酶-IAT技术在红细胞抗体鉴定中的应用研究

目的探讨酶-IAT技术在红细胞抗体鉴定中的应用意义。方法对2018年1—9月本院检验科输血前红细胞血型抗体筛选、抗体鉴定中出现的弱阳性,无法确定抗体特异性的标本28例,使用二步酶处理谱细胞方法分别再做抗体鉴定,通过分析酶处理谱细胞前后的抗体鉴定结果,确定抗体特异性;从中选取有代表性6例,结合血型结果,探讨酶处理技术对于不同血型系统的作用,并分析酶处理技术的应用范围。结果红细胞血型抗体筛选、鉴定为弱阳性的28例标本中,经酶处理谱细胞后71.43%(20/28)标本确定了抗体特异性20例,其中Rh血型系统抗体占60%(12/28),Kidd抗体占15%(3/20),Lewis抗体占10%(2/20),自身抗体15%(3/20);28.57%(8/28)标本仍未检出抗体特异性。结论酶-IAT法可以增强抗体鉴定弱阳性标本的检出能力,明确抗体特异性,有益于保障临床输血安全;但若存在自身抗体,酶处理技术的应用会受到限制,还需考虑结合其他试验解决问题。     

低频率抗Mur抗体引起溶血性输血反应的调查研究

目的研究抗Mur抗体血型血清学特征，调查其在输血医学中临床意义。方法对2例患者的血清，与已知血型的试剂红细胞和4个已知Mur抗原，在盐水介质、低离子间接抗球蛋白介质，分析鉴定出其抗体的特异性。结果这2例患者与已知血型的试剂红细胞在多种反应介质中的反应结果显示患者血清中含有抗Mur抗体，患者血清与4个已知Mur抗原的反应证实该例抗体只与Mur抗原反应。结论该例同种抗体为特异性抗Mur抗体，在临床会引起溶血性输血反应。在东方人群中Mihenberger血型抗体常规筛选鉴定值得探讨。     

一例p表型的血清学分析

目的分析患者血型血清学特点,明确患者血型及血型抗体特异性,为患者寻找配合型血液。方法采用血型血清学方法进行红细胞ABO、p血型鉴定;采用盐水法、微柱凝胶法、Liss-IAT法进行不规则抗体筛查和抗体特异性鉴定。结果患者血型为B型,p表型,血清中检出抗-PP1PK。结论患者为罕见p表型,血清中含有抗-PP1PK。     

联合应用α-半乳糖苷酶和α-N-乙酰半乳糖胺酶实现红细胞AB→O血型转变

目的研究酶解去除AB型红细胞表面的A和B抗原、实现AB→O血型转变的方法,以获得通用型红细胞。方法联合应用α-半乳糖苷酶和α-N-乙酰半乳糖胺酶酶解AB型红细胞,用单克隆抗-A、抗-A1和抗-B抗体检测酶解效果,用稀有血型抗体检测酶解前后红细胞表面的稀有血型,用流式细胞术检测酶解前后红细胞表面A、B、H抗原,原子力显微镜观察酶解前后红细胞形态,主侧配血试验检测酶解红细胞是否符合临床输血要求。结果A1B和A2B亚型红细胞的A、A1和B抗原均能够完全被酶解去除,酶解前后红细胞的稀有血型抗原不变;流式细胞术显示,酶解后AB型红细胞表面的A和B抗原消失,而H抗原明显增加,和O型红细胞相当;酶解不影响红细胞的形态;主侧配血试验证明酶解改造后的AB型红细胞可以安全输给O、A、B、AB等各型受血者。结论联合应用α-半乳糖苷酶和α-N-乙酰半乳糖胺酶成功实现了AB→O血型转变,获得酶解通用型红细胞。     

多血型系统抗体鉴定和交叉配血策略一例抗-f合并抗-M

目的 鉴定一例患者血浆中抗体的特异性并制定交叉配血的策略。方法 采用盐水介质试管法和抗人球蛋白卡(LISS/Coombs卡)做抗体筛选和抗体鉴定,根据谱细胞、酶处理谱细胞及4℃谱细胞反应格局,确定患者血浆中意外抗体的特异性。结果 患者Rh血型为CCDee,MN血型为NN。多种抗体鉴定结果显示,其血浆中存在同种抗体:Rh血型系统抗-f(f:ce复合抗原)合并MNS血型系统抗-M。结论 抗-f仅与具有f抗原(R0、r)的红细胞反应,且能被蛋白水解酶增强;IgG类抗-M在4℃环境的反应性更强。经过输血免疫后,患者产生抗-f和抗-M抗体,交叉配血策略为优先选择无M抗原红细胞进行交叉配血,对配血相合的红细胞成分血进行RhCE抗原检测,选择无f抗原红细胞发血。     

红细胞谱的建立、保存及临床应用

目的 在南昌地区建立一套能鉴定出血型特异性抗体的红细胞谱。方法从无偿献血的人群中挑选O型健康献血员的红细胞，应用血型系统特异性的相关抗血清，先在瓷板上进行血型抗原分型初筛试验，挑选出符合要求的抗原，然后再用试管法做抗原确认试验。结果筛选出20名献血员为谱细胞成员，组成每套为10份的谱细胞，包含C、C、D、E、e、M、N、S、s、Mur、P、JK^a、JK^b、Fy^a、Fy^b、K、k等20余种抗原，可鉴定Rh、MN、P、Lewis、Kidd等8个与输血相关的血型系统的20余种抗体。将谱细胞应用于临床研究，鉴定出血型特异性抗体26例。结论本实验室研究的红细胞谱抗原符合血型抗原分布频率，能有效鉴定出抗体的特异性，可用于血清中不规则抗体筛选及特异性鉴定，并且通过红细胞的冰冻保存技术，完成谱细胞的保存工作。[编者按]     

献血者ABO血型筛查过程中正反定型不符原因分析

常规的ABO定型包括用已知抗体特异性的试剂血清检查红细胞的抗原(正定型)，以及用已知血型的红细胞检查血清中的抗体(反定型)，只有正反定型相符合时，才能正确定型。而ABO抗原的血型或变异型很多，在常规的实验室批量鉴定血型过程中，由于反应时间短、实验室温度低、实验方法的局限，会出现弱抗体、不规则抗体、冷凝集抗     

6例抗-Mur报告分析

目的输血前检测中对抗-Mur进行血型血清学特异性鉴定,分析其在输血中的临床意义。方法采用盐水法与微柱凝胶法分别筛查和鉴定红细胞血型不规则抗体,采用吸收放散试验鉴定抗体特异性,并检测抗体的Ig类型。结果 6例患者血清中均检出抗-Mur。结论闽南地区抗-Mur出现频率较高,抗-Mur常规抗体筛查检测、Miltenberger系列表型筛查可为输血安全提供保障。     

罕见复合抗体抗-A_1Le~b的鉴定及其特征分析——附1例报告

目的探讨罕见复合抗体抗-A1Leb的鉴定方法并分析其特征。方法首先用试管法鉴定患者的ABO及Lewis血型,并做抗体筛选及鉴定;再将患者血清和多个随机献血者红细胞反应,初步判断抗体的特性;最后将患者血清与已知ABO亚型及Lewis血型的献血者红细胞反应,鉴定抗体的特异性。结果患者血型为A1,Le(a-b-);其血清与所有O型红细胞及Leb抗原阴性的红细胞均不反应,仅与Le(b+)的A1型红细胞反应,鉴定为抗-A1Leb。结论当遇到仅与部分A或B型红细胞反应而不与任何O型红细胞反应的不规则抗体时,应考虑该抗体可能是针对Lewis血型系统复合抗原的抗体。     

Rh血型抗-G抗体的综合鉴定方法的建立及初步应用

目的建立Rh血型抗-G抗体的鉴定方法,并初步应用于含有抗-D和抗-C抗体的Rh阴性人员的抗-G抗体分析。方法首先采用卡式法初筛Rh血型,并通过间接抗人球蛋白试验确认Rh血型,其中抗体筛查检测血清中不规则抗体,谱细胞分析法确定抗体特异性,最后采用吸收放散试验对获得阳性抗体进行验证和鉴别。结果经多次吸收和放散法分析,可有效鉴别抗-G抗体。采用该法鉴定临床一例患者,其血型为AB型Rh阴性(ccdee),血清中不规则抗体可以与AB型RhD阳性红细胞凝集,也能凝集AB型RhD阴性含有C抗原的红细胞,经特异性吸收放散试验后发现该患者血清中不规则抗体为Ig-G抗-D和抗-G。结论多次吸收和细胞放散实验是在缺少稀有、特异性红细胞时,鉴定抗-G抗体的简便实用方法。     

Heterophile antibodies. Part I. The specificity of agglutinating and hemolytic sheep red blood cell antibodies in human sera

Absorption and agglutination inhibition studies carried out on human sera with intact red blood cells, secretory substances and soluble red blood cell membrane extracts show that antibodies against sheep red blood cells possess at least two types of specificities. The agglutinating type has specificities in common with human anti-A and anti-B, while the hemolytic antibody does not. Comparative titration studies clearly demonstrate that there is no correlation in the distribution of these two varieties of sheep red blood cell antibodies. Guinea pig and rat erythrocytes share human A- and B-like determinants with sheep red blood cells. Rabbit, horse and goose red blood cells do not possess these blood group factors.     

Heterophile antibodies in the sera from renal transplant recipients.

Microcytotoxicity tests with the sera from renal transplant recipients against cultured B-cell lines were performed by using guinea pig complement. Out of 106 sera, 18 sera showed strong cytotoxicity against cultured cell lines. The same results were obtained by using normal human serum as a complement source. These sera did not show any cytotoxicity against normal peripheral T- and B-lymphocytes. On the other hand, the sera containing the cytotoxicity against cultured B-cell lines were found to have high-titered heterophile antibodies against bovine red blood cells (BRBC) when hemolysis in agar gel with guinea pig complement was used. The sera without cytotoxicity did not have high-titered heterophile antibodies. The sera with cytotoxicity against cultured B-cell lines reacted in the same way against both human tonsilar lymphocytes cultured with the medium containing fetal calf serum and human serum. Therefore, the antigenicity of cultured cells was not affected by the fetal calf serum in the culture medium. After absorption with BRBC, the sera lost their cytotoxicity against cultured cells. Hemolysins in the sera were almost completely removed after absorption of the sera with cultured cells. From these results, it seemed apparent that the sera from renal transplant recipients contained heterophile antibodies which reacted with the cells of some cultured B-cell lines and tonsilar lymphocytes.     

Analysis of the structure and activity of A and A,B immunoglobulin A monoclonal antibodies

A study of the serologic activity and molecular structures of three spleen-derived mouse IgA monoclonal human blood group-specific supernatants was undertaken; this was part of an evaluation of these monoclonals as blood typing reagents. The monoclonal antibodies were eluted through a precalibrated size-exclusion column, and fractions were analyzed by immunoblotting, heavy and light chain-specific enzyme-linked immunosorbent assay, and liquid- and solid-phase serologic tests. Results indicated that one of the supernatants (anti-A specificity) contained tetrameric and monomeric forms of IgA, while the other two (anti-A,B specificity) contained three higher polymeric forms (1000-4000 kDa) and one dimeric form. The tetrameric and polymeric forms showed red cell agglutinating activity, whereas the dimeric and monomeric forms did not. All forms contained heavy and light chains. The monomeric anti-A showed specific binding to appropriate red cells in a solid-phase assay, but the dimeric anti-A,B fractions did not. Purified fractions stored at 4 degrees C did not show any equilibration toward other forms, which indicated that the molecules are stable once secreted. The use of such antibodies as blood grouping reagents requires careful monitoring to ensure that high proportions of nonagglutinating molecular weight forms are not produced, as they could compromise the performance of the reagent by binding to red cell antigen in competition with the agglutinating forms.     

ABO-incompatible bone marrow transplantation: preparation by plasma exchange and in vivo antibody absorption

The threat of fatal hemolytic transfusion reaction has been a major deterrent to the use of ABO-incompatible bone marrow donors. An eighteen year old type O male with acute leukemia was prepared to receive type A HLA identical marrow using large volume plasma exchange prior to and following cyclophosphamide administration. Saline agglutinating anti-A antibody was initially present at a titer of 1:128. The initial antiglobulin titer of dithiothreitol (DTT) treated serum was 1:64. Initially the T 1/2 of 51Cr labeled type A red blood cells was less than five minutes. The first plasma exchange removed large quantities of anti-A antibody but 51Cr survival of type A red blood cells was unchanged. A second plasma exchange after an interval of three days, increased the T 1/2 of 51Cr labeled cells to 20 minutes. Saline agglutinating anti-A was not present at a titer of 1:8 and the antiglobulin titer of DTT treated serum was 1:4. Reaction to subsequent transfusion of type A red blood cells (5 units) was limited to a single febrile episode during the first unit. Survival of 51Cr labeled red blood cells increased to T 1/2 equal to 21 hours. These data indicate that plasma exchange and infusion of ABO incompatible red blood cells effectively reduce antibody concentration and prolong survival of ABO incompatible erythrocytes. It is suggested that the prolongation of 51Cr survival of ABO-incompatible red blood cells to a point that extravascular destruction is predominant be used to establish the safety of ABO-incompatible marrow infusion.     

Serologic activity of fatty acid dependent antibodies in albumin-free systems

Fatty acid dependent agglutinin (FADA) refers to serum with the special ability to cause agglutination of red blood cells in the presence of certain fatty acids. The agglutinating mechanism is unclear. It has been proposed that the agglutinin reacts with albumin that has been conformationally altered by sodium caprylate and that the immune complex is passively adsorbed onto red blood cells. This report presents data that contradicts the proposal assigning a specific role to albumin in the agglutinating mechanism. FADA were isolated by column chromatography of resolubilized euglobulin preparations. No evidence of contamination with albumin was obtained in those IgM fractions possessing FADA activity. We propose, as an alternative explanation, that the serologic activity of FADA depends upon the interaction of IgM agglutinins with haptenic fatty acids.     

The role of fetal calf serum in the primary immune response in vitro

The mode of action of 2-mercaptoethanol (2-ME) on the primary immune response in vitro was investigated. Fetal calf serum (FCS) was preincubated with 2-ME and lyophilized to remove free 2-ME. This 2-ME- treated FCS was able to substitute the function of adherent cells in the primary immune response against sheep red blood cells (SRBC) in vitro; Fractionation of 2-tme-treated FCS on a Sephadex G-100 column showed that 2-ME acted on a high molecular serum component which after activation, could substitute for macrophages. In order to obtain a humoral immune response against SRBC in vitro, spleen cells require selected FCS. These "good" sera could be distinguished from "deficient" sera by their higher content of this 2-ME-activated factor. The height of the in vitro immune response to SRBC was dependent on the amount of activated factor added to the culture medium. FCS normally required in the culture medium could be completely replaced by the factor- containing fraction without deleterious effect on the culture medium. The factor should be added to the spleen cells during the first 24 h of culture and remain there for 72 h in order to obtain an optimal immune response. The factor could be partially absorbed by spleen cells but not by SRBC. The relationship between macrophage, 2-ME, and FCS in eliciting an in vitro primary immune response is discussed.     

A HEMOLYTIC SYSTEM ASSOCIATED WITH ENTERITIS IN RABBITS : I. NATURE OF THE CELL CHANGE AND THE SERUM FACTORS CONCERNED

A disease characterized by frequent association of enteritis and polyagglutinable cells often develops in weanling rabbits. The red cell lesion renders the cells susceptible to agglutination and hemolysis in normal rabbit sera. The degree of red cell abnormality varies among different animals and disappears when the animals recover. The abnormality of the red cells responsible for their polyagglutinability and susceptibility to hemolysis was resistant to the action of trypsin or papain and persisted in heated stroma preparations derived from polyagglutinable cells. The factors necessary for agglutination and hemolysis of the polyagglutinable cells are present in normal rabbit sera but are lacking in the sera of affected rabbits. These factors returned to normal levels as the polyagglutinable cell lesion disappeared. The sera of rabbits with polyagglutinable cells contained normal levels of complement and properdin. Whereas the agglutinating factor in normal sera is heat-stable at 56°C for 30 minutes, the hemolytic factor is heat labile. The hemolytic factor is apparently distinct from complement and properdin since it was adsorbed from normal rabbit serum by zymosan or by polyagglutinable cells at 0°C. However, complement was fixed when normal rabbit serum was reacted with stroma from polyagglutinable cells. Hemolysis of polyagglutinable cells by normal rabbit serum at 25°C was inhibited by preliminary incubation of the mixture at 0°C prior to incubation at 25°C. Evidence was obtained which indicated that this inhibition was due to progression of a reaction involving Ca⁺⁺ independent of a reaction involving Mg⁺⁺.     

An investigation of the relationship between the V(hrv), VS, and hrH antigens

The data from this investigation show that the anti-VS sera studied cannot be separated into anti-hrv (anti-V) and anti-hrH specificities. The antibody specificity is singular and is directed against an antigenic determinant present on VS positive red blood cells. That some anti-VS sera appear to have a separable specificity may be due to the incomplete absorption of those antisera. It was shown that V−, VS+ red blood cells adsorb more anti-VS than do those that are V+, VS+. Several absorption of anti-VS serum with V+, VS+ red blood cells may remove enough of the antibody that the absorbed serum will no longer (visibly) react with V+, VS+ red blood cells, though the same absorbed serum will react with V−, VS+ red blood cells. That this is not a separable specificity can be demonstrated by subsequent absorption to exhaustion of the same serum with V+, VS+ red blood cells. Testing of the original Hernandez serum comfirmed that it defines the antigenic specificity, hrH. The relationship of hrH to VS may be similar to the relationship of the Rh0 (D) antigen to the antigens of the Rh0 (D) mosaic.     

NATURALLY OCCURRING HUMAN ANTIBODY REACTING WITH BENCE JONES PROTEINS

Agglutinating substances having characteristics of naturally occurring macroglobulin antibodies to human Bence Jones proteins have been identified in human sera. By means of hemagglutination and hemagglutination inhibition techniques, common determinants have been demonstrated on the light (L) polypeptide chains of pooled normal human γ₂-globulin and on some Bence Jones proteins of group 1 but not of group 2. Individual human sera serve to delineate subgroups of the two major antigenic groups of the Bence Jones proteins by agglutinating cells coated by one but not another protein of the same antigenic group. The complexity of subgroups, especially of group 2, is established by testing a panel of Bence Jones proteins of the same group for their ability to inhibit hemagglutination. By this means it appeared that different sera recognized different group-specific determinants of cells coated with a single Bence Jones protein. The capacity of the L polypeptide chains and proteolytic fragments of γ₂-globulin to inhibit the hemagglutination reaction between Bence Jones protein or L chain-coated cells and human sera was examined. These studies demonstrated that the determinants, toward which agglutinators of human serum are directed, appear to be blocked in intact γ₂-globulin and in all fragments in which H chain remains in proximity to L chain. It would appear that the presence of H chains bound to L chains by non-covalent bonds completely obstructs the reactivity of the involved L chain groups. The agglutinating capacity of a serum toward Bence Jones proteins or L chains of γ₂-globulin appeared to be independent of its agglutinating capacity for cells coated with intact γ₂-globulin. No correlation of the presence in serum of agglutinators for Bence Jones proteins or L chains with health or disease has been established.     

Normal "irregular" hemagglutinins in the sera of healthy adults found by using increased volumens of serum in agglutination and conglutination tests (author's transl)]

Using increased volumens of serum [in the Multiple-Dosage (MD) or a High Dosage (HD) test] with a sensitive manual reading technique all sera of 37 donors of blood group A2 showed irregular anti-A1(alpha1) antibodies. With the same technique, up to 1/3 of the sera of 417 healthy blood donors agglutinated red cells of a selected antigen pattern at room temperature; at 4 degrees C the ratio of positive reacting sera was much higher. The specificity of the antibodies, as indicated by the antigramme type, could be confirmed in the most cases by reacting with a series of known positive and negative reference red cells. The eluates of some of the sera yielded the same antigramme pattern as the native sera did. Some of the antibodies showed a specificity corresponding to a red cell property of the respective blood donor; but not in each case the antibodies were capable to agglutinate the donor's cells. If "autoagglutinins" were strong, frequently they revealed a character of panagglutinability, particular in the cold. With the antiglobulin test, which followed the NaCl-MD procedure, the number of positive reactions increased; on the other hand, there were some of the previously positive agglutination tests, which became negative during the course of the AHG step.