Direct PCR-Based VNTR Analysis of TPO Intron 10 for Rapid Detection of Maternal Cell Contamination in Prenatal Diagnosis.

Maternal cell contamination (MCC) in fetal specimens poses a major risk for misdiagnosis in prenatal genetic testing. Standard variable number tandem repeat (VNTR) analysis of the thyroid peroxidase (TPO) gene intron 10 is informative for MCC detection but traditionally requires DNA extraction, limiting its use in resource-limited laboratories. This study aimed to develop and validate a direct PCR-based VNTR assay for MCC detection without DNA extraction. Leftover whole blood and amniotic fluid specimens from 90 families undergoing prenatal diagnosis for severe thalassemia were analyzed, with white blood cell and amniotic fluid pellets directly subjected to PCR amplification of the TPO intron 10 VNTR locus using a high-pH buffer to overcome PCR inhibitors. The direct PCR results were compared with standard DNA-based VNTR analysis in a blinded study, and VNTR heterozygosity at the TPO locus was also assessed in 148 unrelated Thai individuals to evaluate marker informativeness. Among 253 specimens, informative VNTR patterns were observed in 65 families (72.2%), with MCC ruled out in 64 families (71.1%) and detected in 1 family (1.1%). Uninformative VNTR patterns occurred in 25 families (27.8%) due to maternal homozygosity or similarity between maternal and fetal VNTRs. Direct PCR showed 100% concordance with standard DNA-based VNTR analysis. The heterozygosity rate of the TPO intron 10 VNTR was 87.8% among unrelated Thai individuals, supporting its suitability for routine MCC screening. The developed direct PCR protocol is a practical, rapid, and reliable tool for MCC detection, streamlining laboratory workflows and ensuring accurate prenatal diagnosis in low-resource settings.