G6PD Deficiency Screening Assay

On each day when the assays were undertaken a set of controls (normal, moderately deficient, severely deficient, no-enzyme) were taken and spotted onto 3 MM filter paper. A single ¹/₁₆ inch diameter disc (FISKARS Hand Punch) was punched out from each blood-spot sample, and placed in a single well of a 96-well flat bottom microplate (Greiner Bio-One). Control spots and an extra bloodspot for no-substrate control were also punched and placed in allocated wells. One well remained empty and served as a "no sample" negative control. 200 μL of working assay was then pipetted and mixed into each well except the substrate negative control well, into which 200 μL of no-substrate assay mix was added. Plates were then incubated for 2 hours at ambient temperature.
96 well plates were first inspected by eye, and then absorbance quantified in a microplate reader (Chromate 4300, Awareness Technology) at OD_450-595 . Normal G6PD activity is indicated by dark yellow to orange colouration, while severe and moderate deficiency appearing as almost colourless, and faintly yellow colouration respectively (Figure 1). Although for the purpose of this study only deficient (severe or moderate)/normal status were recorded, it was possible to distinguish intermediate from normal activity by their intermediate yellow colouration and absorbance.