recommended prior to a subsequent
dose change. See Table 2 for information about interindividual
pharmacokinetic variability.
References
1. Aspeslet LJ, Yatscoff RW. Requirements for therapeutic drug
monitoring of sirolimus, an immunosuppressive agent used in renal
transplantation. Clin Ther. 2000;22 Suppl B:B86-92.
2. Kahan BD, Murgia MG, Slaton J, et al. Potential applications of
therapeutic drug monitoring of sirolimus immunosuppression in
clinical renal transplantation. Ther Drug Monit. 1995;17:672-675.
3. MacDonald A, Scarola J, Burke JT, et al. Clinical pharmacokinetics
and therapeutic drug monitoring of sirolimus. Clin Ther. 2000;22
Suppl B:B101-121.
4. Rapamune (sirolimus) Oral Solution and Tablets. U.S. Package Insert.
Wyeth Laboratories, 2000.
5. Zimmerman JJ, Kahan BD. Pharmacokinetics of sirolimus in stable
240

Section 9 Toxicology

Table 1. Drug Half-life and Time to Steady State

Drug Name Half-life Time to Steady State

Acetaminophen 2–4 hours 10–20 hours (Adult)
Amikacin 2–3 hours (Adult) 15–24 hours (Adult)
1–3 hours (Child) 5–17 hours (Child)
5–7 hours (Neonate) 24–48 hours (Neonate)
Amiodarone 35–68 days (Adult) Approx. 190–390 days or longer
20–47 days (IV, Adult)
Amitriptyline 17–40 hours (Amitriptyline) 4–10 days (Amitriptyline)
15–93 hours (Nortriptyline) 4–19 days (Nortriptyline)
Caffeine 3–12 hours (Adult) Not applicable
40–230 hours (Neonate) Not applicable
Carbamazepine 20–30 hours (Adult) 7–12 days (Adults and neonates)
14–17 hours (Adult, chronic use)
4–14 hours (Child)
5–11 hours (Neonate)
Chlorpromazine 6–30 hours 2–5 days
Clomipramine 16–36 hours 4 weeks
Clonazepam 19–60 hours 5–6 days
Clozapine 6–17 hours 2–4 days
Cyclosporine 17–40 hours (Influenced by underlying disease) Weeks to months (Influenced by underlying disease)
Desipramine 12–76 hours 2–11 days
Digitoxin 4–10 days 25–30 days
Digoxin 30–45 hours (Adult) 7–11 days (Adult)
18–36 hours (Child) 2–10 days (Child)
35–70 hours (Neonate) 2–10 days (Neonate)
Disopyramide 3–11 hours 30–50 hours
Doxepin 8–36 hours 2–8 days
Ethosuximide 48–60 hours (Adult) 12 days (Adult)
30–60 hours (Child) 6–12 days (Child)
Felbamate 11–23 hours 3–6 days
Flecainide 12–27 hours (Longer in renal failure) 3–6 days
Flucytosine (5FC) 3–4 hours 1–2 days
Fluoxetine 1–3 days 6–17 days
Fluphenazine 13–58 hours (Hydrochloride) 3–13 days
3–4 days (Enanthate)
5–12 days (Decandate)
Gabapentin 5–9 hours 1–2 days
Gentamicin 2 hours (Adult) 15–24 hours (Adult)
1–3 hours (Child) 5–16 hours (Child)
3–7 hours (Neonate) 24–48 hours (Neonate)
Haloperidol 14–41 hours (Lactate) 3–9 weeks (Lactate)
14–28 days (Decanoate) Not applicable (Decanoate)
Imipramine 6–24 hours (Imipramine) 2–5 days (Imipramine)
12–76 hours (Desipramine) 2–11 days (Desipramine)
Lamotrigine 6–30 hours 2–7 days
Lidocaine 0.5–5 hours Not applicable
(continued on next page)
 241

Section 9 Toxicology

Table 1. Drug Half-life and Time to Steady State (continued)

Drug Name Half-life Time to Steady State

Maprotiline 51 hours 7–14 days

Mesoridazine 2–9 hours 1–2 days
Methylphenidate 1.4–4.2 hours Not applicable
Mycophenolic acid 11–24 hours (Influenced by underlying disease) Not applicable
Nortriptyline 15–93 hours 4–19 days
Paroxetine 7–37 hours 2–9 days
Perphenazine 8–12 hours 2–3 days
Phenobarbital 4 days (Adult) 25–30 days
48–72 hours (Child)
4–8 days (Neonate)
Phenytoin 8–60 hours (Adult; dose dependent) 6–8 days
20–60 hours (Neonate)
Primidone 6–20 hours (Adult) 2–3 days
5–11 hours (Child)
Procainamide 2–5 hours 12–25 hours
6 hours (NAPA) 30–40 hours (NAPA)
Propranolol 2–3 hours Not applicable
Protriptyline 54–198 hours 10–27 days
Quinidine 5–12 hours 20–30 hours
Risperidone 3 hours (Fast metabolizer) 1 day
20 hours (Slow metabolizer) 5 days
Salicylate 3–6 hours (Adult) 20–30 hours (Adult)
15–30 hours (Child) 30–60 hours (Child)
Sirolimus (Rapamycin) 47–79 hours (Influenced by underlying disease) Not applicable
Tacrolimus (FK506) 12–30 hours (Influenced by underlying disease) Not applicable
Theophylline 4–6 hours (Adult, non-smoking) 2–3 days (Non-smoking)
2–4 hours (Child, older than 6 months)
19–24 hours (Neonate)
14–57 hours (Premature)
20–120 hours (Hepatic or cardiac failure)
Thioridazine 26–36 hours 5–8 days
Tobramycin 2–4 hours (Adult) 15–24 hours
1–2 hours (Child)
4–9 hours (Neonate)
Tocainide 11–17 hours 3 days
Trazodone 4–7 hours 2–3 days
Trifluoperazine 7–18 hours 2–4 days
Trimipramine 16–39 hours 4–9 days
Valproic Acid 8–12 hours (Adult) 2 days
6–15 hours (Child)
Vancomycin 3–8 hours 24–30 hours
Zonisamide 50–68 hours 14 Days
Note: Values listed above are for adults, unless otherwise specified.
242

Section 9 Toxicology

Table 2. Sirolimus Interindividual Pharmacokinetic Variability

Oral Solution Tablet

Time to peak concentration (tmax) 1.4 ± 1.2 h 3.46 ± 2.40 h
Terminal half-life (t1/2) 62.3 ±15.2 h Not established
Oral clearance rate (Cl/F) 208 ± 95 mL/h/kg 139 ± 63 mL/h/kg
Apparent oral steady-state volume (Vss/F) 12.0 ± 4.6 L/kg Not established

renal transplant patients after multiple oral dose administration. J to the effects of lead due to greater gastrointestinal absorption. Severe
Clin Pharmacol. 1997;37:405-415. intoxication results in mental and growth retardation.

9.3 Trace Elements Interpretive Information: Individuals older than 6 years of age
typically have blood lead levels b10 Ng/dL. Those 6 years or younger
9.3.1 Cobalt, Blood can be classified according to blood lead levels as shown in Table 3.

Clinical Use: This test is used to diagnose cobalt toxicity and to
monitor cobalt therapy.
Clinical Background: Cobalt is used in the manufacturing of hard
metal alloys (eg, steel, tungsten carbide), glass, paints, ruminants, and
fertilizers. The allowable atmospheric threshold is 0.02 mg/m3 in the
United States. Cobalt is also an essential element that is supplied in the
diet at an average intake of 280 Ng/day. Dietary cobalt is excreted in
the urine (86%) and feces (14%).
9.3.3 Thallium, Blood
Clinical Use: This test is used to diagnose thallium toxicity.
Clinical Background: Thallium salts are used as insecticides and
rodenticides, as a tracer (201Tl) in myocardial imaging, and in
manufacturing of low-temperature thermometers, photoelectric cells,
dye pigments, and certain cement. In the United States, the industrial
atmospheric time-weighted average (TWA) is 0.1 mg/m3.

The symptoms of acute exposure are nausea, vomiting, pulmonary
fibrosis, allergy, hemorrhage, and cardiomyopathy. Chronic exposure is
associated with allergic dermatitis, nausea, vomiting, pulmonary
fibrosis, cough, dyspnea, and heart disease (including myocardial
failure).
The half-life is not well established.
Method: This inductively-coupled plasma/mass spectrometry (ICP-MS)
method uses an argon plasma at 6,000 to 10,000 oK to destroy the
organic matter in the sample and to ionize the metals. The resulting
metallic ions are detected and quantitated in the mass spectrometer
with an internal standard. Results are reported in Ng/L.
Many thallium compounds are readily absorbed by the digestive tract,
skin, and lungs. Fatal and non-fatal thallium poisonings stem from
medicinal, cosmetic, industrial, and pesticide application. Symptoms of
intoxication include colic, nausea, vomiting, tremors, albuminuria,
sensory changes, polyneuritis, speech impairment, weakness, ataxia,
tachycardia, arrhythmia, paralysis, and convulsions. Alopecia may occur
after 1 to 3 weeks. The lethal adult dose is about 8 to 15 mg/kg of
soluble thallium salt. Although Prussian blue hastens excretion, no
single chelating agent has been shown to be an especially effective
treatment.
The half-life is 2 to 4 days.

Interpretive Information: Non-exposed adults typically have levels

b1.8 Ng/L.
Table 3. Recommended Follow-up for Various Blood Lead
9.3.2 Lead, Blood Classifications
Clinical Use: This test is used to detect lead exposure and/or toxicity. Blood Lead
Class (μg/dL) Follow-up
Clinical Background: Lead is a cumulative protoplasmic poison that
may be ingested, inhaled, or adsorbed directly through the skin. Toxic I b9 None
effects are categorized as gastrointestinal, central nervous system, IIA 10–14 Community-wide lead poisoning prevention
neuromuscular, hematologic, renal, or constitutional. Common symptoms activities; regular screening
include constipation, anorexia, abdominal pain, weight loss, fatigue, and
a characteristic peripheral neuropathy (wrist drop). Severe poisoning IIB 15–19 Nutritional and educational interventions
may lead to nephropathy, encephalopathy, convulsions, and even death. and more frequent screening
III 20–44 Environmental evaluation and remediation
Lead poisoning may result from occupational exposure (mining, and a medical evaluation; medical
smelters, sheet metal, battery manufacture, automobile radiator repair, intervention may be needed
demolition work, alloys, and metal plating). Poisoning may also occur
subsequent to exposure to leaded containers, lead clay, glazed pottery, IV 45–69 Medical and environmental interventions
solder, paints, and bullets (wound). including chelation therapy
V r70 Medical emergency treatment
Infants and children aged 9 months to 6 years are particularly susceptible
Source: CDC: Preventing Lead Poisoning in Young Children (Oct. 1991).
 243

Section 9 Toxicology

Table 4. Industrial Sources of Metal Exposure

Source of Exposure Metal(s)

Agriculture Arsenic, mercury
Alloys and metal plating Arsenic, cadmium, cobalt, lead, mercury
Battery manufacture Cadmium, lead, nickel
Construction Cadmium, lead, mercury
Home renovation Lead, mercury
Mining Arsenic, cadmium, cobalt, lead, mercury
Semiconductor manufacture Arsenic

Method: This inductively-coupled plasma/mass spectrometry (ICP-MS)

method uses an argon plasma at 6,000 to 10,000 °K to destroy the
organic matter in the sample and to ionize the metals. The resulting
metallic ions are detected and quantitated in the mass spectrometer
using an internal standard. Results are reported in Ng/L.

Interpretive Information: Non-exposed adults typically have levels

b5 Ng/L. Toxicity occurs at levels r80 Ng/L.

9.3.4 Comprehensive Toxic Metal Panel, 24-Hour Urine

Clinical Use: This test is used to diagnose toxicity caused by various

trace elements including arsenic, cadmium, cobalt, lead, mercury, and
thallium.

Clinical Background: The symptoms of metal intoxication can mimic

other treatable disorders. Such symptoms include nausea, vomiting,
dementia, anemia, kidney failure, infertility, seizure, neuropathy, learning
disorders, and pulmonary dysfunction. Thus, urine metal analysis may
assist in differential diagnosis as well as in monitoring patients for
elimination of toxic metal(s) during treatment. Elevated metal levels are
associated with diets high in seafood (including shellfish), certain folk
medicines, exposure to contaminated water, lead-based and latex paint,
cigarette smoke, and industrial exposure (Table 4).

Method: This inductively-coupled plasma/mass spectrometry (ICP-MS)

method uses an argon plasma at 6,000 to 10,000 °K to destroy the
organic matter in the sample and to ionize the metals. The resulting
metallic ions are detected and quantitated in the mass spectrometer
using an internal standard. Results are reported in Ng/L. Analytical
sensitivity is reported in Table 5.

Interpretive Information: Reference and toxic ranges are listed in

Table 5.

Table 5. Trace Element Reference and Toxic Ranges for 24-

Hour Urine Samples
Limit of Reference Toxic
Quantitation Range Range
(μg/L) (μg/L) (μg/L)
Arsenic (As) 10 b80 Not established
Cadmium (Cd) 0.5 b5 Not established
Cobalt (Co) 0.5 b2 Not established
Lead (Pb) 10 <80 Not established
Mercury (Hg) 10 b20 r150
Thallium (Tl) 0.5 b2 >200