Evaluation of a flow cytometric test for G6PD‐deficient erythrocytes

Abstract

Objective Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency, an X‐linked recessive disorder, is the commonest erythrocytic enzymopathy worldwide. Reliable diagnosis and severity prediction in G6PD‐deficient/heterozygous females remain challenging. A recently developed flow cytometric test for G6PD deficiency has shown promise in precisely identifying deficient females. This paper presents our experiences with this test in a subtropical setting and presents a modification in flow cytometric data acquisition strategy. Methods The methaemoglobin reduction + ferryl Hb generation‐based flow cytometric G6PD test was compared with the screening methaemoglobin reduction test (MRT) and confirmatory G6PD enzyme activity assay (EAA) in 20 G6PD‐deficient males, 22 G6PD‐heterozygous/deficient females and 20 controls. Stained cells were also assessed for bright/dim G6PD activity under a fluorescent microscope. Results Flow cytometry separated and quantified %bright cells in heterozygous/deficient females, objectively classifying them into 6 normal (>85% bright cells), 14 intermediate (10‐85%) and two G6PD‐deficient (<10% bright cells). Concordance with MRT was 89% (55/62 cases) and with EAA was 77% (48/62 cases). Fluorometrically predicted violet laser excitation (405‐nm) with signal acquisition in the 425–475 nm region was a technical advancement noted for the first time in this paper. Conclusion Flow cytometry/fluorescence microscopy represent technically straightforward methods for the detection and quantification of G6PD‐deficient erythrocytes. Based on our results, we recommend their application as a first‐line investigation to screen females who are prescribed an oxidant drug like primaquine or dapsone.