The impact of hematocrit, punch location, and cellular interference on neonatal screening for biotinidase deficiency
1Department of Medical Biology and Genetics, School of Medicine, Ankara Medipol University, Ankara, Türkiye.
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, p<0.05). Lower hematocrit (HCT34) led to a significant overestimation of activity, yielding >20% 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<0.05), indicating that approximately 10% of the quantified activity in DBS is leukocyte-derived and constitutes a previously unaccounted source of analytical bias.
Conclusion:
This study is the first to definitively quantify the effects of key pre-analytical variables on fluorometric biotinidase testing. Punch location, hematocrit, and cellular content are not merely sources of noise but are significant confounders that can lead to both false-positive and false-negative results. We strongly recommend that DBS calibrators and patient samples be punched from the center of the spot and that second-tier testing interpretations consider individual infant hematological parameters to enhance the clinical sensitivity of newborn screening for biotinidase deficiency.
