马来酰亚胺苯甲氨酯聚乙二醇修饰膜蛋白掩盖AB和RhD抗原制备通用血型红细胞

聚乙二醇（PEG）上的活性基团结合在红细胞表面掩盖血型抗原是制备通用血型红细胞的途径之一，这些PEG链有很强的水合作用，能覆盖红细胞表面的大片区域，阻断血型抗原与抗体结合。甲基氧PEG-5000（mPEG-5000）是常用的红细胞修饰剂，主要修饰蛋白上的氨基基团。在氯化氰脲酸（CnCl）催化下，mPEG-5000与红细胞膜上氨基形成共价键连接，掩盖Rh抗原和A或B抗原。CnCl—PEG-5000浓度越高，血型抗原的覆盖效果越好。由于微环境下A和B血型抗原处无氨基基团或者氨基基团不能被CnCl—PEG-5000修饰，不能完全阻断抗-A、B与A和B血型抗原结合。本文报道用马来酰亚胺苯甲氨酯聚乙二醇（Mal—Phe—PEG）修饰红细胞A、B和RhD抗原的方法。     

聚乙二醇衍生物修饰红细胞的保存性能研究

目的 评估经甲氧基聚乙二醇-琥珀酰亚胺丙酸酯(mPEG-SPA)修饰后红细胞(RBC)的保存性能.方法 以mPEG-SPA体外修饰健康人RBC;观察修饰前后RBC的变形性、渗透脆性、自身溶血率、磷脂酰丝氨酸和CD59,评估修饰对RBC保存性能的影响.结果 mPEG-SPA修饰后RBC的变形性略有下降、渗透脆性和自身溶血率有所增加;修饰后红细胞磷脂酰丝氨酸表达明显增加;红细胞CD59的表达严重下降,其表达率几近于零.结论 mPEG-SPA修饰后红细胞的保存性能下降,可能影响修饰后红细胞的寿命和输入体内的存活率.     

甲氧基聚乙二醇修饰改造RhD(+)血型的初步研究

Rh血型与ABO血型一样是重要的血型系统，Rh血型不符会引起严重的输血反应及新生儿溶血病。中国人群中RhD阴性血型的比例仅为0．2％-0．5％，将RhD阳性红细胞改造成RhD阴性红细胞在临床输血中有重要的意义。本研究用4种不同端基的甲氧基聚乙二醇(mPEG)，修饰A型、B型、AB型和O型的RhD( )红细胞，比较4种mPEG衍生物对RhD抗原的修饰效果。同时观察mPEG修饰对红细胞形态、结构和功能的影响。结果表明，mPEG—BTC(苯并三唑端基PEG)较其他3种PEG的修饰效果好，在1mmol／L时可以有效地遮蔽红细胞表面RhD抗原，而对红细胞形态、渗透脆性、自身溶血、乙酰胆碱酯酶、胆固醇、ATP、2，3-DPG含量以及变形性无影响。结论：初步实现了将RhD( )红细胞修饰改造成RhD(-)红细胞的目的。     

甲氧基聚乙二醇修饰红细胞解决自身免疫性疾病所致配血困难研究

目的观察红细胞(RBC)经甲氧基聚乙二醇-琥珀酰亚胺丙酸酯(mPEG-SPA)修饰后对血型抗原的遮蔽作用,评估其解决自身免疫性疾病等所致配血困难的效果。方法以mPEG-SPA体外修饰正常人RBC,用微柱凝胶血型卡检测对A、B、RhD、RhC、RhE血型抗原的免疫遮蔽作用,并比较不同分子量不同浓度mPEG-SPA的遮蔽效果;观察修饰前后RBC的变形性、渗透脆性、自身溶血率,判断修饰对RBC功能的影响;比较修饰前后自身抗体阳性患者血清与相同ABO血型标本交叉配血时的凝集情况。结果mPEG-SPA修饰不影响RBC的变形性、渗透脆性、自身溶血率等特性,对A和B血型抗原基本没有遮蔽作用,而分子量20000的mPEG-SPA浓度达2mmol/L或分子量10000的mPEG-SPA为6mmol/L时,能有效地遮蔽RhD、RhC和RhE抗原,可有效消除受血者自身抗体和其他相应抗体所致的凝集现象。结论mPEG-SPA修饰可有效遮蔽Rh血型抗原并解决自身抗体所致的配血困难。     

CD55和CD59对聚乙二醇衍生物修饰红细胞保存性能的影响

目的 观察经甲氧基聚乙二醇-琥珀酰亚胺丙酸酯(mPEG-SPA)修饰后红细胞(RBC)CD55和CD59的表达情况,评估其对修饰后红细胞保存性能的影响.方法 以mPEG-SPA体外修饰健康者RBC;观察修饰前后RBC的变形性、渗透脆性、自身溶血率、CD55和CD59,判断修饰后对RBC保存性能的影响.结果 mPEG-...     

N-苯基马来酰亚胺消毒剂的研制及其消毒效果观察

目的研制N-苯基马来酰亚胺消毒剂工艺条件,观察其杀菌效果。方法采用正交设计和载体浸泡定量杀菌试验方法进行了实验室研究。结果N-苯基马来酰亚胺消毒剂工艺条件为脱水剂用量为15 m l、催化剂为20mmol/L、反应温度为55℃、反应时间为60 m in,得到该化合物收率89%。以含N-苯基马来酰亚胺80 mg/L的该消毒液对载体上枯草杆菌黑色变种芽孢作用25 m in,平均杀灭率为99.95%。结论通过本设计工艺,可得到N-苯基马来酰亚结晶状产品且合成收率较高,用该化合物配制的消毒液具有较强的杀灭细菌芽孢的能力。     

HLA-DRB1-12寡核苷酸芯片的制备及优化

生物芯片技术是基于杂交原理发展而来的，是将固相反应的原理和形式应用于大分子识别反应(核酸杂交、抗原抗体结合或酶促的模板依赖性的连接、延伸等反应)中，以达到对大量的目标分子进行快速、平行的特异识别。寡核苷酸芯片的制备过程中关键部分是基片的表面化学处理和探针末端的不同修饰。为了比较不同末端修饰探针在不同化学处理的载玻片上的杂交信号的强弱，本研究根据HLA—DRBl—12的序列设计8种不同类型的探针，即4种5′末端修饰探针，包括末端氨基修饰探针(N)，氨基加四聚乙二醇间隔臂探针(NL)，硫代探针(S)，硫代加四聚乙二醇间隔臂探针(SL)和4种3′末端修饰探针，同样包括末端氨基修饰探针，氨基加四聚乙二醇间隔臂探针，硫代探针，硫代加四聚乙二醇间隔臂探针。将这8种探针分别固定在溴化芯片和醛基芯片上。与末端标记荧光的不对称的PCR产物进行杂交，通过比较杂交结果荧光信号的强弱，筛选出探针同活化基片的最佳组合。从而达到优化寡核苷酸芯片制备的目的。另外，为了进一步比较四聚乙二醇间隔臂对杂交信号的影响。设计末端连接不同数目四聚乙二醇的3′氨基探针。结果显示，3′末端修饰探针杂交信号强于5′末端修饰探针，探针在溴化芯片中的杂交信号强度高于在醛基芯片中杂交信号，3′带有间隔臂的R氨基探针在溴化芯片中的杂交信号最强。另外3′氨基探针中间隔臂可以增强杂交信号强度，但随着数目的增加，杂交信号并不显增加。结论：溴化芯片中3′末端带间隔臂的氨基修饰探针在杂交过程中可以更有效的捕获靶标，提高寡核苷酸芯片的杂交能力；四聚乙二醇的数量不必过多使用；通过这种方法可以达到优化芯片制备的目的，为下一步HLA寡核苷酸分型芯片及其他基因芯片的研制提供有效的手段。     

甲氧基聚乙二醇遮蔽猕猴红细胞表面类人B抗原的研究

目的　观察mPEG对猕猴类人B抗原的修饰效果及mPEG修饰后的红细胞给动物输血的安全性。方法　用 3mg/ml的mPEG修饰遮蔽猕猴红细胞表面的类人B血型抗原 ,吸收放散法检测猕猴血型及mPEG对猕猴红细胞血型的修饰结果 ;红细胞变形仪等检测mPEG修饰对红细胞的变形能力、结构与功能的影响 ;荧光素标记 ,自体回输实验检测红细胞存活率 ;异体回输实验观察修饰后红细胞对受血猴的血液、尿液的影响。结果　mPEG修饰可以遮蔽猕猴类人B抗原的抗原性 ,不影响所修饰红细胞结构、功能及体内存活 ;mPEG修饰的类人B型红细胞输给类人A型血的猕猴 ,未发生输血反应 ;受血猴的血细胞、血液生化及尿液各项指标与输血前相比 ,无明显变化。结论　mPEG修饰可遮蔽猕猴红细胞表面类人B抗原的抗原性 ,mPEG修饰红细胞在受血猴体内是安全的。     

可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体与巨噬细胞的免疫学功能

背景：巨噬细胞进行抗原吞噬、分泌细胞因子以及迁移,均需囊泡转运,而可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族在囊泡转运过程中发挥重要作用。目的：全面了解可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体在巨噬细胞免疫活动中的功能和作用机制。方法：以＂snare proteins,macrophages,SNARE蛋白,巨噬细胞＂为检索词检索PubMed、Embase和维普数据库（2011-11）,语言限定为英文或中文。经阅读题目和摘要进行初筛,排除研究方向与本文无关、内容重复性研究。另补充必要的对理解其作用机制有帮助的文献。结果与结论：最终纳入27篇文献。可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族是保证囊泡物质定向运输和准确卸载的分子基础,在巨噬细胞抗原吞噬、细胞因子分泌、以及移行过程中发挥作用。以可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族为切入点对巨噬细胞免疫活动进行研究是一种合理的策略。     

可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体与巨噬细胞的免疫学功能

背景:巨噬细胞进行抗原吞噬、分泌细胞因子以及迁移,均需囊泡转运,而可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族在囊泡转运过程中发挥重要作用。目的:全面了解可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体在巨噬细胞免疫活动中的功能和作用机制。方法:以"snare proteins,macrophages,SNARE蛋白,巨噬细胞"为检索词检索PubMed、Embase和维普数据库(2011-11),语言限定为英文或中文。经阅读题目和摘要进行初筛,排除研究方向与本文无关、内容重复性研究。另补充必要的对理解其作用机制有帮助的文献。结果与结论:最终纳入27篇文献。可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族是保证囊泡物质定向运输和准确卸载的分子基础,在巨噬细胞抗原吞噬、细胞因子分泌、以及移行过程中发挥作用。以可溶性N-乙基马来酰亚胺敏感因子结合蛋白受体家族为切入点对巨噬细胞免疫活动进行研究是一种合理的策略。     

Hemolytic Disease of the Fetus and Newborn: Modern Practice and Future Investigations

Red blood cell (RBC) sensitization occurs in some women in response to exposure to paternally derived RBC antigens during pregnancy or to nonself antigens on transfused RBCs during their lifetime. Once sensitized, future pregnancies may be at risk for hemolytic disease of the fetus and newborn. Although great strides have been made over the past few decades in terms of identifying blood group antigens and in predicting fetal anemia through the use of noninvasive monitoring, many questions remain in terms of understanding RBC alloimmunization risk factors, preventative therapies, and treatment strategies. At the present time, there is room for improvement in these areas in both developed and developing countries. Evidence-based, universal guidelines describing recommended RBC antigen matching transfusion strategies for girls or women, before pregnancy or during intrauterine transfusions, would be welcomed. A better understanding of the mechanism(s) of action of Rh immunoglobulin, first introduced more than half of a century ago and one of the most successful immunoprophylaxis therapies in existence today, would also be a large step forward. For example, answers to questions of the role(s) that fetal RBC clearance, antigen masking, antigen modulation, and immune suppression play in the effectiveness of Rh immunoglobulin may help to guide the development of novel preventative therapies during pregnancy for immunization to RhD and non-RhD antigens. Furthermore, a better understanding of the importance of anti-RhD or other alloantibody glycosylation patterns may be beneficial not only in developing such novel immunoprophylaxis therapies but also in predicting the clinical significance of existing maternal alloantibodies. One other area of need includes the development of therapies beyond intrauterine transfusions to mitigate the dangers of maternal alloantibodies to developing fetuses. We challenge physicians, scientists, and funding agencies to prioritize studies of RBC alloimmunization and hemolytic disease of the fetus and newborn and to invest in the children of our future.     

Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells

BACKGROUND: The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. STUDY DESIGN AND METHODS: A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RESULTS: RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. CONCLUSIONS: Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.     

Murine monoclonal antibodies reactive with a human monoclonal anti-RhD antibody (BRAD-5)

BRAD-3 and BRAD-5 are human monoclonal antibodies that recognize the RhD antigen on red blood cells. Both antibodies are currently in clinical trials for use as a replacement for polyclonal anti-RhD in the prophylactic treatment of haemolytic disease of the newborn. We have produced three murine IgG1 antibodies that cause agglutination of cells sensitized with BRAD-5 and also block binding of BRAD-5 to its target antigen. Using a haemagglutination assay, these antibodies, 1D7, 2E6 and 3B1, have shown specificity for BRAD-5 as they did not bind to other monoclonal anti-RhD antibodies of differing specificity or derived from other donors. This assay has also been used to show a lack of reactivity with anti-RhD antibodies present in 198 human serum samples from 44 anti-RhD immune individuals. The three anti-BRAD-5 antibodies have been shown to recognize different epitopes on the BRAD-5 molecule using a blocking ELISA. These antibodies appear to recognize private idiotopes on BRAD-5 that were not detectable in RhD immune sera, and therefore they will be of use for monitoring BRAD-5 in clinical trials.     

Decreased immunorejection in unmatched blood transfusions by attachment of methoxypolyethylene glycol on human red blood cells and the effect on D antigen

BACKGROUND: Pegylation of red blood cells (RBCs) has been the primary focus of research on the immunocamouflage of cell. The aim of this study was to demonstrate pegylation homogeneity, its shielding effect on D antigens, and its storage stability. In addition, methoxypolyethylene glycol (mPEG)-modified RBCs (mPEG-RBCs) were tested serologically against a panel of serum samples that was difficult to match to find a solution to the difficulty in matching. STUDY DESIGN AND METHODS: In this study, fluorescein-PEG and a confocal laser scanning microscope were used to monitor PEG attachment on RBC population and observe reaction homogeneity, the stability of mPEG combined with RBCs in vitro was evaluated by the RBC ghost agglutination test, the pegylation sites on membrane were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with two dye methods, and the effect of pegylation on D antigen was detected by immunoblotting techniques. Compatibility tests were carried out between 66 cases of serum with difficulty in blood matching and mPEG-camouflaged RBCs by use of four blood matching methods including direct agglutination, indirect antiglobulin test (IAT), microtyping gel cards (MTS), and the manual polybrene technique (MPT). RESULTS: The results indicated the homogeneity of pegylation, the absence of RhD protein in mPEG-modified D+ RBCs by Western blotting, and attachment of PEG to RBCs after 30 days of storage, while RBCs still remained antigenically silent. All pegylation RBCs showed a negative reaction with ABO-matched patients' serum samples by direct agglutination, IAT, and MTS, which indicated that pegylation RBCs and patients' serum samples were compatible. MPT was not suitable for detecting blood matching of mPEG-RBCs, because modification changed the RBCs' biophysical properties. CONCLUSION: In conclusion, mPEG-RBCs have acceptable in vitro properties and provide a useful solution to problems with clinical blood matching, although such masking leaves much to be desired.     

多克隆抗RhD抗体的纯化和鉴定

摘要：目的：纯化人血清中多克隆抗RhD抗体,并对其进行鉴定。 方法：收集5例RhD阴性者血清,经不规则抗体筛查和确认后,按等比例混合,分别用RhD(-)A型红细胞和RhD(-)B型红细胞对血清中的抗A、抗B抗体于4 ℃过夜吸收处理;用RhD(+)O型红细胞制备血影细胞,以血影细胞作为抗原,吸附血清中抗RhD抗体,再用低pH值缓冲液洗脱、分离纯化抗RhD抗体,用DEAE-Sephadex A-50层析法提取其中IgG组分;分别用western blot、微柱法鉴定。 结果：5名研究对象血清中均含抗RhD抗体;血清中的抗A和抗B血型抗体被完全吸收,纯化后抗RhD抗体IgG组分效价达到1∶32。 结论：本实验获得了高效价的抗RhD多克隆抗体,为进一步利用噬菌体肽库获得RhD血型抗原表位研究等奠定基础。     

流式细胞术检测RhD抗原及其临床应用

目的　探讨流式细胞术 (FCM)检测RhD抗原及其临床应用价值。方法　选取标准的RhD(- )和RhD(+)细胞按 9个不同比例混合 ,分别用直标法 (FITC 抗 D)和间标法 (抗 D和FITC 抗 IgG)染色 ,在FCM上作细胞相对和绝对计数。选择合适检测条件 ,对 2名RhD(- )患者误输RhD(+)红细胞后作RhD(+)细胞计数。结果　与间标法相比 ,直标法检测结果更接近理论值。而单平台和双平台计数法与理论值间无显著性差异 (F =0 .0 32 5 ,P >0 .0 5 )。对 2名RhD(- )的 3份不同时间样本检测发现 ,RhD(+)细胞相对计数分别为 4 .73%、1 0 .87%和 2 0 .86 % ,绝对计数分别为 0 .1 2 4× 1 0 1 2 /L、0 .2 4 5× 1 0 1 2 /L和 0 .5 1 7× 1 0 1 2 /L。结论　FCM能检测RhD(- )细胞中不同浓度的RhD(+)细胞 ,可应用于临床不合血液输注后嵌合态异体细胞的检测     

Resolution by erythrocytapheresis of the exposure of an Rh-negative person to Rh-positive cells: an alternative treatment

Acute trauma with severe bleeding caused the unavoidable transfusion of Rh-positive red cells (RBCs) to a young, childless, Rh-negative woman during surgery. Of the 10 units of RBCs given, 6 were Rh positive. An alternative treatment for large-volume exposure involves the removal of some of the Rh-positive cells transfused at a time when the patient's condition could not be jeopardized by the shortage of Rh-negative RBCs. It is believed that the combination of erythrocytapheresis followed by Rh immune globulin treatment was successful, when immunoprophylaxis alone might not have been. It is strongly recommended that partial RBC exchange for the removal of the unwanted RBCs be considered in addition to Rh immune globulin treatment in cases of large-volume exposure such as the one presented.     

RhD基因检测方法在Rh阴性血型基因鉴定中的应用

目的：探讨RhD基因检测方法在中国汉族人Rh阴血型鉴定中的应用价值。方法：对于68例各种表型的Rh阴性中国汉族样本,用扩增RhD基因外显子3,4,5,7,10的5种方法,检测RhD基因的存在与否。结果：68例样本,总阴性率为52．95,其中29例含有C抗原的Rh阴样性本,5种方法鉴定皆为阴性的仅有1例（3．4％）,而表型为C阴性的38例样本,34例（89．5％）为阴性。结论：以检测RhD基因存在与否为基础的Rh阴性血型基因鉴定方法,不适用于C抗原阳性的中国汉族Rh阴性血型基因鉴定,仅有扩增RhD外子3,4,10的方法,适用于含C抗原的Rh阴性血型的基因鉴定。     

Core temperature changes in resuspended red blood cells (RBCs) and pediatric RBCs removed from refrigerated storage

BACKGROUND: The 30-minute rule, whereby intact red blood cell (RBC) products may be returned to stock if returned to 4°C storage within 30 minutes of issue, was established many years ago. It was based on observations that the core temperature of units of whole blood removed from storage temperatures of 1 to 6°C, and left at room temperature, would reach 10°C at between 45 minutes and 1 hour.
STUDY DESIGN AND METHODS: Forty-one units of RBCs resuspended leukoreduced and 8 units of pediatric RBCs resuspended leukoreduced were exposed to ambient temperature for periods of time between 0 and 60 minutes. Core temperatures of all units were measured at 1-minute or 5-minute intervals.
RESULTS: Resuspended RBCs units reached a mean core temperature of 10°C at 15 minutes, 12.7°C at 30 minutes, and 15°C at 60 minutes. Pediatric RBCs reached a mean core temperature of 12.8°C at 15 minutes, 15.5°C at 30 minutes, and 17.8°C at 60 minutes.
CONCLUSION: In view of our results, and the range of RBC products now available, it may be timely for blood services to review and reduce the 30-minute rule.