輸血實驗 Microsoft PowerPoint 演示文稿.ppt

1、臨床輸血學實驗臨床輸血學實驗Transfusion Medicine(blood transfusion laboratories)上海市第六人民醫(yī)院上海市第六人民醫(yī)院Shanghai Sixth People Hospital 輸血科輸血科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-飲血療飲血療法法The period 1500-1800 治療

2、精神病治療精神病 血液有毒血液有毒 放血療法；人體血液循環(huán)放血療法；人體血液循環(huán)-為輸血奠定基礎；血液為輸血奠定基礎；血液的攜氧功能的攜氧功能-合理治療手段合理治療手段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人類血型的發(fā)現(xiàn)，為安全輸血提人類血型的發(fā)現(xiàn)，為安全輸血提供重要保證。供重要保證。而且，在遺傳學、人類學、法醫(yī)而且，在遺傳學、人類學、法醫(yī)學、免疫學、部分疾病的發(fā)病機學、免疫學、部分疾病的發(fā)病機制探討上也具有重要意義。制探討上也具有重要意義。血型系統(tǒng)定義血型系統(tǒng)定義血型通常定義為血液各成分的遺傳多態(tài)性血型通常定義為血液各成分的遺傳多態(tài)性（genetic polymorphism)大部分

10、血型屬于免疫血液學范疇，但血型的檢大部分血型屬于免疫血液學范疇，但血型的檢出并不都是用免疫學方法。如出并不都是用免疫學方法。如 紅細胞酶型、紅細胞酶型、血清蛋白型血清蛋白型電泳法電泳法HLA-D抗原抗原混合淋混合淋巴細胞保養(yǎng)法巴細胞保養(yǎng)法血型系統(tǒng)血型系統(tǒng)研究方法研究方法 采用群體調(diào)查采用群體調(diào)查，如果某一血型頻率，如果某一血型頻率在另一血型系統(tǒng)各抗原中呈均勻分布，說明這在另一血型系統(tǒng)各抗原中呈均勻分布，說明這兩種血型抗原在遺傳上各自獨立，也可以說這兩種血型抗原在遺傳上各自獨立，也可以說這兩種血型抗原的基因位點在不同對的染色體上，兩種血型抗原的基因位點在不同對的染色體上，服從自由組合規(guī)律；或在同

11、一對染色體的不同服從自由組合規(guī)律；或在同一對染色體的不同位點上，但遺傳距離甚遠位點上，但遺傳距離甚遠 控制控制ABO血型基因位于第九染色體血型基因位于第九染色體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)抗原位點數(shù)和劑量效應抗原位點數(shù)和劑量效應基因為純合子時，抗原點位數(shù)多，與抗體反應基因為純合子時，抗原點位數(shù)多，與抗體反應強強基因為雜合子時，抗原點位數(shù)少，與

13、抗體反應基因為雜合子時，抗原點位數(shù)少，與抗體反應弱弱劑量效應在劑量效應在MN血型系統(tǒng)和血型系統(tǒng)和Rh血型系統(tǒng)比較明血型系統(tǒng)比較明顯，在顯，在ABO血型系統(tǒng)等其他不明顯。血型系統(tǒng)等其他不明顯。血型抗體血型抗體天然抗體天然抗體 :是指沒有可察覺抗原刺激產(chǎn)生的是指沒有可察覺抗原刺激產(chǎn)生的“抗體抗體”，（菌類、花粉塵埃等）多主要有存，（菌類、花粉塵埃等）多主要有存在于在于ABO、MN、P和和Lewis血型系統(tǒng)中，以血型系統(tǒng)中，以IgM和和IgG共同存在，共同存在，A型和型和B型多數(shù)以型多數(shù)以IgM為為主，主，O型人在成年后以型人在成年后以IgG占優(yōu)勢，特別是女占優(yōu)勢，特別是女性性 血型抗體血型抗體規(guī)則

14、抗體；指規(guī)則抗體；指ABO血型系統(tǒng)有規(guī)律產(chǎn)生抗血型系統(tǒng)有規(guī)律產(chǎn)生抗A和和抗抗B，符合，符合Landgteiner規(guī)則規(guī)則 是反定型的依據(jù)是反定型的依據(jù) 免疫抗體：指有可查抗原刺激而產(chǎn)生，常見有免疫抗體：指有可查抗原刺激而產(chǎn)生，常見有輸血、妊娠和注射輸血、妊娠和注射3種方式。種方式。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血型的鑒定血型的鑒定 熟悉熟悉Rh血型的確認試驗血型的確認試驗LOGO.Part one ABO SYSTEMPrinciple of procedure紅細胞上具有紅細胞上具有A抗原者為抗原者為A型型- Red blood cells possess

16、 A blood group antigens有有B抗原者為抗原者為B型型- Red blood cells possess B blood group antigensA和和B抗原都沒有者為抗原都沒有者為O型型- Red blood cells lack A and B blood group antigensA和和B抗原都有者為抗原都有者為AB型型- Red blood cells possess A and B blood group antigensRh SYSTEMRh血型系是最為復雜的一個血型系。血型系是最為復雜的一個血型系。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已發(fā)現(xiàn)的抗原已發(fā)現(xiàn)的抗原40多個。多個。涉及臨床主要是涉及臨床主要是5個抗原：個抗原：C、c、D、E、e及其相應的特異性抗體。及其相應的特異性抗體。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被檢者被檢者RBC用生理鹽水配成用生理鹽水配成2-5%懸液待用懸液待用(2ml 0.9% NS +100ul 壓積紅細胞）壓積紅細胞）TEST PROCEDURE-tube test正定型：正定型： 取小試管三只，分別用記號筆標記后加抗取小試管三只，分別用記號筆標記后加抗A A、抗抗B B、抗、抗D D血清各一滴，然后加受檢者血清各一滴，然后加受檢者RBCRBC懸液懸液一滴。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： 取小試管三只，分別用記號筆標記取小試管三只，分別用記號筆標記AcAc、BcBc、OcOc后，加受檢者血清一滴，然后加標準后，加受檢者血清一滴，然后加標準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將上述混勻后靜置數(shù)分鐘或?qū)⑸鲜龌靹蚝箪o置數(shù)分鐘或3000r/m3000r/m離心離心15s15s肉眼判肉眼判斷凝集度。斷凝集度。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+一個結(jié)實的大凝塊，背景清楚。一個結(jié)實的大凝塊，背景清楚。3+數(shù)個結(jié)實的大凝塊，背景清楚。數(shù)個結(jié)實的大凝塊，背景清楚。2+中等大小的凝塊加小凝塊，背景清楚。中等大小的凝塊加小凝塊，背景清楚。1+小凝塊，背景較渾濁。小凝塊，背景較渾濁。+或或W+細胞有微小凝塊，背景紅色渾濁，通常細胞有微小凝塊，背景紅色渾濁，通常用顯微鏡觀察。用顯微鏡觀察。MF混合外觀凝集，少數(shù)紅細胞形成較大的混

26、合外觀凝集，少數(shù)紅細胞形成較大的凝集塊，而周圍是游離的紅細胞。凝集塊，而周圍是游離的紅細胞。0(negative)陰性，紅細胞呈游離狀態(tài)，無凝集象陰性，紅細胞呈游離狀態(tài)，無凝集象H(hemolysis)溶血，有游離血紅蛋白。溶血，有游離血紅蛋白。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有一種表現(xiàn)自己的性格,行動受感情左右,喜歡政治,好勝心強,對立斗爭有戒心與喜歡交友并存,能看清形勢,很注意別人對自己的好惡.有信念和理想.率直,具有現(xiàn)時與羅曼蒂克雙重性.言行帶有夸張性，講話帶有教育人和教訓人的語氣A內(nèi)心想法不愿為人所知.對別人情緒及環(huán)境變化有敏銳反應,但對別人的想法缺乏客觀理解.思想行動慎重而有規(guī)律,個性內(nèi)向,沒個性謹小慎微固步自封,遵從常理,重視家庭,富于計劃性.擅長以說服和說明的調(diào)子講話B開放型對人缺乏戒心,情緒善變,活動力強

29、,不原受約束,行動奔放光明正大反復無常,富于靈活性,歡熱鬧場合.固執(zhí)己見,善于批評,先下結(jié)論后作說明AB善于待人接物,很注意合情合理,期望在社會上施展能力,注意別人對自己的感情,厭惡表里不一.個性中庸,富有實踐精神計劃性強不喜歡指手劃腳容易疲勞.表情不豐富,正式場合講話呆板,回避感情激烈的講話或事件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： 掌握交叉配血試驗的原理掌握交叉配血試驗的原理 熟悉交叉配血試驗的操作熟悉交叉配血試驗的操作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鹽水介質(zhì)鹽水介質(zhì)-saline酶介質(zhì)酶介質(zhì)-enzyme + liss抗球蛋白介質(zhì)抗球蛋白介質(zhì)-IAT Im

32、mediate spin crossmatch主側(cè)配血主側(cè)配血-Major crossmatch (most important)-comparing donor eryghrocytes to recipient serum antibodies in recipient serum against donor erythrocytes. Immediate spin crossmatch次側(cè)配血次側(cè)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+獻血員獻血員2-5%紅細胞鹽水紅細胞鹽水懸液懸液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%紅細胞鹽水懸液紅細胞鹽水懸液1滴滴+獻血員血獻血員血清清1-2滴滴將上述混勻后靜置數(shù)分鐘或?qū)⑸鲜龌靹蚝箪o置數(shù)分鐘或3000r/m3000r/m離心離心15s15s肉眼判斷凝集度。肉眼判斷凝集度。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、間接抗人球蛋白試驗的原理掌握直接、間接抗人球蛋白試驗的原理熟悉直接、間接抗人球蛋白試驗的操熟悉直接、間接

39、抗人球蛋白試驗的操了解直接、間接抗人球蛋白試驗的臨床應用了解直接、間接抗人球蛋白試驗的臨床應用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 取一試管放一滴受檢者壓積紅細胞，用生理取一試管放一滴受檢者壓積紅細胞，用生理鹽洗滌鹽洗滌3 3次，末次洗滌后，配成次，末次洗滌后，配成2-5%RBC2-5%RBC懸液懸液待用。待用。 取三只試管分別放取三只試管分別放1 1滴多價抗人球蛋白、單價滴多價抗人球蛋白、單價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一滴一滴混勻，混勻，30003000r/min /min 離心離心 1515s。Add one drop of Anti