Detection of HLA Class I-Specific Antibodies by the QuikScreen Enzyme-Linked Immunosorbent Assay

Transcription

1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, May 1997, p Vol. 4, No X/97/$ Copyright 1997, American Society for Microbiology Detection of HLA Class I-Specific Antibodies by the QuikScreen Enzyme-Linked Immunosorbent Assay DONNA P. LUCAS,* MILLIE L. PAPAROUNIS, LESLIE MYERS, JOHN M. HART, AND ANDREA A. ZACHARY Immunogenetics Laboratory, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland Received 13 September 1996/Returned for modification 21 November 1996/Accepted 24 January 1997 The GTI QuikScreen test is an enzyme-linked immunosorbent assay (ELISA) that uses soluble HLA class I antigens as targets. In tests of 5,893 human serum specimens, we evaluated the reliability, sensitivity, and utility of the GTI QuikScreen test for detecting HLA class I-specific antibody. We found that the test could reliably detect HLA-specific antibodies of the immunoglobulin G (IgG) but not the IgM class. The degree of correlation with lymphocytotoxicity testing varied among the different serum sources, with the best correlation achieved with sera from renal transplant (r > 0.7) and the poorest with sera from patients with end-stage liver disease (r 0.26), possibly because of elevated alkaline phosphatase levels in the liver patients. Test reproducibility was high (96%), and test failure rate was low (1.7%). The test sensitivity is comparable to that of the antiglobulin cytotoxicity and, possibly, even flow cytometric tests. There was a highly significant (P < 0.001) correlation between the optical densities obtained in the ELISA and the percent panel reactive antibody determined by cytotoxicity testing. Therefore, although designed only to determine the presence or absence of HLA-specific antibody, GTI QuikScreen test results also provided an indication of the extent of sensitization. The test is one of the most effective and efficient ways to determine if antibodies producing a positive result in crossmatch tests are specific for HLA class I antigens. As an adjunct to serum screening by cytotoxicity testing, the GTI QuikScreen test can produce a substantial savings of time and effort that reduces the cost to the laboratory and to the patient. One of the most important components of histocompatibility testing for transplantation and for platelet and leukocyte transfusion therapy is detection and characterization of antibodies reactive with the transplanted organ or tissue. Of the various alloantibodies that may be produced, those most commonly involved in organ rejection and tissue destruction are HLA specific (reviewed in reference 13). Failure to detect the presence of these antibodies in patients may result in graft loss, destruction of transfused platelets and leukocytes, unnecessary shipment of organs, and increased time and effort spent on crossmatches. On the other hand, the failure to recognize when antibodies are not HLA specific may prevent transplantation inappropriately. Historically, HLA-specific antibodies have been detected by the complement-dependent cytotoxicity (CDC) test, which requires viable lymphocytes from a panel of individuals selected to represent a wide array of HLA antigens. Characterization of the antibodies present in presensitized patients awaiting transplantation is invaluable for the reasons noted above. However, there are several problems inherent in the CDC assay. It utilizes viable lymphocytes, and factors that affect cell viability can compromise the accuracy of the test results. The effectiveness of rabbit serum, used as a source of complement and heterophile antibody, may vary greatly, and this variability in turn affects test sensitivity. The test cannot be applied to sera from patients being treated with cytotoxic antibodies, such as OKT3, without first removing or blocking those antibodies. The test does not differentiate HLA-specific from other cytotoxic antibodies. Finally, most renal transplant have monthly * Corresponding author. Mailing address: Immunogenetics Laboratory, Johns Hopkins University, 2041 E. Monument St., Baltimore, MD Phone: (410) Fax: (410) serum specimens tested to determine the presence and specificity of antibody that may be present. Such large numbers of tests add significantly to the cost of organ transplantation. Because of the extensive time and labor involved in maintaining a panel, testing sera, and analyzing results, there is a high cost:benefit ratio for tests of sera lacking HLA-specific antibodies. Thus, a simpler and less expensive alternative that does not compromise the quality of patient care is desirable. Several investigators (2, 7, 8, 10) have developed enzymelinked immunosorbent assays (ELISA) that use soluble HLA molecules as targets. Such assays have multiple potential advantages over the lymphocytotoxicity test, including (i) elimination of the need for viable lymphocytes and complement, (ii) detection of only HLA-specific antibodies, (iii) partial automation, (iv) increased objectivity when an ELISA plate reader is used, and (v) rapid turn-around-time. The GTI QuikScreen (GTIQS) test (GTI, Inc., Brookfield, Wis.) is a Food and Drug Administration-approved, commercially available ELISA that uses platelet-derived, solubilized HLA class I antigens from a pool of 600 Caucasian, African-American, and Hispanic blood donors. The antigens are affixed to the wells of microtiter plates via a mouse monoclonal antibody specific for an HLA class I monomorphic epitope. In principle, HLA-specific antibodies present in test sera added to the wells bind to the class I molecules and are detected by the addition of an alkaline phosphatase-conjugated antibody reactive with human immunoglobulin. We have tested 5,893 sera using the GTIQS kit and report our findings here. MATERIALS AND METHODS Serum samples and assays employed. A total of 5,893 serum samples were tested in three phases: (i) a pilot study to evaluate the performance of the GTIQS test; (ii) clinical testing with the GTIQS test, using an anti-human immunoglobulin G (IgG)-IgA-IgM (anti-iggam) antiglobulin; and (iii) clinical testing using an anti-human IgG (anti-igg) antiglobulin. The specimens included 252

2 VOL. 4, 1997 QuikScreen ELISA FOR HLA CLASS I-SPECIFIC ANTIBODIES 253 TABLE 1. Number of serum samples tested Pilot study No. of samples Clinical testing for: IgGAM Patients awaiting transplant of: Kidney 245 2,994 1,406 Liver Heart Lung Platelet support patients Normal human serum HLA typing reagents (human alloantisera) Bone marrow transplant Total 485 3,243 2,165 serum samples from various types of patients, including those awaiting organ or bone marrow transplantation, those who had received an organ transplant, and those who were receiving or were for platelet transfusions; HLA typing reagents that were sera obtained from multiparous women or placental eluates; and sera from healthy, nonsensitized males. The racial distribution of the subjects from whom the sera were obtained was approximately 60% Caucasoid and 40% African-American, with only a few specimens obtained from other racial groups. The number and types of specimens tested in each phase of the study are given in Table 1. All sera in the pilot study and selected sera from phases 2 and 3 of the study were tested in a lymphocytotoxicity assay in addition to the GTIQS test. In addition, 26 sera tested by flow cytometry for donor lymphocyte-reactive antibody were also tested by the GTIQS assay. Test methods. (i) GTIQS assay. The GTIQS test was performed according to the manufacturer s instructions. Briefly, 50 l of control or test serum, diluted 1:2 with specimen diluent solution, was added to duplicate wells in a microtest plate and incubated for 40 min at 37 C. The plate was then washed three times with 225 l of wash solution per well, and 50 l of an alkaline phosphatase-conjugated, affinity-purified goat antibody to human immunoglobulins (1:100 dilution) was added to each well. After incubation for 40 min at 37 C and three additional washes, 100 l ofp-nitrophenyl phosphate solution diluted 1:100 in the enzyme substrate buffer was added, and the mixture was incubated in the dark at room temperature (18 to 22 C). The reaction was stopped after 30 min by the addition of 100 l of ELISA stop solution, and the absorbance of each well at a wavelength of 405 nm was measured in an ELISA plate reader. Reagents provided by the manufacturer were pooled, solubilized HLA class I molecules preloaded into 96-well microtiter plates; wash solution; specimen diluent solution; alkaline phosphatase-conjugated, affinity-purified goat antibody to human immunoglobulin; p-nitrophenyl phosphate reagent; positive and negative control sera; ELISA stop solution; and enzyme substrate buffer. Test wells having optical densities (ODs) equal to or greater than twice the mean OD of the negative control wells were regarded as positive. Whenever the OD reading of either of the duplicate test wells exceeded 20% of the mean OD of the two wells, the test of that serum was considered invalid and was repeated. (ii) Lymphocytotoxicity assays. Sera were assayed using T-cell-enriched lymphocyte suspensions in one of three variations of the microlymphocytotoxicity technique (6): basic, one wash, or antiglobulin. T-cell-enriched lymphocyte suspensions were prepared using magnetic beads coated with a mouse monoclonal antibody specific for human CD2 (Dynal, Inc., Lake Success, N.Y.) (5). In the basic microlymphocytotoxicity technique, 1 l of serum and 1 l of lymphocyte suspension were mixed in the wells of microtest plates (Robbins Scientific, Mountain View, Calif.) and incubated for 30 min at room temperature. Then 5 l of rabbit serum was added as a source of complement and heterophile antibody. After 60 min at room temperature, the cells were stained with FluoroQuench (One Lambda, Inc., Canoga Park, Calif.), a commercially available stain containing ethidium bromide and acridine orange, and trays were examined by fluorescence microscopy to determine percent cell death. In the one-wash technique, cells were washed with 10 l of phosphate-buffered saline per well prior to the addition of rabbit serum. The antiglobulin method incorporated three wash steps and the addition of 1 l of goat IgG, anti-human kappa-chain polyclonal antibody (catalog no ; Incstar Corp., Stillwater, Minn.) prior to the addition of rabbit serum. (iii) Flow cytometry assays. Flow cytometric crossmatches (FCXM) were performed using a Becton Dickinson FACSCalibur cytometer following the method of Bray (1). T and B lymphocytes were stained with phycoerythrin-conjugated mouse monoclonal antibodies (Becton Dickinson, San Jose, Calif.) specific for human CD3 and CD19, respectively. The presence of lymphocyte-reactive antibody was detected using a fluorescein isothiocyanate-conjugated anti-human IgG IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Fluorescence was measured on a log scale, and positive reactions were considered to be shifts in the median fluorescence of 50 channels for T cells and 100 channels for B cells compared to the median fluorescence of the negative controls. Antibody characterization. Lymphocytotoxicity tests were performed with cells obtained from individuals whose HLA phenotypes had been well-characterized. A panel of 65 individuals representing nearly all HLA class I antigens was used with the basic and one-wash test methods, and a panel of 20 individuals representing the major class I crossreactive groups (4, 9) was used with the antiglobulin test. A panel reactive antibody (PRA) value, given as the percentage of the panel with which the serum gave a positive reaction (cell death of 20%), was determined for each serum. A serum sample with a PRA of 2% was considered positive for the presence of antibody. Serum reaction patterns were evaluated for HLA specificity by a computerized inclusion analysis program (HLA Vision Software, Biotest, Inc., Denville, N.J.), and the assigned antibody specificities were verified by manual analysis. Putative identification of antibody class was achieved by incubating sera at 63 C for 10 min to inactivate IgM antibodies. Analysis. Chi-squared analysis was used to evaluate the correlation between different techniques for detecting the presence of lymphocytotoxic and/or HLAspecific antibody. Student s t test was used to evaluate differences in the mean OD readings of duplicate wells. RESULTS Pilot study. (i) Correlation between GTIQS and lymphocytotoxicity assays. We compared the results of tests of 485 sera tested by the GTIQS and CDC assays for the detection of antibody. The results, categorized by serum source and type of CDC assay employed, are provided in Table 2. The correlations between the two types of assay were statistically significant for tests of patients awaiting kidney or liver transplant and for tests of sera from platelet support patients, but there were no categories in which the two types of assay were completely concordant. Instances in which reactivity is detected by the GTIQS assay but not by CDC assay (GTI /CDC ) may occur with antibodies that do not activate complement effectively. The data suggest that this may account for some of the occurrences of GTI /CDC results, since the rate of these events was lowest for comparisons with the antiglobulin CDC assay, which permits detection of antibodies that do not activate complement. Instances of GTI /CDC results could be due to lymphocytotoxic antibodies that are not HLA specific or to the failure of GTIQS to detect some HLA-specific antibodies, particularly those of the IgM class that might be removed during the multiple wash steps. To investigate the latter possibility, we incubated several sera suspected of having HLA- Cytotoxicity technique Basic or one wash TABLE 2. Serum screening results: GTIQS versus lymphocytotoxicity No. of samples showing GTIQS/cytotoxicity reactions of: r a P First renal transplant b Renal retransplant b Liver transplant b Heart transplant b Lung transplant b ND Basic HLA typing reagents c All Nonsensitized males Total Antiglobulin Platelet support or patients a Correlation coefficient. ND, not determined. b Patients awaiting transplantation. c Predominantly from multiparous females.

3 254 LUCAS ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 3. GTIQS versus cytotoxicity: known IgM antibodies eliminated a No. of samples showing GTIQS/ cytotoxicity reactions of: Patients awaiting first renal transplant Patients awaiting renal retransplant Platelet support patients HLA typing reagents a Sera are those shown in Table 2, excluding those with IgM class antibodies. b Correlation coefficient. specific antibody(ies) of only the IgM class at 63 C for 10 min to aggregate IgM and then retested the sera in the CDC assay. We found that GTIQS did not detect IgM HLA-specific antibodies consistently. That is, some of the sera whose CDC reactivity was abrogated by heat inactivation were reactive in the GTIQS assay, while others were not. We also found that IgM antibodies accounted for most of the GTI /CDC events. The correlations between the two assays for all serum groups examined improved when sera whose reactivities were abrogated by heat inactivation were excluded from the analyses, as shown in Table 3. The amounts of the different HLA antigens present in the preparation of soluble antigens should not be equal but rather should be proportional to their frequencies in the population from which the platelets were obtained. Also, the amounts of some antigens, notably B8, B44, and the HLA-C antigens, are likely to be disproportionately lower than expected because they usually occur at a lower density on platelets (3, 15). To determine if GTI /CDC results might be obtained with antibodies specific for antigens with lower population frequencies or HLA-C antigens, we tested 29 sera that are operationally mono- or duospecific in the CDC assay and that are used in our laboratory as HLA typing reagents. Of these, 24 were positive in the GTIQS assay. Three of the sera that were negative in the GTIQS assay were found to have HLA-specific antibodies of the IgM class. When these were eliminated from the analysis, the GTIQS assay was positive with 92% (24 of 26) of the sera. The specificities of the sera giving positive and negative reactions are provided in Table 4. We then determined if the strength of reactivity in the GTIQS assay (as measured by the OD ratio) was proportional to the amount of antigen expected from its population frequency. For the 24 sera reactive in the GTIQS assay, we calculated the frequencies of the antigens for which the antibodies were specific in a population having the same ethnic composition as the pool of platelet donors (i.e., one-third each African-American, Hispanic, and Caucasian). Allele frequencies from the United Network for Organ Sharing donor registry (14) were used to calculate the antigen frequencies. We found no correlation (r 0.01, P 0.95) between the antigen frequencies and the OD ratios obtained in the GTIQS test. (ii) Duplicate testing. We performed duplicate tests of 102 sera, testing each on different trays. Fifty-eight of the sera were tested on trays from the same lot, and 47 sera were tested on trays from different lots. The concordancy rate was 96% for both intralot and interlot comparisons. Sera containing only IgM, HLA-specific antibodies were excluded from the evaluation. Clinical tests. Based on the outcome of our pilot study, we implemented the GTIQS assay as an adjunct to our cytotoxicity r b P screening for testing sera that met any of the following criteria: (i) a low probability of reactivity in the CDC assay, (ii) necessity for rapid results (the initial sample obtained from renal transplant, samples from for transplant of organs other than kidneys, samples from platelet support patients, and samples used in a pretransplant crossmatch for live donor transplantation), and (iii) samples containing OKT3. Samples selected for duplicate testing by the one-wash CDC technique were every fourth sample obtained from patients awaiting renal transplantation and all samples from organ transplant that gave positive results on the GTIQS test. During the course of our testing, an antiglobulin reagent specific for human IgG was substituted, by the manufacturer, for the anti-iggam reagent used initially. Below, we present results obtained with each of the two antiglobulin reagents used. (i) CDC reactivity of GTIQS-reactive sera. As noted above, the clinical testing protocol requires that certain sera be tested by both GTIQS and CDC assays. Of interest to us was the level of cytotoxicity occurring among sera that gave positive results in the GTIQS assay. As of this writing, CDC screening data were available from 361 GTIQS-tested sera from renal transplant. We found that the basic or one-wash CDC assay detected reactivity in approximately only one-half (43 to 50%, depending on the specificity of the antiglobulin reagent used in the GTIQS assay) of the sera reactive in the GTIQS assay. We analyzed the degree of correlation between the OD ratio obtained in the GTIQS assay and the PRA value obtained in CDC testing for various groups of patients, and the data, differentiated by specificity of the antiglobulin reagent, are given in Table 5. There was a significant correlation for all comparisons, with the highest correlation among the various patient groups occurring in the renal transplant patients. Among heart and liver patients, a better correlation was achieved with the anti-iggam antiglobulin reagent than with the anti-igg reagent. This may be due, in part, to a higher frequency of IgM antibodies among these patients. When we compared the correlations obtained with all sera versus those with a PRA value greater than 10%, we found better correlations with the first group because of the large proportion of sera in that group that were negative by both assays. When all patient groups were considered collectively, a better correlation between the PRA and OD ratio was obtained with the anti-igg reagent for sera with a PRA of 10%. (ii) Frequency of reactivity detected by the GTIQS assay. Of the sera tested, 5,177 were from patients awaiting renal, liver, or heart transplantation and from platelet support patients. We analyzed differences in the frequency of positive reactions occurring among the different groups of patients and between the two antiglobulin reagents used in the GTIQS assay. The frequency of positive reactions obtained with the GTIQS test and the results of the chi-squared tests are given in Table 6. TABLE 4. Results of GTIQS tests of (IgG) HLA typing reagents Positive by GTIQS Negative by GTIQS A1 B8 B51 B14 A3 B12 B62 C5 A9 B13 B7, B42 A10 B15 B37, B44 A11 B17 B41, B42 A23 B18 C1 A28 B21 C2 A30, A31 B40 B45

4 VOL. 4, 1997 QuikScreen ELISA FOR HLA CLASS I-SPECIFIC ANTIBODIES 255 TABLE 5. Correlation between OD ratio (GTIQS) and PRA (CDC) a All sera Sera with PRA of 10% Anti-IgGAM b Anti-IgG Anti-IgGAM Anti-IgG r c P r P r P r P All 0.78 (366) (212) (84) (39) Heart patients 0.73 (65) (35) NS d NS Kidney patients 0.78 (218) (143) (68) (34) Liver patients 0.69 (83) (34) NS NS a Numbers in parentheses are numbers of specimens. b Specificity of antiglobulin reagent. c Correlation coefficient. d NS, number not sufficient for analysis. The frequency of positive reactions differed significantly among the various groups of patients, regardless of the antiglobulin reagent used. A significant difference in the frequency of positive reactions (8.0 versus 11.6%) with the two antiglobulin reagents was seen only in the renal transplant. (iii) Results of tests in duplicate wells. In the GTIQS test, all sera are tested in duplicate on the same tray. We compared the means of the OD readings of these duplicate wells, and the results with both antiglobulin reagents, differentiated by patient group and test interpretation (positive or negative), are given in Table 7. There were significant differences between the means of duplicate wells for all groups except platelet support patients in tests using the anti-iggam reagent. With the exception of the platelet support patients, the differences between the means were larger for all groups tested with the anti-iggam reagent (5.8 to 12.6%) than for tests with the anti-igg reagent (1.4 to 3.1%). (iv) Test failure rate. There were 15 instances in tests of 880 sera (1.7%) in which the GTIQS assay yielded invalid and thus nonreportable results. In our experience, the failure rate in CDC testing is 1.3%, considering the reaction between a serum and a single lymphocyte preparation to be an individual test. (v) Comparison between GTIQS assay and FCXM. FCXM tests of 27 sera were also tested by the GTIQS assay and, in some cases, by one-wash CDC antibody screening and/or antiglobulin CDC crossmatch. The results of these tests are given in Table 8. In the FCXM, 13 sera were reactive with both T and B lymphocytes, and two sera were reactive with B but not with T lymphocytes. All 13 T-cell-reactive sera gave positive results in the GTIQS assay. Nine of these 13 sera were tested in the antiglobulin CDC crossmatch test, and of these, six were positive in the CDC. The two sera that were positive only with B cells in the FCXM were shown to have HLA class II-specific antibody and were not reactive in the GTIQS assay. Three sera gave positive reactions in the GTIQS assay but not in the FCXM. In all three cases, the patient had been shown, by CDC screening, to have antibody specific for HLA antigens not present in the donors. DISCUSSION We evaluated the GTIQS assay in tests of nearly 6,000 human serum samples for the ability to detect HLA class I-specific alloantibody. In our evaluation, we examined reactivities of sera in the GTIQS assay compared to that in the CDC assay, impact of antibody isotype, intratest and intertest reproducibility, test failure rate, impact of serum source, test sensitivity, and cost. We found a significant correlation (r 0.56 to 0.59) between the GTIQS assay and the CDC assay regardless of the type of CDC assay (basic, one wash, or antiglobulin) used. The degree of correlation varied among the different groups from which the sera were obtained, with the best correlations occurring with sera from renal patients, pre- and posttransplant (r 0.66), and the poorest occurring with patients awaiting liver transplantation (r 0.26). It is likely that the poor correlation in tests of sera from end-stage liver disease patients is due to elevated serum levels of alkaline phosphatase, for which the chromogen used in the assay is a substrate, although this was not proven in our testing. The correlations improved appreciably (r 0.73 to 0.78) when sera with cytotoxic reactivity due to IgM antibodies were excluded from the analysis, primarily because of reduction in the numbers of sera that were positive by the CDC assay and negative by the GTIQS assay. This was not surprising, since the multiple wash steps used in the GTIQS assay probably remove many antibodies of the IgM class. Similarly, reproducibility was high (91 to 96%) and improved (96%) when sera with IgM antibodies were excluded from the evaluation. The test failure rate of the GTIQS assay (1.7%) was comparable to that of the CDC assay (1.3%). When we tested sera that were operationally monospecific in the CDC assay by the GTIQS assay, we found that the GTIQS assay detected the presence of antibody specific for antigens that had frequencies of 1% in the population. By comparison, Shroyer et al. (11), in flow cytometric tests using a mixture of lymphocytes from different subjects, found that reactivity with one individual could be detected with a pool of up to six individuals, a threshold frequency of 16%. In reality, most sera that are monospecific by the CDC assay contain multiple antibodies with different HLA specificities. However, since most sensitized patients have antibodies that are oligo- or multispecific by the CDC assay, the results obtained here suggest that the GTIQS assay identified presensitized patients reliably. Sera from healthy males, shown to be nonreactive in both flow cytometric and antiglobulin CDC assays, were nonreactive in the GTIQS assay. Two different antiglobulin reagents, one specific for IgG, TABLE 6. Frequency of reactivity in GTIQS assay: tests categorized by serum source and antiglobulin specificity No. (%) of specimens with antiglobulin specificity Anti-IgGAM a Anti-IgG b Renal transplant 2,994 (8.0) 1,406 (11.6) Liver transplant 113 (15.9) 54 (14.8) 0.03 Heart transplant 76 (9.2) 53 (9.4) Platelet support patients 43 (27.9) 438 (17.4) a ; P b ; P P

5 256 LUCAS ET AL. CLIN. DIAGN. LAB. IMMUNOL. TABLE 7. Comparison of means of OD readings of duplicate wells Antiglobulin Group Mean OD reading Well 1 Well 2 % Difference r b P a Anti-IgGAM All Negatives Positives Kidney transplant Liver transplant Heart transplant Lung transplant Platelet support patients Anti-IgG All Negatives Positives Kidney transplant Liver transplant Heart transplant Platelet support patients Bone marrow transplant patients a Determined by (OD well 2 OD well 1)/OD well 1, where OD well 1 OD well 2. b Correlation coefficient. IgA, and IgM and the other specific for only IgG, were used in the course of our clinical testing. We found the anti-igg reagent to be more sensitive and more reliable than the anti- IgGAM reagent. In tests of 4,400 patients awaiting kidney transplantation, we found that the frequency of positive reactions increased from 8% with the anti-iggam to 11.6% with the anti-igg. The vast majority of these sera were multiple, sequential, monthly samples obtained over a 7-month period from patients on our renal transplant waiting list. Since we did not observe a similar increase in the frequency of reactivity by CDC testing, it is likely that the increased reactivity seen with the anti-igg reagent reflects an increased test sensitivity obtained with that reagent. Comparisons of the mean ODs of duplicate test wells indicate that the anti-igg reagent is more reliable. The differences in the mean values of duplicate wells range from 2.2 to 12.6% with the anti-iggam reagent but are reduced to 1.4 to 3.1% with the anti-igg reagent. Some of the differences occurring with the anti-igg reagent were statistically significant. However, since small differences can reach statistical significance when large numbers of tests are performed, as was done here, the differences are not necessarily biologically significant. Differences in the OD readings of duplicate wells are important when the interpretation of the test results, i.e., positive versus negative, would be different for the two wells. We found the incidence of this situation to be approximately 3.8%, which is comparable to differences seen in duplicate tests in the CDC assay. In reality, the majority of these cases involved differences of 0.1 or less in the value of the OD ratio (i.e., 1.9 versus 2.0), which could be attributed to normal test variability. Considering the low incidence of these events and the minimal time and cost necessary for repeat testing by the GTIQS assay, we believe that the degree of difference in the OD readings of duplicate wells is within acceptable limits. The GTIQS assay is designed to provide information only about the presence or absence of HLA class I-specific antibody, i.e., a positive or negative result. However, in tests of patients sera, we found that the OD ratios obtained in the GTIQS test correlated significantly with PRA values obtained in cytotoxicity testing. There was no correlation between the OD ratios and the expected frequencies of the specificities in tests of mono- and duospecific sera used as HLA typing reagents. Although these two results appear contradictory, the reasons for the differences in the two comparisons may include differences in the nature of the sera, with sera from patients having a larger number of different antibodies than were present in the reagent sera; chance differences in the actual antigen frequencies of the platelet donors versus those that were predicted; differences between the actual antibody content and the apparent antibody content as defined by CDC; and differences between PRA values obtained with a panel selected for HLA type and the frequency of reactivity that would be seen with a random population (12). However, what is important is that the results of GTIQS tests performed on sera from patients provide an indication of the extent of sensitization of the patient being tested. A serious deficiency in the basic and one-wash lymphocytotoxicity assays, the assays most commonly used for antibody screening, is the failure to detect HLA-specific antibody that does not activate complement, either because there is insufficient antibody present or because the antibody is of an isotype that does not activate complement effectively. This deficiency is overcome with the antiglobulin CDC and the flow cytometric TABLE 8. Comparison of results: GTIQS assay versus FCXM, antiglobulin crossmatch, and antibody screening by lymphocytotoxicity a Serum FCXM GTIQS assay Result in: Antiglobulin crossmatch PRA 1 ND 2 ND ND 11 ND ND 12 ND ND ND 0 15 ND ND 16 ND ND ND 0 24 T, B T, B ND 27 ND 29 a Abbreviations: ND, not done; T, T cells; B, B cells.

6 VOL. 4, 1997 QuikScreen ELISA FOR HLA CLASS I-SPECIFIC ANTIBODIES 257 assays. However, both the time and the cost of these assays are greater than those of the less sensitive CDC assay. Our initial comparisons between the GTIQS assay and the antiglobulin CDC assay indicated that the sensitivity of the GTIQS assay was comparable to that of the antiglobulin CDC test. Preliminary data from flow cytometric testing suggest that the GTIQS assay may be as sensitive as that assay. The sensitivity of the GTIQS test permitted us to detect HLA-specific antibodies that would not have been detected by our routine CDC screening, as only 50% of the GTIQS-reactive sera from renal transplant were also reactive by the CDC assay. We believe this is an important advantage of the GTIQS test. In many histocompatibility laboratories, crossmatch tests are performed by more sensitive assays, such as antiglobulin CDC or flow cytometry, than are antibody screening tests. In these situations, positive crossmatches can and do occur in apparently nonsensitized individuals. It is difficult to interpret the clinical relevance of these positive crossmatches, because it is not known whether the antibody detected is HLA specific or not. Of the various methods that can be used to determine antibody specificity, including absorption, blocking, and antibody screening by antiglobulin CDC, flow cytometry, or GTIQS assay, serum testing by the GTIQS assay is the most rapid and cost-effective. We have instituted a policy of testing all sera used in a final, pretransplant crossmatch test by the GTIQS assay and found this to be a very useful aide to crossmatch interpretation. Also, like the flow cytometry test, we found that the GTIQS assay was useful in tests of sera from patients being treated with OKT3. Such sera are, routinely, panreactive in CDC assays even when HLA-specific antibodies are absent. Tests of sera containing OKT3 but lacking HLA-specific antibodies were consistently negative by GTIQS. Finally, the addition of the GTIQS test system to our antibody testing protocols has provided us with a substantial savings (ca. 37%) in our antibody screening costs that has permitted us to decrease our charges for these tests. Further, the substantial time saved through the use of this test has allowed us to put additional effort into more thorough characterization of antibodies detected. In conclusion, we believe our studies demonstrate that the GTIQS assay is a sensitive, rapid, reliable test for detecting the presence of HLA class I-specific antibodies of the IgG class. Further, utilization of the test as an adjunct to tests that use lymphocyte targets can result in a substantial cost saving, decrease the personnel time needed for antibody testing, and provide information useful in the interpretation of crossmatch test results. REFERENCES 1. Bray, R. 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