RHD Variants Research Articles

Evaluating RhD Assessment By Automated Methodology: A Potential ‘Blind Spot’ For RhD Variant Identification

Abstract Background Automated blood typing platforms are ideal for blood donor centers, given their broad recognition of RhD antigen variants, resulting in products from such donors to be labeled as RhD positive. However, this ‘blind spot’ for variants may be problematic for hospital-based transfusion services, since RhD variants may require provision of RhD- RBC units and Rh immune globulin. The extent to which automated methods accurately characterize variant D antigen expression is not known. As such, the objective of our study was to assess D typing results, as well as RHD genotyping, among two automated testing platforms at our facility. Methods The study site is a single tertiary care center with analysis done from 12/2020-1/2024. At our facility, all females <60 years of age with RhD antigen testing results of ≤2+ agglutination (0-4+ scale) via automated methods [solid phase testing 12/20-4/22 (Neo, Immucor, Norcross, GA) and gel testing 5/22-1/24 (Erytra, Grifols, Emeryville, CA)] are assessed by a tube RhD antigen test (Grifols reagents). If tube testing confirms a ≤2+ result (0-4+ scale), molecular variant D analysis is performed (Versiti, Milwaukee, WI). Patients typed as Rh negative with anti-D antibodies, or those with inconsistent D typing, are also subjected to genetic D testing. The Wilcoxon matched pairs signed rank test was employed for comparative data (P< 0.05 was deemed significant). Results Amongst tested patients, 73 demonstrated ≤2+ agglutination via an automated typing method, leading to variant D genetic analysis. Of these, 71% (n=52) harbored variant D antigens. Within this subgroup, 29 (56%) subjects exhibited variants associated with potential allo-anti-D development (n=5 partial D variants; n=24 weak D variants). Notably, 4 patients with partial D and 1 with weak D demonstrated allo-anti-D antibodies. Of 23 patients with variant D who underwent automated gel testing, 20 concurrent tube testing results were available. Gel testing revealed significantly stronger agglutination compared to tube testing (p < 0.001). In contrast, for paired solid phase and tube testing (n=11), there was no statistically significant difference in agglutination strength (p = 0.28). Conclusion Automated testing, particularly gel-based platforms, exhibits reduced sensitivity to RhD antigen variant identification when compared to tube testing, placing subsets of patients potentially at risk for forming allo-anti-D. Implementing protocols for verification of D status could enhance the sensitivity of variant D detection for facilities that largely rely on automated methods for antigen typing.

Molecular testing in a patient with multiple alloantibodies and warm autoimmune hemolytic anemia: A case study

Abstract Introduction/Objective We present a case of RBC alloimmunization and warm autoimmune hemolytic anemia (WAIHA) in a patient with a recent RBC transfusion. Serological testing followed by molecular testing revealed a rare D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/- predicted phenotype. Methods/Case Report A 75-year-old black male with secondary myelofibrosis presented with a sudden onset of chest pain, dyspnea and weakness during an RBC transfusion. The patient was subsequently admitted for further workup. Labs showed low hemoglobin, low haptoglobin, elevated LDH and a positive direct antiglobulin (DAT) test. The patient was started on intravenous immunoglobulin and steroids for suspected WAIHA. On immunohematology serologic testing, blood typed as B-positive and polyspecific DAT positive with anti-IgG positive and anti-C3 negative. Serum plasma studies showed probable warm autoimmune hemolytic anemia and allo anti-S, -E, and -C by previous history. Eluate studies showed panagglutinin and antibody with D-like specificity. Immucor PreciseTypeTM Human Erythrocyte Antigen (HEA) Molecular BeadChip test predicted a partial e antigen and potential hrB-. Genotyping for RHD variants was performed and revealed RHD*01 (homozygous or hemizygous). Targeted genomic testing did not detect variants associated with common partial or weak RhD antigens. Based on this testing, the patient was not predicted to be at risk of allo-anti-D. An RHCE genotype for C, c, E and e variants including hrB and hrS showed RHCE*ce48C,733G/RHCE*ce733G with predicated phenotype of D+ C- E- partial c+ partial e+ VS+ V+ hrS+ hrB+vw/-. Based on the RHCE testing, the patient was predicted to be at risk for allo-anti-c, -e, -f (ce) and possibly anti-hrB. Results (if a Case Study enter NA) NA Conclusion In this case, serologic testing alone did not reveal a precise diagnosis. Molecular testing aided in the diagnosis of WAIHA and will help guide future transfusions. Pathologists must consider molecular testing when faced with a challenging serologic diagnosis.

Single-exon fetal RHD genotyping: a 31-month follow up in the obstetric population of Western Sweden.

The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHD genotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.

Molecular Background of RhD-positive and RhD-negative Phenotypes in a Saudi Population.

The RHD gene is one of the most complex blood group genes. The molecular background of the RHD gene in RhD-negative and RhD-positive individuals varies within and among different populations. Knowing the molecular basis of the RHD gene in a specific population is required to establish effective genotyping methods. While the molecular basis has been revealed in many ethnicities, such as Caucasians and Black Africans, it still requires elucidation in Arabs. The aim of this study was to gain insights into the molecular basis of RhD-positive and RhD-negative phenotypes in Saudi donors. Conventional serological tests were used to determine the Rh phenotypes in 136 Saudi donors by typing D, C, c, E, and e antigens. Multiplex-PCR and Single Specific Primer-PCR were used to detect the presence of exons 3, 4, and 7 and the hybrid Rhesus box gene, respectively, in RhD-negative and/or RhD-positive samples. Of the 136 samples, 70 were RhD positive and 66 were RhD negative. None of the RhD-negative donors had any of the three tested exons, whereas the hybrid Rhesus box gene was detected in all, indicating the zygosity status of the RHD deletion allele. The hybrid Rhesus box gene was detected in 79% of the RhD-positive individuals, suggesting high frequencies of RHD-negative haplotypes. The study findings indicate that Saudis with the RhD-negative phenotype are likely to have an entire RHD deletion in the homozygous state. However, a more comprehensive analysis of variant RHD alleles in the Saudi population is required to implement effective and dedicated molecular RHD typing strategies.

Serologic profiling of D variants in donor routine: unveiling the impact on false-negative results and alloimmunization.

The high number of D variants can lead to the unnecessary use of Rh immune globulin, overuse of D- RBC units, and anti-D allommunization. D variant prevalence varies among ethnic groups, and knowledge of the main variants present in a specific population, their behavior in serologic tests, and their impact on clinical practice is crucial to define the best serologic tests for routine use. The present study aimed to explore the serologic profile of D variants and to determine which variants are most associated with false-negative D typing results and alloimmunization. Donor samples were selected in two study periods. During the first period, D typing was performed on a semi-automated instrument in microplates, and weak D tests were conducted in tube or gel tests. In the second period, D typing was carried out using an automated instrument with microplates, and weak D tests were performed in solid phase. Samples from patients typed as D+ with anti-D were also selected. All samples were characterized by molecular testing. A total of 37 RHD variants were identified. Discrepancies and atypical reactivity without anti-D formation were observed in 83.4 percent of the samples, discrepant D typing results between donations were seen in 12.3 percent, and D+ patients with anti-D comprised 4.3 percent. DAR1.2 was the most prevalent variant. Weak D type 38 was responsible for 75 percent of discrepant samples, followed by weak D type 11, predominantly detected by solid phase. Among the D variants related to alloimmunization, DIVa was the most prevalent, which was not recognized by serologic testing; the same was true for DIIIc. The results highlight the importance of selecting tests for donor screening capable of detecting weak D types 38 and 11, especially in populations where these variants are more prevalent. In pre-transfusion testing, it is crucial that D typing reagents demonstrate weak reactivity with DAR variants; having a serologic strategy to recognize DIVa and DIIIc is also valuable.

Breakpoint regions of an RHD-CE(4-9)-D allele and a rare JK allele in a Pacific Islander individual.

Among 710 RHD alleles, 3 alleles have been shown to express CcEe antigens and, among 67 hybrid alleles of the RHD gene, 2 alleles have evolved to include RHCE exons 4-9. No breakpoint region had been described for such RHD-CE(4-9)-D hybrid alleles. In the Kidd blood group system, the JK*02N.01 null allele is found with high prevalence in the Polynesian population. We investigated a self-identified Pacific Islander with discrepant serologic and molecular results for his C and Jkb antigens. Another 8 samples with genotype-phenotype discrepancies in the Kidd blood group system were assessed. A combination of published molecular methods and commercial kits were applied to analyze the RHD, RHCE, and SLC14A1 gene sequences, as were hemagglutination tests to determine the serologic phenotypes. Nucleotide sequencing of the RHD gene in the index case, including relevant intron stretches, and cDNA identified an RHD-CE(4-9)-D hybrid allele. Nucleotide sequencing of his RHCE gene confirmed the presence of 2 RHCE*ce alleles despite expressing the C antigen. Sequencing of his SLC14A1 gene documented the JK*02N.01 null allele. In the other 8 samples, 5 previously known SLC14A1 nucleotide substitutions were identified. The JK*02N.17 allele was determined to be Jkb-positive. We determined the 2 breakpoint regions of his RHD-CE(4-9)-D hybrid allele, which was likely distinct from the 2 previously published hybrid alleles due to the differences in the linked RHCE allele. His RHD variant was shown to express the C antigen. An SLC14A1 substitution was underlying his unexpected Jkb-negative phenotype. In a quality improvement project, we resolved 8 samples with similarly discrepant results between Jk serology and red cell genotyping.

Estimating the serological underrecognition of patients with weak or partial RHD variants.

For patients with weak or discrepant RhD RBC phenotypes, RHD genotyping is employed to determine need for RhD-negative management. However, many RHD variants are type D-negative or D-positive. Serological recognition rates (RRs) of weak and partial RHD variants are poorly characterized. Four US studies employing RHD genotyping for weak or discrepant RhD phenotypes provided data for race/ethnicity-specific serological recognition. Three studies used microplate, and 1 used gel and tube; 2 had anti-D data. We obtained White and Hispanic/Latino allele frequencies (AFs) of weak D types 1, 2, and 3 single-nucleotide variants (SNVs) from the Genome Aggregation Database (gnomAD, v4.0.0) and devised Hardy-Weinberg-based formulas to correct for gnomAD's overcount of hemizygous RHD SNVs as homozygous. We compiled common partial RHD AF from genotyped cohorts of US Black or sickle cell disease subjects. From variant AF, we calculated hemizygous-plus-homozygous genetic prevalences. Serological prevalence: genetic prevalence ratios yielded serological RRs. Overall RRs of weak D types 1-3 were 17% (95% confidence interval 12%-24%) in Whites and 12% (5%-27%) in Hispanics/Latinos. For eight partial RHD variants in Blacks, overall RR was 11% (8%-14%). However, DAR RR was 80% (38%-156%). Compared to microplate, gel-tube recognition was higher for type 2 and DAU5 and lower for type 4.0. Anti-D was present in 6% of recognized partial RHD cases, but only in 0.7% of estimated total genetic cases. Based on AF, >80% of patients with weak or partial RHD variants were unrecognized serologically. Although overall anti-D rates were low, better detection of partial RHD variants is desirable.

Prediction of the antigenic regions in eight RhD variants identified by computational biology.

Changes in RHD generate variations in protein structure that lead to antigenic variants. The classical model divides them into quantitative (weak and Del) and qualitative (partial D). There are two types of protein antigens: linear and conformational. Computational biology analyses the theoretical assembly of tertiary protein structures and allows us to identify the 'topological' differences between isoforms. Our aim was to determine the theoretical antigenic differences between weak RhD variants compared with normal RhD based on structural analysis using bioinformatic techniques. We analysed the variations in secondary structures and hydrophobicity of RHD*01, RHD*01W.1, W2, W3, RHD*09.03.01, RHD*09.04, RHD*11, RHD*15 and RHD*21. We then modelled the tertiary structure and calculated their probable antigenic regions, intra-protein interactions, displacement and membrane width and compared them with Rhce. The 10 proteins are similar in their secondary structure and hydrophobicity, with the main differences observed in the exofacial coils. We identified six potential antigenic regions: one that is unique to RhD (R3), one that is common to all D (R6), three that are highly variable among RhD isoforms (R1, R2 and R4), one that they share with Rhce (R5) and two that are unique to Rhce (Ra and Rbc). The alloimmunization capacity of these subjects could be explained by the variability of the antigen pattern, which is not necessarily recognized or recognized with lower intensity by the commercially available antibodies, and not because they have a lower protein concentration in the membrane.

Characterization of Novel RHD Allele Variants and Their Implications for Routine Blood Group Diagnostics.

The Rh system, including the highly immunogenic D antigen, is one of the clinically most important blood group systems in transfusion medicine. Numerous alleles of the RHD gene are associated with variant RhD phenotypes. In case of Rh incompatibility, some of them can induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Thus, accurate blood group diagnostics are critical for safe transfusion therapy. We characterized phenotypes of four individuals revealing weakened D expression during routine pre-transfusion testing. Standard gel card matrix techniques with monoclonal and polyclonal anti-D antibodies were used for serological typing, complemented using D epitope and antigen density analysis. Genotyping employing PCR with sequence-specific primers, genomic and allele-specific Sanger sequencing and in silico protein analysis were performed. Four novel RHD alleles associated with weak D or partial D phenotypes were identified. One of the mutations is predicted to disrupt the terminal stop codon and result in an elongated translation of the mutant D protein that phenotypically exhibits a loss of D epitopes. Furthermore, a hybrid gene formed with the homologue RHCE gene is described. The presented data enhances the understanding of the Rh system and may contribute to continued advances in blood group diagnostics.

Rare occurrence of RHD null alleles with Del expression among serologically D-negative blood donors

Background/Aim: An investigation into the diversity of serologically D-negative blood donors in the Republic of Srpska entity of Bosnia and Herzegovina forms the basis of this research. The primary purpose of the study was the examination of RHD variants over a period of five years. Methods: A comprehensive depiction of the RHD distribution in D-negative blood donors is achieved through a combination of serological observations and DNA testing (PCR-SSP with fluorometric signal detection), involving 74,149 blood donors. The adsorption/elution method was used to confirm the Del phenotype. Results: A small fraction (0.31 %) of the serologically D-negative blood donors was found to contain eight different RHD alleles. The Del phenotype of the RHD*01N.03 and RHD*01EL.44 alleles was highlighted, challenging the common perception that these alleles are associated exclusively with a D-negative expression. Conclusion: The importance of molecular methods in analysing and understanding Del variants, which typically elude conventional serological assays , is underscored by the findings. A group of donors seemingly having the RHD*01 allele but who lacked D antigen expression was encountered, hinting at the potential presence of still unidentified, possibly geographically restricted, RHD variants or alterations in other genes responsible for the expression of Rh proteins in the erythrocyte membrane.

RHD-negative red cells may be avoided for patients with ambiguous serologic typing for the RHD antigen.

Serologic typing with monoclonal anti-D is mandatory for RHD antigen determination before transfusion, but due to aberrant (weak or partial) variants of RHD, results may be ambiguous and molecular RHD-typing is required. Before that, RHD-negative (RHD -) red blood cells concentrates (RBCs) shall be transfused to avoid anti-D formation, which probably leads to wastage of RHD - RBCs. STUDY DESIGNAND METHODS: All patients with ambiguous results in serologic RHD-typing and molecular RHD-typing were assessed retrospectively. The proportions of patients at risk for anti-D formation and the proportion of RHD - RBCs transfused unnecessarily were evaluated for the following transfusion strategies: (1) RHD-positive (RHD + )RBCs for all patients, (2) RHD + RBCs for patients with at least 2+ reaction with anti-D, (3) RHD + RBCs for patients with C and/or E in their RHCE-phenotype, (4) RHD + RBCs for patients with C and/or E and at least 2+ reaction, and (5) RHD - RBCs for all patients. A total of 112 patients were included. Most had weak D type 1-3 and a minority had other, rare RHD variants. The risk of anti-D formation was 4.5%, 2.9%, 1.8%, 1.0%, and 0% for strategies 1-5, respectively. The proportion of RHD - RBCs transfused unnecessarily was 0%, 49.5%, 0.9%, 50.5%, and 95.5%. Transfusing patients with a C and/or E in their RHCE-phenotype with RHD + RBCs resulted in a very low risk of immunization while avoiding wastage of RHD - RBCs. Therefore, this strategy should be used for some patients with ambiguous results in serologic RHD-typing and pending results of molecular RHD-typing.

Genotype analysis to clarify RhD variants in discrepant samples of Chilean population.

The D antigen variants are classified as weak, partial, and extremely weak (DEL) and can be differentiated using molecular tests. In Chile, the laboratories of local blood centers do not identify variants of the D antigen, referring them for study to the Reference Laboratory of the Public Health Institute of Chile. So, our aim was to talk about the results of the molecular analysis of variants of the D antigen in samples that had different results in the serological classification. In the D antigen classification of the Rh system, 479 samples with serological discrepant results were sent for molecular analysis. The Rh phenotype was performed with monoclonal anti-C, anti-c, anti-E, and anti-e antisera by direct agglutination. To find the D antigen, researchers used direct agglutination with monoclonal antisera and indirect antiglobulin testing with the column (gel) agglutination method. Molecular analysis was performed with a polymerase chain reaction with sequence-specific primers (SSP-PCR) and sequencing. The presence of D antigen variants was confirmed in 332 samples (69.3%), with an initial discrepancy in serological classification. In this group of discrepant samples, the frequency of weak RhD variants was 66% (219/332), that of extremely weak RhD was 28% (93/332), and that of partial RhD was 6% (20/332). The weak variants type 2 (27.4%), type 3 (8.4%), type 48 (8.4%), and type 1 (8.1%) were the next most prevalent variants after RHD*DEL43 (28%). The ccEe (R2r) phenotype was the most frequently detected (38.4%) and is present in 87% of the RHD*DEL43 samples. The E antigen is associated with the presence of this variant. Our analyses give the first description of D antigen variants in Chile. The most common variants are DEL type (RHD*DEL43) and weak (weak type 2), which are linked to the ccDEe (R2r) phenotype. These findings allow us to characterize the variants of the D antigen in Chile and, according to the obtained data, to design strategies for the management of donors, patients, and pregnant women.

Real-World Feasibility of RH Genotype-Matched Red Blood Cells for Chronically Transfused Patients with Sickle Cell Disease

Introduction Most red blood cell (RBC) alloimmunization in sickle cell disease (SCD) is caused by sensitization to Rh (D, C, c, E, and e) and K antigens. Provision of serologic Rh and K-matched RBCs has reduced but not eliminated Rh alloantibody formation due to the high genetic variation within the RH loci in Blacks. Over 85% of patients with SCD, as well as Black donors needed to support CEK matching programs, have variant RHD or RHCE alleles that result in loss or alteration of Rh antigens. These variant Rh proteins are not detected by standard serologic tests, and thus, genetically matched RBCs may be necessary to prevent all Rh alloimmunization events. We previously used bioinformatic modeling to show that RH genetic matching was achievable for a cohort of ~200 patients of whom ~50% were chronically transfused, and represented 98 different RHD/RHCE allele combinations, with only 10 additional donations each weekday (~1050 Black donors/month) than required for serologic Rh and K matching. The real-world feasibility of providing RH genotype-matched RBCs to patients requiring regular transfusion has not been tested. Methods We performed a pilot study to evaluate the feasibility of providing RH genotype-matched RBCs from Black and Hispanic donors to non-Rh- and Rh-alloimmunized chronically transfused patients with SCD (1 to 18 years old). Black donor units at the blood center were first screened by serology, HEA BeadChip, and in-house RH SNP assays, and those selected for transfusion to study subjects had comprehensive RHD and RHCE genotyping. Data captured included RH genotypes of matched units identified, transfusion delays, unit age at time of transfusion, and pre-transfusion hemoglobin (Hb) and HbS levels. On-study values were compared to pre-study values obtained in the 6 to 12 transfusion visits prior to study enrollment. Results The study opened in February 2020. Due to the COVID-19 pandemic, donor center collections dropped to a nadir of ~500 Black donor units/month in 2020 but returned to ~1500 units/month currently, similar to pre-COVID numbers. For 14 participants (13 Black, 1 Hispanic), we transfused 327 RH genotype-matched units; 287 (88%) were exact RH genotype-matched (donor with identical genotype, homozygous for a matching RH haplotype, or RH allele match) and 40 (12%) were RH matched with the provision that RHD*RHD and *DAU0 and RHCE*ce and *ce48C are equivalent since no epitopes appear to differ between these alleles. No transfusion visits (n=183) were postponed due to the inability to identify RH genotype-matched units, including during the pandemic and national blood shortages that had a disproportionate impact on Black donations. Using individual patients as factors, ANOVA showed no statistically significant difference in unit age (days, Figure) or pre-transfusion Hb values between pre- and on-study transfusion visits. Although the goal maintenance pre-transfusion HbS levels varied between 30 and 50%, a statistically significant lower mean HbS on- (n=183) vs pre-study (n=139) (ANOVA F-ratio 15.4, p=0.000) (Figure) potentially suggests improved transfused RBC survival with RH genotype matching. This feasibility trial was not powered to detect a difference in alloantibody formation, but no Rh nor non-Rh antibody have been detected. Conclusion Recruitment of minority blood donors is a growing initiative nationally needed to support CEK matching. Our preliminary results suggest that genetic RBC matching at the RH loci may be feasible with Black and Hispanic donors. Donor center RH genotyping can facilitate transfusion of RH genotype-matched RBCs, improve allocation of minority and rare donor units and, at the same time, potentially improve patient care by avoiding Rh alloimmunization for individuals with SCD.

Frequency of the 'Asia type' DEL with weak D phenotype in Chinese.

DEL individuals account for 9-30% of serological RhD negative population in east Asia and majority of them carrying the RHD*DEL1 allele are referred to as 'Asia type' DEL individuals. There is a lack of data on the molecular basis for 'Asia type' DELs with weak RhD phenotype. Therefore, the aim of this study is to unveil 'Asia type' DELs by elucidating the genetic background and analyzing the serological results METHODS: With a microplate typing protocol, RhD characterization was performed in samples from one million blood donors collected at Chengdu blood center during the period from 2019 to 2022. RhD confirmatory test was performed by direct antiglobulin test and indirect antiglobulin test with five anti-D reagents to detect RhD variants. Molecular characterization of samples categorized as RhD variants was studied by using direct genomic DNA sequencing and RHD zygosity analysis, followed by adsorption and elution tests conduced for samples carrying RHD*DEL1 allele to confirm the presence of RhD antigens on the red cells RESULTS: We reported here 21 RhD variant samples were detected by micro-column gel agglutination assay with IgG anti-D antibodies. Moreover, the agglutination reaction was stronger with IgG anti-D reagents in micro-column gel card than with IgM/IgG blended anti-D antibodies. Each of the 21 samples carried the RHD*DEL1 allele, which indicated that they were 'Asia type' DEL. Of the 21 'Asia type' DEL samples, 9 samples were detected to be RHD+/ RHD+ homozygotes, whereas the other 12 samples were RHD+/RHD- hemizygotes. Among the samples phenotyped for RhCE, seven were CCee and four were Ccee. In this study, DEL samples carrying RHD*DEL1 showed weak RhD phenotype with some anti-D reagents in RhD confirmatory test, which suggest that a serology strategy using several anti-D reagents may be helpful to detect this 'Asia type' DEL. Further studies are needed to elucidate whether the 'Asia type' DELs with weak RhD phenotype have stronger antigenicity and could cause serious transfusion reaction.

ANTI-D ALLOIMMUNIZATION IN SICKLE CELL DISEASE (SCD) PATIENTS WITH WEAK RHD VARIANT ALLELES

ANTI-D ALLOIMMUNIZATION IN SICKLE CELL DISEASE (SCD) PATIENTS WITH WEAK RHD VARIANT ALLELES

Fetal RHD Screening in RH1 Negative Pregnant Women: Experience in Switzerland.

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

Noninvasive screening of fetal RHD genotype in Chinese pregnant women with serologic RhD-negative phenotype.

Noninvasive fetal RHD genotyping has been provided to nonimmunized RhD-negative pregnant women to guide anti-D prophylaxis. Among the Chinese, more than 30% of the RhD-negative phenotype is associated with variant RHD alleles, which would limit the accuracy of fetal RHD status prediction; thus, more targeting and proper programs need to be developed. Fluorescence quantitative polymerase chain reaction PCR (qPCR) or Sanger sequencing on all RHD exons was used to detect maternal RHD genotypes. For pregnant women with RHD*01N.01 or RHD*01N.03 alleles, the presence of RHD exons 5 and 10 in cell-free DNA was determined by qPCR. For pregnant women with the RHD(1227G>A) allele, high-throughput sequencing on exon 9 of the RHD gene and RHCE gene was used to predict fetal RhD phenotype. Among 65 cases of Chinese pregnant women with the serologic RhD-negative phenotype, three major genotypes were identified: RHD*01N.01/RHD*01N.01 (61.5%), RHD*01N.01/RHD(1227G>A) or RHD*01N.03/RHD(1227G>A) (20%), and RHD*01N.01/RHD*01N.03 (13.8%), along with three cases of minor genotypes (4.6%). For 43 pregnant women with the RHD*01N.01 or RHD*01N.03 alleles, qPCR on maternal cell-free DNA yielded a 98.5% (42/43) accuracy rate and 100% successful prediction rate. High-throughput sequencing was successfully used to predict fetal RhD phenotypes for 13 pregnant women with RHD(1227G>A). On the basis of maternal RHD genotyping, fetal genotyping through qPCR or high-throughput sequencing can improve the accuracy and success rate of prenatal fetal RhD phenotype prediction among Chinese pregnant women. It plays a potential role in guiding anti-D prophylaxis and pregnancy management in Chinese pregnant women.

Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

Transfusion management of Africans with RHD variants in China.

The presence of D variant among minorities could produce a higher rate of alloimmunization observed in patients from this group. This is partly due to the ethnic and racial specificity of RHD variants and the limited availability of Rh-matched blood donors. Approximately half a million African migrants in China carrying distinct Rh blood type composition have presented to the health care system with an imperative safety requirement of blood transfusion among 1.3 billion Chinese individuals. We depict the clinically significant RHD alleles among African migrants living in China and identify the genetic similarities and disparities to Chinese. We discussed practical strategies to manage the unique transfusion needs of African migrants in China.

A rapid ABO and RhD test demonstrates high fidelity to blood bank testing for RhD typing.

The rapid provision of blood products is life-saving for patients with massive hemorrhage. Ideally, RhD-negative blood products would be supplied to a woman of childbearing potential whose Rh type is unknown due to the risk of D-alloimmunization and the potential for hemolytic disease of the fetus and newborn to occur if RhD-positive blood products are transfused. Therefore, there is a need for a test that rapidly determines her RhD type. This study compared the RhD type determined using a rapid ABO and RhD test to the RhD type determined by an immunohematology reference laboratory. After receiving ethics review board approval, 200 random, unique, deidentified patient samples that had undergone routine pretransfusion testing in an immunohematology reference laboratory using column agglutination technology were collected and tested using a rapid ABO and RhD test (Eldoncard Home kit 2511). The RhD typing results from these two methods were compared to determine the accuracy of the rapid ABO and RhD test. The rapid ABO and RhD test produced results that were concordant with the transfusion service's results in 199/200 (99.5%) of cases, with a negative predictive value of 98.2% and 99.3% sensitivity. The single outlier was likely an RhD variant due to its serological characteristics. These data indicate that this rapid ABO and RhD test could be used for the rapid determination of a patient's RhD type, perhaps even in the emergency department, which could guide the selection of blood products provided during their resuscitation.