G6PD Deficiency, enzyme class, haemolytic triggers, newborn screening and diagnosis

Overview

• Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent human enzyme defect, affecting hundreds of millions of people worldwide, with highest frequencies in sub-Saharan Africa, the Mediterranean basin, the Middle East, and South and Southeast Asia.
• Its geographic distribution closely mirrors that of historical malaria endemicity, consistent with a heterozygote survival advantage against Plasmodium falciparum.
• The condition is X-linked recessive; males (hemizygotes) are predominantly affected clinically, whilst females may be hemizygous (rare), homozygous deficient, or heterozygous carriers with variable phenotypic expression due to X-chromosome inactivation (lyonisation).

Beyond G6PD, clinically important red cell enzymopathies include:

• Pyruvate kinase (PK) deficiency: most common glycolytic enzymopathy; chronic hereditary non-spherocytic haemolytic anaemia (HNSHA)
• Glucose phosphate isomerase (GPI) deficiency: second most common chronic haemolytic glycolytic enzymopathy
• Pyrimidine 5′-nucleotidase-1 (P5′N1) deficiency: chronic haemolysis; marked basophilic stippling
• Glutathione pathway enzyme deficiencies: variable acute or chronic haemolysis with Heinz body formation
• Most red cell enzyme disorders are autosomal recessive (homozygous or compound heterozygous).
• G6PD and phosphoglycerate kinase are X-linked; adenosine deaminase overactivity and prolyl hydroxylase 2 deficiency are autosomal dominant exceptions.

Pathophysiology Relevant to Laboratory Testing

G6PD and the Pentose Phosphate Pathway

The pentose phosphate pathway (hexose monophosphate shunt) is the sole source of NADPH in mature red cells:

$$\text{Glucose-6-phosphate} + \text{NADP}^+ \xrightarrow{\text{G6PD}} \text{6-Phosphogluconate} + \text{NADPH} + \text{H}^+$$

$$\text{6-Phosphogluconate} + \text{NADP}^+ \xrightarrow{\text{6-PGD}} \text{Ribulose-5-phosphate} + \text{CO}_2 + \text{NADPH} + \text{H}^+$$

NADPH sustains glutathione reduction via glutathione reductase:

$$\text{GSSG} + \text{NADPH} + \text{H}^+ \xrightarrow{\text{Glutathione reductase}} 2\,\text{GSH} + \text{NADP}^+$$

• GSH neutralises hydrogen peroxide and other reactive oxygen species (ROS), maintains haemoglobin in its functional reduced state, and preserves membrane integrity.
• In G6PD deficiency, failure to regenerate NADPH depletes GSH.
• Under oxidative stress (drugs, fava beans, infection, diabetic ketoacidosis), haemoglobin is oxidised and precipitates as Heinz bodies (insoluble denatured haemoglobin).
• These rigid inclusions cause splenic trapping and intravascular haemolysis.
• Characteristic blood film findings include blister cells (haemoglobin separated from membrane) and contracted, deeply staining cells.

Glycolytic Pathway and Chronic Haemolysis

• Glycolytic enzyme deficiencies (e.g. PK, GPI) impair ATP generation, leading to chronic haemolysis by mechanisms not fully understood.
• These patients are not susceptible to oxidant-triggered haemolytic crises and red cells show no Heinz bodies.
• P5′N1 deficiency disrupts pyrimidine nucleotide catabolism during reticulocyte maturation; accumulation of pyrimidine nucleotides inhibits glycolytic enzymes and causes chronic haemolysis with prominent basophilic stippling.

Critical Pitfall: Testing Timing

During acute haemolysis, the most severely G6PD-deficient cells are preferentially destroyed, leaving a younger population of reticulocytes and stress reticulocytes with relatively higher G6PD activity. This produces a falsely normal or borderline enzyme assay, most pronounced in:

• Hemizygous males with Class III variants (e.g. G6PD A⁻)
• Heterozygous females (already mosaic; deficient cells selectively destroyed before reticulocyte response)
• Screening tests and quantitative assays should ideally be deferred until 2-3 months after the acute episode when the red cell age distribution normalises.
• If testing during acute haemolysis is unavoidable, molecular analysis (from leucocyte DNA) bypasses this pitfall entirely.
• A "normal" G6PD result in the context of a reticulocyte count >10-15% warrants repeat testing after recovery.

Similarly, for PK deficiency, reticulocytes have higher PK activity, concurrent reticulocytosis may partially mask deficiency.

WHO Classification of G6PD Variants

• Variants are classified by residual enzyme activity and clinical expression.
• Criteria for identification include: red cell G6PD activity, electrophoretic mobility, Michaelis constant ($K_m$) for glucose-6-phosphate and NADP⁺, relative utilisation of 2-deoxy-glucose-6-phosphate (2dG6P), and thermal stability.

• Clinically important variants include G6PD A⁻ (African populations; Class III, ~12% residual activity; milder) and G6PD Mediterranean (Class II, <1% residual activity; more severe, triggered by fava beans).
• Over 200 variants are characterised.
• G6PD variants range in activity from nearly 0% to 500% of normal.

Screening Tests for G6PD Deficiency

• Screening tests exploit the principle that G6PD reduces NADP⁺ to NADPH when glucose-6-phosphate is provided as substrate.
• The method of NADPH detection determines the test type.
• Choice of screening test depends on cost, time required, temperature and humidity conditions, and reagent availability.

Fluorescence Spot Test (Beutler-Mitchell Test)

The most widely used test; recommended by the International Council for Standardisation in Haematology (ICSH).

• Principle: NADPH fluoresces under long-wave UV light (365 nm); NADP⁺ does not.
• A red cell lysate is incubated with glucose-6-phosphate and NADP⁺.
• Adequate G6PD activity produces NADPH, yielding fluorescence.
• Absent or markedly reduced fluorescence indicates G6PD deficiency.
• GSSG is included in the reaction mixture as a NADPH consumer to widen the assay's dynamic range.

Method summary:

1. Whole blood (EDTA- or heparin-anticoagulated) lysate is mixed with reaction mixture containing glucose-6-phosphate, NADP⁺, saponin, and GSSG.
2. Spots are placed on filter paper at defined time points (0, 5, 10 minutes) and dried.
3. Dried spots are examined under long-wave UV light.

Interpretation:

Limitations:

• Semi-quantitative only; does not provide enzyme units
• Cannot reliably detect heterozygous females (mosaic expression due to lyonisation)
• False-normal results during acute haemolysis with reticulocytosis
• Requires UV light source; sensitive to temperature, humidity, and reagent freshness

Methaemoglobin Reduction Test (Brewer Test)

• Principle: Sodium nitrite oxidises haemoglobin to methaemoglobin.
• In normal red cells, NADPH generated by G6PD reduces methylene blue, which in turn reduces methaemoglobin back to oxyhaemoglobin.
• G6PD-deficient cells fail to reduce methaemoglobin.
• Method: Blood incubated with sodium nitrite and methylene blue at 37°C for 60-180 minutes.
• Normal blood returns to red (oxyhaemoglobin); G6PD-deficient blood remains brown (methaemoglobin).

Limitations:

• Less sensitive than the fluorescence spot test
• Subjective colour endpoint
• Cannot reliably detect heterozygotes

Dye Decolourisation / Dye-Reduction Tests

• Artificial dyes such as brilliant cresyl blue, dichlorophenolindophenol (DCPIP), or tetrazolium salts (e.g. diphenyltetrazolium bromide with phenazine methosulphate, forming a blue formazan deposit) are used as electron acceptors.
• Decolourisation or deposit formation indicates NADPH production and functional G6PD.
• These are low-cost and suitable for resource-limited or field settings but are less standardised.

Detection of Heterozygotes for G6PD Deficiency

• Heterozygote identification is challenging due to lyonisation producing a mosaic of normal and deficient cells.
• Population-average enzyme activity may fall anywhere across the normal range, particularly if deficient cells undergo selective haemolysis before a reticulocyte response.

• Cytochemical test detail: Red cells incubated on a film with glucose-6-phosphate, NADP⁺, phenazine methosulphate, and MTT.
• G6PD-normal cells reduce MTT to a blue formazan deposit (visible per cell); G6PD-deficient cells remain unstained.
• In heterozygous females, two populations are visible.
• This test also allows confirmation of deficiency during the post-haemolytic reticulocyte-rich phase, when younger (less deficient) cells still stain and profoundly deficient (Class I/II) cells remain pale.

Quantitative G6PD Enzyme Assay

Required when screening tests are positive or equivocal, for precise variant classification, or borderline heterozygote evaluation.

• Principle: The rate of NADPH generation is measured spectrophotometrically as an increase in absorbance at 340 nm.
• Results are expressed as enzyme units (EU) per gram of haemoglobin (measured at 25°C).

$$\text{G6P} + \text{NADP}^+ \xrightarrow{\text{G6PD}} \text{6-Phosphogluconate} + \text{NADPH}$$

Reference values and clinical interpretation:

Values are illustrative; laboratory-specific reference ranges apply.

Technical considerations:

• Assay must be performed on washed red cells free of leucocytes (which contain abundant G6PD)
• Strict temperature control (25°C standard)
• Interpret in conjunction with reticulocyte count; concurrent reticulocytosis mandates cautious interpretation and possible repeat assay 2-3 months post-episode

Identification of G6PD Variants

Definitive variant characterisation for research, population studies, or complex clinical scenarios requires criteria established by WHO and subsequently revised:

Indications for molecular analysis:

• Large-volume prior transfusion (patient's enzyme level obscured by donor cells)
• Reticulocytosis confounding biochemical assay
• Female heterozygote confirmation
• Variant-specific clinical management decisions

Screening for Other Red Cell Enzyme Deficiencies

Pyruvate Kinase (PK) Deficiency

• PK catalyses conversion of phosphoenolpyruvate (PEP) + ADP → pyruvate + ATP.
• Deficiency impairs ATP generation, causing chronic HNSHA.
• Haemolysis is continuous, not triggered by oxidant stress; no Heinz bodies.
• Blood film shows poikilocytosis and "prickle" cells, especially post-splenectomy.
• Treatment includes mitapivat (small-molecule oral PK activator; haemoglobin increase in 40-50% of adults with durable responses); splenectomy alleviates but does not cure anaemia.

• Critical caveat: Leucocytes contain the M2-PK isoform in abundance; red cell preparations must be thoroughly leucocyte-depleted to avoid falsely elevated results.
• Reticulocytes also have higher PK activity, reticulocytosis may partially mask deficiency.

Pyrimidine 5′-Nucleotidase-1 (P5′N1) Deficiency

• P5′N1 degrades pyrimidine nucleotides generated during reticulocyte maturation.
• Deficiency leads to accumulation of pyrimidine nucleotides, which inhibit glycolytic enzymes, causing chronic haemolysis.
• A hallmark is prominent basophilic stippling (differential includes lead poisoning and thalassaemia).
• Screening: Red cell lysate UV absorbance.
• Normal red cell nucleotides peak at 257 nm (adenine nucleotide predominance).
• In P5′N1 deficiency, accumulated pyrimidine nucleotides shift the peak toward 270 nm.
• An elevated 270/257 nm absorbance ratio is diagnostic.

Glutathione-Related Enzyme Deficiencies

Deficiencies of glutathione synthetase, γ-glutamylcysteine synthetase, or glutathione reductase impair oxidative defence, producing intermittent or chronic haemolysis with Heinz body formation.

Screening:

• Reduced glutathione (GSH) estimation: measures absolute GSH content of red cell lysate
• Glutathione stability test: GSH measured before and after incubation with acetylphenylhydrazine (an oxidant). Significant GSH depletion (>60% reduction) indicates instability; normal cells retain >70-80% of GSH

Note: 6-phosphogluconate dehydrogenase (6PGD) deficiency, although documented, appears to have little or no clinical significance for red cell viability, likely because NADPH is already generated by the upstream enzyme G6PD.

Diagnostic Approach to Suspected Red Cell Enzymopathy

Special Considerations

Neonatal Testing

• G6PD-deficient neonates are prone to exaggerated unconjugated hyperbilirubinaemia.
• This is not solely haemolytic, G6PD deficiency in hepatocytes impairs neonatal bilirubin conjugation.
• Co-existing Gilbert syndrome (UGT1A1 promoter variant) substantially worsens neonatal hyperbilirubinaemia.
• Severe cases require phototherapy or exchange transfusion to prevent kernicterus.
• In endemic populations, screening neonates (cord blood or heel-prick) using the fluorescence spot test is important.
• G6PD-deficient babies should be monitored for neonatal jaundice.

Blood Transfusion and Enzyme Testing

• Transfusion with G6PD-normal donor cells dilutes and masks the patient's deficiency in biochemical assays.
• Molecular analysis from leucocyte DNA remains valid in this context.
• In endemic areas, donor blood should be screened for G6PD deficiency before transfusion to premature neonates or use in neonatal exchange transfusion, given the risk of haemolysis and severe hyperbilirubinaemia, including increased serum LDH and bilirubin that may mimic a transfusion reaction.

Drugs and Oxidant Triggers

Many common drugs (aspirin, quinine, penicillin) have been reported to precipitate haemolysis in some patients but not at conventional dosage.

Summary of Key Principles for Examination

• The fluorescence spot test (Beutler-Mitchell; ICSH-recommended) detects NADPH generation under long-wave UV light and is the standard first-line G6PD screen
• False-normal results during acute haemolysis occur because severely deficient cells are selectively destroyed and residual reticulocytes have higher enzyme activity, a critical diagnostic pitfall for both haemizygous males and heterozygous females
• Cytochemical testing identifying two red cell populations (normal and deficient using formazan-based MTT reduction) is the most reliable method for detecting female heterozygotes
• Quantitative spectrophotometric assay (expressed in EU/g Hb, measured at 340 nm) is required for definitive activity measurement and WHO variant classification; leucocyte removal is mandatory
• Molecular analysis (targeted sequencing or NGS) is gold standard for variant identification, heterozygote confirmation, and testing when transfusion confounds biochemical results
• WHO variant classification (Classes I-V) is based on residual enzyme activity and clinical expression; criteria include activity, electrophoretic mobility, $K_m$, 2dG6P utilisation, and thermal stability
• For PK deficiency, the fluorescence spot test shows persistence (not loss) of NADH fluorescence; leucocyte depletion of specimens is mandatory; mitapivat is an approved treatment
• P5′N1 deficiency produces prominent basophilic stippling and an elevated UV absorbance ratio (270/257 nm) in red cell lysates
• Enzyme deficiency testing should ideally be performed outside an acute haemolytic episode with a normal or near-normal reticulocyte count; if testing cannot be deferred, molecular analysis is preferred