Haemoglobin Electrophoresis and HPLC

Haemoglobin electrophoresis and high performance liquid chromatography separate and quantify the haemoglobins in a haemolysate: HbA, HbA2, HbF and variants. Electrophoresis separates by charge on cellulose acetate at alkaline pH, on citrate agar at acid pH, by isoelectric focusing or by capillary zone electrophoresis. Cation exchange HPLC separates by retention time and gives a quantified trace for each fraction.

With the full blood count and film, these methods diagnose thalassaemia syndromes, sickle cell disease and trait, and other haemoglobin variants. They are the basis of antenatal and newborn screening.

About 5 to 7% of the world's population carries a clinically significant haemoglobin variant. HbS is most common in sub-Saharan Africa, parts of India, the Middle East and the Mediterranean, with trait frequencies up to about 20 to 30% in some regions of sub-Saharan Africa. HbE is frequent in South-East Asia.

Indications. Microcytosis with normal iron studies, unexplained haemolysis, suspected sickle cell disease or thalassaemia, antenatal booking in at risk women, partner and family testing, newborn screening, pre-conception counselling, monitoring of HbS percentage in exchange transfusion programmes, and HbF measurement during hydroxycarbamide therapy.

Specimen. EDTA blood. Record transfusion history, ethnicity, iron status and age with the request.

Adult reference values (method specific). HbA 95 to 98%, HbA2 2.0 to 3.3% (up to 3.5% in some methods), HbF under 1%.

Newborns. HbF is 60 to 80% at birth and falls to near adult values by about 12 months. HbA appears from about 6 weeks. Dried blood spot HPLC or isoelectric focusing is used for screening, and results are reported as patterns such as FA (normal), FAS (trait), FS (disease or sickle beta thalassaemia), FSC and FAC.

Method limits. On alkaline electrophoresis HbS co-migrates with HbD Punjab and HbG Philadelphia, and HbC co-migrates with HbE, HbA2 and HbO-Arab. HPLC retention time windows overlap for some variants. A major or unusual finding needs confirmation by a second method (acid agar, isoelectric focusing, capillary electrophoresis, mass spectrometry or DNA analysis).

• Beta thalassaemia trait: HbA2 over 3.5% (usually 4 to 6%), HbF normal or slightly raised (up to about 5%), microcytosis with a high red cell count and mild or no anaemia.
• Beta thalassaemia major and intermedia: HbF 20 to 100%, HbA absent (beta zero) or reduced (beta plus), HbA2 variable.
• Borderline HbA2 (3.3 to 3.5%): needs DNA analysis, since it can represent a silent beta mutation or coexisting alpha thalassaemia.
• Delta-beta-thalassaemia: HbF about 5 to 20% unevenly distributed, with normal or low HbA2.
• Hereditary persistence of foetal haemoglobin: HbF about 10 to 30% evenly distributed, with normal red cell indices and no anaemia.
• Alpha thalassaemia: adult electrophoresis is normal in alpha thalassaemia trait, so DNA analysis is needed. HbH (beta-4 tetramers) appears in HbH disease, and Hb Bart's at birth reflects alpha gene loss, rising with the number of genes lost.
• HbS trait: HbS about 35 to 45% with HbA predominating. HbS is lower (about 25 to 35%) with coexisting alpha thalassaemia.
• Sickle cell anaemia (HbSS): HbS about 80 to 95%, HbF 2 to 20%, HbA absent, HbA2 normal.
• HbSC disease: HbS and HbC each about 45 to 50%.
• Sickle beta thalassaemia: HbS predominant with HbA2 over 3.5%. HbA is absent in beta zero and about 5 to 30% in beta plus.
• HbC trait and disease: HbC about 30 to 40% in trait and over 90% in HbCC.
• HbE trait and disease: HbE about 25 to 35% in trait. HbE with beta thalassaemia gives HbE about 40 to 60% with raised HbF and can be transfusion dependent.
• Raised HbF in adults: hereditary persistence, beta thalassaemia, hydroxycarbamide, pregnancy (modest), myeloproliferative neoplasms, juvenile myelomonocytic leukaemia, and marrow recovery.

Factors altering HbA2. Lowered by iron deficiency, sideroblastic anaemia, alpha thalassaemia and delta thalassaemia. Raised by hyperthyroidism and some antiretroviral drugs. Iron deficiency can lower HbA2 enough to hide beta thalassaemia trait, so the test should be repeated after iron replacement.

Critical findings needing immediate action.

• A significant variant in a pregnant woman whose partner carries a variant needs early counselling so that prenatal diagnosis options are available in time.
• A neonatal screening pattern of FS, FSC or FE needs prompt confirmation and referral.
• A young child with severe anaemia and HbF predominant needs urgent referral for transfusion dependent thalassaemia.

Pitfalls.

• Transfusion in the previous 3 months dilutes variants with donor HbA. Interpret HbS percentage with this in mind during exchange programmes.
• Glycated haemoglobin, carbamylated haemoglobin (uraemia), acetylated haemoglobin and aged samples add non-variant peaks on HPLC.
• Unstable haemoglobins can be missed and need isopropanol or heat stability testing and DNA sequencing.
• Co-migrating variants are misattributed without a second method.
• Alpha thalassaemia trait and silent carriers cannot be excluded by electrophoresis.
• Neonatal samples cannot exclude beta thalassaemia, and a repeat at 6 to 12 months is needed when the result is uncertain.

Special groups.

• Pregnancy: test at booking in at risk women, with partner testing. Prenatal diagnosis by chorionic villus sampling is possible from about 11 weeks.
• Neonates: screening within the first week, with repeat testing at 6 to 12 months for borderline patterns.
• Transfused patients: use a stored pre-transfusion sample when available, or DNA analysis.

Follow up tests. Full blood count and film, iron studies, DNA analysis of the beta and alpha globin genes (sequencing and deletion analysis), sickle solubility test, isopropanol stability test, partner and family testing, prenatal diagnosis, transcranial Doppler in children with HbSS.