Question

Q/ summarise the principle of qualitative and quantitive methods to measure G6PD in the laboratory.

Answer 1

Qualitative Test for measurement of G6PD

Fluorescent Spot Test

G6PD catalyzes a reaction in the pentose phosphate pathway, a metabolic pathway that supplies reducing energy to cells such as RBC through retaining the intensity of the reduced type of the co-enzyme NADPH. G6PD is critical for defending erythrocytes in the existence of oxidizing agents. The fluorescent spot test (FST) for G6PD deficiency engrosses this reaction:

Glucose-6-Phosphate + NADP (Not fluorescent)

                                 G6PDH

6-Phosphogluconate + NADPH (Fluorescent)

A miniature quantity of blood is incubated with glucose-6-phosphate and NADP in the substrate reagent, and then is spotted on filter paper. Once desiccated, the spots are observed under long-wave ultraviolet (UV) light—the by-product of the reaction (NADPH) is fluorescent. NADPH fluorescence is directly proportional to G6PD activity and lack of fluorescence signals G6PD deficiency.

Quantitative Test for evaluation of G6PD activity

WST8/1-methoxy PMS Test

The principle of the WST8/1-methoxy PMS assay depends on reducing hydrogen from NADPH translating WST8 to WST8-formazan in the occurrence of the hydrogen carrier 1-methoxy-PMS. This reaction gives in a tough straightforwardly measurable orange colour, with colour strength openly relative to G6PD activity. After a 2 hr incubation at room temperature, samples with typical G6PD activity demonstrate sturdy orange colour, scarce samples illustrate faint colour (moderate deficiency liable to characterize heterozygotes) or no colour (stern shortage & negative controls). The test has been modified for quantitative batch testing in a 96 well microplate and can be interpret by ELISA reader.