ORIGINAL ARTICLE

Some observations on the measurement of haemoglobin A₂ and S percentages by high performance liquid chromatography in the presence and absence of thalassaemia

C E Head¹, M Conroy¹, M Jarvis², L Phelan³ and B J Bain³

¹ St Mary’s Hospital Campus, Imperial College Faculty of Medicine, Norfolk Place, London W2 1PG, UK
² Department of Haematology, North Middlesex Hospital, Sterling Way, London N18 1QX, UK
³ Department of Haematology, St Mary’s Hospital, Praed Street, London W2 1NY, UK

Correspondence to:
Correspondence to:
Dr B J Bain
Department of Haematology, St Mary’s Hospital, Praed Street, London W2 1NY, UK; b.bain{at}ic.ac.uk

Aims: To assess the accuracy and precision of measuring haemoglobin A₂ by high performance liquid chromatography (HPLC) in the presence and absence of sickle cell trait, with or without thalassaemia trait.

Methods: The haemoglobin A₂ percentage and the haemoglobin A₂ plus S percentages were determined by HPLC on 82 normal controls and 78 patients with sickle cell trait, respectively; the thalassaemia status of each patient was determined by polymerase chain reaction. Red cell indices and haemoglobin A₂ and S percentages were compared in patients with two, three, or four genes.

Results: Of the 78 patients with sickle cell trait, 17 were heterozygous for ⁺ thalassaemia (-^3.7/) and 13 were homozygous (-^3.7/-^3.7). Microcolumn chromatography showed that the haemoglobin A₂ percentage was slightly, but significantly, higher than normal in sickle cell trait. HPLC determinations of haemoglobin A₂ percentage in patients with sickle cell trait are precise but inaccurate, the percentage being appreciably overestimated. The measured haemoglobin A₂ percentage is stable for one week, but inaccuracy increases by two weeks in most samples. Despite this inaccuracy, there are significant differences in the HPLC "haemoglobin A₂ percentage" between groups of individuals with two, three, and four genes.

Conclusions: Haemoglobin A₂ determinations by HPLC are precise but inaccurate. Nevertheless, there are significant differences in the haemoglobin A₂ percentage in subjects with two, three, and four genes. Although there is some overlap between groups, this can be useful, together with the red cell indices, in predicting the likelihood of coexisting thalassaemia.

Keywords: high performance liquid chromatography; haemoglobin A2; haemoglobin S; thalassaemia

Abbreviations: HPLC, high performance liquid chromatography

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