Validating the assessment of glucose 6 phosphate dehydrogenase g6pd kentuckyfishdating com
Upon testing this method on a cohort of Malian children, it was found to exhibit strong agreement with established methods of genetic and gross biochemical typing.
This assay represents a useful addition to the screening and research toolkit for G6PD deficiency, especially in malaria-endemic areas.
Genetic typing of G6PD variants has a long history that began shortly after the development of gel electrophoresis, with variant G6PD enzymes first identified on the basis of differential migration rate on starch gels.
Today, this has been largely superseded by DNA-based genotyping and sequencing, which enable the identification of hundreds of nonsynonymous coding mutations associated with G6PD deficiency worldwide, including many region-specific common variants.
Cytochemical typing provides a categorical assessment of G6PD enzyme activity (i.e., ‘normal’ or ‘deficient’) at the level of an individual RBC, and thus is the biochemical technique with greatest sensitivity for detecting mosaicism in female heterozygotes, the earliest assay developed, is a light microscopic technique that extends the aforementioned MRT by submerging cyanide-treated thin blood smears in an ethanol-based solution of peroxide and citric acid prior to conventional staining, with individual RBCs labeled according to their relative methemoglobin content.
Another more recently developed assay detects the quenching of glutaraldehyde-induced autofluorescence by formazan crystals produced in cells with normal G6PD activity, neither technique is particularly well suited to high-throughput applications.
Cytochemical assays can obviate these shortcomings, but at the expense of added technical complexity and labor.
We describe here a simple, novel cytofluorometric method that extends the classic methemoglobin reduction test, assessing G6PD deficiency at the level of an individual erythrocyte.
A straightforward cytochemical typing assay is presented here, termed the ‘cytofluorometric method’, which provides a fluorometric readout of the classic MRT at the level of an individual RBC.One indirect test with shorter incubation times and more consistent results is the methemoglobin reduction test (MRT), once a widely used screening tool in resource-poor areas.In this colorimetric assay, G6PD activity is assessed by first treating RBCs with nitrite (converting oxyhemoglobin [red] to methemoglobin [brown]), and then examining the rate of NADPH-dependent methemoglobin reduction in the presence of an appropriate redox catalyst (Nile blue or methylene blue) and substrate (glucose).Gross biochemical typing of G6PD involves either direct or indirect measures of enzyme activity in hemolysates.Direct tests are those that assess the enzyme's production of NADPH, which absorbs (340 nm) and fluoresces (450 nm) at characteristic wavelengths of light.