G6PD Assay

The glucose-6-phosphate dehydrogenase (G6PD) assay measures the activity of the first enzyme of the pentose phosphate shunt in red cells. The enzyme produces NADPH, which protects haemoglobin and the red cell membrane from oxidative injury. Deficient cells haemolyse after oxidant exposure, and the diagnosis depends on showing low enzyme activity at the right time.

The quantitative method measures NADPH formation spectrophotometrically at 340 nm in a haemolysate and reports units for each gram of haemoglobin. Qualitative methods include the fluorescent spot test and cytochemical or formazan ring tests, and handheld quantitative point-of-care meters are used where antimalarial safety testing is needed.

About 400 million people are affected worldwide. The gene is X-linked, so hemizygous males and homozygous females are deficient, and heterozygous females show a mosaic of normal and deficient cells. The condition is common in sub-Saharan Africa, the Mediterranean, the Middle East and Asia, where it likely reflects protection against malaria. The A- variant is common in people of African ancestry and the Mediterranean variant is common around the Mediterranean and in the Middle East.

Indications. Acute haemolysis with bite cells or Heinz bodies, haemolysis after drugs or fava beans, neonatal jaundice (particularly early and severe), unexplained haemolytic anaemia, before primaquine or tafenoquine, before rasburicase in at risk patients, and family screening.

Specimen. EDTA blood. Test promptly, since enzyme activity decays on storage.

Reference activity. Assay specific. A typical quantitative adult range is about 4.6 to 13.5 U/g Hb, so use the local laboratory range.

Activity classes. Activity is graded by residual percentage of normal. Class I deficiency (severe, with chronic non-spherocytic haemolytic anaemia) and class II (severe, activity under about 10%) cause marked haemolysis. Class III (about 10 to 60%) causes haemolysis with oxidant triggers, for example in the A- variant. Class IV is normal activity.

Adjusted male median. For antimalarial decisions, results are expressed as a percentage of the adjusted male median. Deficiency is under about 30%, intermediate activity about 30 to 70%, and normal activity over about 70%. Tafenoquine is withheld unless quantitative activity is at least 70%.

Qualitative tests. They detect activity under about 30 to 40% in males. They miss most heterozygous females, so a quantitative test is needed before an oxidant drug in women.

Typical presentation. Acute intravascular haemolysis 24 to 72 hours after exposure, with jaundice, dark urine, a falling haemoglobin, and a film showing bite cells, blister cells, hemighosts and Heinz bodies (shown with supravital staining). The direct antiglobulin test is negative.

High risk triggers. Fava beans (favism, particularly in the Mediterranean variant), primaquine, tafenoquine, dapsone, rasburicase, pegloticase, methylthioninium chloride (methylene blue), and naphthalene.

Lower or dose dependent risk. Sulphonamides including co-trimoxazole, nitrofurantoin, nalidixic acid, and infection, which is the commonest trigger.

Variants. The A- variant (activity about 10 to 20%) produces self-limited haemolysis, since young red cells retain enzyme. The Mediterranean variant (activity under 10%) produces more severe haemolysis that persists while the oxidant is present.

Neonatal disease. Jaundice in the first 24 to 72 hours, sometimes with a normal haemoglobin. It can reach levels that cause kernicterus.

Critical findings needing immediate action.

• Rasburicase and methylthioninium chloride are contraindicated in G6PD deficiency, and testing is needed before their use in at risk patients.
• Severe acute haemolysis with haemoglobinuria or acute kidney injury needs urgent management.
• Neonatal jaundice with deficiency needs urgent bilirubin assessment.

Pitfalls.

• During and soon after acute haemolysis, reticulocytes and young cells have higher activity, which gives a falsely normal result. Repeat 2 to 3 months later when the result is borderline.
• Recent transfusion gives a falsely normal result, so wait at least 3 months.
• Leucocytosis and thrombocytosis raise activity unless cells are washed.
• Heterozygous females can have normal or borderline activity despite a risk of haemolysis, and mosaicism complicates interpretation.
• A low enzyme activity may reflect a lowered haemoglobin, so adjust results for haemoglobin concentration.

Special groups.

• Neonates: cord blood or early screening is used in some programmes, and early jaundice needs prompt assessment.
• Pregnancy and breastfeeding: oxidant drugs (primaquine, sulphonamides, nitrofurantoin) need review, and fava beans are avoided.
• Women: quantitative testing is needed before primaquine or tafenoquine because of mosaicism.

Follow up tests. Full blood count, reticulocyte count, blood film with supravital stain for Heinz bodies, haemolysis screen, direct antiglobulin test, methaemoglobin level, G6PD gene sequencing for the variant, family testing, repeat enzyme assay after recovery.