Factors affecting the evaluation and use of a hemoglobin A2 method - Lot-to-lot variation, long-term deviation and carry-over

Abstract

Background: We compared two Hb A₂ methods, high pressure liquid chromatography and capillary zone electrophoresis, at two occasions. In 2014 the methods showed good agreement, while in 2020 HPLC results were clearly higher than CE results. This finding prompted us to investigate the external quality assessment (EQA) outcome and our total patient results obtained by high pressure liquid chromatography over several years.

Methods: The methods compared were Bio-Rad Variant II Beta-Thal Short Program (HPLC) and Sebia Capilllarys Flex Piercing (CE).

Results: Our annual patient results obtained by HPLC increased significantly from 2014 to 2020. A similar trend was also seen in our EQA results. When patient results were grouped according to different reagent lots it became evident that method comparisons might be severely affected by lot-to-lot variation. We also noticed that samples analyzed with the HPLC method following a sample containing a high proportion of Hb E where prone to give falsely increased Hb A₂ results.

Conclusion: Standardization of the measurement of Hb A₂ is urgently needed. Furthermore, the lot-to-lot variation must be minimized. While waiting for these improvements each laboratory ought to repeatedly evaluate their distribution of patient Hb A₂ results.

Keywords: Capillary zone electrophoresis; EQAS; Hemoglobin A(2); High pressure liquid chromatography; Lot-to-lot variation; Reference intervals; Standardization.