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Q1. Estimation of Hemoglobin

Principle

Methods Used

Cyanmethemoglobin Method - Procedure

• Potassium ferricyanide K3[Fe(CN)6]: 200 mg
• Potassium cyanide (KCN): 50 mg
• Potassium dihydrogen phosphate (KH2PO4): 140 mg
• Non-ionic detergent (Sterox/Triton X): 0.5 mL
• Distilled water to 1000 mL
• pH 7.0-7.4; stable for months in amber bottle

1. Pipette 5.0 mL of Drabkin's reagent into a labeled tube
2. Add 20 µL (0.02 mL) of well-mixed EDTA anticoagulated blood using a calibrated pipette
3. Mix well; let stand at room temperature for 3-5 minutes (ensures complete conversion)
4. Read absorbance at 540 nm against a reagent blank in a spectrophotometer
5. Calculate Hb concentration using calibration curve or standard

Normal Values

Calculation/Reporting

Sources of Error

• Improper mixing of blood before sampling
• Lipemia/turbid plasma (falsely elevated absorbance)
• Very high WBC count (>30 × 10⁹/L) causes turbidity
• Improper blood-to-anticoagulant ratio

• Incomplete conversion (reading before 3 minutes)
• Dirty cuvette; fingerprints on optical surfaces
• Wavelength miscalibration
• Reagent deterioration (yellowed Drabkin's = discard)
• Carboxy-hemoglobin (HbCO) converts slowly - wait 10 min in heavy smokers

• Pipetting inaccuracies (most common with Sahli)
• Failure to zero spectrophotometer properly

Automated Estimation of Hb

1. SLS method (Sysmex): Sodium lauryl sulfate lyses RBCs and converts all Hb forms to stable SLS-Hb; measured photometrically at 540 nm. Advantages: no cyanide (environmentally safer), stable reagent.
2. Impedance + photometric combination: RBC count and Hb measured simultaneously; MCV derived from pulse height; indices calculated automatically.
3. Direct spectrophotometry at multiple wavelengths to correct for interfering substances.
4. Quality control: Automated analyzers use 3-level commercial controls (normal, low, high) daily; Levey-Jennings charts and Westgard rules applied. External QA via proficiency testing programs (EQAS).

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Q2. Total Leukocyte Count (TLC)

Principle

Manual Method

Diluting Fluids

Procedure (Improved Neubauer Hemocytometer)

1. Mix blood well; draw 0.5 mL Turk's fluid into a WBC pipette (or use appropriate dilution tube)
2. Add 0.02 mL blood → dilution = 1:20 (or 20 µL blood + 380 µL Turk's = 1:20)
3. Mix thoroughly for 2-3 minutes (figure-8 rotation if using pipette)
4. Charge the hemocytometer: touch tip to edge of coverslip and allow capillary action to fill the chamber
5. Allow cells to settle for 2-3 minutes
6. Count under 10× objective (low power)
7. Count WBCs in all 9 large squares (entire grid, but conventionally 4 corner squares + central square = 5 squares, each 1 mm²)

Calculation

• Volume = 4 × (1 × 1 × 0.1) = 0.4 mm³
• WBC/µL = (N × 20) / 0.4 = N × 50

• Volume = 9 × 0.1 = 0.9 mm³
• WBC/µL = (N × 20) / 0.9

Normal Range

Sources of Error (Manual)

• Underfilling/overfilling the hemocytometer chamber
• Uneven distribution - improper mixing
• Counting nucleated RBCs as WBCs (correction required: if NRBC seen on smear)
• Platelet clumps mistaken for WBCs
• Lysis failure - cells too lysed in acetic acid if left too long
• Parallax errors - counting cells on boundary lines (count top and left lines only, not bottom and right - the "L rule")
• Dry edges of chamber - avoid counting peripheral rows
• Hemocytometer not horizontal during filling - uneven distribution

Automated TLC

1. Electrical Impedance (Coulter Principle)

• Cells suspended in electrolyte pass through an aperture
• Each cell displaces electrolyte, causing a change in electrical resistance (pulse)
• Pulse height ∝ cell volume; pulse frequency = cell count
• After RBC lysis (by lysing reagent), WBCs counted in impedance channel
• Separates WBC into 3 populations: lymphocytes (small), mid-cells (monocytes, eosinophils, basophils), granulocytes (large) → 3-part differential

2. Light Scattering (VCS / Flow Cytometry-based)

• Cells pass through laser beam
• Forward scatter (FSC) = cell size
• Side scatter (SSC) = granularity/internal complexity
• Fluorescence channels (for DNA/RNA staining) separate immature cells
• 5-part differential: neutrophils, lymphocytes, monocytes, eosinophils, basophils

3. Multi-parameter Analysis (e.g., Sysmex XN-series)

• Uses multiple channels: WBC/DIFF, RET (reticulocyte), PLT channels
• Fluorescent dyes label nucleic acids; immature granulocytes (IG), blasts flagged
• Reports: absolute counts for all 5 types + IG count + atypical lymphocyte flags

Interpretation

Leukocytosis (TLC >11.0 × 10⁹/L in adults)

Leukopenia (TLC <4.0 × 10⁹/L)

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Q3. Peripheral Blood Smear (PBS) - Preparation and Examination

Preparation of Blood Smear

Materials

• Fresh EDTA blood (within 2 hours) or finger-prick capillary blood
• Clean, grease-free glass slides (75×25 mm)
• Spreader slide (edges polished or with 45° angle)

Wedge (Push) Method - Standard Procedure

1. Place a small drop (2-3 µL) of blood 1 cm from one end of the slide
2. Hold the spreader slide at 30-45° angle in front of the blood drop
3. Draw spreader back to touch the blood drop; allow blood to spread along the edge by capillary action
4. Push the spreader forward in one smooth, even, rapid stroke
5. The smear should be 2-3 cm long, with a feathered tail and central body (not touching edges)
6. Air dry immediately (wave the slide or use fan); do not heat-fix

Features of a Good Smear

• Gradual transition from thick (head) to thin (tail/feathered edge)
• No holes, streaks, or ridges
• Occupies 2/3 of the slide length
• RBCs just separate at the feathered edge
• Body of smear: slight overlapping; counting area: single-cell layer

Common Errors in Preparation

Staining Methods

1. Romanowsky Stains (Gold Standard)

• Contains: Leishman's powder (eosin + methylene blue) in absolute methanol
• Methanol: fixes and stains simultaneously
• Procedure:
  1. Flood air-dried smear with Leishman's stain for 2 minutes (fixation)
  2. Add equal volume of buffered water (pH 6.8) for 10-15 minutes
  3. Wash with buffered water; drain; air dry; do not blot
• Result: RBCs - pink/red; WBC nuclei - blue/purple; neutrophil granules - pink-purple; eosinophil granules - bright red; basophil granules - deep purple; platelets - purple granules

• May-fix separately in methanol, then stain in 10% Giemsa (pH 7.2) for 20-30 minutes
• Excellent for parasites (malaria, trypanosomes) and lymphoid cells
• Often used as May-Grünwald-Giemsa (MGG) combination

• Most common in North America
• Fixed and stained simultaneously in Wright's stain (methanol-based)
• Procedure: flood 1-3 min, add buffer, stain 10-15 min, rinse

• Rapid, 2-solution method for thick malaria films
• Solution A (basic) + Solution B (acidic); dip 1-2 seconds each
• Not suitable for detailed morphology

Staining Defects and Causes

Examination Sequence

1. Macroscopic: Check smear quality (good/acceptable/poor)
2. Low power (10×): Overall cellularity, assess area for counting, look for large cells/parasites
3. High power / Oil immersion (100×): Detailed morphology

Differential Leukocyte Count (DLC)

Normal DLC (Adults)

Neutrophil Features

• Segmented: 2-5 lobes connected by thin filaments; pink cytoplasm; azurophilic (primary) granules + specific (secondary) granules
• Band form: Horse-shoe nucleus; no filament visible (lobe > 1/3 width)
• Hypersegmentation: >5 lobes; seen in megaloblastic anemia
• Left shift: Increased band forms, metamyelocytes, myelocytes on smear (infection, leukemoid reaction, CML)

RBC Morphology Assessment

Key Poikilocytes and Their Significance

Platelet Morphology on Smear

• Normal platelets: 1.5-3.5 µm; pale blue with central purple granules (2-4/oil immersion field)
• Giant platelets (megathrombocytes): ITP, MYH9 disorders, CML, myeloproliferative neoplasms
• Platelet clumps: EDTA-induced pseudothrombocytopenia; use citrate tube or warm blood
• Platelet satellitism: Platelets clustering around neutrophils; cause of spurious neutrophilia
• Hypogranular platelets: Myelodysplastic syndrome, grey platelet syndrome

Automated Differential Count

1. 3-part differential (older analyzers): Impedance only; separates lymphocytes, mid-cells, granulocytes (not useful for individual 5-part DLC)
2. 5-part differential:

• VCS technology (Beckman Coulter): Volume (V) by DC impedance, Conductivity (C) by RF, Scatter (S) by laser - gives 5-population separation
• Flow cytometry-based (Sysmex): Fluorescent dye (polymethine/oxazine) + forward/side scatter; separate neutrophils, lymphocytes, monocytes, eosinophils, basophils with flags for blasts/atypical lymphs/IG (immature granulocytes)
• Abbott CELL-DYN Sapphire: 4-angle light scatter + fluorescence

1. Digital morphology systems (CellaVision DM96, Morphogo):

• High-resolution image capture of blood smear
• AI/neural network classifies cells automatically with human review
• Excellent for morphology documentation and educational use

1. Limitations of automated DLC:

• Cannot reliably identify atypical/reactive lymphocytes, blasts, dysplastic cells
• All flagged/abnormal samples require manual microscopy review
• ICSH recommends microscopy review criteria (>1% blasts, flags, clinical context)

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Q4. Erythrocyte Sedimentation Rate (ESR)

Principle

1. Lag phase (10 min): Initial rouleaux formation; little sedimentation
2. Sedimentation phase (40 min): Constant, rapid settling
3. Packing phase (10 min): Slowing as RBCs pack at bottom

Methods

1. Westergren Method (ICSH/WHO Recommended)

• Anticoagulant: Trisodium citrate 3.8% (1 part + 4 parts blood = 1:4 ratio; 0.5 mL citrate + 2.0 mL blood) OR EDTA blood diluted 4:1 with citrate/saline
• Tube: Westergren tube - length 300 mm, internal bore 2.55 mm, graduated 0-200 mm
• Procedure:
  1. Mix blood well with citrate; fill Westergren tube to 0 mark by suction
  2. Place vertically in Westergren stand at room temperature (18-25°C)
  3. Read the level of clear plasma at exactly 1 hour (read from top - 0 mark downward)
  4. Report as mm in 1st hour

2. Wintrobe Method

• Anticoagulant: EDTA or double oxalate blood (no dilution)
• Tube: Wintrobe tube - 110 mm long, 3 mm bore, graduated 0-10 cm
• Fill to 0 mark; read at 1 hour
• Less sensitive than Westergren; also used for PCV simultaneously
• Normal values lower than Westergren

3. Micro-ESR (Pediatric)

• Uses capillary blood; Landau tube (75 mm); read at 1 hour

Normal Values

Factors Affecting ESR

Factors That Increase ESR

• Fibrinogen (most potent rouleaux promoter)
• Immunoglobulins (IgG, IgM, IgA)
• Alpha-2 globulins, acute phase proteins (CRP, haptoglobin)
• Paraproteinemia (myeloma)

• Anemia (fewer RBCs = less hindrance to sedimentation)
• Macrocytosis (larger RBCs form better rouleaux)

• Female sex (higher fibrinogen)
• Pregnancy (increased fibrinogen; ESR not useful in pregnancy)
• Obesity
• Temperature increase

Factors That Decrease ESR

• Polycythemia (high RBC mass - steric hindrance)
• Sickle cell disease (abnormal shape impedes rouleaux)
• Hereditary spherocytosis (spheres don't form rouleaux)
• Hypofibrinogenemia/DIC
• Hyperviscosity (paradoxically slows settling)
• Low temperature
• Congestive cardiac failure (reduced fibrinogen)

Technical Errors That Increase ESR

• Tube not perfectly vertical (even 3° tilt increases ESR by 30%)
• Blood-to-citrate ratio error (wrong dilution)
• Prolonged delay (>2 hours from collection)
• Room temperature >25°C

Technical Errors That Decrease ESR

• Clotted/hemolyzed sample
• Improper filling (below 0 mark)
• Cold temperature

Clinical Significance

• Monitoring disease activity (SLE, RA, IBD)
• Screening for occult disease
• Distinguishing organic from functional disease

Automated ESR Methods

1. TEST 1 System (Greiner Bio-One): Photometric measurement of RBC column at multiple time points; ESR reported in Westergren-equivalent values; results in 20-30 minutes
2. Alifax RED system: Laser-based optical aggregometry; measures kinetics of RBC aggregation in capillary tubes; fast result (20 sec)
3. ISOCOMP analyzer: Thermostatic control; corrects for temperature variations
4. Ves-Matic (Menarini): Infrared sensor tracks sedimentation; provides final Westergren-equivalent

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Q5. Packed Cell Volume (PCV) / Hematocrit - Microhematocrit Method

Principle

Procedure

Equipment

• Heparinized capillary tubes (red band at one end) - for EDTA blood or plain for fresh capillary blood
• Plain capillary tubes (blue band) - for capillary blood (heparinized)
• Microcentrifuge (12,000-15,000 rpm; radius ~8 cm)
• Reading card/chart or special PCV reader

Steps

1. Fill capillary tube 2/3 to 3/4 full with well-mixed EDTA blood by capillary action (hold at 45° angle in blood)
2. Seal the dry end with Cristaseal (plasticine/sealant) - apply flat to seal completely
3. Place in microcentrifuge with sealed end toward the periphery (outward)
4. Balance the centrifuge (pairs of tubes diametrically opposite)
5. Centrifuge at 12,000-15,000 rpm for 5 minutes
6. Read PCV using the reading chart immediately (before the buffy coat disperses)

Reading

• Total blood column (from inside of seal to top of plasma)
• RBC column height
• PCV = Red cell column height / Total blood column height
• Read from the reading card: align bottom of red cell column with 0, top of plasma with 100, read off % at top of red cell column
• Do NOT include buffy coat in the red cell column (buffy coat = WBCs + platelets, appears as grey-white layer above RBCs)

Normal Values

Calculation

1. Hb (g/dL) ≈ PCV (%) / 3 (rough estimate; Hb × 3 = PCV)
2. MCV (fL) = (PCV/L/L × 1000) / RBC (×10¹²/L)
3. MCHC (g/dL) = Hb (g/dL) / PCV (L/L)

Sources of Error

False High PCV

• Trapped plasma: Incomplete centrifugation; high MCV (macrocytes trap more plasma); sickle cells and irregularly shaped cells trap plasma (up to 2-3% error); microhematocrit has ~1-2% trapped plasma
• Polycythemia (genuine)
• Blood from IV line with fluid contamination
• Including buffy coat in reading

False Low PCV

• Overcoagulation (excess EDTA causes RBC shrinkage - water shifts out) - most important error
• Over-centrifugation (partial hemolysis)
• Hemolysis (lysis of RBCs reduces their volume)
• Reading before spin complete

Other Errors

• Capillary tube not sealed properly → blood expelled
• Air bubbles in tube
• Prolonged storage before centrifugation (glucose consumption, cell swelling)
• Tilted centrifuge

Interpretation

• High PCV: Polycythemia vera, secondary polycythemia (altitude, COPD, EPO-secreting tumors), dehydration (relative polycythemia)
• Low PCV: Anemia (all causes), overhydration, dilutional anemia of pregnancy

Automated Estimation of Hematocrit

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Q6. RBC Indices

MCV - Mean Corpuscular Volume

MCV (fL) = PCV (L/L) × 1000 / RBC count (×10¹²/L)

Classification by MCV

Automated Measurement of MCV

MCH - Mean Corpuscular Hemoglobin

MCH (pg) = Hb (g/dL) × 10 / RBC count (×10¹²/L)

Clinical Significance

• Low MCH (<27 pg): Microcytic hypochromic anemia (iron deficiency, thalassemia, sideroblastic)
• High MCH (>32 pg): Macrocytic anemias (megaloblastic); also in hereditary spherocytosis (spherocytes have high MCHC/MCH despite small size)
• MCH closely parallels MCV; rarely gives independent information beyond MCV

Automated Calculation

MCHC - Mean Corpuscular Hemoglobin Concentration

MCHC (g/dL) = Hb (g/dL) / PCV (L/L) OR: MCHC = (MCH/MCV) × 100

Clinical Significance

• Low MCHC (<32 g/dL): Hypochromia - Iron deficiency anemia (most common cause), thalassemia
• High MCHC (>36 g/dL): Hyperchromia - Classically seen in hereditary spherocytosis (spherocytes lack central pallor due to reduced surface:volume ratio); also in sickle cell disease
• MCHC is physiologically constrained (max ~38 g/dL as Hb precipitates above this); any MCHC >38 g/dL = flag as error (lipemia, cold agglutinins, hemolysis in sample → falsely high Hb without proportional rise in RBC/PCV)
• Very low MCHC in combination with low MCV = strong indicator of iron deficiency

Automated Calculation

RDW - Red Cell Distribution Width

RDW-CV (%) = (Standard deviation of MCV / Mean MCV) × 100

Clinical Significance

• Iron deficiency: RDW HIGH + low MCV
• Thalassemia trait: RDW NORMAL + low MCV (Mentzer index = MCV/RBC count; <13 = thalassemia, >13 = iron deficiency)

Automated Determination

Summary Table of RBC Indices

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Q7. Reticulocyte Count

Principle

Supravital Stains

Procedure (Manual - New Methylene Blue)

1. Mix equal volumes of blood and NMB stain solution (0.5 mL each) in a small tube
2. Incubate at 37°C for 15-20 minutes (or room temperature for 30 min)
3. Make thin films from the mixture; air dry
4. Optionally, counterstain with Leishman's for 1-2 min (for better background)
5. Examine under oil immersion (100×)

• Count 1000 RBCs; note cells with blue reticulum (≥2 granules counts as reticulocyte per ICSH)
• Alternative: Count reticulocytes in each field, count until 1000 RBCs total

• Class I: Dense clump/network
• Class II: >3 granules, no clump
• Class III: 2-3 granules
• Class IV: 1 granule (NOT a reticulocyte by ICSH criteria)

Normal Values

ARC = Reticulocyte % × RBC count (×10¹²/L) × 10

Corrected Reticulocyte Count (CRC)

CRC (%) = Reticulocyte % × (Patient PCV / Normal PCV) Normal PCV used: 0.45 (45%)

• CRC >3% = Hyperproliferative marrow (hemolytic anemia, blood loss, treatment response)
• CRC <2% = Hypoproliferative marrow (iron/B12 deficiency, aplastic anemia, marrow infiltration)

Reticulocyte Production Index (RPI)

RPI = CRC / Maturation factor

• RPI >3 = Adequate marrow response (hyperproliferative)
• RPI <2 = Inadequate marrow response (hypoproliferative)
• RPI 2-3 = Borderline/equivocal

Clinical Significance

Automated Reticulocyte Counting

1. Thiazole orange (Sysmex, Beckman Coulter): Fluorescent dye stains RNA; reticulocytes fluoresce more than mature RBCs; laser excitation; fluorescence intensity proportional to RNA content
2. Oxazine 750 + polymethine (Sysmex RET channel): Stain RNA; cells classified by fluorescence level

• %Reticulocytes and Absolute reticulocyte count
• IRF (Immature Reticulocyte Fraction): Fraction of highly fluorescent (RNA-rich) reticulocytes = reflects most recent marrow output; rises earliest in erythropoietic stimulation
• LFR, MFR, HFR: Low, medium, high fluorescence reticulocytes
• RBC/Retic histograms

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Q8. Absolute Eosinophil Count (AEC)

Principle

Diluting Fluids

Procedure

1. Mix blood well; take 0.38 mL Dunger's fluid in a small tube (or use WBC pipette)
2. Add 0.02 mL (20 µL) blood → dilution 1:20
3. Mix gently for 5 minutes (do not over-shake as it may lyse eosinophils)
4. Charge the improved Neubauer hemocytometer
5. Allow to settle for 5 minutes (eosinophils take longer to settle)
6. Count eosinophils in both chambers (both rulings of the hemocytometer)
7. Count all 9 large squares in both chambers = 18 squares total
   • Volume = 18 × 0.1 mm³ = 1.8 mm³

Calculation

• = (N × 20) / 1.8 = N × 11.1

Normal Range

• Normal AEC: 40-440 cells/µL (0.04-0.44 × 10⁹/L)
• Some sources: 100-400/µL (0.1-0.4 × 10⁹/L)

Causes of Eosinophilia (AEC >500/µL or >0.5 × 10⁹/L)

NAACP Mnemonic:

• N - Neoplastic: CML, lymphoma (Hodgkin's), eosinophilic leukemia (CEL), myeloproliferative neoplasms
• A - Allergic/Atopic: Asthma, allergic rhinitis, urticaria, atopic dermatitis
• A - Addison's disease (adrenocortical insufficiency)
• C - Collagen vascular/autoimmune: Polyarteritis nodosa, eosinophilic granulomatosis with polyangiitis (Churg-Strauss), SLE, dermatomyositis
• P - Parasites: Intestinal (Ascaris, hookworm, Toxocara) and tissue-invasive parasites (Trichinella, Toxocara, Filaria, Schistosoma) especially those with tissue-invasive phase

Eosinophilia Classification by Severity:

• Mild: 500-1500/µL
• Moderate: 1500-5000/µL
• Severe/Hypereosinophilic: >5000/µL (risk of organ damage: cardiac Löffler endocarditis, neuropathy)

Causes of Eosinopenia (AEC <40/µL)

• Cushing's syndrome / exogenous corticosteroids (steroids cause eosinophil sequestration in tissues)
• Acute bacterial infections (cortisol response)
• Post-surgery, acute stress
• Typhoid fever (classic: eosinopenia a feature)
• Excess ACTH stimulation

Automated Eosinophil Counting

• Sysmex: Eosinophils identified in WBC/DIFF channel by their characteristic high side-scatter (large, refractile granules) + intermediate fluorescence; also the "EO" channel uses ammonium chloride-based lysis
• Beckman Coulter DxH: VCS technology - eosinophils have distinct conductivity signature due to large granule content
• Abbott CELL-DYN: 4-angle scatter; eosinophil granules give unique 90° depolarized light scatter

• Automated analyzer not available
• Verifying automated count when eosinophilia suspected on smear
• Counting after automated flags

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Q9. Laboratory Diagnosis of Sickling

Sickling Test (Sodium Metabisulfite Test)

Principle

Reagents

• Fresh sodium metabisulfite solution (2%): 200 mg Na₂S₂O₅ dissolved in 10 mL distilled water; must be freshly prepared for each test session (deteriorates within 4 hours)

Procedure

1. Place 1 drop of 2% fresh sodium metabisulfite on a glass slide
2. Add 1 drop of blood; mix well
3. Cover with a sealed coverslip (seal all edges with nail varnish/petroleum jelly to maintain anoxia)
4. Incubate at room temperature for 30 minutes to 1 hour (some protocols: examine at 1 hour and again at 24 hours)
5. Examine under low power (10×) and high power (40×)

Interpretation

• Positive: Presence of sickle-shaped (crescent), oat-shaped, or holly-leaf shaped cells = HbS present
• Negative: All cells remain disc-shaped
• Rate: Most sickle cells by 1 hour; sensitivity improves with longer incubation

Controls

• Positive control: Known sickle cell blood (HbSS)
• Negative control: Normal blood (HbAA)
• Positive and negative controls must be run with every batch

Solubility Test (Sickledex / Itano Solubility Test)

Principle

Procedure

1. Add 2 mL sodium dithionite (Na₂S₂O₄) reducing solution (high phosphate buffer + saponin for hemolysis)
2. Add 20 µL fresh blood
3. Mix and allow to react for 5 minutes at room temperature
4. Examine turbidity: Hold tube in front of a ruled card/paper

Interpretation

• Positive (turbid): Cannot read the lines through the tube = HbS present (sickle cell trait OR disease)
• Negative (clear): Lines clearly visible = HbS absent

Advantages and Limitations

• Advantages: Simple, rapid, no microscopy needed, good screening test
• Limitations:
  • Does not distinguish HbSS from HbAS (trait vs. disease)
  • False positives: Very high WBC count (>50 × 10⁹/L), very high Hb (polycythemia), lipemia, cord blood (HbF)
  • False negatives: Anemia (Hb <7 g/dL - insufficient HbS to cause turbidity), infants <6 months (high HbF), recent transfusion (dilution with HbA)
  • Does not detect other sickling hemoglobins (HbC, HbD, HbO-Arab) unless combined with HbS

Comparison of Tests

Confirmatory Tests for Sickle Hemoglobin

1. Hemoglobin Electrophoresis (Gold Standard)

• HbA: Most anodal
• HbS: Migrates less than HbA (slower)
• HbC, HbE, HbO-Arab: Co-migrate with each other at alkaline pH
• HbF: Migrates between HbA and HbS

• Separates hemoglobins that co-migrate at alkaline pH
• HbS ≠ HbD, HbG (D and G migrate like A at acid pH)
• Complementary to alkaline electrophoresis
• HbS gives a characteristic pattern UNIQUE to acid electrophoresis

2. High-Performance Liquid Chromatography (HPLC) - Bio-Rad Variant II

• Currently the gold standard in many labs
• Separates Hb fractions by ion-exchange chromatography
• Reports retention time and % for each fraction
• Identifies HbA, HbA2, HbF, HbS, HbC, HbE, HbD and rare variants
• HbS: Retention time ~4.4 min (on Variant II program); HbA2 measured accurately
• Also used for neonatal screening

3. Isoelectric Focusing (IEF)

• Separates Hb by isoelectric point
• High resolution; used in neonatal screening programs

4. DNA Analysis

• PCR-based mutation detection for definitive diagnosis, especially in neonatal/prenatal diagnosis
• Detects specific GAG→GTG mutation at codon 6 of β-globin gene

5. Peripheral Blood Smear

• HbSS: Sickle cells, target cells, polychromasia, nucleated RBCs
• HbAS: Occasional target cells; sickle cells rare on routine smear

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Q10. Myeloperoxidase (MPO) Cytochemistry

Principle

H₂O₂ + Chromogen → Colored precipitate (MPO-catalyzed)

Reagents (Kaplow Method - Most Common)

1. Buffered formalin acetone (fixative): 100 mL citrate buffer (pH 6.6) + 10 mL formaldehyde + 90 mL acetone; fix fresh air-dried smears for 30 seconds at 4°C (cold fixation preserves MPO)
2. Incubation medium:
   • Benzidine dihydrochloride (1%) - chromogen (but carcinogenic; largely replaced)
   • Alternative chromogen: 3,3'-diaminobenzidine (DAB) or naphthol ASD chloroacetate
   • Hydrogen peroxide 0.3%
   • Giemsa stain (counterstain)
3. Modified method using 3-amino-9-ethylcarbazole (AEC) as chromogen (produces red precipitate)

Sudan Black B (SBB) - Related Stain

• Not an enzyme stain but stains phospholipids in myeloid granules (similar distribution to MPO)
• More sensitive than MPO for myeloid differentiation; detects cells where MPO may be denatured
• Produces black precipitate in myeloid cells

Procedure (Kaplow MPO)

1. Prepare fresh blood/bone marrow smears; air dry (do NOT heat fix)
2. Fix in buffered formalin-acetone at 4°C for 30 seconds
3. Rinse with distilled water; air dry
4. Immerse in incubation medium (benzidine/DAB + H₂O₂ + buffer) for 30 seconds to 1 minute
5. Rinse with distilled water
6. Counterstain with Giemsa for 1-2 minutes
7. Rinse, air dry, mount with DPX, examine under oil immersion

Interpretation

• 0: No granules
• 1+: Few scattered granules
• 2+: Moderate granules
• 3+: Heavy granule staining
• 4+: Dense, confluent granules

Clinical Applications

1. AML vs. ALL: Most important use; MPO+ = AML (myeloid lineage); MPO- = ALL or non-myeloid
2. AML subtyping:
   • M1 (minimally differentiated myeloid): MPO+ in ≥3% blasts
   • M2 (AML with maturation): MPO strongly positive
   • M3 (APL): MPO extremely strongly positive (hypergranular promyelocytes)
   • M4 (myelomonocytic): MPO+ (neutrophilic component); NSE+ (monocytic component)
   • M5 (monocytic): MPO weak/negative; NSE+++
3. Blast crisis of CML: MPO helps confirm myeloid blast crisis vs. lymphoid
4. Myeloid sarcoma: Confirms myeloid nature of extramedullary mass
5. Bone marrow failure states: Identifies residual myeloid cells

Limitations

• False negative MPO:
  • Prolonged storage of slides (MPO degrades)
  • Hot fixation (heat destroys MPO)
  • Wrong fixative
  • Some M5 (monocytic) AML = MPO negative (use NSE instead)
  • M7 (megakaryoblastic) and ALL = both MPO negative
• False positive:
  • Eosinophil peroxidase (not MPO) - eosinophils always stain
  • Benzidine is carcinogenic (lab hazard); modern labs use DAB or other safer chromogens
• Cannot distinguish M5 from ALL (both MPO negative) - need NSE, CD3, CD20 by IHC/flow cytometry

MPO Detection by Flow Cytometry

Principle

Advantages over Cytochemical MPO

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Q11. Platelet Counting

Manual Platelet Count

Methods

• Count platelets per 100 RBCs in the monolayer zone; multiply by RBC count/µL / 100
• OR: Average number of platelets per oil immersion field × 15,000 = platelet count/µL
• Rough estimate only; not for clinical decisions

• Diluting fluid: Rees-Ecker fluid = sodium citrate 3.8 g + brilliant cresyl blue 0.1 g + formalin 40% (0.2 mL) + distilled water to 100 mL
• Formal citrate acts as anticoagulant + platelet fixative
• BCB stains platelets pale blue

• Diluting fluid: 1% ammonium oxalate solution
• Lyses RBCs selectively (osmotic); WBCs lyse slowly; platelets preserved
• Cleaner preparation than Rees-Ecker

1. Mix blood; add 0.02 mL to 0.38 mL diluting fluid (1:20 dilution)
2. Charge the hemocytometer with a clean cover glass
3. Place in a humid chamber for 20-30 minutes (allows platelets to settle)
4. Count under phase contrast microscopy (40×): Platelets appear as small, bright, refractive discs
5. Count platelets in central large square (1 mm²) = 25 small squares in both chambers = 50 small squares

• Volume of 25 small squares = 25 × (0.2 mm × 0.2 mm × 0.1 mm) = 0.1 mm³
• Platelets/µL = (Platelets in 25 squares × 20) / 0.1 = Platelets in 25 squares × 200
• If counting 50 squares (both chambers): Platelets/µL = Total × 100

Normal Range

• Normal platelet count: 150-400 × 10⁹/L (150,000-400,000/µL)
• Some sources: 150-450 × 10⁹/L

Automated Platelet Count

Impedance Method

• Platelets (<30 fL) counted separately from RBCs (>36 fL) by cell volume threshold
• Problem: Microcytes, RBC fragments, large platelets may be misclassified; giant platelets (>30 fL) may not be counted

Optical/Light Scatter Method

• Platelet channel uses low-angle forward scatter to count and size platelets
• Better discrimination of platelets from fragments/debris

Fluorescent Platelet Count (Sysmex XN PLT-F channel)

• CD61 fluorescent antibody-based counting (or CD41 equivalent fluorescent dye)
• Gold standard for accuracy in thrombocytopenic patients
• Accurately counts even at very low levels (<10 × 10⁹/L)
• Unaffected by fragments, microcytes, or large platelets

Platelet Parameters Generated by Automated Analyzers

Causes of Thrombocytopenia (<150 × 10⁹/L)

Decreased Production

• Aplastic anemia
• Bone marrow infiltration (leukemia, myeloma, lymphoma, metastatic carcinoma)
• Megaloblastic anemia (B12/folate deficiency)
• Myelodysplastic syndrome
• Viral infections (parvovirus B19, CMV, EBV, Hepatitis B/C, HIV)
• Drugs (chemotherapy, thiazides, alcohol, valproate)
• Radiation

Increased Destruction

• Immune:
  • ITP (Immune Thrombocytopenic Purpura) - most common cause of isolated thrombocytopenia in outpatients
  • Drug-induced immune thrombocytopenia (heparin-induced = HIT, quinine, quinidine, sulfonamides)
  • Post-transfusion purpura
  • Neonatal alloimmune thrombocytopenia
  • SLE, APS
• Non-immune (consumption):
  • DIC
  • TTP (Thrombotic Thrombocytopenic Purpura)
  • HUS (Hemolytic Uremic Syndrome)
  • HELLP syndrome (pregnancy)
  • Giant hemangioma (Kasabach-Merritt)

Sequestration / Distribution

• Hypersplenism (splenomegaly from any cause - sequesters up to 90% of platelets)

Dilutional

• Massive transfusion (stored blood has no viable platelets)

Causes of Thrombocytosis (>400 × 10⁹/L)

Pseudothrombocytopenia

1. EDTA-induced platelet clumping (most common): EDTA activates cryptic platelet antibodies (IgG/IgM) that cause platelet aggregation at room temperature → clumps counted as single events or not counted → falsely low count
   • Solution: Repeat count on citrate tube or heparin tube, or warm the blood to 37°C before running
2. Platelet satellitism: Platelets adhere around neutrophils → platelets counted with neutrophil = falsely low platelet count + falsely high neutrophil count
3. Giant platelets: (Bernard-Soulier syndrome, MYH9 disorders, ET) - exceed the upper size threshold and are excluded from platelet count
4. Fragmented RBCs/microcytes - counted as platelets → pseudothrombocytosis

• Examine peripheral smear: platelet clumps visible
• Repeat in citrate/heparin (no EDTA)
• Platelet count returns to normal in alternative anticoagulant

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Q12. Complete Routine Urine Examination

Urine Collection

• Specimen: Midstream clean-catch urine (MSCCU); 10-30 mL
• Container: Wide-mouthed, sterile, dry, clean, labeled container
• Timing: First morning specimen preferred (concentrated; casts/cells better preserved)
• Storage: Examine within 2 hours; if delayed, refrigerate at 4°C (up to 4-6 hours); chemical preservatives (boric acid, formalin) for selected tests
• Avoid contamination from vaginal secretions, periurethral flora

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A. Physical Examination

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B. Chemical Examination (Reagent Strip Tests / Dipstick)

1. Protein (Albustix)

• Normal: Nil to trace (<30 mg/dL or <150 mg/24h)
• Dipstick detects albumin preferentially; does not detect Bence Jones protein (BJP), globulins, or tubular proteins well
• Sensitivity: ~15-30 mg/dL albumin
• False positives: Alkaline/concentrated urine, contamination with chlorhexidine, prolonged immersion, highly buffered urine
• False negatives: Dilute urine, acidic urine, globulinuria (BJP)
• Positive protein (≥1+): Nephropathy, nephrotic syndrome, UTI, cardiac failure; requires 24-hour protein quantification and further workup

2. Glucose (Clinitest / Glucostix)

1. Glucose oxidase oxidizes glucose → gluconic acid + H₂O₂
2. Peroxidase uses H₂O₂ to oxidize a chromogen (tetramethylbenzidine/KI) → colored product

• Normal: Negative (glucose <180 mg/dL in serum = below renal threshold)
• Glycosuria: Blood glucose >180 mg/dL (renal threshold) in diabetes mellitus; also renal glycosuria (normal blood glucose but low tubular threshold - Fanconi syndrome)
• False positives: Chlorine/hypochlorite contamination
• False negatives: Ascorbic acid (vitamin C) competitively inhibits peroxidase → most important cause; highly specific for glucose (non-glucose reducing substances like galactose, fructose, lactose, pentoses are NOT detected by enzymatic strip)
• Reducing substances test (Clinitest tablets - copper reduction): Detects ALL reducing substances including non-glucose sugars; useful in children for galactosemia, fructosuria

3. Ketones

• Detects acetoacetate >5 mg/dL; also acetone (less sensitive); does NOT detect beta-hydroxybutyrate (predominant ketone in DKA)
• Positive in: Diabetic ketoacidosis (DKA), starvation ketosis, low-carbohydrate diets, vomiting, fever in children
• False positive: Mesna, captopril, levodopa

4. Blood / Hemoglobin

• Hematuria: Intact RBCs → spot pattern (speckling on pad)
• Hemoglobinuria: Free hemoglobin (hemolysis) → uniform color change
• Myoglobinuria: Myoglobin also has pseudoperoxidase activity; positive result
• False positives: Povidone-iodine (Betadine) contamination, bacterial peroxidases (UTI), menstrual contamination
• False negatives: Ascorbic acid (most important), nitrite, high SG

5. Bilirubin

• Only conjugated bilirubin is filtered by glomerulus (water-soluble); direct bilirubin in urine = bilirubinuria
• Positive in: Hepatocellular jaundice, obstructive (cholestatic) jaundice
• Negative in: Hemolytic jaundice (unconjugated bilirubin not filtered)
• False positives: Chlorpromazine, rifampicin (orange discoloration)
• False negatives: Prolonged exposure to light (bilirubin photo-oxidized), ascorbic acid, nitrite

6. Urobilinogen

• Normal: 0.1-1.0 Ehrlich units/dL (trace positive on dipstick is normal)
• Increased (>1.0): Hemolytic anemia (increased RBC breakdown → more urobilinogen), hepatocellular disease (impaired conjugation → spillover to urine)
• Absent/decreased: Complete biliary obstruction (no bile reaching gut → no urobilinogen formed), antibiotic therapy (kills gut flora that convert bilirubin to urobilinogen)
• False positives: Porphobilinogen (reacts with Ehrlich's reagent), sulfonamides

7. Nitrites

• Positive: Significant bacteriuria (>10⁵ organisms/mL) with gram-negative bacteria (E. coli, Klebsiella, Proteus, Enterobacter)
• Negative/false negative: Gram-positive organisms (Staphylococcus, Enterococcus) lack nitrate reductase; dilute urine; ascorbic acid; urine not incubated in bladder for >4 hours

8. Leukocyte Esterase

• Positive in: UTI, interstitial nephritis, sterile pyuria (TB, SLE, Chlamydia infection), contamination
• Normal: Negative (≤5 WBCs/hpf)
• False negative: Glycosuria (high glucose slows reaction), proteinuria (>3 g/L), high SG, tetracycline, cephalexin, gentamicin
• Combined positive nitrite + LE = high sensitivity and specificity for UTI

9. Specific Gravity (Density Pad)

10. pH

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C. Microscopic Examination (Urine Sediment)

Preparation

1. Mix urine well; pour 10 mL into conical centrifuge tube
2. Centrifuge at 1500 rpm for 5 minutes (400-500 × g; high speed destroys casts)
3. Decant supernatant; leave ~0.5-1 mL deposit
4. Resuspend deposit gently; place a drop on glass slide; cover with coverslip
5. Examine: Low power (10×) for overview and casts; High power (40×) for cells, bacteria

Normal Findings

• RBCs: 0-3/hpf
• WBCs: 0-5/hpf
• Epithelial cells: Occasional
• Casts: 0-2 hyaline casts/lpf
• Bacteria: Nil (fresh, MSCCU)
• Crystals: Variable (clinically relevant only if pathological type)

Cells

Casts

Crystals

Organisms

• Bacteria (rod or cocci): UTI (require culture + sensitivity)
• Yeast (Candida): UTI in diabetics, immunocompromised, indwelling catheter
• Trichomonas vaginalis: Motile flagellated protozoa; contamination or vaginal trichomoniasis

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D. Automated Urine Analyzers

1. Reflectance Photometry (Strip Readers)

• Urine Dipstick Reader: Clinitek (Siemens), Urinalyzer (Arkray), Aution Max
• Standardized reflectance reading of dipstick pads; eliminates reader subjectivity
• Reports: LE, nitrite, protein, glucose, pH, blood, bilirubin, urobilinogen, SG, ketones, creatinine
• IQ200/IRIS system: Combines reflectance + digital microscopy

2. Automated Urine Microscopy Analyzers

• Uses fluorescent dyes (to label DNA, RNA, cell membranes) + laser light scatter
• 5 populations identified: RBCs, WBCs, epithelial cells, bacteria, casts, yeast
• Results in <90 seconds per sample
• Flags samples for manual review if abnormal populations detected

• Bright-field and/or phase contrast digital imaging
• Automated particle classification by machine learning
• Reports: RBCs (dysmorphic vs. isomorphic), WBCs, epithelial cells, casts (typed), bacteria, crystals, yeast
• High-resolution images stored for review

3. Automated Urine Chemistry Analyzers

• Aution Max AX-4280 (Arkray)
• Clinitek Atlas (Siemens)
• Rapid results; reduces manual reading errors; throughput: 200-500 samples/hour

Advantages of Automated Urine Analysis

• Eliminates observer bias
• Standardized reporting
• High throughput
• Digital image archive
• Reduces manual microscopy workload (only abnormal flagged samples reviewed manually)
• Better detection of dysmorphic RBCs for glomerulonephritis screening

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Summary - Clinical Significance Patterns

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