人类白细胞抗原分型技术的进展

学术背景：以往人类白细胞抗原分型主要采用血清学和细胞学方法，随着PCR技术、基因芯片技术及分子生物学技术的发展，已建立从基因水平上进行的人类白细胞抗原分型技术。目的：介绍人类白细胞抗原分型方法，为实际应用提供依据。检索策略：应用计算机检索PubMed1997—07／2007—07期间的相关文章，检索词“HLA technology”，并限定文章语言种类为English。同时检索万方数据库1997—07／2007—07相关文章，检索词为“人类白细胞抗原”，并限定文献语种为中文。共检索到相关文献142篇，选择与人类白细胞抗原分型技术有关的文献55篇。将初步筛选的文章进一步查找全文，排除综述和重复文献，内容相似的选择发表在权威杂志或近3年发表的文献，最后共纳入30篇进行综述。文献评价：文献对不同的人类白细胞抗原分型方法进行汇总分析，符合纳入标准的30篇文献中，6篇为中文文献，24篇为英文文献。资料综合：传统的人类白细胞抗原分型方法主要是血清学和细胞学方法，分型准确性较差。随着PCR技术的广泛应用，人类白细胞抗原基因分型方法得到了迅速的发展。序列特异性引物、序列特异性寡核苷酸探针、直接测序、基因芯片等各具特点的人类白细胞抗原分型技术不断出现，使人类白细胞抗原的分型更加准确、精细、简便、实用，为临床推广应用打下了基础。结论：各种人类白细胞抗原基因分型方法各有优势，不能相互替代，这也是由人类自细胞抗原系统的高度多态性和复杂性所决定的，根据不同的用途可选择相应的方法。     

Demographic characteristics and prevalence of serologic markers among donors who use the confidential unit exclusion process: the Retrovirus Epidemiology Donor Study

BACKGROUND: Most blood centers utilize a confidential unit exclusion (CUE) process, intended to reduce the risk of transfusion-associated infectious diseases by allowing high-risk donors confidentially to exclude their blood from use for transfusion. The effectiveness of this method remains controversial. STUDY DESIGN AND METHODS: Confirmatory or supplemental test results for antibodies to human immunodeficiency virus, human T-lymphotropic virus type I, and hepatitis C virus, as well as hepatitis B surface antigen and syphilis and screening test results for antibodies to hepatitis B core (antigen) and alanine aminotransferase levels were obtained for approximately 1.8 million units donated during 1991 and 1992 at five blood centers within the United States. The prevalences of these infectious disease markers in units that the donors confidentially excluded (CUE+) and units that the donors did not exclude (CUE-) were calculated and examined within demographic subgroups. RESULTS: Units that were CUE+ were 8 to 41 times more likely to be seropositive for antibodies to human immunodeficiency virus and hepatitis C virus, hepatitis B surface antigen, and syphilis and three to four times more likely to react for antibody to hepatitis B core (antigen) or to have elevated alanine aminotransferase levels than units that were CUE- (p < 0.001). The positive predictive value of CUE (the percentage of CUE+ units that were confirmed seropositive for any marker) was 3.5 percent, and the sensitivity of CUE (the percentage of confirmed-seropositive units that were CUE+) was 2.3 percent. CONCLUSION: The current CUE process has low sensitivity and apparently low positive predictive value, and in many cases, it appeared that donors misunderstood it. Yet, CUE was not a “random process,” as CUE+ units were more likely to be seropositive for any infectious disease marker than CUE- units. This suggests that efforts to improve the CUE system may be warranted. As risk factors for transfusion-transmitted infection become more difficult to identify by history-based screening, however, such efforts may have limited effect.     

碱性成纤维细胞生长因子基因转移对成骨细胞生长特性的影响

目的:碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)对骨组织的损伤有修复作用,许多体内外实验均表明外源性植入碱性成纤维细胞生长因子能明显促进骨形成过程。观察碱性成纤维细胞生长因子基因转移对成骨细胞生长特性的影响。方法:实验于2003-01/07在解放军广州军区总医院医学实验科完成。成年雄性新西兰白兔,体质量1.5～2.5kg,采用聚乙烯亚胺(jetPEITM)介导真核分泌表达载体pcDNA3.1-bFGF及非分泌表达载体pEGFP-C3-bFGF转染原代培养的成骨细胞,消化收集转染成骨细胞以及未经转染的成骨细胞,利用血细胞计数器进行细胞计数,绘制细胞生长曲线,同时测定DNA浓度、碱性磷酸酶及骨钙素含量的变化。结果:①未转染的细胞在第8天进入稳定期,而经转染的细胞在第10天才进入稳定期。未转染细胞最终达到的细胞数量为(1.70±0.02)×109L-1,而经转染的细胞数量为(2.10±0.03)×109L-1,差异显著(P<0.05)。②经pcDNA3.1-bFGF转染的成骨细胞的DNA量最高,约为(255±20)mg/L,而经pEGFP-C3-bFGF转染的成骨细胞的总DNA量约为(225±20)mg/L,未经转染的成骨细胞的总DNA量为(100±10)mg/L。③pcDNA3.1-bFGF转染的成骨细胞培养至第9天后,碱性磷酸酶分泌量为(8.0±0.22)IU/mL;而未转染的成骨细胞碱性磷酸酶分泌量为(13.12±0.18)IU/mL。经pEGFP-C3-bFGF转染的成骨细胞所分泌的碱性磷酸酶与未经转染的细胞的分泌量相当,约为(12.56±0.24)IU/mL。④随着培养时间的延长,骨钙素分泌量的变化趋势与碱性磷酸酶相同,但重组质粒转染成骨细胞与未转染细胞最终分泌的骨钙素量差异无显著性(P>0.05)。结论:①经pcDNA3.1-bFGF及pEGFP-C3-bFGF重组质粒转染后,成骨细胞的生长特性有一定的变化,与添加外源碱性成纤维细胞生长因子的影响基本相同。②pcDNA3.1-bFGF转染的细胞分泌的碱性成纤维细胞生长因子能够促进细胞分裂增殖,降低碱性磷酸酶的表达,而pEGFP-C3-bFGF转染的细胞不能分泌表达蛋白,难以发挥其生物学功能。     

健胃愈疡颗粒干预下大鼠胃溃疡黏膜乳癌相关肽和血小板活化因子的表达及与胃黏膜疏水性的相关性研究

目的:观察对胃溃疡复发有较好疗效的健胃愈疡颗粒对溃疡黏膜乳腺癌相关肽和血小板活化因子表达的影响,分析其可能的作用机制。方法:实验于2005-07/2006-07在湘雅医院中心实验室完成。SD大鼠110只,雌雄各半,随机抽签法分为5组,即正常对照组、假手术组、雷尼替丁组、健胃愈疡组,各20只;模型组30只。以Okabe改良法复制大鼠实验性胃溃疡,假手术组仅以生理盐水代替乙酸注入玻管内。造模后24h,雷尼替丁组、健胃愈疡组大鼠分别灌服盐酸雷尼替丁和健胃愈疡颗粒(药物组成为:柴胡、党参、白芍、延胡索、白芨、珍珠层粉、青黛、甘草,湖南湘雅制药有限公司生产)药液10mL/kg,分别相当于2.70,1.62g/kg,1次/d。假手术组、模型组灌服蒸馏水10mL/kg。10d后各组中随机取出10只大鼠剖腹取胃(处死前大鼠禁食24h),90d时将模型组20只大鼠再分为模型复发组和模型非复发组,各10只;除正常对照组、假手术组、模型非复发组大鼠腹腔内注射生理盐水外,其余各组大鼠腹腔内注射白细胞介素1,1μg/kg;在注射48h,大鼠禁食24h后,剖腹取胃。观察其对胃溃疡大鼠胃黏膜氨基己糖及磷脂含量、溃疡指数和胃黏膜血流的影响,并用RT-PCR观察乳癌相关肽乳癌相关肽和血小板活化因子表达的变化。结果:实验动物110只,全部进入结果分析。①模型组10,92d胃黏膜血流均低于正常对照组(P<0.01);健胃愈疡组同期胃黏膜血流均高于模型组(P<0.01)。②健胃愈疡组和雷尼替丁组10d溃疡指数均低于模型组(P<0.01,P<0.05);模型复发组、健胃愈疡组和雷尼替丁组92d溃疡指数均高于模型组(P<0.01);健胃愈疡组10,92d溃疡指数及复发率均低于雷尼替丁组(P<0.05,P<0.01)。③模型组10,92d氨基己糖和磷脂含量均低于正常对照组(P<0.01)。健胃愈疡组10,92d氨基己糖和磷脂含量均高于模型组和雷尼替丁组(P<0.01)。溃疡指数与氨基已糖、磷脂含量呈负相关(r=-0.957,-0.960,P<0.01)。④健胃愈疡组和雷尼替丁组10d乳癌相关肽mRNA表达较正常组和假手术组提高,血小板活化因子mRNA的表达下调(P<0.01),健胃愈疡组两指标表达变化较雷尼替丁组显著(P<0.01);模型复发组、健胃愈疡组和雷尼替丁组92d乳癌相关肽mRNA、血小板活化因子mRNA的表达同组10d比较差异无显著性意义(P>0.05);模型组乳癌相关肽mRNA、血小板活化因子mRNA的表达同组10d比较差异有显著性意义(P<0.01)。结论:健胃愈疡颗粒可提高乳癌相关肽mRNA及下调血小板活化因子mRNA的表达,影响胃黏膜氨基己糖及磷脂含量,可能是其促进溃疡愈合的机制之一。     

织构复合水凝胶体系的假说

生物软组织可视为具有多层次结构的织构复合水凝胶体系(TCHS)、以水凝胶复合元件(HCE)为基本的构件(CP),通过一定的组合、排列方式构筑一系列多层次结构的不同软组织。软组织中任何层次的结构单元既可视为织构复合水凝胶体系又可视为构件。任何层次的TCHS中,构件的结构及其组合排列方式决定着该层次单元的功能。以织构复合水凝胶体系的观点考察了真核细胞、角膜和骨骼肌的多层次结构。双层网络水凝胶、皮芯复合水凝胶纤维人工肌肉模型、时空匹配可降解细胞支架等研究成果初步地证明了提出织构复合水凝胶体系观点的合理性。     

场电子发射器件中实际电子发射点密度达到实用程度的标准

目前薄膜场致电子发射平板显示方面的研究越来越深入,其制备的器件场发射性能也越来越接近实用的程度。但有关其发射点密度达到何值时,即可达到最基本可以应用的程度至今也没有一个可以直接参考的实用标准。因此提出一个这样的客观标准对于场发射显示器今后的应用来讲具有重要的实际意义。作者结合自身10多年从事碳基薄膜场致电子发射研究的经历和相应的光学理论,提出一个场电子发射器件中实际电子发射点密度可达到实用程度的标准。当薄膜场电子发射点密度在场发射器件全部发射区域内都超过106sites/cm2时,肉眼将无法在屏幕上分辨出各个发射点,此时电子发射点已连成肉眼无法分辨出的面,也就是说这时单纯从场发射均匀性上来讲,场发射器件已达到可以应用的程度。     

新复合纤维蛋白胶可注射性磷酸钙人工骨的理学特性

目的:检测纤维蛋白胶复合β-磷酸三钙/磷酸二氢钙复合人工骨材料的物理学性能,评价纤维蛋白胶对β-磷酸三钙/磷酸二氢钙骨水泥性能的影响,以及其作为注射型复合人工骨用于修复骨缺损的可行性。方法:实验于2006-12/2007-06在南方医科大学珠江医院中心实验室和华南理工大学生物材料实验室完成。①材料:β-磷酸三钙由上海瑞邦生物材料有限公司提供,磷酸二氢钙为东泰化工赠,纤维蛋白胶购自广州倍绣生物技术有限公司。②复合材料制备:将β-磷酸三钙/磷酸二氢钙骨水泥按3∶1的比例充分混合后,与纤维蛋白胶按凝固后的体积2∶1体积比混合,制成复合人工骨材料。③观察指标:测定复合材料的凝固时间,抗压强度,抗稀散性能,并用扫描电镜观察其煅烧前后的显微结构特征,以未加纤维蛋白胶的磷酸钙水泥为对照(CPC组)。结果:复合人工骨材料的平均初凝时间长于CPC组(P<0.004),终凝时间在初凝时间后2～4 min;复合材料的抗压强度为(14.72±1.81)MPa。复合材料较CPC组有良好的抗稀散性能,扫描电镜发现,纤维蛋白胶贯穿于磷酸钙水泥晶体间,并将磷酸钙水泥晶体紧密连接。煅烧后复合材料的孔径有增大,空隙率为57.28%,并且微孔之间有空隙互相贯通。结论:该骨水泥复合材料凝固时间符合临床操作的需要;抗压强度达到松质骨强度的要求;煅烧后磷酸钙水泥的空隙率明显提高,有利于材料的降解。     

Practical guidelines for process validation and process control of white cell-reduced blood components: report of the Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion (ISBT)

Background: The increased use of white (WBC)-reduced blood components has prompted many institutions to develop quality assurance programs directed to such component preparation processes. For consistent preparation of WBC-reduced blood components that meet clinical needs as well as national standards, a program of process validation and control should be instituted. This involves controlling key factors that affect WBC reduction as well as periodic monitoring of the residual cellular content of components. Practical guidelines for the implementation of such a program are provided. Study Design and Methods: A program involving three phases of monitoring was developed by individuals belonging to an international working party of the International Society of Blood Transfusion. Results: The first phase, process validation, evaluates a minimum of 20 consecutive units (a minimum of 60 units when nonparametric measurements are used) to document the successful local implementation of a new or substantially modified process. Ongoing process control employing Levey-Jennings type control charts is used to demonstrate that the process remains stable over time. Process capability assessment and conformance with standards are evaluated once residual WBCs are counted in a sufficient number of units. This enables a facility to claim with a specified degree of confidence that a stated proportion of WBC-reduced units will meet national standards. Two approaches to determine the number of units that should be selected for counting are presented. The first approach considers units as either acceptable or not acceptable and assumes that the distribution of failed (or nonconforming) units approximates the Poisson distribution. The second approach takes into consideration the observed WBC content of the tested units, with the assumption that the residual WBC content in WBC-reduced components follows a lognormal distribution. A method to assess the lognormal distribution of residual WBCs is presented. Specific tables based on each of these approaches are provided to guide the reader in the design of a program that will verify conformance with any national standard at specific confidence levels. The approach can be generalized to other process control applications. Conclusion: Guidelines are presented for process validation, process control, and assessment of conformance in the production of WBC-reduced blood components. Policy makers retain the responsibility to establish, on the basis of the expected use of WBC- reduced components, requirements for the frequency of testing and for the proportion of prepared units that are expected with a stated degree of confidence to meet the standards. Facilities preparing WBC-reduced components can monitor key factors that influence the preparation of WBC-reduced blood, can periodically assess their conformance with the standards, and can intervene to correct adverse changes in the process. This approach can be used to ensure the consistent quality of WBC- reduced blood components.     

Microdroplet fluorochromatic assay for the enumeration of white cells (WBCs) in WBC-reduced blood components: validation and application for evaluating newly developed WBC-reduction filters

BACKGROUND: Sensitive and accurate counting methods are required to assess the residual white cells (WBCs) in WBC-reduced blood components. The Nageotte hemocytometer, widely used for this purpose, is cumbersome, and its efficacy is dependent upon the skill of the operator. The performance of a simple fluorochromatic assay using tissue-typing microdroplet trays is presented here. STUDY DESIGN AND METHODS: Undiluted samples of blood components were mixed with a fluorochromatic dye in trays. WBCs were counted under an epifluorescence microscope. The accuracy and sensitivity of this method were compared with those of the reference Nageotte hemocytometer method by using serial dilution of samples of platelets and red cells containing known concentrations of WBCs and by calculating the standard curves. The Nageotte hemocytometer and the microdroplet fluorochromatic assay (MFA) were also compared in terms of count correlation and reproducibility in 320 paired counts of plateletpheresis samples. MFA was used to evaluate a newly developed WBC-reduction red cell filter. RESULTS: The MFA for platelets and red cells was linear to 0.1 and 0.03 WBCs per microL, respectively. The linear regression line of log10 MFA versus log10 Nageotte method had a slope of 0.963, intercept of -0.04, and r2 of 0.968. The Nageotte method gave an estimation of WBC content 12 to 20 percent greater than that of the MFA. The MFA, with a larger neat sample volume, showed precision comparable to that of the Nageotte method. The filters demonstrated a median WBC reduction of 4.8 log10. CONCLUSION: The MFA is a sensitive and accurate method for quality control processes to assess the residual WBCs in WBC-reduced blood components.     

Sampling errors and the precision associated with counting very low numbers of white cells in blood components

Attention to the accurate and precise measurement of the white cell (WBC) content of transfused products has risen in response to awareness of the potential benefits of WBC-depleted components and the development of technical capabilities to produce these components. The techniques thus far reported have focused on the reliability of detecting a WBC, provided it is present in the test system. The likelihood of selecting a WBC from the product of interest for counting in the analytical system--that is, the sampling error--must also be considered. The occurrence of a WBC in a WBC-depleted component is a rare event and may be modeled with the binomial or the Poisson distribution. Several assay techniques were analyzed by using these distribution models to determine the confidence intervals of the WBC content. The 95-percent confidence intervals spanned more than 2 logs10 for some methods at 3 x 10(5) WBCs per product. It is concluded that the reporting of WBC content for research provides not only the estimate of the mean but also a confidence interval for this estimate. Quality control procedures should be designed to verify that the WBC content is less than the targeted amount and should provide an associated statement of confidence.