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PA13.2-4 | Performing Common Haematological Tests: Hb, RBC/WBC, DLC — Part 1

Hemoglobin Estimation

Why Hb matters: Hemoglobin concentration is the single most clinically relevant CBC parameter. Two methods are in widespread use in Indian labs.

Hemoglobin Estimation Methods: Cyanmethemoglobin vs Sahli's Acidhaematin

Method 1 — Cyanmethemoglobin (Gold Standard)

Principle: Blood is diluted in Drabkin's reagent (potassium ferricyanide + potassium cyanide + sodium bicarbonate). Ferricyanide oxidises haemoglobin → methaemoglobin; cyanide converts it → cyanmethemoglobin, a stable compound that absorbs maximally at 540 nm.

Step-by-step procedure:
1. Pipette 5 mL of Drabkin's reagent into a cuvette.
2. Add 20 µL of well-mixed EDTA blood (dilution = 1:251, effectively 1:250).
3. Mix gently; wait 3 minutes at room temperature for complete conversion.
4. Read absorbance at 540 nm on the spectrophotometer against a Drabkin's blank.
5. Read Hb from the calibration curve (or apply: Hb g/dL = absorbance × factor from standard curve).

Calibration: Use certified cyanmethemoglobin standard (e.g., 18 g/dL standard diluted to a 5-point curve). Run calibration at the start of each batch and whenever a new reagent lot is opened.

Advantages: Measures all haemoglobin forms except sulphemoglobin. Internationally standardised (ICSH reference method). Coefficient of variation (CV) < 1% on a well-maintained photometer.

Limitation: Requires spectrophotometer; reagent contains cyanide (toxic waste disposal rules apply).

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Method 2 — Sahli's (Acidhaematin) Method

Principle: Blood is mixed with 0.1N HCl → haemoglobin converted to brown acidhaematin → colour compared visually to a brown glass comparator.

Quick steps: 20 µL blood into tube with HCl, wait 5 min, dilute with distilled water until colour matches comparator, read from calibration tube markings (g/dL).

Use case: Field settings, resource-limited labs, no electricity.

Inaccuracy caveats (critical for exams): Only converts oxyHb partially; foetal Hb (HbF) is more resistant to acid — falsely low in neonates. Subjective colour matching varies between observers (CV 5–10%). Fails in jaundiced or lipaemic samples. Never use Sahli's for precise clinical decisions when a spectrophotometer is available.

Parameter	Cyanmethemoglobin	Sahli's
CV	< 1%	5–10%
HbF accurate?	Yes	No
Equipment	Spectrophotometer	Comparator tube
Toxic waste	Yes (cyanide)	No
WHO standard	Yes	No

RBC Count — Manual and Automated

Neubauer Hemocytometer Structure and RBC Counting Technique

The Improved Neubauer Hemocytometer

The chamber has a central platform 0.1 mm below the cover slip. Each of the 9 large squares has an area of 1 mm². The central large square is subdivided into 25 medium squares, each into 16 small squares.

Volume per small square = 1/400 mm² × 0.1 mm = 1/4000 µL

Manual RBC Count

Reagent: Hayem's fluid (mercuric chloride + sodium sulphate + sodium chloride). Isotonic, fixes cells, prevents clumping.

Dilution: 1:200 (20 µL blood + 3.98 mL Hayem's, or use RBC Thoma pipette to the 0.5 mark, Hayem's to the 101 mark).

Counting area: Count RBCs in 5 small squares of the central RBC area (one central + four diagonal corner small squares of the central medium square). These 5 squares have a combined area of 5/400 mm² = 1/80 mm².

Formula:
$$\text{RBC count} = N \times \frac{200}{1} \times \frac{1}{(5/400 \times 0.1)} \div 1000 = N \times 10{,}000$$

Simplified: RBC/µL = cells counted × 10,000

Example: Count 200 cells in 5 small squares → RBC = 200 × 10,000 = 2,000,000/µL (2.0 × 10⁶/µL)

Normal ranges:
- Men: 4.5–5.9 × 10⁶/µL
- Women: 3.8–5.2 × 10⁶/µL

Automated RBC Count

Modern analyzers use two principles in tandem:
- Electrical impedance (Coulter principle): Cells suspended in electrolyte solution pass through an aperture; each cell causes a brief resistance change proportional to its volume — counts and sizes simultaneously.
- Light scatter (laser): Forward scatter ≈ size; side scatter ≈ internal complexity (granularity). Differentiates cell types beyond what impedance alone achieves.

Why you still need to know manual: Analyzer flags (e.g., 'RBC fragments', 'schistocytes') must be verified on the smear. If the sample is haemolysed, the automated Hb is unreliable and the RBC count may be spuriously low.

WBC Count — Total and Differential

Total WBC Count (Manual)

Reagent: Turk's fluid (glacial acetic acid + crystal violet/gentian violet + distilled water). Acetic acid lyses RBCs; crystal violet stains WBC nuclei pale blue so they are visible.

Dilution: 1:20 (50 µL blood + 950 µL Turk's, or WBC Thoma pipette to 0.5, Turk's to the 11 mark).

Counting area: Count all nucleated cells in the 4 corner large squares of the hemocytometer (each 1 mm²; total area = 4 mm²; volume = 4 × 0.1 = 0.4 µL after dilution correction).

Formula:
$$\text{WBC/µL} = N \times \frac{20}{4 \times 0.1} = N \times 50$$

Example: Count 160 cells in 4 large squares → WBC = 160 × 50 = 8,000/µL

Normal WBC range: 4,000–11,000/µL (WHO; paediatric ranges differ — neonates can be 9,000–30,000/µL normally).

WBC Count Methodology: Manual Counting and Blood Smear Preparation

Sources of Falsely Abnormal WBC Counts

Error	Effect on WBC
Clotted/partially clotted sample	Falsely low (cells trapped in fibrin)
Nucleated RBCs (NRBCs) counted as WBCs	Falsely high — automated analyzer cannot distinguish NRBCs without specific channel; manual: do not count NRBCs in WBC square
Platelet clumps (large aggregates)	Flagged as WBCs in some channels — falsely high
Cryoglobulin precipitate at RT	Falsely high WBC (protein aggregates look like cells) — warm sample to 37°C

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Differential Leukocyte Count (DLC)

Why DLC is done on the smear, not the chamber: The morphology of each cell — nucleus shape, cytoplasm granularity, toxic granulation — is invisible in the hemocytometer. A Romanowsky-stained smear is mandatory.

Smear preparation — Wedge method:
1. Place a small drop of blood (~3 µL) on a clean, grease-free slide near one end.
2. Hold a second slide (spreader) at 30–45° to the first slide, touch the drop, and push steadily forward in one smooth motion.
3. Ideal smear: thumb-sized, fish-tail tail, thins to a feather edge. Bone-dry before staining.

Staining — Leishman stain (Romanowsky-type):
1. Flood air-dried smear with Leishman stain; wait 2 min.
2. Add twice the volume of phosphate buffer (pH 6.8); mix by tilting; wait 12–15 min.
3. Wash gently with buffer, drain, air-dry. Do NOT blot — blotting distorts cells.
4. Result: RBCs pink-salmon; WBC nuclei blue-purple; granules specific colours (neutrophil = pink-purple, eosinophil = bright red-orange, basophil = dark blue-black).

Systematic scanning — the 'battlements' technique:

Scan in the monolayer body (mid-smear, where cells just touch but do not overlap). Use 40× objective. Move in a regular grid pattern (up-across-down, like battlements). Count at least 100 consecutive WBCs, classifying each.

Normal DLC:

Cell	Normal %	Absolute count (normal WBC 5,000–10,000)
Neutrophils (segmented)	50–70%	2,500–7,000/µL
Lymphocytes	20–40%	1,000–4,000/µL
Monocytes	2–8%	200–800/µL
Eosinophils	1–6%	40–600/µL
Basophils	0–1%	0–100/µL

Absolute counts are more clinically useful than percentages. A lymphocyte percentage of 45% means nothing if the WBC is 3,000 (absolute = 1,350, which is borderline low — not high).

The left shift rule: Bands (immature neutrophils) > 10% = left shift → bacterial infection, sepsis, tissue damage. A 'leukaemoid reaction' is WBC > 50,000/µL with marked left shift, mimicking leukaemia but reactive in cause (e.g., severe sepsis, tuberculosis).