Molecular analysis of patients with weak D and serologic analysis of those with anti-D (excluding type 1 and type 2).

Whether or not patients whose red blood cells (RBCs) carry certain weak D types produce anti-D, and if they do whether it is allo- or auto anti-D, remains controversial. The aim of this study was to determine the serologic features of anti-D in individuals expressing a weak D other than type 1 or type 2 and to assess whether the anti-D was an allo- or autoantibody. Serologic D typing and molecular analyses were performed on 748 individuals.Serologic characterization of anti-D included autologous controls,direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum onto autologous RBCs. From molecular analyses, 459 individuals exhibited a weak D type. We described seven novel RHD variant alleles. The most frequent types of weak D were type 1 (30.1%), type 2 (23.7%), type 4.0 (10.2%), type 4.2.2(20.3%), type 11 (3.9%), and type 15 (3.7%). Anti-D was identified in the sera of 9 of 47 individuals with weak D type 4.0, in 14 of 93 with weak D type 4.2.2, in 1 of 18 with weak D type 11, in 1 of 17 with weak D type 15, and in 1 weak D type 33 individual.Anti-D was demonstrated to be an alloantibody in weak D type 4.0, type 4.2.2, and type 15 individuals, but an autoantibody in weak D type 11 and type 33 individuals. In conclusion, only a complete serologic investigation of individuals with a given weak D type identified by molecular analysis allows concluding on the nature of the antibody. Transfusing weak D type 4.2.2 and type 15 patients with D- RBC units and proposing anti-D immunoprophylaxis to women with these weak D types should be considered.

RHD*DOL1 and RHD*DOL2 encode a partial D antigen and are in cis with the rare RHCE*ceBI allele in people of African descent.

BACKGROUND: Several studies showed in people of African descent the existence of a genetic linkage between RHD alleles encoding a variant D antigen and a given altered RHCE*ce allele. RHCE*ceBI is a rare allele encountered in people of African descent, that encodes a Hr- hr(S) - Rhce protein. Our study shows that RHCE*ceBI appears to be genetically linked to two very similar variant RHD alleles, RHD*DOL1 and RHD*DOL2, and demonstrates for the first time that DOL-2 is a partial D antigen. STUDY DESIGN AND METHODS: After finding out an individual with both RHCE*ceBI and RHD*DOL presumed to be in cis, we hypothesized a genetic linkage between those two genes. All individuals (n = 7) known to carry RHCE*ceBI in our laboratory, including the index case, were fully investigated at the serologic and molecular level. RESULTS: One individual with alloanti-D, being homozygous for RHCE*ceBI and RHD*DOL2, allowed us to confirm the genetic linkage between those two genes, as well as the partial D status of DOL-2. In the six RHCE*ceBI remaining individuals, three were found with RHD*DOL2 and 3 with RHD*DOL1, likely in cis. Three of them made an alloanti-D; one was DOL-1 and two were DOL-2. CONCLUSION: The rare RHCE*ceBI allele appears to be in cis either with RHD*DOL1 or with RHD*DOL2 in people of African descent. DOL-1 and DOL-2 must be considered as partial D antigens. We recommend a systematic search for RHD*DOL1 and RHD*DOL2 in people found to carry RHCE*ceBI and vice versa, especially in patients with sickle cell disease.

Anti-D investigations in individuals expressing weak D Type 1 or weak D Type 2: allo- or autoantibodies?

BACKGROUND: Whether anti-D produced by individuals with a weak D phenotype are allo- or autoantibodies remains a matter of debate even though blood transfusion practice is impacted. The aim of our study was to determine the serologic features of anti-D in individuals expressing the most frequent weak D type in Caucasians that are weak D Type 1 or weak D Type 2, to assess whether anti-D were allo- or autoantibodies. STUDY DESIGN AND METHODS: Serologic D typing and molecular analysis enabled the including of 121 weak D Type 1 individuals and 99 weak D Type 2 individuals in our study. Serologic features of anti-D included autologous controls, direct antiglobulin test, elution, and titration of anti-D before and after adsorption of serum on autologous red blood cells (RBCs). RESULTS: Serologic D typing showed a variable reactivity of RBCs expressing weak D Type 1 or weak D Type 2 (4+ to 0). Anti-D was identified in six weak D Type 1 and six weak D Type 2 individuals, respectively. The serologic data were in favor of autoantibodies. CONCLUSION: A complete anti-D investigation in individuals with a D variant (weak D or partial D identified by molecular analysis) should be systematically performed before any valid conclusion on the nature of the antibody. Transfusing weak D Type 1 or weak D Type 2 patients with D+ RBC units should be recommended. Weak D Type 1 or weak D Type 2 pregnant women do not need anti-D immunoprophylaxis.

Analysis of RhCE variants among 806 individuals in France: considerations for transfusion safety, with emphasis on patients with sickle cell disease.

BACKGROUND: DNA testing has enabled the documenting of numerous variants of RHCE alleles, especially in individuals of African origin. The risk for production of clinically significant alloantibodies to Rh antigens of patients carrying variant RHCE alleles has led us to analyze the different RhCE variants investigated by molecular biology. Alloimmunization was analyzed regarding the RHCE genetic profile. STUDY DESIGN AND METHODS: Samples from 806 individuals with altered expression of RhCE antigens and/or producing anti-RhCE in the presence of the corresponding antigen were analyzed. RESULTS: A total of 572 individuals were shown to express RhCE variants. Variant RHCE*ce alleles and RH haplotypes were identified in 83% of cases, the most frequent ones being the R(N) haplotype, the ceMO allele, the (C)ce(s) haplotype/ce(s) 1006 allele, and the ceAR allele identified in 36, 23, 20, and 17% of the tested samples, respectively. The absence of a high-prevalence Rh antigen was documented in 93 individuals. Partial C and partial e were expressed by 53% of individuals with RhCE variants. Rh antibodies were identified in 127 (20%) of 623 patients. They were found to be alloantibodies in 48 (38%) of these 127 patients. Alloimmunization against a high-prevalence Rh antigen was detected in 25% of cases. CONCLUSION: The challenge in clinical red blood cell (RBC) transfusion of patients with sickle cell disease, notably, would be to provide not only phenotypically matched, but also genetically matched, RBC units regarding RhCE variants.

Anti-HrB and anti-hrb revisited.

BACKGROUND: Since their description in the 1970s, anti-Hr(B) (antibody against a high-prevalence Rh antigen) and anti-hr(B) (anti-e-like antibody) are still a subject of debate about representing two aspects of a global immune response or being two independent antibodies. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the immune response against the antigens of Rh system of 30 individuals presenting a hr(B)(RH31)- phenotype. Genomic analysis of RH genes was performed in all individuals. RESULTS: Among the 30 individuals, 27 had a Hr(B)(RH34)- phenotype. No immunization against Rh antigens was found in 16 individuals. Three individuals made anti-D only, whereas six individuals made anti-Hr(B) (four with anti-hr(B) and two without anti-hr(B)) and two individuals made anti-hr(B) without anti-Hr(B). Among the 30 individuals, three had a Hr(B)+ phenotype. No immunization against Rh antigens was found in one individual, whereas two individuals made anti-hr(B); the genomic analysis of selected individuals showed the presence of a (C)ce(s) haplotype, either Type 1 or Type 2, and a DIII Type 5 ce(s) haplotype, in the homozygous state, in compound heterozygosity with each other or in heterozygosity with a DcE haplotype. Genomic data were in accordance with serologic data. CONCLUSION: Our data provide the evidence that anti-Hr(B) and anti-hr(B) are independent antibodies, defining two different specificities. These antibodies may be produced by individuals expressing variants of RhCE protein. Serologic and molecular data indicate that e antigen encoded by the (C)ce(s) haplotype is a partial antigen. In individuals carrying a (C)ce(s) haplotype, the risk and the type of alloimmunization to Rh antigens are related to the second Rh haplotype.

RhD variants in Caucasians: consequences for checking clinically relevant alleles.

BACKGROUND: Weak D type carriers cannot be immunized against D except when antigen density is below 400 antigens per RBC, whereas partial D carriers can produce anti-D. STUDY DESIGN AND METHODS: A total of 168 blood samples from Caucasian individuals were studied because of weak D expression and/or anti-D production. Serologic analysis and molecular analysis were performed. RESULTS: In total, 70 partial D and 62 weak D were identified. Among weak D samples, 30 weak D Type 1 and 21 weak D Type 2 alleles were found. Five new alleles were characterized carrying 399G > T, 680T > C, 833G > A, 851C > T, and 1015G > A, respectively. According to previous studies, antigen density was up to 500 for weak D Type 1 and 2, except when there was a dCe haplotype in trans. Antigen density was below 400 antigens per red blood cell for the new variants and most other weak D variants. CONCLUSION: These results provide molecular characterization of five new D variants. They also suggest that it would be advantageous to develop in routine laboratories weak D Type 1 and 2 genotyping for serologically depressed D antigen. It will help to avoid wasting of D- red blood cell units because carriers may safely receive D+ units.

Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety.

The molecular backgrounds of variants encountered in Afro-Caribbean black individuals and associated with the production of clinically significant antibodies against high-incidence antigens (anti-RH18, anti-RH34) and against Rhe epitopes were determined. We showed that RH:-18 phenotypes are produced by 3 distinct RHCE alleles: ceEK carrying 48G>C (exon 1), 712A>G, 787A>G, 800T>A (exon 5); ceBI carrying 48G>C (exon 1), 712A>G (exon 5), 818C>T (exon 6), 1132C>G (exon 8); and the already known ceAR allele carrying 48G>C (exon 1), 712A>G, 733C>G, 787A>G, 800T>A (exon 5), and 916A>G (exon 6). The RH:-34 phenotype is produced by the (C)ce(s) haplotype described previously and composed of a hybrid D-CE(3-8)-D gene with 4 extra mutations next to a ce(s) allele (733C>G; exon 5) with an extra mutation in exon 7 (1006G>T). Partial Rhe with risk of immunization against lacking epitopes can be produced by the new ce(s) allele carrying an extra mutation in exon 3 (340C>T) and by the ceMO allele described previously. A population of sickle cell disease patients was screened to estimate the incidence of these rare alleles, with the conclusion that a procedure is required to detect the associated phenotypes in black donors to ensure transfusion safety for patients. We also described a new variant [ce(s)(748)] and variants carrying different altered alleles in nonimmunized patients and for whom the risk of immunization is discussed.

Molecular background of D(C)(e) haplotypes within the white population.

BACKGROUND: D(C)(e) and D(C)e haplotypes may be encountered in the white population. Few data are available on the molecular backgrounds responsible for depressed expression of C and e. STUDY DESIGN AND METHODS: Individuals of white origin carrying a D(C)(e) genotype resulting in depressed expression of C or both C and e were subdivided into two categories based on the RBC reactivity with the human sera Mol and Hor, which contain antibodies against low-frequency antigens of the Rh (RH) system and other non-Rh low-frequency antigens. Neither Hor+, Mol+ nor Hor+, Mol- RBCs expressed the V (RH10), VS (RH20), and/or Rh32 (RH32) low-frequency antigens. These results suggested that Hor+, Mol+ variants expressed Rh33 (RH33 or Har) and FPTT (RH50), whereas Hor+, Mol- variants might express an undefined low-frequency antigen. Further serologic and molecular analyses were performed. RESULTS: Molecular analysis of Hor+, Mol+ variants revealed a hybrid gene structure RHCe-D(5)-Ce, in which exon 5 of RHCE (RHCe allele) was replaced by exon 5 of RHD (the so-called RHCeVA allele). The presence of exon 5RHD resulted in several amino acid alterations predicted in the external loop 4 of the CeVA polypeptide. Molecular analysis of Hor+, Mol- variants revealed the presence of a new RHCe allele characterized by a single point mutation C340T within exon 3 (the so-called RHCeMA allele), resulting in a R114W substitution predicted on the external loop 2 of the CeMA polypeptide. A serologic study showed a different pattern of reactivity with C and e MoAbs. CONCLUSION: Two types of mutations resulted in amino acid substitutions predicted in external loops 4 and 2, respectively, which altered both the C and e reactivity, and indicated conformation changes or defective interaction between nonadjacent loops of the Ce polypeptide. Serologic analysis showed that together with Hor and Mol sera testing, the use of different C and e MoAbs could help to identify these variants within the white population.