B-210 Performance Evaluation of Different Analytical Methods for Fetal Gender Diagnosis

Background The fetal gender exam detects male genetic material in maternal blood using real-time PCR technique (Polymerase Chain Reaction), targeting Y chromosome-specific genes. According to the latest research, accurate results can be obtained from the 7th week of pregnancy, with over 90% accuracy. High laboratory demand has driven the search for highly sensitive and automated methodologies to provide early and reliable pregnancy results. The aim of this study was to compare the performance of three quantitative PCR methodologies for detecting male genes in maternal plasma samples. Methods This study evaluated 57 maternal blood samples collected in EDTA tubes with gel separators. Total DNA was extracted from plasma using the QIAsymphony DSP Virus/Pathogen Midi Kit® (Qiagen) on QIAsymphony automated equipment, following the manufacturer*s guidelines. Extracted DNA was amplified using the following methodologies: (i) in-house, with targets and protocol described by Ahmadi and collaborators; (ii) commercial method Fetal Gender® (MicoBiomed); and (iii) commercial method Investigator Quantiplex Pro® (IQP) (Qiagen). The in-house methodology targeted the SRY gene, responsible for activating male gonadal development, and DYS-14, expressed exclusively in testicles. The Fetal Gender® methodology used only the DYS-14 target. The methodologies differ about the housekeeping gene: in-house targets the ß-globin gene, while Fetal Gender® targets the GAPDH gene. The IQP method targets only human male genetic material, validated for diagnostic use according to CLSI guidelines. Of the 57 samples, 42 were analyzed with the IQP and in-house methods, and 15 with the IQP and Fetal Gender® methods. Comparisons were analyzed based on the presence or absence of the male gene and inconclusive results, with kappa index analysis and acceptance criteria =75%. The limit of detection (LoD) of the IQP methodology was calculated to measure the minimum detectable target amount. Results For the 42 samples compared between in-house and IQP methods, a concordance of 88.1% (37/42) and a kappa index of 79.8% were obtained of which six samples showed initially inconclusive results (in-house), which were reduced to three when processed by the IQP methodology. For 15 samples processed by Fetal Gender® and compared with IQP, 100% concordance and kappa index were achieved, and there were no inconclusive results. The LoD for the IQP methodology was 2.97 ng/µL, while the in-house and Fetal Gender® methods presented LoD of 2.7 ng/µL and 3.5 ng/µL, respectively. Conclusion Compared to the in-house method, the IQP method demonstrated a similar LoD but greater specificity, reducing the rate of inconclusive results by 50%. Regarding the Fetal Gender® method, the LoD was lower, indicating higher sensitivity. The specificity was the same for both methods, with no inconclusive results for either.