Hemoglobin Variant Analysis and Its Comparison between Conventional High-performance Liquid Chromatography Using Whole Blood versus Dried Blood Spot: High-performance Liquid Chromatography.

Background: High-performance liquid chromatography (HPLC) using whole blood (WB) samples is considered gold standard for screening and diagnosis of sickle cell, beta-thalassemia, and other hemoglobinopathies. Collection of WB and temperature-controlled logistics to a laboratory limits the use of HPLC in population screening hemoglobinopathies, especially in remote, tribal regions, having limited resources for venous blood sample collection and transport.

Objectives: Use of dried blood spot (DBS) sample for HPLC analysis (DBS-HPLC), as an alternative to WB can ease the process of sample collection, decrease the time and resources utilized, and save substantial time and cost on the hemoglobinopathy screening program operations for all age groups.

Materials and methods: We compared the results from HPLC analysis of hemoglobin (Hb) variants on DBS (DBS-HPLC) stored and transported at ambient temperatures to laboratory at 3, 5, 11, and 24 days post collection, to results from fresh WB analyzed on the same day by HPLC.

Results: The results showed accurate identification and quantitation of fetal hemoglobin, HbA, HbS, HbA2, HbE, and HbD by DBS-HPLC even after 3 weeks of storage and transport at ambient temperature, with accurate interpretation of all major hemoglobinopathies, i.e., homozygous, and heterozygous cases of sickle cell, beta-thalassemia, HbE, HbD, and compound heterozygous cases of these variants, when compared to the conventional WB HPLC results.

Conclusion: DBS sample collection combined with HPLC analysis offers a cost-effective, operationally efficient, and accurate method for unified, integrated, and comprehensive population screening test for hemoglobinopathies in resource poor, remote and geographically vast regions in India.