Annals of Clinical & Laboratory Science 37:167-169 (2007)
© 2007 Association of Clinical Scientists
Eluate Testing Following Microscopically Positive Direct Antiglobulin Tests with Anti-IgG
Elie Richa,
Georgette Benidt,
Craig Tauscher,
Robert Stowers,
Sandra Bryant and
James Stubbs
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
Address correspondence to Elie Richa, M.D., 200 1st Ave SW, Rochester, MN 55905, USA; tel 507 284 9010; fax 507 284 1339; e-mail: richa.elie{at}mayo.edu.
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Abstract
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The direct antiglobulin test (DAT) demonstrates the presence of immunoglobulin (eg, IgG) or complement on the surface of red blood cells (RBCs). Immunoglobulin can be removed from RBCs by elution. The liquid end-product of elution procedures, the eluate, can be evaluated by antibody identification procedures. Antibody identification studies following acid/EDTA elution and DATs performed in our immunohematology laboratory during 2005 were evaluated to determine the usefulness of eluate testing following a microscopically positive IgG DAT. In total, 310 eluates were prepared during the year 2005; 146 of these eluates were derived from RBC samples with a microscopically positive DAT. The remaining eluates were derived from RBC samples with macroscopically positive IgG DATs (85 were weakly +; 32 were 1+; 40 were 2+; and 7 were 3+). Data were collected for the number and types of new antibodies (warm autoantibodies or alloantibodies) that were identified as a consequence of eluate testing. The incidence of new alloantibodies in the microscopically positive group (0.7%) was significantly lower than the combined incidence in the macroscopically positive groups (5.5%, p = 0.02). Based on these results, the authors conclude that performing antibody identification procedures on acid/EDTA eluates derived from RBC samples with microscopically positive IgG DATs has limited utility.
Keywords: blood transfusion, blood bank procedures, direct antiglobulin test, antibody identification
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Introduction
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The direct antiglobulin test (DAT) [1] is used to demonstrate the presence of immunoglobulin (eg, IgG) or complement molecules on the membranes of red blood cells (RBCs). The DAT is often used for evaluation of transfusion reactions, autoimmune hemolytic anemia, or hemolytic disease of the newborn. The detection thresholds for the DAT are 100 to 500 IgG molecules or 400 to 1100 complement molecules per RBC [2]. The results of the DAT are typically reported according to a 0 to 4+ grading system, where 0 indicates no agglutination and 4+ means solid agglutination. When negative results are obtained by macroscopic inspection, technologists in many laboratories examine the contents of the test tube microscopically (at 100x magnification) for agglutination. If there is microscopic evidence of agglutination, the DAT is reported as microscopically positive (micro+). Antibodies can be removed from RBC membranes by a variety of elution procedures [3]. The solution containing antibody recovered from RBC surfaces is the eluate. Preparation and testing of eluates is time-consuming and may delay the final interpretation of serologic evaluations. To ascertain the value of such testing, all antibody identification procedures involving RBC eluates that were performed in our laboratory during 2005 were evaluated in relation to DAT reaction strength.
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Materials and Methods
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To perform the DAT, a 2% suspension of patient RBCs was added to a set of 4 test tubes. The RBCs were washed 3 times with normal saline and the final wash was completely decanted. Polyspecific antiglobulin, anti-C3b, anti-IgG, and normal saline (control) were added to their respective test tubes and the mixtures were centrifuged. The RBCs were examined for macroscopic agglutination. If no macroscopic agglutination was observed, the RBCs were examined microscopically at x100 magnification. After the DAT results were recorded, IgG-coated RBCs were then added to the polyspecific and anti-IgG tubes and complement-coated RBCs were added to the anti-C3b tubes as positive controls. The tubes were centrifuged and examined for agglutination.
The antibody elution procedure was performed under the following circumstances: (1) a first-time positive DAT in a patient, (2) an increase in the DAT reaction strength, or (3) when 12 mo had elapsed since the patient’s last eluate. After 3 wash cycles to remove unbound antibody, the patient’s RBCs were resuspended and incubated in a test tube containing acid/EDTA solution (2 ml of 0.1 M glycine-HCl (pH 1.5) plus 0.5 ml of 10% (w/v) EDTA). The acid/EDTA incubation removed bound antibody from the surface of RBCs. Buffer solution (1 M Tris, pH 7) was added and the test tube was centrifuged. The supernatant was transferred to a clean test tube and adjusted to neutral pH with the same buffer. After the eluate was recentrifuged and transferred to a clean test tube, it was ready for use in antibody identification.
All antibody identification evaluations utilizing RBC eluates (ie, eluate tests) were reviewed for the period from 1 January to 31 December 2005. For data analysis, the evaluations were all considered as separate tests, even when performed on multiple occasions for an individual patient. The total number of evaluations in the study was 310. The results were categorized based on DAT reaction strength as follows:
- Micro+ (ie, positive on microscopic examination),
- Weak+ (ie, weakly positive on macroscopic inspection),
- 1+,
- 2+, or
- 3+.
The results of eluate tests were compared to the corresponding results of antibody identification studies utilizing serum or plasma. The outcomes were categorized as follows:
- No new findings,
- Any new antibody (alloantibody or warm autoantibody)
- A new alloantibody.
Statistical analyses was performed by Fisher’s exact test, assuming that the 310 eluates analyzed in the study were from 310 individual patients.
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Results
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DAT reaction strength was associated with a borderline statistically significant difference in the percentage of any new antibodies identified by eluate testing (p = 0.052) (Table 1
). When only new alloantibodies were considered, the statistical significance of DAT strength relative to elution study findings was greater (p = 0.009) (Table 2).
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Table 1. Percentage of new findings (alloantibody or warm autoantibody) after eluate testing following a direct antiglobulin test (DAT).
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The one alloantibody identified following a microscopically positive (micro+) DAT was anti-K. In the group that was weakly positive on macroscopic inspection (weak+), 8 alloantibodies were identified (anti-C, anti-E, anti-K, anti-A1, anti-Fya, anti-Jkb, and 2 maternal anti-D). In the 1+ group, one new alloantibody was identified (anti-D). No new alloantibodies were identified in 2+ or 3+ groups.
Micro+ DATs yielded a lower rate of new antibody detection (5.5%) than the combined groups of macroscopically positive DATs, 12.2% (p = 0.047) (Table 1
).
When only new alloantibodies were considered, the micro+ DATs were again associated with a significantly lower rate of detection compared to the macroscopically positive DATs (0.7% vs 5.5%; p = 0.02) (Table 2).
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Discussion
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The important question is whether eluate testing performed on micro+ DAT RBC samples yields clinically significant results. If so, this suggests that eluate testing should be done following microscopically positive DATs. We hypothesized that the percentage of micro+ DAT samples yielding new alloantibodies would be significantly lower than the percentage of new alloantibodies identified in association with macroscopically positive DATs. Such findings would argue against the routine performance of eluate testing in the setting of a micro+ DAT.
Seven of the 8 new antibodies identified with eluate testing following a micro+ DAT were warm autoantibodies. Patients with new warm autoanti-bodies and no co-existent clinically significant alloantibodies are eligible for an electronic RBC crossmatch. One of the 8 new antibodies was an anti-K alloantibody, which is clinically significant. The detection of this antibody would have been delayed if eluate testing had not been performed. The question is whether one such case should drive standard practice. Because our analysis revealed such a low percentage of new clinically significant alloantibodies in the setting of a micro+ DAT and a statistically significant difference with regard to detection of such alloantibodies compared to macroscopically positive DATs, we concluded that eluate testing in the setting of micro+ DATs should not be a standard practice.
In conclusion, the results of this study led to a change in standard practice in our immunohematology laboratory. Eluate testing is no longer routinely performed following a microscopically positive direct antiglobulin test.
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References:
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- Mollison PL Engelfriet CP, Contreras M. Blood Transfusion in Clinical Medicine, 10th ed, Blackwell Scientific, Oxford, England, 1997; pp 222.
- Engelfriet CP, Pondman KW, Wolters G, von dem Borne AE, Beckers D, Misset-Groenveld G, van Loghem JJ. Autoimmune hemolytic anemias: III. Preparation and examination of specific antisera against complement components and products and their use in serological studies. Clin Exp Immunol 1970;6:721.[Medline]
- Landsteiner K, Miller, CP. Serologic studies on the blood of primates: II. The blood group of anthropoid apes. J Exp Med 1925;42:853.[Abstract/Free Full Text]
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Abstract
Introduction
