G6PD screening (fluorescent spot test)

Principle

• G6PD catalyses oxidation of glucose-6-phosphate, reducing NADP+ to NADPH.
• NADPH fluoresces under long-wave UV light (about 365 nm); NADP+ does not. Lack of fluorescence indicates deficient enzyme activity.

Specimen

• EDTA, heparin or ACD blood; stable for several days at 4 °C.

Reagents and equipment

• Reagent containing glucose-6-phosphate, NADP+, oxidised glutathione, saponin (to lyse cells) and buffer (Beutler method).
• Whatman filter paper, long-wave UV lamp, water bath at 37 °C (or room temperature per kit).

Procedure

• Mix a small volume of blood with reagent.
• Spot onto filter paper immediately (0 min) and after incubation (commonly 5 and 10 min).
• Allow spots to dry completely and view under UV light in a dark room.
• Test normal and deficient controls in each run.

Results

• Normal: bright fluorescence of later spots.
• Deficient: little or no fluorescence.

Quality control and pitfalls

• False normal: during or soon after a haemolytic episode (older deficient cells destroyed; young cells and reticulocytes have higher activity), after transfusion, and with high WBC count.
• Heterozygous females may have intermediate activity and be missed; use a quantitative assay.
• Severe anaemia can reduce fluorescence; follow kit guidance.
• Repeat testing about 2–3 months after haemolysis if the first result is normal.

Clinical use

• Screening for G6PD deficiency in neonatal jaundice, acute haemolysis after oxidant drugs (e.g. primaquine, dapsone, rasburicase), infection or fava beans.
• Required before primaquine or tafenoquine malaria radical cure.
• Abnormal screens are confirmed by quantitative spectrophotometric assay (NADPH at 340 nm).