Frederik Banch Clausen

Report of the first nationally implemented clinical routine screening for fetal RHD in D- pregnant women to ascertain the requirement for antenatal RhD prophylaxis

BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D− women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening.

High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery

To assess whether spontaneous preterm delivery can be predicted from the amount of cell free fetal DNA (cffDNA) as determined by routine fetal RHD genotyping at 25 weeks’ gestation.

Next-generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell-free fetal DNA in maternal plasma.

Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next‐generation sequencing (NGS) technology.

Is current serologic RhD typing of blood donors sufficient for avoiding immunization of recipients

BACKGROUND: Avoiding immunization with clinically important antibodies is a primary objective in transfusion medicine. Therefore, it is central to identify the extent of D antigens that escape routine RhD typing of blood donors and to improve methodology if necessary.

Integration of noninvasive prenatal prediction of fetal blood group into clinical prenatal care

Incompatibility of red blood cell blood group antigens between a pregnant woman and her fetus can cause maternal immunization and, consequently, hemolytic disease of the fetus and newborn. Noninvasive prenatal testing of cell-free fetal DNA can be used to assess the risk of hemolytic disease of the fetus and newborn to fetuses of immunized women. Prediction of the fetal RhD type has been very successful and is now integrated into clinical practice to assist in the management of the pregnancies of RhD immunized women. In addition, noninvasive prediction of the fetal RhD type can be applied to guide targeted prenatal prophylaxis, thus avoiding unnecessary exposure to anti-D in pregnant women. The analytical aspect of noninvasive fetal RHD typing is very robust and accurate, and its routine utilization has demonstrated high sensitivities for fetal RHD detection. A high compliance with administering anti-D is essential for obtaining a clinical effect. Noninvasive fetal typing of RHC/c, RHE/e, and KEL may become more widely used in the future.Incompatibility of red blood cell blood group antigens between a pregnant woman and her fetus can cause maternal immunization and, consequently, hemolytic disease of the fetus and newborn. Noninvasive prenatal testing of cell‐free fetal DNA can be used to assess the risk of hemolytic disease of the fetus and newborn to fetuses of immunized women. Prediction of the fetal RhD type has been very successful and is now integrated into clinical practice to assist in the management of the pregnancies of RhD immunized women. In addition, noninvasive prediction of the fetal RhD type can be applied to guide targeted prenatal prophylaxis, thus avoiding unnecessary exposure to anti‐D in pregnant women. The analytical aspect of noninvasive fetal RHD typing is very robust and accurate, and its routine utilization has demonstrated high sensitivities for fetal RHD detection. A high compliance with administering anti‐D is essential for obtaining a clinical effect. Noninvasive fetal typing of RHC/c, RHE/e, and KEL may become more widely used in the future.

Pre-Analytical Conditions in Non-Invasive Prenatal Testing of Cell-Free Fetal RHD

Background Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening. Methods Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104). Results The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10°C to 28°C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10–39, n = 1317). Conclusion The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.

Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis.

Objective: To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis. Methods: We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative women in gestational weeks 6–37. Results: Dilution experiments revealed no inhibition of amplification in the multiplex assays. For plasma samples, the duplex assay was significantly more sensitive than the triplex assay (p < 0.0001). For the duplex assay (exon 7/10), accuracy was 99.0%. For the triplex assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable. Conclusion: The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay needs further optimization.

Noninvasive fetal RhD genotyping

Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10-11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women.

Quantitation of RHD by real‐time polymerase chain reaction for determination of RHD zygosity and RHD mosaicism/chimerism: an evaluation of four quantitative methods

BACKGROUND: Determination of RHD zygosity of the spouse is crucial in preconception counseling of families with history of hemolytic disease of the fetus and newborn caused by anti‐D. RHD zygosity can be determined by quantitative real‐time polymerase chain reaction (PCR) basically by determining RHD dosage, and this feature is relevant in investigating RHD mosaicism and chimerism.

Study of compliance with a new, targeted antenatal D immunization prevention programme in Denmark.

Background and Objectives  A targeted routine antenatal anti‐D prophylaxis programme was implemented in Denmark where anti‐D immunoglobulin is given based on the result from noninvasive antenatal screening for fetal RHD. Our objective was to evaluate compliance with this new programme right after its initiation.