B-361 Method Validation of Human Chorionic Gonadotropin (hCG) and a-Fetoprotein (AFP) in Cerebrospinal Fluid (CSF)

Abstract Background Intracranial germ cell tumors (GCTs), accounting for 2%–3% of pediatric brain tumors, are diagnostically challenging. GCTs are classified into germinomas and nongerminomatous germ-cell tumors (NGGCTs), which include yolk sac tumors, choriocarcinomas, and mixed tumors. Neuroimaging alone often cannot differentiate GCTs from other tumors, making biomarkers like a-fetoprotein (AFP) and human chorionic gonadotropin (hCG) in cerebrospinal fluid (CSF) crucial for diagnosis. Elevated AFP indicates yolk sac tumors, while increased hCG suggests choriocarcinomas. Measuring these markers in CSF is more reliable than in serum and aids in monitoring treatment response and detecting recurrence. Matrix effects can impact test results when alternative sample types are used; therefore, validation is essential before clinical implementation. In this study, we validated AFP and hCG assays on the Abbott Alinity I platform. Methods To prepare the samples, clear and colorless CSF samples were initially tested to confirm the absence of alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG). Subsequently, serum samples with high concentrations of AFP and hCG were spiked into the CSF to create a range of concentrations. Linearity/Recovery Linearity was initially evaluated by testing singlet CSF samples with varying AFP and hCG concentrations across the serum AMR, which spans 0.1 to 2000 µg/L for AFP and 1.2 to 5000 IU/L for hCG, to determine the proposed linearity range. The linearity range was then confirmed by testing newly prepared samples within the proposed range in duplicate or triplicate. Precision Within-day imprecision was assessed by measuring two pools 10 times in a single day at target concentrations of 25, 500, and 1500 µg/L for AFP and 25, 1000, and 4000 IU/L for hCG. Day-to-day imprecision was evaluated by assaying individual aliquots four times daily over five days at the same target concentrations. Limit of quantitation LOQ was defined as the lowest concentration measured with a coefficient of variation (CV) <20% and total error <20%. Samples were assayed eight times per day over five days. Four CSF specimens containing AFP and hCG at concentrations ranging from 0.1 to 1.0 µg/L (0.1, 0.2, 0.5, 1.0) and 1.2 to 3 IU/L (1.2, 1.5, 2, 3), respectively, were analyzed to determine the LOQ. Sample Stability Sample stability was evaluated at 25°C (48 hours) and 4°C (12 days) to assess potential changes in analyte concentration over time. Stability was considered acceptable if the variation in concentration remained within 15% of the original test result. Carryover The carryover is assessed by comparing the response of a high-concentration sample (approximately 80% of the ULOQ) following a CSF blank. Results The method showed a linear range of 0.1–2000 µg/L for AFP and 2–5000 IU/L for hCG in CSF, with no significant matrix effect. Precision was <5% CV, and CSF samples remained stable for 48 hours at 25°C and 12 days at 2–8°C. LOQ was 0.1 µg/L for AFP and 2 IU/L for hCG, with no significant carryover. Conclusions: The Abbott Alinity I AFP and hCG assays accurately quantify AFP and hCG in CSF.

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