The G6PD flow-cytometric assay is a reliable tool for diagnosis of G6PD deficiency in women and anaemic subjects

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson’s r 0.918–0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

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

“…The comparison of enzyme activity to genotype and intracellular RBC G6PD activity profiles has been described elsewhere 12, 20, 21, 22. In this methods article, using a quantitative method, percent bright cells, generated from the same cytofluorometric assay, are compared to results from DNA sequencing.…”

“…This tool allows interlaboratory data comparison and broader cross‐geographical and genotypic analysis of G6PD deficiency, especially in females 21. An important next step is to attempt to associate this phenotypic data to risk of hemolysis on exposure to an oxidative stress such as primaquine and its metabolites.…”

“…Additionally, 2 datasets, 1 from Thailand and 1 from the USA, are used to demonstrate the utility of the tool for determination of heterozygosity for G6PD in females. Association between mosaic profiles, G6PD activity, and hemoglobinopathies has been published separately …”

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 ].…”

Primaquine-induced haemolysis in females heterozygous for G6PD deficiency

“…In heterozygous females this will measure a weighted average of the activities of the two red blood cell populations (G6PD normal and G6PD deficient). The flow-cytometric read-out of the MRT [ 35 ] is a promising assay for the detection of G6PD activity at the single red blood cell level that assesses the actual proportion of G6PD normal and deficient red cell populations [ 36 ]. The spectrophotometric assay is quite straightforward, but it requires skilled laboratory technicians, specialized laboratory equipment, and reagents.…”