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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,Testing Specimen Requirement2ml EDTA whole blood Fill in the tube label(s)with name,HistoryAncient 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
2、携氧功能-合理治疗手段The discovery of blood groups 血型之父-Landsteiner,Karl 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 of blood transfusion1666 Richard Lower(Oxford)conducts experiments involving
3、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 another,Landmarks in the history of blood transfusion,1901 Karl Landsteiner(Vienna)disco
4、vers 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.Awarded Nobel Prize for Medicine in,Landmarks in the history of blood transfusion,1915 Richa
5、rd 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 Bernard Fantus,1940 Landsteiner and Wiener(New York)identify Rhesus antigens in man1940 Edwi
6、n 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 Harbour,Landmarks in the history of blood transfusion,Landmarks in the history of blood transfusion,194
7、5 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 al,1950)1948 National Blood Transfusion Service(NBTS)established in the UK,1951 Edwin Cohn(Boston)and
8、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 anti-Rh antibody to prevent haemolytic disease of the newborn,Landmarks in the history of blood transfusion,人
9、类血型的发现，为安全输血提供重要保证。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。,血型系统定义,血型通常定义为血液各成分的遗传多态性（genetic polymorphism)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 红细胞酶型、血清蛋白型电泳法HLA-D抗原混合淋巴细胞保养法,血型系统,研究方法 采用群体调查，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，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 控制ABO血型基因位
10、于第九染色体,Red blood cell bloodgroup,255 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 intermediated molecule,usually an enzyme that creates the antigenic specificity
11、 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,基因为纯合子时，抗原点位数多，与抗体反应强基因为杂合子时，抗原点位数少，与抗体反应弱剂量效应在MN血型系统和Rh血型系统比较明显，在ABO血型系统等其他不明显。,血型抗体,天然抗体:是指没有可察觉抗原刺激产生的“抗体”，（菌类、花粉尘埃等）多主要有存在于ABO、MN、P和Lewis血型系统中
12、，以IgM和IgG共同存在，A型和B型多数以IgM为主，O型人在成年后以IgG占优势，特别是女性,血型抗体,规则抗体；指ABO血型系统有规律产生抗A和抗B，符合Landgteiner规则 是反定型的依据 免疫抗体：指有可查抗原刺激而产生，常见有输血、妊娠和注射3种方式。,Platelets（22）:Red cell antigens：ABOHLA antigensHPAWhile blood cell antigens:HLA antigensGranulocytes-specific antigens,Before transfusion,ABO and Rh typing of donor
13、 and recipient red cellsAntibody Detection Test(Direct/Indirect Antiglobulin or Coombs Test)Red cell compatibility testing,Part oneRed blood cell group,目的要求：掌握ABO、Rh血型的鉴定 熟悉Rh血型的确认试验,Part one,ABO SYSTEMPrinciple of procedure,红细胞上具有A抗原者为A型-Red blood cells possess A blood group antigens有B抗原者为B型-Red bl
14、ood cells possess B blood group antigensA和B抗原都没有者为O型-Red blood cells lack A and B blood group antigensA和B抗原都有者为AB型-Red blood cells possess A and B blood group antigens,Rh SYSTEM,Rh血型系是最为复杂的一个血型系。The D antigen is capable of stimulating production of Anti-D in persons lacking the D antigen.Anti-D is a
15、 clinically significant antibody capable of causing RBC destruction and may result in hemolytic disease of the newborn and transfusion reaction.,Rh SYSTEM,Therefore,the D antigen is commonly considered in the routine selection of blood for transfusion,已发现的抗原40多个。涉及临床主要是5个抗原：C、c、D、E、e及其相应的特异性抗体。,Rh S
16、YSTEMFour 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.,The principle of blood group Antigen-antibody reaction,Red cell agglutination
17、 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 Antigen-antibody reaction,The red cell serologic tests are designed to enhance and speed the cells reaction with
18、IgM or IgG antibodies and to detect the reaction by looking for direct cell agglutination or using reagents or conditions to enhance red cell agglutination,Methods,Solid phase adherence testTube testGel testManualSemi-automated systemAutomated system,Materials Required,Centrifuge0.9%salinepipetterre
19、agents:The monoclonal antibody(IgM)include Anti-A、Anti-B、Anti-D 5%Standard RBC,PROCEDURE,Specimen collection and preparationRed blood cell suspensions(4%RBC)can be prepared using the following combinations of saline and packed red blood cells:Saline Volume-2mlPacked RBC Volume-100ul被检者RBC用生理盐水配成2-5%
20、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 separatelyAdd 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Ac、Bc、Oc后，加受检者血清一滴，然后加标
21、准A、B及O型RBC各一滴。reverse groupingAdd one drop of serum/plasma to three tubes separatelyAdd one drop of 5%standard RBC to three tubes separately,TEST PROCEDURE将上述混匀后静置数分钟或3000r/m离心15s肉眼判断凝集度。Shake gently to mix contents of reaction tubes.Centrifuge six tubes with 3000r/s,15m.Shake gently with 45-60 angl
22、es.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,Grouping anomalies,Cold auto-antibodiesAcquired BUnexpected mixed field reactionssubgroupPartial or weak DD negative,
23、RhD negative verification,Reagents:three different Anti-D human(IgG)monoclonal antibody MethodIndirect antiglobulin test(IAT)Gene test,The relationship between ABO group and personality,ABO group and amativeness,female,male,Part twoRed cell compatibility testing-Crossmatch,目的要求：掌握交叉配血试验的原理 熟悉交叉配血试验的
24、操作,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 blood typing is not available against every blood group,just the majo
25、r 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-saline酶介质-enzyme+liss抗球蛋白介质-IAT,Immediate spin crossmatch,主侧配血-Major crossmatch(most important)-comparing donor eryghroc
26、ytes to recipient serum antibodies in recipient serum against donor erythrocytes.,Immediate spin crossmatch,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,Crossmatch procedure,Major
27、crossmatch:取受血者血清1-2滴+献血员2-5%红细胞盐水悬液1滴Add 1d of 5%suspension of donor RBCs and 1d recipient plasma/serum into the tube,Crossmatch procedure,Minor crossmatchAdd 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。Cen
28、trifuge six tubes with 3000r/s,15m.Shake softly with 45-60 angles.Evaluate two tubes for agglutination under the microscope.,Crossmatch procedure,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 i
29、ncompatible reaction 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.,some questions,Red cell components(donors)the same ABO and RhD gr
30、oup as the patient If ABO identical blood is not available(patient)group O RBC+group AB plasmaIf RhD-negative blood are limited RhD positive blood may be selected,some questions,Patient with clinically significant red cell antibody the blood cells related antigen is negative Patient with autoimmune
31、haemolytic anaemia analyse the major and minor crossmatch,Plasma/Platelet/Granulocyte/Cryoprecipitate,Plasma/Platelet/Granulocyte/Cryoprecipitate Transfusion-Selection of ABO and Rh Type Not usually done Red Cell Compatibility in Plasma/Platelet/Cryoprecipitate TransfusionRed cell compatibility test
32、ing must be done in Granulocyte(20ml red cell),Part threeCoombstest,目的要求：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了解直接、间接抗人球蛋白试验的临床应用,Antihuman globulinprinciple,Antihuman globulin is prepared from the serum of rabbits immunized with human IgG or human complement,usually the C3 complement.These reagents hav
33、e reactivity only against IgG or C3 and are called monospecific.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 Coom
34、bs test detects the two different antigens that might 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.,
35、techniques,SalineHigh-proteinLow-ionic strength solutionPolythylene glycolPolybrene test,Tube testsSolid-phase testGel test Affinity column,Normal 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
36、results:If the test is positive,the antigens that 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 problem,TEST PROCEDURE,1、direct antiglobulin t
37、est 取一试管放一滴受检者压积红细胞，用生理盐洗涤3次，末次洗涤后，配成2-5%RBC悬液待用。取三只试管分别放1滴多价抗人球蛋白、单价抗人球及抗C3血清，再各加待用RBC一滴混匀，3000r/min 离心 15s。Add one drop of Anti-IgG+C3、Anti-IgG、Anti-C3 to three tubes separatelyAdd one drop of a 2-5%RBC suspension to three tubes separately,将上述混匀后3000r/m离心15s肉眼判断凝集度.Shake gently to mix contents of
38、reaction tubes.Centrifuge six tubes with 3000r/s,15m.Shake gently with 45-60 angles.Evaluate three tubes for agglutination and/or hemolysis upon test completion.Record the reaction strength on the paper.,TEST PROCEDURE,2、indirect antiglobulin test 取四只试管分别加入受检者血清一滴，记号为1、2、3、4号。再按顺序分别加入筛选红细胞I、II、III号。
39、最后一管加入受检者5%RBC悬液。37度水浴箱放置30m，将四管红细胞洗涤3次，最后每管加多价抗人球蛋白血清一滴，混匀3000r/min 离心 15s。,2、indirect antiglobulin testAdd one drop of serum to four tubes separately.Add one drop of a spreen RBC suspension to three tubes separately,and one drop of receive 5%RBC suspension to the fourth tube.Warm in the 37C water
40、30mWash four tubes three timesAdd one drop of anti-IgG to four tubes separately.Shake gently to mix contents of reaction tubes.,2、indirect antiglobulin testCentrifuge six tubes with 3000r/s,15m.Shake gently with 45-60 angles.Evaluate four tubes for agglutination and/or hemolysis upon test completion.Record the reaction strength on the paper.,A 1944 poster urging civilians to donate blood for military casualties.The Army Blood Supply Depot was based in Bristol,and blood from the south-west region was processed for military use,
