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.

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2 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

3 Testing Specimen Requirement 2ml EDTA whole blood Fill in the tube label(s) with name

4 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

5 Karl Landsteiner (186-1943), who was awarded the Nobel Prize for Medicine and Physiology in 1930 for his discovery of the ABO antigen system.

6 Landmarks in the history of blood transfusion 1666 Richard Lower (Oxford) conducts experiments 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

7 Landmarks in the history of blood transfusion 1901 Karl Landsteiner (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

8 Landmarks in the history of blood 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

9 1940 Landsteiner and Wiener (New York) identify 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

10 Landmarks 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 (NBTS) established in the UK

11 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 of the newborn Landmarks in the history of blood transfusion

12 人类血型的发现，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。

13 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

14 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 血型基因位于第九染色体

15 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 intermediated 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)

16 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。

17 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，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

18 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。

19 Platelets （ 22 ） :  Red cell antigens ： ABO  HLA antigens  HPA While blood cell antigens:  HLA antigens  Granulocytes-specific antigens

20 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

21 Part one Red 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

22 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  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

23 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 clinically significant antibody capable of causing RBC destruction and may result in hemolytic disease of the newborn and transfusion reaction.

24 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。

25 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 transfusion reaction.

26 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.

27 The principle of blood group Antigen-antibody reaction  The red cell serologic tests are designed to enhance and speed the cell’s 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

28 Methods  Solid phase adherence test  Tube test  Gel test  Manual  Semi-automated system  Automated system

29 Materials Required  Centrifuge  0.9% saline  pipetter  reagents: The monoclonal antibody(IgM) include Anti-A 、 Anti-B 、 Anti-D 5% Standard RBC

30 PROCEDURE Specimen collection and preparation Red blood cell suspensions(4%RBC) can be prepared using the following 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）

31 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 separately  Add one drop of a 2-5% RBC suspension to three tubes separately

32 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

33 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 angles.  Evaluate six tubes for agglutination and/or hemolysis upon test completion.  Record the reaction strength on the paper.

34 4+ 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。 2+ 中等大小的凝块加小凝块，背景清楚。 1+ 小凝块，背景较浑浊。 + 或 W+ 细胞有微小凝块，背景红色浑浊，通常 用显微镜观察。 MF 混合外观凝集，少数红细胞形成较大的 凝集块，而周围是游离的红细胞。 0(negative) 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。 Evaluate the agglutination strength

35 Anti-AAnti-BAnti-DAcBcOc grouping +-+-+-A RhD positive -+++--B RhD positive --+++-O RhD positive +++---AB RhD positive Interpretation of results

36 Grouping anomalies  Cold auto-antibodies  Acquired B  Unexpected mixed field reactions  subgroup  Partial or weak D  D negative

37 RhD negative verification Reagents: three different Anti-D human (IgG) monoclonal antibody Method  Indirect antiglobulin test (IAT)  Gene test

38 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, 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 ABAB 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

39 OA BAB ABO group and amativeness female male

40 Part two Red 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操作

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

42 Choice of techniques  盐水介质 ---saline  酶介质 ---enzyme + liss  抗球蛋白介质 ---IAT

43 Immediate spin crossmatch  主侧配血 ---Major crossmatch (most important)---comparing donor eryghrocytes to recipient serum antibodies in recipient serum against donor erythrocytes.

44 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

45 Crossmatch procedure Major 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

46 Crossmatch procedure 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

47 将上述混匀后静置数分钟或 3000r/m 离心 15s 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 procedure

48 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 substantially to the plasma volume of the recipient, the serum should be removed from the donor whole blood.

49 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 RhD positive blood may be selected

50 some questions  Patient with clinically significant red cell antibody the blood cell’s related antigen is negative  Patient with autoimmune haemolytic anaemia analyse the major and minor crossmatch

51 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 Transfusion  Red cell compatibility testing must be done in Granulocyte(20ml red cell)

52 Part three Coombs’test 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

53 Antihuman globulin principle 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 and/or C3 can be detected on the surface of red cells.

54 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 patient's red blood cells. An indirect Coombs' test looks for antibodies to someone else's red blood cells in the patient's serum (the blood without the cells). Combining the two tests gives clues to the origin of the hemolysis.

55 techniques  Saline  High-protein  Low-ionic strength solution  Polythylene glycol  Polybrene test  Tube tests  Solid-phase test  Gel test  Affinity column

56 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 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

57 TEST PROCEDURE 1 、 direct antiglobulin test 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， 3000 r /min 离心 15 s 。  Add one drop of Anti-IgG+C3 、 Anti-IgG 、 Anti-C3 to three tubes separately  Add one drop of a 2-5% RBC suspension to three tubes separately

58 将上述混匀后 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 angles.  Evaluate three tubes for agglutination and/or hemolysis upon test completion.  Record the reaction strength on the paper. TEST PROCEDURE

59 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。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 3000 r /min 离心 15 s 。

60 2 、 indirect antiglobulin test  Add 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 37°C water 30m  Wash four tubes three times  Add one drop of anti-IgG to four tubes separately.  Shake gently to mix contents of reaction tubes.

61 2 、 indirect antiglobulin test  Centrifuge 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.

62 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

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