An improved, simple screening method for detection of glucose-6-phosphate dehydrogenase deficiency

We established a new, simple and rapid screening method for detection of glucose-6-phosphate dehydrogenase (G6PD)-deficiency by using a new formazan substrate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) with a hydrogen carrier of 1-methoxyphenazine methosulfate (1-methoxy PMS), instead of a combination of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and phenazine methosulfate (PMS), as used in many previous formazan methods. WST-8 does not react with haemoglobin, and the formed formazan is highly water-soluble, differing from MTT. Thus, the whole procedure can be performed in aqueous solution in a tube or well without any special equipment other than micropipettes. Within 1 h at room temperature, the strong orange colour of the WST-8 formazan formed in normal blood samples could be distinguished, by naked eye, from G6PD-deficient blood samples with less than 50% residual activity. We also found that reagents in the WST-8/1-methoxy PMS method were more resistant against exposure to sunlight than those in an MTT/PMS method. As the new method is both qualitative and quantitative, it is possible to express G6PD activity as increase of NADPH concentration by reading absorbance at 460 nm after incubation for 30 or 60 min.