The impact of hematocrit, punch location, and cellular interference on neonatal screening for biotinidase deficiency

BACKGROUND: Biotinidase deficiency is a core condition in newborn screening programs worldwide. While fluorometric enzyme activity assays from dried blood spots (DBS) are the standard first-tier test, their accuracy can be susceptible to pre-analytical variation. To date, the specific impact of hematocrit (HCT), punch location, and cellular interference on fluorometric biotinidase measurements have not been systematically examined. METHODS: We prepared blood pools to isolate specific variables: a reference pool at 50% HCT (HCT50), a low-HCT pool at 34% (HCT34), and a leukocyte-depleted pool (HCT50(-W)). Secondary pools were created with biotinidase activities of 0, 50, 100, and 200 U. DBS samples were prepared from all pools. Biotinidase activity was measured fluorometrically from central and peripheral punches (n=12 per condition). Statistical analysis included t-tests and Cohen's d effect sizes, with a >10% deviation set as the threshold for clinical significance. RESULTS: Our results demonstrated a significant systematic bias: Peripheral punches yielded higher biotinidase activity than central punches across all sample types (13.36%–16.61% difference, p20% higher values at the critical biotinidase activity-50 U level. Crucially, leukocyte depletion resulted in a statistically significant decrease in measured activity (~10.6%, p