ABSTRACT
【Objective】 To study the blood group serology and molecular biology of patients with RhD--, so as to guide clinical blood use. 【Methods】 The EDTA-K2 anticoagulant blood of the patient was detected for Rh antigens and antibodies. Meanwhile, DNA was extracted, and the 1-10 exon of RHCE and RHAG gene was sequenced by Sanger sequencing. 【Results】 The serological test showed O type RhD--, and all spectral cells were positive. RHCE gene sequencing showed RHCE*02/RHCE*02, RHAG gene sequencing showed mutations at site 808 G > A and site 861 G > A of exon 6. 【Conclusion】 When patients were with RhD--, and related immune conditions such as pregnancy and/or transfusion history were present, autologous blood transfusion or plasma exchange could be an option for emergency blood use.
ABSTRACT
【Objective】 To conduct serological and molecular study of Del type in RhD-negative donor population in Zhongshan area, so as to improve the diagnosis of Del type. 【Methods】 A total of 102 initially RhD-negative samples, collected from December 2017 to February 2019, were classified by RHCE and PCR-SSP genotyping. And 95 cases of truly negative RhD were confirmed by IAT, 28 cases of Del type were identified by absorption and elution test. The phenotype and genotyping characteristics of Del type in Zhongshan area were summarized based on domestic data of relative literature. 【Results】 Among 102 initially RhD-negative samples by serological test, 95 were truely RhD-negative, 28 were DELRHD 1227A without any other Del allele. Among them, RHCE antigen type were Ccee in 20(71.4%) cases, CCee in 8(28.6%), with no difference in comparison with other regions in China. The frequency of Del in RhD-negative blood donors was 29.5% (28/95), with difference between Shanghai, Taiwan, and Fuzhou, but no difference between Nanchang, Zhejiang, and Wuhan. 【Conclusion】 The study showed that the Del phenotype was closely related to Ce haplotype, and has no difference with other regions in China. The frequency of Del type in RhD negative donors was 29.5%, with regional differences. RHD1227A was the main allele of Del.
ABSTRACT
【Objective】 To analyze the genetic background of RhD-negative blood donors by detecting RHD and RHCE genes of those donors. 【Methods】 From March 2021 to May 2022, the blood samples of RhD-negative blood donors, who had been screened out by RhD primary screening and confirmatory experiments in the Yaan Blood Center, were firstly identified whether the RHD allele was completely deleted, then whether there were deletions in 10 exons of non-RHD allele complete deletion samples, finally, the remaining samples without RHD alleles and exon deletions were further analyzed by DNA sequencing. RHCE gene was detected by SSP-PCR method. 【Results】 Among the RHD gene test results of 104 RhD-negative samples, 65 cases were completely deleted (d/d), 33 were RHD partially deleted (one allele deletion), and 6 were without RHD gene deletion. The RHD alleles of 33 samples with partial deletion were detected by 10 exons, 13 had partial exon deletion, with genotype as RHD*D-CE(3-9)-D/d and phenotype as RhD negativity, and the remaining 20 samples had no exon deletion. The exon sequencing results of the non-deletion samples showed RHD*1227A/RHD*1227A in 6 samples, RHD*1227A/d in 19, RHD*3A/d in 1; both of the last two were considered Del by ISBT. The RHCE gene test results showed that all cc genotype blood donors were RhD true negative, while Del blood donors had no cc genotype. 【Conclusion】 Through the genetic background study of RhD negative blood donors, it is found that there is a high proportion of Del with weak expression of RhD antigen, whether this blood type affects clinical blood safety needs further researches.
ABSTRACT
D antigens are clinically significant, and routine tests on the D antigen requires the inclusion of weak D testing, which is performed using indirect antihuman immunoglobulin methods. On the other hand, exact typing of the D type of an individual can be done more precisely with RHD genotyping, which is a useful tool in cases where the RHD gene is intact. The majority of weak-D or partial-D cases are from single nucleotide changes or hybridization of RHD and RHCE genes. Nevertheless, frameshift mutations can also result in weak or partial-D. The characteristics of a frameshift mutation is typically a change in protein product after a problematic mutation and early termination of transcription, leading into truncated protein products. This paper reports a D-variant case with RHD 711delC along with a review of the relevant literature. In addition, the results of software analysis are reported.
Subject(s)
Frameshift Mutation , Genotype , Hand , ImmunoglobulinsABSTRACT
BACKGROUND: The Rh blood group includes several antigens, of which D, C, E, c, and e are clinically important. Although nucleic acids from whole blood can be used for Rh blood group genotyping, it is also possible to genotype free circulating fetal nucleic acids from plasma and serum. We performed Rh blood group phenotyping and genotyping using nucleic acids from whole blood and free circulating nucleic acids from plasma and serum, respectively. The results were compared. METHODS: Forty-four blood samples were phenotyped and genotyped for RhD and RhCE blood groups. Phenotyping was performed by hemagglutination assay. Further tests were performed on RhD-negative samples. Nucleic acids were extracted from whole blood, plasma, and serum. Plasma and serum were prepared after filtration and genotyped by real-time polymerase chain reaction. RESULTS: RhD blood group results showed one (2.3%) discrepant case in which the DEL phenotype appeared wild RHD genotype. Among nucleic acids, there were seven discrepant results: two from plasma and five from serum based on whole blood nucleic acids. RhCE blood group results showed three (6.8%) phenotype-genotype discordances. Among nucleic acids, seven (15.9%mpared to phenotypes. Kappa coefficients of serum were lower than those of plasma. CONCLUSION: RHD and RHCE genotype could be identified by assaying free circulating nucleic acids in plasma or serum. This study suggests that plasma is more reliable than serum as a specimen for RHD and RHCE genotyping of free circulating nucleic acids.
Subject(s)
Blood Group Antigens , Filtration , Genotype , Hemagglutination , Nucleic Acids , Phenotype , Plasma , Real-Time Polymerase Chain ReactionABSTRACT
BACKGROUND: RhC/c blood group antigens are of clinical importance and molecular genotyping for them can be useful when serological typing is difficult. A method to determine the RhC/c genotype, by targeting exon 1 nt48 and exon 2 nt307, has been used. However, this approach is not accurate for the RHc(cyt48) variant allele. We applied a more accurate genotyping method, using the intron 2 109 bp insert of the RHCE gene, and evaluated its performance in comparison with the standard method. METHODS: RhD and RhC/c serotypes of 236 subjects were determined. We compared two genotype results with the serological phenotype. One method examined the allele-specific exon 1 nt48 and exon 2 nt307 polymorphism area (Method 1), while the other method detected the intron 2 insert instead of the exon 1 nt48 (Method 2) by polymerase chain reaction with sequence-specific primers (PCR-SSP). RESULTS: The predicted phenotypes by Method 1 were not matched with the true phenotypes in 24 cases (24/236, 10.2%). By contrast, the predicted results by Method 2 matched with true phenotypes in all cases except one. The RHc(cyt48) variant was suspected in 22 cases (23.7%) of the 93 Rhc cases. CONCLUSION: For the determination of the RhC/c genotype in Koreans, the method that analyzes exon 1 nt48 is inaccurate. Instead, intron 2 insert analysis with exon 2 nt307 by PCR-SSP appears to be a more accurate alternative.
Subject(s)
Alleles , Blood Group Antigens , Exons , Genotype , Introns , Phenotype , Polymerase Chain ReactionABSTRACT
Objective To analyze the genomic structure of a novel RHD allele. Methods Through polymerase chain reaction (PCR), sequence specific primer-PCR(SSP-PCR) and genomic DNA sequencing,the RHD gene in a D-negative individual was detected. Results In SSP-PCR tests, the sample was tested negative for exons 3~7, 9~10 and intron 2(Din 2), but was positive for the Rh downstream Box3. The RHCE genotype was Ccee. Three PCR methods were used to detect intron 10(Din10) of RHD gene;all gave negative results. Through a RHD full length coding region sequencing method, the sample was found to have sequences identical to normal RHD in exons 1 and 2, while exons 3~10 were negative. Conclusion The sample is D antigen negative, RHD gene positive, with amalgamative allele of RHD-CE(2~10).
ABSTRACT
A mutation is predominant in weak D individuals,and DⅥⅢ mutation in partial D individuals.