Haemoglobinopathies Laboratory Diagnosis
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Direct answer
A haemoglobinopathy is a structural (qualitative) or synthetic (quantitative) defect in globin: sickle haemoglobin and haemoglobin C are variants, while the thalassaemias are underproduction of alpha or beta chains. The MLT workup moves from the CBC picture (microcytic hypochromic anaemia with raised red cell count and normal iron studies suggesting beta thalassaemia trait) through screening with NESTROFT, to cellulose acetate electrophoresis at alkaline pH 8.6 for separation, citrate agar at pH 6.2 for confirmation of HbS, and quantification of HbA2 (above 3.5 per cent defines beta thalassaemia trait) and HbF by elution or HPLC. Family studies complete the picture where patterns are ambiguous.
What you must remember
- Alkaline electrophoresis (pH 8.6, cellulose acetate): mobility order from anode HbA fastest, then HbF, then HbS with D and G, and HbC slowest — HbS, D, G and Lepore co-migrate, so alkaline position alone never confirms HbS.
- Confirmation at acid pH: citrate agar electrophoresis at pH 6.2 separates true HbS from D and G, which migrate with HbA at acid pH — the standard confirmatory step.
- Beta thalassaemia trait numbers: HbA2 3.5-7 per cent (normal under 2.5-3.5), HbF normal under 1 per cent, red cell count high for the degree of anaemia, MCH low — iron deficiency, by contrast, drops the red cell count.
- Beta thalassaemia major: HbF 70-95 per cent, HbA nearly absent, electrophoresis showing dominant F with variable A depending on genotype; transfusion-dependent from infancy.
- NESTROFT (naked eye single-tube red cell osmotic fragility test): the Indian field screening test for beta thalassaemia trait — test cells in 0.36 per cent saline; persistence of turbidity suggests the resistant, hypochromic population of trait.
- HbH disease and alpha thalassaemia: HbH (beta tetramers) appears as fast-migrating bands; brilliant cresyl blue supravital staining shows golf-ball inclusions — alpha thalassaemia needs DNA studies for confirmation beyond HbH disease.
- Other discriminators: HbD Punjab and HbE are common Indian variants; HbE migrates with HbA2/C position at alkaline pH and is abundant in the north-east.
Working through a microcytic anaemia panel
Picture a 22-year-old woman from Nagpur with Hb 9.8 g/dL, MCV 64 fL, red cell count 6.1 million per microlitre, ferritin normal. The high red cell count with marked microcytosis argues against iron deficiency and for beta thalassaemia trait. NESTROFT is positive — the tube stays turbid against the control line. Electrophoresis at pH 8.6 shows a heavy HbA band with a small band at the A2 position; elution quantification reads HbA2 4.8 per cent. That single number, above 3.5 per cent, closes the diagnosis of beta thalassaemia trait, and genetic counselling follows — because the real patient is the future couple: if her partner also carries a beta thalassaemia mutation, each pregnancy carries a one-in-four risk of thalassaemia major.
Contrast the eight-month-old with failure to thrive, Hb 5.6 g/dL and target cells everywhere: electrophoresis shows HbF 85 per cent with almost no HbA. Beta thalassaemia major — the laboratory confirms what the smear suggested, and the report triggers referral for a transfusion and chelation programme, and discussion of haematopoietic stem cell transplantation, the only curative option.
How exams frame it
MLT papers love the co-migration question: which haemoglobins travel with S at pH 8.6 (answer D, G, Lepore), and which test resolves them (citrate agar pH 6.2). The second favourite is the trait versus iron deficiency table reduced to one discriminating number each — red cell count and HbA2. A practical viva angle: a patient on repeated transfusions shows a near-normal electrophoretic pattern because donor HbA dominates, so thalassaemia major workup should precede or account for transfusion. Finally, screening programme questions — which test mass-screens couples at community level — expect NESTROFT as the Indian low-cost answer, with HPLC as the reference quantification method in referral centres.
Frequently asked questions
At what pH is cellulose acetate haemoglobin electrophoresis performed and why?
At alkaline pH 8.6, where haemoglobins are negatively charged and migrate toward the anode at rates determined by their charge, separating A, F, S and C.
Which haemoglobins co-migrate with HbS at alkaline pH, and how is this resolved?
HbD, HbG and HbLepore travel with HbS at pH 8.6; citrate agar electrophoresis at pH 6.2 separates true HbS, while D and G move with HbA.
What HbA2 value defines beta thalassaemia trait?
HbA2 above 3.5 per cent (typically 4-7 per cent) in the presence of microcytosis and a high red cell count defines the trait, provided iron deficiency is not coexisting, as it can lower HbA2.
What is NESTROFT and what is its role in India?
The naked eye single-tube osmotic fragility test using 0.36 per cent saline; it mass-screens for beta thalassaemia trait because trait red cells resist hypotonic lysis, keeping the tube turbid.
How is HbH demonstrated in the laboratory?
Brilliant cresyl blue supravital staining shows multiple small golf-ball-like inclusions in red cells, and electrophoresis reveals fast-moving HbH bands, indicating alpha thalassaemia with three-gene deletion.