Fetal RhD genotyping: A more efficient use of anti-D immunoglobulin

The most important application of blood group genotyping by molecular genetics is the prediction of fetal RhD phenotype in pregnant women with anti-D, in order to assess the risk of haemolytic disease of the fetus and newborn. This diagnostic test performed on cell-free fetal DNA in the maternal plasma, is now a routine procedure in some countries. High-throughput modifications of this form of fetal D-typing would be valuable for testing fetuses of all D-negative pregnant women to avoid unnecessary antenatal treatment with anti-D immunoglobulin in the 40% of D-negative pregnant women with a D-negative fetus. The results of trials in Bristol and Amsterdam suggest that such routine testing is feasible and accurate.     

Obstetric outcome after RhD and Kell testing

The study was conducted to report on the use of molecular biology methods and pregnancy outcome in women sensitized to either Rhesus D (RhD) or Kell 1 (K1) antigens. Paternal RhD genotype was determined by DNA amplification of an RhD-specific sequence from single sperm cells. Paternal Kell phenotype was determined by serologic assays using peripheral blood samples, and the fetal RhD or Kell-type status were established by the polymerase chain reaction (PCR) with amniotic cells. Thirteen women (14 pregnancies, one with twins) sensitized to RhD and four sensitized to K1 antigens, comprised the study group. All had paternal heterozygosity to either D or K1 antigens. Nine fetuses were RhD positive and five were RhD negative. An additional woman underwent early spontaneous abortion. The nine RhD-positive fetuses underwent a total of 41 invasive procedures. One fetus with hydrops fetalis died in utero after intrauterine blood transfusion. All the remaining RhD- positive fetuses were delivered after 33 weeks gestation, and all those who were RhD negative were delivered at term. Four women were sensitized to the K1 antigen; in three, the fetus was found to be K1 negative, and in one, K1 positive, necessitating intrauterine blood transfusion. In all cases, the results of RhD or K1 genotype analyses from amniotic fluid were compatible with fetal/neonatal red blood cell RhD or Kell phenotypes. In conclusion, the use of molecular biology techniques represents a major advance in the clinical management of RhD and Kell alloimmunization.      

Prenatal screening

In most countries, pregnant women are early in pregnancy typed for ABO and RhD and screened for the presence of red cell antibodies. Maternal red cell alloantibodies of IgG class are actively transported by the placenta to the fetus and destruct the fetal red cells, if these carry the involved antigen leading to haemolytic disease of the fetus and newborn, HDFN. HDFN is a disease which –if untreated– can cause perinatal mortality and morbidity, with a substantial risk for long-term sequelae. In most cases of HDFN, alloimmunization against the RhD antigen is involved, although introduction of anti-D immunoglobulin (Ig) prophylaxis drastically decreased anti-D-mediated HDFN. In the last decade, non-invasive fetal genotyping, with cell-free fetal DNA present in maternal plasma, for RhD, RhC, Rhc, RhE and K became a diagnostic reality. In a few countries non-invasive fetal RhD genotyping has already been implemented to target antenatal and postnatal anti-D Ig prophylaxis to RhD-negative women carrying RhD-positive fetuses. Both applications of non-invasive fetal genotyping are summarized in this article.     

Determination of Rh type and gender using circulating cell-free fetal DNA in early pregnancy of Rh negative women in turkey

IntroductionChoosing the right clinical approach for early and reliable diagnosis/screening is becoming more important day by day. The aim of the study was to determine the early RhD type with cff-DNA obtained from maternal plasma, especially in the light of recent developments. In this way, it is aimed to apply Rh Ig only to mothers who are determined to have RhD (+) fetuses and to prevent unnecessary further tests that may possess a risk for RhD (-) fetuses.MethodsPrediction of fetal gender and RH genotype was performed by using RT-qPCR method. With simultaneous amplification of sequences of SRY, DYS14 and RH genes (exon 7 and exon 10). Fetal gender and RhD were determined in 30 RHD (-) pregnant women with cfDNA.ResultsAs a result of genotyping, the gender of 67% (20/30) fetuses was determined as male; the gender of 33% (10/30) fetuses was determined as female in a sample group of 30 pregnancies. It was determined that the DYS14 100% (20/20) gene was more sensitive than the SRY 97% (18/20) gene in gender determination after examining prenatal and postnatal results. As a result of the analysis, the presence of 17% (5/30) RhD (-) fetuses and 83% (25/30) RhD (+) fetuses were determined which is 100% compatible with postnatal results.DiscussionDetecting fetal RhD gene in maternal plasma made an important contribution to its use in non-invasive prenatal screening. This study shows that unnecessary intervention and cost can be avoided with successful genotyping analysis performed with RT-qPCR.     

The importance of antenatal immunoprophylaxis for prevention of hemolytic disease of the fetus and newborn

Hemolytic disease of the fetus and newborn (HDFN) is a consequence of maternal alloimmunization against fetal red blood cell antigens. Alloimmunization against D antigen from Rhesus (Rh) blood group system is particularly important because of its strong immunogenicity. During the last few decades, the introduction of RhD prophylaxis by postpartum administration of anti-D immunoglobulin to RhD negative women, now improved with antenatal prophylaxis, has led to a dramatic decrease in perinatal mortality and morbidity from HDFN. However, severe cases have not disappeared, mostly due to prophylaxis failure. In our case, inappropriate prenatal care during the first pregnancy in an RhD negative mother resulted in primary immunization. In the next pregnancy with an RhD positive child, the mother's secondary immune response was extremely strong and led to early development of severe fetal anemia. The fetus survived thanks to the treatment with intrauterine transfusions (IUT), but they caused suppression of erythropoiesis, which lasted for months after birth. The long lasting, late anemia was treated with repeated postnatal red cell transfusions and recombinant human erythropoietin (rHuEPO). Despite the severity of HDFN in our case, the short-term outcome is good. The boy has normal growth until now, but due to the possibility of an adverse long-term neurodevelopmental outcome, this case requires continuous follow up. It also reminds of the fact that RhD alloimmunization remains an actual problem in daily routine. Antenatal prophylaxis is a crucial step in quality care of those who are at a risk of HDFN.     

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.     

Molecular RH blood group typing of serologically D-/CE+ donors: the use of a polymerase chain reaction-sequence-specific primer test kit with pooled samples

The known presence of RHD blood group alleles in apparently D– individuals who are positive for C or E antigens leads to an appropriate investigation for the RHD gene on the red blood cells (RBCs) of D– blood donors, thus preventing their RBCs from immunizing D– recipients. Ready-to-use polymerase chain reaction–sequence-specific primer (PCR-SSP) typing kits are available and allow single-sample results. The need to perform this testing on a large number of donors affiliated with the Transfusion Department of Udine (Northern Italy) led to the use of molecular genetic RH blood group typing with PCR-SSP test kits and DNA samples mixed in pools. From a population of 35,000 blood donors screened for D antigen by serologic typing, a total of 235 samples, distributed in pools of 5 DNA samples, were investigated. Positive results were reevaluated by opening the pools and retesting single samples. Validation of DNA-pool typing with commercial kits was done. Among 235 genotyped samples, 12 were found to be PCR positive (5.1%), exhibiting DEL genotype and RHD-CE-D hybrid alleles. Our data demonstrate that the use of a PCR-SSP commercial test kit with pooled samples is a helpful and valid method to correctly detect RHD alleles. As a consequence, we reclassified our donors as carriers of potentially immunogenic alleles.     

Technologies involved in molecular blood group genotyping

Background Until the 1990s, blood screening, typing and diagnostics depended entirely on serological techniques. For over a century, agglutination has been the gold standard for red blood cell (RBC) antigen detection used in all blood services. However, haemagglutination has certain limitations, such as, the difficulty to phenotype recently and multi-transfused patients or direct antiglobulin test (DAT)-positive patients. The haemagglutination test provides only indirect indications of risk or severity of haemolytic disease of the new born. In part, to overcome these limitations, nucleic acid-based technologies have been used in immunohematology reference laboratories. Methods There are many molecular methods available for red cell genotyping: PCR, PCR-RFLP, PCR-SSP or PCR-ASP, real time PCR, DNA sequencing and pirosequencing and methods with microarrays-based systems. Other molecular techniques that are under development and may be available for red cell genotyping in the next decade include fluidic or open microarrays; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS); and mini-sequencing. Conclusions Although serology may be superior for some blood group typing, genotyping assays offer a good alternative for problems encountered by serology. In many laboratories, blood group genotyping is already used at a low-throughput level for diagnostics in cases of problematic serology. Especially in case of weak expression of antigens, the presence of rare antigens or auto-antibodies or after multiple transfusions, genotyping is superior. The non-invasive determination of the foetal RHD analysis in maternal plasma by real-time PCR is well established and already offered as a clinical service in a number of countries. The recent availability of automated, high throughput, DNA-array platforms, allows to introduce into the hospital and donor centres this DNA-based typing methodology. The evolution of molecular methods combined with automation and high-quality standards will make large-scale screening a cost-effective reality.     

Evaluation of the enzyme test for the detection of clinically significant red blood cell antibodies during pregnancy

In the Croatian transfusion medicine, no general agreement has yet been achieved whether red blood cell (RBC) Rhesus (Rh) antibodies detected during pregnancy only by enzyme tests can cause hemolytic disease of the newborn (HDN). Results of the detection of clinically significant RBC antibodies by low-ionic-strength additive solution antiglobulin test (LISS-IAT) and trypsin enzyme test in 22,947 pregnant women are presented. All pregnant women in whom clinically significant RBC antibodies (RBC-CSA) were detected by LISS-IAT and/or enzyme tests were followed and observed during pregnancy. The women who had enzyme-only anti-D antibodies in their serum were followed up during subsequent pregnancies. Out of 302 positive results obtained by both techniques, irregular clinically significant enzyme-only antibodies (anti-RhD and anti-RhE specificity) were detected in 14 (4.6%) pregnant women. None of 11 RhD positive newborns whose mothers had enzyme-only anti-D antibodies, had signs of HDN after delivery. In these 11 women, anti-D antibodies were detected by LISS-IAT in the first trimenon of subsequent pregnancy. Nine infants born from subsequent pregnancies to women who had previously had enzyme-only anti-D, had clinical signs of HDN. The authors concluded that there is no need for enzyme tests in prenatal testing because enzyme tests are not reliable in the prediction of HDN.     

Development of a flow cytometric test for the detection of D-positive fetal cells after fetomaternal hemorrhage and a survey of the prevalence in D-negative women

A sensitive test for the presence of D-positive fetal red blood cells (RBCs) in the maternal circulation of D-negative women has been developed. It was used to investigate the possibility that the occasional failure in preventing alloimmunization might be due to the administration of inadequate amounts of prophylactic anti-D Rh immune globulin. The standard dose in Australia contains 125 microg of antibody, and can suppress immunization by an estimated 6 mL of packed D-positive RBCs. A fetomaternal hemorrhage (FMH) of this volume is detectable in the maternal circulation as approximately 0.25 percent of the total RBCs. Our test utilizes a commercially available human monoclonal IgG anti-D that has been biotinylated and used with a dye-conjugated streptavidin. Flow cytometry is used to quantitate fluorescing D-positive RBCs. To date, 2,288 tests have been performed on blood samples from D-negative women attending local antenatal clinics or at the time of delivery. Evidence for an FMH has been obtained in six cases (0.26%). In one case, the FMH was only 0.1 percent, and in another (confirmed by the Kleihauer-Betke method), fetal cells constituted only 0.2 percent. Additional Rh immune globulin was not given to these patients. In the other four cases, the D-positive fetal cells were estimated to be 0.7,0.5,0.5, and 0.4 percent, and additional prophylactic Rh immune globulin was administered. Although the prevalence of FMH is low, screening D- negative women at risk of alloimmunization has proved to be simple, fast, and inexpensive.     

中国汉族群体RhD阴性个体D基因外显子多态性研究

目的 研究中国汉族RhD阴性[RhD(-)]个体Rh血型系统D基因8个外显子的构成情况。方法 应用聚合酶链反应－序列特异性引物（PCR－SSP）方法,对50名汉族人RhD(-)个体RHD基因及RHCE基因进行了研究。扩增RHD基因的第2,3,4,5,6,7,9,10外显子和内含子4,以及RHCE基因的第1,2,4,5外显子和内含子4。结果 50名RhD(－）供者的Rh表型为ccdee 22人,ccdEe 3 w ,CE3w ,在表型为ccdee或ccdEe的25人中,D基因8个外显子全部缺失,在表型为Ccdee或CcdEe的25人中,RHD基因则表现出多态性：9人存在全部基因8个外显子,7人缺失全部D基因8个外显子,5人存在D基因第2外显子,3人存在D基因第6外显子,1人存在D基因第2,6,10外显子,结论 中国汉族RhD(-)个体D基因外显子具有多态性,Rh表型为cc的个体D基因8个外显子全部缺失,在表型为Cc的个体中观察到5种D基因多态性,中国汉族人D基因结构不同于高加索人,故在应用RHD基因定型结果时,应慎重对待。     

Alloimmunity to RhD in humans.

The RhD protein is expressed only on human red blood cells (RBC), and is one of the most immunogenic of the blood groups. It is of clinical importance since the alloantibody (anti-D) can hemolyse D positive RBC after blood transfusion, or cause hemolytic disease of the newborn. The immunogenicity of D is better understood with the knowledge of the genetic basis of the protein(s) involved, the molecular orientation in the RBC membrane, and the nature of the cellular immune response to proteins. The adaptive humoral response consists of antigen presenting cells, T-cells and B-cells, which interact cooperatively to result in antibody against the antigen in question. The anti-D that B-cells produce is targeted against surface membrane determinants (B-cell epitopes) and are conformational i.e. non-contiguous amino acids. The antigen specific T-cells recognize short linear peptides in the context of MHC class II, and these T-cell epitopes can reside anywhere in the protein. Since the RhD protein in D-positive individuals differs by some 35 amino acids from the RhCcEe protein in D-negative individuals, the opportunity for generating immunogenic T-cell epitopes is much greater than that for alleles characterized by a single amino acid difference e.g. E and e. Multiple conformational B-cell epitopes are also created by the presence of several D-specific amino acids in extracellular loops on the red cells surface, which may stimulate several B-cell clones and develop a strong polyclonal antibody response. With greater understanding comes the possibility of manipulating the immune response to D in clinical situations.     

RHD 1227A is an important genetic marker for RhD(el) individuals

Approximately 30% of apparently Rh- Taiwanese people actually were RhD(el), a rare variant of the Rh system that might carry a grossly intact RHD gene. Several studies have indicated that the RhD(el) trait might be generated by multiple molecular mechanisms. In this study, a total of 294 Taiwanese serologically RhD- blood donors were tested for Rh phenotypes and RHD genotypes. Among them, total RHD deletion, partial RHD gene, and RhD(el) were found in 185 (62.9%), 15 (5.1%), and 94 (32.0%), respectively. The 1227A allele and exon 9 of the RHD gene were found in all 94 RhD(el) donors. The Ccee was the most prevalent phenotype in the RhD(el) group (78/94 [83%]), and the ccee phenotype was highly prevalent in the true D- group (87.6%). RHD 1227A can be used as an important and useful genetic marker for RhD(el). It can be detected easily by a simple, rapid, specific sequence primer-polymerase chain reaction method.     

贵州黔东南地区侗族人群RHD基因合子型检测

目的 调查贵州黔东南地区侗族人群RHD基因组合情况,了解RHD基因遗传特点.方法 根据中国人Rh分子特点和RHD基因缺失原理设计2对引物,对301名侗族无血缘关系的血样分别检测RHD基因第1外显子和融合盒子,再引入1对内对照引物以确保结果的可靠性.结果 292例RhD阳性样本中58例(19.86%)为RHD+/RHD-型,234例(80.14%)为RHD+/RHD+型.9例血清学初筛RhD阴性样本中:5例RHD+/RHD-型(2例弱D,3例Del型);3例RHD+/RHD+型(1例弱D,2例DeD;1例RHD-/RHD-(1例D阴性).结论 本组RhD阳性人群中存在的RHD+/RHD-杂合型高于其它地区的报道,9例血清学初筛D阴性的人群中存在RHD基因单倍体比例也较高.     

Noninvasive prenatal screening for RHD: the 1st national antenatal directed anti-D prophylaxis program – the Danish model or a guide to robust prediction of need of anti-D

Background As of January 1, 2010, Denmark implemented a national antenatal directed anti-D prophylaxis program. Prior to this, only postnatal prophylaxis directed by serology of cord blood was practiced. A preparatory work for modernization and optimization of fetal care was carried out by the National Board of Health with participation of leading obstetricians, specialists in fetal medicine, general practitioners, midwives, clinical immunologists and administrators. The promising results of non-invasive prenatal diagnosis based on cell free fetal DNA enabled an antenatal directed use of anti-D to those with a D positive fetus, thus eliminating approximately 40% of the antenatal use of anti-D. The complete program now includes an antenatal RHD screening in gestational week 25, and a postnatal serological RhD typing of cord blood, in both instances followed by administration of anti-D to those found positive. Aim The explicit aim of the RHD screening was to obtain the highest possible sensitivity to recommend anti-D in all cases where it might be indicated whereas specificity was of secondary priority. Methods Health services in Denmark are organized in five regions; accordingly screening has been implemented in every region, each using its own in-house screening methodology. However, all regions have implemented automated DNA purification and the assays are variations over a theme of multiple PCRs covering several exons. A control reaction for DNA purification is obligatory. However, a control for fetal DNA is not included. A working group with members from all regions has implemented a national scheme for quality control. Digital on-line delivery of results to general practitioners, midwives and obstetricians is implemented in most regions thus cutting down on cost and time. Results The continuation of postnatal serology has allowed a quality control of RHD screening. During the first six months of screening only two false negative results occurred out of 2312 tested (0·087%) suggesting a sensitivity of 99·9%. Due to the sacrifice of specificity some unnecessary use of anti-D occurred, 39 cases or 1·7%. Discussion The elimination of 38·3% of the use of anti-D, that is unnecessary and the related clinical contacts balances the expenses for the screening. An unforeseen benefit of the screening has emerged. The availability of the antenatal RHD results at the time of delivery has simplified logistics and preliminary clinical action is taken based on the antenatal result. Only in the very rare occasions of a (false negative) discrepancy between antenatal and postnatal results is communication needed between laboratory, clinician, and patient. The challenge will be to maintain the high standards in a possible future setting without serology serving as a benchmark for quality control.     

Prenatal typing of fetal DNA in cases of potential alloimmune haemolytic disease of the newborn: clinical benefit outweighs disadvantage.

Haemolytic disease of the newborn (HDN) is caused by the action of maternal antibodies on paternally derived antigens present on fetal erythrocytes. In its most severe form it may lead to death in utero. Although the introduction of anti-D prophylaxis has greatly reduced the incidence of the disease, a significant number of cases appear each year. Current antenatal management aims to predict whether the fetus will be severely affected, correct the fetal anaemia if present and deliver the baby at the optimal time. Indirect methods include antibody quantitation performed on maternal blood samples, and amniocentesis to determine bilirubin levels in the amniotic fluid. Fetal blood sampling offers a direct method to determine the antigen status of the fetus, and enables measurement of fetal haemoglobin and haematocrit if appropriate. Both fetal blood sampling and amniocentesis are invasive procedures and have the potential to produce further antibody stimulation which can compound existing problems and compromise the pregnancy. The ultimate aim is to develop an accurate, non-invasive technique for fetal DNA typing which can be carried out in the first trimester. Research methods outlined here suggest that this is feasible. Pinpointing those pregnancies where further intervention is not required will reduce the demand on NHS resources.     

The fetal and neonatal outcomes of Rhesus D antibody affected pregnancies in Northern Ireland

It has been suggested that routine antenatal prophylactic anti-D should be introduced for prevention of Rhesus D (RhD) haemolytic disease. Before making changes to the current prevention program it is important, therefore, to have up-to-date data on affected infants. Pregnant women with anti-D antibodies between September 1994 and February 1997 were identified by the Northern Ireland Blood Transfusion Service. The records of 124 women and 130 babies were examined. 26% of planned deliveries occurred in hospitals without paediatric cover. Rhesus isoimmunisation affected 78 babies. Fifty-nine infants were admitted to one of seven neonatal units. There were 2 stillbirths and 1 neonatal death. Two infants have severe neurodevelopmental delay. There is still significant morbidity associated with RhD haemolytic disease. Care for RhD affected pregnancies should be centralised to guarantee training opportunities and maintenance of expertise. The current management of these pregnancies should be re-examined before changing the Rhesus prevention program.     

贵州黔东南地区侗族人群RHD基因合子型检测

目的 调查贵州黔东南地区侗族人群RHD基因组合情况,了解RHD基因遗传特点.方法 根据中国人Rh分子特点和RHD基因缺失原理设计2对引物,对301名侗族无血缘关系的血样分别检测RHD基因第1外显子和融合盒子,再引入1对内对照引物以确保结果的可靠性.结果 292例RhD阳性样本中58例(19.86%)为RHD+/RHD-型,234例(80.14%)为RHD+/RHD+型.9例血清学初筛RhD阴性样本中:5例RHD+/RHD-型(2例弱D,3例Del型);3例RHD+/RHD+型(1例弱D,2例DeD;1例RHD-/RHD-(1例D阴性).结论 本组RhD阳性人群中存在的RHD+/RHD-杂合型高于其它地区的报道,9例血清学初筛D阴性的人群中存在RHD基因单倍体比例也较高.

Abstract：

Objective To investigate the RHD zygosity distribution and the genetic characteristics of RHD gene in the Dong ethnic population in southeast area of Guizhou province. Methods Based on the characteristics of Chinese RHD and the RHD-specifie deletion, two pairs of primers specific for hybrid Rhesus box and exon 1 of RHD respectively, were designed, combined with a pair of internal control primers. Polymerase chain reaction was performed to genotype the samples. Results In the 292 RhD positive samples, 58 (19. 86%) were RHD+/RHD- heterozygotes, and the others (80. 14%) were RHD+/RHD+ homozygotes. In the 9 D-negative samples, 5 were RHD+/RHD- heterozygotes (2 weak D, 3 Del), 3 were RHD+/RHD+ homozygotes (lweak D, 2 Del), and 1 was RHD-/RHD- homozygote.Conclusion RHD+/RHD- heterozygosity is higher (19. 86%) in D-positive individuals of Dong ethnic group in Guizhou province than that in other areas; RHD gene heterozygosity is also high in the D-negative individuals in this ethnic group.     

贵州黔东南地区侗族人群RHD基因合子型检测

目的 调查贵州黔东南地区侗族人群RHD基因组合情况,了解RHD基因遗传特点.方法 根据中国人Rh分子特点和RHD基因缺失原理设计2对引物,对301名侗族无血缘关系的血样分别检测RHD基因第1外显子和融合盒子,再引入1对内对照引物以确保结果的可靠性.结果 292例RhD阳性样本中58例(19.86%)为RHD+/RHD-型,234例(80.14%)为RHD+/RHD+型.9例血清学初筛RhD阴性样本中:5例RHD+/RHD-型(2例弱D,3例Del型);3例RHD+/RHD+型(1例弱D,2例DeD;1例RHD-/RHD-(1例D阴性).结论 本组RhD阳性人群中存在的RHD+/RHD-杂合型高于其它地区的报道,9例血清学初筛D阴性的人群中存在RHD基因单倍体比例也较高.     

Serologic and molecular characterization of D variants in Brazilians: impact for typing and transfusion strategy

Rh discrepancies are a problem during routine testing because of partial D or weak D phenotypes. Panels of monoclonal antibodies (MoAb) are being developed to identify D variants such as partial D and weak D when there are anomalous D typing results; however, molecular characterization offers a more specific classification of weak and partial D. The weak D and partial D phenotypes are caused by many different RHD alleles encoding aberrant D proteins, resulting in distinct serologic phenotypes and the possibility of anti-D immunization. We evaluated currently used serologic methods and reagents to detect and identify D variants and correlated the results with molecular analyses. A total of 306 blood samples from Brazilian blood donors and patients with discrepant results in routine D typing were analyzed. In total, 166 (54.2%) weak D, 136 (44.4%) partial D, 3 (1%) DEL, and 1 (0.3%) DHAR variants were identified. Among weak D samples, 76 weak D type 1 (45.8%), 75 weak D type 2 (45.2%), 13 weak D type 3 (7.8%), and 2 weak D type 5 (1.2%) alleles were found. Among the partial D samples, 49 type 4.0 weak partial D (36%), 9 DAR (6.6%), 24 DFR (17.6%), 6 DBT (4.4%), 1 DHMi (0.73%), 26 DVI (19%), 14 DVa (10.3%), 5 DIVb (3.7%), and 2 DVII (1.5%) were observed. Two samples identified as DEL by adsorption-elution were characterized by molecular analyses as RHD(IVS5–38DEL4) and one sample was characterized as RHD(K409K). One sample was characterized as DHAR, a CE variant positive with some monoclonal anti-D. Our results showed that the use of different methods and anti-D reagents in the serologic routine analysis revealed D variants that can be further investigated. Molecular methods can help to differentiate between partial D and weak D and to characterize the weak D types, providing additional information of value in the determination of D phenotypes. This distinction is important for optimized management of D– RBC units and for the prevention of anti-D–related hemolytic disease of the fetus and newborn.