Cation Exchange High Performance Liquid Chromatography for Diagnosis of Haemoglobinopathies.

Background: Cation exchange high performance liquid chromatography (HPLC) is emerging as the method of choice for initial screening and diagnosis of haemoglobinopathies. The use of alkaline and acid gel electrophoresis in the developing countries may result in incorrect diagnosis of haemoglobinopathies. The aim of the study is to assess the accuracy and precision of diagnosis of haemoglobinopathies by HPLC and its possible advantage over conventional techniques.

Methods: Over a two year period, 955 patients presenting with anaemia were evaluated by HPLC for diagnosis of haemoglobinopathies. All cases showing 'unknown peaks' and other rare haemoglobin variants on HPLC were further analyzed by agar gel electrophoresis at alkaline pH (8.6) and at acid pH (6.0).

Results: A total of 137 (14.3%) patients showed different abnormal haemoglobins variants. Of these 91 (66.4%) were diagnosed to have beta - heterozygous thalassaemia based on high level of HbA2 (>3.9%), five (3.7%) as beta - homozygous thalassaemia (HbF 25 - 91%), 15 (10.9%) as sickle cell trait, two (1.5%) as compound heterozygous state of sickle - β(+) thalassaemia and three (2.2%) patients as homozygous sickle cell anaemia (HbSS). One (0.7%) patient had unknown peak on HPLC with retention time of 4.78 minutes, constituting 16.8% of total haemoglobin. Sickling test was negative. He was diagnosed as HbQ - India heterozygous. Thirteen (9.5%) patients were diagnosed as HbE syndrome and were further sub classified as HbE trait (five cases) and HbE disease (eight cases). Seven (5.1%) patients were diagnosed as Hb - D Punjab heterozygous.

Conclusions: The simplicity of the sample preparation, superior resolution of the method and accurate quantitation of haemoglobin concentration, combined with complete automation, makes this an ideal methodology for diagnosis of haemoglobinopathies.