Antiglobulin Reagents Research Articles

Historical Note: Past, Present and Future of the Antiglobulin Test

This is a review of two invited talks on the antiglobulin reaction on the occasion of the 50th Anniversaries of the International Blood Group Reference Laboratory, now at Bristol, and the National Blood Service, London, and coincidentally the 51st anniversary of the antiglobulin test! The first talk (as specially requested) is a very personal reminiscence of the discovery, with Rob Race and Arthur Mourant, of the antiglobulin test in blood group serology. The second talk traces developments in antiglobulin testing in general immunology using, of course, isotype-specific antiglobulin reagents as and when they became available. Special emphasis was given to: (1) testing for poorly agglutinating bacterial antibodies; (2) special procedures for measuring reaginic (IgE) antibodies to various allergens; (3) the incorporation of an antiglobulin step or stage in many of the routine automated immunoassay procedures; (4) the special experimental procedures in the form of mixed antiglobulin reactions to reveal antibodies to the surface antigens on tissue cells, and, finally, (5) by chemically coupling antiglobulin to red blood cells, a distinctive integrated immunoassay system was developed, enabling reverse passive haemagglutination as an assay for immunoglobulins, rosetting reactions to reveal native IgG receptors on lymphocytes or antibody immunoglobulin reacting with CD marker antigens; this same reagent, namely red-cell-labelled antiglobulin, can be used to detect antibodies reacting with either bacteria or antigens adsorbed on the walls of wells of microtitre plates. The need for improved stabilization and preservation of the antiglobulin-linked red cells to prolong their 'shelf-life' is stressed.

Complement‐Binding Anti‐Jka Not Detectable by DiaMed Gels

An anti-Jka was not detectable by the DiaMed-ID Micro Typing System but was strongly reactive when tested using other indirect antiglobulin test (IAT) methods. However, it was detectable in the DiaMed-ID system when the recommended diluent ID-Diluent 2, was replaced by other low ionic Strength saline (LISS) solutions. This led us to believe that the problem lay with the ID-Diluent 2. The anti-Jka was investigated using various low ionic strength diluents in the IAT against both polyspecific and monospecific antiglobulin reagents. We tested the ID-Diluent 2 for its ability to inhibit complement activation, and bind calcium. The anti-Jka was shown to be a 'complement-only' binding antibody that reacted in all IATs performed except when tested with ID-Diluent 2. The ID-Diluent was shown to prevent haemolysis of ABO lytic antibodies, and further testing shows that it binds calcium. The use of a diluent which prevents haemolysis and binds calcium in an IAT system that contains anti-complement as well as anti-IgG must be questioned.

Measurement of red blood cell-bound C3b and C3d using an enzyme-linked direct antiglobulin test

Complement has a complex role in immune mediated red blood cell (RBC) destruction and usually induces extravascular hemolysis of C3b-coated RBCs by erythrophagocytosis and by acting synergistically with cell-bound immunoglobulins. A sensitive two-stage enzyme-linked direct antiglobulin test (ELDAT) was developed and used to measure RBC-bound C3b and C3d in 120 healthy adult individuals and in 60 patients suffering from a variety of conditions, including warm- and cold-type autoimmune hemolytic anemia, neoplasia, and collagen diseases. The results were compared with those of standard agglutination tests employing polyclonal and monoclonal antiglobulin reagents. Small amounts of C3b and C3d were detected on RBCs of the healthy individuals only by the ELDAT and probably reflected the continuing low-grade activation of complement necessary for the maintenance of homeostasis of a variety of physiological systems. The quantity did not vary with age or gender. In the patients, increased amounts of RBC-bound C3b and C3d were relatively common and probably resulted from autoantibody activity, immune-complexes, and nonspecific adsorption. There was no association between positive ELDAT results and the presence of active hemolysis. The ELDAT was far more sensitive than the agglutination tests for detecting RBC-bound C3b and also for C3d if the monoclonal reagent was employed.

7.1#127 - Anti-kappa and anti-lambda specific murine monoclonal antibodies as antiglobulin reagents

7.1#127 - Anti-kappa and anti-lambda specific murine monoclonal antibodies as antiglobulin reagents

Antibody screening and direct antiglobulin testing using BioVue™ column agglutination technology versus standard tube tests

The BioVue™ column agglutination technology (CAT) was evaluated simultaneously with standard tube test (STT) methodology for use in indirect antiglobulin testing (IAT) and direct antiglobulin testing (DAT). One thousand thirty-five blood specimens were used for the IAT comparison, and 44 blood specimens were used for the DAT comparison. Both polyspecific antiglobulin and anti-IgG antiglobulin reagents were used in the tube testing and the CAT testing. For IAT, specificity was 100 percent for CAT and 99.6 percent for STT; sensitivity was 97.9 percent for CAT and 100 percent for STT. In addition, a 67 percent labor savings was realized with CAT versus STT. Specificity and sensitivity of both methodologies were 100 percent for the DAT. BioVue™ proved to be a reliable and efficient alternative to standard test tube methods for doing IATs and DATs. Immunohematology 1993;9:78.

A prospective study to determine the safety of omitting the antiglobulin crossmatch from pretransfusion testing.

Transfusion medicine laboratories routinely perform a series of pretransfusion serological tests including: ABO grouping, Rh typing, and investigation of the recipient's serum to detect antibodies against blood group antigens (antibody screen). As a final check, most laboratories also perform a crossmatch in which the recipient's serum is incubated with the donor's red cells followed by the addition of an antiglobulin reagent (antiglobulin crossmatch). The need for the antiglobulin crossmatch when the antibody screen is negative has been questioned because there are few antibodies that are detected by this test. Such antibodies are usually directed against low incidence antigens that are not expressed on the screening cells and in many cases the clinical importance of these antibodies is uncertain. For these reasons, we performed a prospective study in which patients requiring red cell transfusion had a group and screen performed. If the antibody screen was negative the antiglobulin crossmatch was omitted. Following the transfusion of the blood, the antiglobulin crossmatch was performed to look for any potential incompatibility. All patients were monitored both serologically and clinically. Over the 2-year interval of the study 9128 patients were entered. There were 8936 patients (97.9%) with a negative antibody screen and 26.9% (2404 patients) were transfused a total of 10,899 red cell concentrates. The antiglobulin crossmatch performed after the transfusion indicated that 168 red cell concentrates (1.5%) would have been incompatible if the antiglobulin crossmatch had been performed pretransfusion. These 168 red cell concentrates were transfused to 119 patients during 130 transfusion episodes (defined as all transfusions administered within 24 h). Of the 130 transfusion episodes, 79.2% (103/130) were false positive laboratory results. There were 27 transfusion episodes where the antiglobulin crossmatch on blood transfused was positive due to an IgG antibody. Even though these transfused red cell concentrates were designated incompatible by the antiglobulin crossmatch, none of the patients receiving this blood had clinical or serological evidence of haemolysis. We concluded that the antiglobulin phase of the crossmatch can be omitted from pretransfusion testing without putting patients at risk.

Method for studying anti-complement reagents: use of freeze-thawed sensitized erythrocytes

The use of red blood cells coated with complement fractions using low ionic strength saline conditions is part of the evaluation of anti-complement antibodies, specially monoclonal antibodies used for antiglobulin reagents. This work shows that such cells stored in liquid nitrogen allow satisfactory long-term studies except for the anti-C3g antibodies.

抗ヒトＩｇＧ単クロン抗体の抗グロブリン試薬への応用：抗ヒトＩｇＧ単クロン抗体の作製とその性状解析

Ten mouse monoclonal antibodies (mAbs) against human IgG were produced to apply to antiglobulin reagent. Of these 10mAbs, 5 reacted with heavy (H) chains and 4 recognized light (L) chains of human IgG in Western blot analysis. The specificity of a remained mAb, which gave strong positive reaction in enzyme-linked immunosorbent assay using human IgG as antigen, was not defined by Western blot analysis. All mAbs reacted with human IgG sensitized red blood cells on membrane immunofluorescence assay, but anti-H chain mAbs only exhibited strong positivity on antiglobulin tests using human IgG sensitized red blood cells. These results suggest that the anti-H chain mAbs are important as an antiglobulin reagent.

An auto‐anti‐M causing hemolysis in vitro

A 64-year-old white man, who had never received a transfusion, was found to have anti-M in his serum. The antibody agglutinated all M+ red cells in room-temperature tests. When the ionic strength of the test milieu was reduced by use of an additive solution and the tests were incubated at 37 degrees C, the antibody hemolyzed M + N- but not M+N+ red cells. All M+ red cells reacted in indirect antiglobulin tests using polyspecific antiglobulin reagents when such tests followed an initial incubation at room temperature. When red cells and the patient's serum were warmed to 37 degrees C before being mixed, no antibody activity was demonstrable. The antibody was adsorbed to exhaustion onto M+N- and M+N+ red cells (including the patient's own), and its activity was destroyed by dithiothreitol. There was no evidence of in vivo red cell destruction by the autoantibody. No previously reported example of anti-M has been shown to activate complement in conventional in vitro tests. This example was extraordinary in that it caused sufficient complement activation to present as an in vitro hemolysin.

Western Blot Identification of Platelet Proteins That Bind Normal Serum Immunoglobulins. Characteristics of a 95-Kd Reactive Protein

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2-mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.

Western blot identification of platelet proteins that bind normal serum immunoglobulins. Characteristics of a 95-Kd reactive protein

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2- mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.

Investigation into factors influencing performance of the canine antiglobulin test

Antiglobulin (Coombs') reagents were assessed for their ability to detect immunoglobulin and complement attached to red cells. Polyspecific and monospecific reagents were prepared using a number of immunisation protocols. Performance of these antisera against control red cells was compared, in a direct Coombs' test, with samples from cases of canine autoimmune haemolytic anaemia (AIHA). A combined reagent containing two monospecific antisera (anti-IgG + anti-C3) gave optimum results. Positive control red cells were required to standardise canine Coombs' reagents for the laboratory diagnosis of AIHA. The optimum incubation temperature for the canine Coombs' test was shown to be 37 degrees C.

Vancomycin‐induced red cell aggregation

Vancomycin, an antibiotic similar in structure to ristocetin, is used to treat staphylococcal infections. However, vancomycin-induced hemagglutination complicated red cell (RBC) serologic testing in the blood bank. At concentrations greater than 3.0 mg per ml, vancomycin caused spontaneous macroscopic red cell (RBC) aggregation; concentrations of 2.0 and 2.5 mg per ml were associated with weakly positive aggregation with anti-IgG and polyspecific antiglobulin reagents negative with anti-complement; and concentrations less than 1.5 mg per ml had no apparent effect. Ficin-treated RBCs demonstrated negative reactions with the antiglobulin reagents. Vancomycin-induced aggregation was reversed partially with 0.2 M trisodium citrate, and supernatant transfer studies showed that normal RBCs retained a significantly (p less than 0.025) greater percentage of vancomycin than did ficin-treated RBCs. Vancomycin causes the aggregation of RBCs, which can be a source of confusion in the blood bank. The mechanism(s) through which vancomycin enhances aggregation may be related to its polycationic properties and to its direct protein binding to the RBC membrane, although other nonimmunologic mechanisms may be operative.

Electroblotting and immunohistochemical staining for identification of platelet antibodies.

The Western blot procedure has been adapted for use with a biotinylated antiglobulin reagent and a horseradish peroxidase complex (Vectastain ABC) to determine specific sites of antigen binding and permit discrimination between various types of platelet antibodies. Monospecific anti-PlA1 antisera or sera containing mixtures of multispecific HLA and unidentified platelet specific antibodies were tested with PlA1 phenotyped platelets. Using monospecific anti-PlA1, one intense band with relative molecular weight (Mr) of 88,000 and corresponding to glycoprotein IIIa was seen with the PlA1+ platelets. With mixtures of antibodies, reactions were seen with platelet specific antibodies without interference from the HLA antibodies; with one serum a band of Mr approximately 135,000 was identified with Baka+, but not Baka- platelets, indicating the presence of an anti-Baka in the serum. The location of the Baka antigen corresponded to the area for GP IIb. With another serum, a band of Mr 88,000 was revealed with PlA1- and some PlA1+ platelets suggesting the presence of an anti-PlA2. Two additional bands of Mr 160,000 and 200,000 were present on all preparations including autologous controls, probably due to the presence of non-specific IgG. Thus, immunohistochemical staining is readily adaptable to the Western blot technique, and antibodies to platelet-specific antigens can be easily differentiated from HLA antibodies.

A survey of anti-platelet antibody detection--report of the UK Platelet and Granulocyte Serology Working Group, 1985.

In 1985, serum samples were distributed to 27 laboratories within the UK and Eire with the objective of determining the current state of anti-platelet antibody detection. The sera included five unknown samples for blind assessment and four well-characterized reference reagents. The laboratories involved used a wide range of different methods but the most commonly used techniques were the fluorescent antiglobulin test and the enzyme-linked immunosorbent assay. In the trial, all responding laboratories were able to detect potent anti-platelet alloantibodies but there was a 43% incidence of 'false positive' results with samples which had been designated as inert. There were no apparent differences in performance between assays using fluorescein- or enzyme-labelled antiglobulin reagents or between assays which utilized PFA-treated rather than untreated platelets.

Is the indirect antiglobulin crossmatch justified?

Pretransfusion testing has undergone a rationalization, resulting in a more efficient transfusion service but with no compromise to patient safety. Current debate centres on the necessity of performing an antiglobulin crossmatch if a suitable screening test for detecting red cell antibodies is negative. A retrospective study of all pretransfusion testing was undertaken to determine the specificities and possible clinical significance of antibodies detected by the crossmatch alone. The results indicate that omission of the crossmatch will not compromise patient safety. This will have benefits to both the patient and the laboratory. The patient will benefit because of less time needed to provide homologous donor blood. The laboratory will benefit from cost savings as a result of less antiglobulin reagent being used, less time taken for providing blood (and hence further cost savings) and less blood needing to be held in stock. The cost savings generated could be channelled into areas that will result in greater patient safety at a lower cost than crossmatching. Two of these areas are prevention of patient and specimen identification errors by the use of a unique transfusion number system and the full utilization of an autologous blood service.

Fatal Immune Hemolytic Anemia and Hepatic Failure Associated with a Warm-Reacting IgM Autoantibody

Autoimmune hemolytic anemia (AIHA) caused by warm-reacting IgM autoantibodies is rare. We report a fatal case of primary AIHA with a warm-reacting IgM autoantibody. Recurrent episodes of intravascular hemolysis, unresponsive to all therapy and progressive hepatic dysfunction characterized the patient’s clinical course. Despite corticosteroid therapy, splenectomy and multiple blood transfusions, the patient died from liver failure. The IgM autoantibody caused autoagglutination of the patient’s red cells at 37 °C. Eluates prepared from the patient’s red cells agglutinated saline-suspended test cells without the addition of antiglobulin reagent. We propose that warm-reacting IgM antibodies may lead to in vivo autoagglutination and may be associated with hepatic failure.

Antigenic stimulation with proteins of cow's milk via the oral route in guinea pigs and rats. 2. Antibodies to beta-lactoglobulin secreted into the alimentary canal and serum.

The intestinal contents of guinea pigs and rats on a milk-drinking diet were examined for mucosal-secreted antibody to beta-lactoglobulin. Assay of the immunoglobulin content of duodenum, jejunum and ileum of the guinea pig gave geometric means of 70, 90 and 95 micrograms/ml. In the rat, where isotype-specific antiglobulin reagents were available, similar figures were obtained but it could be shown that the vast majority was IgA. Passive haemagglutination titres up to 1,000 of secreted anti-beta-lactoglobulin antibody (resistant to the endogenous proteases in the intestinal contents) were measured in guinea pigs. Fifteen days after cessation of milk-drinking, the antibody had dropped to undetectable levels. No such antibody could be shown in rats. Rats also failed to develop detectable serum antibody whereas, the guinea pigs had passive haemagglutination titres in the order of 16,000.

The use of hapten-modified antigens instead of solid-phase-coupled antigens in a RAST-type assay

We developed a modification of the solid-phase immunoassay for the detection of antibodies of specified isotype. This modified assay involves interaction of the antibody in solution with a haptenated antigen, followed by binding of the antigen-antibody complex to an anti-hapten immunoadsorbent. The antibody is subsequently detected with a suitably labelled antiglobulin reagent. The advantages of this test are: (1) The procedure is more convenient, especially in test protocols where multiple antigens are used. Because the antigens are in solution, computer-controlled dispensation of the antigens is possible. (2) The efficiency of antibody binding is increased, presumably because of the higher avidity of the antigen-antibody interaction in homogeneous solution compared to that in a heterogeneous system. Therefore, less antigen per test is required, and a solid phase with a lower capacity can be used.

An investigation into the effect of reagent volume on the spin indirect antiglobulin test.

Three polyspecific antiglobulin reagents were tested in a spin antiglobulin technique with 51 antibodies of varying blood group specificities in which the volume of antiglobulin reagent used ranged from 1 volume (35 microliters) to 3 volumes (105 microliters). Statistical analysis of results showed that a significant decrease in avidity and agglutination scores occurred as the volume of antiglobulin reagent used was increased ('volume effect'). The volume effect was shown by 33/51 (65%) of antibodies with all three antiglobulin reagents and by 48/51 (94%) of antibodies with at least one antiglobulin reagent. Only 3/51 (6%) failed to show the volume effect with all three antiglobulin reagents.