Background: Red cell (RBC) blood group alloimmunization remains a major problem in transfusion medicine. Patients with sickle cell disease (SCD) are at particularly high risk for developing alloantibodies to RBC antigens compared to other multiply transfused patient populations. Hemagglutination is the classical method used to test for blood group antigens, but depending on the typing methods and reagents used may result in discrepancies that preclude interpretation based on serologic reactivity alone. Molecular methods, including customized DNA microarrays, are increasingly used to complement serologic methods in predicting blood type. The purpose of this study was to determine the diversity and frequency of RH alleles in African Americans and to assess the performance of a DNA microarray for RH allele determination. Material and methods: Two sets of samples were tested: (i) individuals with known variant Rh types and (ii) randomly selected African American donors and patients with SCD. Standard hemagglutination tests were used to establish the Rh phenotype, and cDNA- and gDNA-based analyses (sequencing, PCR-RFLP, and customized RHD and RHCE microarrays were used to predict the genotype. Results: In a total of 829 samples (1,658 alleles), 72 different alleles (40 RHD and 32 RHCE) were identified, 22 of which are novel. DNA microarrays detected all nucleotides probed, allowing for characterization of over 900 alleles. Conclusions: High-throughput DNA testing platforms provide a means to test a relatively large number of donors and potentially prevent immunization by changing the way antigen-negative blood is provided to patients. Because of the high RH allelic diversity found in the African American population, determination of an accurate Rh phenotype often requires DNA testing, in conjunction with serologic testing. Allele-specific microarrays offer a means to perform high-throughput donor Rh typing and serve as a valuable adjunct to serologic methods to predict Rh type. Because DNA microarrays test for only a fixed panel of allelic polymorphisms and cannot determine haplotype phase, alternative methods such as Next Generation Sequencing hold the greatest potential to accurately characterize blood group phenotypes and ameliorate the clinical course of multiply-transfused patients with sickle cell disease.
BACKGROUND: In the Rh blood group system, variant RhD and RhCE express several partial antigens. We investigated RH in samples with partial DIVa that demonstrated weak and variable reactivity with anti‐C. STUDY DESIGN AND METHODS: Standard hemagglutination techniques, polymerase chain reaction–based assays, and RH sequencing were used. RESULTS: DNA analysis showed that six red blood cell (RBC) samples with weak and inconsistent reactivity with anti‐C lacked RHCE*C, but all had RHD*DIVa, which encodes partial D and Goa. We then tested RBCs from 19 Go(a+) cryopreserved samples (confirmed to have RHD*DIVa) with four anti‐C and observed weak variable reactions. RHCE genotyping found all but one of the samples with RHD*DIVa also had RHCE nt 48G>C and 1025C>T, named RHCE*ceTI. Lookback of samples referred for workup and found to have either allele revealed 47 of 55 had both RHD*DIVa and RHCE*ceTI, four had RHD*DIVa without RHCE*ceTI, and four had RHCE*ceTI without RHD*DIVa. Alloanti‐c was found in a patient with c+ RBCs and RHCE*ceTI in trans to RHCE*Ce, and alloanti‐e was found in a patient with e+ RBC and RHCE*ceTI in trans to RHCE*cE. RHD*DIVa in trans to RHD erroneously tested as RHD hemizygous. CONCLUSIONS: RHD*DIVa and RHCE*ceTI almost always, but not invariably, travel together. This haplotype is found in people of African ancestry and the RBCs can demonstrate aberrant reactivity with anti‐C. RHCE*ceTI encodes partial c and e antigens. We confirm that RHD zygosity assays are unreliable in samples with RHD*DIVa.
Background RHCE*ceMO has nucleotide changes 48G>C and 667G>T, which encode, respectively, 16Cys and 223Phe associated with altered expression of e antigen. RHD*DAU0 has nt1136C>T, which encodes 379Met associated with normal levels of D. We compiled serologic and DNA testing data on samples with RHCE*ceMO to determine the RBC antigen expression, antibody specificity, RHD association, and the prevalence in African-Americans. Methods Serologic testing was performed by standard methods. Genomic DNA was used for PCR-RFLP and RH- exon sequencing, and for some, Rh-cDNA was sequenced. Seventy-seven (50 donor and 27 patient) samples with RHCE*ceMO were studied, and 350 African-Americans were screened for allele prevalence. Results RBCs from RHCE*ceMO homozygotes (or heterozygotes with RHCE*cE in trans) were weakly or non-reactive with some anti-e , and were non-reactive with polyclonal anti-hrS and anti-hrB. Twenty-three transfused patients homozygous for RHCE*ceMO/ceMO or with RHCE*ceMO in trans to RHCE*cE or *ce had allo anti-e, anti-f, anti-hrS/hrB, or an antibody to a high prevalence Rh antigen. Three patients with allo-anti-c had RHCE*ceMO in trans to RHCE*Ce. RHD*DAU0 was present in 30% of African-Americans tested and in 69 of 77 (90%) of samples with RHCE*ceMO. Conclusions RHCE*ceMO encodes partial e, as previously reported, and also encodes partial c, a hrS– and hrB– phenotype, and the absence of a high prevalence antigen (RH61). The antibody in transfused patients depended on the RHCE allele in trans. RHCE*ceMO was present in 1 in 50 African-Americans with an allele frequency of 0.01, is often linked to RHD*DAU0, and is potentially of clinical significance for transfusion.
Background RH43 (Crawford) is encoded by RHCE*ce with nucleotide changes 48G>C, 697C>G, and 733C>G (RHCE*ceCF). We investigated the Rh antigen expression and antibody specificities in four patients with this allele. Methods Hemagglutination tests, DNA extraction, PCR-RFLP, AS-PCR, reticulocyte RNA isolation, RT-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures. Results RBCs from two patients typed D+C−E−c+e+/−, hrS−/+W, hrB− and their serum was reactive (3+) with all RBC samples of common Rh phenotype tested, but non reactive with Rhnull or D- - RBCs (apparent alloanti-Rh17). At the RHCE locus, Patient 1 was homozygous for RHCE*ceCF, and Patient 2 inherited RHCE*ceCF in trans to a silenced RHCE*cE. Cross testing of serum and RBCs from these two samples showed mutual compatibility, indicating that both antibodies define the same novel high prevalence antigen on Rhce. Two additional patients, one whose serum contained alloanti-c but the RBCs typed C+c+, and one whose serum contained anti-e but the RBCs typed E+e+, also had RHCE*ceCF. RHCE*Ce was present in trans in the former and RHCE*cE in the latter patient. Conclusion We report that amino acid changes on RhceCF (Trp16Cys, Gln233Glu, and Leu245Val) alter the protein to the extent that c and e antigens are partial, and a high prevalence antigen, we have named CELO (provisional ISBT number 004058; RH58) is not expressed. CELO is antithetical to RH43 (Crawford).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.