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Unit 2 of 3 · B.Sc MLS Sem 3

Unit 2: Sample collection and core haematology tests

Basic Haematology & Haematological Techniques-I notes · PTU syllabus (BMLS301-18)

3 min read10 topics10 exam questions
On this page
  1. Unit summary
  2. Collection and preservation of blood
  3. Haemoglobin estimation
  4. Total leucocyte count
  5. Differential leucocyte count
  6. Erythrocyte sedimentation rate (ESR)
  7. Packed cell volume (haematocrit)
  8. Red cell indices
  9. Absolute eosinophil count
  10. Reticulocyte count
  11. Platelet count
  12. Key terms
  13. Quick revision
  14. Important questions

Unit summary

Reliable results begin with a good sample and correctly performed tests. This unit covers collection and preservation of blood, and the principle, procedure, normal values, clinical significance and sources of error of haemoglobin estimation, total and differential leucocyte counts, ESR, packed cell volume, red cell indices, absolute eosinophil count, reticulocyte count and platelet count.

After this unit you can

  • Collect and preserve blood with the right anticoagulant
  • Estimate haemoglobin and count leucocytes
  • Perform ESR, PCV and calculate red cell indices
  • Perform eosinophil, reticulocyte and platelet counts

PTU syllabus topics

  • Collection and preservation of blood samples
  • principle
  • procedure
  • normal values
  • clinical significance and sources of error for haemoglobinometry
  • total leucocyte count (TLC)
  • differential leucocyte count (DLC)
  • erythrocyte sedimentation rate (ESR)
  • packed cell volume/haematocrit
  • red cell indices
  • absolute eosinophil count
  • reticulocyte count
  • platelet count
ComparisonCore haematology tests
Normal range (adult)
Significance

Haemoglobin

Men 13–17, women 12–15 g/dL

Low in anaemia

TLC

4,000–11,000/µL

High in infection

ESR (Westergren)

Men 0–15, women 0–20 mm/hr

Raised in inflammation

Platelet count

1.5–4.5 lakh/µL

Low in dengue, ITP

PCV

Men 40–50%, women 36–46%

Low in anaemia

1

Topic 1

Collection and preservation of blood

ComparisonAnticoagulants
Action
Use

EDTA (lavender top)

Chelates calcium

Complete blood count, blood films

Trisodium citrate 3.2% (blue top)

Binds calcium; reversible

Coagulation (9 : 1 blood to citrate); ESR (4 : 1)

Heparin (green top)

Activates antithrombin

Osmotic fragility, some chemistry

Double oxalate

Precipitates calcium

Older haematology use

Fluoride–oxalate (grey top)

Stops glycolysis

Blood glucose

Key termsCollection methods
Capillary (skin puncture)
Fingertip or heel (infants); wipe the first drop
Venous (venepuncture)
Median cubital vein; tourniquet under 1 minute; correct order of draw
Arterial
Blood gases (radial artery)
  • Preservation: CBC within 6 hours at room temperature (up to 24 hours at 4 °C); films made within 2–3 hours of EDTA collection to avoid artefacts. Avoid haemolysis and clotting.
2

Topic 2

Haemoglobin estimation

ComparisonHb methods
Principle
Note

Sahli's (acid haematin)

Blood + 0.1 N HCl → brown acid haematin, diluted until it matches a standard

Simple; error up to ±10%

Cyanmethaemoglobin (reference method)

Drabkin's reagent converts all Hb (except sulphHb) to stable cyanmethaemoglobin; read at 540 nm

Accurate; standardised

Oxyhaemoglobin

Blood in dilute ammonia; read at 540 nm

Quick

Example

Cyanmethaemoglobin: 20 µL blood + 5 mL Drabkin's (1 : 251). Hb (g/dL) = (Test OD ÷ Standard OD) × standard concentration (mg/dL) × 251 ÷ 1,000.

  • Significance: low in anaemia; high in polycythaemia and dehydration. Errors: lipaemia, high WBC counts, poor mixing.
3

Topic 3

Total leucocyte count

  • Principle: blood diluted 1 : 20 in Türk's fluid (2% acetic acid lyses red cells; gentian violet stains nuclei); cells counted in the four large corner squares of the Neubauer chamber.
Key formulasTLC calculation

  • Formula

    TLC/µL = cells counted × dilution ÷ (area × depth)

  • Four corner squares

    Area 4 mm², depth 0.1 mm → cells × 20 ÷ 0.4 = cells × 50

Example

140 cells counted → 140 × 50 = 7,000/µL.

  • Leucocytosis (more than 11,000): bacterial infection, leukaemia. Leucopenia (below 4,000): viral infections, typhoid, marrow failure. Correct for nucleated red cells.
4

Topic 4

Differential leucocyte count

  • Procedure: stain a thin film (Leishman or Giemsa), scan the body–tail junction under oil, count 100 white cells using a battlement track and express as percentages. Absolute count = % × TLC.
  • Significance: neutrophilia (bacterial infection), lymphocytosis (viral infection, TB), eosinophilia (allergy, worms), monocytosis (TB, malaria).
5

Topic 5

Erythrocyte sedimentation rate (ESR)

ComparisonESR methods
Westergren (reference)
Wintrobe

Anticoagulant

3.8% citrate, 4 : 1

EDTA or double oxalate (undiluted)

Tube

200 mm long, 2.5 mm bore

100 mm long

Normal (1 hour)

Men 0–15 mm; women 0–20 mm

Men 0–9 mm; women 0–20 mm

  • Principle: red cells form rouleaux and settle; raised fibrinogen and globulins increase rouleaux. High ESR: infections, TB, rheumatoid arthritis, multiple myeloma, pregnancy, anaemia. Errors: tilted tube, temperature, delay, bubbles.
6

Topic 6

Packed cell volume (haematocrit)

  • Principle: centrifuging blood separates red cells from plasma; PCV = height of packed red cells ÷ total height × 100. Microhaematocrit: capillary tube spun at 12,000 rpm for 5 minutes; macro (Wintrobe): 3,000 rpm for 30 minutes. The buffy coat holds white cells and platelets.
  • Significance: low in anaemia, high in polycythaemia and dehydration (burns).
7

Topic 7

Red cell indices

Key formulasRed cell indices

  • MCV (fL)

    PCV (%) × 10 ÷ RBC (millions/µL); normal 80–100

  • MCH (pg)

    Hb (g/dL) × 10 ÷ RBC (millions/µL); normal 27–32

  • MCHC (g/dL)

    Hb (g/dL) × 100 ÷ PCV (%); normal 32–36

  • RDW

    Variation in red cell size; normal 11.5–14.5%

Example

Hb 9 g/dL, PCV 30%, RBC 4.0 million: MCV = 75 fL, MCH = 22.5 pg, MCHC = 30 g/dL — microcytic hypochromic anaemia (iron deficiency).

  • Classification of anaemia: microcytic (iron deficiency, thalassaemia), normocytic (blood loss, chronic disease), macrocytic (B12 or folate deficiency).
8

Topic 8

Absolute eosinophil count

  • Diluting fluid: Dunger's or Hinkelman's fluid (eosin stains granules; acetone and water lyse other cells); count in the Fuchs–Rosenthal or Neubauer chamber. Normal: 40–440/µL. Raised: allergy, asthma, parasitic infections, Hodgkin lymphoma; low: steroid therapy, Cushing's syndrome.
9

Topic 9

Reticulocyte count

  • Principle: supravital stain (new methylene blue or brilliant cresyl blue) precipitates residual RNA as a blue reticulum. Mix equal volumes of blood and stain, incubate 15 minutes, make films, count reticulocytes per 1,000 red cells.
  • Normal: 0.5–2.5% in adults (2–6% in newborns). High: haemolysis, after bleeding, response to iron or B12 treatment; low: aplastic anaemia, marrow failure.
Key formulasCorrected reticulocyte count

  • Formula

    Retic % × patient PCV ÷ normal PCV (45)

10

Topic 10

Platelet count

  • Manual: 1 : 20 dilution in 1% ammonium oxalate; count in the central large square of the Neubauer chamber after the cells settle (10–15 minutes in a moist chamber); phase contrast improves accuracy. Normal: 1.5–4.1 lakh/µL.
  • Thrombocytopenia: dengue, ITP, leukaemia, aplastic anaemia, DIC; thrombocytosis: after splenectomy, inflammation, myeloproliferative disease. Errors: platelet clumps (EDTA-induced), poor mixing, giant platelets.

Key terms

Anticoagulant
Substance preventing blood clotting
Türk's fluid
Diluting fluid for leucocyte counts
Rouleaux
Stacks of red cells increasing ESR
MCV
Average red cell volume
Supravital stain
Stain applied to living cells, used for reticulocytes

Quick revision

  • EDTA, citrate, heparin, oxalate, fluoride; collection and storage.
  • Sahli's and cyanmethaemoglobin; TLC × 50; DLC on 100 cells.
  • ESR Westergren and Wintrobe; PCV micro and macro.
  • MCV, MCH, MCHC; morphological classification of anaemia.
  • Absolute eosinophil count; reticulocyte count; platelet count; sources of error.

Important exam questions

Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).

Short-answer questions

  1. Q1.Which anticoagulant is used for a CBC?
  2. Q2.What is the principle of the cyanmethaemoglobin method?
  3. Q3.What is the composition of Türk's fluid?
  4. Q4.Name two causes of a high ESR.
  5. Q5.Calculate MCHC for Hb 12 g/dL and PCV 40%.
  6. Q6.Which stain is used for reticulocytes?

Long-answer questions

  1. Q1.Describe anticoagulants and the collection of blood samples.
  2. Q2.Describe methods of haemoglobin estimation.
  3. Q3.Describe the total and differential leucocyte counts.
  4. Q4.Describe ESR, PCV and red cell indices.

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