目的 调查分析该站连续3年无偿献血者血型鉴定经过初检、复检和确认后的准确率,为东莞地区实现电子交叉配血提供必要数据支持.方法 收集2006～2008年186 069人次无偿献血者的血型检测结果,分析ABO/RhD血型经过初检、复检和确认后发生血型错误的概率和非技术因素导致错误的频率,比较重复献血者和初次献血者血型鉴定的错误率,文献调查血型初检发生错误的频率范围.结果 ABO血型鉴定复检后错误率(0.009%)明显低于初检的错误发生率(0.08%)(P<0.05),确认试验后发生错误率为0,且明显低于初检和复检(P<0.05).重复献血者血型鉴定错误率明显低于初次献血者(P<0.05).文献调查ABO血型初检发生错误的频率范围为0.04%～1.16%.结论 血型鉴定经过初检、复检和确认后发生错误的概率为0,重复献血者血型鉴定准确率高于初次献血者,满足电子配血对献血者血型鉴定准确率100%的要求,该地区具备开展电子配血的首要条件.

Objective Through research on T cell numbers and cell activation,cytokine secretion of regular whole blood volunteers and regular platelet volunteers,we evaluate the impact of regular donating blood as a stimulating factor to the body's T cells immune function.Methods The numbers of T cells,CD4+ T cells and CD8+ T cells in peripheral blood of three groups of people(regular whole blood volunteers,regular platelet volunteers,control group)were counted by flow cytometer(FCM).Peripheral blood lymphocytes were separated and cultured in vitro,then phytohemagglutinin(PHA) added to stimulate the activation of original T-cells,CD4+CD25+Activated T cells groups were counted by FCM.Secretion of IL-2 was detected by Luminex 100,a multifunctional platform.Results There was no significant statistical difference of in the total number of T cells and,the number of the CD4+ T cells and CD8+ Tcells between the group of free volunteer regular whole blood donations,the group of free to donate volunteer platelet donation as compared to the control group(P0.05).T cell activation expresses showed the highest in the fourth day(72 h) of culture.There was no significant difference of in CD4+CD25+ activated T cells numbers,and IL-2 secretion level between a regular basis to donate volunteer whole blood donation group and the control group(P0.05).The CD4+CD25+ activated T cells number and IL-2 secretion level of regular volunteer platelet donation group crowd was significantly higher than the control group's(P0.05).Conclusion Intensely positive and negative immune regulation keeps the number and function of T cell of regular blood donors in balance.

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.

Objective To analysis NK cell count and cytotoxic activity in regular whole blood and platelet donors and to evaluate their immunity. Methods The NK cell count was analyzed by flow cytometer in peripheral blood in three crowds, viz free regular whole blood, platelet, and the first time whole blood donors. The last crow was the control group. The NK cell cytotoxic activity was analyzed by mixed culture of effecter cell/ target cell. Results The NK cell numbers and its anti-ratio to target cells in free regular whole blood donors had no significant differences with the control group(P>0.05). The platelet donors' NK cell numbers was significantly increased compared to control group(P<0.05), but their NK cell anti-ratio was significant lower than control group(P<0.05). Conclusion Free regular whole blood donation have a long interval (> 3 months), and have enough time to recovery their NK cell numbers and cytotoxicity. In contrast, platelet donation interval is shorter than other groups, so they must increase their NK cell numbers to regulate the effect caused by low function.

目的 实验评估异型输血产生的危害,为异型输血提供实验依据.方法 建立模拟供者受者血液,受者进行ABH分泌型和非分泌型分类,混合后检测红细胞发生凝集的强度,测定血清中K+含量和血红蛋白含量以评价溶血程度.结果 非分泌型受者红细胞凝集强度强于分泌型受者,K+浓度和血红蛋白含量随着输入的异型血液越多发生溶血的程度越高.结论 异型输血后会发生红细胞凝集溶血现象,ABH非分泌型受者红细胞凝集强度强于分泌型受者,激活补体发生溶血的程度无明显差异,分泌型受者血清中的血型物质不能减轻异型输血发生的溶血,输入的异型血液越多,溶血越严重,不主张临床异型输血.

AIM:To compare the effect of ZZAP and CDP elution tests on DAT(+) red blood cells and the absorption effect of the eluted red blood cells. METHODS:Twenty anti-IgG type and twenty anti-(IgG+C3d)type DAT(+) red blood cell samples were obtained from clinic patients and ZZAP and CDP elution tests were conducted respectively. The effects of autoantibody elution on red blood cell members by DAT test and the absorption effects of autoantibody absorption in serum with eluted red blood cells by crossmatch tests were compared. RESULTS:No significant difference was observed in anti-(IgG+C3d) type red blood cells using ZZAP test or CDP elution test. The effect of autoantibody elution in anti-IgG type was significantly better than that in anti-(IgG+C3d) type (P<0.05)but using CDP elution test,no significant difference was found between anti-(IgG+C3d)type and anti-IgG type red blood cells. The absorption effect of anti-(IgG+C3d) type and anti-IgG type red blood cells by ZZAP was better than that by CDP (P<0.05),but using ZZAP test or CDP test,no significant difference was found in anti-IgG type red blood cells. No significant difference was observed in anti-(IgG+C3d)type and anti-IgG type red blood cells using CDP. The absorption effect of anti-IgG type red blood cells was significantly better than that of anti-(IgG+C3d) type using ZZAP (P<0.05). CONCLUSION:Anti-(IgG+C3d)type red blood cells are not effectively eluted by ZZAP test or CDP test,but ZZAP is better than CDP in eluting anti-IgG type DAT(+) red blood cells.

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.

An unknown HLA-B allele which was similar to HLA-B*270401 was detected by FLOW-SSOPCR-SSP and heterozygous sequence-based typing (SBT) in Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. Amplifying exon 2-5(include intron 2-4) of the HLA-B*27 allele separately by using allele-specific primers and sequencing in both directions. Identifying the difference between the novel B*27 allele and B*270401. The sequence of novel B*27 from exon 2 to partial exon 5 is 1 815 bp. There are 10 nt changes from B*270401 in exon 3-4, at nt634where A-->C(codon130 AGC-->CGC, 130 S-->R); nt670 where A-->T (codon142 ACC-->TCC, 142 T-->S); nt683 where G-->T (codon146 TGG-->TTG, 146 W-->L); nt698 where A-->T (codon151 GAG-->GTG, 151 E-->V); nt774 where G-->C (codon176 GAG-->GAC, 176 E-->D); nt776 where C-->A (codon177 ACG-->AAG, 177 T-->K); nt781 where C-->G (codon179 CAG-->GAG, 179Q-->E); nt789 where G-->T (codon181 GCG-->GCT) resulting no coding change; nt1438 where C-->T (codon206 GGC-->GGT) resulting no coding change; nt1449 where G-->C (codon210 GGG-->GCG, 210G-->A). In IMGT/HLA database, only three alleles (B*270502/2706/2732) have sequences of introns. The same sequence in intron 2 showed homology between the novel HLA-B*27 allele and B*2706, but their homology could not be supported in intron 3-4. Comparing the sequence of the novel B*27 allele in intron 3 and 4 with B*27 group, it showed there are three mutations at nt106 C-->G, nt179 G-->A, nt536 G-->A and one deletion at nt168 in intron 3 and one mutations at nt82 T-->C in intron 4, but the sequence of the novel B*27 allele in intron 3 and 4 was all the same to B*070201. The sequence was submitted to Gen-Bank and the accession number was DQ915176. The allele has been confirmed as an extension of B*2736 by the WHO Nomenclature committee in November 2006.

Objective To develop and establish a effective method for high-volume automatically extracting genomic DNA from whole blood samples,and utilize this method in HLA genotyping by luminex flow array assay.Methods Genomic DNA was extracted automatically from 600 μl blood samples by TECAN DNA workstation.The yield and purity of each sample DNA was tested by UV-spectrophotometer,the integrality of these DNA samples were runelectrophoresis by agarose gel,and the fluorescent intensity of HLA-A,B andDRB1 PCR products hybridized with specific probes were calculated and analyzed.Results The mean yield and purity(A260nm/A280nm)of genomic DNA extracted from whole blood samples were 10.052±0.824 μg and 1.821±0.201 respectively,the molecular weight was approximate 21kb.The fluorescent intensity for HLA-A,B and DRB1 PCR products hybridized with specific probes were 2 183.84±478.12(exon2),2 168.28±338.59(exon3);2057.27±397.41(exon2),1 959.78±283.24(exon3)and 3 643.38±327.85 respectively.Conclusion This highly qualitied genomic DNA canbe extracted automatically from whole blood samples by using TECAN DNA workstation,and the isolated DNA samples were suitable for HLA genotyping by luminex flow array assay and other downstream molecular biological experiments.

Objective To compare the quality and quantity from whole blood samples for genomic DNA extraction by the workstation and two manual methods.Methods Three methods were used to extract genomic DNA from same thirty-six samples,and the results were compared in quantity,quality and the cost.Results The three methods all could give high field of DNA,but there were very different in operation procedure and the cost.Conclusion Workstation is fit for extration genomic DNA from lots of samples because of a little blood and low cost.The Gentra method and the hydrochloric acid carbamidine method are fit for fresh and frozen whole blood,and the hydrochloric acid carbamidine method can be used to extract genomic DNA from blood clots.

Objective To utilize the optimum DNA extraction method in routine HLA genotyping of bone marrow donor program by comparing the yield and quality of genomic DNA extracted from obsolete whole blood samples by the magnetic beads method and the salt out method. Methods Based on the TECAN DNA workstation platform,the genomic DNA of 48 obsolete whole blood samples were extracted automatically by Promega magnetic beads method and Gentra salt out method,respectively; the extracted genomic DNA of each sample was finally dissolve in 100μl TE solution. The concentration and purity of each DNA sample was tested by UV-spectrophotometer,the integrality of these DNA sample were run electrophoresis by agarose gel. Results The mean concentration and purity (A260/A280 ) of genomic DNA extracted by Gentra salt out method were 28.6±8.7 ng/μl and 1.63±0.209,respectively; the mean concentration and purity (A260-A320/A280-A320) of genomic DNA extracted by Promega magnetic beads method were 94.2±10.3 ng/ul and 1.821±0.102,respectively. The genomic DNA extracted by two methods were run by electrophoresis on the agarose gel and indicated that the molecular weight for 48 DNA samples were all 15kb in length. Conclusions Our results indicate that the yield and purity of genomic DNA extracted by magnetic beads method are better than those of GentraTM salt out method,genomic DNA extracted from the obsolete whole blood by magnetic beads method is suitable for routine HLA typing and other downstream molecular biological experiments.

目的研究流式磁珠反向SSO法HLA-A、B、DRB1分型结果判读中出现的常见问题,并提出相应的解决策略。方法对3 219名随机抽取的深圳市无关造血干细胞供者及组织配型患者的DNA采用流式磁珠反向SSO法进行HLA-A、B、DRB1低分辨基因分型,模棱两可结果用PCR-序列特异性引物(Sequence Specific Primer,SSP)法及PCR-SBT测序方法等方法复核确认。结果3 219份样本中发现有95份(95/3 219)28种类型的模棱两可结果,其中HLA-A位点有3(3/28)种,HLA-B位点有25(25/28)种,91.6%的模棱两可结果其样本的荧光值在阳性控制线上,本研究观察到的模棱两可结果通过基因频率分析、PCR-SSP法及PCR-SBT基因测序等方法得以定型。结论建立流式磁珠反向SSO法HLA-A、B、DRB1基因分型模棱两可结果解决策略,对提高HLA数据分析的速度和分型的准确性有重要的意义。

Objective To study the effect of DNA extraction from whole blood frozen and thawn out repeatly on quantity and purity. Methods 100 samples were frozen and thawn outre- peatly every otherday. DNA were extracted and surveyed concentration and purity from the thawn outsamples. Results Compared with the different times of repeat frost and thaw,theef- fectoffrozen and thawn outrepeatlycould decrease the quantity of DNA (P0.01), but there were no obvious effect on purity (P0.05). Conclusion The sample with repeat frost and thaw can decrease the quantity of DNA extraction,but there is noeffect on purity.

目的比较五种HLA低分辨率分型试剂的检测效果.方法用五种HLA分型试剂分别检测7份标准品,分析比较各种试剂的检测结果.结果五种分型试剂基因检出率均为100%,无漏检.分辨率较好,均能达到低分辨率的要求.但试剂A和D特异性略低于其它试剂.结论储备两种分型试剂以弥补彼此的不足.

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, 2μg/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.