An Antibody Screening Test Based on the Antiglobulin Gel Technique, Pooled Test Cells, and Plasma

The recently introduced gel technique offers significant advances compared to traditional tube tests. The purpose of the present study was to design an antibody screening test based on the gel technique, pooled cells, and plasma and to evaluate this test by comparison with a conventional spin-tube indirect antiglobulin test (IAT) combined with a two-stage papain technique. Pilot studies were performed to establish optimal parameters during the design phase, and finally 5,446 consecutive samples were screened for irregular antibodies by the gel technique in parallel with routine techniques. Irregular erythrocyte antibodies were detected in 151 samples, and the gel technique proved equal or superior to the tube test in the detection of all antibodies except 'enzyme-only' anti-E and anti-Le^a. We conclude from this study that screening for unexpected antibodies using the gel IAT in our set-up, which includes: (1) omission of enzyme technique; (2) the use of stabilized (EDTA) plasma instead of serum as test material in order to facilitate automation; and (3) pooling 2 by 2 of 4 test cells (to make the gel technique price competitive), is a fast, reliable and sensitive procedure that maintains transfusion safety and compares favourably with our previous routine of saline IAT combined with a two-stage papain technique.     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

Erythrocyte Antibody Screening in Solid-Phase: A Comparison of Two Solid-Phase Microplate Assays with the Indirect Antiglobulin Test in Polyethylene Glycol for the Detection of Irregular Erythrocyte Antibodies

The screening of a serum for irregular erythrocyte antibodies in the indirect antiglobulin test is a well-established technique. We compared the test results of two different solid-phase microplate indirect antiglobulin tests with a liquid-phase indirect antiglobulin test in tubes. Antibody screening with both solid-phase microplate techniques proved to be more sensitive than the liquid-phase indirect antiglobulin test. In addition, a difference in sensitivity between the two solid-phase techniques was observed: prior immobilization of test erythrocytes on the microplate followed by incubation with a serum and detection of sensitization with antihuman IgG-coated detector cells gave better test results than secondary immobilization on the microplate of test erythrocytes sensitized with antibodies and an antihuman globulin serum.     

EA Rosette Formation: a Simple Means to Increase Sensitivity of the Antiglobulin Test in Patients with Anti Red Cell Antibodies

S ummary . Erythrocyte antibody (EA) rosette formation with the Fc-receptor on the K-562 erythro-myeloid cell line was employed for the detection of subagglutinating amounts of Ig molecules bound to red cells. The sensitivity of this method exceeds that of the conventional direct and indirect antiglobulin tests without any alteration of the incubation media or pretreatment of red cells. The increased sensitivity did not diminish the specificity of the test, which can detect IgG, IgM and complement as well. This method may demonstrate the presence of antibodies on red cells in patients with suspected autoimmune haemolytic anaemia and negative antiglobulin test.     

Antibody elutions in Thai patients with a positive direct antiglobulin test

Background

The direct antiglobulin test is performed to determine whether an anaemic patient with evidence of haemolysis has autoimmune or alloimmune haemolytic anaemia.

Materials and methods

We determined the antibody specificity of eluted IgG antibodies from patients’ blood samples with a positive direct antiglobulin test. Overall, 134 Thai patients were included in this study. EDTA blood samples were obtained from recently transfused patients, patients with unexplained anaemia and patients who had serum antibodies detected during routine pre-transfusion tests from different hospital blood banks. These complicated samples were sent to the National Blood Centre of the Thai Red Cross Society for investigation and to find compatible blood components. Each blood sample underwent a direct antiglobulin test with the gel technique using polyspecific antihuman globulin and mononospecific anti-IgG and anti-C3d. Acid eluates were prepared from the samples for which the direct antiglobulin test was positive and the specificities of the eluted antibodies were determined by the gel technique.

Results

Of the samples tested, 101 showed a positive direct antiglobulin test result (75.4%) using polyspecific antihuman globulin sera whereas only 95 samples (70.9%) were positive with anti-IgG or anti-IgG and anti-C3d. Moreover, 54 of 95 eluates (56.8%) were positive for antibody screening and tested with the reagent panel cells. Twenty-one eluates had specific alloantibodies, which were concordant with the findings in the patients’ sera and all patients had a history of blood transfusion. Additionally, 33 eluates contained pan-agglutinins. Interestingly, alloantibodies could be determined using titration studies in 5 of 26 eluates with pan-agglutinins.

Conclusion

Although the direct antiglobulin test is not routinely performed in pre-transfusion screening, this test and elution studies would be useful in patients with a history of previous transfusions, and in those for whom compatible blood cannot be found.     

Direct Comparisons between a Radio-Immune Antiglobulin Test, an Enzyme-Linked Antiglobulin Test, and a Haemagglutination Assay: Application to the Screening of Anti-RBC Sera and Monoclonal Antibodies

Abstract. Direct comparative studies between a radio-immune antiglobulin test (RIAT), an enzyme-linked antiglobulin test (ELAT) and a haemagglutination assay (HA) were carried out using anti-RBC hybridoma culture supernatants, monoclonal antibodies of established specificity, and immune mouse sera. The direct comparisons revealed that in many cases, RIAT and ELAT were slightly more sensitive than HA for the detection and study of anti-RBC antibodies. RIAT and ELAT detect non-agglutinating antibodies and sub-agglutinating concentrations of antibodies which would be missed if HA is used as the only screening test for hybridoma supernatants.     

Comparison of conventional tube test with diamed gel microcolumn assay for anti-D titration

Anti-D titration is the first step in the evaluation of the RhD-sensitized patient. Traditionally, anti-D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti-D titer needs to be established. Seventy-nine known blood samples with anti-D (titers 1-32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R0r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2-2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti-D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4-fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti-D titration by the gel microcolumn assay.     

A prospective study of routine antenatal enzyme antibody screening demonstrates lack of clinical value in predicting haemolytic disease of the newborn

A prospective study of 7065 consecutive new pregnancies identified 230 with a positive screen, of which 27% (62/230) were 'enzyme-only' antibodies. 32 of these (52%) were potentially clinically important and were all of Rh specificity: 22 anti-E, seven anti-Cw, two anti-D and one anti-c. However, only three of these enzyme-only antibodies (one anti-D, one anti-c and one anti-E) became reactive by the indirect antiglobulin test (IAT) during the course of pregnancy, and all were detected in the routine 34-36-week maternal sample. No babies were affected, and we reaffirm that routine antibody screening by enzyme techniques is unnecessary.     

Comparison of conventional tube test with diamed gel microcolumn assay for anti‐D titration

Summary Anti‐D titration is the first step in the evaluation of the RhD‐sensitized patient. Traditionally, anti‐D titration has been performed by tube agglutination. Gel microcolumn assay is a method that has gained widespread usage throughout the world, mainly for ABO/Rh typing, unexpected antibody screening and direct antiglobulin tests. As gel assay has become widely used as a routine method to detect red blood cell alloantibodies, a critical anti‐D titer needs to be established. Seventy‐nine known blood samples with anti‐D (titers 1–32) were titrated simultaneously by the conventional tube test and the gel microcolumn assay. Red blood cells (R₀r phenotype) were used, with a final concentration of 3% for tube and 0.8% for gel. Serial twofold dilutions (2–2.048) were prepared for each technique, followed by reading in antiglobulin phase. Anti‐D titration in the gel microcolumn assay showed significantly higher titers (mean 3.4‐fold) than the conventional tube test in all samples studied. Based on these data, it was not possible to determine a critical titer for anti‐D titration by the gel microcolumn assay.     

An Autoantibody with Activity Dependent on Red Cell Age in the Serum of a Patient with Autoimmune Haemolytic Anaemia and a Negative Direct Antiglobulin Test

A red cell IgM autoantibody with anti-e specificity was identified in the serum of a rhesus-negative (rr) patient presenting with haemolytic anaemia and a negative direct antiglobulin test. The autoantibody strongly agglutinated normal allogeneic rhesus-negative (rr) red cells in saline at 37 degrees C but had only weak activity for autologous red cells. Incubation of the patient's serum with subpopulations of normal allogeneic red cells obtained by density fractionation, demonstrated that the strong agglutinating activity of the autoantibody was for red cells with density greater than 1.090 g/ml. Young red cell subpopulations of lower density gave weak reactions and low titration scores equivalent to those obtained with autologous red cells during the haemolytic episode. The patient's red cells during remission however, when the patient's haemoglobin level had returned to normal, were strongly agglutinated by serum samples taken during the haemolytic episode; as was the case with normal allogeneic red cells, the strong activity was for patient red cells with density greater than 1.090 g/ml, red cell populations of lower density giving low titration scores. The findings in this case indicate that the patient's red cells which had survived haemolysis during the haemolytic episode were young red cells (density less than 1.090 g/ml), the weak sensitization of these cells being insufficient to cause their destruction by macrophages. Furthermore, these findings, together with observations that IgG autoantibodies may also bind less strongly to young red cells [Gray et al., Br. J. Haemat., 55: 335-345, 1983; Branch et al., Blood 63: 177-180, 1984].(ABSTRACT TRUNCATED AT 250 WORDS)     

5种不规则抗体筛查方法的检测阈值比较

目的:了解各种不规则抗体筛查方法的检测阈值,以避免不规则抗体漏检而引起输血不良反应。方法:选取献血者不规则抗体筛查阳性样本3例和配血不合疑难样本9例,采用盐水法、酶介质法、凝聚胺法、抗人球蛋白法、微柱凝胶法平行检测,比较5种方法的检测阈值。结果:盐水法只能检出IgM类的不规则抗体;木瓜酶法在检测IgG抗体效价时敏感性略低,而且有漏检的可能;凝聚胺法检测结果不易判断;抗人球蛋白法操作较繁琐;微柱凝胶法对IgG类抗体检测效价较高,高于凝聚胺法和抗人球蛋白法。结论:微柱凝胶法操作简单,检出率较高,是值得推广的不规则抗体筛查方法。     

A Modified Radio-Immune Antiglobulin Test for the Screening of Anti-Red Blood Cell Antibodies in Hybridoma Supernatants

A radio-immune antiglobulin test was developed using mouse monoclonal anti-human A red blood cell (RBC) antibodies, and applied to the screening and study of hybridoma supernatants containing anti-human RBC antibodies. The latter antibodies were provided either as known monoclonal antibodies from culture supernatants and ascitic fluids, or as culture supernatants to be screened. The technical feasibility of the test was greatly facilitated by the use of remova-well disposable polystyrene plates, multichannel pipettes, and multiple-plate centrifuge holders. The sensitivity of the test and its relevance for the screening of hybridoma anti-human RBC antibodies is discussed.     

The value of gel test and ELAT in autoimmune haemolytic anaemia

Summary Three methods for detection of warm type IgG autoantibody were evaluated using 400 blood samples from 147 patients suspected of autoimmune haemolytic anaemia (AIHA). Three direct antiglobulin techniques (DAT) were used: conventional tube DAT, gel-DAT by micro-method and gel-DAT enzyme linked antiglobulin test (ELAT). Eluate examinations confirmed the presence of autoantibodies on red cells. These tests were compared directly using 126 selected blood samples from 85 patients with IgG molecules on their red cells detected by the gel test. In 106 of these samples, collected from 65 patients with clinical symptoms of AIHA, the presence of autoantibody was confirmed by acid elution. The ELAT was positive in 100 samples (94%), 87 samples for tube DAT (82%). The ELAT as well as the tube DAT was negative in 20 samples with non-reactive eluates by gel test. The gel-DAT was therefore not fully specific and detected IgG on red cells of patients with hypergammaglobulinaemia. However, due its higher sensitivity it proved useful as a screening test. The ELAT allowed changes in the number of IgG molecules per red cell to be monitored quantitatively. Both methods play a part in the diagnosis and monitoring patients with warm type IgG autoantibody.     

母婴Rh血型不合引起的新生儿溶血病血清学分析

目的：对12例母婴Rh血型不合引起的新生儿溶血病进行血清学分析。方法：采用直接抗球蛋白试验、游离抗体试验、放散试验检测ABO以外的抗体,采用谱细胞与患儿血清和母亲血清进行间接抗球蛋白试验鉴定抗体。结果：12例新生儿RhHDN中检出抗-D 7例,抗-E 3例,抗-c 1例,抗-E,c 1例,其中3例溶血病患儿为Rh阳性孕妇所生产。结论：应推广产前孕期夫妇Rh血型鉴定分型（包括RhD阴性和RhD阳性）及孕妇Rh免疫性抗体筛查,以减少胎儿受害,保证优生优育。     

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.     

Red cell alloimmunization in multitransfused patients and multiparous women

Purpose  To determine the incidence of alloimmunization against red cell antigens and the thermal amplitude and specificity of antibodies in multitransfused patients and multiparous women. Methods  Antibody screening was performed in 30 nontransfused orthopedic surgery cases (control), 504 multitransfused patients and 325 multiparous women. Antibody screening at 4°C, 22°C and 37°C was carried out in a saline phase, by indirect antiglobulin technique (IAT), using papain cystein, low ionic strength solution (LISS) and polyethylene glycol (PEG). Results  In multitransfused patients IgM antibodies were more frequently detected at 4°C and the IgG antibody incidence was 7.1% by enzyme method, 7.7% IAT, 9.4% LISS, 10.2% using PEG & 10.2% multiparous women. Bad obstetric history cases had significantly higher incidence of alloimmunization. The antibody specificity of antibodies was mainly in Lewis, Rh, Kidd and MN systems. Conclusion  Antibody screening before transfusion, at set time intervals after transfusion and during antenatal period is recommended.     

A comparison of conventional tube test and gel technique in evaluation of direct antiglobulin test

In vivo coating of red cells by antibody and/or complement is detected using various sensitive techniques, however most hospitals even today rely on the conventional tube technique (CTT). We compared the performance of the CTT and recently introduced gel test (GT) in the evaluation of direct antiglobulin test (DAT). The CTT and GT were first compared using in-house prepared control cells. The polyspecific DATs were performed simultaneously by CTT and GT on 170 consecutive blood samples. Positive samples were further tested for monospecific IgG and C3d by both techniques. GT demonstrated stronger agglutination scores (60 vs. 43) compared to CTT using control cells. The sensitivity and specificity of the GT was 98.4 and 95.2%, respectively as compared to CTT for polyspecific DAT. Discordance between the two test systems was seen in 6/170 patients. Of these, 5 were missed by CTT while GT failed to detect in vivo coating in only 1 case. The agreement between two methods of DAT was 96.4% (kappa = 0.926) using polyspecific AHG and 95.7% (kappa = 0.379) with monospecific anti-IgG. We conclude that GT is a better alternative to CTT for detecting red cell bound antibodies in various clinical conditions.     

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.     

Immune hemolytic anemia associated with negative routine serology

Immune hemolytic anemia can occur in patients who have no antibodies detectable by routine procedures (direct [DAT] and indirect [IAT] antiglobulin tests). DAT-negative autoimmune hemolytic anemias (AIHAs) represent 5% to 10% of all AIHAs. Three causes have been identified: (1) small numbers of red blood cell (RBC)-bound IgG molecules below the threshold of the DAT; (2) IgA and IgM autoantibodies; and (3) low-affinity autoantibodies. Antibody-independent cytotoxic events caused by natural killer (NK) cells have also been implicated. DATs are sometimes found to be positive when tested by reference laboratories, due to poor technique in reading antiglobulin tests in hospital laboratories. Hemolytic transfusion reactions also can occur when no alloantibodies are detectable by routine procedures. In some cases antibodies can be detected by special serologic procedures (such as the Polybrene test); in other instances phenotypically matched RBCs survive well and a specific antigen can be shown to be involved, suggesting a specificity (like anti-C) that is undetectable by any technique. Antibodies other than to blood group antigens, such as human leukocyte antigen (HLA) antibodies, may sometimes be involved.     

Analysis of islet cell antibodies on frozen sections of human pancreas

The sensitivity and specificity of the assay for islet cell cytoplasmic antibodies in human serum were examined using cryostat sections from fresh frozen pancreas. The specificity of the assay was close to 100% while the sensitivity was 40%-98% depending on the pancreas used. Inter-observer variation was 12-27%. End-point titres of islet cell antibodies varied with the sensitivity of each pancreas. End-point titration of the antibodies in two different laboratories using the same pancreas was significantly correlated (Spearman test p less than 0.001). We conclude that a reliable determination of islet cell antibody titres in human serum requires careful characterization of the sensitivity and specificity of each pancreas used as a source of frozen sections, in the indirect immunofluorescence assay.