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

Abstract

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.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings

“…It should be stressed that analysis of the Rh system requires a deep knowledge of the existing variants and the relationship between the homologous RHD and RHCE genes, whatever the methodological developments and progress made in that field. In some situations, Rh phenotype identification requires analysis of both genes; for example, a Cw phenotype, in the absence of the 109pb insertion in exon 2 of RHCE , may be due to an RHD * DAU5 allele [ 19 ]; an RhD phenotype, in the absence of the RHD gene, may be due to an RHCE*ceHAR allele [ 44 ]; identification of an RH:-18 phenotype in an RHD*DAR context requires definition of the RHCE genotype, i.e., RHCE*ceAR/RHCE*ce or RHCE*ceAR / RHCE*ceAR .…”

“…First-line RhCE phenotyping was a microplate technique (Qwalys EVO; DIAGAST, Loos, France), the second-line technique was performed using a microfiltration method (OCD Auto-Vue Innova; Ortho Clinical Diagnostics, Raritan, NJ, USA), and the third-line technique used saline-filled test tubes with monoclonal antibodies (DIAGAST, Loos, France). All methods were performed in accordance with the manufacturer’s recommendations and as described in [ 19 ].…”

“…In the context of routine immunohematology, serological techniques enable us to characterize most of the Rh phenotypes, and within the last decade, molecular biology solutions have helped compensate for the main limitations of serological techniques [ 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Still, in some situations, ambiguities or discrepancies cannot be resolved, especially for RhCE, and uncertainties remain regarding the transfusion instructions; in this context, the French Blood Center, whose main mission is to ensure France’s self-sufficiency in blood products under optimal safety and quality conditions, is continuously setting up molecular typing strategies in line with technological developments [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ].…”