The CSG involved placing 2–3 μL whole blood into a receptacle within a plastic cassette,

followed by a few drops of hemolyzing reaction buffer provided with the kit. The cassette was visually read after standing 10 minutes at room temperature. Development of a distinct purple color in the cassette window represented a negative test outcome, whereas development of no color, or color distinctly lighter than most others, constituted evidence of a positive test outcome, that is, positive for G6PD deficiency. Fig 1 illustrates this distinction in color development. The CSG is this website composed of a cellulose strip impregnated with the G6P substrate of G6PD and a colorless tetrazolium compound salt (patent pending). Reduction of that compound yields a purple formazan dye. In the strip containing hemolysate and G6P substrate, the extent of reduction depends on G6PD activity. The package insert for this product specifies that a tested concentration of 0.156 mM

(2.5 mg/dL) CuCl did not impact with the assay system. The highest final concentration of CuCl in the G6PD activity assays did not exceed 0.04 mM (after dilution of RBC suspension in lysates). We thus considered CuCl interference in the assays by direct redox disturbance (as opposed to its known G6PD enzyme inhibitory properties) very unlikely. A total of 9 separate experiments over the course of several months using 2 known G6PD normal blood donors were conducted (see Fig 2). On each occasion a suspension of 0.45 mL whole blood mixed with 0.05 mL water served as the normal (no CuCl) G6PD activity control. In the case of the hemizygote model, 5 other tubes contained Belnacasan the same except with the addition of CuCl to water to provide final whole blood suspension of CuCl concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mM. In the case of the heterozygote model, blood was incubated with 1.0 mM CuCl in water or water only.

These were placed in Fludarabine cell line a 37°C water bath and incubated for 24 hours. After gentle mixing, these tubes were immediately treated essentially as whole blood in the conduct of the quantitative and qualitative G6PD assays as outlined previously in accordance with the standard instructions. In case of the hemizygote model, single tubes representing each of the inhibition treatments were aliquoted into 5 tubes. Each of those tubes was then used for all 3 of the G6PD assays that immediately followed: quantitative, FST, and CSG. Each of these 6 experiments thus generated 30 measurements of G6PD activity, 30 FST readings, and 30 CSG readings, or a total of 180 each. In the heterozygote model, each of the 10 distinct CuCl treatments (see Fig 2) were aliquoted into 3 vials, each generating a separate G6PD assessment, or 30 for each of the 3 separate experiments for a total of 90 assessments. In all, 270 separate assessments were conducted for each of the 3 distinct G6PD assays in both models.