Haematology

In short

Haematology is the branch of laboratory medicine that studies blood cells, the blood-forming (haemopoietic) organs and haemostasis — counting and describing red cells, white cells and platelets, measuring haemoglobin, and testing clotting. It matters because the complete blood count is the single most ordered test in any Indian hospital and because the morphology on a stained smear still decides diagnoses that no analyser can make. It is examined heavily in B.Sc MLT university papers (Haematology I and II), in DMLT practicals, and in recruitment tests for Dr Lal PathLabs, SRL, Metropolis, Thyrocare, Apollo, AIIMS/PGI technologist posts and NABL-accredited hospital labs. Practice in India is governed by NABL 112 / ISO 15189 for medical laboratories, the Clinical Establishments Act, 2010 in adopting states, and the Bio-Medical Waste Management Rules, 2016 (as amended) for sharps and blood disposal.

Explained

Blood, haemopoiesis and specimen handling

• Blood composition: plasma ~55%, formed elements ~45%. Normal adult volume about 5–5.5 L (roughly 70–80 mL/kg).
• Haemopoiesis: pluripotent stem cell → myeloid lineage (RBC, granulocytes, monocytes, platelets) and lymphoid lineage (T, B, NK cells). Sites: yolk sac (first weeks) → liver and spleen (mid-fetal) → bone marrow from about the fifth month of fetal life, and the only site after birth; in adults confined to vertebrae, sternum, ribs, pelvis, skull and proximal femur.
• Erythropoiesis sequence: proerythroblast → basophilic → polychromatic → orthochromatic normoblast → reticulocyte → mature RBC; takes about 5–7 days, driven by erythropoietin from the renal peritubular cells in response to hypoxia.
• RBC: biconcave disc, 7–8 µm, no nucleus, lifespan ~120 days, destroyed in spleen (reticuloendothelial system). Platelet: 2–4 µm fragment of megakaryocyte cytoplasm, lifespan 7–10 days. Neutrophil: circulates 6–10 hours.
• Anticoagulants (learn the colour code — commonly asked):

• Why EDTA: chelates calcium, preserves cell morphology and volume best; excess EDTA shrinks cells and falsely lowers PCV/MCV. Why citrate for coagulation: reversible calcium binding, so calcium can be added back in the test.
• Sample rules: fill to the mark, mix 8–10 gentle inversions, never shake; run CBC within 4–6 hours at room temperature (up to 24 h at 2–8 °C, but morphology degrades); make the smear within 1–2 hours. Reject clotted, haemolysed and under-filled samples.

Haemoglobin, PCV and the red cell indices

• Haemoglobin structure: 4 globin chains + 4 haem (Fe²⁺ + protoporphyrin IX). HbA (α₂β₂) ~97% in adults, HbA₂ (α₂δ₂) 2–3.5%, HbF (α₂γ₂) <1%.
• Haemoglobin estimation methods:
  • Cyanmethaemoglobin (HiCN) — the reference method; Drabkin's reagent (potassium ferricyanide + potassium cyanide + bicarbonate/phosphate) converts all Hb forms to cyanmethaemoglobin, read at 540 nm. Accurate, includes all derivatives; drawback — cyanide is toxic and sulphaemoglobin is not measured.
  • Sahli / acid haematin — visual comparison after conversion to acid haematin in 0.1 N HCl; cheap and field-usable but ±10% error, subjective, misses HbF/carboxy-Hb. Still in many teaching labs.
  • Alkaline haematin, oxyhaemoglobin (direct, 540 nm) — simple; oxyHb method is unstable.
  • Automated analysers — mostly cyanide-free sodium lauryl sulphate (SLS-Hb) method.
• PCV / haematocrit: packed red cell volume as a fraction or percentage. Wintrobe tube (100 mm, EDTA, 3,000 rpm for 30 min) or microhaematocrit capillary (12,000 rpm for 5 min); analysers compute PCV = RBC × MCV. Trapped plasma causes a 1–3% overestimate in manual methods.
• Red cell indices classify anaemia morphologically — the examiner's favourite link between numbers and diagnosis.
• Reference intervals (adult, typical Indian lab; always quote your own lab's range): Hb male 13–17 g/dL, female 12–15 g/dL; RBC 4.5–5.5 and 3.8–4.8 ×10¹²/L; PCV 40–50% and 36–46%; MCV 80–100 fL; MCH 27–32 pg; MCHC 32–36 g/dL; RDW 11.5–14.5%; platelets 150–410 ×10⁹/L; TLC 4–11 ×10⁹/L; ESR (Westergren) 0–15 mm/h male, 0–20 mm/h female; reticulocytes 0.5–2.5%.
• WHO anaemia cut-offs: <13 g/dL adult men, <12 g/dL non-pregnant women, <11 g/dL pregnant women and children 6–59 months. India's Anaemia Mukt Bharat programme uses these; the NFHS-5 survey found anaemia in about 57% of women aged 15–49.

Cell counts: manual and automated

• Haemocytometer (Neubauer chamber): 3 mm × 3 mm ruled area, 9 large squares of 1 mm², depth 0.1 mm. Central large square = 25 medium squares = 400 small squares.
  • RBC count: dilute 1:200 in Hayem's or formol-citrate diluent; count 5 medium squares (80 small squares) in the central square, area 0.2 mm², volume 0.02 µL.
  • WBC count: dilute 1:20 in Turk's fluid (2% glacial acetic acid + gentian violet — lyses RBCs, stains nuclei); count 4 corner large squares, area 4 mm², volume 0.4 µL.
  • Platelet count: dilute 1:20 in 1% ammonium oxalate (lyses RBCs, preserves platelets); count the whole central 1 mm² under phase contrast or high dry.
• Charging and counting rules: fill in one flow with no overflow or air bubbles, let platelets settle 10–15 min in a moist chamber, follow the boundary rule (count cells touching the top and left lines, not bottom and right).
• Automated counters:
  • Impedance (Coulter) principle — cells in an electrolyte pass through an aperture and each displaces electrolyte, giving a voltage pulse; pulse number = count, pulse height ∝ cell volume. Gives a 3-part differential (lymphocyte / mixed / granulocyte).
  • Light scattering and flow cytometry — forward scatter (size), side scatter (granularity), fluorescence after nucleic-acid staining; gives a 5-part differential and reticulocyte counts.
  • VCS — volume, conductivity (nucleus:cytoplasm), scatter.
  • Analysers report PCV, MCV, RDW directly or by computation and flag abnormal populations; every flagged sample needs a manual smear review.
• Known interferences: cold agglutinins and rouleaux falsely lower RBC and raise MCV; nucleated RBCs, giant platelets and cryoglobulins falsely raise TLC; platelet clumps and EDTA-dependent pseudothrombocytopenia falsely lower platelets (re-collect in citrate and correct by 1.1); lipaemia and very high WBC falsely raise Hb.

Peripheral smear, staining and differential count

• Making the smear: a drop 2–3 mm from one end of a clean slide, spreader at 30–45°, one smooth push; a good film is 3–4 cm long, tongue-shaped, with a feathered edge and a monolayer.
• Romanowsky stains combine an acidic dye (eosin, stains basic cytoplasm/haemoglobin pink) and a basic dye (methylene blue/azure, stains acidic nuclei blue): Leishman (contains methanol, fixes and stains — stain 2 min, then add double the buffer, 7–10 min), Giemsa (best for malarial parasites, needs prior methanol fixation), Wright, May-Grünwald-Giemsa (MGG), Jenner. Buffer pH 6.8 — too acidic gives everything pink, too alkaline gives everything blue.
• Differential leucocyte count (DLC): examine the monolayer by the battlement (crenellation / zig-zag) method, count 100 cells (200 if abnormal). Adult normals: neutrophils 40–75%, lymphocytes 20–45%, monocytes 2–10%, eosinophils 1–6%, basophils 0–1%.
• RBC morphology to report: anisocytosis, poikilocytosis, microcytic hypochromic (iron deficiency, thalassaemia), macrocytic (B12/folate deficiency), target cells, pencil cells, tear-drop cells (myelofibrosis), spherocytes (hereditary spherocytosis, autoimmune haemolysis), sickle cells, basophilic stippling (lead poisoning, thalassaemia), Howell-Jolly bodies (post-splenectomy), rouleaux (myeloma, high ESR), nucleated RBCs, Heinz bodies (G6PD deficiency, supravital crystal violet).
• WBC changes: toxic granules, Döhle bodies and left shift in bacterial sepsis; hypersegmented neutrophils in megaloblastic anaemia; atypical lymphocytes in viral infection; blasts with Auer rods in AML.
• Special stains: supravital new methylene blue / brilliant cresyl blue for reticulocytes; Perls' Prussian blue for iron and ring sideroblasts; myeloperoxidase / Sudan black for myeloid blasts; PAS for lymphoblasts; LAP score (low in CML, high in leukaemoid reaction); NAP/TdT by immunophenotyping now.
• Reticulocyte count: percentage and absolute count tell you whether the marrow is responding — the central question in every anaemia work-up.

Anaemias, leukaemias and the bone marrow

• Anaemia — definition: haemoglobin (or red cell mass) below the reference range for age, sex and physiological state.
• Morphological classification (by MCV/MCHC): microcytic hypochromic — iron deficiency, thalassaemia trait, sideroblastic, anaemia of chronic disease; normocytic normochromic — acute blood loss, haemolysis, renal disease, aplastic; macrocytic — B12/folate deficiency (megaloblastic), liver disease, hypothyroidism, alcohol, myelodysplasia.
• Aetiological classification: (a) blood loss, (b) decreased production (nutritional deficiency, marrow failure, marrow replacement), (c) increased destruction (haemolysis).
• Iron deficiency anaemia — commonest in India: low Hb, MCV, MCH, MCHC, high RDW, low serum ferritin and iron, raised TIBC, low transferrin saturation, pencil cells on smear.
• Megaloblastic anaemia: macro-ovalocytes, hypersegmented neutrophils, pancytopenia with raised LDH and indirect bilirubin, low B12 or folate; marrow shows nuclear-cytoplasmic asynchrony.
• Haemolytic anaemias: raised reticulocytes, unconjugated bilirubin and LDH, low haptoglobin, urobilinogen in urine.
  • *Intracorpuscular:* membrane (hereditary spherocytosis — osmotic fragility raised, EMA binding test), enzyme (G6PD deficiency — commonest enzymopathy, X-linked, screened by methaemoglobin reduction / fluorescent spot test), haemoglobinopathy (sickle cell — sickling test with 2% sodium metabisulphite, solubility test, confirmed by HPLC or electrophoresis; thalassaemia — raised HbF/HbA₂, Mentzer index <13 suggests trait).
  • *Extracorpuscular:* autoimmune (direct Coombs positive), malaria, drugs, mechanical (MAHA — schistocytes), incompatible transfusion.
• India-specific burden: sickle cell disease is concentrated in central and western tribal belts (Madhya Pradesh, Chhattisgarh, Maharashtra, Odisha, Gujarat, Jharkhand) and the National Sickle Cell Anaemia Elimination Mission (2023) screens the 0–40 age group there; β-thalassaemia carrier rate is roughly 3–4% nationally and higher in some communities. Screening uses CBC indices + HPLC.
• Aplastic anaemia: pancytopenia with reticulocytopenia and a hypocellular (fatty) marrow — diagnosis needs a trephine biopsy, not just aspiration.
• Leukaemias: malignant clonal proliferation of leucocyte precursors.

• Classification systems: the older FAB scheme (ALL L1–L3; AML M0–M7) is morphology-based; the WHO classification (current editions, latest revision 2022) is the standard and combines morphology, immunophenotype, genetics and clinical features.
• Bone marrow aspiration: posterior superior iliac spine (sternum in adults only with care); assess cellularity, M:E ratio (normal 2:1 to 4:1), maturation, iron stores, parasites (Leishman-Donovan bodies), and blast percentage (≥20% blasts = acute leukaemia by WHO).
• Other important counts: absolute eosinophil count (allergy, parasites, tropical eosinophilia), absolute neutrophil count (neutropenia risk grading), LE cell and malaria parasite detection (thick and thin films, QBC, rapid antigen).

Haemostasis and coagulation testing

• Three stages: (1) vascular — vasoconstriction; (2) platelet plug — adhesion via vWF–GPIb, activation, aggregation via GPIIb/IIIa and fibrinogen; (3) coagulation cascade producing fibrin, then fibrinolysis by plasmin.
• Pathways: *intrinsic* (XII, XI, IX, VIII) measured by APTT; *extrinsic* (tissue factor + VII) measured by PT; *common* (X, V, II prothrombin, I fibrinogen) in both. Vitamin-K-dependent factors: II, VII, IX, X plus proteins C and S. Factor VII has the shortest half-life, so PT rises first in liver disease and warfarin therapy.
• Screening tests:
  • Bleeding time (BT) — Ivy (normal 2–7 min) or Duke (1–3 min); tests platelet and vessel function. Largely obsolete, replaced by platelet function analysers.
  • Clotting time (CT) — Lee-White, 5–10 min; insensitive.
  • Prothrombin time (PT) — citrated platelet-poor plasma + tissue thromboplastin + calcium at 37 °C; normal 11–14 s. Reported as INR for oral anticoagulant monitoring.
  • APTT — plasma + phospholipid + an activator (kaolin/silica) + calcium; normal 25–40 s. Monitors unfractionated heparin.
  • Thrombin time, fibrinogen (Clauss) 200–400 mg/dL, D-dimer (raised in DVT, pulmonary embolism, DIC), mixing studies (corrects = factor deficiency; fails to correct = inhibitor, e.g. lupus anticoagulant), factor assays, platelet aggregometry.
• Pre-analytical rules for coagulation: 9:1 blood-to-citrate ratio exactly, no underfilling, centrifuge 1,500–2,500 g for 15 min for platelet-poor plasma (<10 ×10⁹/L), test within 24 h (PT) / 4 h (APTT, longer only if frozen at −70 °C), never draw from a heparinised line. If PCV >55%, the citrate volume must be reduced or the PT/APTT will be falsely prolonged.
• Pattern interpretation:

• Haemophilia A — X-linked recessive factor VIII deficiency, prolonged APTT with normal PT, platelets and BT; haemarthrosis is typical. von Willebrand disease — commonest inherited bleeding disorder, prolonged BT/PFA with variable APTT, low vWF antigen and ristocetin cofactor activity.
• DIC — prolonged PT and APTT, low platelets and fibrinogen, high D-dimer, schistocytes on the smear.
• ESR: rate of fall of red cells in 1 hour. Westergren (200 mm tube, 1:4 citrate, the reference method) or Wintrobe (100 mm, EDTA, no dilution). Raised by fibrinogen and globulins (infection, TB, malignancy, pregnancy, anaemia); low in polycythaemia and sickle cell disease. The tube must be exactly vertical — a 3° tilt can raise the result by 30%.

Quality, safety and Indian regulation

• Internal quality control (IQC): run commercial tri-level controls each shift, plot on Levey-Jennings charts, apply Westgard rules (1₃s, 2₂s, R₄s, 4₁s, 10x) and the moving average (Bull's algorithm) on patient MCHC/indices for analyser drift.
• Delta check — compare a patient's result with their previous one; critical values (for example Hb <7 g/dL, platelets <20 ×10⁹/L, TLC <1 ×10⁹/L, INR >5) must be telephoned and logged.
• EQAS / PT: external programmes such as CMC Vellore's EQAS, AIIMS and the NABL-recognised proficiency schemes; participation is mandatory for accreditation.
• Accreditation and law: NABL 112 (specific criteria for medical labs, built on ISO 15189), the Clinical Establishments (Registration and Regulation) Act, 2010 where adopted, the Drugs and Cosmetics Act for reagents and kits, and Bio-Medical Waste Management Rules, 2016 (yellow bag for blood-soaked waste, red for contaminated plastics, white puncture-proof container for needles, blue for glass). Needle-stick and sharps policy, HBV vaccination for staff and universal precautions are mandatory. Prenatal haemoglobinopathy work must respect the PCPNDT Act, 1994 rules on prenatal diagnostic procedures.
• Calibration and traceability: analyser calibration with the manufacturer's calibrator traceable to the ICSH HiCN standard; log maintenance, background counts and carryover checks.

Key points

• Haemopoiesis - blood cell formation from a pluripotent stem cell; adult site is the axial bone marrow.
• EDTA - anticoagulant of choice for CBC (chelates calcium, preserves morphology); citrate 3.2% at 1:9 for coagulation.
• HiCN method - reference haemoglobin method using Drabkin's reagent, read at 540 nm.
• Neubauer chamber depth is 0.1 mm - RBC counted in 80 small squares (0.02 µL), WBC in 4 corner squares (0.4 µL).
• Turk's fluid for WBC, 1% ammonium oxalate for platelets, Hayem's for RBC - each diluent lyses or preserves what the count needs.
• MCV classifies anaemia - <80 fL microcytic, 80–100 normocytic, >100 macrocytic.
• MCHC is the only index measuring concentration - raised only in spherocytosis or a lipaemic/cold-agglutinin artefact.
• RDW - red cell size variation; high in iron deficiency, normal in thalassaemia trait (the key discriminator).
• Reticulocyte count - distinguishes a hypoproliferative marrow (low) from haemolysis or blood loss (high).
• Romanowsky stain - eosin (acid dye) + methylene blue/azure (basic dye), buffer pH 6.8; Leishman is self-fixing, Giemsa needs methanol.
• PT measures the extrinsic and common pathway; APTT the intrinsic and common - PT/INR monitors warfarin, APTT monitors unfractionated heparin.
• Vitamin-K-dependent factors II, VII, IX, X - and proteins C and S; VII has the shortest half-life.
• Haemophilia A - X-linked factor VIII deficiency; prolonged APTT, normal PT, normal platelets and BT.
• ≥20% blasts in blood or marrow - defines acute leukaemia under the WHO classification; Auer rods point to AML.
• BCR-ABL1 / Philadelphia chromosome t(9;22) - diagnostic of CML and the target of imatinib.
• Westergren ESR is the reference method - 200 mm tube, strictly vertical, read at 60 minutes.
• Levey-Jennings chart + Westgard rules - the backbone of daily internal quality control.
• NABL 112 / ISO 15189 and BMW Rules, 2016 - accreditation and waste-segregation framework for every Indian haematology lab.

Formulas and examples

Example 1 — WBC count from a Neubauer chamber. 1:20 dilution in Turk's fluid; 120 cells counted in the 4 corner squares.

• Area = 4 × 1 mm² = 4 mm², depth 0.1 mm → volume = 0.4 µL (0.4 mm³).
• WBC = (120 × 20) / 0.4 = 2,400/0.4 = 6,000/µL = 6.0 ×10⁹/L — normal.
• Shortcut check: N × 50 = 120 × 50 = 6,000/µL. ✓

Example 2 — red cell indices and interpretation. Hb 8.4 g/dL, PCV 28%, RBC 4.0 ×10¹²/L.

• MCV = 28 × 10 / 4.0 = 70 fL → microcytic.
• MCH = 8.4 × 10 / 4.0 = 21 pg → hypochromic.
• MCHC = 8.4 × 100 / 28 = 30 g/dL → low.
• Mentzer index = 70 / 4.0 = 17.5 (>13) → iron deficiency anaemia more likely than thalassaemia trait; confirm with serum ferritin (low) and RDW (high).
• Contrast: Hb 10.5, PCV 33%, RBC 5.8 → MCV = 33 × 10/5.8 = 56.9 fL, Mentzer = 56.9/5.8 = 9.8 (<13) → suspect β-thalassaemia trait; confirm by HPLC (raised HbA₂).

Example 3 — reticulocyte count and marrow response. Observed reticulocytes 6%, patient PCV 21%, RBC 2.5 ×10¹²/L.

• Corrected retic % = 6 × (21/45) = 6 × 0.4667 = 2.8%.
• Maturation factor at PCV ~21% ≈ 2.0 → RPI = 2.8 / 2.0 = 1.4.
• Absolute retic = 6 × 2.5 × 10 = 150 ×10⁹/L (normal 25–75 ×10⁹/L).
• Interpretation: the absolute count is raised, but RPI <2 means the marrow response is less than adequate for this degree of anaemia — think haemolysis with a coexisting deficiency, or partially treated blood loss.

Example 4 — INR and corrected WBC. Patient PT 32 s, mean normal PT 12.5 s, thromboplastin ISI = 1.2.

• PT ratio = 32/12.5 = 2.56; INR = 2.56^1.2.
• ln 2.56 = 0.9400; × 1.2 = 1.1280; e^1.1280 ≈ 3.09 → INR ≈ 3.1, above the usual 2.0–3.0 warfarin range — report and inform the clinician.
• Same patient's analyser TLC = 24.0 ×10⁹/L with 50 nucleated RBCs per 100 WBC on the smear: corrected WBC = (24.0 × 100)/(100 + 50) = 2,400/150 = 16.0 ×10⁹/L. Reporting 24.0 would overstate the leucocytosis by 50%.

Example 5 — absolute counts from a DLC. TLC 14.0 ×10⁹/L; neutrophils 82%, lymphocytes 10%, monocytes 4%, eosinophils 4%.

• ANC = 14.0 × 0.82 = 11.5 ×10⁹/L (neutrophilia with a left shift → bacterial infection).
• Absolute lymphocytes = 1.4 ×10⁹/L; monocytes = 0.56 ×10⁹/L; AEC = 14.0 × 0.04 = 0.56 ×10⁹/L = 560/µL (raised; normal 40–440/µL) → consider allergy or parasitic infestation.

Where it is used

• Diagnostic chains and hospital labs: Dr Lal PathLabs, SRL, Metropolis, Thyrocare, Agilus, Apollo, Fortis, Manipal and all government medical colleges run 24×7 haematology sections — CBC is the highest-volume test in the lab.
• Technologist roles: haematology bench technologist, phlebotomist, lab in-charge (CBC analyser operation, smear review, coagulation bench), QC coordinator and NABL documentation officer.
• National programmes: Anaemia Mukt Bharat (Hb screening with digital haemoglobinometers and HemoCue at HWCs), the National Sickle Cell Anaemia Elimination Mission's screening in tribal districts, and thalassaemia carrier screening before marriage and in antenatal clinics.
• Blood centres and oncology units: pre-transfusion CBC and platelet counts, CD34 counts for stem-cell harvest, chemotherapy monitoring for neutropenia (ANC-based dose decisions).
• Industry roles: application and service engineers for Sysmex, Beckman Coulter, Horiba, Mindray, Transasia/Erba and Agappe analysers; reagent QC in diagnostic manufacturing.
• Research and clinical trials: CRO central laboratories (Syneos, Parexel, Veeda, Lambda) where haematology safety panels are run under GCP, and ICMR institutes studying haemoglobinopathies.

Interview questions

• Q: Why is EDTA preferred for a CBC and citrate for coagulation tests? A: EDTA chelates calcium irreversibly and preserves cell size and morphology best; citrate binds calcium reversibly so calcium can be added back to start the clotting reaction in vitro.
• Q: What is the depth of a Neubauer chamber and why does it matter? A: 0.1 mm; the count is cells per unit volume, so area counted × 0.1 mm gives the volume, and any error in depth (a chipped cover-glass or an overfilled chamber) changes every result.
• Q: A patient's platelet count is 40 ×10⁹/L but there is no bleeding. What do you do? A: Review the smear for platelet clumps and giant platelets — EDTA-dependent pseudothrombocytopenia; re-collect in sodium citrate, count and multiply by 1.1 to correct for dilution.
• Q: Which single index best separates iron deficiency from thalassaemia trait on a CBC? A: RDW with the RBC count — thalassaemia trait has a high RBC, normal RDW and Mentzer index <13; iron deficiency has a low RBC, high RDW and index >13. HPLC confirms.
• Q: Why is MCHC rarely above 36 g/dL, and what does a high value usually mean? A: Haemoglobin cannot be packed much more densely in a red cell, so a high MCHC usually means spherocytosis or an artefact — lipaemia, cold agglutinins, haemolysis or a wrong PCV.
• Q: Normal PT, prolonged APTT, normal platelets and bleeding time in a boy with knee swelling — diagnosis and next test? A: Haemophilia A or B; confirm with a mixing study (should correct) followed by factor VIII and IX assays.
• Q: Why is INR used instead of plain PT in seconds? A: Thromboplastin reagents differ in sensitivity, so the same plasma gives different PTs; INR normalises the ratio using the reagent's ISI, making results comparable between laboratories.
• Q: Why must the ESR tube stand exactly vertical? A: Tilting lets cells slide down the wall instead of settling, so even a few degrees of tilt can raise the result substantially — up to about 30%.
• Q: What is the reference method for haemoglobin estimation and why is Sahli's not acceptable for reporting? A: The cyanmethaemoglobin (HiCN) method with Drabkin's reagent at 540 nm; Sahli's relies on visual colour matching, has about ±10% error and does not convert all haemoglobin derivatives.
• Q: Which Westgard rule would you treat as a random error and which as systematic? A: 1₃s and R₄s indicate random error; 2₂s, 4₁s and 10x indicate systematic error or drift, so you check calibration, reagent lot and the analyser.
• Q: How does an impedance counter distinguish cell size from cell number? A: Each cell passing the aperture gives a voltage pulse — the number of pulses is the count and the pulse height is proportional to cell volume.
• Q: What defines acute leukaemia on a marrow examination and how do you tell AML from ALL at the bench? A: 20% or more blasts (WHO); AML blasts are myeloperoxidase and Sudan black positive and may show Auer rods, ALL blasts are PAS positive and MPO negative — immunophenotyping confirms.

Common mistakes

• Using the wrong diluent — Turk's fluid for a platelet count or ammonium oxalate for WBCs — and getting a count with nothing to count.
• Forgetting the dilution factor or the volume (area × 0.1 mm) in the manual count formula, usually by an order of 10.
• Reporting an analyser result with a flag without reviewing the smear, so nucleated RBCs, platelet clumps or blasts go unreported.
• Under-filling a citrate tube or ignoring a high PCV, which falsely prolongs PT and APTT and can trigger a wrong anticoagulant decision.
• Confusing which pathway each screening test covers — writing APTT for the extrinsic pathway, or expecting PT to detect haemophilia.
• Making a thick, short smear with no monolayer or feathered edge, then counting the differential at the thick end where cells are clumped and distorted.
• Quoting textbook reference ranges instead of the laboratory's own validated ranges, and ignoring age, sex, pregnancy and altitude variation.

Related topics

Clinical pathology · Blood banking and transfusion · Immunology and serology tests · Histopathology and cytology · Clinical biochemistry: organ function tests · PCR and DNA sequencing