Improved Specificity of NRBC Detection in Chorionic Villus Sample Supernatant Fluids Using Anti-Zeta and Anti-Epsilon Monoclonal Antibodies

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Abstract
Objective: Fetal erythrocytes leak from fetal capillaries at the time of chorionic villus sampling (CVS). It has been reported that in approximately 60% of CVS cases fetal nucleated red blood cells (NRBC) can be isolated from the supernatant fluid by immunophenotyping with monoclonal antibody (Ab) against the γ-chain of fetal hemoglobin and used as an additional source for confirmation of the fetal karyotype. However, the increased prevalence of β-thalassemia mutations in countries such as Greece results in many pregnant women who produce γ-positive cells. This makes it difficult to distinguish between the fetal and maternal origin of the NRBC. Use of Abs against embryonic hemoglobin chains ζ and ε may increase specificity for fetal NRBC detection. Methods: Mouse monoclonal Abs against Hb-ζ and Hb-ε were used in order to examine if specificity for fetal NRBC detection in CVS supernatant fluids could be improved. 41 samples were studied using anti-ζ and 20 using anti-ε monoclonal Abs. Results: Anti-ζ or anti-ε positive erythrocytes were, respectively, identified in 52 of 61 CVS samples and anti-ζ or anti-ε positive NRBC were present in all cases. The mean number of Hb-positive erythrocytes identified with the anti-ζ Ab was 58 and the mean number of NRBC 29. The mean number of anti-ε positive erythrocytes was 30 and of NRBC 23. FISH with X and Y chromosome specific probes was performed in 26 cases and the results were concordant with the CVS karyotype. Statistical analysis using the correlation test showed that anti-ζ and anti-ε were more specific for the detection of embryonic NRBCs. Conclusions: Since embryonic monoclonal Abs show increased specificity, they should be preferentially used for NRBC detection in CVS supernatant fluids. Furthermore, the increased specificity of anti-ζ and anti-ε Abs may considerably improve prenatal diagnosis from fetal cells isolated from maternal circulation.