ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC ...

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ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC ...

  1. 1. 1 ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC DIALYSIS Andrea A. Zachary1, Donna P. Lucas1, Barbara Detrick 2, Mary S. Leffell1 Departments of Medicine1 and Pathology2 The Johns Hopkins University School of Medicine, Baltimore, MD Corresponding Author Andrea A. Zachary, PhD, D(ABHI) Immunogenetics Laboratory The Johns Hopkins University School of Medicine 2041 E. Monument Street Baltimore, MD 21205 aaz@jhmi.edu phone: 410-614-8978 fax: 410-955-0431 Keywords IgM Elimination Antibody testing Non-specific reactivity Crossmatch testing Abbreviated title: Effect of IgM Elimination on Antibody Assays page 1 of 22
  2. 2. ELIMINATION OF INTERFERENCE IN ANTIBODY ASSAYS BY HYPOTONIC DIALYSIS Abbreviations SPI solid phase immunoassays L liter MFI mean fluorescence intensity PBS phosphate buffered saline Abstract Non-HLA-specific antibodies, IgM antibodies, and immune complexes can result in increased background reactivity and can interfere with binding of HLA-specific antibodies, abrogating clinical utility of the antibody test results. We present data here showing that immunoglobulins of the IgM class can block binding of IgG HLA-specific antibodies and that precipitation and removal of IgM by hypotonic dialysis can restore normal reactivity to some sera that have high background in solid phase assays or unexplainable positive reactions in crossmatch tests. The IgM-depleted fractions showed normal reactivity of negative controls and clear specificity and reaction strengths that correlated with those of crossmatch tests. We also show that removal of IgM by filtration reduces non-specific background but also reduces the strength of specific reactions compared to those of IgM-depleted fractions obtained by hypotonic dialysis. Hypotonic dialysis of sera with normal background levels and patterns of reactivity, does not alter the specificity but may slightly reduce the strength of some specific reactions. Introduction Two types of assays, differentiated by target, are used for the detection and identification of HLA-specific antibodies: [1] the cytotoxicity and flow cytometric assays that use lymphocyte targets and [2] solid phase immunoassays (SPI) that use soluble HLA molecule targets. Lymphocyte-based assays are fraught with multiple technical issues including inappropriate reactions due to reduced cell viability, variability in antigen expression, and the presence of lymphocyte binding antibodies that are not specific for HLA. False positive reactions have been particularly problematic when B lymphocytes are used as targets (1,2). SPI have resolved many of the problems associated with lymphocyte-based assays, have superior sensitivity and specificity, require small serum volumes, and can be partially or fully automated (3-7). The result is the ability to obtain results very rapidly, detect the presence of antibody to donor HLA page 2 of 22
  3. 3. before clinical signs of antibody-mediated rejection appear, and more readily identify epitope- specific antibodies. The high degree of sensitivity and specificity and the ease with which antibody specificity can be determined permits differentiation of clinically relevant and irrelevant crossmatches enhancing the clinical interpretation of test results. Although SPI have fewer technical problems than do cell-based assays, like any immunologic assay, they are not completely problem free (7-9). Binding of antibodies not-specific for HLA antigens or of immune complexes may result in high background reactivity and may interfere with binding of HLA-specific antibodies. The effects may be increased values for negative controls, decreased values for positive controls, and/or alteration of the HLA specificities detected. In turn, this may result in a failure to detect antibodies present and confound interpretation of crossmatch results. We have found that high background in SPI often occurs when there are high levels of IgM autoantibodies or immune complexes present. Commercially available products designed to reduce background resolve the issue in a some cases, but not most cases. Others have shown that neither reducing agents nor heat aggregation eliminate IgM immunoglobulins completely (10). IgM immunoglobulins are water insoluble euglobulins while IgG immunoglobulins are water soluble pseudoglobulins. Dialyzing serum against distilled water results in precipitation of IgM permitting an effective elimination of IgM (11). We present here data showing that removal of IgM by hypotonic dialysis eliminates or reduces non-specific reactivity from SPI and crossmatch tests sufficiently to provide clinically meaningful results. Materials and Methods Sera Sera were from 16 renal renal transplant candidates. Selection of sera was based on the following criteria: negative controls values above expected (4 sera), specificity and/or strength of donor-specific antibody that did not correlate with crossmatch results (3 sera), or both (7 sera). Two sera that did not meet these criteria were also tested as controls. All sera were tested as untreated sera and as the IgM-depleted pseudoglobulin fraction by multianalyte bead assay on the Luminex® platform using panels appropriate for determining antibody specificity page 3 of 22
  4. 4. and strength. The total number of such tests exceeds the number of sera as some sera were tested with both phenotype and single antigen panels and some sera were tested for both class I and class II HLA specificity. Some of IgM-depleted fractions were also tested in crossmatch tests against donor cells previously tested with untreated sera. A subset of sera had immunoglobulin fractionation performed using spin columns and the IgG portion of each was tested on the Luminex platform. Antibody Characterization Sera were tested using phenotype panels (Lifematch class I, class II,, Tepnel LifeCodes, Stamford, CT) and/or single antigen panels (Single antigen beads, One Lambda, Canoga Park, CA) on the Luminex platform, according to the manufacturers’ instructions. Results were expressed as specificity and as mean fluorescence intensity (MFI) values. A subset of sera were tested with the DynaChip® ELISA system ( Invitrogen, Corp., Carlsbad, CA) according to manufacturer’s instructions. Specificity analysis was performed manually by two to three highly experienced individuals. Reaction strength in single antigen panel tests were made using raw values to avoid the impact of elevated negative control values of the untreated sera. Crossmatch Testing Cytotoxicity tests were performed with positively selected T and B lymphocytes using antiglobulin-enhanced and one wash procedures, respectively, as described previously (1). Three color flow cytometric crossmatches were performed on a Becton-Dickinson FACSCalibur as described, previously (5). The second antibody used was an IgG heavy chain-specific, mouse monoclonal antibody (BD Biosciences, San Jose, CA). Results were expressed as a ratio of the median channel fluorescence of the test serum to that of the negative control. Hypotonic Dialysis Dialysis of serum was performed using Pierce Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific, Rockford, IL), according to manufacturer’s instructions. Briefly, hydrated cassettes were loaded with 500w of the serum of interest and dialyzed against 4L of deionized water, l page 4 of 22
  5. 5. overnight (14-16 hours) in the cold (2-8oC), with stirring. After overnight dialysis and when a euglobulin precipitate was visible, the contents of the cassette was removed and centrifuged for one hour at 15-20K x g at 2-8oC to collect the precipitated IgM-containing euglobulin fraction. The precipitated euglobulin fraction was resolubilized in isotonic, buffered saline. The supernatant fluid was removed and placed into a new dialysis cassette and back dialyzed against 2 changes of 4L of phosphate buffered saline overnight at room temperature to restore isotonicity. At the end of the second dialysis, the IgM-depleted fraction was removed from the cassette and reserved for antibody testing. Spin Column Immunoglobulin Fractionation Separation of IgG and IgM immunoglobulin was performed using the 300K Nanosep® Centrifugal Device (Pall Corporation, East Hills, NY). Briefly, 50Cl of serum was loaded into the sample reservoir, covered, and spun at 14,000xg in a fixed angle centrifuge for 12 minutes. The IgG portion of the serum was recovered from the lower chamber of the device. Measurement of IgM Depletion IgM immunoglobulin levels were measured in the serum and IgM-depleted fraction by rate nephelometry (IMMAGE 800 System, Beckman Coulter, Inc, Fullerton, CA, USA) (12). These tests showed that while there was some IgM remaining in the pseudoglobulin fraction, the amount had been diminished by 67-82%. RESULTS Unless otherwise specified, IgM-depleted fraction refers to the soluble fraction remaining after hypotonic dialysis. Comparison of Control Bead Results Both the phenotype and single antigen panels have a positive control bead. The phenotype panel has three negative control beads and the single antigen panel has one. Comparison of the results obtained with these control beads for untreated sera and the IgM-depleted fractions are given in Table 1. Some of the IgM-depleted fractions yielded different specificities than did page 5 of 22
  6. 6. the untreated sera. Therefore, the results in Table 1 have been separated into two categories according to whether or not dialysis treatment resulted in a change in antibody specificity. For some sera, changes were observed for only one of the two classes of specificity and the control bead results were categorized, accordingly. In all categories, the mean values of the positive controls were higher for the IgM-depleted fractions than for the untreated sera while the mean values of the negative controls were lower. This was true not only for mean values but for values of each individual serum. Specificity Comparisons The specificity assignments obtained with phenotype and single antigen panels, for cases where the specificities of the untreated sera and IgM-depleted fractions differed, are given in tables 2 and 3, respectively. The specificity data for sera with no change in specificity after IgM depletion by dialysis are not shown. In tests with phenotype panels, four sera, 5, 6, 8, and 16, had no specificity identifiable in the untreated serum but had clearly identifiable specificities in the IgM-depleted fraction. For serum 12, class I specificity could be determined only in the IgM-depleted fraction. Class II specificity was determined in both the untreated and IgM- depleted fractions but the specificities were substantially different (DR1 and DR4 in the untreated serum and DR1 and DR52 in the IgM-depleted fraction). When a specificity was determined, the MFI value given in Table 2 is for the highest ranking phenotype with reactivity due to the assigned specificity. When specificity could not be assigned, the MFI is for the highest ranking phenotype overall. In all cases where specificity changes occurred, the reactivity strength was greater with IgM-depleted fraction than with untreated serum. There were several sources of data indicating that the dialysis procedure did not produce an artifactual change in specificity or reaction strength. Two control sera which did not have increased background reactivity in the negative controls nor disparities in the strength of antibody determined by antibody screening and crossmatch tests, showed no specificity differences between the IgM-depleted fractions and untreated sera tested with phenotype panels. As noted above, some sera demonstrated high background in tests for only one of two HLA antibody specificity classes. There were two sera, 5 and 8, which had high background in tests for class I-specific but not in tests for class II-specific antibodies. For both sera, IgM depletion resulted in specificity changes only for class I-specific antibodies - i.e., those tests in which high background had occurred with untreated sera. Further, specificity changes did not page 6 of 22
  7. 7. always occur after IgM-depletion of a serum with increased background. There was one serum (serum 10) that had increased reactivity in the negative controls but had no specificity differences between the IgM-depleted fraction and the untreated serum (data not shown). In all cases except one, in which there was no specificity change, the reaction strength was less for IgM-depleted fraction than for untreated serum. In the one exception, only one of two specificities had lower strength in the IgM-depleted fraction than in the untreated serum. This is in contrast to the results given above when the specificities obtained with the two components were different and the reactions were uniformly stronger with the IgM-depleted fraction. Ten untreated sera and corresponding IgM-depleted fractions were tested with single antigen panels and all showed differences in the specificities of the five highest ranking beads (ranked in order of descending strength). Seven of these pairs of components, sera 5-9, 13, and 16, had complete differences - i.e., there were no beads in common among the top five - while for 3 pairs, 2, 3, and 11, there were some beads in common. In all but one case (serum 7) the reactions were stronger with the IgM-depleted fractions than with the untreated sera, even for beads that were the highest ranking with both fractions. In some cases the specificity differences were not significant. For example, for serum 5, four of the five strongest reacting beads of the untreated serum were not among the 5 highest ranking beads of the IgM-depleted fraction but had strong reactions with the IgM-depleted fraction with MFI values ranging from 11,767 to 16,899. However, the A3002 bead was among the five highest ranking beads for the untreated serum 5 (MFI = 13271) but was one of the lower ranking beads for the IgM-depleted fraction (MFI= 5278). Importantly, the rank order of the beads was changed in all cases. Correlation With Crossmatch Results We have established correlations between MFI values and crossmatch strength based on numerous comparisons. Our investigation was undertaken, in part, because of cases in which the strength of reactions in the antibody identification tests could not be reconciled with those of the crossmatch tests. There were 10 sera tested in solid phase assays and used in 12 crossmatch tests. The IgM-depleted fractions of nine sera were tested in solid phase assays page 7 of 22
  8. 8. and seven of the crossmatches were repeated using IgM-depleted fractions. Table 4 provides the MFI values for donor-specific antibodies and the strength of crossmatches with the respective donors. Reactivity of the untreated sera included: [1] MFIs that were too low to account for the strength of ten crossmatch tests - nine CDC (sera 3, 5, 6 in 2 crossmatches, 7, 8, 9, 11, and 16) and one flow cytometric (serum 12) crossmatch; [2] a positive flow cytometric crossmatch with T cells in the absence of class I-specific antibody (serum 7); and [3] positive allogeneic and autologous crossmatch test results (serum 14). Using the IgM-depleted fractions, antibody identification tests were repeated for nine sera and seven crossmatch tests were repeated. The MFI values for the IgM-depleted fractions were increased for 8 sera (sera 3, 5, 6, 8, 9, 12, and 16) to become proportional to the strength of the crossmatches performed with the untreated sera. The IgM-depleted fraction showed no change in titer in three repeated crossmatch tests involving sera 5, 11, and 12, and a slight decrease in the titer of serum 16, all of which were consistent with the MFIs obtained with the IgM-depleted fractions of those sera. The IgM-depleted fraction of serum 7 yielded a negative cytotoxicity crossmatch and a flow cytometric crossmatch that was negative with T cells and of reduced strength, compared to that obtained with untreated serum, with B cells. The crossmatch results for the IgM-depleted fraction of serum 7 were now consistent with both the untreated serum and the IgM-depleted fraction of serum 7. Both allogeneic and autologous crossmatches performed with untreated serum 14 were positive while those performed with the IgM-depleted fraction were negative and correlated with the low MFI values obtained in the solid phase assays of the untreated serum. For this serum, the solid phase immunoassays were not repeated with the IgM- depleted fraction since the results of control beads with the untreated serum were within normal ranges. In all cases, the IgM depleted fractions permitted reconciliation of the results of crossmatch and solid phase tests and meaningful clinical interpretation. Tests of Sequential Sera We compared the results of tests of the untreated serum and IgM-depleted fraction of serum 12 with the results of tests of two previous sera from the same serum donor. These data are given in Table 5. The MFIs for the class I phenotype panel control beads and for the B locus antigens for the six strongest reacting phenotypes are provided. The patient had demonstrated antibody to HLA-B7 for several months up to the day before transplantation (T-1). Two days page 8 of 22
  9. 9. after transplantation (T+2) we observed an increase in the reactivity of the negative control beads of the class I phenotype panel and an even greater increase in the reactivity of the negative control beads of the cII phenotype panel (data not shown). Four days after transplantation (T+4), the increased background reactivity continued and no specificity could be determined for class I reactivity. The HLA-B7-bearing phenotypes were now found among the 15 lowest reacting phenotypes. The predominant class II-specific antibody, which had been DR17 was now DR1 and DR4 (data not shown). A test of the IgM-depleted fraction of the T+4 serum showed a restoration of normal reactivity in the negative controls and of the B7, DR17 specificity patterns seen in previous sera. It further revealed a very substantial increase in the strength of those antibodies. Procedure Modifications In an attempt to shorten the procedure time, we evaluated spin columns that provide separation of IgG and IgM fractions and also tested the impact of reducing the times of the two incubations of the hypotonic dialysis procedure. Results obtained with the IgM-depleted fraction and IgG fraction obtained by spin column separation of nine sera are shown in Table 6. Spin column separation was as effective as hypotonic dialysis in reducing background and providing appropriate levels of reactivity with control beads in the solid phase assays. However, in all cases, the HLA-specific reactivity of the IgM-depleted fraction obtained by dialysis was stronger than that of the IgG fraction obtained by spin column separation. Also, there were 3 sera (3, 6, and 16) for which the 5 strongest reacting single antigens beads were not the same for the two different preparations. We have found that precipitation of the euglobulin fraction requires the overnight (ca. 16 hour) dialysis in the cold. To assess the time of back dialysis against PBS needed to restore isotonicity, we tested aliquots of the pseudoglobulin fraction taken immediately after dialysis against distilled water and taken at two hour intervals after the start of dialysis against PBS. Washed, packed red cells were resuspended in the various fractions and examined for lysis. We found that after 4 hours of dialysis against PBS, no red cells lysis occurred. Further, lymphocytotoxicity tests using the preparation obtained after 4 hours of back dialysis against PBS yielded no inappropriate lymphocyte death. page 9 of 22
  10. 10. Tests of Sera in ELISA DynaChip™ is an automated ELISA performed on glass microchips that contain single class I and class II phenotypes. We tested 5 untreated sera on the DynaChip™ platform. For all 5 sera, the background reactivity was significantly reduced compared to tests of the untreated sera on the Luminex platform. Four of the five sera were panreactive making it difficult to determine antibody specificity precisely. However, the fifth serum was the untreated T+4 serum shown in Table 6. Tests of this serum on the DynaChip™ showed almost no reactivity in the negative control and a clear pattern of specificity with B7 being the predominant antibody. That is, the DynaChip™ results of tests of the untreated serum were very similar to the results of Luminex tests of the IgM-depleted fraction of that serum. Discussion Cell-based crossmatch tests have been used for more than 40 years to determine the strength of donor-reactive antibody. Increasingly, it is being demonstrated that solid phase immunoassays can be used to assess both the specificity and strength of antibodies to HLA and, thus, provide an interpretive guide for cell-based crossmatches and, in many cases, a virtual crossmatch (7, 13-16). We have shown here that solid phase assays can be confounded by the presence of blocking factors comprised of IgM immunoglobulin and/or soluble immune complexes. This interference may appear as inappropriately high reactivity with negative control beads, as reactivity that lacks a definable pattern of HLA specificity, and/or as reactivity that does not correlate in strength with that of crossmatch tests. We present a simple method utilizing hypotonic dialysis that effectively depletes IgM blocking factors which, in turn, provides improved specificity determination and more accurate correlation with donor crossmatches. Dialysis of serum against distilled water precipitates the euglobulin fraction of serum and has been used historically as a means of reducing or eliminating IgM (11). We have demonstrated here that IgM can produce substantial test interference making it difficult or impossible to determine antibody specificity and strength. We have further shown that the IgM-depleted fraction obtained by hypotonic dialysis has a substantially reduced background level of reactivity and has antibody specificity and strength that correlate with crossmatch results. page 10 of 22
  11. 11. Several lines of evidence indicate that the results obtained with the IgM-depleted fraction were correct. First, in the multianalyte bead assays, IgM-depleted fractions had higher values for the positive control beads and lower values for negative control beads than did the euglobulin fractions. These values were more in line with expected values for valid assays. Second, the IgM-depleted fractions yielded antibody identification test results that correlated with those of crossmatch tests in both antibody specificity and strength. In most cases reported here, the reactivity of the IgM-depleted fraction was different from that of untreated serum in the solid phase assays while crossmatch reactivity was the same for both fractions. However, in two cases, the opposite occurred. That is, the reactivity of the crossmatch test was different for the IgM-depleted fraction. Third, tests of sequential sera from an individual patient showed that the IgM-depleted fraction of a serum with high background reactivity yielded results that were in line with those of other sera that were from the same patient but that did not have high background reactivity. The IgM-depleted fraction from sera with high background reactivity had HLA specificity that differed from that of the untreated serum. However, no change in specificity occurred following dialysis of sera with normal reactivity with control beads. The effect on specificity was variable. For sera with high background in tests of either class I-specific antibodies or class II-specific antibodies but not both, changes in specificity occurred only with the tests that had high background with untreated serum. There was also variability in the degree of impact on specificity. For some sera, the same beads were positive with both untreated serum and the IgM-depleted fraction but the ordering of the beads, when listed in descending order of strength, was changed and often clustering of specificities was evident only with the IgM- depleted fraction. For some sera, the ordering of the beads was affected more drastically. For example, with serum 2, the three strongest reacting beads for the IgM-depleted fraction were alleles of HLA-A2, with MFI values of 21-23,000. These three beads were also positive with the untreated serum but were ranked 13th, 26th, and 36th, in bead order with MFI values of 4-8,000. The most drastic differences occurred with serum 6 where the five highest ranking beads in tests of the IgM-depleted fraction, all with MFI values ca. 23,000, were negative with the untreated serum with raw MFI values of 312-585. In all cases, the predominant antibodies gave stronger reactions with the IgM-depleted fraction than with untreated serum. This was not true for all antibodies. Some “apparent” antibodies that gave moderate reactions with untreated serum, had weaker reactions with the IgM-depleted fraction. page 11 of 22
  12. 12. The hypotonic dialysis procedure is simple but takes two days. For the small volumes of serum treated here, we found that isotonicity sufficient for tests of cells, was restored in four hours of dialysis against phosphate buffered saline and that no such back dialysis was necessary for solid phase assays. However, we found that a clear euglobulin precipitate was evident only after a minimum of 18-20 hours of dialysis against distilled water. Separation of immunoglobulins using spin columns with a filter permeable to IgG but not to IgM requires less than two hours. The results of the IgG fractions obtained with these columns yielded specificities comparable to the IgM-depleted fractions obtained by hypotonic dialysis but with appreciably lower reactivity strength. This may have been due to IgM overloading the filter and blocking permeation of some of the IgG. This suggests that the spin column procedure would be helpful in clarifying antibody specificity but not with determining antibody strength. Traditional methods to reduce IgM reactivity involve aggregation through heating or the reduction of disulfide bonds by agents such as dithiothreitol and dithioerythritol. Heat-inactivation of serum is not suitable for solid phase testing since the aggregates formed may bind non-specifically while IgM reduction involves the use of carcinogenic agents and may cause the loss of some IgG. Further, neither technique will eliminate IgM reactivity completely (10). As has been described by others, a small amount of IgM was left in the IgM-depleted portion of the dialyzed serum. However, the IgM-depleted portion of the serum was free from the background activity of the untreated serum and provided clear specificity determination. Several questions remain to be answered. It appears that some component in the euglobulin fraction is binding to the Luminex beads and blocking the binding of HLA-specific antibody. The euglobulin portion contains other serum components in addition to IgM and the relative contribution of those components, if any, is unknown. Another issue is why the non-specific binding may occur with assays for class I only, class II only, or both. This does not appear to be due to binding of an HLA-specific IgM antibody since this should not result in reactivity with negative control beads. Additionally, we have tested the euglobulin fraction of two sera and did not find HLA-specific antibody. Reduced reactivity seen in the DynaChip™ assay which is performed on a glass microchip, suggests that nonspecific reactivity in the Luminex assay may be the result of binding of a serum component to the polystyrene of the beads. However, one would expect that binding would occur equally with beads bearing class I and those bearing class II antigens. Further, treatment of these sera with commercially available reagents to page 12 of 22
  13. 13. abrogate non-specific binding to beads did not reduce background nor restore normal reactivity to the sera. The results obtained here indicate that there are differences in the negative control beads in the class I and class II kits for both the phenotype and single antigen panels. In summary, we have demonstrated that hypotonic dialysis is an effective way to reduce or eliminate non-specific binding to Luminex beads and permit specific binding of antibodies to the HLA antigens. A more rapid method for achieving this is via filter separation however, this method appears to reduce the amount of HLA-specific antibody present. Utilizing a matrix of glass rather than polystyrene may also be an option for reducing non-specific binding to plastic but this approach needs further investigation. page 13 of 22
  14. 14. References 1. Hopkins KA. The basic lymphocyte microcytotoxicity tests: standard and AHG enhancement. In: Hahn AB, Land GA, Strothman RM, eds. The ASHI Laboratory Manual, 4th ed. Mt. Laurel, NJ: The American Society for Histocompatibility and Immunogenetics; 2000, p 1.C.1.1-1.C.1.5. 2. Zachary AA and Hart JM. Relevance of antibody screening and crossmatching in solid organ transplantation. In: Leffell MS, Donnenberg AD, Rose NR, eds. Handbook of Human Immunology. Boca Raton, FL: CRC Press; 1997, p 478-519. 4. Kao KJ, Scornik JC, Small SJ. Enzyme-linked immunoassay for HLA antibodies- An alternative to panel studies by lymphocytoxicity. Transplantation. 1993;55:192-196. 5. Zachary AA, Delaney NL, Lucas DP, Leffell MS. Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets. I. Evaluation of the GTI QuikID assay and analysis of antibody patterns. Human Immunol. 2001; 62: 228-235. 6. El-Awar N, Terasaki PI. HLA antibody identification with single antigen beads compared to conventional methods. Human Immunol. 2005;66:989-987. 7. Zachary AA and Leffell MS. Detecting and monitoring donor-specific antibodies. Human Immunology. 2008. In press. 8. Jackson AM, Zachary AA The problem of transplanting the sensitized patient: Whose problem is it? Frontiers in Bioscience. 2008; 13: 1396-1412. 9. Gebel HM, Harris SB, Zibari G, Bray RA. Conundrums with Flow-PRA beads. Clinical Transplant. 2002;16 (Suppl 7):24-29. 10. Thorne N, Klingman LL, Teresi GA, Cook DJ. Effects of heat inactivation and DTT treatment of serum on immunoglobulin binding. Human Immunol. 1993;37 (Suppl 1):123. page 14 of 22
  15. 15. 11. Andrew SM, Titus JA, Coico R, Amin A. Purification of Immunoglobulin M and Immunoglobulin D. Curr Protoc Immunol. 2001 May; Chapter 2: Unit 2.9. 12. Warren JS. Immunoglobulin quantification and viscosity measurement. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of Molecular and Clinical Laboratory Immunology, 7th edition. Washington, D.C.:ASM Press; 2006, pp 69-74. 13. Leffell MS, Montgomery RA, Zachary AA. The changing role of antibody testing in transplantation. Clin Transpl. 2005:259-271. 14. Bray RA, Nolen JD, Larsen C, et al. Transplanting the highly sensitized patient: The Emory algorithm. Am J Transplant. 2006;6: 2307-2315. 15. Vaidya S. Clinical importance of anti-human leukocyte antigen-specific antibody concentration in performing calculated panel reactive antibody and virtual crossmatches. Transplantation. 2008;85:1046-1050. 16. Reinsmoen NL, Lai C-H, Vo A, Cao K, Ong G, Naim M, et al. Acceptable donor specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation. In press. Table 1. Control Value Means for Untreated Sera and IgM-Depleted Fractions Group Sera with No Specificity Change1 Sera with Changes IgM Ab Class Panel Type Control2 No. Untreated No. Untreated Depleted3 PC 131954 18027 13856 I Phenotype 4 NC 4 81-129 55-90 322-866 PC 14745 18740 14254 II Phenotype 6 3 NC 117-324 71-245 367-1139 PC 10780 13827 10702 I Single Antigen NC 2 371 94 4 420 PC:NC 31 190 33 page 15 of 22
  16. 16. PC 11173 12421 9792 II Single Antigen NC 4 186 124 3 147 PC:NC 147 107 152 1 Refers to whether or not there was a difference in the antibody specificities identified in the untreated serum and IgM-depleted fractions. 2 PC: positive control, NC: negative control. For the phenotype panels, the means of the range of the three negative controls is provided. 3 Water soluble fraction obtained after hypotonic dialysis 4 Values are means of the mean fluorescence intensity (MFI) values No. refers to the number of pairs of untreated sera and IgM-depleted fractions. page 16 of 22
  17. 17. Table 2. Sera With Changed Specificity - Phenotype Panels Untreated Sera IgM-Depleted Fraction1 serum Specificity Maximum MFI2 Specificity Maximum MFI2 5 no pattern 17473 B7 CREG 23710 6 no pattern 20758 B7 CREG 25774 panreactive - DR9, DR10 7 19397, 17930 panreactive - DR7, DR9 strongest 22873, 20256 strongest 8 no pattern 15582 A3, A11, B7 24342, 24690, 24787 9 DR1, 10, 53 6008, 9808, 15057 DR10, 53 22284, 17301 12 no pattern for cI, DR1, 4 17129, 11355, 11898 B7 CREG, DR1, DR52 20464, 24752, 24172 16 no pattern 15754 DR1, 2, 9 22250, 21501, 21501 1 Water soluble fraction obtained after hypotonic dialysis 2 Values are for the highest reaction due to antibody of the listed specificity or, when not specificity was determined, for the highest reaction overall. page 17 of 22
  18. 18. Table 3. Sera With Changed Specificity - Highest Reacting Beads on Single Antigen Panels Serum Data Untreated Sera IgM-Depleted Fraction Specificity A6802 B2708 A6901 A3402 A6801 A0201 A0206 A0203 A6901 A6801 2 MFI 13771 12020 11962 11380 10526 23003 21676 21477 19752 18721 Specificity DQA0401 DQA0401 DQA0303 DQA0201 DQA0102 DQA0503 DQA0505 DQA0501 DQA0401 DQA0601 3 MFI 8782 6409 3920 3072 2792 20499 20614 19898 19414 19586 Specificity B5401 B3901 B1402 A3002 Cw1402 B4201 B5501 B0801 B8201 Cw0702 5 MFI 12891 12644 12602 13271 12254 20527 20252 19665 19760 19376 Specificity A4301 A3301 B1302 A1101 B2705 B4201 B5501 B0801 B1510 B3501 6 MFI 10450 10095 9852 9762 9730 23617 23231 23108 22942 22926 Specificity DQB0202 DQB0502 DQB0402 DPB1001 DPB0501 DQB0201 DR53a DRB0701 DQB0501 DPB0101 7 MFI 14885 14795 14316 14058 13603 14520 13617 13416 13302 12710 Specificity B4101 Cw0501 B5601 B1301 B4801 B2708 A7401 B5501 B2705 A1102 8 MFI 16679 14971 14192 13908 13887 22329 22232 22313 22115 22476 Specificity DQB0501 DQB0304 DQB0303 DQB0302 DR10 DQB0301 DQB0201 DQB0301 DQB0301 DQB0301 9 MFI 13981 14429 11459 11482 10302 17155 16650 16866 16322 16159 Specificity DQB0202 DQB0201 DQB0201 DQB0201 DQB0402 DQB0201 DQB0603 DQB0601 DQB0609 DQB0201 11 MFI 13455 13055 12578 11507 10852 19203 18357 17995 17641 16429 Specificity A2301 A2402 A0101 B1512 A3601 A0201 A0203 A0206 A1102 A7401 13 MFI 13501 13487 13228 13139 12938 24645 24128 22970 22197 21770 Specificity DRB1301 DRB1201 DRB1602 DRB0901 DRB1202 DQB0601 DQB0501 DQB0502 DQB0609 DQB0603 16 MFI 10652 10565 10335 10297 10589 21578 21226 20889 21095 20795 page 18 of 22
  19. 19. a DRB4*0103 MFI values are the raw values; to conserve space, all DRB1 alleles are given as DRB and no asterisks are included for any allele. Differences between the untreated serum and the IgM-Depleted Fraction are shown in bold. page 19 of 22
  20. 20. Table 4. Crossmatch and Antibody Identification Data Untreated Sera IgM-Depleted Fraction2 Pt Mismatches Pan 1 MFIs CDCXM3 FCXM4 MFIs CDCXM3 FCXM4 3 DQA5 S 1878 B: 256 19898 5 B35 S 5729 T>512 17301 T>512 6 B37, B49 S 8612, 9585 T: 32 12065, 12883 A29, A68, B27, 9688, 1495, 9730, 10258, 20374, 6 S T: 128 Cw2 4849 21096, 6079 7 DR11, DR15, DQ7 S 938, 1199, 9853 B: 2 T4, B7 459, 461, 9032 NEG T- B:4 4798, 13254, 19000, 13721, 8 A30, B58, Cw10 S T:128 NT 5766 4002 9 DR4, DR53, DQ7 S 1354, 5862, 584 B: 512 722, 14084, 17155 9 DR1, DQ5 S 7716, 8770 NEG 4693, 6197 11 DR1, DR11, DQ5 S 568, 4114, 5121 B: 8 114, 1200, 15265 B: 8 T:21, 12 B7, DR17 P 9986, 4036 T:25, B:90 20464, 22930 B:112 T-, B- allo: T64, B88, 14 A2, B7, B13 P 533, 1887, 533 auto auto: T73, B82 neg 16 DR1, DQ5 S 6442, 1873 B>512 17303, 21226 B: 256 1 Panel from which MFI values were determined, P: phenotype, S: single antigen. Values are given as maximum MFI for the specificity. 2 Water soluble fraction obtained after hypotonic dialysis 3 Titers for cytotoxicity crossmatches 4 Values are ratios of median channel of test serum to that of the negative control page 20 of 22
  21. 21. Table 5. Patterns of Sequential Sera from a Single Patient T-11 T+21 T+41 T+41 Untreated Serum Untreated Serum Untreated Serum IgM-Depleted Fraction Pos Pos Pos Neg Con3 Neg Con Neg Con Pos Con Neg Con Con2 Con Con 130064 45, 98, 1003 16762 233, 428, 518 14378 189, 424, 466 18422 48, 122, 123 MFI Phenotype MFI Phenotype MFI Phenotype MFI Phenotype 2693 B7 B72 2937 B7 B51 17129 B42 B65 20464 B7 B72 2594 B7 B51 2821 B7 B37 15087 B50 B72 20080 B7 B51 2592 B7 B13 2802 B42 B44 14963 B50 B57 19139 B7 B37 2489 B7 B27 2780 B7 B72 14847 B18 B38 18974 B7 B27 2211 B42 B44 2558 B7 B27 14463 B13 B27 18611 B42 B44 2122 B7 B58 2209 B7 B58 14184 B8 B39 18366 B60 B13 1 Time, relative to transplantation date, in days; positive control; negative control; MFI values 2 3 4 page 21 of 22
  22. 22. Table 6. Reactivity of IgM-Depleted Fractions Obtained by Hypotonic Dialysis vs. Spin Column Results After Dialysis Separation Results After Spin Column Separation Pt Pan1 Specificities MFIs Specificities MFIs 1 P A2, DR4 529, 11625 A2, DR4 236, 7776 12828, 22873, 20256, 6817, 17780, 12134, 7 P DR4, 7, 9, 10 DR4, 7, 9, 10 7199 2762 24342, 24784, 19276, A3, B7, DR52, DR53 22320, 23009, 10379, 8 P A3, B7, DR52, DR53 5634 1834 18641, 25711, 21698, A1, A2, A3, A9, A11, A68, 10936, 19374, 12858, 13 P 21345, 23094, 24784, A1, A2, A3, A9, A11, A68, B17 B17 12897, 14760, 18280, 24136 17468 15 P DR1, DR51 1406, 759 DR1, DR51 531, 314 A: 0201, 0206, 0203, 6901, 22711, 21401, 21199, A: 0201, 0206, 0203, 6901, 14389, 12292, 11683, 2 S 6801, 6802 19498, 18482, 18393 6802, 6801 8969, 4191, 4043 DQA: 0503. 0505, 0501, 20036, 20026, 19713, DQA: 0401, 0501, 0401, 0601, 19463, 11672, 16908, 3 S 0401, 0601 19130, 19079 DQB: 0301 9902, 16065 B: 4201, 5501, 0801, 23453, 22951, 22933, A: 0201, B: 4201, 5501, 15063, 14279, 15684, 6 S 1510, 3501 22809, 22733 5101, 7801 14139, 14078 DQB: 0601, 0501, 0502, 21186, 21226, 20889, DRB: 0902, 1601, 0901, 16667, 15865, 15546, 16 S 0609, 0603 21095, 20765 0102, DQB: 0402 15347, 15022 1 Panel from which MFI values were determined, P: phenotype, S: single antigen. Values are given as maximum MFI for the specificity. Bolded numbers indicate where differences, in specificity, between the two fractions, occurred. page 22 of 22

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