A Orzińska1, K Guz, E Brojer, B Zupańska. 1. Department of Immunohematology and Immunology of Transfusion Medicine, Institute of Hematology and Transfusion Medicine, 14 Gandhi Street, 02 776 Warsaw, Poland. orzinska@ihit.waw.pl
Abstract
OBJECTIVES: Anti-Rhc antibodies may be the reason for the hemolytic disease of the newborn, therefore, noninvasive Rhc determination is important for pregnancy monitoring. For this purpose, we decided to introduce real-time polymerase chain reaction (PCR) method. METHODS: Blood from 200 donors, plasma and whole-blood from 11 Rhc-negative mothers, as well as blood from fathers and newborns were examined. Rhc sensitivity and specificity were first determined by real-time PCR using genomic DNA from donors. The same Rhc genotyping method was used for fetal Rhc detection in maternal plasma. To confirm the fetal Rhc-negative result, plasma was tested with a panel of biallelic insertion/deletion polymorphisms for the presence of fetal DNA. RESULTS: The c allele assay showed full specificity. The mean Ct value for one copy of c allele diluted in C-negative DNA was determined from extrapolating the correlation curve as 39.9. Full concordance was observed between the fetal Rhc genotypes from maternal plasma and the newborn phenotypes. CONCLUSIONS: Preliminary results show that it is possible to examine fetal c allele of RHCE gene in the plasma of pregnant women with anti-c by means of a noninvasive method. The diagnostic accuracy of the procedure, however, has yet to be confirmed in a larger group. Copyright (c) 2008 John Wiley & Sons, Ltd.
OBJECTIVES: Anti-Rhc antibodies may be the reason for the hemolytic disease of the newborn, therefore, noninvasive Rhc determination is important for pregnancy monitoring. For this purpose, we decided to introduce real-time polymerase chain reaction (PCR) method. METHODS: Blood from 200 donors, plasma and whole-blood from 11 Rhc-negative mothers, as well as blood from fathers and newborns were examined. Rhc sensitivity and specificity were first determined by real-time PCR using genomic DNA from donors. The same Rhc genotyping method was used for fetal Rhc detection in maternal plasma. To confirm the fetal Rhc-negative result, plasma was tested with a panel of biallelic insertion/deletion polymorphisms for the presence of fetal DNA. RESULTS: The c allele assay showed full specificity. The mean Ct value for one copy of c allele diluted in C-negative DNA was determined from extrapolating the correlation curve as 39.9. Full concordance was observed between the fetal Rhc genotypes from maternal plasma and the newborn phenotypes. CONCLUSIONS: Preliminary results show that it is possible to examine fetal c allele of RHCE gene in the plasma of pregnant women with anti-c by means of a noninvasive method. The diagnostic accuracy of the procedure, however, has yet to be confirmed in a larger group. Copyright (c) 2008 John Wiley & Sons, Ltd.