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OBJECTIVE@#To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-).@*METHODS@#Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors.@*RESULTS@#The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰.@*CONCLUSION@#A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.
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Humanos , Antígenos de Grupos Sanguíneos/genética , Genotipo , Técnicas de Genotipaje , Heterocigoto , Alelos , Donantes de Sangre , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
【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.
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【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).
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【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.
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【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.
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【Objective】 To discuss the interference of anti-CD47 in pre-transfusion test and the mitigation measures. 【Methods】 Blood sample of one patient received anti-CD47 treatment was collected to conduct routine serological tests including ABO/Rh phenotype, direct anti-human globulin test, irregular antibody screening, antibody identification and cross-match. Packed platelet from multiple type O blood donors was used to absorb with patient′s plasma. The patient′s plasma was absorbed with CCDee, ccDEE and ccdee red cells, respectively. Anti-IgG monoclonal Gamma-clone which lacks reactivity with human subclass IgG4 was used to perform antibody screening and cross-match. Capture-R was used to perform antibody screening. 【Results】 The direct anti-human globulin test was positive(1+ ), the reactivity in all phases was strong positive(3+ -4+ ). The anti-CD47 was eliminated after platelet and red cells absorption. Antibody screening became negative using Gamma-clone and Capture-R, and cross-match successfully using Gamma-clone. 【Conclusion】 Anti-CD47 monoclonal antibody can interfere with pre-transfusion test and cross matching. To remove the interference of anti-CD47 requires the use of Gamma-clone anti-IgG in the indirect antiglobulin testing or Capture-R.
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【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.
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【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.
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We report a case of a newborn baby who suffered from hemolytic disease of fetus and newborn (HDFN) caused by anti-Di a. The baby presented with worsening jaundice started at three hours after birth and was transferred to Dongguan Maternal and Child Health Care Hospital. The newborn's hemoglobin (Hb) was 82 and 76 g/L at five and nine hours after birth, and the total bilirubin (TBIL) was 243.2 and 309.8 μmol/L, respectively. Blood samples of the newborn and the parents were collected for HDFN immunohematology test twelve hours after birth. They showed that the newborn and the father's blood type was A and RhDCCee, while the mother was A and RhDCcee. Direct antiglobulin test (DAT) indicateda strong positive for the newborn and negative for the parents. The reaction of the reagent to red blood cells for antibody screening with the patient's plasma, red cells eluate, and the mother's plasma were all negative, but were positive with the father's red blood cells. The newborn was recovered after treating with phototherapy, intravenous immunoglobulins and urgent blood exchange (the exchanged blood was the same ABO and RhD blood type and cross-matched). The newborn's plasma and red cells eluate were collected before blood exchange, and the mother's plasma were used to assess the red blood cells reaction, and IgG anti-Di a was identified in each sample. Di a blood typing was positive for the newborn and the father, and negative for the mother. Therefore, the newborn was diagnosed as HDFN caused by anti-Di a.
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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.
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Humanos , Sistema del Grupo Sanguíneo ABO , Alelos , Exones , Genotipo , FenotipoRESUMEN
<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>
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Femenino , Humanos , Persona de Mediana Edad , Sistema del Grupo Sanguíneo ABO , Genética , Alelos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Primary intraocular lymphoma (PIOL) is mostly composed by diffuse large B-cell lymphoma with a high degree of malignancy and often masquerades as uveitis.Moreover,it has a poor prognosis,therefore the early diagnosis and treatment are very important.Cytology,cytokine analysis,immunohistochemistry and genetic testing are often used in combination to improve the diagnosis rate of PIOL,however,eye tissue biopsy is the gold standard for diagnosis of PIOL.Currently,since there is no unified treatment program,the main treatment is given priority to local chemotherapy and orbital radiotherapy,and the use of multiple therapies can improve the efficacy of refractory PIOL.Base on these,this article reviews the current diagnosis and treatment for providing certain reference.
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<p><b>OBJECTIVE</b>To investigate the frequency of anti-Mur and Mur antigen among blood donors in Guangzhou to provide evidence for guiding clinical transfusion and prenatal screening.</p><p><b>METHODS</b>DG Gel Coombs cards were used to screen active anti-Mur at 37 degrees celsius; from 2725 blood donors. Multiplex ligation-dependent probe amplification (MLPA) was used to genotype Mur antigen from 91 blood donors, and human anti-Mur serum was used to verify the phenotypes deduced from the genotypes.</p><p><b>RESULTS</b>The frequency of anti-Mur and genotyped Mur antigen was 0.04% (1/2725) and 6.59% (6/91), respectively, and the phenotyping results were consistent with the genotyping results.</p><p><b>CONCLUSION</b>The blood donors in Guangzhou show a low frequency of anti-Mur and a relatively high frequency of Mur antigen. Genotyping using MLPA allows Mur antigen genotyping when commercial anti-Mur is not available.</p>
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Humanos , Donantes de Sangre , Antígenos de Grupos Sanguíneos , Genética , Alergia e Inmunología , China , Genotipo , Técnicas de Genotipaje , Reacción en Cadena de la Polimerasa Multiplex , FenotipoRESUMEN
<p><b>OBJECTIVE</b>To report a rare case of hemolytic disease of the newborn (HDN) with kernicterus caused by anti-Di(a) diagnosed using high-throughput genotyping multiplex ligation-dependent probe amplification (MLPA).</p><p><b>METHODS</b>Conventional serological methods were used to detect the antibodies related with HDN. The genotypes of more than 40 red blood cell antigens for the newborn and her parents were obtained using the high-throughput MLPA assay. The antibody titers were tested using a standard serological method.</p><p><b>RESULTS</b>The unknown antibody against the low-frequency antigens was predicted based on the primary serological tests. The genotyping results for more than 40 red blood cell antigens of the newborn and her parents showed incompatible antigens of MNS and Diego blood group system, indicating the existence of anti-N or anti-Di(a). Further serological tests confirmed anti-Di(a) existence in the plasma of the newborn and her mother. The titer of anti-Di(a) in the mother's plasma was 1:32.</p><p><b>CONCLUSION</b>Severe HDN including kernicterus can result from anti-Di(a). High-throughput genotyping MLPA assay can help type some rare antigens in complicated cases. The reagent red cell panels including Di(a)-positive cells are necessary in routine antibody screening test in Chinese population.</p>
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Femenino , Humanos , Recién Nacido , Incompatibilidad de Grupos Sanguíneos , Genética , Eritroblastosis Fetal , Diagnóstico , Alergia e Inmunología , Recambio Total de Sangre , Genotipo , Técnicas de Amplificación de Ácido Nucleico , Métodos , Sistema del Grupo Sanguíneo Rh-Hr , Genética , Alergia e Inmunología , Globulina Inmune rho(D) , Genética , Alergia e InmunologíaRESUMEN
ObjectiveTo study the molecular genetic mechanism of para-bombay phenotype in two individuals.MethodsThe proband was a female.When the proband donated blood,because the forward blood group wasn't coincident with her reverse blood group,the blood and saliva specimen from proband and her family members were sent to Guangzhou Blood Center for further identification.Routine serological techniques were used to determine proband's and her family members' blood group and ABH antigen in saliva.The coding regions of FUT1 and FUT2 gene,exon 6 and exon 7 of ABO gene were amplified by polymerase chain reaction using proband's and her family members' genomic DNA.All amplified products were analyzed after being directly sequenced.The two-base deletion regions of FUT1 gene were certified by cloning and haplotype sequencing.Results Proband's and her little brother's blood group were identified as para-bombay while other family members' blood group were normal.Two-base deletion heterozygous mutations of FUT1 gene were found in proband and her brother,AG deletion at position 547-552 and TT deletion at position 880-882,which caused a reading frame shift and a premature stop eodon.Meanwhile,880-882del TT heterozygous mutation was found in proband's grandfather and her father and 547-552del AG heterozygous mutation was found in proband's mother and her little sister.ResultsOf cloning and haplotype sequencing certified that these two-base deletion mutations occurred at 547-548 and 881-882 position respectively.Three new mutations were found in FUT2 gene,390C ＞ T,418A ＞ T and 749G ＞ A,which could cause the change of amino acid at position 140Ile ＞ Phe and 250Arg ＞ Gln.Conclusions Two-base deletion heterozygous mutations in different positions in FUT1 gene were found in 2 individuals,which maybe the molecular genetic mechanism of para-bombay phenotype.Heterozygous deletion mutation in one-strand DNA wouldn't change the ABO blood group.Three new mutations were also found in FUT2 gene.( Chin J Lab Med,2012,35:815-819)