Strategies for Laboratory and Patient Management

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1 Strategies for Laboratory and Patient Management Evaluation and Implementation of the Gel Test for Indirect Antiglobulin Testing in a Community Hospital Laboratory John C. Cate, IV, MD; Nancy Reilly, MPA, MT(ASCP)SBB Background. The gel test, developed by Lapierre in 98, was designed to standardize antiglobulin testing while improving sensitivity and specificity of the method. Principle. Anti human serum immunoglobulin G (IgG) mixed with Sephadex G00 (gel phase) in a microtube traps red cell IgG agglutination complexes during migration through the gel in a centrifugation step. Agglutination complexes are visibly detectable at various levels in the microtube as an inverse function of antibody coated on red cells. Unsensitized red cells form a cell pellet at the base of the microtube. Objective. To determine if indirect anti human globulin testing could be standardized and simplified by replacing the tube test with the gel test without compromising quality or increasing costs. Setting. A medium-sized community hospital. Results. In a blinded retrospective study, we used patient sera (n 0), which included 0 positive specimens containing 8 known antibodies. Sixteen antibodies were detected and identified with the tube method ( anti-d and anti-c not detected). By the gel test, 8 antibodies were detected and identified. All negative samples showed 00% concordance. Favorable results were obtained in a nonblinded prospective correlation study (n 2). Our technologists found the gel test easier to read and more reproducible and reliable than the tube method; they also found increased sensitivity for detecting weakly reacting antibodies. We successfully introduced the gel test into our laboratory as the standard method for indirect antiglobulin testing. Following implementation, improved personnel management was achieved. Conclusions. The gel test is a reliable and advantageous method and is appropriate for routine use for detection and identification of alloantibodies in a community hospital transfusion service laboratory. (Arch Pathol Lab Med. 999;2:69 697) The indirect anti human globulin (AHG) test, developed by Coombs et al in 95, received strong support from Grove-Rasmussen in 96 as a reliable screening procedure for the detection of clinically significant antibodies in patient sera. It was clear from that study that the indirect antiglobulin test was highly effective in detecting serum alloantibodies; of 96 antibodies screened by this method, 99.6% were detected. Concurrently, the American Association of Blood Banks, in its third edition of the Standards, introduced the indirect AHG test as an acceptable alternate to the Coombs phase cross-match in a carefully controlled testing environment. 5 National practices of red cell compatibility testing did not change Accepted for publication January 27, 999. From the Trident Medical Center Clinical Laboratories, Charleston, SC (Dr Cate and Ms Reilly), and the Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston (Dr Cate). Ms Reilly is now with the Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston. Presented in part at the annual session of the Academy of Clinical Laboratory Physicians and Scientists, Minneapolis, Minn, June 6, 997. Reprints: John C. Cate, IV, MD, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 65 Ashley Ave, Suite 09, PO Box , Charleston, SC significantly 6,7 until type and screen 8 0 and the abbreviated cross-match,2 became an accepted part of the red cell compatibility testing repertoire. By 990, approximately 0% of hospital laboratories had adopted these procedures, which included the elimination of the antiglobulin cross-match. Moreover, investigators were exploring technologies to standardize and improve AHG testing. In 98, Lapierre discovered that dextran acrylamide gel placed in microtubes containing anti-immunoglobulin (Ig) G could detect accurately and reproducibly the presence of red cells sensitized with alloantibodies. Shortly thereafter, a commercial kit for AHG testing by the gel test was field-tested and introduced to the European market. In 990, Lapierre et al 5 reported that the gel test showed improved reliability when correlated with tube results for detecting a variety of clinically significant known antibodies. Bromilow et al 6 and Lillevang et al 7 subsequently confirmed those results in 2 comprehensive studies. The first, an evaluation of 900 random antenatal samples, showed that the gel system detected.7% red cell antibodies compared to 2.% for the tube system. Specificity was increased with the gel system. The second study, using the gel test and tube method to screen 56 consecutive samples for irregular antibodies, reported similar advantages for the gel test over the tube method. Arch Pathol Lab Med Vol 2, August 999 Gel Test for Indirect Antiglobulin Testing Cate & Reilly 69

2 Figure. Gel method for indirect antiglobulin test. Either serum or plasma can be used as patient sample. a and b are positive and negative reactions, respectively. indicates strong positivity for serum alloantibodies; negative indicates a lack of serum alloantibodies. Modified with permission from Transfusion (990;0:0), published by the American Association of Blood Banks. Figure 2.* Gel cards with negative, positive, and false-positive gel reaction patterns. A, Gel card with 0 to reactions. For sera positive for alloantibodies, agglutination reaction location from gel surface is an inverse function of the degree of red sensitization. Highly sensitized cells form a tight agglutination pattern close to the gel surface ( ; tube 5). Moderately sensitized cells form a widely dispersed agglutination pattern from gel surface to half the length of tube ( ; tube ) or dispersed throughout the microtube (2 ; tube ). Minimally sensitized cells form a loosely dispersed agglutination pattern from half the length of microtube to tip ( ; tube 2). Unsensitized cells form normal cell pellets at the tip (0; tubes and 6). B, False-positive gel reactions caused by fibrin particulate matter or rings. False-positive reactions with fibrin ring at gel surface and normal cell pellet at the tip are seen in tubes, 5, and 6. Negative reactions with no fibrin at surface and normal cell pellet at tip are seen in tubes, 2, and. C, Gel reactions with hemolysis and hemolytic antibodies. Hemolyzed sample from traumatic venipuncture with gel reaction chamber or upper gel shading, no agglutination reaction, and normal cell pellet at the tip (tube 5). Hemolytic antibody with gel reaction chamber shading, no agglutination reaction, and trace cell pellet at the tip (tube 6). Intermediate positive reaction with no gel reaction chamber shading, mid-zone agglutination reaction, and trace cell pellet at the tip (nonhemolytic; ; tube ). Negative reaction with no gel reaction chamber shading, no agglutination reaction, and normal cell pellet at the tip (tube ). Unsampled microtubes with no gel reaction chamber shading, no agglutination reaction, and no cell pellet at the tip (tubes and 2). * 0 indicates lack of serum alloantibodies; through indicate weak to strong positive reactions for serum alloantibodies. Normal indicates well-defined, prominent. Numbers through 6 indicate microtube positions on gel cards A, B, and C. Shading indicates presence of hemolysis. The evaluation of the gel test reported in this study was performed in response to the following requests: () to provide a more consistent and reproducible AHG testing methodology for use by nonspecialized technologists; (2) to provide for next-day review by experienced transfusion service technologists of equivocal AHG testing results obtained during off-hours; () to provide for batch immunohematology testing from a remote-site laboratory; and () to provide for improvement of transfusion service testing efficiency while reducing the number of full-time equivalents at the remote location, if feasible. PRINCIPLE OF GEL TEST The gel test (Figure ) is performed with a dextran acrylamide gel containing broad-spectrum anti-igg placed in 6 microtubes on a plastic card measuring approximately 6 cm. Red cell suspensions and patient serum are pipetted onto the gel surface and incubated at 7 C for 5 minutes. The microtubes have a wide mouth to provide optimum surface area for anti-igg reaction with red cell antigens during the incubation phase. The gel is located in the long narrow neck of the microtube. As centrifugation occurs, this geometry provides prolonged contact of the red cells with the gel phase during migration for 0 minutes at centrifugation settings of 70g. Precise centrifuge settings for time and speed must be followed because the long axis of the microtubes must achieve a parallel position to the direction of the centrifugal force during centrifugation for proper red cell migration and antigenantibody reaction to occur. During centrifugation, sensitized red cells aggregate and become trapped in the gel, forming distinctive patterns of agglutination during the gel. Following centrifugation, the cards are read against a white surface and graded according to the pattern of agglutination reactions. Negative reactions yield a well-defined, prominent cell button at the tip of the microtube. Positive reactions ( ) form a well-defined, prominent agglutinated cell layer near the gel surface. Reactions (0 ) are graded according to the pattern of agglutinated cells dispersed through the length of the microtube (Figure 2, A). Reactions are stable for at least days if gel cards are kept upright, covered with tape, and refrigerated. A conventional blood bank workstation space is suffi- 69 Arch Pathol Lab Med Vol 2, August 999 Gel Test for Indirect Antiglobulin Testing Cate & Reilly

3 cient for testing. Required equipment includes multichannel pipettes, 7 C incubator, workrack, and centrifuge designed to accommodate the reaction cards. No special equipment is required to read the reactions. Currently, cards licensed by the Food and Drug Administration for testing include anti-igg (broad spectrum), anti-a, anti-b, anti-ab, anti-d, anti-e, anti-c, anti-c, anti-e, and a neutral buffer card. MATERIALS AND METHODS Gel Test Method Indirect antiglobulin antibody testing was performed using commercially available gel cards (ID MTS Anti-IgG; Ortho, Raritan, NJ) containing broad spectrum anti-human IgG. Fifty microliters of 0.8% red cell suspension in a modified low-ionic saline solution buffer and 25 L of serum or plasma were pipetted into the upper reaction chamber of the microtube and incubated for 5 minutes at 7 C in an MTS incubator designed to accommodate gel cards. Gel cards were then centrifuged for 0 minutes (70g) ata90 angle to the axis of the specially designed MTS centrifuge, removed from the centrifuge, read against a white background, and graded using the negative to scale. For this technology, the test reactant s wash and red cell button resuspension steps are eliminated. Tube Method The tube method was performed in glass 2 75-mm tubes. Two drops of sera were pipetted using 6-inch polyethylene plastic transfer pipettes. One drop of % to % red blood cell suspension and 2 drops of low-ionic saline solution buffer were incubated for 0 minutes at 7 C. Test reactants were washed times before adding 2 drops of anti-igg. Reactions were read macroscopically. Retrospective Study Forty patient serum samples were tested for irregular antibodies, correlating the tube antiglobulin method with the gel test. Ten of the samples contained 8 known antibodies with 8 specificities. Four aliquots of each sera were prepared, numbered, and given to each of 2 technologists to test each set of 2 sera by the gel test and the tube method in a blinded, parallel protocol. Testing was performed on the same day to provide consistent results and to avoid deterioration of antibody reactivity. Antibody identification was performed in tube and gel using standard cell panels. Sera were obtained from patients and blood donors, except for 2 samples, which contained commercially obtained antibodies diluted in patient sera containing no alloantibodies. Prospective Study A prospective study for alloantibody detection and identification was performed on 2 fresh patient samples, comparing the tube method with the gel test. Fresh serum was used for all testing; comparative testing was performed using both methods on the same day. In this study, samples were not blinded. RESULTS For the retrospective study, each technologist independently detected 6 of the 8 known antibodies by the tube method, one missing a weak anti-d and both missing an anti-c. In contrast, both technologists detected all 8 known antibodies using the gel test (Table ). All negative samples were 00% concordant. In the prospective study using fresh patient sera (n 2), the technologists identified 20 samples as negative for alloantibodies and positive by the tube method. The positive sample contained an anti-le a and a cold autoantibody. By the gel test, 6 samples were identified as negative for alloantibodies and 5 were identified as positive. The antibody specificity in the 5 positive samples included Table. Comparison of Tube vs Gel for Indirect Anti Human Globulin Test* Antibody n Tube Gel Arch Pathol Lab Med Vol 2, August 999 Gel Test for Indirect Antiglobulin Testing Cate & Reilly 695 D C E c K V Kp a Fy b Total 8 * Retrospective study. 2 6 (89%) 8 (00%) Table 2. Comparison of Tube vs Gel for Indirect Anti Human Globulin Test (n 2)* Test Negative Positive Antibody Type (No.) Gel 6 5 Cold antibody (); nonspecific reaction (); suspicious low frequency antibodies () Tube 20 Anti-Le a and cold antibody * Prospective study. cold autoantibody, nonspecific reaction, and probable low-frequency antibodies (Table 2). Following the correlation study, the gel test was introduced into our laboratory as the routine test for indirect antiglobulin testing, and 05 additional gel tests were performed on fresh sera in parallel with tube testing. A falsepositive rate of.7% (7/05) was obtained in the first month of testing due to fibrin present in fresh serum trapped at the top of the gel, suggesting a positive reaction (Figure 2, B). After switching to EDTA plasma for patient samples, false-positive results became infrequent. Total costs were moderately lower for the gel method owing to decreased personnel and equipment maintenance costs. Biomedical costs were reduced by the elimination of 2 cell washers, 2 immufuges, and dri-baths. Personnel were reduced by 0.5 full-time equivalents at a remote site laboratory as a result of routine immunohematology requests being transferred to our facility for testing. Following conversion to batch analysis for all routine indirect antiglobulin testing, we observed that time was saved when testing multiple specimens using the gel test, owing to a decrease in the number of sample processing steps. The gel test analytical phase was not found to be faster than that of the tube test: both require approximately 0 minutes for performance of antiglobulin testing. However, technologists could attend to other duties while waiting for the gel test to be completed, since some handson testing (wash step and reading the 7 C phase) is eliminated. Overall, testing efficiency was improved following introduction of the gel test into routine use. Implementation Prior to the onset of our study, a defined protocol was established to optimize the outcome if implementation were to occur. An initial demonstration of the methodology to our laboratory personnel was given by a manufacturer s representative. Following the demonstration, correlation testing was performed between our in-house tube method and the gel test. A cost analysis was instituted, which included labor, reagent, and equipment costs. Pro-

4 cedures were written for the gel test, reagents and equipment were ordered, and transfusion service personnel were trained on the method. The tube method and the gel test were tested in parallel until problems could be worked out in the transition phase. The gel test was then introduced as our routine method for transfusion service technologists only. Finally, laboratory generalists were trained in gel technology, with little difficulty. Improved morale and reduced stress levels were observed as the gel method was introduced into our routine testing menu. COMMENT This article describes our evaluation and implementation of the gel technique for indirect AHG testing. We were guided by the premise that a practical, robust red cell alloantibody screening test should maximize sensitivity for clinically significant antibodies, minimize sensitivity for insignificant antibodies, maximize blood transfusion safety, and standardize and simplify technical performance. With these objectives in mind, we compared tube and gel methods with the understanding that no one technique is ideal. 8 The gel test was easy to perform and gave excellent results for clinically significant alloantibodies (D, E, K, c, Jka, etc). Clearly delineated negative to agglutination reactions promoted standardized interpretation among technologists. Gel column technology was found to improve detection yield of clinically significant, weakly reacting antibodies when tested against homozygous screening cells. Similar results have been obtained by others. 9 Additionally, test performance variability was reduced by elimination of technique-dependent steps such as red cell button resuspension. Stable gel reactions ( days) allow for equivocal results obtained during offhours to be reviewed and interpreted by experienced transfusion service technologists. The no-wash procedure may reduce bloodborne pathogen exposure in the laboratory testing environment. These advantages are of particular importance for the smaller laboratory, where less experienced generalists may perform red cell compatibility testing. The problem identified with fibrin particulates causing false-positive gel results was most commonly encountered with incompletely clotted specimens associated with stat requests and specimens collected from dialysis and patients receiving anticoagulants. Following conversion to EDTA plasma, no further positive reactions due to fibrin occurred. The use of EDTA plasma for AHG testing, as suggested by Lillevang et al, 7 is preferable to serum for gel testing and does not appear to hinder the detection of some complement activating antibodies. Furthermore, failure to use polyvalent antiglobulin-containing anticomplement, in their experience, did not result in false-negative detection of complement-activating hemolytic antibodies. In our study, we experienced no false-negative reactions using gel microtubes incorporated with anti-igg only. Neppert et al 20 reported a case of a clinically significant transfusion reaction due to anti-vel, which was unsatisfactorily detected by the gel test. The authors attributed the problem to the presence of EDTA in the gel diluent. However, gel diluent approved for use in the US market does not contain EDTA. 2 Recently, Stevens et al 22 reported a case of false-negative gel reaction associated with a hemolytic transfusion reaction that was not EDTA related; multiple serologic methods failed to reveal the presence of hemolytic anti-k (detected only by a solid-phase red cell adherence [SPRCA; Immucor, Norcross, Ga] technique). Rare false-positive reactions due to rouleaux associated with hyperglobulinemia have been reported from the laboratory of Lillevang et al. 7 In contrast, Fabijańska-Mitek et al 2 reported no false-positive gel reactions when testing 80 patients sera containing monoclonal proteins (unagglutinated red cells with rouleaux passed through the gel to the microtube base). We observed no false-positive gel reactions associated with rouleaux during our study. Our technologists expressed concern that a hemolyzed sample obtained from a traumatic venipuncture might be confused with the presence of hemolytic antibodies and misinterpreted as a positive reaction using the gel test. Samples were compared between a negative gel reaction, a positive gel test, a traumatic hemolyzed specimen, and a strongly reacting hemolytic antibody (Figure 2, C). False-positive reactions due to traumatic venipuncture are determined by noting the presence of hemolyzed plasma in the reaction chamber, the absence of agglutinates in the gel tube, and a normal cell pellet at the base of the microtube. In contrast, hemolytic antibodies will give a faintly visible or absent cell pellet at the base and will show pink to red discoloration in the reaction chamber when hemolysis is triggered during the plasma red cell incubation phase. Following implementation, we observed that newly developing warm autoantibodies are detected earlier by the gel method than with the tube test. In these cases, an attempt is made to exclude all common red cell antibodies using select cells. If this is not possible, an antibody screen is performed using the tube test. If the tube test yields negative results, we continue to use the tube method to perform antibody screening and compatibility testing, including the 7 C and AHG phases, until the problem is resolved clinically. Occasionally, we encountered a positive antibody screen that either did not react when tested against panel cells or reacted against miscellaneous cells with no apparent specificity. Using the tube method, these specimens tested negative. Specimens with inconclusive antibody specificity were referred to an independent reference laboratory. Using the tube and gel test for antibody identification studies, they obtained concordant results, demonstrating that miscellaneous reactions were reproducible but without apparent specificity. The following policy for miscellaneous gel reactions was established: perform antibody identification studies to determine the antibody specificity; if all common blood group antibodies can be excluded, issue compatible, gel-crossmatched blood units for transfusion. These miscellaneous reactions, also detected by gel test in our prospective correlation study, remain unexplainable and without apparent specificity. A recent communiqúe from Ortho-Clinical Diagnostics 2 gives a possible explanation for these unexpected reactions. Ortho announced the withdrawal of certain red cell donors whose cells exhibit very weak, inconsistently reactive positive direct antiglobulin test when tested against ID-MTS Anti-IgG gel cards. Gel users were also advised to carefully follow manufacturer instructions when preparing reagent red blood cell suspensions. Cell suspensions more concentrated than 0.8% can affect the sensitivity of the gel test and may result in false-positive results. The smaller sample volume used in gel testing has con- 696 Arch Pathol Lab Med Vol 2, August 999 Gel Test for Indirect Antiglobulin Testing Cate & Reilly

5 tributed to the efficiency of specimen procurement and processing for AHG testing. Smaller sample size is an advantage when phlebotomizing pediatric, oncology, and dialysis patients with smaller or sclerotic veins and has all but eliminated requests for repeat phlebotomies due to insufficient sample quantity. Through intense vendor training, correlation studies, and parallel testing, unanticipated problems were discovered and eliminated prior to the introduction of the gel test for routine patient testing. Our study suggests that the gel test is suitable for routine indirect antiglobulin testing in a community hospital environment. Improved simplicity and quality of testing, with more efficient use of general laboratory personnel, prompted favorable responses from technologists involved with the study and implementation phases and from hospital administrators monitoring costs and personnel management. Lapierre s original goals for the gel test, 5,25 that it be easy to perform, sensitive, and reproducible without sacrificing quality or increasing costs, were achieved in our study. We thank Trident Medical Center, Charleston, SC, for technical and financial support for this study. The proficient secretarial assistance of India Arbenz is greatly appreciated. References. Coombs RRA, Mourant AE, Race RR. Detection of weak and incomplete Rh agglutinins: a new test. Lancet. 95;2:5. 2. Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and incomplete Rh agglutinins. Br J Exp Pathol. 95;26: Coombs RRA. Historical note: past, present and future of the antiglobulin test. Vox Sang. 998;7: Grove-Rasmussen M. Routine compatibility testing: standards of the AABB as applied to compatibility tests. Transfusion. 96;: Standards for Blood Banks and Transfusion Services.rd ed. Chicago, Ill: American Association of Blood Banks; 962:. 6. Garratty G. The role of compatibility tests. Transfusion. 982;22: Beck ML, Tilzer LL. Red cell compatibility testing: a perspective for the future. Transfus Med Rev. 996;0: Mintz PD, Nordine RB, Benry JB, et al. Expected hemotherapy in elective surgery. NY State J Med. 976;76: Friedman BA, Oberman HA, Chadwick AR, et al. The maximum surgical blood order schedule and surgical blood use in the United States. Transfusion. 976;6: Boral LI, Henry JB. The type and screen: a safe alternative and supplement in selected surgical procedures. Transfusion. 977;7: Oberman HA, Barnes BA, Friedman BA. The risk of abbreviating the major crossmatch in urgent or massive transfusion. Transfusion. 978;8:7. 2. Heistø H. Pretransfusion blood group serology: limited value of the antiglobulin phase of the crossmatch when a careful screening test for unexpected antibodies is performed. Transfusion. 979;9: Cooper ES, Walker RH, Schmidt PJ, et al. The 990 Comprehensive Blood Bank Surveys of the College of American Pathologists. Arch Pathol Lab Med. 99;7: Lapierre Y. Introduction. In: Present and Future of the Gel Test: First International Symposium. Morat, Switzerland: DiaMed AG; 99:0. 5. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion. 990;0: Bromilow IM, Adams KE, Hope J, et al. Evaluation of the ID-gel test for antibody screening and identification. Transfus Med. 99;: Lillevang ST, Georgsen J, Kristensen T. An antibody screening test based on the antiglobulin gel technique, pooled test cells, and plasma. Vox Sang. 99; 66: Garratty G. What is the best technique for the detection of red cell antibodies? [International Forum]. Vox Sang. 995;69: Contreras M, Knight R. What is the best technique for the detection of red cell antibodies? [International Forum]. Vox Sang. 995;69: Neppert J, Bartz L, Clasen C. Unsatisfactory detection of an in vivo haemolytic anti-vel by the gel test. Vox Sang. 998;75: MTS Diluent 2 [package insert]. Pompano Beach, Fla: Micro Typing Systems, Inc; Stevens J, Smith JW, Gilcher RO, et al. A clinically significant anti-k not identified by the MTS gel method [abstract]. Transfusion. 998;8(suppl):8S. 2. Fabijańska-Mitek J, Żupańska B, Pogłód R, et al. Evaluation of the microcolumn technology for pretransfusion testing in multiple myeloma patients. Vox Sang. 998;7: MTS Anti-IgG Card [communiqué]. Raritan, NJ: Ortho-Clinical Diagnostics; Malyska H, Weiland D. The gel test. Lab Med. 99;25:8 85. Arch Pathol Lab Med Vol 2, August 999 Gel Test for Indirect Antiglobulin Testing Cate & Reilly 697