Actual status and perspectives of the noninvasive prenatal diagnostics in feto-maternal incompatibilities

Nieinwazyjna diagnostyka prenatalna jest oparta na analizie pozakomórkowego DNA płodu obecnego w osoczu kobiet w ciąży. W niniejszej pracy poglądowej autorki przeanalizowały przyczyny stosowania obecnie tej metodologii tylko do genotypowania niektórych antygenów krwinek czerwonych płodu. Podsumowały też doświadczenia Instytutu Hematologii i Transfuzjologii w prenatalnej diagnostyce konfliktów matczyno-płodowych w zakresie antygenów RhD, RhC+D, Rhc, RhE i K oraz omówiły perspektywy rozwoju badań prenatalnych na podstawie nowoczesnych technologii: digital PCR, spektrometrii mas MALDI-TOF, sekwencjonowania nowej generacji. J. Transf. Med. 2010; 4: 144–154Non-invasive prenatal diagnostics is based on the analysis of cell free fetal DNA present in the plasma of pregnant women. In this review we analysed the reasons why this methodology can be currently used only for genotyping of some fetal red blood cell antigens. We also summerised the experience of the Institute of Haematology and Transfusion Medicine in prenatal diagnostics of feto-maternal incompalibilities against RhD, RhC+D, Rhc, RhE and K antigens and discussed the perspectives of development of prenatal tests based on modern technologies: digital PCR, mass spectrometry MALDI-TOF, new generation sequencing. J. Transf. Med. 2010; 4: 144–15

RHD variant in RhD/-/ mother with anti-D makes noninvasive fetal RHD genotyping impossible

Abstract Objectives: Noninvasive fetal RHD genotyping from maternal plasma of RhD(/-) pregnant women of Caucasian race may be used for predicting the risk of hemolytic disease because the RHD gene is usually absent in such populations. If detected in plasma of such women, the RHD gene originates from the RhD(+) fetus. The number of fetal copies of the gene in maternal plasma is extremely small. In the presented case of the RhD(/-) pregnant woman with anti-D it was impossible to give a fetal RHD result due to mother’s RHD(+) genotype. The fetal RHD was determined from amniocytes. Aim: to present the difficulties related to the interpretation of results of invasive and noninvasive procedures. Material and methods: whole blood, plasma and amniotic fluid of the RhD(-) woman with anti-D (14 week of pregnancy) as well as whole blood of the newborn. RHD and RHCE*c were genotyped by real-time PCR in DNA isolated from maternal plasma and amniocytes and the RHD and d-genotypes were tested by SSP methods in DNA isolated from whole blood and amniocytes . Results: RHD and RHCE*c were detected in DNA isolated from plasma. The high level of RHD suggested its origin from the mother’s DNA therefore it was impossible to determine the fetal RHD. The d-little test identified a RHD(IVS3+1G>A) variant in the mother’s genome. A weak signal of real-time PCR for the RHD was obtained in amniocytes but the RHD was not detected by SSP. The RHCE*c was detected by both methods. Results were inconclusive; the fetal RHD status remained unknown. The child was RhD(-) with RHD in its DNA undetected by either method. Conclusions: 1/ The RHD(IVS3+1G>A) variant in the RhD(-) mother precluded formal noninvasive fetal RHD genotyping. 2/Real-time PCR is too sensitive for amniocyte testing and may lead to false results as it detects trace maternal DNA in amniotic fluid 3/ The frequency of RHD(IVS3+1G>A) occurrence in Poland requires further studies