Variant RHCE Research Articles

Genotyped RhD+ red cells for D-positive patients with sickle cell disease with conventional RHD and unexpected anti-D

AbstractAnti-D can occur in D-positive patients who inherit RHD genetic variants encoding partial D antigen expression, but unexpected anti-D is also found in the plasma of patients with sickle cell disease who have conventional RHD gene(s) and are transfused with units from Black donors. These anti-D are likely stimulated by variant Rh expressed on donor cells; however, patients with anti-D, regardless of cause, are transfused for a lifetime with D-negative (Rh-negative) blood. This results in significant increased use of Rh-negative units, especially for those requiring chronic transfusion, which can strain Rh-negative blood inventories. We tested whether D-positive patients who made anti-D and had conventional RhD by RHD genotyping could safely be returned to D-positive transfusions without anti-D reappearance or compromised red blood cell survival using RHD genotype-matched units from Black donors. Five patients receiving chronic red cell exchange received an increasing number of D-positive units per procedure with a total of 72 D-positive RHD genotyped units transfused, with no anti-D restimulation. Unexpected anti-C and anti-E were identified during the study associated with donors with variant RHCE alleles. RH genotyping of D-positive units for transfusion may improve use and allocation of valuable Black donor units and reduce demand for Rh-negative blood. This trial was registered at www.clinicaltrials.gov as NCT04156906.

Investigation of Delayed Transfusion Reactions in Sickle Cell Disease Patients Polytransfused in the Brazilian Amazon.

Sickle cell disease (SCD) affects approximately 100,000 people in the United States and millions worldwide, with the highest prevalence of 70% of SCD being found in individuals of African ethnicity. Delayed hemolytic, alloimmunization, and anamnestic transfusion reactions in multiple transfusion patients need to be investigated and managed to avoid a worsening of the patient's clinical status. This paper aims to investigate delayed transfusion reactions in SCD patients who were polytransfused in the Brazilian Amazon. The clinical and laboratory indicators of SCD patients with more than four transfusions were investigated. The patients were treated at the Fundação Hospitalar de Hematologia e Hemoterapia do Estado do Amazonas, Brazil. A total of 44 polytransfused patients with SCD were followed. Regarding Rh phenotype, it was possible to observe a frequency of 26.6% (12) patients with the RZRZ (DCE/DCE) phenotype, in addition to 4.5% (two) patients with RH and RHCE variants. It was also possible to observe 20.5% (nine) patients with an alloimmunization reaction, who presented the following alloantibodies: anti-RhD, anti-E, anti-K, anti-Jkb, anti-N, anti-S, and anti-Dia, two of which are unidentified. Of these, four (44.4%) patients also presented autoantibodies, anti-e, and three unidentified antibodies, and four (44.4%) patients presented an anamnestic reaction, with anti-RhD, K, and Jkb antibodies. Of the 44 patients monitored, 54.4% (24) had clinical and laboratory indicators of a delayed hemolytic reaction. Delayed transfusion reactions, often neglected, occur frequently. Therefore, transfusions need to be monitored for at least 28 days, with medical investigation of clinical and laboratory indicators to make greater use of this therapeutic resource.

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

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.

RHCE variant alleles and risk of alloimmunization in Brazilians.

Variant RHCE alleles are found mainly in Afro-descendant individuals, as well as in patients with sickle cell disease (SCD). The most common variants are related to the RHCE*ce allele, which can generate partial e and c antigens. Although RHCE variant alleles have been extensively studied, defining their clinical significance is a difficult task. We evaluated the risk of RhCE alloimmunization as a consequence of partial antigens in patients with a positive phenotype transfused with red blood cell (RBC) units with the corresponding antigen. A retrospective study was performed with Brazilian patients, evaluating the number of antigen-positive transfused RBC units (incompatible due to partial antigen) in 27 patients with SCD carrying RHCE variant alleles who did not develop antibodies as well as evaluating the variants present in 12 patients with partial phenotype and correlated antibody (one patient with SCD and 11 patients with other pathologies). Two patients showed variant alleles with molecular changes that had not yet been described. Variant RHCE alleles were identified in a previous study using molecular methods. RHCE*ceVS.01 was the most frequent allele found among the patients without antibodies. Six patients with partial c antigen had a mean of 3.8 c+ RBC units transfused, and 10 patients with partial e antigen were exposed for a mean of 7.2 e+ RBC units. Among the variant alleles found in alloimmunized patients, the most frequent was RHCE*ceAR, which was found in five patients; the antibodies developed were anti-hrS and/or anti-c. Our results showed that RHCE*ceVS.01 is indeed the most frequent variant allele in our cohort of patients with SCD, but the partial antigens that were identified have low risk of alloimmunization. RHCE*ceAR is the most impactful variant in the Brazilian population with high risk of alloimmunization and clinically significant anti-hrS formation.

ALLOIMMUNIZATION PREVALENCE IN PATIENTS WITH SICKLE CELL DISEASE (SCD): ASSESSMENT OF THREE TRANSFUSION PROTOCOLS

RBC alloimmunization is a major complication associated with RBC transfusion in patients with SCD with serious complications for the patient including life-threatening delayed hemolytic transfusion reactions and an increase in the risks inherent to transfusion such as TRALI, infectious diseases and the hyperhemolysis syndrome. In addition, the presence of alloantibodies, which is often associated with the concomitant presence of autoantibodies, can lead to difficulty in finding compatible units, which can cause transfusion delays. The prevalence of alloimmunization vary according to the transfusion protocols used and may be reduced by matching for RBC antigens. Based on this, the aim of this study was to assess the prevalence of RBC alloimmunization in patients receiving transfusions with ABO and D matching, with phenotype matching for C, E and K antigens and with extended minor RBC antigen matching. A total of 368 patients with SCD, homozygous for HbS were enrolled. In this period, 81 patients received RBC units matched for ABO and RhD, 188 patients were matched for C, E and K and 99 patients were matched for ABO, Rh, K, Fya/Fy/b, Jka/Jkb and S/s antigens. Transfusion and antibody histories were obtained from our records, and antibody screening was done to assess the alloimmunization prevalence. Molecular typing was performed in all patients who had recent transfusions or a positive direct antiglobulin test by Laboratory Developed Tests (LDT) and HEA BeadChip (Immucor) to predict their antigen profile. RH variant alleles were determined in patient's antigen-positive who developed Rh antibodies using RHD and RHCE BeadChips (Immucor). Adsorption onto autologous RBC was also performed to aid the differentiation of autoantibodies from alloantibodies. Of the 368 patients evaluated, 128 were male and 240 female. Mean age was 37.4 years (range 18-60) and they received an average of 74 RBC units. Prevalence of alloimmunization in the SCD patients with ABO/RhD matched only units (n=81) was 54%. For individuals transfused with C, E and K matched units (n=188), alloimmunization prevalence was 13% and patients transfused with extended-matching units (n=99) the prevalence of alloimmunization was 0.8%. Antibody specificities developed by the patients receiving Rh and K matched units were: Anti-e, -D, -Jk a , -Jk b , -S, -Di a , -V and by the patients receiving extended matching were anti-C, -e, -V, -VS. The variant RHCE*ceS , RHCE*ceMO , RHD*DIIIa-CE(4-7)-D and RHD*DAU3 alleles were associated with the production of Rh antibodies in these patients and 9.8% of the alloimmunized patients developed autoantibodies. RBC alloimmunization rates shown to decrease to 24 % in patients with SCD transfused with RBCs matched for C, E and K antigens and to 1.5 % to patients with extended matching. Provision of RBC transfusions with limited and extended antigen matching is essential to reduce the rates of alloimmunization and hemolytic transfusion reactions in patients with SCD.

Five-Years Review of RHCE Alleles Detected after Weak and/or Discrepant C Results in Southern France.

Immunohematology laboratories are regularly facing transfusion issues due to serological weaknesses. Altered (partial) RH antigens account for most of them. In some situations, RHCE variant alleles are involved. Herein we present our three-step molecular exploration, with allele frequencies, that has efficiently untangled RH2 phenotype weaknesses and discrepancies in our 2017–2021 cohort. In the last 5 years, the PACA Corse EFS molecular platform received 265 samples from healthy blood donors or patients with C and C/e typing difficulties. The first-intention technique (DNA array and real time PCR for RHCE*CeRN research) detected RHCE variant alleles in 143 cases (54%). The RHCE alleles classically found in African populations were the most frequent, with RHCE*CeRN allele in 40 cases (15%) and (C)ces haplotype type 1 and 2 in 26 cases (10%). A “CE” effect haplotype was suspected in 56 cases, due to the uncommon DCE haplotype that may explain the low C expression. When there were no RHCE*Ce or RHCE*CE alleles, we then searched for RHD polymorphisms by DNA array. We detected the RHD*DAU5 and RHD*DIVa in 18 and 7 cases respectively, suggesting that C ambiguity is related to the presence of these alleles which has never been described with DAU5. If no variant RHCE and RHD alleles were detected, we finally sequenced the 10 exons of both RHCE and RHD genes according to the clinical context and found seven new RHCE alleles. Thus, this molecular strategy would improve the knowledge of RHCE variants’ expression and, thus, optimize the transfusion management.

From the investigation of RHD-CE hybrid genes to the recognition of RHCE variants and RHD zygosity. Expanding the analysis by QMPSF in Brazilian donors and in patients with sickle cell disease.

Hybrid genes are responsible for the formation of Rh variants and are common in patients with sickle cell disease (SCD). However, it is not usually possible to detect them by conventional molecular protocols. In the present study, hybrid genes were investigated using the Quantitative Multiplex Polymerase chain reaction of Short Fluorescent Fragments (QMPSF), a molecular protocol that quantifies the copy number of RHD and RHCE exons. In addition, we explored additional relevant information obtained with QMPSF, such as recognition of variant RHCE and RHD zygosity. Three groups of subjects were selected for the study: patients with SCD, self-declared African descent donors (SDA), and D-negative donors. RHD and RHCE hybrids genes were investigated by the QMPSF method. Real-time multiplex polymerase chain reaction (PCR) assay was used to confirm the copy number of the RHD in two samples. Cloning was performed to investigate the allele. Relative RhD antigen density was investigated by flow cytometry, and RhCE phenotyping was performed with both tube and gel methods. In the 507 samples analysed, hybrid allele frequencies were found in 20.08% of patients with SCD, in 18.22% of individuals in the SDA group, and 3.67% of D-negative donors. The SCD and SDA groups had a higher frequency of hybrid alleles, most commonly involving exon 8, with which we found an association with c.733C>G, a common polymorphism observed in individuals of African descent. Of note, two patients with SCD were shown to carry three gene copies, as confirmed by quantitative PCR; no increase in D expression was observed in these patients. In addition, the QMPSF guided the investigation of 144 RHCE variants and RHD zygosity, and two novel alleles were identified. The QMPSF was shown to identify hybrid alleles involved in altered Rh phenotypes in Brazilian donors and patients with SCD. The association of the hybrid RHCE-D(8)-CE allele with c.733C>G suggests this hybrid allele may be used as a marker to detect the most frequent variants found in patients with SCD.

RH VARIANTS IN BRAZILIAN PATIENTS WITH SICKLE CELL DISEASE (SCD) AND AFRICAN BRAZILIAN DONORS

The transfusion therapy with packed red blood cells (RBCs) is a common practice on prevention and treatment of patients with SCD. However, RBC alloimmunization, especially against Rh antigens in patients with Rh variants, is still a challenge, as antibodies developed against these variants may have clinical significance. Knowing the prevalence of Rh variants can help in assessing the risks of alloimmunization and developing strategies to find compatible blood donors to chronically transfused patients. Based on this, our aim was to determine the occurrence of variant Rh alleles in patients with SCD undergoing regular transfusion and in African Brazilian blood donors to define a strategy for searching compatible blood for patients with Rh variants. Simultaneously, we analyzed 82 DNA samples of patients with SCD and 68 DNA samples of repeated blood donors self-declared as Afro descendants. All samples were genotyped using the HEA, RHD and RHCE BeadChip (Bioarrays, Immucor, USA) platforms. From the 82 patients with SCD evaluated, 21 (25.6%) had RH variant alleles, from which 6 (38.9%) were alloimmunized against Rh antigens. Thirteen patients (61.90%) had only RhCE variants, 5 patients (23.81%) presented only RhD variants and 3 patients (14.29%) had RhCE and RhD variants. The most common RHCE variant alleles found in patients were RHCE*ce733G and RHCE*ce S while the most common RHD variants were RHD*weak D type 4.0 and RHD*DAR. From the group of 68 blood donors analyzed, 25 (36.76%) had RH variant alleles. Three donors (12.0%) presented only RhCE variants, 20 donors (80.0%) had only RhD variants and 2 donors (8.0%) had both, RhCE and RhD variants. The most common RHCE variant alleles found in donors were RHCE*ce48C and RHCE*ce733G,1006T while the most common RHD variants were RHD*weak D type 2 and RHD*DAR. As for the RHD variant alleles, homozygous, heterozygous and compound heterozygous alleles were observed. As for the RHCE altered alleles found in patients and donors, we observed heterozygous variant alleles with wild type RHCE alleles, homozygous RHCE variant alleles and compound heterozygous alleles. Knowledge of Rh variants in recipients and donors can help to assess the risk of Rh alloimmunization and to develop an adequate strategy for selecting blood donors with compatible RH haplotypes.

RHCE diversity among Brazilian patients with sickle cell disease (SCD) and selected groups of blood donors.

Several centers have selected Black donors to prevent Rh alloimmunization of patients with sickle cell disease (SCD). As the Brazilian population is considered very admixed and race definition by self-declaration is questionable, this study aimed to compare RHCE diversity among patients with SCD and selected groups of Brazilian blood donors to define which group of donors would be the adequate red cell supply for patients with SCD. We compared RHCE allele frequencies between patients with SCD and four groups of Brazilian blood donors: self-declared Black donors (SDB), donors with predominant African genetic markers (AAM), donors with weak D expression (WDD), and random donors (RDs). Variant RHCE alleles were identified using molecular protocols. Among patients with SCD, 47% had at least one variant RHCE, in SDB and WDD this frequency was higher, 53% and 58.6%, respectively. In AAM and in RD the frequencies were 32% and 27.6%, respectively. In patients with SCD and SDB, the most common alleles were RHCE*ce.01, RHCE*ceVS.01, and RHCE*ceVS.02. WDD had a high frequency of RHCE*ceAR and highest frequency of variant RHCE in both alleles, followed by patients with SCD and SDB. This study showed that even in an admixed population the selection of SDB donors is the best choice of matching for transfusion support in patients with SCD. For specific RHCE alleles, selection of donors with weak D expression could be a good option.

Molecular genetics of the Rh blood group system: alleles and antibodies—a narrative review

Objective: This work proposes a review of the antibodies which have been associated with variant RHD and RHCE alleles, except null alleles.

Impact of Extended Molecular RBC Antigen Matching between Blood Donors and Patients with Hematological Diseases

Background: Programs to prevent alloimmunization against red RBC antigens aims to provide antigen-compatible blood transfusions for patients with hematological diseases. Recently, there is an emerging role of molecular methods in donor-receptor compatibility for major blood group polymorphisms and for Rh variants to improve transfusion safety.Aims: We aimed to determine the genotype and allele frequencies of major blood-group systems and RHD and RHCE variants in blood donors and patients with SCD, Myelodysplastic Syndromes (MDS), Acute Myeloid Leukemia (AML) or Autoimmune Hemolytic Anemia (AIHA) to verify if the genotyping results are in concordance with serologic typing results and to perform molecular compatibility between donors and patients.Methods: Samples from 198 blood donors were phenotyped for five clinically relevant blood-group system antigens (RH, KEL, JK, FY and MNS). Samples from 158 patients with hematological diseases (101 with SCD, 14 with MDS, 26 with AIHA and 17 with AML) and from 198 blood donors were evaluated molecularly for 18 blood-group systems alleles (RH, CO, CROM, DI, DO, FY, GE, IN, JK, KEL, KN, LE, LU, LW, MNS, OK, SC and YT) using the Blood-MLPA assay (Multiplex Ligation-dependent Probe Amplification). RHD and/or RHCE exon-specific sequencing was performed to discriminate RHD and RHCE subtypes not distinguished by MLPA. Computerized molecular matching was performed between all patients and donors for ABO, RH, MNS, KEL, FY, JK and DI blood-group systems (Level 1). Afterwards, we extended the matching degree [ABO, RH (further variants), MNS, KEL, FY, JK and DI (Level 2)] for patients who inherit clinically relevant RHD and/or RHCE genotypes predicting partial Rh antigens.Results: We found statistically significant differences in the frequencies of the blood group alleles MNS, FY, JK, DO, CO, GE and RH (RHD*DAU0, RHCE*ceVS.01, RHCE*Ce and RHCE*CE) between donors and patients with hematological diseases. The allelic profile of AML patients was the most similar to the allelic profile of donors, while that of SCD patients was the most different. Eight donors and nine patients had clinically relevant RHD genotypes, while 14 donors and 22 patients had clinically relevant RHCE genotypes. There was 99.2% concordance between the results of the serologic typing and the results of the predicted phenotypes by the blood-MLPA. Considering the Level 1 matching It would be possible to find at least one compatible blood donor for 91.1% patients with hematological diseases. From recently transfused patients (i.e., those transfused up to one year prior to participation in the study), 70.3% would have enough blood units for an annual transfusion cycle. The MDS patient group were the least attended with matched blood (55.6%) and the one with the lowest number of compatible donors (85.7%). Taking into consideration the Level 2 matching the average number of available donors per patient reduced from 19.7 to 3.5 when the degree of matching was extended (i.e., from Level 1 to Level 2). Besides, the number of compatible donors was not sufficient to fulfil the transfusion needs of patients with Rh variants.Conclusion: We reliably determine the genotype and allele frequencies of clinically relevant blood-group systems in donors and in patients using molecular methods. It would be possible to meet the transfusion needs of most patients with hematological diseases through molecular compatibility, but the current donor population would not fill the transfusion requirements of patients with Rh variants. Molecular RBC matching increases the safety of patients with SCD, MDS, AML and AIHA by providing more accurate compatibility with genotyped donors and by reducing the risk of transfusion reactions. DisclosuresNo relevant conflicts of interest to declare.

Antigen matching for transfusion support in Brazilian female patients with sickle cell disease to reduce RBC alloimmunization.

Red blood cell (RBC) alloimmunization is a complication of patients with sickle cell disease (SCD) and it has a greater impact on pregnancy, leading to a risk of hemolytic disease of the newborn and reducing blood availability for pregnant women. This study proposed to evaluate antigen matching transfusion protocols, aiming to reduce RBC alloimmunization in Brazilian female patients with SCD. Samples from female patients with SCD (153) and self-declared Afro-Brazilian donors (307) were genotyped for RBC antigens and RH variants were investigated. The transfusion needs of patients during 1-year period and the number of compatible donors were assessed using three antigen-matching transfusion protocols: prophylactic CEK antigen-matched RBCs, prophylactic extended antigen-matched RBCs, and extended-matched red blood cells (RBCs) only for alloimmunized patients. In addition, RH molecular matching has been proposed for patients carrying variant RHCE. Provision of CEK antigen-matched donors would have been possible in 92.4% of transfusion events while provision of prophylactic extended antigen-matched RBCs would cover 88.7% of the transfusion events. Extended antigen matching for alloimmunized patients would be efficient in 99% of the cases. The presence of partial D in 10 patients increased the need of D-negative donors. Compatible donors could be enough for four of the five patients with altered RHCE genotypes in both alleles. In Brazilians, screening African descent donors allows the implementation of prophylactic CEK and extended antigen-matching transfusion protocols to female patients with SCD to reduce RBC alloimmunization; however, the supply of compatible blood can be impaired for patients with Rh variants.

Serological screening and genetic analysis of RhCE variants in the Chinese Southern Han donors.

To screen RhCE variants in the Chinese Southern Han donors for molecular genetic analysis. More than hundreds of RhCE variant alleles have been described to resulting in weak and/or partial expression of RhCE antigens, generation of low-prevalence antigens and/or absence of a high-prevalence antigen of Rh system, which mainly reported in the people of African origin. In this study, the serological screening and molecular genetic analysis of RhCE variants were performed in the Chinese Southern Han donors. The blood samples of E(+) donors were preliminarily collected. Then, RhCE antigens of the E(+) samples were further typed by using two sets of monoclonal anti-C, anti-c, anti-e and another anti-E. When weak expression of RhCE antigens was found, direct sequencing for 10 exons of RHCE gene, RH genotyping analysis by using multiplex ligation-dependent probe amplification, flow cytometric analysis and even cDNA sequencing were performed. A total of 4487 E(+) samples were collected and four samples with weak expression of antigens were detected. RHCE*Ce375G and RHCE*Ce667T variant alleles were identified in two samples with weak expression of e antigen, respectively. But no variant alleles were found in another two samples with weak expression of C antigen. The variant RHCE*Ce375G validated by mRNA sequencing and the deduced RHCE*Ce667T alleles were firstly identified in the Chinese population. The DCE haplotype might account for the weak expression of C antigen in two donors.

Variant genotypes associated with reduced expression of RhCE antigens among Brazilian blood donors.

The genetic diversity of the RHCE gene locus has been explored in diverse populations of different racial backgrounds. Data referring to the diversity of RHCE encoding weakened expression of C, c, E, and e in multiethnic populations is still incomplete. Samples from Brazilian blood donors presenting reduced expression of C, c, E, or e on gel method were selected for the study. All exons and flanking introns of RHCE were genotyped though direct Sanger sequencing for the included donors. Sixty-six donors were included: 23 with weak C, 22 with weak c, 6 with weak E, 14 with weak e, and 1 with weak c and E. Among the samples with weak C, the following altered RH*C were encountered: RHCE*CeMA (n=3), RHCE*Ce941C (n=1), and RHCE*CeVA (n=1). RHD*D-CE(4-7)-D was detected in six cases, RHCE*CE was presumably present in five cases, and seven cases were unexplained. Two altered alleles underlay the weak c phenotype: RHCE*ceJAL (n=20) and RHCE*ce340T (n=2), and two altered RHCE justified weak e: RHCE*ceMO (n=6) and RHCE*ceJAL (n=8). Three variant RHCE were associated with weak E: RHCE*cEJU (n=4), RHCE*cE382C (n=1), and RHCE*cEIV (n=1). The RHCE*cE905A justified one case of weak c and E. We describe the distribution of RHCE variants found in association with weak expression of C, c, E, and e in blood donors of multiethnic origin, which differs in comparison to that previously reported for people of African or Caucasian descent.

Overcoming the challenges of interpreting complex and uncommon RH alleles from whole genomes.

Rh is one of the most diverse and complex blood group systems. Recently, next generation sequencing (NGS) has proven to be a viable option for RH genotyping. We have developed automated software (bloodTyper) for determining alleles encoding RBC antigens from NGS-based whole genome sequencing (WGS). The bloodTyper algorithm has not yet been optimized and evaluated for complex and uncommon RH alleles. Twenty-two samples with previous polymerase chain reaction (PCR) and Sanger sequencing-based RH genotyping underwent WGS. bloodTyper was used to detect RH alleles including those defined by structural variation (SV) using a combination of three independent strategies: sequence read depth of coverage, split reads and paired reads. bloodTyper was programmed to identify D negative and positive phenotypes as well as the presence of alleles encoding weak D, partial D and variant RHCE. Sequence read depth of coverage calculation accurately determined RHD zygosity and detected the presence of RHD/RHCE hybrids. RHCE*C was determined by sequence read depth of coverage and by split read methods. RHD hybrid alleles and RHCE*C were confirmed by using a paired read approach. Small SVs present in RHCE*CeRN and RHCE*ceHAR were detected by a combined read depth of coverage and paired read approach. The combination of several different interpretive approaches allowed for automated software based-RH genotyping of WGS data including RHD zygosity and complex compound RHD and RHCE heterozygotes. The scalable nature of this automated analysis will enable RH genotyping in large genomic sequencing projects.

How do I incorporate red cell genotyping to improve chronic transfusion therapy?

Children with transfusion dependent anemia, such as sickle cell disease (SCD) and thalassemia, are at an increased risk for developing red blood cell (RBC) alloantibodies due to their lifelong need for transfusion therapy. With the advent of genotyping, extended RBC antigen typing can be incorporated into chronic transfusion therapy programs (CTTPs) to improve patient care and provide antigen matched blood for this population of patients. The hospital, blood center (BC), and hematology clinic caring for children requiring long-term transfusion support developed a CTTP. Genotyping was performed at entry to determine patient RBC antigen type. Limited versus extended antigen matching of transfusions was provided based on known RBC antibodies. Fifty patients with the following disorders were enrolled: 20 with SCD, 23 with thalassemia, and 7 with other disorders. At enrollment, nine (18%) had RBC alloantibodies, including six (30%) of patients with SCD and three (13%) with thalassemia. Two children developed antibodies after enrollment; one warm autoantibody following limited "CEK" matched RBCs and one patient with a hemizygous variant RHD allele developed anti-D. Six (30%) patients with SCD had variant RHCE alleles; two had homozygous variant alleles and four had a variant present along with a wild type allele. We demonstrate how a CTTP can be developed in a community hospital through collaboration with the blood supplier, hospital, and clinical care team. A model of incorporating RBC genotyping informs risk for alloimmunization and allows consideration of transfusion strategy for providing prophylactic antigen matched blood.

Challenges in providing compatible blood with Rh genotype-matching in Brazilian patients with sickle cell disease.

The aim of this study was to verify the possibility of performing prophylactic Rh genotype-matching in Brazilian patients with sickle cell disease (SCD) and identify the genotypes that are lacking or insufficient in our donor cohort. Rh alloimmunisation is still a challenge in transfused patients with SCD. Rh genotype-matching may mitigate Rh alloimmunisation. We examined the transfusion requests for antigen-matched donor units in SCD patients with Rh variants and compared the Rh altered alleles in the patients to the Rh allele frequency in a selected donor population. For each patient and donor, RBC antigen genotyping was performed using HEA, RHD and RHCE BeadChip arrays. Sequencing was used to clarify inconclusive results. Twenty-one patients and 956 Brazilian blood donors were genotyped. According to our matching strategies, 47·6% of patients filled most of their unit requests, but 52·4% of patients had insufficient donors to fill their annual transfusion needs. We found different combinations of RHCE variant alleles in patients and donors, but the most frequent genotypes that are lacking or insufficient in our donor cohort are those associated with the lack of hrB and hrS high prevalence antigens and those co-inherited with altered RHD alleles. Our study shows that the provision of compatible blood with Rh genotype-matching in Brazilian patients with SCD can be feasible but challenging and, efficient strategies of recruitment of African-Brazilian donors must be developed.

Molecular matching for patients with haematological diseases expressing altered RHD-RHCE genotypes.

The high homology and the inverted orientation of RHD and RHCE may give rise to non-functional and aberrant RH alleles. RH genotyping is used to screen RH matched donors to African descent patients. This study aimed to define a strategy for testing RHD and RHCE variants in blood donors to provide compatible units for transfusion of patients with haematological diseases. Samples from 132 patients [101 Sickle cell disease (SCD), 14 myelodysplastic syndrome (MDS), 17 acute myelogenous leukaemia (AML)] and 198 Brazilian donors were studied. Major blood group alleles, RHD, RHCE alleles and RHD zygosity were determined by the blood-MLPA assay. Sequencing was performed to determine RHD and RHCE variant subtypes. A match was an RH genotype that did not encode Rh antigens absent in the patient, along with matching for ABO, MNS, KEL, FY, JK and DI antigens. Overall, 7·6% of blood donors and 17.4% of patients presented RH genotypes that predict expression of partial Rh antigens or lack of high prevalence Rh antigens. From 23 patients with clinically relevant RH genotypes, 15 had available matched donors. We report the presence of clinically relevant RH genotypes in SCD and in non-SCD patients. In our admixed population, many patients carry variant RHCE alleles in heterozygosity with normal RHCE alleles. Thus, our results suggest that donors could be selected based on the normal RH allele.

RHD genotyping of serologic RhD-negative blood donors in a hospital-based blood donor center.

Serologic RhD-negative blood donors are tested by a method known to detect weak D antigen expression. Serology does not detect all red blood cells with RhD expression and RHD genotyping has been used to identify variant RHD alleles, which may lead to some RhD expression. The aim of this study was to determine the frequency of RHD variant alleles in serologic RhD-negative blood donors at a hospital-based donor center in Los Angeles. RHD genotyping of serologic RhD-negative blood donors over a 20-month period was performed using the Immucor RHD BeadChip assay. DNA sequencing was performed when the RHD BeadChip assay failed to assign a genotype. For RHD variants known or suspected to result in RhD expression, recipients of previous blood donations were investigated for alloimmunization. RHD genotyping was performed in 1174 RhD-negative blood donors, and 1122 were genotyped for RHCE variants. Eleven donors (0.94%) harbored mutations predicted to yield RhD expression. The predicted phenotypes were, in decreasing frequency, DEL, partial, and weak D phenotypes. Anti-D was not detected in 16 patients who had received blood from these donors after an average follow up of 182 days. Genotyping can be used to identify donors with the potential to sensitize RhD-negative recipients. In this limited study, 0.94% of serologic RhD-negative blood donors were found to have variant RHD alleles that might cause alloimmunization in RhD-negative recipients. To our knowledge, a study of this nature has not been reported in the United States.