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.

The establishment of a high-resolution melting curve genotyping method for ABCG2*376T allele of Jr(a-) and its distribution frequency study / 中国输血杂志

【Objective】 To establish a high-throughput detection method for ABCG2*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. 【Methods】 The specific primers were designed and synthesized, the sample carrying homozygous ABCG2*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2*376T alleles were sequenced to confirm the accuracy of the HRM method. 【Results】 A HRM method, which can detect ABCG2*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. 【Conclusion】 The HRM method can be used to accurately screen and type ABCG2*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

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.

Anovel solid-phase agglutination reagent for detecting IgG irregular alloantibodies of red cell blood groups / 中国输血杂志

【Objective】 To develop a novel solid-phase agglutination reagent for detecting IgG irregular antibodies of red cell blood groups and evaluate its performance. 【Methods】 Monoclonal anti-RBC antibody was coated on the bottom of the microwell strips, then RBCs were bonded to the antibody and formed the monolayer by dispensing 100 μL RBC suspesion to microwell strips.RBC antigen membrane monolayer was formed by lysing RBC layer with ddH2O, then the drying medium was added to the strips and dried under reduce pressure in a vacuum dryer, thus the dried reagent microplate was obtained.Serial diluted solutions of polyclonal sheep anti-human globulin(IgG+ C3d/4)was used to react with IgG anti-D sensitized O group RBCs to select out the best indicator.Stability of membrane antigen was tested by detecting IgG anti-D and anti-E with the lowest titer by different batches of regeats. Sensitivity of the novel reagent, Capture-R Ready Screen and microcolumn gel card was carried out by detecting irregular antibodies with different titer.350 plasma samples were tested by the novel reagent and Capture-R Ready Screen to evaluate their detection ability. 【Results】 Anti-RBC solution with concentration of 20 μg/mL could fix the membrane monolayer very well on the bottom of microstrips. Sixteentimes dilution of polyclonal sheep anti-human globulin and anti-D sensitized RBCs were selected out as the best indicator.Antigen reactivity of dried RBC membrane was not weakened during the 6-monthstorage period.Sensitivity of the novel reagent was higher than Capture-R Ready Screen and microcolumn gel card. The positive consistence ratio of the novel reagent and Capture-R Ready Screen was 98.0%, the negative consistence ratio was 99.66%, and the total consistence ratio was 99.43%. 【Conclusion】 A novel solid-phase agglutination reagent with higher sensitivity and longer storage time has been developed successfully and it has an equal detection ability compared with Capture-R Ready Screen for detecting irregular alloantibodies of red cell blood groups.

Identification and characterization of Cromer blood group anti-IFC / 中国输血杂志

【Objective】 To explore the identification method and characteristics of anti-IFC against Cromer blood group. 【Methods】 ABO blood group was identified by tube method, and Rh blood group was identified by Rh typing card. Irregular antibody screening and antibody identification were carried out using microtube column agglutination technology(MCAT). The serum of the patient reacted with panel cells treated with antitrypsin, trypsin and bromelain respectively to determine the specificity of the antibody. The serum was inhibited with pure CD55 protein for antibody identification. The DAF, the regulatory gene of Cromer blood group system, was amplified and sequenced. The expression of CD55 on cell membrane was analyzed by flow cytometry. 【Results】 The patient′s blood type was B, CcDEe. The patient′s serum reacted with all the untreated panel red cells, bromelain-treated red cells, trpsin-treated red cells, and DTT treated red cells.It was negative with chymotypsin-treated cells and could be neutralized by CD55 protein. No mutation was found by DAF sequencing. The expression of CD55 on patient′s cell membrane was deficient. 【Conclusion】 This high frequency antibody was identified as anti-IFC. The transient depression in CD55 protein may due to the patient′s GI system abnormalities.

Correlation between special A/O genotype and the O phenotype / 中华医学遗传学杂志

OBJECTIVE@#To explore the correlation between special A/O genotype and the O phenotype.@*METHODS@#Group O samples with partially reduced or lack of isoagglutinins were collected to determinate the ABO genotype with a PCR-sequence specific primer (PCR-SSP) assay. Seven samples with A/O genotype were selected for further study. Serological tests including forward and reverse typing, H antigen determination and adsorption/elution were carried out with a tube method. Genomic DNA was genotyped by amplifying and sequencing of the coding regions of exons 1 to 7 of the ABO gene.@*RESULTS@#Seven samples were serotyped as group O by the forward typing test. However, reduced anti-A activity was found in 5 samples by the reverse typing test, reduced anti-A and anti-B activities were found in 1 sample, and no anti-A isoagglutinin activity was found with 1 sample. H antigen was determined in all samples by routine serologic method. Neither anti-A nor anti-B was eluted from red cells derived from all samples. Three samples were genotyped as Ael02/O02, whilst the remainders were Ael02/O13, Ael02/O65, Am04/O75, Ael06/O02, respectively.@*CONCLUSION@#Special A/O genotype may not express the A antigen, leading to the generation of group O red cells. Reduced or missed anti-A activity is the typical serological feature of this special group of O phenotype, for which ABO*Ael02 and ABO*O02 are the major alleles. Group O individuals with isoagglutinin detection problem should be grouped by serological tests and genomic DNA analysis.

Identification of a novel ABO B(A)07 allele / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis for a novel B(A) phenotype.</p><p><b>METHODS</b>Genomic DNA was abstracted from peripheral blood sample from the proband. ABO genotyping were carried out with sequence specific primer PCR (PCR-SSP). Exons 6 and 7 of the ABO gene were amplified with PCR and sequenced.</p><p><b>RESULTS</b>Anti-A serum could not be adsorbed or eluted by the donor's red blood cells, and no irregular antibodies were found in the plasma. PCR-SSP showed that the ABO genotype of the donor was ABO *B/O. Sequencing results showed that one of the alleles was ABO *O02, while the other could not be defined but contained the following mutation points, 297A>G, 526C>G, 657C>T, 701C>T, 703G>A, 796C>A, 803G>C, and 930G>A. The data was accepted by the GenBank (the loading code was KM974887) and the Blood Group Antigen Mutation Database, and was confirmed to be a novel allele of B(A).</p><p><b>CONCLUSION</b>A novel allele ABO *B(A)07 with 701C>T has been identified, which may facilitate further study on blood antigen variants and typing of the blood groups.</p>

Efficacy and safety on upper ureteric lithotripsy by Shuo Tong ureteroscopy / 中华泌尿外科杂志

Objective To investigate the efficacy and safety of Shuo Tong ureteroscopy in the treatment of upper ureteric stones.Methods The data of 832 patients,who underwent Shuo Tong ureteroscopy combined with holmium laser for the treatment of upper ureteric stones were collected between July 2014 and February 2017.The patients accepted general anesthesia under lithotomy position.After dilating the ureter,a ureteral access sheath was inserted along the guide wire.Finally,stones were broken and clean up by Shuo Tong ureteroscope.According to the stone size,823 patients were divided into ≤2.0 cm group (n=112,13.6％),2.1-3.0 cm group (n =395,48.0％),3.1-4.0 cm group (n =257,31.2％),＞ 4.0 cm group (n =59,7.2％)The stone free rate(SFR) and post-operative complications were observed.Results Among 983 operations,663 cases accepted one-stage operations and 160 cases accepted second stage operations.the operative time and postoperative hospital stay were 23-145 min(mean 74.8 ± 35.3) and 1-5 days(mean 1.9 ±0.8),respectively.The SFR in the first day and one month after operation was 75.6％ and 83.8％,respectively.The stone free rate in each group were 89.3％,82.5％,61.1％,52.5％ at first day after operation respectively(P ＜ 0.05).While,the SFR after one month were 95.5％,87.6％,72.0％,61.0％ in each group,respectively(P ＜ 0.05).Fever rate after operation was 11.1％,ureteral injury rate was 1.9％,severe hematuria rate was 0.6％,stone street formation rate was 1.2％,perirenal hematoma rate was 0.4％.All patients were improved after conventional treatment.Conclusion Shuo Tong ureteroscopy is a safe treatment with high stone free rate for upper ureteric stones.

Efficacy and safety on upper ureteric lithotripsy by Shuo Tong ureteroscopy / 中华泌尿外科杂志

Objective To investigate the efficacy and safety of Shuo Tong ureteroscopy in the treatment of upper ureteric stones.Methods The data of 832 patients,who underwent Shuo Tong ureteroscopy combined with holmium laser for the treatment of upper ureteric stones were collected between July 2014 and February 2017.The patients accepted general anesthesia under lithotomy position.After dilating the ureter,a ureteral access sheath was inserted along the guide wire.Finally,stones were broken and clean up by Shuo Tong ureteroscope.According to the stone size,823 patients were divided into ≤2.0 cm group (n=112,13.6％),2.1-3.0 cm group (n =395,48.0％),3.1-4.0 cm group (n =257,31.2％),＞ 4.0 cm group (n =59,7.2％)The stone free rate(SFR) and post-operative complications were observed.Results Among 983 operations,663 cases accepted one-stage operations and 160 cases accepted second stage operations.the operative time and postoperative hospital stay were 23-145 min(mean 74.8 ± 35.3) and 1-5 days(mean 1.9 ±0.8),respectively.The SFR in the first day and one month after operation was 75.6％ and 83.8％,respectively.The stone free rate in each group were 89.3％,82.5％,61.1％,52.5％ at first day after operation respectively(P ＜ 0.05).While,the SFR after one month were 95.5％,87.6％,72.0％,61.0％ in each group,respectively(P ＜ 0.05).Fever rate after operation was 11.1％,ureteral injury rate was 1.9％,severe hematuria rate was 0.6％,stone street formation rate was 1.2％,perirenal hematoma rate was 0.4％.All patients were improved after conventional treatment.Conclusion Shuo Tong ureteroscopy is a safe treatment with high stone free rate for upper ureteric stones.