Principle
G6PD is the first enzyme in the HMP shunt pathway, which protects red cells from any oxidant injury by providing source of NADPH. G6PD enzyme deficiency is X-linked recessive disease and is more common in male. Sodium nitrite converts Hb to Hi. When methylene blue is not added, methaemoglobin persists, but incubation of the samples with methylene blue allows stimulation of the pentose phosphate pathway in subject with normal G6PD level. The Hi is reduced during the incubation period. In G6PD-deficient subject, the block in the pentose phosphate pathway prevents this reduction.
Primary sample
4 cc fresh (< 1 hour) whole blood in EDTA vaccute. Wash 3 times in normal saline and prepare 50% cell suspension.
Reagents
1) Sodium nitrite
2) Dextrose
3) Methylene blue: 150 mg in 1000 ml distilled water
Combined reagent: 1.25 grams of sodium nitrite + 5 grams of dextrose dissolved in 100 ml of distilled water.
0.2 ml of the combined reagent is placed into individual glass tubes. Glass tubes must be used because plastic may adsorb some reagents. The contents of the tubes are allowed to evaporate to dryness at room temperature (15-25o C) and the tubes must be tightly sealed.
Method
1) Add 2 ml of whole / 50% red cell suspension to the tube containing 0.1 ml of the combined reagent either freshly prepared or dried. Add 0.1 ml of methylene blue. Close the tube with a stopper and gently mix the contents by rotating by hand for 5 minutes.
2) Prepare control tubes
Normal reference tube: 2 ml of blood in a similar tube without reagents.
Deficient reference tube: 2 ml of blood to a tube containing 0.1 ml sodium nitrite dextrose mixture.
3) Incubate the sample at 37o C for 3 hours.
4) After 10 minutes of incubation, inspect the test sample tube to check if the color is brown. This indicates the conversion of Hb to methemoglobin and that the reagent is working.
5) After the incubation, pipette 0.1 ml volumes from the test sample, the normal reference tube, and the deficient reference tube into 10 ml of water in separate, clear glass test tubes of identical diameter.
6) Mix the contents gently.
7) Compare the colors in the different tubes.
Result
Normal blood yields a colour similar to that in the normal reference tube (clear red). The brown colour at 10 minutes is re-converted to a red colour if G6PD is normal.
Deficient subjects give a brown color similar to that in the deficient reference tube at 3 hours.
Potential source of variability
1) After 1 hour, the G6PD enzyme activity of RBC starts decreasing. So result is unreliable, if fresh sample is not used.
2) A low hematocrit level can lead to a false negative result.
Image source: Horiba