Cytochemical flow analysis of intracellular G6PD and aggregate analysis of mosaic G6PD expression

Abstract

BackgroundMedicines that exert oxidative pressure on red blood cells (RBC) can cause severe hemolysis in patients with glucose‐6‐phosphate dehydrogenase (G6PD) deficiency. Due to X‐chromosome inactivation, females heterozygous for G6PD with 1 allele encoding a G6PD‐deficient protein and the other a normal protein produce 2 RBC populations each expressing exclusively 1 allele. The G6PD mosaic is not captured with routine G6PD tests.MethodsAn open‐source software tool for G6PD cytofluorometric data interpretation is described. The tool interprets data in terms of % bright RBC, or cells with normal G6PD activity in specimens collected from 2 geographically and ethnically distinct populations, an African American cohort (USA) and a Karen and Burman ethnic cohort (Thailand) comprising 242 specimens including 89 heterozygous females.ResultsThe tool allowed comparison of data across 2 laboratories and both populations. Hemizygous normal or deficient males and homozygous normal or deficient females cluster at narrow % bright cells with mean values of 96%, or 6% (males) and 97%, or 2% (females), respectively. Heterozygous females show a distribution of 10‐85% bright cells and a mean of 50%. The distributions are associated with the severity of the G6PD mutation.ConclusionsConsistent cytofluorometric G6PD analysis facilitates interlaboratory comparison of cellular G6PD profiles and contributes to understanding primaquine‐associated hemolytic risk.

Addressing the gender-knowledge gap in glucose-6-phosphate dehydrogenase deficiency: challenges and opportunities

“…The resulting overall levels of G6PD enzyme activity in heterozygous females mainly range from 30% to 80% of normal G6PD activity; values within this range are considered as intermediate (Figure 1). 6–11…”

“…Microscopy or flow cytometry-based assays that determine G6PD levels in individual RBCs are extremely informative for understanding the mosaic expression of g6pd alleles in individual heterozygous females. 7,9,10,19,20 These are not practical clinical assays, however. Likewise, genetic tests are deterministic regarding an individual’s G6PD genotype, but are not clinically useful for understanding the phenotypic (clinically relevant) G6PD status of heterozygous females with one normal and one deficient G6PD allele.…”

“…The severity of the underlying prevalent g6pd -deficient allele affects the population distribution for the G6PD activity levels of heterozygous females (e.g., leading to female populations being skewed toward higher or lower G6PD activity levels), but it does not significantly impact the male distributions. 20…”

Implications of current therapeutic restrictions for primaquine and tafenoquine in the radical cure of vivax malaria

“…: 51–86) of heterozygous females test as G6PD deficient at a 70% threshold. Both models conclude that an increasing level of G6PDd severity is associated with lower mean enzyme activities in heterozygotes [ 33 , 34 ].…”

Optimizing G6PD testing for Plasmodium vivax case management and beyond: why sex, counseling, and community engagement matter

“…Where G6PD testing is driven by malaria case management, often rural and lower socioeconomic populations will benefit, as these populations often experience higher rates of malaria infection. Recent renewed attention to the characterisation at a cellular level of heterozygous G6PD deficiency may help to raise awareness of the clinical implications of intermediate G6PD activity in females [35][36][37] .…”