Fetal RHD genotyping by analysis of maternal plasma in a mixed population

Background: Maternal plasma analysis for the determination of the fetal RHD status is an exciting tool for the management of RhD‐negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi‐ethnic population. Methods: We analyzed plasma samples from 88 RhD‐negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Fourteen patients (16%) had anti‐D alloantibody. We used Taqman primers and probes to detect by real‐time PCR, exons 4, 5, and 10 of RHD. As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective nenonates were collected during delivery and hemagglutination was performed. Results: Fifty‐eight samples (66%) were genotyped as RHD+, 27 samples (31%) showed complete absence of RHD and 3 samples (3 %) presented a D variant (RHDψ). All the results agreed with the neonatal typing, including the three fetuses with the RHDψ, phenotyped as RhD‐negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Conclusion: Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5, and 10) can be sufficient for clinical application in a multi‐ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma for our population. J. Clin. Lab. Anal. 25:100–104, 2011. © 2011 Wiley‐Liss, Inc.     

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. STUDY DESIGN AND METHODS: In each of the five Danish health care regions, blood samples were drawn from D? women in Gestational Week 25. DNA was extracted from the maternal plasma and analyzed for the presence of the RHD gene by real‐time polymerase chain reaction targeting two RHD exons. Prediction of the fetal RhD type was compared with serologic typing of the newborn in 2312 pregnancies, which represented the first 6 months of routine analysis. RESULTS: For the detection of fetal RHD, the sensitivity was 99.9%. The accuracy was 96.5%. The recommendation for unnecessary antenatal RhD prophylaxis for women carrying a D? fetus was correctly avoided in 862 cases (37.3%), while 39 women (1.7%) were recommended for antenatal RhD prophylaxis unnecessarily. Two RHD+ fetuses (0.087%) were not detected, and antenatal RhIG was not given. CONCLUSION: These data represent the first demonstration of the reliability of routine antenatal fetal RHD screening in D?, pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Our findings should encourage the implementation of such screening programs worldwide, to reduce the unnecessary use of RhIG.     

Lessons learned from the implementation of non-invasive fetal RHD screening

Introduction: In the fight against hemolytic disease of the fetus and newborn, pregnant RhD negative women are offered antenatal and postnatal anti-D immunoglobulin prophylaxis to prevent the development of antibodies against the fetal D antigen. Antenatal prophylaxis has traditionally been provided to all D negative pregnant women, as the fetal RhD type remains unknown until birth. With noninvasive prenatal testing of cell-free DNA, predicting the fetal RhD type has become highly feasible based on analysis of the fetal RHD gene. Fetal RHD screening can guide targeted antenatal prophylaxis, treating only women who carry an RhD positive fetus, thereby avoiding the unnecessary treatment of approximately 40% of the RhD negative women.

Areas covered: Areas covered are the current clinical practice, performance, and challenges of fetal RHD screening, and relevant issues for its implementation.

Expert commentary: Fetal RHD screening is highly accurate, with sensitivities of 99.9%, as reported from clinical programs. From gestational week 10, sensitivities are approximately 99%. Despite challenges in assay design, low levels of cell-free fetal DNA in the maternal plasma, and concerns regarding implementation and medical necessity, the clinical experience with fetal RHD screening and targeted prophylaxis has generated encouraging results, and its future widespread use is expected.     

Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women.

BACKGROUND: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis. AIM: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women. METHODS: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth. RESULTS: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results. CONCLUSION: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.     

Non-invasive prenatal testing for management of haemolytic disease of the fetus and newborn induced by maternal alloimmunisation

Anti-D immunoglobulin prophylaxis reduces the risk of RhD negative women becoming alloimmunised to the RhD antigen and is a major preventative strategy in reducing the burden of haemolytic disease of the fetus and newborn (HDFN).HDFN also arises from other maternal red cell antibodies, with the most clinically significant, after anti-D, being anti-K, anti-c and anti-E. Among the 39 human blood group systems advanced genomic technologies are still revealing novel or rare antigens involved in maternal alloimmunisation.Where clinically significant maternal antibodies are detected in pregnancy, non-invasive prenatal testing (NIPT) of cell-free fetal DNA provides a safe way to assess the fetal blood group antigen status. This provides information as to the risk for HDFN and thus guides management strategies.In many countries, NIPT fetal RHD genotyping as a diagnostic test using real-time PCR has already been integrated into routine clinical care for the management of women with allo-anti-D to assess the risk for HDFN. In addition, screening programs have been established to provide antenatal assessment of the fetal RHD genotype in non-alloimmunised RhD negative pregnant women to target anti-D prophylaxis to those predicted to be carrying an RhD positive baby. Both diagnostic and screening assays exhibit high accuracy (over 99 %).NIPT fetal genotyping for atypical (other than RhD) blood group antigens presents more challenges as most arise from a single nucleotide variant. Recent studies show potential for genomic and digital technologies to provide a personalised medicine approach with NIPT to assess fetal blood group status for women with other (non-D) red cell antibodies to manage the risk for HDFN.     

Routine fetal RHD genotyping with maternal plasma: a four-year experience in Belgium

BACKGROUND: The objective was to evaluate the diagnostic value of RHD fetal genotyping from the plasma of D- mothers as soon as 10 weeks' gestation in a routine clinical practice in Belgium. STUDY DESIGN AND METHODS: A prospective study was conducted between November 2002 and December 2006. DNA extraction was performed in an automated closed tube system. Fetal RHD/SRY genotypes were detected in the plasma of 563 pregnant mothers by real-time polymerase chain reaction (PCR) targeting multiple exons 4, 5, and 10 of the RHD gene and targeting an SRY gene sequence. These were compared to the D phenotypes determined in the 581 babies they delivered. RESULTS: By combining amplification of three exons, the concordance rate of fetal RHD genotypes in maternal plasma and newborn D phenotypes at delivery was 100 percent (99.8% including one unusual false-positive). The presence of nonfunctional RHD genes and the absence of a universal fetal marker, irrespective of fetal sex, did not influence the accuracy of fetal RhD status prediction. The RHD genotyping from 18 twin pregnancies was also assessed. Five weak D women were excluded from the RHD fetal genotyping prediction. Three discrepant results (0.5%) between predicted fetal genotype and cord blood phenotype were not confirmed by the baby phenotypes from venipuncture blood. CONCLUSION: Prenatal prediction of fetal RHD by targeting multiple exons from the maternal plasma with real-time PCR is highly sensitive and accurate. Over 4 years, this experience has highly modified our management of D- pregnant women.     

Evaluation of single‐nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups

BACKGROUND: Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single‐nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. STUDY DESIGN AND METHODS: DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single‐base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real‐time PCR of fetal DNA from maternal plasma. RESULTS: The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false‐negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. CONCLUSION: Coamplification of 52 SNPs with RHD‐specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false‐negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples.     

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.     

RHD gene polymorphisms in alloimmunized RhD-negative individuals with high rate of racial admixture

BackgroundThe D-negative phenotype is the result of the total RHD gene deletion in almost all Caucasians, but it accounts for only about 20% in Africans and 70% in Asians. In Africans the RHDΨ that is one of the most important causes of the D-negative phenotype. We investigated the RHD polymorphisms in D-negative phenotype mixed Brazilians who have anti-D alloantibody.Study design and methodsBlood samples from 130 individuals previously typed as D-negative were phenotyped again using: (a) two tube reagents (Anti-D blend reagent, Cellular line TH-28, MS-26; and Anti-D polyclonal); (b) one gel test ID-Card for Rh subgroups including C^w and K antigen; and (c) ABO/Rh (Anti-D blend reagent, Cellular line 175-2, LDM3). The method used for RHD screening detected the presence of RHD exon 10 and intron 4. Sequence analysis was performed on PCR products amplified from genomic DNA for all 10 exons RHD gene.ResultsWe found that 118/130 (90.8%) of D-negative tested individuals had total RHD gene deletion, while 12/130 (9.2%) showed RHD gene polymorphisms. The RHDΨ was found in 10 (7.7%) individuals, one sample (0.77%) hybrid RHD-CE-D^s /RHDΨ, and another (0.77%) weak D type 4.2.ConclusionsThe results showed that the RHD gene was present in 9.2% of racially mixed Brazilians who produced usually clinically significant anti-D alloantibodies. Therefore, the data showed that careful attention is necessary for clinicians in applying RhD genotyping to transfusion medicine in populations with high rate of racial admixture.     

Frequency and characterization of RHD variant alleles in a population of blood donors from southeastern Brazil: Comparison with other populations

BackgroundThe correct determination of D antigen could help to avoid alloimmunization in pregnant women and patients receiving blood transfusions. However, there are limitations in the identification of D variants as the partial and weak D phenotypes make the determination of D antigen a great challenge in the transfusion routine.’Study design and methodsThe molecular characterization of D variants was performed on blood donors from southeastern Brazil with atypical D typing. Furthermore, the serological profile of all RHD variant alleles identified was analyzed using different Anti-D clones. The prevalence of RHD alleles and genotypes found was compared with those described in other countries and in other regions from Brazil.ResultsAtypical serologic D typing occurred in 0.79 % of blood donors. The majority of RHD variant alleles (88 %) were first characterized by multiplex PCR and PCR-SSP as RHD*weak partial 4 (47 %), followed by RHD*weak D type 3 (29.9 %), RHD*weak D type 2 (3.9 %) and RHD*weak D type 1 (3.1 %). Genomic DNA sequencing characterized the RHD*weak partial 4 variants found in RHD*DAR1.2 (weak 4.2.2) (22 %), RHD*DAR3 (weak 4.0.1) (2.4 %), RHD*DAR3.1 (weak 4.0) (22 %) and RHD*DAR4 (weak 4.1) (0.8 %). RHD variant alleles associated with partial D, such as, RHD*DAU-4 (1.6 %), RHD*DAU-5 (2.4 %), RHD*DAU-6 (1.6 %), RHD* DIII type 8 (1.6 %), RHD*DVII (3.9 %) and RHD* DMH (0.8 %) were also observed.ConclusionThe prevalence of RHD variant alleles observed in this cohort differ from those found in other populations, including Brazilians from other regions. RHD allele distribution in specific regions should be considered for implementation of algorithms and genotyping strategies aiming at a more effective and safe transfusion.     

Comparison of PCR methods for detecting fetal RhDin maternal plasma

Background: Aim of this study was to establish the method yielding the highest sensitivity routinely used to determine fetal RhD type and gender from maternal cell‐free plasma DNA in different periods of gestation. Methods: Plasma DNA concentrations were measured from 46 pregnant women in different gestational periods and tested for RhD using three different PCR methods on exon 7: Thermal Cycler, Taqman method on LightCycler, and melting curve analysis on LightCycler. In addition, fetal gender was determined by PCR. Cell‐free plasma DNA was measured in 100 healthy volunteers as a reference group. Results: The mean value of cell‐free plasma DNA in the reference group was 10.9 pg/µL mean, (standard deviation (SD): 3.66) in 50 healthy women and 12.7 pg/µL (SD: 8.2) in 50 healthy men. In the firsttrimester of pregnancy cell‐free plasmaDNA was 14.9 pg/µL mean, (SD: 4.2), in the second trimester 15.4 pg/µL mean, (SD: 4.96), and the maximum was achieved in the third trimester of pregnancy 15.6 pg/µl mean, (SD: 6.49). TaqMan probes had the same accuracy, when compared with Thermal Cycler technology (46 samples, 6 failures). Using real‐time PCR with melting curve analysis 12 of 17 samples were correctly tested. Gender determination was correctly in 41 of 46 samples. Conclusion: RhD determinations with TaqMan and Thermal Cycler technology are useful methods for fetal RhD prediction. To increase the accuracy of RhD determination it is necessary to test on other exons in addition. J. Clin. Lab. Anal. 23:24–28, 2009. © 2009 Wiley‐Liss, Inc.     

Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service

BACKGROUND: Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell-free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D- genomes. STUDY DESIGN AND METHODS: Real-time PCR assays were designed to distinguish RHD from RHDpsi (possessed by the majority of D- black Africans). Plasma-derived DNA from 137 D- women was subjected to real-time PCR to detect fetal RHD and Y chromosome-associated SRY sequences. The accuracy of RHD genotyping from maternal plasma was investigated by comparing results with those obtained by conventional RHD genotyping from fetal tissue or serologic tests on the infant's RBCs. The quantity of fetal DNA in maternal plasma was investigated in 94 pregnancies. RESULTS: Fetal D status was predicted with 100-percent accuracy from maternal plasma. The number of copies of fetal DNA in maternal plasma was found to increase with gestation. CONCLUSION: Combination of the sensitivity of real-time PCR with an improved RHD typing assay to distinguish RHD from RHDpsi enables highly accurate prediction of fetal D status from maternal plasma. This has resulted in the implementation of a clinical noninvasive fetal RHD genotyping service.     

Determination of RhD zygosity: comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach

BACKGROUND: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity. METHODS: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, DeltaCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage. RESULTS: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the DeltaCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P: <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay. CONCLUSION: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.     

Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles

BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians. STUDY DESIGN AND METHODS: Preliminary profiling was carried out on single‐nucleotide polymorphisms (SNPs) associated with 13 prevalent RHD alleles, that is, weak D Types 1, 2, 3, 4.0, 4.0.1, 4.1, 4.2, 5, 11, 15, and 17; RHD(IVS3+1g>a); and RHD(K409K). Multiplex PCR was used to amplify six RHD regions encompassing 14 SNPs. Identification was obtained by incorporation of the complementary dye single base at the 3′‐end of each probe‐primer. A prospective analysis was then carried out on 152 blood samples from patients (n = 53) and donors (n = 88) with equivocal RhD serology and pregnant women (n = 11). RESULTS: After validation, our SNaPshot assay allowed direct genotyping of 82.9% of samples overall and 100% of samples harboring weak D Types 1, 2, 3, and 4.1 alleles. In the remaining 17.1% of samples overall, sequence investigation allowed accurate genotyping. In addition, four novel RHD alleles were identified, that is, RHD(S256P), RHD(L390L), RHD(F410V), and RHD(IVS4‐2a>g). CONCLUSION: The SNaPshot assay described herein is a helpful supplementary tool for resolving doubtful RhD serology. By allowing accurate identification of weak D and DEL alleles this assay should allow better management of the donors and the patients genotyped weak D Types 1, 2, 3, and 4.1 who can receive D+ blood units.     

山东地区汉族RhD阴性献血者RHD基因的纯合性分析

目的：Rhesus血型（简称Rh血型）因其抗原众多、变种各异和临床疾病相关性而备受重视。该血型系统中与临床疾病关联最密切的抗原即RhD抗原具有较高的免疫原性。编码RhD抗原的RHD基因两侧各有一个序列高度相似的Rh盒子基因,RhD阴性即是由上、下游Rh盒子基因之间的基因重组引起。分析RhD阴性孕妇丈夫RHD基因的纯合性可预测胎儿患新生儿溶血病的几率。本研究的目的是分析山东地区汉族人RhD阴性表型形成的分子机制,并对Rh盒子基因的扩增产物进行分析以确定RHD基因的纯合性。方法：74例RhD阴性献血者的DNA样品首先进行多重聚合酶链反应-序列特异性引物（PCR-SSP）分析。然后对Rh盒子基因进行PCR基因扩增,其扩增产物采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法进行RHD基因的纯合性测定。结果：46例（62%）样品在多重PCR-SSP分析中显示缺失RHD基因,在PCR-RFLP分析中显示为纯合的RHD基因阴性。22例（30%）样品显示存在RHD基因,其中19例显示为杂合的RHD基因,3例显示为纯合的RHD基因。5例（7%）样品缺失RHD基因,但PCR-RFLP分析显示存在一个RHD基因,进一步的分析表明它们至少存在RHD基因第1和10外显子。1例（1%）样品显示存在RHD基因,但缺失第6外显子。结论：HD 基因缺失是引起中国汉族人RhD阴性表型形成的主要分子机制。RhD阴性个体主要表现为纯合的RHD基因阴性,少部分RhD阴性个体存在杂合的RHD基因和纯合的RHD基因。     

荧光定量PCR检测Rh阴性孕妇外周血浆游离DNA中胎儿RhD血型

目的 探讨荧光定量PCR(fluorescence quantitative polymerase chain reaction,FQ-PCR)技术对Rh阴性孕妇血浆中游离胎儿DNA进行非创性产前诊断胎儿RhD血型的可行性.方法 选取78份妊娠11～40周、B超确诊为单胎的Rh阴性孕妇血浆.采用9个短串联重复序列(short tandem repeat,STR)多态性位点及Y染色体性别决定区基因(sex-determining region Y chromosome,SRY)确定胎儿DNA的存在;运用FQ-PCR技术对血浆中游离胎儿DNA进行RHD基因外显子5、7、10和内含子4定量分析,以确定胎儿RhD血型的基因型;其基因型结果与产后新生儿脐血血清学检测结果进行对比分析,回顾性评价胎儿基因定型结果的准确性.结果 78份标本中,41份检测到SRY基因,平均浓度为(214.7±120.9)拷贝/ml,产后证实皆为男性.70份FQ-PCR基因定型结果与血清学结果相符,另有5份确定为假阳性,3份基因定型结果不可确定,检测结果总符合率为90%(70/78).5份假阳性标本通过检测RHD1227A等位基因鉴定了4份RhD放散型,FQ-PCR最终结果准确率达到95%(74/78).结论应用FQ-PCR方法进行非创性胎儿RhD血型检测可用于新生儿溶血病的预防和诊断.     

The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible

BACKGROUND: Noninvasive fetal RHD genotyping might become a valuable tool in decision making on antenatal Rh prophylaxis, which is currently in routine practice for all D? pregnancies in several countries. This study provides a large‐scale validation study of this technology to address questions concerning feasibility and applicability of its introduction into clinical routine. STUDY DESIGN AND METHODS: Real‐time polymerase chain reaction (PCR) targeting RHD Exons 5 and 7 was applied for the detection of fetal‐specific RHD sequences in maternal plasma. A total of 1113 women in 6 to 32 weeks (median, Week 25) of pregnancy were recruited. All of them were serologically typed as D? according to current German guidelines. DNA was extracted via a spin‐column method and a novel automated approach using magnetic tips. Real‐time PCR results were compared with postnatal serology and discrepancies further elucidated by DNA sequencing from a newborn's buccal swab. RESULTS: Sensitivities of fetal RHD genotyping were 99.7 percent (spin columns) and 99.8 percent (magnetic tips), thus comparable with serology (99.5%). The detection of weak D variants was more reliable by real‐time PCR. Specificities of fetal RHD genotyping were 99.2 percent (spin columns) and 98.1 percent (magnetic tips), which is lower than serology (>99.7%). Automation achieved significantly higher yields of cell‐free fetal DNA. CONCLUSION: This prospective clinical trial revealed that routine determination of the fetal D status from maternal plasma is feasible. Noninvasive fetal RHD genotyping can be considered as sensitive as the traditional postnatal serologic assay.     

Noninvasive determination of fetal RHD status by examination of cell-free DNA in maternal plasma

BACKGROUND: Cell-free fetal DNA in maternal plasma opens the way for routine risk-free diagnosis of fetal D status of D- mothers. The focus was on accuracy of RHD typing and confirmation of fetal DNA in maternal plasma while RHD was not detected. STUDY DESIGN AND METHODS: Plasma DNA was extracted (by manual and/or automatic method) from 255 D- pregnant women and amplified in exons 7 and 10 and intron 4 of RHD gene with real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY and, when negative, by one of 11 different polymorphisms found in the father but not in the mother. The results were compared with the D status of the newborns. RESULTS: After exclusion of 25 cases (10%) because of material shortage, in 230 cases (90%) available for complete study, the predictive value of the procedure of fetal RHD testing (RHD genotyping plus confirmation of fetal DNA) was 99.6 percent. SRY detection confirmed fetal DNA presence in maternal plasma in all boys, whereas the detection of various polymorphisms in all girls but one. CONCLUSIONS: Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).     

中国人群RhD阴性个体中D基因多态性的研究

目的 建立RHD基因分型技术，分析中国人群RhD阴性个体的RHD基因多态性分布状况。方法 设计8对特异性引物扩增RHD7个外显子(3，4，5，6，7，9，10)和内含子4，应用PCR—SSP技术，对l15例经常规血清学试验和吸收放散试验鉴定的“Rh阴性个体”，进行D基因多态性的研究。结果 l15例RhD阴性个体，用吸收放散试验检测后分成2组，一组被确认为RhD阴性表现型共88例(76．5％)，另一组是Del(放散)表现型共27例(23．5％)。应用PCR—SSP法对l15例RhD阴性个体作RHD内含于4检查，结果Del型个体都显示有RHD内含于4；对l15例中的17例进一步作RHD的7个外显子的鉴定，其中6例Del带有完整的RHD基因，ll例经吸收放散试验确认为RhD阴性表现型的个体则显示4种情况：7例全部缺失RHD基因，2例(1例ccEe和1例Ccee)带有完整的RHD基巴，1例缺失RHD的第5外显子和1例仅携带第10外显子。结论 PCR—SSP技术可用于RHD基因的研究。常规血清学鉴定的RhD阴性中存在较高比例的Del型，且有可能带有完整的RHD基因。而被吸收放散试验确认为RhD的阴性个体有可能携带RHD基因并显示出多态性，这些个体中有Rhc抗原表达，有别于献中认为全局RhC抗原表达的报告。