Spectrum of haemoglobinopathies diagnosed by cation exchange-HPLC & modulating effects of nutritional deficiency anaemias from north India

BACKGROUND & OBJECTIVES: The usefulness of cation exchange high performance liquid chromatography (CE-HPLC) as a tool for detection of thalassaemia/haemoglobin variants was evaluated in a prospective study in a tertiary care centre in north India. We also tried to evaluate the effect of concurrent nutritional deficiency on the HPLC pattern in the local ethnic population. METHODS: A total of 800 blood samples were analyzed on the Bio-Rad Variant HPLC system by β-thal short program. The retention times, proportion of the haemoglobin (%), and the peak characteristics for all haemoglobin fractions were recorded. Alkaline and acid haemoglobin electrophoresis was performed to document the identities of the haemoglobin variants, wherever necessary. Many cases were subjected to family studies for a definitive diagnosis. RESULTS: Among 800 samples tested, 553 (69.1%) were found to have normal HPLC pattern. Apart from β- thalassaemia, nine additional variants were encountered; HbS (2.8%), HbE (2.5%) and HbD (1.1%) being the most common variants present. Other variants included Hb Q-India, Hb-Lepore, δβ-thalassemia/ HPFH, HbD-Iran, HbJ-Meerut and HbH disease. There was a significant decrease in the level of HbA(2) associated with iron deficiency anaemia (IDA) (P=0.004) and increase in megaloblastic anaemia (P<0.001) among subjects with normal HPLC pattern. INTERPRETATION & CONCLUSIONS: HPLC was found to be a simple, rapid and reliable method for the detection of hemoglobin variants. An accurate diagnosis can be provided in majority of cases by use of retention time, proportion of total haemoglobin, and peak characteristics of HPLC. Haemoglobin electrophoresis and family studies play a valuable role in difficult cases. Concurrent nutritional deficiency also has an effect on HbA(2) levels.