Analysis of Haemoglobin Profile in Haemoglobinopathies by High Performance Liquid Chromatography and Capillary Zone Electrophoresis Methods: A Cross-sectional Study

Introduction: Mutation or deletion of one of the globin genes of haemoglobin results in thalassaemias and haemoglobinopathies. Two diagnostic modalities that can be used to study haemoglobin abnormalities are High-Performance Liquid Chromatography (HPLC) and Capillary Zone Electrophoresis (CZE). Aim: To compare HPLC and CZE for evaluating haemoglobinopathies. Materials and Methods: The present prospective cross- sectional analytical study was done over a period between 1st December 2018 to 30th May 2020 in department of Pathology (Central laboratory), BLDEDU’s Shri BM Patil Medical College, Hospital and Research Centre, Vijayapura, Karnataka. The blood samples were taken from 48 adults and children and were examined for routine thalassaemia screening using both the modalities (HPLC and CZE), excluding those patients who had undergone blood transfusion within a span of 12 weeks. All samples were analysed using an automated cell counter (Sysmex XN-1000), HPLC (Biorad D-10), and CZE. Parameters studied included: Complete Blood Count (CBC), Haemoglobin A (HbA), Haemoglobin A2 (HbA2), Foetal Haemoglobin (HbF). Chi-square test for association, comparison of means using the test, ANOVA (Analysis of variance) for comparison between and among groups, and sensitivity and specificity analysis were made. Results: Out of 48 cases, 24 cases were of thalassaemia trait, nine cases were normal, three cases of β-thalassaemia major, two cases of homozygous/double heterozygous for β-thalassaemia and δβ-thalassaemia, one case of sickle cell anaemia, one case of compound heterozygous sickle cell anaemia and β-thalassaemia and one case of compound heterozygous for HbE/ β-thalassaemia. In the remaining seven cases only HPLC was done. When the HbF value is >16.5%, it merges with HbA1c values in HPLC (Bio-Rad D-10), because of which it is challenging to comment about exact values of HbF with the help of HPLC. So for such cases, CZE helped us to evaluate the exact value of HbF. Conclusion: Both the methods were fairly useful in analysing haemoglobinopathies. However, CZE is preferred over HPLC method. The advantage of Hb electrophoresis over HPLC is that it gives exact values of HbF, which is essential to differentiate thalassaemia intermedia from thalassaemia major. Also, it exactly evaluates the percentage of HbE, which elutes with HbA in the case of HPLC. CZE is complimentary to HPLC, which helps assess exact values of HbF, HbS, and HbE.