Optimizing Biotinidase Activity Assays: Insights From Clinical and Laboratory Evaluation From a Low-Middle Income Country.

Objective This study aimed to verify the methods used for biotinidase deficiency (BTD) assays, including fluorometric and colorimetric techniques, measure biotinidase (BT) activity in dried blood spots (DBS) and serum samples, and explore the clinical spectrum of patients with BTD based on low serum BT activity. Methods A cross-sectional study was conducted at the Newborn Screening Lab, Aga Khan University, Karachi, following ethical approval from August 2021 to December 2024. The study was conducted in three phases. Phase 1 consisted of verification of performance characteristics (precision, accuracy, analytical measurement range, and linearity), according to Clinical Laboratory Improvement Amendments standards, of DBS-BT activity using a fluorometric enzyme immunoassay method. In phase 2, a colorimetric assay verified the performance characteristics of serum BT activity. Clinical evaluation of serum BT was assessed using data collected from 2021 to 2024 in phase 3. Results Phase 1: Precision of the DBS-BT assay, performed using level one and two controls, was acceptable, with a low coefficient of variation (CV) of 5.6%. Accuracy with proficiency specimens showed 100% agreement and an excellent correlation (r = 0.98). Phase 2: Precision of serum BT assay, performed using level one control, was acceptable, with a low CV of 4.86%. Accuracy with proficiency specimens showed 100% agreement and a correlation of r = 0.97. Phase 3: Clinical performance of DBS-BT samples (n = 438) and serum BT samples (n = 228) were conducted during the third phase. Clinical evaluation of serum BT samples revealed that 12.7% (n = 29) showed low BT activity and were diagnosed with BTD. These cases (82%, n = 23) were diagnosed after the neonatal/infantile period. In these patients, seizures were the most common clinical symptom (62%, n = 18), and high consanguinity was observed in 15 (51.7%) patient families. Conclusion These findings highlight the robustness of both DBS and serum BT tests in terms of precision, accuracy, and linearity. For diagnosing BTD, serum BT showed a good clinical detection rate. The delayed diagnosis and high consanguinity were noted, which emphasizes the need for enhanced screening programs and awareness, particularly in populations with high rates of consanguineous marriages.