Hematology: Principles & Clinical Procedures

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Hematology: Principles & Clinical Procedures

Comprehensive Study Notes for Medical Laboratory Science

Hematology Study Notes 1.6

Hematology: Principles & Clinical Procedures 1.6

Comprehensive Study Notes for Medical Laboratory Science

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1.6.1 Hemoglobin Estimation & Standard Curve Calibration

Principle

Cyanmethemoglobin (HiCN) Method: Blood is diluted 1:201 in Drabkin’s reagent (containing potassium ferricyanide K₃Fe(CN)₆ and potassium cyanide KCN). Potassium ferricyanide oxidizes all hemoglobin forms to methemoglobin, which then reacts with potassium cyanide to form stable cyanmethemoglobin (HiCN) complex. The intensity of the colored complex is measured at 540 nm wavelength, which is directly proportional to hemoglobin concentration.

Procedure

Standard Curve Preparation:

  1. Prepare serial dilutions of reference solution (Hb standard: 25 g/dL) using Drabkin’s reagent
  2. Make dilutions: 1:1 (25 g/dL), 1:2 (12.5 g/dL), 1:4 (6.25 g/dL), 1:8 (3.125 g/dL)
  3. Allow tubes to stand for 10 minutes at room temperature
  4. Transfer dilutions to cuvettes (start with blank/Drabkin’s solution)
  5. Zero spectrophotometer with blank solution
  6. Measure absorbance at 540 nm for each dilution
  7. Plot absorbance (Y-axis) vs. Hb concentration (X-axis)
  8. Draw a straight-line curve through all points

Patient Sample Preparation:

  1. Pipette 5 ml of Drabkin’s reagent into a clean test tube
  2. Add 20 µl of EDTA anticoagulated blood sample
  3. Mix well and incubate for 10 minutes
  4. Transfer to cuvette
  5. Read absorbance at 540 nm (zeroing with blank)
  6. Determine Hb concentration from standard curve

Calibration of Spectrophotometer

  • Blank Calibration: Use Drabkin’s solution to set baseline absorbance to zero
  • Wavelength Accuracy: Ensure 540 nm is accurately set
  • Standard Curve Validity: Prepare fresh standard curve if absorbance readings show irregularities
  • Regular Recalibration: Every 100 samples or daily, whichever is more frequent

Advantages & Disadvantages

Advantages Disadvantages
✓ All Hb forms except sulfhemoglobin converted to HiCN ✗ Requires spectrophotometer (expensive equipment)
✓ No visual error; objective measurement ✗ Affected by high WBC count (causes turbidity)
✓ Stable color solution; no fading over time ✗ Cyanide handling hazard (chemical safety)
✓ Linear relationship between absorbance & Hb ✗ Incomplete hemolysis with very thick blood

Normal Values

Adult Reference Range:
  • Males: 13.5–17.5 g/dL
  • Females: 12.0–15.5 g/dL
  • Children (6-12 yrs): 11.5–15.5 g/dL

Common Sources of Error

  • Pipetting Error: Inaccurate volume measurement
  • Dirty/Scratched Cuvettes: Affects light transmission
  • Deteriorated Reagents: Use fresh Drabkin’s solution
  • High WBC Count (>20,000/µL): Centrifuge specimen first
  • Improper Mixing: Incomplete hemolysis
  • Temperature Variation: Read at room temperature (20–25°C)

1.6.2 Total Cell Counts: WBC, RBC, Platelets & Reticulocytes

1.6.2A: Total WBC Count (Leukocyte Count)

Principle: Blood is diluted 1:20 using a diluting solution (WBC diluting fluid containing acetic acid to lyse RBCs and highlight nuclei). Cells are counted in a hemocytometer chamber under microscope.

Procedure:

  1. Make 1:20 dilution: Add 20 µL EDTA blood to 380 µL WBC diluting fluid
  2. Mix thoroughly by rolling tube or vortexing
  3. Discard first 3 drops; load hemocytometer chamber with drop 4
  4. Count WBCs in all four corner squares (1 mm²) using low-power objective (10×)
  5. Formula: Cells/mm³ = (Total counted ÷ No. of squares) × Dilution factor × Depth correction
  6. Standard: Count ≥ 4 corners; difference ≤ 10 cells between counts
Normal Range: 4,500–11,000 cells/µL (4.5–11.0 × 10³/µL)

1.6.2B: Total RBC Count (Erythrocyte Count)

Principle: Blood is diluted 1:200 in isotonic saline. RBCs are counted in hemocytometer using high-power objective. RBCs appear as small, colorless discs.

Procedure:

  1. Make 1:200 dilution: Add 20 µL EDTA blood to 3.98 mL isotonic saline
  2. Mix well; allow 5 minutes for stabilization
  3. Load hemocytometer carefully without air bubbles
  4. Count RBCs in all 5 small squares (center square + 4 corner small squares) using high-power objective (40×)
  5. Formula: RBCs/mm³ = (Total counted ÷ 5) × Dilution factor (200)
  6. If total count < 100 in 5 squares, use 25 small squares (5 rows)
Normal Range:
  • Males: 4.5–5.9 × 10⁶/µL
  • Females: 4.1–5.1 × 10⁶/µL
  • Children: 4.5–5.1 × 10⁶/µL

1.6.2C: Platelet Count (Thrombocyte Count)

Principle: Blood is diluted 1:20 or 1:100 in Rees-Ecker diluting fluid (containing formaldehyde to preserve platelets and ammonium oxalate to lyse RBCs). Platelets appear as small, refractive bodies in hemocytometer.

Procedure:

  1. Make 1:20 dilution: Add 20 µL EDTA blood to 380 µL Rees-Ecker fluid
  2. Mix and allow 10–15 minutes for RBC lysis
  3. Load hemocytometer; count platelets in all four corner squares
  4. Use high-power objective (40×) with reduced light
  5. Formula: Platelets/mm³ = (Total counted ÷ 4) × 20
  6. Count minimum 100 platelets; perform duplicate counts
Normal Range: 150,000–400,000/µL (150–400 × 10⁹/L)

1.6.2D: Reticulocyte Count (Young RBC Count)

Principle: Reticulocytes are immature RBCs containing residual RNA precipitated as reticulum (stained dots). Blood is stained with supravital stains (new methylene blue or brilliant cresyl blue). Count is expressed as percentage of total RBC and absolute count.

Procedure:

  1. Prepare two equal volumes: Blood + supravital stain in small tube
  2. Incubate 15 minutes at room temperature
  3. Make thin smear; allow to dry
  4. Examine under oil immersion objective (100×) without coverslip
  5. Count 500–1000 RBCs; note those with reticular precipitate (0–8 fine dots)
  6. Percentage: (Reticulocytes counted ÷ Total RBCs counted) × 100
  7. Absolute Count: Reticulocyte % × Total RBC count ÷ 100
Normal Range:
  • Percentage: 0.5–2.5% of RBCs
  • Absolute: 25,000–75,000/µL

Clinical Significance of Elevated Reticulocyte Count:

  • Indicates effective bone marrow response to anemia
  • Suggests hemolysis or recent blood loss (RBC destruction)
  • Shows treatment response in nutritional anemias (B₁₂, folate)
  • Delayed response in bone marrow suppression or aplastic anemia

1.6.3 ESR, BT, CT & RBC Indices

1.6.3A: Erythrocyte Sedimentation Rate (ESR)

Principle: RBCs settle (sediment) in anticoagulated blood under gravity. Elevated proteins (fibrinogen, globulins) increase rouleaux formation, accelerating sedimentation. ESR is a non-specific inflammatory marker.

Westergren Method (Preferred):

  1. Mix 2 parts blood + 1 part 3.8% sodium citrate
  2. Fill Westergren tube (200 mm, graduated) to 0 mark exactly
  3. Place vertically on stand
  4. Read sedimentation at 1 hour
  5. Distance fallen = ESR in mm/hour
Normal Values:
  • Males < 50 yrs: 0–15 mm/hr
  • Males > 50 yrs: 0–20 mm/hr
  • Females < 50 yrs: 0–20 mm/hr
  • Females > 50 yrs: 0–30 mm/hr

1.6.3B: Bleeding Time (BT) and Clotting Time (CT)

Bleeding Time (Assessment of Primary Hemostasis):

Duke’s Method:
  1. Puncture earlobe with sterile lancet (2 mm deep)
  2. Wipe first drop with gauze
  3. At 30-second intervals, gently touch blood drops with filter paper
  4. Stop when bleeding ceases; bleeding time is elapsed time
  5. Normal: 2–3 minutes (Duke’s)
Ivy’s Method (More Standardized):
  1. Puncture volar forearm (9 mm × 2 mm, 2.5 mm deep)
  2. Blot blood with filter paper every 30 seconds
  3. Record time when bleeding stops
  4. Normal: 3–9 minutes (Ivy’s)
Abnormal BT Indicates:
  • Thrombocytopenia (low platelet count)
  • Platelet dysfunction
  • Von Willebrand disease
  • Aspirin use
  • Does NOT assess coagulation factors

Clotting Time (CT) – Assessment of Coagulation Factors:

Lee-White Method:
  1. Take 1 mL venous blood in clean, dry glass tube
  2. Record time (start)
  3. Keep tube at 37°C (body temperature)
  4. Tilt tube every 30 seconds until clot forms completely
  5. Record time when clot stops flowing (end time)
  6. CT = End time − Start time
  7. Normal: 5–8 minutes
Prolonged CT Suggests:
  • Deficiency of coagulation factors
  • Anticoagulant therapy (warfarin, heparin)
  • Liver disease
  • DIC (Disseminated Intravascular Coagulation)

1.6.3C: RBC Indices (Morphometric Data)

RBC indices describe the size, hemoglobin content, and concentration of red blood cells. These are calculated from RBC count, hemoglobin, and hematocrit.

1. Mean Corpuscular Volume (MCV)

Formula: MCV (fL) = (Hematocrit × 10) ÷ RBC count (millions/µL)

Normal: 80–100 fL (femtoliters)

Classification:

  • Macrocytic (> 100 fL): B₁₂ deficiency, folate deficiency
  • Normocytic (80–100 fL): Normal or hemolytic anemia
  • Microcytic (< 80 fL): Iron deficiency, thalassemia

2. Mean Corpuscular Hemoglobin (MCH)

Formula: MCH (pg) = (Hemoglobin × 10) ÷ RBC count (millions/µL)

Normal: 27–31 pg (picograms)

Indicates: Average amount of Hb per RBC

3. Mean Corpuscular Hemoglobin Concentration (MCHC)

Formula: MCHC (%) = (Hemoglobin × 100) ÷ Hematocrit

Normal: 32–36 g/dL

Indicates: Average Hb concentration within RBC (reflects hemoglobin saturation)

Index Formula Normal Range Clinical Use
MCV (Hct × 10) ÷ RBC 80–100 fL Classify anemia by RBC size
MCH (Hb × 10) ÷ RBC 27–31 pg Hb content per RBC
MCHC (Hb × 100) ÷ Hct 32–36 g/dL Hb concentration in RBC
RDW CV of RBC distribution 11.5–14.5% RBC size variation (anisocytosis)

1.6.4 Coombs Test (Antiglobulin Test)

Principle: Coombs test detects antibodies (immunoglobulins or complement proteins) attached to RBC surface. Uses anti-human globulin (Coombs reagent) to cause agglutination of coated RBCs, making antibodies visible. Discovered by Coombs, Mourant & Race in 1945.

Types of Coombs Test

1. Direct Coombs Test (Direct Antiglobulin Test – DAT)

Purpose: Detects antibodies and complement already attached to RBC surface
Clinical Use: Diagnose hemolytic anemia (autoimmune hemolytic anemia – AIHA)
Procedure:
  1. Prepare 5% RBC suspension in isotonic saline
  2. Add 1 drop RBC suspension to clean test tube
  3. Wash 3 times with normal saline (removes plasma & unbound antibodies)
  4. Decant completely after final wash
  5. Add 2 drops anti-human globulin (Coombs reagent)
  6. Mix well; centrifuge 1 minute at 1500 RPM
  7. Examine for agglutination macroscopically and microscopically
Result Interpretation:
  • Positive: Visible agglutination (clumping) of RBCs → Antibodies present on RBC surface
  • Negative: No agglutination → No antibodies on RBC surface
Positive Direct Coombs Suggests:
  • Autoimmune hemolytic anemia (AIHA) – warm antibodies (IgG, 37°C)
  • Cold agglutinin disease – cold antibodies (IgM, < 32°C)
  • Hemolytic disease of newborn (HDN) – maternal IgG antibodies
  • Transfusion reaction – incompatible transfused RBCs
  • Drug-induced hemolytic anemia (methyldopa, penicillin, quinidine)

2. Indirect Coombs Test (Indirect Antiglobulin Test – IAT)

Purpose: Detects free antibodies in plasma/serum (circulating unbound antibodies)
Clinical Use: Pre-transfusion testing, prenatal screening, antibody detection
Procedure:
  1. Mix patient’s serum with test RBCs (panel cells with known antigens)
  2. Incubate 37°C for 15–30 minutes (allows antibody-antigen binding)
  3. Wash 3 times with isotonic saline
  4. Add anti-human globulin (Coombs reagent)
  5. Centrifuge 1 minute at 1500 RPM
  6. Examine for agglutination
Result Interpretation:
  • Positive: Agglutination → Antibodies present in patient’s serum
  • Negative: No agglutination → No antibodies or very low titer
Positive Indirect Coombs Indicates:
  • Presence of alloimmunization (antibodies against foreign RBC antigens)
  • Hemolytic transfusion reaction risk
  • Hemolytic disease of newborn (maternal antibody screening)
  • Incompatible blood for transfusion

Comparison Table

Parameter Direct Coombs (DAT) Indirect Coombs (IAT)
Antibody Location On RBC surface (bound) In serum/plasma (free)
Clinical Purpose Diagnose hemolysis Pre-transfusion compatibility
Primary Use AIHA, HDN, transfusion reaction Blood bank screening, antibody ID
Sample Patient’s RBCs Patient’s serum + test RBCs
Incubation Direct (no preincubation) 37°C for 15–30 min

Quality Control & Interpretation

Important Notes:

  • Use Coombs control cells (presensitized RBCs) to verify reagent reactivity
  • Insufficient washing → false positive (residual serum remains)
  • Over-incubation → RBC lysis (false negative)
  • Positive in SLE, RA, hepatitis (not only hemolytic anemia)
  • Negative Coombs does NOT rule out hemolysis (may need other tests)

1.6.5 Blood Banking & Transfusion: Pre-Transfusion Testing

Compatibility Testing Overview

Goal: Ensure blood compatibility between donor and recipient to prevent hemolytic transfusion reactions and hemolytic disease of the newborn. Involves serological testing and electronic crossmatching.

Components of Pre-Transfusion Testing

1. ABO & Rh Typing

Forward Grouping (Patient RBCs + Reagent Antibodies):
  • Test RBCs with anti-A serum → Agglutination indicates A antigen present
  • Test RBCs with anti-B serum → Agglutination indicates B antigen present
  • Test RBCs with anti-D serum → Agglutination indicates Rh positive
Reverse Grouping (Patient Serum + Test RBCs):
  • Confirms serum contains expected naturally occurring antibodies
  • Type A serum: contains anti-B
  • Type B serum: contains anti-A
  • Type AB serum: no anti-A or anti-B
  • Type O serum: contains both anti-A and anti-B
ABO & Rh Distribution:
  • O positive (37%)
  • A positive (35%)
  • B positive (9%)
  • AB positive (3%)
  • O negative (6%)
  • A negative (6%)
  • B negative (2%)
  • AB negative (1%)

2. Antibody Screening (Indirect Coombs Method)

Purpose: Detect unexpected alloantibodies in recipient’s serum
Method: Patient serum incubated with panel RBCs expressing all major blood group antigens
Result:
  • Negative screen: No unexpected antibodies (routine transfusion possible with ABO/Rh match)
  • Positive screen: Alloantibodies detected → Antibody identification required → Antigen-negative RBCs selected

3. Crossmatching (Final Compatibility Check)

Serological Crossmatch:
  1. Mix donor RBCs with recipient serum/plasma
  2. Incubate 37°C for 15–30 minutes
  3. Add Coombs reagent (anti-human globulin)
  4. Centrifuge and observe for agglutination
Electronic (Computer) Crossmatch:
  • Used when antibody screen is negative
  • Computer verifies ABO/Rh compatibility between donor & recipient
  • Faster turnaround (~5 minutes vs. 30–45 min for serological)
Result Interpretation:
  • Compatible: No agglutination → Blood safe for transfusion
  • Incompatible: Agglutination present → Blood NOT safe (risk of hemolytic reaction)

Type & Screen vs. Crossmatch

Procedure Components Turnaround Time When Used
Type & Screen ABO/Rh grouping + Antibody screening ~10–15 min Elective surgery, pre-operative preparation
Full Crossmatch Type & Screen + Serological crossmatch ~30–45 min Intended transfusion (blood to be issued)
Emergency Transfusion ABO/Rh match only (O Rh- universal) < 5 min Life-threatening hemorrhage

Critical Patient Identification Requirements

Most transfusion errors are due to patient misidentification! Must have:
  • Correct patient identification at phlebotomy
  • Labeled specimen with 2 patient identifiers
  • Proper chain of custody throughout testing
  • Final verification before transfusion at bedside
  • Blood bank and recipient identification bands matched

Transfusion Reactions & Prevention

Reaction Type Cause Prevention
Acute Hemolytic ABO incompatibility; IgG antibody binding Proper crossmatching & patient ID
Delayed Hemolytic Alloimmunization to minor RBC antigens Antibody screening; negative crossmatch
Febrile WBC or platelet antibodies Leukocyte-reduced products
Allergic Donor plasma proteins Washed RBCs; antihistamine premedication

1.6.6 Coagulation Profile: Mechanism, Disorders & Investigations

Overview of Hemostasis & Coagulation Cascade

Hemostasis: Process of blood clot formation to stop bleeding. Has three phases:
1) Primary: Platelet adhesion/aggregation
2) Secondary: Coagulation cascade (factors I–XIII)
3) Tertiary: Fibrinolysis (clot breakdown)

Coagulation Cascade (Simplified)

Three Pathways:

  • Extrinsic Pathway: Tissue factor (TF) + Factor VII → Activates Factor X
  • Intrinsic Pathway: Factors XII, XI, IX, VIII → Activates Factor X
  • Common Pathway: Factors X, V, II (thrombin), I (fibrinogen) → Fibrin clot formation

Final Steps:

  1. Factor X activation (junction point)
  2. Prothrombin (Factor II) → Thrombin
  3. Fibrinogen (Factor I) → Fibrin monomer
  4. Fibrin monomer → Cross-linked fibrin clot

Screening Tests for Coagulation

1. Prothrombin Time (PT / INR)

Principle: Measures time for citrated plasma to clot after adding tissue factor (TF) & calcium
Assesses: Extrinsic pathway (TF) + Common pathway
Factors Measured: I, II, V, VII, X
Procedure:
  1. Collect blood in 3.2% sodium citrate (9:1 blood:citrate ratio)
  2. Centrifuge to obtain platelet-poor plasma
  3. Add tissue thromboplastin (TF) + calcium to plasma
  4. Record time for clot formation (in seconds)
  5. Compare with control normal plasma
Normal Range: 11–13.5 seconds
INR (International Normalized Ratio): 0.8–1.1 (standardized measure independent of reagent)
Clinical Interpretation:
  • Prolonged PT: Deficiency of factors I, II, V, VII, or X
  • Early vitamin K deficiency
  • Warfarin (anticoagulant) therapy
  • Liver disease (cholestatic, failure)
  • DIC (Disseminated Intravascular Coagulation)

2. Activated Partial Thromboplastin Time (aPTT)

Principle: Measures time for citrated plasma to clot after adding phospholipid + activator (kaolin) + calcium
Assesses: Intrinsic pathway + Common pathway
Factors Measured: I, II, V, VIII, IX, X, XI, XII + Contact factors
Procedure:
  1. Collect plasma as in PT (citrated)
  2. Add phospholipid + contact activator (kaolin/silica)
  3. Incubate 3–5 minutes
  4. Add calcium; measure clotting time
Normal Range: 25–35 seconds
Clinical Interpretation:
  • Prolonged aPTT: Deficiency of intrinsic factors (VIII, IX, XI, XII)
  • Hemophilia A (Factor VIII deficiency)
  • Hemophilia B (Factor IX deficiency)
  • Heparin therapy (anticoagulant)
  • Von Willebrand disease
  • Lupus anticoagulant (inhibitor)

3. Thrombin Time (TT) / Thrombin Clotting Time (TCT)

Principle: Measures time for citrated plasma to clot after direct addition of thrombin (bypasses all coagulation factors)
Assesses: Fibrinogen function
Factors Measured: Fibrinogen (Factor I) only
Procedure:
  1. Add thrombin directly to citrated plasma
  2. Measure time for fibrin clot formation
Normal Range: 14–16 seconds
Clinical Interpretation:
  • Prolonged TT: Low fibrinogen or fibrinogen dysfunction
  • Severe hypofibrinogenemia (< 100 mg/dL)
  • DIC (both decreased fibrinogen & increased FDPs)
  • Heparin therapy (direct effect on thrombin)
  • Liver failure
  • Paraproteinemia (inhibits fibrin polymerization)

Interpretation of Coagulation Results

Result Patterns & Defects:

PT aPTT TT Probable Defect Example Disorders
↑ N N Factor VII deficiency Early vitamin K deficiency; warfarin
N ↑ N Intrinsic pathway Hemophilia A/B; heparin; vWD
N N ↑ Fibrinogen defect Hypofibrinogenemia; dysfibrinogenemia
↑ ↑ N Common pathway Severe DIC; liver failure; vitamin K deficiency
↑ ↑ ↑ Multiple defects Overt DIC; massive transfusion; liver failure
N N N Possibly normal vWD (mild); platelet disorder; fibrinolytic defect

Common Coagulation Disorders

Vitamin K Deficiency:

  • Factors Affected: II, VII, IX, X (vitamin K-dependent)
  • PT: Prolonged (Factor VII is most sensitive)
  • aPTT: Normal initially, prolonged if severe
  • Treatment: Vitamin K (phytomenadione) IV/IM

Hemophilia A (Factor VIII Deficiency):

  • Inheritance: X-linked recessive
  • PT: Normal
  • aPTT: Prolonged (corrects with normal plasma)
  • Treatment: Factor VIII concentrates, fresh frozen plasma (FFP)

DIC (Disseminated Intravascular Coagulation):

  • PT: Prolonged
  • aPTT: Prolonged
  • Fibrinogen: Decreased
  • Platelets: Decreased
  • FDPs (Fibrin Degradation Products): Elevated

Additional Tests in Coagulation Disorders

  • Fibrinogen assay: Measures fibrinogen concentration (normal 200–400 mg/dL)
  • FDP (Fibrin Degradation Products): Detects fibrinolysis (elevated in DIC, liver disease)
  • D-Dimer: Specific FDP marker (elevated in DIC, thrombosis, PE)
  • Platelet count: Assess primary hemostasis
  • Individual factor assays: Measure specific factor levels (VIII, IX, etc.)

Clinical Pearl:

Never order coagulation studies WITHOUT clinical indication. Most routine preoperative PT/aPTT are unnecessary. Reserve for: known bleeding history, liver disease, anticoagulation therapy, or family history of bleeding disorders.

1.6.7 LE Cell Preparation & Testing

LE Cell Phenomenon: In vitro test demonstrating antinuclear antibodies in patient serum by showing characteristic LE cells. Primarily used to diagnose Systemic Lupus Erythematosus (SLE), though less specific than modern ANA testing.

Principle of LE Cell Formation

  1. Nuclear Material Exposure: WBCs are damaged/lysed, releasing homogenous nuclear material (LE bodies)
  2. Antigen-Antibody Reaction: Antinuclear factors (anti-DNA, anti-histone antibodies) from patient serum bind to exposed nuclear material
  3. Opsonization: Complement proteins coat the antibody-antigen complex
  4. Phagocytosis: Neutrophils phagocytose the coated LE bodies
  5. LE Cell Formation: Neutrophil containing phagocytosed LE body = Lupus Erythematosus (LE) cell
  6. Rosette Form: Multiple neutrophils clustering around single LE body = rosette formation

Procedure

Step 1: Cell Damage (Traumatization)

  1. Collect 5–10 mL venous blood (no anticoagulant) into clean, sterile tube
  2. Allow clotting at room temperature for 1–2 hours
  3. Add clotted blood to fine wire sieve in glass dish
  4. Using glass rod, crush clot through sieve → allows RBC lysis, WBC traumatization
  5. Collect crushed blood in 13 × 100 mm tube; add 2–3 glass beads

Step 2: Incubation

  1. Shake tube vigorously on mechanical shaker for 5 minutes (further WBC trauma)
  2. Incubate at 37°C for 15–30 minutes (optimal temperature for antibody-antigen reactions)
  3. Can also incubate at room temperature 22°C (takes longer, ~1 hour)

Step 3: Preparation & Staining

  1. Using Pasteur pipette, withdraw buffy coat layer (interface between plasma & packed RBCs)
  2. Transfer to clean glass slides
  3. Cover with coverslip; make smear by tilting/sliding coverslip
  4. Air-dry slides completely
  5. Stain with Wright’s stain (standard hematology stain)
  6. Rinse with buffer solution; air-dry

Step 4: Microscopic Examination

  1. Examine under 40× objective for overall stain quality & rosette forms (screening)
  2. Use 100× oil immersion objective for definitive LE cell identification
  3. Count minimum 1000 WBCs or search at least 100 fields

LE Cell & Rosette Morphology

Characteristic Appearances:

  • LE Cell: Neutrophil containing ingested, round, homogenous pale-staining nucleus (LE body). The ingested nucleus appears as a large amorphous mass filling the neutrophil cytoplasm
  • LE Body (Hematoxylin Body): Destroyed nucleus with homogenous, dark-purple appearance on Wright’s stain. Composed of denatured nuclear material
  • Rosette Form: 3–5+ neutrophils clustered around central LE body, resembling flower petals. More easily recognized than single LE cells
  • Tart Cell: WBC ingesting fragmented (rough) nuclear material (NOT homogenous). This is NOT an LE cell – avoid false positive

Result Interpretation

Positive LE Cell Preparation:

  • Presence of ≥ 1 LE cell or rosette form per 100 WBCs examined
  • Suggests SLE or related autoimmune disease
  • Sensitivity: 50–80% in active SLE (lower in early disease)
  • Specificity: 95–98% (rarely positive in non-SLE)

Negative LE Cell Preparation:

  • No LE cells or rosette forms found after extensive search
  • Does NOT rule out SLE (low sensitivity)
  • May be negative in inactive/well-controlled SLE

Conditions Associated with Positive LE Cells

SLE-Related SLE-Unrelated
• Systemic Lupus Erythematosus (80%) • Rheumatoid Arthritis (10–30%)
• Drug-induced SLE (hydralazine, procainamide) • Sjögren’s Syndrome
• Neonatal SLE (maternal antibodies) • Hepatitis
• Certain medications (antibiotics, anticonvulsants)

Advantages & Limitations

  • Advantages: Simple, inexpensive, visible morphology, historically significant
  • Limitations: Subjective interpretation, low sensitivity, largely replaced by ANA testing, requires skilled microscopist, false positives possible (Tart cells)

Modern Alternative: ANA (Antinuclear Antibody) Testing

  • More sensitive & specific than LE cell test
  • Uses immunofluorescence or ELISA to detect circulating antinuclear antibodies
  • Shows staining pattern: homogenous, speckled, nucleolar, centromere
  • LE cell test is now largely historical; ANA is standard for SLE diagnosis

1.6.8 Tissue & Blood Parasites: Identification on Blood Smears

Blood Parasites: Parasitic organisms found in circulating blood. Detection requires examination of thick smears (high sensitivity) and thin smears (better morphology identification) stained with Giemsa, Wright, or Wright-Giemsa stain.

Specimen Collection & Preparation

Thick Smear:

  • Purpose: Increased sensitivity (more blood examined, ~20× more RBCs than thin smear)
  • Preparation: Large drop of blood (~10 µL) spread on slide; allowed to air-dry without fixing
  • Staining: RBCs are lysed during staining (no hemoglobin present)
  • Advantage: Detect very low parasitemia (< 0.1%)
  • Disadvantage: Difficult morphology; parasites may distort

Thin Smear:

  • Purpose: Better parasite morphology & identification
  • Preparation: Small drop of blood spread with coverslip to thin layer
  • Staining: Wright-Giemsa stain (after methanol fixation); RBCs remain intact
  • Advantage: Clear morphology; identify species
  • Disadvantage: Lower sensitivity; requires more blood to find rare parasites

Major Tissue/Blood Parasites

1. Malaria Parasites (Plasmodium species)

Plasmodium falciparum (Severe Malaria):
  • RBC Preference: All RBC ages (young to old)
  • RBC Changes: Maurer’s clefts; Schüffner’s dots (fine, sparse)
  • Characteristic: Multiple ring-forms in single RBC; high parasitemia possible (up to 50%)
  • Gametocytes: Crescent or banana-shaped (diagnostic)
Plasmodium vivax (Tertian Malaria):
  • RBC Preference: Young RBCs (reticulocytes)
  • RBC Changes: Large, pale; Schüffner’s dots (coarse, prominent)
  • Characteristic: Rings, trophozoites, schizonts clearly visible
  • Parasitemia: Usually < 5%
Plasmodium malariae (Quartan Malaria):
  • RBC Preference: Old RBCs (senescent)
  • RBC Changes: Small (smaller than normal RBC)
  • Characteristic: Band-like trophozoites; rosette schizonts
  • Parasitemia: Usually < 1% (low)
Plasmodium ovale:
  • RBC Preference: Young RBCs
  • RBC Shape: Fimbriated (irregular borders); oval shape
  • Schüffner’s Dots: Coarse, prominent

2. Babesia (Intraerythrocytic Protozoan)

  • Appearance: Ring forms; characteristic “Maltese cross” (tetrad form) pathognomonic
  • Location: Within RBCs
  • Clinical: Babesiosis (hemolytic anemia, fever); transmitted by Ixodes tick
  • Detection: Thin blood smear with Giemsa or Wright stain

3. Microfilariae (Filaria Parasites)

  • Wuchereria bancrofti: Nocturnal periodicity (appear in blood evening–morning)
  • Brugia malayi: Subperiodic form; long, thin with pointed tail
  • Appearance: Long, thread-like worms in thick smear; sheath absent (in most species)
  • Clinical: Filariasis (lymphatic damage, elephantiasis)

4. Leishmania (Intracellular Protozoan)

  • Location: Within monocytes, macrophages, neutrophils
  • Appearance: Round, oval amastigotes with eccentric nucleus & kinetoplast
  • Staining: Giemsa – appears as small dots with characteristic morphology
  • Clinical: Leishmaniasis (cutaneous, visceral)

5. Trypanosoma (Flagellate Protozoan)

  • Trypanosoma brucei: Large, motile, flagellated organisms in blood plasma
  • Characteristic: Undulating membrane, posterior kinetoplast
  • Clinical: African sleeping sickness (African trypanosomiasis)
  • Detection: Wet mount examination (motility); Giemsa smear

Examination Technique for Parasites

Microscopic Examination:

  1. Thick Smear: Screen with 10× objective; confirm with 40× and 100× oil immersion
  2. Thin Smear: Examine systematically with 100× oil immersion objective
  3. Count minimum 300–1000 RBCs or 100 microscopic fields
  4. For malaria: Count parasites per 100 RBCs to determine parasitemia percentage
  5. Identify parasite morphology: rings, trophozoites, schizonts, gametocytes

Normal Values & Reporting

Negative: No parasites seen after examination of adequate blood volume
Positive: Organism identified + species identified + parasitemia level (for malaria: percentage of infected RBCs)
Reporting: “Plasmodium falciparum detected; parasitemia 2.5%” or “No blood parasites seen”

Sensitivity Comparisons

  • Blood Smear (Thick & Thin): 50–500 parasites/µL detectable
  • Rapid Diagnostic Tests (RDTs): 100 parasites/µL detectable
  • PCR (Polymerase Chain Reaction): 2–5 parasites/µL (gold standard; species identification)
  • Culture: Not practical for Plasmodium (long turnaround)

Quality Assurance in Parasite Detection

  • Use fresh blood samples; avoid hemolysis
  • Proper fixation & staining of smears
  • Trained microscopists (parasitology expertise)
  • Dual examination (two technologists) for positive cases
  • Regular quality control with known positive & negative controls

1.6.9 Absolute Cell Count (Absolute Differential Count)

Absolute Cell Count: Determines the actual number of each WBC type per unit volume of blood, rather than percentage. More clinically accurate than relative (percentage) counts because it reflects true cell concentration.

Differential vs. Absolute Count

Parameter Relative (Differential) Count Absolute Count
What it measures Percentage of each WBC type Actual number of each WBC type
Calculation Count 100 WBCs; record % % × Total WBC count ÷ 100
Unit Percentage (%) Cells/µL (cells/mm³)
Accuracy Low (can be misleading if total WBC is very high/low) Higher (reflects true pathology)
Clinical Use Screening only Diagnosis, treatment decisions

Formula for Absolute Cell Count

General Formula:

Absolute Count = (% of Cell Type ÷ 100) × Total WBC Count

Or alternatively:

Absolute Count = (% × Total WBC) ÷ 100

Example:

Patient Results:

  • Total WBC: 8,000 cells/µL (normal)
  • Differential: 60% Neutrophils

Calculation: Absolute Neutrophil Count = (60 ÷ 100) × 8,000 = 4,800 cells/µL

Clinical Interpretation: Normal neutrophil count (normal range 2,000–7,500 cells/µL)

Normal Absolute Cell Counts

Standard Reference Ranges (Adults):
WBC Type Absolute Count (cells/µL) Percentage (%)
Neutrophils 2,000–7,500 40–75%
Lymphocytes 1,000–4,800 20–40%
Monocytes 200–900 2–8%
Eosinophils 0–400 0–4%
Basophils 0–100 0–2%

Clinical Significance: Interpretation of Abnormalities

Absolute Neutrophil Count (ANC)

Elevated Absolute Neutrophils (Neutrophilia > 7,500/µL):
  • Infection (bacterial, some viral, fungal)
  • Inflammation (rheumatoid arthritis, IBD)
  • Leukemia (chronic myeloid, acute myeloid)
  • Stress (physical trauma, surgery)
  • Corticosteroid therapy (demargination)
  • Smoking
Decreased Absolute Neutrophils (Neutropenia < 2,000/µL):
  • Bone marrow suppression (chemotherapy, radiation)
  • Aplastic anemia
  • Drug reactions (antibiotics, anticonvulsants)
  • Severe infections (sepsis exhausts reserve)
  • Autoimmune disease (SLE, Felty syndrome)
  • HIV/AIDS (CD4 < 200)

Absolute Lymphocyte Count (ALC)

Elevated Absolute Lymphocytes (Lymphocytosis > 4,800/µL):
  • Viral infections (EBV, CMV, measles)
  • Bacterial infections (TB, whooping cough)
  • Lymphocytic leukemia (acute, chronic)
  • Autoimmune diseases
Decreased Absolute Lymphocytes (Lymphopenia < 1,000/µL):
  • HIV/AIDS (CD4 < 200 is severe)
  • Steroid therapy (cortisol suppresses T cells)
  • Severe infections
  • Lymphoma
  • Congenital immunodeficiency

Absolute Eosinophil Count (AEC)

Elevated Absolute Eosinophils (Eosinophilia > 400/µL):
  • Parasitic infections (roundworms, hookworms)
  • Allergic disorders (asthma, hay fever)
  • Eosinophilic leukemia
  • Autoimmune diseases
  • Medication reactions

Clinical Importance: When to Use Absolute Counts

Absolute Counts are Essential For:

  • Chemotherapy Decisions: Absolute neutrophil count < 500 = high infection risk; chemotherapy held
  • HIV Monitoring: CD4 (absolute lymphocyte count subset) guides antiretroviral therapy & opportunistic infection prophylaxis
  • Infection Risk Assessment: Can have normal total WBC but low absolute neutrophils (high risk)
  • Allergy/Parasitic Disease: Absolute eosinophil count > 1,500 suggests parasites or severe allergy
  • Sepsis Monitoring: Absolute neutrophil count response indicates bone marrow capacity to fight infection

Pitfall Example: Why Percentage Alone Can Mislead

Case 1: Normal WBC with Abnormal Absolute Count

Lab Results:

  • Total WBC: 3,000 cells/µL (LOW)
  • Neutrophils: 75% (appears “normal”)

Absolute Neutrophil Count: (75 ÷ 100) × 3,000 = 2,250 cells/µL

Interpretation: NEUTROPENIA! Patient at risk despite normal percentage. If only looking at percentage, this would be missed.

Case 2: Elevated WBC with Apparent Lymphocytosis

Lab Results:

  • Total WBC: 30,000 cells/µL (HIGH)
  • Lymphocytes: 20% (appears “low”)

Absolute Lymphocyte Count: (20 ÷ 100) × 30,000 = 6,000 cells/µL

Interpretation: LYMPHOCYTOSIS! Despite low percentage, absolute count is elevated (normal max 4,800), indicating true increase. Likely leukemia or infectious mononucleosis.

Modern Automated Analysis

  • Hematology analyzers automatically report both percentage AND absolute counts
  • Always order & review absolute counts for clinical decisions
  • Never make clinical decisions based on percentage alone
  • Verify unusual results with manual differential on blood smear

Medical Laboratory Science Hematology Study Notes 1.6

Comprehensive reference for principles & clinical procedures. For educational purposes.

Last Updated: December 2025 | Kathmandu, Nepal | Mero Healthline Study Resources

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