输血实验MicrosoftPowerPoint演示文稿

1、临床输血学实验临床输血学实验Transfusion Medicine(blood transfusion laboratories)上海市第六人民医院上海市第六人民医院Shanghai Sixth People Hospital 输血科输血科Transfusion DepartmentTesting Specimen Requirement2ml EDTA whole blood Fill in the tube label(s) with name HistoryAncient times 敬畏血液敬畏血液-生命的源泉生命的源泉-饮血疗饮血疗法法The period 1500-1800 治疗

2、精神病治疗精神病 血液有毒血液有毒 放血疗法；人体血液循环放血疗法；人体血液循环-为输血奠定基础；血液为输血奠定基础；血液的携氧功能的携氧功能-合理治疗手段合理治疗手段The discovery of blood groups 血型之父血型之父-LandsteinerKarl Landsteiner (186-1943), who was awarded the Nobel Prize for Medicine and Physiology in 1930 for his discovery of the ABO antigen system. Landmarks in the history

3、 of blood transfusion1666 Richard Lower (Oxford) conducts experiments involving transfusion of blood from one animal to another1667 Jean Denis (Paris) transfuses blood from animals to humans1818 James Blundell (London) is credited with being the first person to transfuse blood from one human to anot

4、herLandmarks in the history of blood transfusion1901 Karl Landsteiner (Vienna) discovers ABO blood groups. Awarded Nobel Prize for Medicine in 19301908 Alexis Carrel (New York) develops a surgical technique for transfusion, involving anastomosis of vein in the recipient with artery in the donor. Awa

5、rded Nobel Prize for Medicine inLandmarks in the history of blood transfusion1915 Richard Lewinsohn (New York) develops 0.2% sodium citrate as anticoagulant1921 The first blood donor service in the world was established in London by Percy Oliver1937 Blood bank established in a Chicago hospital by Be

6、rnard Fantus1940 Landsteiner and Wiener (New York) identify Rhesus antigens in man1940 Edwin Cohn (Boston) develops a method for fractionation of plasma proteins. The following year, albumin produced by this method was used for the first time to treat victims of the Japanese attack on Pearl HarbourL

7、andmarks in the history of blood transfusionLandmarks in the history of blood transfusion1945 Antiglobulin test devised by Coombs (Cambridge), which also facilitated identification of several other antigenic systems suchas Kell (Coombs et al, 1946), Duffy (Cutbush et al, 1950) and Kidd (Cutbush et a

8、l, 1950)1948 National Blood Transfusion Service (NBTS) established in the UK1951 Edwin Cohn (Boston) and colleagues develop the first blood cell separator1964 Judith Pool (Palo Alto, California) develops cryoprecipitate for the treatment of haemophilia1966 Cyril Clarke (Liverpool) reports the use of

9、 anti-Rh antibody to prevent haemolytic disease of the newbornLandmarks in the history of blood transfusion人类血型的发现，为安全输血提人类血型的发现，为安全输血提供重要保证。供重要保证。而且，在遗传学、人类学、法医而且，在遗传学、人类学、法医学、免疫学、部分疾病的发病机学、免疫学、部分疾病的发病机制探讨上也具有重要意义。制探讨上也具有重要意义。血型系统定义血型系统定义血型通常定义为血液各成分的遗传多态性血型通常定义为血液各成分的遗传多态性（genetic polymorphism)大部分

10、血型属于免疫血液学范畴，但血型的检大部分血型属于免疫血液学范畴，但血型的检出并不都是用免疫学方法。如出并不都是用免疫学方法。如 红细胞酶型、红细胞酶型、血清蛋白型血清蛋白型电泳法电泳法HLA-D抗原抗原混合淋混合淋巴细胞保养法巴细胞保养法血型系统血型系统研究方法研究方法 采用群体调查采用群体调查，如果某一血型频率，如果某一血型频率在另一血型系统各抗原中呈均匀分布，说明这在另一血型系统各抗原中呈均匀分布，说明这两种血型抗原在遗传上各自独立，也可以说这两种血型抗原在遗传上各自独立，也可以说这两种血型抗原的基因位点在不同对的染色体上，两种血型抗原的基因位点在不同对的染色体上，服从自由组合规律；或在同

11、一对染色体的不同服从自由组合规律；或在同一对染色体的不同位点上，但遗传距离甚远位点上，但遗传距离甚远 控制控制ABO血型基因位于第九染色体血型基因位于第九染色体Red blood cell bloodgroup255 antigens, 29 blood group systemsCarbohydrate-defined antigens:ABO, Lewis, Hh, P and Ii (these antigens is determined by sugars, and thus the genes responsible for these antigens code for an i

12、ntermediated molecule, usually an enzyme that creates the antigenic specificity by transferring sugar molecules on the protein or lipid). Antigen determined by amino acid sequences of proteins(directly determined by genes)抗原位点数和剂量效应抗原位点数和剂量效应基因为纯合子时，抗原点位数多，与抗体反应基因为纯合子时，抗原点位数多，与抗体反应强强基因为杂合子时，抗原点位数少，与

13、抗体反应基因为杂合子时，抗原点位数少，与抗体反应弱弱剂量效应在剂量效应在MN血型系统和血型系统和Rh血型系统比较明血型系统比较明显，在显，在ABO血型系统等其他不明显。血型系统等其他不明显。血型抗体血型抗体天然抗体天然抗体 :是指没有可察觉抗原刺激产生的是指没有可察觉抗原刺激产生的“抗体抗体”，（菌类、花粉尘埃等）多主要有存，（菌类、花粉尘埃等）多主要有存在于在于ABO、MN、P和和Lewis血型系统中，以血型系统中，以IgM和和IgG共同存在，共同存在，A型和型和B型多数以型多数以IgM为为主，主，O型人在成年后以型人在成年后以IgG占优势，特别是女占优势，特别是女性性 血型抗体血型抗体规则

14、抗体；指规则抗体；指ABO血型系统有规律产生抗血型系统有规律产生抗A和和抗抗B，符合，符合Landgteiner规则规则 是反定型的依据是反定型的依据 免疫抗体：指有可查抗原刺激而产生，常见有免疫抗体：指有可查抗原刺激而产生，常见有输血、妊娠和注射输血、妊娠和注射3种方式。种方式。Platelets（22）:Red cell antigens：ABOHLA antigensHPAWhile blood cell antigens:HLA antigensGranulocytes-specific antigensBefore transfusionABO and Rh typing of do

15、nor and recipient red cellsAntibody Detection Test(Direct/Indirect Antiglobulin or Coombs Test)Red cell compatibility testingPart oneRed blood cell group目的要求：目的要求： 掌握掌握ABO、Rh血型的鉴定血型的鉴定 熟悉熟悉Rh血型的确认试验血型的确认试验LOGO.Part one ABO SYSTEMPrinciple of procedure红细胞上具有红细胞上具有A抗原者为抗原者为A型型- Red blood cells possess

16、 A blood group antigens有有B抗原者为抗原者为B型型- Red blood cells possess B blood group antigensA和和B抗原都没有者为抗原都没有者为O型型- Red blood cells lack A and B blood group antigensA和和B抗原都有者为抗原都有者为AB型型- Red blood cells possess A and B blood group antigensRh SYSTEMRh血型系是最为复杂的一个血型系。血型系是最为复杂的一个血型系。The D antigen is capable of

17、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. Rh SYSTEMTherefore, the D antigen is commonly considered in the routine selec

18、tion of blood for transfusion已发现的抗原已发现的抗原40多个。多个。涉及临床主要是涉及临床主要是5个抗原：个抗原：C、c、D、E、e及其相应的特异性抗体。及其相应的特异性抗体。Rh SYSTEMFour 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

19、 cause the transfusion reaction.The principle of blood group Antigen-antibody reactionRed cell agglutination occurs in two stages: first the antibody binds to red cell surface; then the antibodies interact to bring the cells in approximation, and agglutination occurs. The principle of blood group An

20、tigen-antibody reactionThe red cell serologic tests are designed to enhance and speed the cells reaction with IgM or IgG antibodies and to detect the reaction by looking for direct cell agglutination or using reagents or conditions to enhance red cell agglutinationMethodsSolid phase adherence testTu

21、be testGel testManualSemi-automated systemAutomated systemMaterials RequiredCentrifuge0.9% salinepipetterreagents:The monoclonal antibody(IgM) include Anti-A 、 Anti-B 、 Anti-D 5% Standard RBCPROCEDURESpecimen collection and preparationRed blood cell suspensions(4%RBC) can be prepared using the follo

22、wing combinations of saline and packed red blood cells:Saline Volume-2mlPacked RBC Volume-100ul被检者被检者RBC用生理盐水配成用生理盐水配成2-5%悬液待用悬液待用(2ml 0.9% NS +100ul 压积红细胞）压积红细胞）TEST PROCEDURE-tube test正定型：正定型： 取小试管三只，分别用记号笔标记后加抗取小试管三只，分别用记号笔标记后加抗A A、抗抗B B、抗、抗D D血清各一滴，然后加受检者血清各一滴，然后加受检者RBCRBC悬液悬液一滴。一滴。blood groupin

23、g Add one drop of Anti-A、Anti-B、Anti-D to three tubes separatelyAdd one drop of a 2-5% RBC suspension to three tubes separatelyTEST PROCEDURE反定型：反定型： 取小试管三只，分别用记号笔标记取小试管三只，分别用记号笔标记AcAc、BcBc、OcOc后，加受检者血清一滴，然后加标准后，加受检者血清一滴，然后加标准A A、B B及及O O型型RBCRBC各一滴。各一滴。reverse groupingreverse groupingAdd one drop o

24、f serum/plasma to three tubes separatelyAdd one drop of 5% standard RBC to three tubes separatelyTEST PROCEDURE将上述混匀后静置数分钟或将上述混匀后静置数分钟或3000r/m3000r/m离心离心15s15s肉眼判肉眼判断凝集度。断凝集度。Shake gently to mix contents of reaction tubes.Centrifuge six tubes with 3000r/s,15m.Shake gently with 45-60 angles.Evaluate

25、six tubes for agglutination and/or hemolysis upon test completion.Record the reaction strength on the paper. 4+一个结实的大凝块，背景清楚。一个结实的大凝块，背景清楚。3+数个结实的大凝块，背景清楚。数个结实的大凝块，背景清楚。2+中等大小的凝块加小凝块，背景清楚。中等大小的凝块加小凝块，背景清楚。1+小凝块，背景较浑浊。小凝块，背景较浑浊。+或或W+细胞有微小凝块，背景红色浑浊，通常细胞有微小凝块，背景红色浑浊，通常用显微镜观察。用显微镜观察。MF混合外观凝集，少数红细胞形成较大的混

26、合外观凝集，少数红细胞形成较大的凝集块，而周围是游离的红细胞。凝集块，而周围是游离的红细胞。0(negative)阴性，红细胞呈游离状态，无凝集象阴性，红细胞呈游离状态，无凝集象H(hemolysis)溶血，有游离血红蛋白。溶血，有游离血红蛋白。Evaluate the agglutination strengthEvaluate the agglutination strengthAnti-AAnti-AAnti-BAnti-BAnti-DAnti-DAcAcBcBcOcOcgrouping+ +- -+ +- -+ +- -A RhD A RhD positive- -+ + + +- -

27、 -B RhD B RhD positive- - -+ + + +- -O RhD O RhD positive+ + + +- - - -AB RhD AB RhD positiveInterpretation of results Grouping anomaliesCold auto-antibodiesAcquired BUnexpected mixed field reactionssubgroupPartial or weak DD negativeRhD negative verification Reagents: three different Anti-D human (

28、IgG) monoclonal antibody MethodIndirect antiglobulin test (IAT)Gene test O有一种表现自己的性格,行动受感情左右,喜欢政治,好胜心强,对立斗争有戒心与喜欢交友并存,能看清形势,很注意别人对自己的好恶.有信念和理想.率直,具有现时与罗曼蒂克双重性.言行带有夸张性，讲话带有教育人和教训人的语气A内心想法不愿为人所知.对别人情绪及环境变化有敏锐反应,但对别人的想法缺乏客观理解.思想行动慎重而有规律,个性内向,没个性谨小慎微固步自封,遵从常理,重视家庭,富于计划性.擅长以说服和说明的调子讲话B开放型对人缺乏戒心,情绪善变,活动力强

29、,不原受约束,行动奔放光明正大反复无常,富于灵活性,欢热闹场合.固执己见,善于批评,先下结论后作说明AB善于待人接物,很注意合情合理,期望在社会上施展能力,注意别人对自己的感情,厌恶表里不一.个性中庸,富有实践精神计划性强不喜欢指手划脚容易疲劳.表情不丰富,正式场合讲话呆板,回避感情激烈的讲话或事件The relationship between ABO group and personality OABAB ABO group and amativenessfemalemalePart twoRed cell compatibility testing-Crossmatch 目的要求：目的要

30、求： 掌握交叉配血试验的原理掌握交叉配血试验的原理 熟悉交叉配血试验的操作熟悉交叉配血试验的操作L L OO GG OOPaart two CROSSMATCH 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

31、blood typing is not available against every 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 Choice of techniques盐水介质盐水介质-saline酶介质酶介质-enzyme + liss抗球蛋白介质抗球蛋白介质-IAT Im

32、mediate spin crossmatch主侧配血主侧配血-Major crossmatch (most important)-comparing donor eryghrocytes to recipient serum antibodies in recipient serum against donor erythrocytes. Immediate spin crossmatch次侧配血次侧配血-Minor crossmatch-This compares donor serum to recipient erythrocytes and checks for preformed

33、antibodies in donor serum that could hemolyse recipient red cells Crossmatch procedure Major crossmatch: 取受血者血清取受血者血清1-2滴滴+献血员献血员2-5%红细胞盐水红细胞盐水悬液悬液1滴滴Add 1d of 5% suspension of donor RBCs and 1d recipient plasma/serum into the tubeCrossmatch procedureMinor crossmatchAdd 1d of 5% suspension of recipi

34、ent RBCs and 1d donor plasma/serum into the tube,取受血者取受血者2-5%红细胞盐水悬液红细胞盐水悬液1滴滴+献血员血献血员血清清1-2滴滴将上述混匀后静置数分钟或将上述混匀后静置数分钟或3000r/m3000r/m离心离心15s15s肉眼判断凝集度。肉眼判断凝集度。Centrifuge six tubes with 3000r/s,15m.Shake softly with 45-60 angles.Evaluate two tubes for agglutination under the microscope.Crossmatch proc

35、edureResultWhen 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 on the minor crossmatch, the transfusion can go ahead. However, if the donated serum is likely to contribute substantia

36、lly to the plasma volume of the recipient, the serum should be removed from the donor whole blood. some questionsRed cell components(donors) the same ABO and RhD group as the patient If ABO identical blood is not available(patient) group O RBC + group AB plasmaIf RhD-negative blood are limited RhD p

37、ositive blood may be selected some questionsPatient with clinically significant red cell antibody the blood cells related antigen is negative Patient with autoimmune haemolytic anaemia analyse the major and minor crossmatch Plasma/Platelet/Granulocyte/Cryoprecipitate Plasma/Platelet/Granulocyte/Cryo

38、precipitate Transfusion- Selection of ABO and Rh Type Not usually done Red Cell Compatibility in Plasma/Platelet/Cryoprecipitate TransfusionRed cell compatibility testing must be done in Granulocyte(20ml red cell)Part threeCoombstest目的要求：目的要求：掌握直接、间接抗人球蛋白试验的原理掌握直接、间接抗人球蛋白试验的原理熟悉直接、间接抗人球蛋白试验的操熟悉直接、间接

39、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了解直接、间接抗人球蛋白试验的临床应用CompanyLOGOPzret threeAntihuman globulinprinciple Antihuman globulin is prepared from the serum of rabbits immunized with human IgG or human complement, usually the C3 complement. These reagents have reactivity only against IgG or C3 and are called mono

40、specific. Depending on the kind of antihuman globulin used, IgG and/or C3 can be detected on the surface of red cells. Coombs test/Antiglobulin Coombs tests are blood tests that identify the causes of anemiaThere are two Coombs tests. A direct Coombs test detects the two different antigens that migh

41、t induce hemolysis in the patients red blood cells. An indirect Coombs test looks for antibodies to someone elses red blood cells in the patients serum (the blood without the cells). Combining the two tests gives clues to the origin of the hemolysis.techniquesSalineHigh-proteinLow-ionic strength sol

42、utionPolythylene glycolPolybrene testTube testsSolid-phase testGel test Affinity columnNormal results:If the Coombs tests are negative, the anemia is unlikely to be autoimmune, and the hematologist will have to search elsewhere for a cause.Abnormal results:If the test is positive, the antigens that

43、react will narrow the search for a cause. Coombs tests are also done for blood transfusion reactions to determine why the transfused blood did not match, and when there is a chance a newborn may have an Rh problemTEST PROCEDURE1、direct antiglobulin test 取一试管放一滴受检者压积红细胞，用生理取一试管放一滴受检者压积红细胞，用生理盐洗涤盐洗涤3 3次，末次洗涤后，配成次，末次洗涤后，配成2-5%RBC2-5%RBC悬液悬液待用。待用。 取三只试管分别放取三只试管分别放1 1滴多价抗人球蛋白、单价滴多价抗人球蛋白、单价抗人球及抗抗人球及抗C3C3血清，再各加待用血清，再各加待用RBCRBC一滴一滴混匀，混匀，30003000r/min /min 离心离心 1515s。Add one drop of Anti