Objective Study on fetomaternal immuno state and RHD type of a pregnant woman of weak D phenotype.MethodsThrough polymerase chain reaction (PCR)、direct genomic DNA sequencing and flow cytometry.ResultsIn both sequence specific promer (SSP) PCR and the sequencing PCR tests, the sample was detected negative in exons 3 6 of the RHD gene, whereas all other exons (exons 1 2,7 10) were tested positive. And the sequence of detected exons (exons 1 2,7 10) are the same with normal RHD in GenBank (accession no. AJ299020 1 and AJ299026 9). Serologically and genetically, the sample can be designated as D category VI type Ⅲ. Through a duce tube PCR method, the RhD zygosity of this individual was typed CD VI e/cde。In flow cytometry, a few fetal erythrocytes were detected in peripheral blood of the mother. However there were no anti D detected in sera and hemolytic disease of the newborn(HDN) observed at all.ConclusionSevere cases of HDN have occurred in D positive babies born to partial D mother with anti D, although HDN don't take place in this case. We may still consider D VI phenotype individuals as D positive donors and D negative receiptions in our transfusion practice and in clinical anti D allo immune prophylaxis and monitoring.

Objective To prepare B-lymphoblastoid cell lines of HLA novel allele B*5610 in a family for further study and identification . Method Isolate mononuclear cells under aseptic conditions from the peripheral blood. After infection with Epstein-Barr virus, the cells were cultured in 20% FBS, 2?g/ml CsA RPMI 1640. Results Immortalized B-lymphoblastoid cell lines of five B *5610 carriers in a family were achieved, and the new genes were inherited stably. Conclusion Our work is important for storing and breeding the precious material of biomedicine because the B *5610 genes in the immortalized B-lymphoblastoid cell lines were inherited stably.

Objective To gain purified platelets for further research of the immune function of platelet.Methods Platelets were purified by positive(MS) and negative(LD) magnetic cell sorting(MACS) separation.The percentage of activated platelets was detected by flow cytometry and nucleated cell clearance was evaluated by white cell count.Results The percentage of activated platelets before separation was(2.39?1.10)%,and increased to(2.56?1.08)% and(16.76?4.04)%,respectively,after MS and LD MACS.The clearances of nucleated cells after MACS MS and LD were(98.44?0.24)% and(98.47?0.18)%,respectively.The recovery rate of purified platelet after MS and LD was(76.50?1.49)% and(79.20?2.61)%.Conclusion MACS LD method was more suitable for the platelet purification.

ObjectiveTo analyze the polymorphism of HLA-A,B,and DRB1 alleles and their haplotypes in Chinese Man population.Methods Frequencies of HLA-A,B and DRB1 alleles and haplotypes were estimated by maximum-likelihood estimation method based on the genotypes of 2183 Chinese Man bone marrow donors.ResultsA total of 18 HLA-A alleles,44 HLA-B alleles and 15 HLA-DRB1 alleles were detected in Man population,and the most frequent alleles were A*02,A*11,A*24,A*30,A*33,B*13,B*35,B*46,B*51,B*40(B60),B*40(B61),B*15(B62),DRB1*04,DRB1*07,DRB1*08,DRB1*09,DRB1*11,DRB1*12,DRB1*13,DRB1*14 and DRB1*15.A*30-B*13,A*02-DRB1*15,B*13-DRB1*07 and A*30-B*13-DRB1*07 were the most frequent haplotypes in Man population for A-B,A-DRB1,B-DRB1 and A-B-DRB1 haplotype,respectively.The number of haplotypes with frequency ≥ 0.01 for A-B,A-DRB1 and B-DRB1 haplotype was 31,24 and 27,respectively,and ≥ 0.005 for A-B-DRB1 haplotype was 32.There were 14 in A-B,3 in A-DRB1,14 in B-DRB1 and 38 in A-B-DRB1 haploypes that showed strong linkage disequilibrium with ALD≥0.40.ConclusionsThe distribution of HLA-A,B and DRB1 alleles and haplotypes in Man population was similar to that in Northern Chinese Han population.

Objective To study the inter-allelic recombination event occurring in the HLA-C locus in a family of Chinese Han nationality, and to evaluate the molecular genetic background of the new HLA allele.Methods Peripheral blood samples were collected from a Chinese leukemia woman patient, as well as her healthy parents and two brothers.HLA-A, C, B, DRB1 and DQB1 alleles were typed by high-resolution PCR-sequence-based typing (SBT) method using Atria Genetic AlleleSEQR HLA SBT kits.The Protrans S4 HLA-C single allele-specific sequencing strategy was used to separate the two HLA-C alleles and to determine novelty of the allele.The full length sequences of HLA-C alleles of the patient and her parents were further analyzed using cloning and haplotype sequencing method. The HLA five loci linked haplotypes and the recombination site were analyzed by family study, meanwhile the full length sequences of the five HLA-C alleles were compared with the IMGT/HLA database by the program BLAST.Results The two haplotypes of the father and mother were a:A*0207-C*010201-B*550201-DRB1*090102-DRQ1*030302 and b:A*240201-C*120202-B*5201-DRB1*1502-DRQ1*0601, c:A*300101-C*060201-B*130201-DRB1*0405-DRQ1*0401 and d:A*110101-C*070201-B*4001-DRB1*080302-DRQ1*0601,respectively.The two brothers inherited their parent′s haplotypes a, d and b, c respectively.The two haplotypes of the patient were the maternal c and paternal recombinant a/b haplotype.The recombinant a/b haplotype A*240201-C*new-B*550201-DRB1*090102-DRQ1*030302, A*240201 came from the paternal haplotype b,while B*550201-DRB1*090102-DRQ1*030302 came from the other paternal haplotype a.When comparing the full length sequences of the HLA-C new allele with the father′s allele C*010201 and C*120202, it could deduce that the recombinant a/b haplotype derived from a recombination event occurring between the paternal chromosome 6 during meiosis.The crossover site was between genomic nt273 and nt330 of HLA-C alleles, which created a HLA-C new allele and the fifth haplotype of the family, and inherited it to the patient.The full length sequences of the new allele had been submitted to Genbank, and officially named C*0121 by WHO nomenclature committee.Conclusion This study demonstrates a rare inter-allelic recombination event occurring in the HLA-C locus within a Chinese Han family and illustrates the process of novel allele and haplotype, and provides direct theory for further studying the mechanisms of gene recombination and HLA polymorphism.

Objective To compare efficacy of female sterilization by modified Uchida technique and silver clips and to evaluate the influence on operation procedure and clinical effect with or without surgery training of service providers. Methods A comparative, multicenter clinical trial was performed in 18 county and township-level service centers. Totally 2198 women underwent sterilization from these 18 study center were divided into 1116 women sterilized by modified Uchida technique and 1082 women by silver clips.Those 18 centers were classified into 9 training groups which provide surgical skills of sterilization and other contents and 9 non training groups. Clinical documents of sterilization were recorded. All women were followup at 3, 6 and 12 months after surgery. Results There were no complications during surgery by both sterilization. The failure rate was 2.03% (22/1082) in silver clip method and the mean operative time were ( 12. 4 ± 6. 4 ) minutes in training group and ( 14. 4 ± 8. 1 ) minutes in non training group. In modified Uchida method, the failure rate was 0. 18% (2/1116) and the mean operative time were (16. 2 ± 4. 9)minutes in training group and (19.0 ±8.6) minutes in non training group. The mean operative time between two groups reached statistical difference ( all P ＜ 0. 05 ). Total ended rate in modified Uchida technique were 2. 2/hundred women year in training group and 2. 5/hundred women year in non training group, and the rate of silver slips were 3. 9/hundred women year and 4. 8/hundred women year, which did not show significant difference ( all P ＞ 0. 05 ). There was no significant difference in acceptability and side effects of all women between two methods (P ＞ 0. 05). The training of service providers could influence acceptability of women (P ＜ 0. 05). Conclusions Clinical efficacy was not influenced by those two methods. The operative time and acceptability were improved by training surgeons in silver clips method.

BACKGROUND: Umbilical cord blood (UCB) with limited karyocytes is mainly used in child patients. Recently, physicians have tried to mix two units of cord blood in the treatment of adults with hematological system diseases.OBJECTIVE: To monitor quantitatively the dynamic changes and the development rules of engraftment, chimera types and relative amount after allogeneic transplantation of mixed UCB from two units in adults with leukemia.DESIGN: Donors and the recipient were regarded as observational subjects in umbilical cord blood transplantation (UCBT). DNA extracted from blood samples of donors and the recipient before and after transplantation was considered as detecting samples. Short tandem repeat (STR) loci were as observational measures.SETTING: Key Laboratory of Immunology and Genetics of Institute of Transfusion Medicine of Shenzhen Blood Center.PARTICIPANT: A 43-year male patient with acute myeloid leukemia (AML), 75 kg, who was hospitalized at Shenzhen Hospital of Peking University, was enrolled in June 2005. The patient received two units of human leucocyte antigen (HLA), one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2) at month 6 after complete remission from first chemotherapy. UCB was collected from Guangzhou umbilical cord blood bank. The patient signed the informed consent.METHODS: The adult with AML received two units of HLA, one locus mismatched unrelated UCBT (2.5×107 kg-1 karyocytes in UCB 1, and 1.53×107 kg-1 karyocytes in UCB 2). Nine STR loci of the blood sample were determined before and after transplantation by quantitative technique of fluorescence labeling with multiplex polymerase chain reaction (MPCR), while the engraftment and chimera types were qualitatively evaluated by comparing differential loci between the recipient and the donors. The relative amount of two units of UCB was calculated in the patient after transplantation according to the differential gene peak areas of two donors with 377XL DNA sequencer after fluorescence scanning. The engraftment level and the development rules of donors' cells were analyzed quantitatively. In addition, the results were also compared with that of HLA loci distinct analysis for engraftment.MAIN OUTCOME MEASURES: After UCBT, transition process of nine STR loci of the recipient and two donors was observed, and engraftment was quantitatively and qualitatively described.RESULTS: Two units of UCB at day 15 after transplantation were engrafted simultaneously and revealed a complete chimera of the two. The relative amounts of UCB 1 and UCB 2 were 51.3% and 48.7%, respectively. Subsequently, UCB 1 went up to 70.0% and UCB 2 declined to 30.0% at day 30. However, only the genotype of UCB 1 was detected at day 52, and engraftment turned to a complete chimera of a single donor. The one with fewer karyocytes was rejected and the one with more karyocytes was engrafted for a long term.CONCLUSION: To detect quantitatively STR chimera with fluorescence labeling and MPCR can show precisely the engraftment level and the change of two units of UCB. It provides an accurate and reliable experimental basis for clinical UCB application and donor selection. It is proved that adult transplantation at the same time with mixed UCB from two units HLA one locus mismatched unrelated donors is feasible.

Objective To assess the clinical characteristics of postoperative myocardial infarction (PMI) in patients undergoing off-pump coronary artery bypass grafting(OPCAB).Methods Two hundred and sixty-six patients undertook OPCAB in the Shandong Provincial Chest Hospital from January 2008 to June 2015 were selected,there were 22 cases clinical diagnosis of PMI as PMI group,44 cases patients whose general information matched MI group were selected as no PMI group.The data of two groups including preoperative records and postoperative symptoms,electrocardiogram (ECG),cTnI and echocardiography change were compared.Results There were no significant differences about preoperative indexes between the two groups(P ＞0.05).Incidences of severe chest pain and new pathological Q-waves and elevated ST segments were significantly higher in PMI group than those in no PMI group (90.9％ (20/22) vs.18.2％ (8/44),27.3％ (6/22) vs.4.5％(2/44),95.4％(21/22) vs.27.3％(12/44)),the differences were significant(P＜0.005).Peak serum levels of cTnI during the first 24 h after operation were significantly higher in patients of PMI group than those in no PMI group,the difference was significant((4.52±2.81) μg/L vs.(0.26±0.22) μg/L,P=0.04).There was no significant difference in the incidence of myocardial segmental motion.Conclusion It is difficult to predict coronary artery bypass grafting after myocardial infarction.It has great value of postoperative ECG,the patient complained in diagnosis of postoperative myocardial infarction.CTn is a very sensitive indicator,but its diagnosis clinical myocardial infarction boundary value still need to be open to question.

BACKGROUND:As the sequencing technology has been widely used and high-resolution confirmation of organ transplant matching has been gradualy developed, new human leukocyte antigen (HLA) aleles are emerging. However, the gene frequency of some genes cannot be calculated accurately, and there are rare reports. These genes are often ignored, and it is easy to misjudge their genotypes only according to gene frequency. OBJECTIVE:To test and analyze a rare alele, HLA-C*08:99, from a volunteer donor of hematopoietic stem cel transplantation. METHODS: Genomic DNA was extracted automaticaly from the blood sample by using quick DNA purified kit and amplified by HLA-C locus commercial sequence-based typing kit. The purified PCR product was utilized as the DNA template in the sequencing reaction, and six direct sequencing reactions of PCR product covering exons 2, 3 and 4 in both directions were performed using commercial kit. Four direct sequencing reactions of PCR product covering exon 5 in both directions, exon 6 in forward direction and exon 7 in reverse direction were performed using in-house BigDye terminator cycle sequencing reaction kit. Sequencing reaction products purified by ethanol/sodium acetate/ ethylenediaminetetraacetic acid method were sequenced by ABI PrismTM3730 DNA Sequencer. RESULTS AND CONCLUSION:The alele assignment was analyzed with Assign-SBT 3.6+ software, and the sample HLA-C typing result was C*07:04, 08:99. Increasing the sequencing analysis at exons 5, 6 and 7 of HLA-C locus wil help to make clear the ambiguous SBT result and improve the accuracy of HLA-C typing when it is necessary, which shows important significance in clinical tissue matching. Cite this article:Wang DM, Zou HY, Nie DM, Gao SQ, Wang F.Sequencing analysis of a rare human leukocyte antigen, C*08:99, from a volunteer donor of hematopoietic stem cel transplantation. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):102-106.