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1、临床输血学实验 Transfusion Medicine (blood transfusion laboratories) 上海市第六人民医院 Shanghai Sixth People Hospital 输血科 Transfusion Department,History Ancient times 敬畏血液-生命的源泉-饮血疗法 The period 1500-1800 治疗精神病 血液有毒 放血疗法-人体血液循环，为输血奠定基础-血液的携氧功能，合理治疗手段 The discovery of blood groups 血型之父-Landsteiner,人类血型的发现，为安全输血提供重要保
2、证。 而且，在遗传学、人类学、法医学、免疫学、部分疾病的发病机制探讨上也具有重要意义。,While blood cell antigens: Red cell antigens HLA antigens Granulocytes-specific antigens Platelets（22）,Part one Red blood cell group,目的要求： 掌握ABO血型的鉴定 熟悉Rh血型的鉴定,ABO SYSTEM Principle of procedure,红细胞上具有A抗原者为A型- Red blood cells possess A blood group antigens
3、有B抗原者为B型- Red blood cells possess B blood group antigens A和B抗原都没有者为O型- Red blood cells lack A and B blood group antigens A和B抗原都有者为AB型- Red blood cells possess A and B blood group antigens,Rh SYSTEM,The most complex in the red cells antigen Individuals who possess the D antigen are Rh positive, who l
4、ack it are Rh negative. The D antigen is capable of stimulating production of Anti-D in persons lacking the D antigen. Anti-D is a clinically significant antibody capable of causing RBC destruction and may result in hemolytic disease of the newborn and transfusion reaction.,已发现的抗原40多个。 涉及临床主要是5个抗原：
5、C、c、D、E、e及其相应的特异性抗体。 Four other antigens(C、c、E、e) account for almost all of the Rh-related transfusion problems. They are less antigenic than D, and/or the antibodies are less clinically dangerous. But they can cause the transfusion reaction.,Materials Required,Centrifuge 0.9% saline pipetter reagen
6、ts: The monoclonal antibody(IgM) include Anti-A 、 Anti-B 、 Anti-D 5% Standard RBC-Affirmagen,Methods,Solid phase adherence test Tube test Gel test Manual Semi-automated system Automated system,PROCEDURE,Specimen collection and preparation Red blood cell suspensions(4%RBC) can be prepared using the f
7、ollowing combinations of saline and packed red blood cells: Saline Volume-2ml Packed RBC Volume-100ul 被检者RBC用生理盐水配成2-5%悬液待用 (2ml 0.9% NS +100ul 压积红细胞）,TEST PROCEDURE-tube test,正定型： 取小试管三只，分别用记号笔标记后加抗A、抗B、抗D血清各一滴，然后加受检者RBC悬液一滴。 blood grouping Add one drop of Anti-A、Anti-B、Anti-D to three tubes separa
8、tely Add one drop of a 2-5% RBC suspension to three tubes separately,TEST PROCEDURE,反定型： 取小试管三只，分别用记号笔标记后加受检者血清一滴，然后加标准A、B及O型RBC各一滴。 reverse grouping Add one drop of serum/plasma to three tubes separately Add one drop of 5% standard RBC to three tubes separately,将上述混匀后静置数分钟或3000r/m离心15s肉眼判断凝集度。 Shak
9、e gently to mix contents of reaction tubes. Centrifuge six tubes with 3000r/s,15m. Shake gently with 45-60 angles. Evaluate six tubes for agglutination and/or hemolysis upon test completion. Record the reaction strength on the paper.,Evaluate the agglutination strength,Interpretation of results,Grou
10、ping anomalies,Cold auto-antibodies Acquired B Unexpected mixed field reactions Partial or weak D D negative Intrauterine transfusions,RhD negative verification,Reagents: three different Anti-D human (IgG) monoclonal antibody Method Indirect antiglobulin test (IAT) Gene test,Indirect antiglobulin te
11、st,Incubating the donors red cells with the Rh typing sera Washing the cells three times Adding AHG Centrifuging the mixture Observing it for agglutination,The relationship between ABO group and personality,ABO group and amativeness,female,male,Part two Red cell compatibility testing-Crossmatch,目的要求
12、： 掌握交叉配血试验的原理 熟悉交叉配血试验的操作,CROSSMATCHING,The crossmatch is defined as a procedure to exclude incompatibility between donor and recipient. Crossmatching will pick up incompatibilities between the donor and recipient that will not be evident on blood typing (as blood typing is not available against eve
13、ry blood group, just the major ones). In addition, the crossmatch procedure will not pick up low titer antibodies and thus will not prevent delayed-type hemolytic transfusion reactions,Immediate spin crossmatch,主侧配血-Major crossmatch (most important)-comparing donor eryghrocytes to recipient serum an
14、tibodies in recipient serum against donor erythrocytes. 次侧配血-Minor crossmatch This compares donor serum to recipient erythrocytes and checks for preformed antibodies in donor serum that could hemolyse recipient red cells,Choice of techniques,盐水介质-saline 酶介质-enzyme + liss 抗球蛋白介质-IAT,Crossmatch proced
15、ure,Major crossmatch: Add 1d of 5% suspension of donor RBCs and 1d recipient plasma/serum into the tube 取受血者血清1-2滴+献血员2-5%红细胞盐水悬液1滴 Minor crossmatch Add 1d of 5% suspension of recipient RBCs and 1d donor plasma/serum into the tube, 取受血者2-5%红细胞盐水悬液1滴+献血员血清1-2滴,将上述混匀后静置数分钟或3000r/m离心15s肉眼判断凝集度。 Centrif
16、uge six tubes with 3000r/s,15m. Shake softly with 45-60 angles. Evaluate two tubes for agglutination under the microscope.,Result,When there is an incompatible reaction on the major crossmatch, the donor blood should not be transfusion under any circumstances. When there is an incompatible reaction
17、on the minor crossmatch, the transfusion can go ahead. However, if the donated serum is likely to contribute substantially to the plasma volume of the recipient, the serum should be removed from the donor whole blood.,Selection of blood,Red cell components-the same ABO and RhD group as the patient I
18、f ABO identical blood is not available-group O RBC + group AB plasma If RhD-negative blood are limited-RhD positive blood may be selected Patient with clinically significant red cell antibody Patient with autoimmune haemolytic anaemia Massive blood transfusion,Part three Coombstest,目的要求： 掌握直接、间接抗人球蛋
19、白试验的原理 熟悉直接、间接抗人球蛋白试验的操 了解直接、间接抗人球蛋白试验的临床应用,一、抗人球蛋白试验的原理,倘若用提纯的或在人的全血清内的人的球蛋白给动物（如兔和山羊）注射，动物对外来的蛋白质将会产生抗体，即抗人球蛋白。,抗人球蛋白血清，在适当处理之后将会与人球蛋白发生特异性反应。如果球蛋白分子，或者抗体或补体附着于红细胞膜上，抗球蛋白血清将会与球蛋白结合。因为球蛋白附着于红细胞上，抗球蛋白血清与球蛋白分子的结合就引起红细胞凝集反之，将不会被凝集。,直接抗球蛋白试验 证实体内被致敏的红细胞。 洗涤后的患者红细胞可直接进行抗人球蛋白血清检查。 间接抗球蛋白试验 证实体外被红细胞的致敏。,血
20、清与红细胞孵育，然后用生理盐水洗涤红细胞以除去未结合到红细胞上多余的抗体。若加入抗人球蛋白血清导致的红细胞凝集，可证明是红细胞表面具有与血清中的抗体相同特异性的表面抗原。,意义： 检查受检红细胞是否在体内已被抗体或补体致敏。 用于： 新生儿溶血病 溶血性输血反应 自身免疫溶血性贫血 药物诱导溶血性贫血,二、操作,1、直接抗球蛋白试验操作 取一试管放一滴受检者血液，用生理盐洗涤3-4次，末次洗涤后，配成2-5%RBC悬液待用。 取一试管放1-2滴多价抗人球蛋白血清（IgG+C3），再加待用RBC一滴混匀，3000r/min 离心 15s。,持一定的角度轻轻地摇动试管直到松动试管底细胞。倘若观察到均匀的细胞悬液为阴性；有凝集物为阳性。,（4） 抗IgG阳性对照同（2）、抗C3阳性对照细胞（一份ACD抗凝血+九份10%蔗糖，37。C 15m，洗三次，37。C水浴30m，配成2-5%红细胞悬液）阴性对照用正常人“O”型红细胞。,2、 间接抗球蛋白试验,意义：检查受检者血清中有否有红细胞的不规则抗体，适用于抗体筛查和鉴定及交叉配合试验等。,（2） 上述试管中血清于红细胞混匀，离心（3000r/min 15s）倘若阳性对照管呈现凝集，阴性对照管呈现不凝集，受检管出现凝集为阳性，不凝集则为阴性。,谢谢,