G6PD activity patterns and a stable adjusted male median derived from 16 years of clinical laboratory data

Purpose: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy worldwide, poses significant clinical and public health challenges, particularly in malaria-endemic regions. Despite the importance of quantitative G6PD activity assessments, clinically meaningful deficiency thresholds remain difficult to define owing to biological variability related to age, sex, and X-linked inheritance. The adjusted male median (AMM) is a recommended framework for defining 100% activity and deriving assay- and region-specific thresholds. However, large-scale evaluations of AMM stability and G6PD activity patterns in Pakistan are limited. Methods: We retrospectively analyzed all quantitative G6PD assays performed at a national reference laboratory in Pakistan between January 2009 and December 2024. Records with valid G6PD activity (U/g hemoglobin (Hb)), age, and sex information were included. Age- and sex-stratified distributions were examined across predefined clinical age groups. The calculated AMM was based on adult males aged 18–45 years (excluding those with ≤ 10% of the initial median). Sensitivity analyses were used to assess AMM stability across alternative adult male age windows (15–50 and 18–60 years). The G6PD activity categories were defined as severe (< 10%), moderate (10–30%), intermediate (30–60%), and normal (> 60%) relative to the AMM. Results: In total, 51,748 specimens (41,397 from males and 10,351 from females) were analyzed. The median (interquartile range) G6PD activity was 10.3 (7.8–12.6) U/g Hb among males and 10.7 (8.5–12.9) U/g Hb among females. The AMM derived from 7,436 adult males was 9.2 U/g Hb and remained highly stable across alternative age windows (9.2–9.3 U/g Hb; ~ 1% variation). Based on AMM thresholds, 15.4% (95% CI: 15.1–15.7%) of the tested population had < 60% activity, including 6.8% with severe and 4.9% with moderate deficiency. G6PD activity was highest in neonates and infants and declined progressively with age. Conclusions: The locally derived AMM provides a robust reference standard that reflects the enzymatic capacity within the specific clinical catchment area and serves as a foundation for future clinical validation studies. Age- and sex-specific activity patterns aligned with known physiological and genetic determinants. Although this study provided a locally validated laboratory reference framework, prospective studies correlating the enzymatic categories with clinical outcomes and genotypic data are needed before its broader clinical or public health implementation.