G6PD qualitative screening test

Physiological role
G6PD is a key enzyme in the hexose monophosphate pathway (HMP) and is required for the formation of NADPH. NADPH is essential for maintaining the integrity of the erythrocyte membrane.

Specimen collection and preparation
- Process specimen collected in EDTA tube
- Before analysis, check that the specimen is not clotted

Clinically significance
G6PD deficiency may be cause of hemolytic disease of newborns in Asian Mediterranean. Drug-induced hemolytic anaemia is most commonly associated in erythrocyte deficiencies of G6PD.
This deficiency is due to presence of a labile G6PD enzyme that is present in young cells but rapidly disappears with cell aging. A range of variant defective enzymes have been found in different racial group.

Performing G6PD screening test by R&D diagnostic G6PD deficiency screening test kit

- Label a filter paper (in test kit) with the running number

- Label 4 spaces in filter paper for Blank, Normal, Intermediate and Deficient control

- Pipette 100µ of substrate into all the tubes. Substrate must be at room temperature for test

- Mix the Normal control (a previous day’s specimen with G6PD present). Pipette 5µl into its labeled test tube containing the substrate, mix and immediately start the timer

- Using a sufficient time interval between sample, mix the Intermediate control and likewise pipette 5µl into its labeled test tube of substrate and mix.

- With the same time interval between samples, pipette the Deficient control and test samples accordingly

- When the timer reaches 10minutes, mix and pipette 10µl of the Normal control mixture onto its labeled portion on the filter paper

- Following the same time interval between samples, likewise mix and pipette the Deficient, Intermediate controls and the rest of the samples into their labeled position on the filter paper

- Pipette 10µl of the working substrate onto the position labeled “Blank”. Allow filter paper to dry completely and place it in the ultraviolet viewing cabinet

- Close the “Frontal Access Door” and switch on the “Long wave switch”

- View fluorescent through the viewing port which has ultraviolet absorbing filter to protect the eyes and increased fluorescent contrast

- Specimen from patient with normal G6PD activity will show strong fluorescence. Failure to fluorescence after 10minutes incubation suggests a total lack or marked deficiency of G6PD

- Strong fluorescence as compared to the intensity of the positive control, report as present. Report doubtful for weak or no fluorescence as compared with the intensity of the Intermediate deficient control

- Check doubtful specimen for blood clots
o If clot, repeat test with new specimen
o If no clot, repeat test
o All doubtful specimen are required to do G6PD quantitative determination

Yeo Sok Kian Jocelyn
0703359J