Molecular genetic analysis of a patient with double population of red blood cells for RhD antigen / 中国输血杂志

【Objective】 To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. 【Methods】 Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. 【Results】 The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. 【Conclusion】 This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

Effect of RHAG variants identified in Chinese population on RHAG mRNA splicing in vitro / 中国输血杂志

【Objective】 To study the effect of RHAG variants identified in Chinese population on mRNA splicing by minigene splicing assay(MSA) in vitro. 【Methods】 The pSplicePOLR2G minigene expression plasmids were constructed for 10 RHAG mutations with relatively high distribution frequency in Chinese population near splicing sites or synonymous mutations by analyzing the RHAG gene data in the KMxD database. Then, the wild-type and mutant plasmids were transfected into HEK 293T cells, and RNA was extracted 48 hours after transfection. After reverse transcription, specific primers were used for PCR amplification, and then agarose gel electrophoresis and capillary electrophoresis were performed to determine whether the mutations will affect the normal splicing of exons. 【Results】 MSA in vitro showed that 2 mutations (c.158-5delT, c. 807+ 3A>C) near the splicing site reduced the amount of normal transcripts slightly. The remaining 8 synonymous mutations(c.312G>A, c. 341+ 3G>A, c. 609C>T, c. 681G>A, c. 861G>A, c. 957T>A, c. 984T>C and c. 1139-7G>A) had no impact on the splicing of RHAG mRNA. 【Conclusion】 This study showed that RHAG gene was conservative in terms of splicing, and the mutations near splicing sites and synonymous mutations were less likely to cause abnormal splicing of RHAG gene.

Identification of pregnant women with DFR genotype and discussion of prenatal examination strategy / 中国输血杂志

【Objective】 To identify three cases of pregnant women with the D variant phenotype using serological and molecular tests, and discuss the strategy of prenatal examination. 【Methods】 The peripheral blood samples from three pregnant women with the D variant phenotype were collected. RhD variant phenotype was determined using routine serological methods with two different kinds of monoclonal anti-D. The serological characteristic for the epitope of D antigen was further analyzed using the commercial panel anti-D reagents (D-Screen, Diagast). The hybrid RHD-CE-D allele was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) assay and polymerase chain reaction with sequence specific primers (PCR-SSP) method. Further Sanger sequencing of RHD gene exons was also performed. 【Results】 DFR phenotype was primarily determined by serological characteristic for the epitope of D antigen. RHD*DFR2/01N.01(n=2) and RHD*DFR1/1227A(n=1) genotypes were identified by the MLPA assay, PCR-SSP and Sanger sequencing. 【Conclusion】 Two pregnant women with RHD*DFR2/01N.01 genotype should be treated as D negative patients clinically, while the pregnant woman with RHD*DFR1/1227A genotype can be treated as Asia type DEL to avoid unnecessary antibody screening and anti-D prophylaxis.

In vitro method establishment for detecting monocyte phagocytosis of sensitized RBCs by flow cytometry / 中国输血杂志

【Objective】 To establish an experimental method for detecting phagocytosis of sensitized red blood cells in vitro by flow cytometry. 【Methods】 Mononuclear cells were isolated from the peripheral blood of blood donors and cultured in a cell incubator for 1 hour, and then adherent monocytes were isolated and obtained. Dib-positive red blood cells (RBCs) were labeled with PKH26 and then sensitized with IgG anti-Dib. The sensitized RBCs were added to monocytes for in vitro phagocytosis assay. Monocytes were labeled with FITC anti-human CD14, then phagocytosis was measured by flow cytometry, and the phagocytic efficiency was calculated. The method was used to detect the phagocytic efficiency of monocytes on human IgG anti-D sensitized RBCs with different titers. 【Results】 The phagocytic efficiency of monocytes was averaged at 5% (1.2%~7.6%, SD 3.30) versus 81% (71.4%~92.7%, SD 8.65) in the negative versus positive control group, respectively. Phagocytic activity of monocytes mediated by anti-D was correlated with the antibody titer. The phagocytosis efficiency was within 10% when the antibody titer was lower than 32 and increased sharply when the titer was between 32 to 128, it entered a plateau and stabilized at 80% at the titer above 256. 【Conclusion】 A detection platform for detecting phagocytosis-sensitized RBCs in vitro by flow cytometry has been successfully established. It can be used to assess the clinical significance of red blood cell allotype or autologous IgG antibodies.

Anti-Jra developed in Jra(-) pregnant woman, case report with molecular genetic analysis / 中国输血杂志

【Objective】 To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. 【Methods】 ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. 【Results】 The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. 【Conclusion】 In this case, the development of anti-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

Epitope analysis and molecular study of weak D type 136: A case report / 中国输血杂志

【Objective】 To identify the blood group epitope of a D variant individual and analyze its molecular characteristics. 【Methods】 The saline test and indirect antiglobulin test (IAT) were used to identify the RhD serologically. The anti-human globulin gel card was used for direct antiglobulin test (DAT). RhD epitopes were detected using the epitope detection kit (D-Screen). RhCE antigens were typed using Rh typing Card. The RHD gene zygomorphism was further analyzed by PCR-RFLP. Ten exons of RHD gene were amplified by PCR and analyzed by direct sequencing. 【Results】 DAT test was negative, and the serological results showed weak expression of RhD, which was D variant. The RhD epitope test results showed that the red blood cells of this patient had a weak agglutination with 4 monoclonal anti-D against epD6.4, epD6.1, epD2.1, and epD5.4 (w+ to 2+ ), and reacted negatively with other epitope antibodies. RhCE antigen typing was Ccee; The RHD gene zygomorphism result was D+ /D-, the sequencing of RHD exons revealed that the first exon carried c. 41C>T (p.Pro14Leu) missense mutation, and its genotype was RHD*01W.136/01N.01. 【Conclusion】 This D variant is the first weak D type 136 reported in the Chinese population, and its phenotype is weak partial D.

Serological characteristics and genotype analysis of D epitope blood group of RhD variant individuals in Guangzhou / 中国输血杂志

【Objective】 To explore the characteristics of the D antigen epitope of individuals with RhD variants and the genetic molecular mechanism of gene mutations in Guangzhou. 【Methods】 A total of 59 samples of RhD variants were collected from blood donors and hospitals in Guangzhou from January to August 2019. Serological characteristics of D epitopes were further analyzed using two kinds of monoclonal anti-D reagents and D epitope detection kits, and RHCE phenotypic typing was performed. QuickGene DNA extraction kit was used to extract the genomic DNA of the samples, and PCR-RFLP method was used to analyze the RHD gene zygote type. The RHD gene sequence was detected by multiple ligation-dependent probe amplification(MLPA) genotyping, and the RHD exon(1~10) Sanger sequencing was performed on the samples still in doubt after the above detection. DNAStar/SeqMan analysis software was used for comprehensive analysis. 【Results】 In this group of individuals with RhD variants in Guangzhou, 27.12%(16/59) were detected from blood donors [accounting for 0.007%(16/232 793) of blood donors in Guangzhou during the same period], and difficult samples of patients sent by hospitals for determination accounted for 72.88%(43/59). RHD genotype detection: 40.68%(24/59) were RHD*weak partial 15, 25.42%(15/59) were RHD* DⅥ.3 and 33.90%(20/59) were rare RHD variants [76.92%(10/13) were RhD variants with 2 different alleles]. Serological D-screen revealed a relatively fixed pattern of RHD*DⅥ.3 in anti-D antibody(clone: P3*212 23B10), while the others was negative. The phenotypic distribution of RhD variant CE was Ccee 38.98%(23/59), ccEe 35.59%(21/59), CcEe 25.42%(15/59). 【Conclusion】 Weak partial D15 and DⅥ.3 were the most common RhD variants in Guangzhou Han population, and DⅥ can be preliminarily identified by serological methods such as D-Screen anti-D reagent, while the remaining RhD variants can only be identified by molecular biological methods, and >95% of the RhD variants were C+ or E+ phenotypes.