BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c-E-e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression. ABBREVIATIONS: MPLA = multiplex ligation-dependent probe amplification; NGS = next-generation sequencing; RT = room temperature; wt = wild type. From the

Background The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary. Study Design and Methods Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three‐step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon–intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti‐D investigation was performed. Results DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty‐two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti‐D was detected in two DVI type 3 pregnant women. Discussion The three‐step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.

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Enzyme-linked immunosorbent assay (ELISAs) specific for Epstein-Barr virus nuclear antigen 1 (EBNA1)-immunoglobulin A (IgA) are most commonly used in the clinical diagnosis of EBV infection. But they have a low sensitivity and the enzyme-labeled antibodies are unstable. In this study, a novel immunoassay based on an indirect time-resolved fluoroimmunoassay (TRFIA) was developed. Microtiter plates were coated with recombinant EBNA1. We used Eu(3) (+)-labeled anti-human IgA as probe. The precision, sensitivity, specificity, and stability were evaluated, and comparison with traditional and commercially available ELISAs was also made. The cut-off value for our TRFIA was 2.7. Intra- and inter-assay coefficients of variation for the TRFIA were 1.56-4.99% and 3.92-6.95%, respectively; whereas those for the ELISA were 4.54-8.16% and 7.07-10.52%, respectively. Sensitivity was obviously better than traditional ELISA when diluted positive samples serially. Additionally, stability, specificity test and comparison of sensitivity and specificity between the TRFIA and commercial ELISAs all proved satisfactory. In conclusion, the results demonstrated that EBNA1 IgA TRFIA was a sensitive immunoassay and had potential value in large-scale screening of human serum samples in developing countries.

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