Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of alpha thalassaemia.
OBJECTIVE
To assess the accuracy and precision of measuring haemoglobin A(2) by high performance liquid chromatography (HPLC) in the presence and absence of sickle cell trait, with or without alpha thalassaemia trait.


METHODS
The haemoglobin A(2) percentage and the haemoglobin A(2) plus S percentages were determined by HPLC on 82 normal controls and 78 patients with sickle cell trait, respectively; the alpha thalassaemia status of each patient was determined by polymerase chain reaction. Red cell indices and haemoglobin A(2) and S percentages were compared in patients with two, three, or four alpha genes.


RESULTS
Of the 78 patients with sickle cell trait, 17 were heterozygous for alpha(+) thalassaemia (-alpha(3.7)/alphaalpha) and 13 were homozygous (-alpha(3.7)/-alpha(3.7)). Microcolumn chromatography showed that the haemoglobin A(2) percentage was slightly, but significantly, higher than normal in sickle cell trait. HPLC determinations of haemoglobin A(2) percentage in patients with sickle cell trait are precise but inaccurate, the percentage being appreciably overestimated. The measured haemoglobin A(2) 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 A(2) percentage" between groups of individuals with two, three, and four alpha genes.


CONCLUSIONS
Haemoglobin A(2) determinations by HPLC are precise but inaccurate. Nevertheless, there are significant differences in the haemoglobin A(2) percentage in subjects with two, three, and four alpha genes. Although there is some overlap between groups, this can be useful, together with the red cell indices, in predicting the likelihood of coexisting alpha thalassaemia.
St Mary's Hospital Campus, Imperial College Faculty of Medicine, Norfolk Place, London W2 1PG, UK.
C E
CE
Head
M
M
Conroy
M
M
Jarvis
L
L
Phelan
B J
BJ
Bain
D000465:Algorithms; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003720:Densitometry / Q000379:methods; D004909:Erythrocyte Indices; D017353:Gene Deletion; D005838:Genotype; D006443:Hemoglobin A2 / Q000032:analysis*; D006451:Hemoglobin, Sickle / Q000032:analysis*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012805:Sickle Cell Trait / Q000097:blood* / Q000150:complications / Q000235:genetics; D017085:alpha-Thalassemia / Q000097:blood* / Q000150:complications / Q000235:genetics
D006451:Hemoglobin, Sickle; D006443:Hemoglobin A2