Systematic Approach in Anemia Evaluation and Review of Peripheral Smears

Jun W. Kim, MD Family Medicine Residency Dewitt Army Community Hospital

Objective
Recognize abnormal peripheral blood smear Review differentials through systematic approach

Approach to Dx

Hx- age, duration, onset, subjacent illness, blood loss (GI, menstruation, surgery), diet, medications, toxic exposure, occupation, Family Hx, Social Hx PE- complete exam including skin (jaundice, petechiae), HEENT, Abdomen (hepatosplenomegally), lymphatics, rectal, and pelvic

Basic Labs to Start

Repeat CBC w/ manual differential

(WBC, RBC, HCT, HGB, PLT, indices- MCH, MCHC, MCV, RDW)

Peripheral Smear Reticulocyte count

Reticulocyte count

Retic count = % immature RBC Normal 0.5-1.5% (for non-anemic) <1% Inadequate production >=1% Increased production (? adequacy)

Reticulocyte Correction

%Retic count frequently overestimates Retic count should be compared to nonanemic RBC count to assess adequacy of response Corrected Retic count = %Retic X HCT/45

Reticulocyte Production Index

Correction for left shift Retic lifespan is increased in blood RPI = % Retic X Hct/45 X 1/CF Hct Correction factor (CF) 40-45 1.0 35-39 1.5 25-34 2.0 15-24 2.5 Normal RPI = 1 (for non-anemic pt) RPI < 2 : hypoproliferative RPI >=2 : hyperproliferative

Retic Production Index

Hypoproliferative

Hyperproliferative

- Iron def. anemia - B12/folate def. - Chronic disease - Sideroblastic anemia - Aplastic anemia - Myeloproliferative

- Hemolytic disease - Hemoglobinopathy (including thalassemia)

Peripheral smear
Optimal area for review RBC morphology, WBC differential, PLT (clumping?)

RBC morphology
7-9 m with 1/3 central palor Lifespan of 110-120 days About the size of nucleus of normal lymphocyte Poikilocytosis & Anisocytosis

Basophilic stippling

Precipitated RNA lead or heavy metal poisoning ETOH abuse Hemolytic anemia

Burr cells

Altered lipid in cell membrane artifact Uremia Renal failure gastric CA transfused old blood

Elliptocytes/ovalocytes

Abnormal cytoskeletal proteins Hereditary elliptocytosis

Howell Jolly body

Nuclear remnant DNA hemolytic anemia absent or hypofunction spleen

Schistocyte/helmet cells

Fragmented (mechanical or phagocytosis) DIC TTP HUS Vasculitis prosthetic heart valve severe burns

Sickle cells

Molecular aggregation of Hgb-S SS, SC, S-thal rarely S-trait

NRBC

Common in newborn severe degree of hemolysis

Spherocyte

Absent central palor look smaller Hereditary spherocytosis immune hemolytic anemia

Stomatocyte

Mouth like Membrane defect Smear artifact Hereditary stomatocytosis Liver disease

Target cells

Increased redundancy of membrane hemoglobinopathies thalassemia liver disease

Tear drop cells

Distorted drop shaped Smear artifact myelofibrosis promyeloblastic leukemia space occupying lesions of marrow

Differentials
H&P Indices (MCV, MCHC, RDW) RBC Morphology Retic response Other labs as needed

Anemia Differential Dx by Flow Chart

MCV/smear

Micro

Normo

Macro

Iron panel

Retic

Retic

Low

High

Iron/B12/Folate

Go to *Occult Blood Loss

**Normal

Low

Bone Marrow Bx

B12/Folate

Anemia of Chronic Dis.

B12 Low

High

Folate/Low

*Occult Blood Loss

Normal

Yes

MMA/Homocysteine MMA high B12 Low

No

Coombs (+)

Homocysteine high Folate Low

Coombs (-)

Normal Go to **

First use size (MCV) to sort the Differential Dx

MCV Micro Normo Macro

Microcytic anemia

Get Iron panel- serum iron, TIBC, ferritin

Iron def. anemia Siderobla -stic anemia Thalasse mia -mia Chronic disease

dec inc

inc dec

dec inc

inc/nl dec

dec/nl inc/nl dec inc

Iron def. Anemia

Low Retic count High RDW Due to chronic blood loss Diet deficiency

Thalassemia

Normal to inc. RPI Normal RDW Target cells Mentzer index <13 =MCV/RBC Youdens index - using RDW & Mentzer index
- sensitivity = 82% - specificity = 80%

confirm w/ Hgb electrophoresis

Thalassemia continues
Alpha-thalassemia SE Asia & Africa aaaa - normal aaaa^ - silent carrier aaa^a^ - trait (mild) aa^a^a^ - HbH (Bart) hemolytic disease a^a^a^a^ - hydrops fetalis (stillborn) Beta-thalassemia Mediterranean Beta-thal minor one beta gene, increased HbA2/HbF Beta-thal major 2 beta genes, severe, failure to thrive, sig HbF

Sideroblastic anemia

Accumulation of mitochondrial iron in erythroblasts Hereditary Drugs - INH, lead, zinc, alcohol, chloramphenicol, cycloserine, plavix Hypothermia Confirm w/ BM Bx

Sample question #1

Anemia of chronic disease is due to inadequate production of, or poor response to, which one of the following?
A. B. C. D. E. Iron Folate Erythropoietin Ferritin Hemosiderin
AFP, Nov. 15, 2000

Anemia of chronic disease

Infections: TB, SBE, osteo, chronic UTI or pyelo, fungal Malignancy: mets, leukemia, lymphoma, myeloma

Chronic inflammatory disorders: RA, SLE, Sarcoid, collagen vascular disease, polymyalgia rheumatica, chronic hepatitis, decubitus ulcer

Macrocytic anemia
Macro

RPI >= 2

RPI < 2

Check Occult Blood Loss

Check B12 and folate

No

Coombs test

Yes

Macrocytic: RPI < 2

B12/Folate B12 Low Normal MMA Homocysteine Normal Folate Low

MMA High

Homocysteine High

Consider Liver, Renal, Thyroid, Alcohol, Chronic dis.

Consider Bone Marrow Bx

Macrocytic: RPI < 2 Megaloblastic Anemia

Folate B12 Inadequate absorption Inadequate intake Synthesized by plants Synthesized by and micro-organism bacteria Meat, fish, dairy (strict Green leafy veges Fruits vegans) Absorbed in jejunum Absorbed as B12-IF complex in ileum (gastrectomy) Ca++ and pH dependant (PPI)

Sample question #2

Which of the following tests can be useful in determining if an elderly patient has folate deficiency?
A. B. C. D. RBC folate concentration Serum homocysteine level Serum ferritin level Serum methylmalonic acid level
AFP, Oct. 1, 2000

Macrocytic: RPI < 2 Megaloblastic Anemia

Smear Macro-ovalocytic Polychromasia Hypersegmented neutrophil Other Labs Homocysteine Folate def. Methylmalonic acid B12 def. Intrinsic Factor Ab test very specific for pernicious anemia but only 50% sensitive Parietal cell AB test quite sensitive (90%) but not specific Schilling test

Macrocytic: RPI < 2 Non-megaloblastic

Consider Liver, Renal, Endocrine (thyroid), alcohol, drugs Consider anemia of chronic disease Get Bone Marrow Biopsy Myelodysplastic Myeloproliferative Leukemia, Lymphoma, Multiple Myeloma

Macrocytic: RPI < 2 continues Aplastic Anemia

Fanconi anemia congenital Direct stem cell destruction external radiation Drugs - chloramphenicol, gold, sulfonamides, felbamate Other Toxins - Solvents, degreasing agents, pesticides Viral infection - parvovirus B19, HIV, other Idiopathic

Macrocytic: RPI >= 2

Occult Blood Loss?

Yes

No

Investigate source

Check for Hemolysis

Peripheral smear

Coombs (DAT)

Sample question #3

Of the following laboratory results, which one does not occur in hemolytic anemia?
A.
B. C.

D.
E.

Reticulocytosis Increased unconjugated bilirubin Increased haptoglobin Increased LDH Hemosiderinuria

AFP, June 1, 2004

Macrocytic: RPI >= 2

Hemolytic Anemia
Other Lab Characteristics

RBC morphology Serum haptoglobin Serum LDH Unconjugated bilirubin Hemoglobinuria Hemosiderinuria

Macrocytic: RPI >= 2

Hemolytic Anemia
Coombs (DAT)

Positive

Negative

Immune Hemolysis Drug related Hemolysis Transfusion, Infection, Cancer

Hemoglobinopathy, G6PD, PK, Spherocytosis, Eliptocytosis, PNH, TTP, DIC

Coombs positive with Spherocytes Autoimmune hemolytic anemia

Warm AIHA Abrupt onset IgG Anti-Rh, e, C, c, LW, U Jaundice Splenomegaly SLE, CLL, Lymphoma Drugs: methyl-dopa, mefenamic acid, cimetidine, cefazolin Cold AIHA Insidious onset IgM, complement Anti-I, I, Pr Cold agglutinin titer Absent jaundice Mycoplasma Virus

Coombs positive with Spherocytes Other immune hemolytic anemia

Alloantibody hemolytic anemia Transfusion reaction Feto-maternal incompatibility (Kleihauer-Betke test) Drug related Hemolytic anemia Toxic immune complex (drug+Ab+C3) - Quinine, Quinidine, Rifampin, INH, Sulfonamides, Tetracyclin Hapten formation (anti-IgG) - PCN, methicillin, ampicillin

Coombs Negative Hemolytic anemia

Episodic - G6PD def., PNH Hemoglobinopathy - Sickle, crystals or target cells Elliptocytosis Spherocytosis DIC, TTP

Coombs Negative Hemolytic Anemia Membrane Defects

Spherocytosis Common among Northern European Autosomal dominant Decreased spectrin Osmotic fragility test Autohemolysis test Elliptocytosis 90% with no clinically significant hemolysis Abnormal membrane protein

Coombs Negative Hemolytic Anemia Deficiency of RBC Enzymes

G6PD Def.

Pyruvate Kinase Def.

X-linked Mediterranean, African American, and Asian Oxidant drugs ASA,

quinine, primaquine, chloroquine, sulfacetamide, sulfamethoxazole, nitrofurantoin, chloramphenicol, procainamide, quinidine

Infections Quantitative test

Severe anemia in newborns Adults symptomatic Jaundice Splenomegaly Fluorescent screening test Quantitative test

Coombs Negative Hemolytic Anemia Hemoglobinopathy

HbS disease Valine substitution for Glutamic acid at the 6th position of b-chain Sickle crises Severe anemia Screening test - Na Metabisulfite solubility Hgb electrophoresis

Coombs Negative Hemolytic Anemia Hemoglobinopathy continues

HbC disease

HbSC disease

Mild hemolysis Splenomegaly Lysine substitution HbC crystals bar of gold Hgb electrophoresis

Sickle and SC crystals Washington monument Less crises More retinopathy/aseptic necrosis

Coombs Negative Hemolytic Anemia Paroxysmal Nocturnal Hemoglobinuria

Rare chronic condition Recurrent abdominal pain, vomiting, headaches, eye pain, thrombophlebitis Episodic Hgb in urine, Hemosiderinuria Abnormal cell membrane - increased lysis by complement Screening - Sucrose hemolysis test Confirm - Acid hemolysis test (Hams test)

Coombs Negative Hemolytic Anemia

Fragmented RBCs & Thrombocytopenia
TTP-HUS

Thrombocytopenia Microangiopathic hemolytic anemia Neurologic symptoms and signs Renal failure Fever
Idiopathic - 37 % Drug-associated - 13 % Autoimmune disease - 13 % Sepsis - 9 % Pregnancy - 7 % Bloody diarrhea - 6 % Hematopoietic cell transplantation - 4 %

DIC Depletion of clotting factor (TTP normal) Thrombocytopenia Bleeding (64%) Renal dysfunction (25%) Hepatic dysfunction (19%) Respiratory dysfunction (16%) Shock (14%) Thromboemboli (7%) Central nervous system involvement (2%) Sepsis, trauma, malignancy

TTP-HUS / DIC

Normocytic Anemia Hyperproliferative (RPI >= 2)

Use same flow chart as macrocytic hyperproliferative

Occult Blood Loss?

Yes

No

Investigate source

Check for Hemolysis

Peripheral smear

Coombs (DAT)

Normocytic Anemia Hypoproliferative (RPI < 2)

1. Get iron panel (ferritin)/B12/folate - some clue from RBC indices to check early disease, high RDW, peripheral smear. 2. Consider liver, renal, drugs, toxin, endocrine (thyroid), and anemia of chronic disease.
3. Get BM bx - Leukopenia, thrombocytopenia, CRI < 0.1 - Aplastic anemia/pancytopenia - Abnormal (immature) cells on smear

Questions?