1、臨床輸血學實驗Transfusion Medicine (blood transfusion laboratories) 上海市第六人民醫院 Shanghai Sixth People Hospital 輸血科 Transfusion Department,Testing Specimen Requirement 2ml EDTA whole blood Fill in the tube label(s) with name,History Ancient times 敬畏血液-生命的源泉-飲血療法 The period 1500-1800 治療精神病 血液有毒 放血療法；人體血液循環-為輸血

2、奠定基礎；血液的攜氧功能-合理治療手段 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 transfusion 1666 Richard Lower (Oxford) conducts experimen

3、ts involving transfusion of blood from one animal to another 1667 Jean Denis (Paris) transfuses blood from animals to humans 1818 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 Lands

4、teiner (Vienna) discovers ABO blood groups. Awarded Nobel Prize for Medicine in 1930 1908 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 b

5、lood transfusion,1915 Richard Lewinsohn (New York) develops 0.2% sodium citrate as anticoagulant 1921 The first blood donor service in the world was established in London by Percy Oliver 1937 Blood bank established in a Chicago hospital by Bernard Fantus,1940 Landsteiner and Wiener (New York) identi

6、fy Rhesus antigens in man 1940 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 Harbour,Landmarks in the history of blood transfusion,Landmarks

7、in the history of blood transfusion,1945 Antiglobulin test devised by Coombs (Cambridge), which also facilitated identification of several other antigenic systems such as Kell (Coombs et al, 1946), Duffy (Cutbush et al, 1950) and Kidd (Cutbush et al, 1950) 1948 National Blood Transfusion Service (NB

8、TS) established in the UK,1951 Edwin Cohn (Boston) and colleagues develop the first blood cell separator 1964 Judith Pool (Palo Alto, California) develops cryoprecipitate for the treatment of haemophilia 1966 Cyril Clarke (Liverpool) reports the use of anti-Rh antibody to prevent haemolytic disease

9、of the newborn,Landmarks in the history of blood transfusion,人類血型的發現，為安全輸血提供重要保證。 而且，在遺傳學、人類學、法醫學、免疫學、部分疾病的發病機制探討上也具有重要意義,血型系統定義,血型通常定義為血液各成分的遺傳多態性（genetic polymorphism) 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 紅細胞酶型、血清蛋白型電泳法HLA-D抗原混合淋巴細胞保養法,血型系統,研究方法 采用群體調查，如果某一血型頻率在另一血型系統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

10、兩種血型抗原的基因位點在不同對的染色體上，服從自由組合規律；或在同一對染色體的不同位點上，但遺傳距離甚遠 控制ABO血型基因位于第九染色體,Red blood cell bloodgroup,255 antigens, 29 blood group systems Carbohydrate-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 intermedi

11、ated 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,抗原位點數和劑量效應,基因為純合子時，抗原點位數多，與抗體反應強 基因為雜合子時，抗原點位數少，與抗體反應弱 劑量效應在MN血型系統和Rh血型系統比較明顯，在ABO血型系統

12、等其他不明顯,血型抗體,天然抗體 :是指沒有可察覺抗原刺激產生的“抗體”，（菌類、花粉塵埃等）多主要有存在于ABO、MN、P和Lewis血型系統中，以IgM和IgG共同存在，A型和B型多數以IgM為主，O型人在成年后以IgG占優勢，特別是女性,血型抗體,規則抗體；指ABO血型系統有規律產生抗A和抗B，符合Landgteiner規則 是反定型的依據 免疫抗體：指有可查抗原刺激而產生，常見有輸血、妊娠和注射3種方式,Platelets（22）: Red cell antigens：ABO HLA antigens HPA While blood cell antigens: HLA antigen

13、s Granulocytes-specific antigens,Before transfusion,ABO and Rh typing of donor and recipient red cells Antibody 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血型的鑒定 熟悉Rh血型的確認試驗,Part one,ABO SYSTEMPrinciple of proc

14、edure,紅細胞上具有A抗原者為A型- Red blood cells possess A blood group antigens 有B抗原者為B型- Red blood cells possess B blood group antigens A和B抗原都沒有者為O型- Red blood cells lack A and B blood group antigens A和B抗原都有者為AB型- Red blood cells possess A and B blood group antigens,Rh SYSTEM,Rh血型系是最為復雜的一個血型系。 The D antigen is

15、 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,Rh SYSTEM,Therefore, the D antigen is commonly considered in the r

16、outine selection of blood for transfusion,已發現的抗原40多個。 涉及臨床主要是5個抗原： C、c、D、E、e及其相應的特異性抗體,Rh SYSTEM 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 tra

17、nsfusion reaction,The principle of blood group Antigen-antibody reaction,Red 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 Antigen-antibody

18、 reaction,The 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 agglutination,Methods,Solid phase adherence test Tube test Ge

19、l test Manual Semi-automated system Automated system,Materials Required,Centrifuge 0.9% saline pipetter reagents: The monoclonal antibody(IgM) include Anti-A 、 Anti-B 、 Anti-D 5% Standard RBC,PROCEDURE,Specimen collection and preparation Red blood cell suspensions(4%RBC) can be prepared using the fo

20、llowing combinations of saline and packed red blood cells: Saline Volume-2ml Packed RBC Volume-100ul 被檢者RBC用生理鹽水配成2-5%懸液待用 (2ml 0.9% NS +100ul 壓積紅細胞,TEST PROCEDURE-tube test,正定型： 取小試管三只，分別用記號筆標記后加抗A、抗B、抗D血清各一滴，然后加受檢者RBC懸液一滴。 blood grouping Add one drop of Anti-A、Anti-B、Anti-D to three tubes separate

21、ly Add one drop of a 2-5% RBC suspension to three tubes separately,TEST PROCEDURE,反定型： 取小試管三只，分別用記號筆標記Ac、Bc、Oc后，加受檢者血清一滴，然后加標準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,TEST PROCEDURE 將上述混勻后靜置數分鐘或300

22、0r/m離心15s肉眼判斷凝集度。 Shake gently to mix contents of reaction tubes. Centrifuge six tubes with 3000r/s,15m. Shake gently with 45-60 angles. Evaluate six tubes for agglutination and/or hemolysis upon test completion. Record the reaction strength on the paper,Evaluate the agglutination strength,Interpret

23、ation of results,Grouping anomalies,Cold auto-antibodies Acquired B Unexpected mixed field reactions subgroup Partial or weak D D negative,RhD negative verification,Reagents: three different Anti-D human (IgG) monoclonal antibody Method Indirect antiglobulin test (IAT) Gene test,The relationship bet

24、ween ABO group and personality,ABO group and amativeness,female,male,Part twoRed cell compatibility testing-Crossmatch,目的要求： 掌握交叉配血試驗的原理 熟悉交叉配血試驗的操作,CROSSMATCH,The crossmatch is defined as a procedure to exclude incompatibility between donor and recipient. Crossmatching will pick up incompatibilitie

25、s 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 major ones). In addition, the crossmatch procedure will not pick up low titer antibodies and thus will not prevent delayed-type hemolytic transfusion react

26、ions,Choice of techniques,鹽水介質-saline 酶介質-enzyme + liss 抗球蛋白介質-IAT,Immediate spin crossmatch,主側配血-Major crossmatch (most important)-comparing donor eryghrocytes to recipient serum antibodies in recipient serum against donor erythrocytes,Immediate spin crossmatch,次側配血-Minor crossmatch-This compares d

27、onor serum to recipient erythrocytes and checks for preformed antibodies in donor serum that could hemolyse recipient red cells,Crossmatch procedure,Major crossmatch: 取受血者血清1-2滴+獻血員2-5%紅細胞鹽水懸液1滴 Add 1d of 5% suspension of donor RBCs and 1d recipient plasma/serum into the tube,Crossmatch procedure,Mi

28、nor crossmatch Add 1d of 5% suspension of recipient RBCs and 1d donor plasma/serum into the tube, 取受血者2-5%紅細胞鹽水懸液1滴+獻血員血清1-2滴,將上述混勻后靜置數分鐘或3000r/m離心15s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 p

29、rocedure,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 on the minor crossmatch, the transfusion can go ahead. However, if the donated serum is likely to contribute subst

30、antially 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 group as the patient If ABO identical blood is not available(patient) group O RBC + group AB plasma If RhD-negative blood are limited

31、 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 haemolytic anaemia analyse the major and minor crossmatch,Plasma/Platelet/Granulocyte/Cryoprecipitate,Plasma/Platelet/Granulocy

32、te/Cryoprecipitate Transfusion- Selection of ABO and Rh Type Not usually done Red Cell Compatibility in Plasma/Platelet/Cryoprecipitate Transfusion Red cell compatibility testing must be done in Granulocyte(20ml red cell,Part threeCoombstest,目的要求： 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 了解直接、間接抗人球蛋白試驗的臨床

33、應用,Antihuman globulinprinciple,Antihuman globulin is prepared from the serum of rabbits immunized with human IgG or human complement, usually the C3 complement. These reagents have reactivity only against IgG or C3 and are called monospecific. Depending on the kind of antihuman globulin used, IgG an

34、d/or C3 can be detected on the surface of red cells,Coombs test/Antiglobulin,Coombs tests are blood tests that identify the causes of anemia There are two Coombs tests. A direct Coombs test detects the two different antigens that might induce hemolysis in the patients red blood cells. An indirect Co

35、ombs 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,techniques,Saline High-protein Low-ionic strength solution Polythylene glycol Polybrene test,Tube tests Solid-phase

36、test Gel 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 results:If the test is positive, the antigens that react will narrow the search for a cause. Coombs tests a

37、re 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 test 取一試管放一滴受檢者壓積紅細胞，用生理鹽洗滌3次，末次洗滌后，配成2-5%RBC懸液待用。 取三只試管分別放1滴多價抗人球蛋白、單價抗人球及抗C3血清，再各加待用RBC一滴混勻，3000r/min 離心 15s。 Add one drop of Anti-IgG+C3、Anti-IgG、Anti-C3 to three tubes separately Add one drop of a 2-5% R