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.
Highlights
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by mutations that impair the stability of the protein; the enzymatic capacity of the protein to reduce NADP+ in red blood cells is used, among other biochemical properties, to characterize the deficient variants
Flow-cytometric analysis should be performed within one hour otherwise the relative proportion of normal and deficient red blood cells can drastically change over time, especially in blood from heterozygous women
In this study we confirm those findings and highlight the differences observed when activity is calculated with respect to Hb concentration or number of red blood cells (RBCs) in blood samples coming from subjects with anaemia and other blood conditions
Summary
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by mutations that impair the stability of the protein; the enzymatic capacity of the protein to reduce NADP+ in red blood cells is used, among other biochemical properties, to characterize the deficient variants. The technique is influenced by the concentration of haemoglobin in the blood sample and can result in an estimated value of G6PD activity that is falsely high in subjects with anaemia or iron deficiency. Factors such as reduced RBCs volume, increased number of reticulocytes or a combination of both are believed to contribute to the increase in assessed activity. Whilst it is not expected that subjects with severe G6PD deficiency would be mis-diagnosed even when anaemic, a correct quantitative characterization of phenotype in heterozygous women, including those with anaemia, is essential for the safe deployment of radical curative treatment for P. vivax using high dose primaquine or other 8-amoniquinolines. In the study populations a sizable proportion of healthy subjects were expected to be anaemic and/or carriers of haemoglobinopathies it was possible to analyse haematologic factors that may influence quantitative assessment of G6PD enzymatic phenotype with the two methodologies used
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