Evaluation of pre-analytical and analytical variables affecting galactose-1-phosphate uridyltransferase assay performance in the diagnosis of classical galactosemia.

Introduction: Accurate and rapid assessment of galactose-1-phosphate uridyl transferase (GALT) activity is critical for the diagnosis of Classical Galactosemia (CG). However, the performance of GALT assays is variable, and there is a lack of evidence to support best practices.

Materials And Methods: We established a rapid quantitative fluorometric GALT assay to evaluate the impact of blood collection-tubes, assay-composition, and sample storage conditions using whole blood (WB) and dried blood spot (DBS) specimens from control individuals, CG patients and individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency.

Results: Mean (SD) WB GALT activity was 3.1 (0.9) µmol/hr/mL in controls (n = 90), 0.08 (range 0.05-0.11) µmol/hr/mL in CG patients (n = 3) and 2.7 (0.8) µmol/hr/mL in G6PD deficient samples (n = 36). GALT activity in G6PD-deficient samples increased significantly (p < 0.01) with 0.04 U/L G6PD added to the reaction-mixture. DBS GALT activity was 2.2 (0.6) µmol/hr/mL in controls (n = 39) and 0.04 (range 0.0-0.11) µmol/hr/mL in CG patients (n = 3). WB and DBS assays showed excellent linearity (r > 0.99). Assay reaction-mixture containing magnesium chloride prevented EDTA-mediated inhibition of GALT. DBS samples remained stable at -20 °C for 28 days, but showed a 16 % decrease at room temperature (RT) after 7 days. WB was stable for 14 days at 4 °C and -20 °C, but only 2 days at RT. Two freeze-thaw cycles reduced WB activity by 17 %.

Conclusion: The assay accurately measured GALT activity in WB and DBS specimens without the need for Hb correction. Optimisation of legacy GALT assays and understanding of sample stability will ensure accurate results, preventing misdiagnosis and/or misinterpretation.