DIAGNOSTICS

HEMATOLOGY &
IMMUNOLOGY
OUTLINE:

•RBC tests
•WBC tests
•Platelets
•Serology
 Routine Hematologic Procedures
• Complete Blood Count (CBC
• RBC count
• WBC count
• Hemoglobin
• Hematocrit
• WBC Differential count
• RBC indices (MCV, MCH, MCHC)
• Platelet count
 UNITS
TRADITIONAL CF S.I.
RBC __X 106 cells/mm3 6 __X 10 12 cells/L

WBC __X 103 cells/mm3

10 __X 109 cells/L
 NORMAL VALUES
MALE FEMALE
RBC 4.5 to 6.0 x 106 /mm3 4.0 to 4.5 106 /mm3
4.5 to 6.9 x 1012 /L 4.0 to 4.5 1012/L
WBC 5,000 to 10000 cells/mm3
5 to 10 x 103 cells/mm3
5 to 10 x 109 cells/L
 HEMOCYTOMETER

Neubauer’s Counting Chamber

HEMOCYTOMETER
 RBC count WBC count
Diluting fluid Isotonic – preserve Hypotonic – destroy all
the shape of cells cells except WBC, and
it stains the nucleus for
easier identification.
Example NSS Acetic acid
Dilution factor 1:200 1:20
Pipette Thoma pipette
Has RED bead Has WHITE bead
Has 101 mark Has 11 mark
Count 5 small squares in 4 corner large squares
central large
square
 INTERPRETATION:
Increased Decreased
RBC ERYTHROCYTOSIS ERYTHROPENIA
• Hemoconcentration due • Anemia
to dehydration, burns • Pregnancy
• Polycythemia vera • Old age
• Cyanosis (Lung disease,
heart disease)
WBC LEUKOCYTOSIS LEUKOPENIA
• Infections • Typhoid fever
• Pregnancy • Tuberculosis
• Severe pain • Measles
• Influenza
• Leukemia
 WBC Differential Count
• Relative count – expressed in percent
• Absolute count – more accurate
- expressed in ___ x109/L
• Peripheral Blood Smear
• Count 100 WBCs (all kinds)
• Differentiate each WBC.
• Expressed as relative count
• Shift to the LEFT: increased number of IMMATURE
WBC (including BANDS)
 Increased Decreased
Neutrophils • ALL bacterial • Typhoid fever
Neutrophilia infection • Measles
Neutropenia • Influenza
Lymphocytes • VIRAL infections • ACUTE infection
Lymphocytosis • Excess
Lymphopenia irradiation
Monocytes • Tuberculosis
• Malaria
Eosinophils • Asthma
• Hypersensitivity
• Parasites
Basophils • Hypersensitivity
 HEMOGLOBIN
• Part of the RBC that carries the OXYGEN
molecule.
• Each hemoglobin carries 4 molecules of oxygen.
• Normal values:
• Male: 14 to 18 g/dL
• Female: 12 to 16 g/dL
 HEMATOCRIT
• Volume percentage of RBCs in blood.
• Normal value:
• Male: 42-52%
• Female: 37%-47%
• Rule of three:
• 3 x RBC = Hemoglobin
• 3 x HgB = Hematocrit
 INTERPRETATION
Increased Decreased
MCV MACROCYTIC MICROCYTIC
• Megaloblastic • Iron Deficiency
anemia Anemia (IDA)
• Thalassemia
MCH*** • Megaloblastic • IDA
anemia • Thalassemia
MCHC HYPERCHROMIC HYPOCHROMIC
• Spherocytes • IDA
• Thalassemia
ERYTHROCYTE SEDIMENTATION
RATE (ESR)
• The test measures the distance that RBCs
have fallen after one hour.
• Used for monitoring inflammatory conditions
such as rheumatoid arthritis.
• Rouleaux formation – stacking of RBCs
OSMOTIC FRAGILITY TEST (OFT)
• This test demonstrates increased RBC fragility
in which the RBCs have decreased surface
area-to-volume ratios.
• Specimen is exposed to increasingly
HYPOTONIC solutions.
 ASSESSMENT
•Primary Hemostasis
•Platelet studies
•Vascular Fragility Tests
•Secondary Hemostasis
•Coagulation studies
PLATELET STUDIES
Platelet NV: 150-450 X 109/L
Count Significant bleeding – less than
60x109/L
Most serious site – CNS (Intracranial
bleeding)
Platelet Reagents:
Aggregation • Ristocetin
• ADP
• Collagen
• Epinephrine
CLOT RETRACTION TIME
• Clot retraction depends on normal levels of
platelets, calcium, ATP and fibrinogen.
• Abnormal results begins at levels less than 60 x 109/L
PLATELET AND VESSELS
BLEEDING Prolongation of bleeding time begins
TIME at levels lower than 100 x109/L
• Duke Method – fingertip/earlobe
• Ivy Method – pressure cup at 40
mmHg
CAPILLARY aka Tourniquet test or Rumpel-Leede
RESISTANCE Test
TEST • 100mmHg for 5 minutes.
• Read after 15-30 mins.
• Used for screening test in Dengue
COAGULATION STUDIES
CLOTTING TIME Obsolete test
• Capillary blood method
• NV: 2-4 minutes
• Whole blood/Lee and White
Method
• NV: 7-15 minutes
PROTHROMBIN Extrinsic and Common Pathway
TIME Used to monitor Warfarin Therapy
NV: 10-12 secs
INR International Normalized Ratio
• Used to standardized the PT results.
• Variable results are caused by inherent
variation in reagents and machine system
• It is better to used PT/INR to monitor
Warfarin Therapy.
• ISI – International Sensitivity Index =
assigned by the WHO reagent

Formula:
INR = (PTpatient/PTnormal)ISI
APTT Intrinsic and Common Pathway
(Activated Used to monitor Heparin Therapy
Partial Rgt:
Thromboplastin • Activator – Kaolin, ellagic acid
Time) and celite (Activates FXII)
NV: 25-35 secs.
STYPVEN TIME aka Russell Viper Venom Time
Test for Common Pathway
Rgt:
• East Indian Viper Venom (Vipera
russelli) – Activate clotting factor
X
NV: 6-10 secs
THROMBIN Measures thrombin-induced cleavage
TIME of fibrinogen to form fibrin monomers
and eventually INSOLUBLE fibrin clot
Rgt: Thrombin
NV: 10-14 secs
Prolonged in:
• HYPOfibrinogenemia
UREA aka Duckert Test
SOLUBILITY Detects FXIII deficiency
TEST NV: Clot should be insoluble to Urea at
24°C
 WARFARIN vs HEPARIN
WARFARIN HEPARIN
Used as anticoagulant/blood thinner drug (MI,
Stroke)
Overdosage cause BLEEDING
Monitor using PT/INR Monitor using aPTT
Antidote: activated Antidote: Protamine
Vitamin K Sulfate
 SOLVE THE PROBLEMS:
↑ PT
N APTT
N PT
↑ APTT
↑ PT
↑ APTT
N PT
N APTT
ABN Duckert Test
↑ PT
↑ APTT
↑ TT
SEROLOGY
• Study of the non-cellular portion of blood
• BLOOD CELL – contains the Antigen
• SERUM – contains the Antibodies
AFFINITY vs AVIDITY
• AFFINITY:
• Initial force of attraction between
antibodies and its corresponding
antigen.

• AVIDITY:
• Sum of all attractive forces between
antigen and antibody.
 AFFINITY
• Four bonds:
1. Ionic bonds: attraction of 2 opposite charges
2. Hydrogen bonds: attraction of Hydrogen to either
nitrogen or oxygen
3. Hydrophobic bonds: attraction of nonpolar
molecules with a non-aqueous molecule
4. Van der Waals forces: attraction of two molecules
that are very close to each other
 Precipitation VS Agglutination
• PRECIPITATION
• Involves combination of SOLUBLE antigen with
SOLUBLE antibody to produce INSOLUBLE
complexes that are visible.
• AGGLUTINATION
• Process by which PARTICULATE antigens
aggregate with specific antibody to for LARGER
complexes.
 Types of Agglutination Reactions
1. Direct/Active Agglutination
• Slide agglutination
• Tube agglutination
• Coombs test
2. Passive Agglutination
• Reverse passive agglutination
3. Hemagglutination
4. Co-agglutination
 DIRECT/ACTIVE AGGLUTINATION
• Antigen and antibody reaction forms
complexes are directly observed.
• Methods:
SLIDE METHOD TUBE METHOD
For qualitative For quantitative
purpose (presence or purpose (determine
absence) the level)
 COOMBS or ANTIGLOBULIN
AGGLUTINATION TEST
• Antiglobulin – non-agglutinating antibodies
• This test makes use of Coombs reagent which
binds to non-agglutinating antibodies.
• Types:
1. Direct Coombs/Direct Antiglobulin Test (DAT)
2. Indirect Coombs/Indirect Antiglobulin Test (IAT)
 Passive Agglutination
• Agglutination reaction involves particles that are
coated with antigens (carrier particle).
 Reverse Passive Agglutination
• Agglutination reaction involves particles that are
coated with ANTIBODIES.
 Hemagglutination
• Agglutination reaction involves red blood cells
• Used for detection of VIRAL infection.
 Co-agglutination
• Agglutination reaction involves bacteria.
• Staphylococcus aureus – most commonly used due
to presence of protein A, which bind at the Fc
region of antibody.
 Immunoassays
• QUANTITATIVE study of levels of either antigen or
antibodies.
• Makes use of LABELS
• Radioactive Immunoassay
• Fluorescent Immunoassay
• Enzyme Immunoassay
• Enzyme Linked Immuno-Sorbent Assay (ELISA)
• Labels are used as a marker to facilitate counting.
 Immunoassays
• Forms:
COMPETITIVE NON-COMPETITIVE
Patient’s antigen Label couples with
competes with the patient’s antigen that
label for the antibody.coupled to the
antibody.
Amount of label is Amount of label is
INVERSELY proportional DIRECT proportional to
to the antigen. the antigen.
 COMPLEMENT LEVELS
COMPLEMENT ASSOCIATED DISEASE
C1(q,r,s), C2, C4 Lupus
C1INH HAE (Hereditary
Angioedema)
DAF, MIRL Paroxysmal nocturnal
hemoglobinuria
C5-8 Neisseria
Only C9 deficiency has no known disease
association
TUMOR MARKERS
Tumor marker Associated cancers
Bence Jones Proteins Multiple Myeloma
Alpha-Fetoprotein (AFP) Liver cancer
Human Chorionic Choriocarcinoma
Gonadotropin (hCG)
BRCA-1 Breast and ovarian
cancer
HER-2/NEU Breast cancer
Prostate Specific antigen Prostate cancer
(PSA)
 Tumor marker Associated cancers
Calcitonin Medullary thyroid cancer
CA-125 Ovarian cancer
CA-19.9 Colon and pancreatic cancer
CA-15.3 Breast cancer
CEA Gastrointestinal tract cancers
 HLA Testing
HLA Disease Association
HLA-DR4 • Rheumatoid Arthritis
HLA-DR2, HLA-DR3 • Systemic Lupus
Erythematosus
HLA-DR3 • Scleroderma
• Sjogren’s Disease
Abnormal Antibodies
Antibodies Disease Association
• Anti-GAD (Glutamic acid Type I Diabetes
decarboxylase) Mellitus
• Anti-insulin
• Antibodies against Fc region of IgG Rheumatoid Arthritis
• Anti-ANA (Anti-Nuclear Antibodies) SLE
• Anti-ENA(Anti-Extractable Nuclear
Antibodies)
• Anti-dsDNA
• Lupus anticoagulant
 Antibodies Disease Association
• Anti-TSH (Anti-Thyroid Grave’s disease
Stimulating Hormone)
• Anti-TPO (Anti-Thyroid Hashimoto’s disease
Peroxidase)
• Anti-parietal cell Pernicious anemia
• Anti-intrinsic factor
 Indirect Immunofluorescence
• Visualization of a specific antibody by binding
with a fluorescent dye.
• Staining patterns in the nucleus can
determine the diagnosis.
END