Evaluation of high performance liquid chromatography for routine estimation of haemoglobins A2 and F.

AIMS

To compare high performance liquid chromatography (HPLC) with conventional methods for the estimation of blood haemoglobin A2 (HbA2) and haemoglobin F (HbF) concentrations in routine thalassemia screening.

METHODS

An HPLC system (the VARIANT Hemoglobin Testing System) was tested for precision and reproducibility in the measurement of HbA2 and HbF, and reference ranges were obtained for a local healthy adult population. HPLC was compared with column anion exchange chromatography for HbA2 measurement, and radial immunodiffusion, or alkaline denaturation for HbF measurement. The reliability of HbA2 measurement by HPLC for the detection of beta thalassaemia and HbE was assessed in 200 consecutive samples for routine thalassaemia screening.

RESULTS

HPLC was rapid, technically easy, and gave good precision and reproducibility. In all comparisons linear regression analysis showed good correlation between HbA2 or HbF concentrations determined by HPLC and by the respective conventional methods. All beta thalassaemia or haemoglobin E carriers presumptively identified by conventional methods in 200 consecutive samples were detected by HbA2 measurement using HPLC, without any false positive or false negative results.

CONCLUSIONS

The measurement of HbA2 and HbF by HPLC is rapid, reproducible, and precise. It is as reliable as column chromatography for the measurement of HbA2 and radial immunodiffusion or alkaline denaturation for the measurement of HbF. HPLC may be an appropriate method for rapid screening in population surveys for beta thalassaemia and HbE carriers.