HPLC in characterization of hemoglobin profile in thalassemia syndromes and hemoglobinopathies: a clinicohematological correlation.

Abstract

High-performance liquid chromatography (HPLC) is a technique introduced for the accurate diagnosis of hemoglobinopathies and thalassemias. The advantage of the HPLC system is the excellent resolution, reproducibility & quantification of several normal & abnormal hemoglobin resulting in accurate diagnosis of thalassemia syndromes. The purpose of this study is to evaluate the HPLC technique in diagnosis of thalassemia syndromes and also correlate it with clinicohematological profile in these cases. A total of 110 cases were diagnosed as thalassemias and hemoglobinopathies by Bio- Rad variant II HPLC system by β-thal short program. The retention times, proportion of the haemoglobin (%), and peak characteristics for all hemoglobin (Hb) fractions were recorded. Alkaline Hb electrophoresis was performed in each case. Other tests performed were HbF estimation by Betke's method, brilliant cresyl blue preparation for HbH inclusion bodies, sickling tests using 2% metabisulphite and serum Ferritin estimation. Family studies were carried out wherever necessary. Of 110 cases included in the study, 87 cases were of thalassemic disorders and 23 cases were of hemoglobinopathies. Four Hb variants were identified including HbD, HbE, HbS, HbJ Oxford. There was a significant decrease in the level of HbA2 associated with iron deficiency anemia. The mean HbA2 levels in both iron deplete and iron replete groups were clearly>4%, suggesting that HPLC identified nearly all high HbA2 β-thalassemia trait even in spite of iron deficiency.