- Email: [email protected]
The detection and definition of IgM alloantibodies in the presence of IgM autoantibodies using flowPRA beads
The Detection and Definition of IgM Alloantibodies in the Presence of IgM Autoantibodies Using FlowPRA Beads Naheed Khan, Amanda J. Robson, Judith E. Worthington, and Susan Martin ABSTRACT: We have developed a flow cytometry– based screening method using FlowPRA (One Lambda) human leukocyte antigen (HLA) class I panel beads and FlowPRA (One Lambda) HLA class I specificity beads for the detection and definition of immunoglobulin (Ig)M HLA–specific antibodies in the presence of IgM autoantibodies. Forty-six autoantibody-positive patients who were on the waiting list for a renal transplant (56 sera) were tested in parallel with FlowPRA (One Lambda) HLA class I beads and FlowPRA (One Lambda) control beads. Sera that were positive for IgM HLA class I antibodies were subsequently tested with FlowPRA HLA class I specificity beads to determine the HLA specificities. Thirteen of the 46 patients were positive for IgM HLA class
INTRODUCTION Immunologic evaluation of prospective renal transplant recipients requires the determination of preexisting alloantibody specificities. Sensitization to human leukocyte antigen (HLA) alloantibodies can be caused by three major stimuli: blood transfusions, pregnancy, and transplantation. The method of antigen presentation, dose of the antigen, and the nature of the response differ for each of these stimuli. Multiple transfusions induce significant clonal expansion, which could result in a sustained level of antibody production [1]. Alloimmunization caused by pregnancy is a result of stimulation by paternal HLA antigens expressed on the fetus. Transplantation can cause alloimmunization if an allograft recipient is exposed to donor-recipient HLA-mismatched antigens and consequently develop HLA donor-specific antibodies. Immunoglobulin (Ig) M antibodies in renal patients From the Transplantation Laboratory, Manchester Royal Infirmary, Manchester, United Kingdom. Address reprint requests to: N. Khan, Transplantation Laboratory, Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Oxford Road Manchester M13 9WL, UK. Tel: (⫹44) 161 276 6397; Fax: (⫹44) 161 276 6148; E-mail: [email protected]. Received November 28, 2002; revised February 25, 2003; accepted February 27, 2003. Human Immunology 64, 593–599 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Inc.
I–specific antibodies. Eleven of the 13 had previous failed transplants and 2 were awaiting a primary transplant. For 9 of the 13 positive patients, IgM HLA class I specificities were defined. We have demonstrated the presence of IgM HLA–specific antibodies in patients with IgM autoantibodies. This study demonstrates the value of FlowPRA HLA class I panel and specificity beads for the detection and definition of IgM HLA class I–specific antibodies. Human Immunology 64, 593–599 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc. KEYWORDS: IgM; flow cytometry; FlowPRA
may be directed against HLA or non-HLA targets. Lymphocytotoxic IgM antibodies directed against non-HLA targets are known as IgM autoantibodies. The non-HLA target of these antibodies renders them non– graft-damaging. IgM autoantibodies react with both random donor lymphocytes and autologous lymphocytes [2]. Their presence can cause a false positive pretransplant crossmatch. To avoid a false positive cytotoxic crossmatch, patients are crossmatched in the presence and absence of dithiothreitol (DTT). DTT abrogates IgM by reducing the intersubunit disulphide bonds of the IgM pentamer but does not effect the interchain disulphide bonds of IgG antibody. The role of IgM HLA–specific alloantibodies in graft survival is controversial, but studies have shown their association with early transplant rejection [3]. Other studies have suggested the relevance of IgM alloantibodies in regraft patients [4]. In contrast, a study by Tardif and McCalmon in 1995 indicated that IgM alloantibodies were not a contraindication to transplantation; the authors also reported successful renal transplantation in the presence of donor HLA-specific IgM antibody [5]. However, class switching from IgM to IgG alloantibody 0198-8859/03/$–see front matter doi:10.1016/S1098-8859(03)00065-X
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is detrimental [6]. A recent study showed that 8.4% of an IgM autoantibody-positive population had IgM alloantibodies in the absence of IgG alloantibodies [7]. For patients who have both IgM autoantibodies and IgM alloantibodies, there is a possibility that IgM autoantibodies may mask the presence of IgM donor–specific antibodies [8]; therefore, the latter must be clearly identified. The objective of this study was to develop a flow cytometry method for the detection of IgM HLA class I–specific antibodies in an IgM autoantibody–positive patient population and to define HLA antibody specificities that would be considered unacceptable in a donor for a given patient.
MATERIALS AND METHODS Patients Forty-six autoantibody-positive patients on the renal transplant list were investigated. The patients were categorized according to their previous sensitization and screening results. The nontransplanted study group comprised nine patients (10 sera) who were waiting for a primary transplant. The posttransplant study group comprised 37 patients (46 sera) who had received a renal transplant that had failed. Methods Fifty-six sera were investigated for IgM alloantibodies. The sera were selected on the basis of previous HLA antibody screening results by Quikscreen (GTI Inc., Brookfield, WI, USA), QuikID (GTI), flow cytometry, and complement-dependent cytotoxicity assay. Quikscreen (QS) is a solid-phase enzyme-linked immunoassay (ELISA)-based assay that was used to detect the presence or absence of IgG/A/M HLA class I–specific antibodies [9]. QuikID (QID) is a solid-phase ELISAbased assay that was used for the detection and definition of IgG HLA class I–specific antibodies [9]. Flow cytometry screening (FCS) used a locally developed panel of Epstein Bar virus (EBV)-transformed lymphoblastoid cell lines that detected the presence or absence of IgG HLA class I– and class II–specific antibodies [10]. Complement-dependent cytotoxicity (CDC) employed a selected panel of peripheral blood lymphocytes that aimed to detect and define complement fixing HLA class I specific antibodies. Sera were selected for the study when they met one of the following criteria. (1) QS positive, indicating the presence of IgG, IgM, or IgA HLA class I–specific antibodies but negative for IgG HLA class I–specific antibodies by FCS or QID.
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(2) CDC positive but with a reduced reactivity after treatment with DTT. (3) CDC positive and QS positive but negative for IgG HLA–specific antibodies by flow cytometry. FlowPRA IgM: Assay Procedure The assay employed FlowPRA (One Lambda) HLA class I panel beads and FlowPRA (One Lambda) control beads. FlowPRA HLA class I panel beads are 2– 4 m microparticles coated with HLA class I antigens purified from a panel of 30 cell lines. FlowPRA control beads are the same microparticles, but that are not coated with HLA antigen. Control beads are used to monitor the background level of nonspecific binding for the test serum and the test controls. A fluorescein isothiocyanate (FITC)-conjugated F (ab⬘)2 fragment goat antihuman IgM (FITC-IgM, Jackson Immunoresearch, West Grove, PA, USA) was used to identify IgM alloantibodies bound to FlowPRA beads. The FITC-IgM antibody was initially titered to determine the optimal dilution for the detection of HLA class I antibodies. Test controls included a pool of sera from patients with high titer, broadly reactive alloantibodies as a positive control and pooled human AB serum from nontransfused males as a negative control. Each serum sample and the controls were tested in parallel with FlowPRA class I beads and FlowPRA control beads. A total of 5 l of FlowPRA class I beads or 1 l of FlowPRA control beads were mixed with 20 l of neat serum and then incubated at 22 °C in the dark for 30 minutes. The beads were then washed twice in 1 ml FlowPRA wash buffer. 100 l of the FITC IgM at a 1:100 dilution was added to each test, vortexed to mix, then incubated at 22 °C in the dark for 30 minutes. The samples were then washed with 1 ml FlowPRA wash buffer and resuspended in 0.5 ml of FlowPRA wash buffer. The samples were analysed using a XL Coulter flow cytometer. Data Analysis The beads were gated on the forward scatter (FSC) versus side scatter (SSC) dot plot and a FL2 v FSC dot plot was obtained for the bead population. Separate FL1 histograms were obtained for FlowPRA class I beads and FlowPRA control beads (Figure 1). FL1 histogram for FlowPRA class I beads reacting with negative control serum was initially set between a range of 3% and 8% (manufacturer’s suggested value 5%) at the beginning of each run. HLA-specific IgM-positive sera showed a fluorescent shift with the FlowPRA HLA class I panel beads compared with the FlowPRA control beads and negative control serum. The following equation was used
Detection and definition of IgM alloantibodies
FIGURE 1 Histogram of FlowPRA control beads and FlowPRA class I panel beads reacting with negative control serum and positive patient serum. FlowPRA control beads reacting with (a) negative and (b) positive sera. FlowPRA class I panel beads reacting with (c) negative and (d) positive sera. HLA-specific IgM positive serum showing a fluorescent shift with FlowPRA HLA class I panel beads (d) compared with FlowPRA control beads (b), which indicate HLA-specific binding.
to determine positivity for IgM HLA class I–specific antibodies. % pos FlowPRA class I beads ⫺ % pos FlowPRA control beads ⫽ %TEST (%TEST ⫺ % negative control) ⬎ 2% ⫽ positive The TEST value for each serum indicates the % of FlowPRA class I beads bound by IgM HLA class I–specific antibodies. This was repeated for all test samples and negative and positive control. Test sera that were 2% above the negative control were denoted positive for IgM HLA class I–specific antibodies because this was the minimum IgM class I positivity for which IgM antibody specificity had been determined.
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FlowPRA IgM Specificity Beads: Assay Procedure FlowPRA class I specificity beads are microparticles coated with HLA class I antigens purified from a panel of 32 cell lines that cover all the major and many of the minor HLA specificities. FlowPRA specificity beads are supplied as four groups of eight different colored beads, which can be distinguished by the flow cytometer because of their differing fluorescent properties. These beads can be excited at 488 nm, generating a maximum emission of approximately 580 nm. FITC-IgM was used to identify IgM alloantibodies bound to FlowPRA specificity beads. Controls included a pool of sera from patients with high-titer, broadly reactive alloantibodies as a positive control and pooled human AB serum from nontransfused males as a negative control. Four tubes for each test serum, negative and positive controls, were set up. A total of 5 l of each group of FlowPRA specificity beads were mixed with 20 l of serum and incubated at 22 °C in the dark for 30 minutes. The beads were then washed twice in 1 ml of FlowPRA wash buffer. A total of 100 l of FITC-IgM at a 1:100 dilution was added to each test. After a vortex mix, the tubes were incubated at 22 °C in the dark for 30 minutes. The samples were then washed with 1 ml FlowPRA wash buffer and then resus-
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FIGURE 2 Histograms for the four group reactions of FlowPRA-specific class I beads, with Patient 5 illustrating the presence of IgM HLA-A30 and HLA-A31 antibody. *positive bead population
pended in 0.5 ml of FlowPRA wash buffer. The samples were analyzed using a XL Coulter flow cytometer. Data Analysis Different beads generate different FL2 channel shifts and therefore different colored beads in a group can be separated on the FL2 channel. Positive beads showed an FL1 channel shift on the FL1 versus FL2 dot plots when compared with the negative control serum. The median fluorescence values were recorded for each bead reaction and compared with the specificities of the antigens bound to the corresponding bead (Figure 2). Figure 2 shows there is one bead in group 1 that is positive. This bead has an HLA-A30 coated on its surface. There is one bead in group 2 that is positive. This bead has an HLA-A31 coat on its surface. There are three beads in group 3 that are positive. These beads in common have HLA-A30 and HLA-A31 coated on their surface. This illustrates the presence of IgM HLA-A30 and HLA-A31 antibody in patient 5. RESULTS Nontransplant Study Group Two of the nine patients (22%) were shown to have IgM HLA class I-specific antibodies (Table 1); for one of these antibody specificities were defined. The level of positivity for each patient is shown in Table 1, and specificities defined in Table 2.
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Posttransplant Study Group Eleven of the 37 (30%) patients were shown to have IgM HLA class I antibodies, and for 8 of the 11 specificities were defined. The level of positivity for each patient is shown in Table 1, and specificities defined in Table 2. Posttransplant sera from patients 3, 4, and 5 had been consistently QS positive with the combined IgG/A/M conjugate, but IgG negative by FCS. For patients 3 and 4, HLA class I–specific antibodies were defined by CDC. For each of these patients IgM alloantibodies were defined using FlowPRA-IgM (Table 2). Patient 3 had received an HLA-B44 and HLA-B62 mismatched renal graft. CDC had defined posttransplant antibodies to the B7 CREG, but none of these antibodies were donor-specific. IgM antibodies to the HLA-B7 CREG were detected by FlowPRA-IgM; in addition, an IgM donor–specific HLA-B44 antibody was found (Table 2). Patient 4 received an HLA-A1, -A11, and -B55 mismatched renal transplant. We found IgM HLA-A1, -A3, -A9, -A11, and -A36 antibodies in the posttransplant serum by FlowPRA-IgM. HLA-A1 and -A11 specificities related to the transplant mismatches and the HLA-A3 specificity related to a previous pregnancy. HLA-A1, -A3, and -A11 share an epitope with HLA-A9 and HLA-A36; IgM antibodies to these HLA specificities were also found (Table 2). For patient 5 an IgG HLA-B12 specificity had been defined, but reduction in panel reactivity by DTT suggested the additional presence of IgM antibodies. Using FlowPRA-IgM, IgM antibodies to HLA-A30 and HLAA31 were defined (Table 2). Patient 6 received an HLA-A28 and HLA-B27 mis-
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TABLE 1 Patients positive with FlowPRA-immunoglobulin M
Patient No. Posttransplant patients 1 2 3 4 5 6 7 8 9 10 11 Nontransplanted patients 12 13
Class I beads positivity A (%)
Control beads positivity B (%)
Test C (%)a
Negative control D (%)b
IgM class I positivity E (%)c
28.6 31.5 64 37.1 10.9 75.9 32.5 17.6 81.7 36.5 13.3
15.7 4.74 21.1 6.67 2.17 1.90 2.66 9.58 0.21 21.5 4.51
12.9 26.76 42.9 30.43 8.73 74 29.84 8.02 81.49 15 8.79
6.91 3.58 3.58 3.58 3.58 4.93 3.58 3.28 6.61 3.58 6.61
5.99 23.18 39.32d 26.85d 5.15d 69.07d 26.26d 4.74 74.88d 11.42d 2.18d
73.5 35.2
0.88 2.14
76.62 33.06
8.63 4.93
67.99d 28.13
A ⫺ B ⫽ C. D ⫽ class I bead positivity of negative control serum– control bead positivity of negative control serum. c C ⫺ D ⫽ E. d Immunoglobulin M antibody specificities determined (see Table 2). a
b
matched renal graft. Posttransplant sera had been positive by QS using combined IgG/A/M conjugate but negative by QID with the IgG conjugate. Using FlowPRAIgM we identified IgM donor–specific antibodies to HLAA28 and HLA-B27. HLA-B27 shares an epitope with HLA-B13, -B40, -B22, -B48, -B41, and -B42 to which antibodies were also detected and defined (Table 2). Patient 7 received a cadaver HLA-A31 mismatch kidney transplant and received anti-thymocyte globulin treatment in the postoperative period. Patient 7 had been positive by CDC, but we were unable to define a specificity. HLA-A31 is a split of the broad antigen HLAA19. The positive CDC result may have been due to autoantibodies or possibly the ATG treatment, which
can cause false-positive CDC results. However using FlowPRA-IgM, IgM antibodies to HLA-A30 and HLAA32 were defined (Table 2). These antigens are also the splits of the broad HLA-A19. A 1 month posttransplant serum from patient 10 had reacted with 90% of the panel when tested by CDC, but no HLA antibody specificity had been defined. The QID test was negative. Using FlowPRA-IgM, IgM antibodies to the A1 CREG were found (Table 2). These did not relate to the HLA-B7 transplant mismatch but may have resulted from blood transfusions. Patient 9 received an HLA-B7 and HLA-B60 mismatched renal transplant. CDC had defined donor-specific antibodies in posttransplant serum. When tested by
TABLE 2 IgM HLA class I–specific antibodies as determined by Flow PRA-IgM Patient no.
HLA antibody defined by CDC
HLA IgG antibody specificity
3
B7, B22, B27, B41, B60, B61, B81
Negative
4 5 6
A1, A3, B5, B12, B15, B21, B22 B12 No specificity defined
Negative B12 Negative
7 9 10 11 12
No specificity defined B7, B55, B56, B60, B61, Bw6 No specificity defined B40 A1, A3, A10, A11, A19
Negative Not tested Negative Not tested Not tested
HLA IgM antibody specificity B7, B8, B13, B27, B41, B42, B44, B45, B48, B55, B60, B61 A1, A3, A11, A23, A24, A36 A30, A31 A28, B7, B13, B27, B22, B40, B41, B42 A30, A32 B27, B44, B45 A3, A9, A10, A29, A30, A31, A32 B40 A1, A3, A10, A11, A19
Abbreviations: IgM ⫽ immunoglobulin; HLA ⫽ human leukocyte antigen; CDC ⫽ complement-dependent cytotoxicity.
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FlowPRA-IgM, we identified additional antibodies to HLA-B27, ⫺44 and ⫺45. For patients 11 and 12, FlowPRA-IgM identified the specificities defined by CDC (Table 2). For patient 11, reduction in panel reactivity by DTT suggested the presence of an IgM antibody, which was confirmed by FlowPRA-IgM.
DISCUSSION This study has shown that the FlowPRA-IgM assay is a more sensitive technique for the detection and definition of IgM antibodies than is CDC. FlowPRA-IgM detected and defined IgM antibodies in three patient samples when the CDC assays had proved unsuccessful and QID was negative for IgG antibodies. In another CDC-positive patient, the antibody defined by CDC was confirmed as IgG HLA-B12 by the QID test. When this sample was tested by FlowPRA-IgM, IgM-HLA-A30 and HLAA31 antibodies were also detected. CDC had failed to detect and define these IgM specificities. In one patient the CDC assay had defined HLA class I–specific antibodies but the patient was negative for IgG alloantibodies by QID. When tested using FlowPRA-IgM, the CDC-defined specificities were confirmed, and further IgM specificities were also defined. There were no particular specificities missed by FlowPRA-IgM, and there was only one patient for whom CDC-defined specificities were not confirmed as either IgG by QID or IgM by FlowPRA-IgM. The role of IgM alloantibodies in graft survival is controversial. Previous studies have shown their association with early graft loss and their relevance in retransplant patients. One study showed that 8.4% of an IgM autoantibody–positive population had IgM alloantibodies in the absence of IgG alloantibodies [7]. In addition they reported a case study suggesting that IgM donor–specific antibodies may contribute to early failure of a graft [3]. In contrast there have also been reports of cases of successful renal transplantation in the presence of HLA donor–specific IgM antibody [5]. The low affinity and limited intravascular distribution of IgM may make IgM alloantibodies relatively nonhazardous. We found donor-specific IgM HLA class I antibodies in three patients with failed transplants. These antibodies may have contributed to failure of the grafts, which may suggest that specificity rather than isotype is of key importance. One reason why IgM antibodies have generally not been considered to be graft damaging is because, usually, after relevant tests, they have been defined as autoantibodies. In this study we have established the existence of IgM alloantibodies in the presence of IgM autoantibody.
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Thirty percent of posttransplant patients with existing IgM autoantibodies selected for this study have been shown to have IgM HLA class I–specific antibodies. Whether these antibodies are deleterious for the transplant or not and whether a patient should be denied a transplant because of these antibodies in current or historic sera is debatable. The policy employed at our center is to take both IgG and IgM HLA–specific antibodies into account. IgM HLA class I antigens to which a patient is sensitized are avoided when selecting a kidney donor. Transplanting across these mismatch antigens could induce the risk of isotype switching from IgM to IgG HLA–specific antibodies, which could prove to be detrimental to the graft. We therefore consider it important that routine screening in the laboratory detects and defines these IgM HLA class I antibodies. In our current screening strategy, QS positive sera are investigated for IgG alloantibodies by QID. As a result of this study, If the QID test were negative, the sera would now be screened for the presence of IgM HLA class I antibodies using FlowPRA panel and specificity beads consecutively. CONCLUSION We have demonstrated the presence of IgM HLA–specific antibodies in patients with IgM autoantibodies. Studies have shown the relevance of IgM alloantibodies in graft loss; therefore, we take into account the presence of these antibodies in patient sera when selecting a kidney donor. This study has demonstrated the value of FlowPRA-IgM for the simultaneous detection and definition of IgM antibodies. REFERENCES 1. Scornik JC, Brunson ME, Howard RJ, Pfaff WW: Alloimmunisation, memory and the interpretation of crossmatch results for renal transplantation. Transplantation 54:389, 1992. 2. Taylor CJ, Ting A, Morris PJ: Production and characterisation of human monoclonal lymphocytotoxic autoantibodies from a renal dialysis patient. Tissue Antigens 37: 112, 1991. 3. Worthington JE, Thomas AA, Dyer PA, Martin S: Detection of HLA-specific antibodies by PRA-STAT and their association with transplant outcome. Transplantation 65:12, 1998. 4. Smith JD, Danskine AJ, Rose ML, Yacoub MH: Successful renal transplantation in the presence of donor specific HLA IgM antibodies. Transpl Proc 27:664, 1992. 5. Tardif GN, McCalmon RT Jr: Successful renal transplantation in the presence of donor specific HLA IgM antibodies. Transpl Proc 27:664, 1995.
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6. Sumethkul V, Mongkolsuk T, Sujirachato K, Chiewsilp P: Immunoglobulin class switch of posttransplant panel reactive antibody and the impact on kidney allograft outcome. Transpl Proc 30:1167, 1998. 7. Worthington JE: Investigation of the incidence of IgM alloantibodies in renal patients with IgM autoantibodies. Manchester, UK: Transplantation Laboratory, Manchester Royal Infirmary, 1997. 8. Chapman JR, Taylor CJ, Ting A: Immunological class and specificity of antibodies causing positive T-cell cross-
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matches: relationship to renal transplant outcome. Transplantation 42:608, 1986. 9. Worthington JE, Robson AJ, Sheldon S, Langton A: A comparison of enzyme-linked immunoabsorbent assays and flow cytometry for the detection of HLA specific antibodies. Hum Immunol 62:1178, 2001. 10. Robson AJ, Langton A, Worthington JE, Martin S: A comparison of flow cytometry screening methods. Eur J Immunogen 26:47, 1999.
INTRODUCTION Immunologic evaluation of prospective renal transplant recipients requires the determination of preexisting alloantibody specificities. Sensitization to human leukocyte antigen (HLA) alloantibodies can be caused by three major stimuli: blood transfusions, pregnancy, and transplantation. The method of antigen presentation, dose of the antigen, and the nature of the response differ for each of these stimuli. Multiple transfusions induce significant clonal expansion, which could result in a sustained level of antibody production [1]. Alloimmunization caused by pregnancy is a result of stimulation by paternal HLA antigens expressed on the fetus. Transplantation can cause alloimmunization if an allograft recipient is exposed to donor-recipient HLA-mismatched antigens and consequently develop HLA donor-specific antibodies. Immunoglobulin (Ig) M antibodies in renal patients From the Transplantation Laboratory, Manchester Royal Infirmary, Manchester, United Kingdom. Address reprint requests to: N. Khan, Transplantation Laboratory, Manchester Institute of Nephrology and Transplantation, Manchester Royal Infirmary, Oxford Road Manchester M13 9WL, UK. Tel: (⫹44) 161 276 6397; Fax: (⫹44) 161 276 6148; E-mail: [email protected]. Received November 28, 2002; revised February 25, 2003; accepted February 27, 2003. Human Immunology 64, 593–599 (2003) © American Society for Histocompatibility and Immunogenetics, 2003 Published by Elsevier Inc.
I–specific antibodies. Eleven of the 13 had previous failed transplants and 2 were awaiting a primary transplant. For 9 of the 13 positive patients, IgM HLA class I specificities were defined. We have demonstrated the presence of IgM HLA–specific antibodies in patients with IgM autoantibodies. This study demonstrates the value of FlowPRA HLA class I panel and specificity beads for the detection and definition of IgM HLA class I–specific antibodies. Human Immunology 64, 593–599 (2003). © American Society for Histocompatibility and Immunogenetics, 2003. Published by Elsevier Inc. KEYWORDS: IgM; flow cytometry; FlowPRA
may be directed against HLA or non-HLA targets. Lymphocytotoxic IgM antibodies directed against non-HLA targets are known as IgM autoantibodies. The non-HLA target of these antibodies renders them non– graft-damaging. IgM autoantibodies react with both random donor lymphocytes and autologous lymphocytes [2]. Their presence can cause a false positive pretransplant crossmatch. To avoid a false positive cytotoxic crossmatch, patients are crossmatched in the presence and absence of dithiothreitol (DTT). DTT abrogates IgM by reducing the intersubunit disulphide bonds of the IgM pentamer but does not effect the interchain disulphide bonds of IgG antibody. The role of IgM HLA–specific alloantibodies in graft survival is controversial, but studies have shown their association with early transplant rejection [3]. Other studies have suggested the relevance of IgM alloantibodies in regraft patients [4]. In contrast, a study by Tardif and McCalmon in 1995 indicated that IgM alloantibodies were not a contraindication to transplantation; the authors also reported successful renal transplantation in the presence of donor HLA-specific IgM antibody [5]. However, class switching from IgM to IgG alloantibody 0198-8859/03/$–see front matter doi:10.1016/S1098-8859(03)00065-X
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is detrimental [6]. A recent study showed that 8.4% of an IgM autoantibody-positive population had IgM alloantibodies in the absence of IgG alloantibodies [7]. For patients who have both IgM autoantibodies and IgM alloantibodies, there is a possibility that IgM autoantibodies may mask the presence of IgM donor–specific antibodies [8]; therefore, the latter must be clearly identified. The objective of this study was to develop a flow cytometry method for the detection of IgM HLA class I–specific antibodies in an IgM autoantibody–positive patient population and to define HLA antibody specificities that would be considered unacceptable in a donor for a given patient.
MATERIALS AND METHODS Patients Forty-six autoantibody-positive patients on the renal transplant list were investigated. The patients were categorized according to their previous sensitization and screening results. The nontransplanted study group comprised nine patients (10 sera) who were waiting for a primary transplant. The posttransplant study group comprised 37 patients (46 sera) who had received a renal transplant that had failed. Methods Fifty-six sera were investigated for IgM alloantibodies. The sera were selected on the basis of previous HLA antibody screening results by Quikscreen (GTI Inc., Brookfield, WI, USA), QuikID (GTI), flow cytometry, and complement-dependent cytotoxicity assay. Quikscreen (QS) is a solid-phase enzyme-linked immunoassay (ELISA)-based assay that was used to detect the presence or absence of IgG/A/M HLA class I–specific antibodies [9]. QuikID (QID) is a solid-phase ELISAbased assay that was used for the detection and definition of IgG HLA class I–specific antibodies [9]. Flow cytometry screening (FCS) used a locally developed panel of Epstein Bar virus (EBV)-transformed lymphoblastoid cell lines that detected the presence or absence of IgG HLA class I– and class II–specific antibodies [10]. Complement-dependent cytotoxicity (CDC) employed a selected panel of peripheral blood lymphocytes that aimed to detect and define complement fixing HLA class I specific antibodies. Sera were selected for the study when they met one of the following criteria. (1) QS positive, indicating the presence of IgG, IgM, or IgA HLA class I–specific antibodies but negative for IgG HLA class I–specific antibodies by FCS or QID.
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(2) CDC positive but with a reduced reactivity after treatment with DTT. (3) CDC positive and QS positive but negative for IgG HLA–specific antibodies by flow cytometry. FlowPRA IgM: Assay Procedure The assay employed FlowPRA (One Lambda) HLA class I panel beads and FlowPRA (One Lambda) control beads. FlowPRA HLA class I panel beads are 2– 4 m microparticles coated with HLA class I antigens purified from a panel of 30 cell lines. FlowPRA control beads are the same microparticles, but that are not coated with HLA antigen. Control beads are used to monitor the background level of nonspecific binding for the test serum and the test controls. A fluorescein isothiocyanate (FITC)-conjugated F (ab⬘)2 fragment goat antihuman IgM (FITC-IgM, Jackson Immunoresearch, West Grove, PA, USA) was used to identify IgM alloantibodies bound to FlowPRA beads. The FITC-IgM antibody was initially titered to determine the optimal dilution for the detection of HLA class I antibodies. Test controls included a pool of sera from patients with high titer, broadly reactive alloantibodies as a positive control and pooled human AB serum from nontransfused males as a negative control. Each serum sample and the controls were tested in parallel with FlowPRA class I beads and FlowPRA control beads. A total of 5 l of FlowPRA class I beads or 1 l of FlowPRA control beads were mixed with 20 l of neat serum and then incubated at 22 °C in the dark for 30 minutes. The beads were then washed twice in 1 ml FlowPRA wash buffer. 100 l of the FITC IgM at a 1:100 dilution was added to each test, vortexed to mix, then incubated at 22 °C in the dark for 30 minutes. The samples were then washed with 1 ml FlowPRA wash buffer and resuspended in 0.5 ml of FlowPRA wash buffer. The samples were analysed using a XL Coulter flow cytometer. Data Analysis The beads were gated on the forward scatter (FSC) versus side scatter (SSC) dot plot and a FL2 v FSC dot plot was obtained for the bead population. Separate FL1 histograms were obtained for FlowPRA class I beads and FlowPRA control beads (Figure 1). FL1 histogram for FlowPRA class I beads reacting with negative control serum was initially set between a range of 3% and 8% (manufacturer’s suggested value 5%) at the beginning of each run. HLA-specific IgM-positive sera showed a fluorescent shift with the FlowPRA HLA class I panel beads compared with the FlowPRA control beads and negative control serum. The following equation was used
Detection and definition of IgM alloantibodies
FIGURE 1 Histogram of FlowPRA control beads and FlowPRA class I panel beads reacting with negative control serum and positive patient serum. FlowPRA control beads reacting with (a) negative and (b) positive sera. FlowPRA class I panel beads reacting with (c) negative and (d) positive sera. HLA-specific IgM positive serum showing a fluorescent shift with FlowPRA HLA class I panel beads (d) compared with FlowPRA control beads (b), which indicate HLA-specific binding.
to determine positivity for IgM HLA class I–specific antibodies. % pos FlowPRA class I beads ⫺ % pos FlowPRA control beads ⫽ %TEST (%TEST ⫺ % negative control) ⬎ 2% ⫽ positive The TEST value for each serum indicates the % of FlowPRA class I beads bound by IgM HLA class I–specific antibodies. This was repeated for all test samples and negative and positive control. Test sera that were 2% above the negative control were denoted positive for IgM HLA class I–specific antibodies because this was the minimum IgM class I positivity for which IgM antibody specificity had been determined.
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FlowPRA IgM Specificity Beads: Assay Procedure FlowPRA class I specificity beads are microparticles coated with HLA class I antigens purified from a panel of 32 cell lines that cover all the major and many of the minor HLA specificities. FlowPRA specificity beads are supplied as four groups of eight different colored beads, which can be distinguished by the flow cytometer because of their differing fluorescent properties. These beads can be excited at 488 nm, generating a maximum emission of approximately 580 nm. FITC-IgM was used to identify IgM alloantibodies bound to FlowPRA specificity beads. Controls included a pool of sera from patients with high-titer, broadly reactive alloantibodies as a positive control and pooled human AB serum from nontransfused males as a negative control. Four tubes for each test serum, negative and positive controls, were set up. A total of 5 l of each group of FlowPRA specificity beads were mixed with 20 l of serum and incubated at 22 °C in the dark for 30 minutes. The beads were then washed twice in 1 ml of FlowPRA wash buffer. A total of 100 l of FITC-IgM at a 1:100 dilution was added to each test. After a vortex mix, the tubes were incubated at 22 °C in the dark for 30 minutes. The samples were then washed with 1 ml FlowPRA wash buffer and then resus-
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FIGURE 2 Histograms for the four group reactions of FlowPRA-specific class I beads, with Patient 5 illustrating the presence of IgM HLA-A30 and HLA-A31 antibody. *positive bead population
pended in 0.5 ml of FlowPRA wash buffer. The samples were analyzed using a XL Coulter flow cytometer. Data Analysis Different beads generate different FL2 channel shifts and therefore different colored beads in a group can be separated on the FL2 channel. Positive beads showed an FL1 channel shift on the FL1 versus FL2 dot plots when compared with the negative control serum. The median fluorescence values were recorded for each bead reaction and compared with the specificities of the antigens bound to the corresponding bead (Figure 2). Figure 2 shows there is one bead in group 1 that is positive. This bead has an HLA-A30 coated on its surface. There is one bead in group 2 that is positive. This bead has an HLA-A31 coat on its surface. There are three beads in group 3 that are positive. These beads in common have HLA-A30 and HLA-A31 coated on their surface. This illustrates the presence of IgM HLA-A30 and HLA-A31 antibody in patient 5. RESULTS Nontransplant Study Group Two of the nine patients (22%) were shown to have IgM HLA class I-specific antibodies (Table 1); for one of these antibody specificities were defined. The level of positivity for each patient is shown in Table 1, and specificities defined in Table 2.
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Posttransplant Study Group Eleven of the 37 (30%) patients were shown to have IgM HLA class I antibodies, and for 8 of the 11 specificities were defined. The level of positivity for each patient is shown in Table 1, and specificities defined in Table 2. Posttransplant sera from patients 3, 4, and 5 had been consistently QS positive with the combined IgG/A/M conjugate, but IgG negative by FCS. For patients 3 and 4, HLA class I–specific antibodies were defined by CDC. For each of these patients IgM alloantibodies were defined using FlowPRA-IgM (Table 2). Patient 3 had received an HLA-B44 and HLA-B62 mismatched renal graft. CDC had defined posttransplant antibodies to the B7 CREG, but none of these antibodies were donor-specific. IgM antibodies to the HLA-B7 CREG were detected by FlowPRA-IgM; in addition, an IgM donor–specific HLA-B44 antibody was found (Table 2). Patient 4 received an HLA-A1, -A11, and -B55 mismatched renal transplant. We found IgM HLA-A1, -A3, -A9, -A11, and -A36 antibodies in the posttransplant serum by FlowPRA-IgM. HLA-A1 and -A11 specificities related to the transplant mismatches and the HLA-A3 specificity related to a previous pregnancy. HLA-A1, -A3, and -A11 share an epitope with HLA-A9 and HLA-A36; IgM antibodies to these HLA specificities were also found (Table 2). For patient 5 an IgG HLA-B12 specificity had been defined, but reduction in panel reactivity by DTT suggested the additional presence of IgM antibodies. Using FlowPRA-IgM, IgM antibodies to HLA-A30 and HLAA31 were defined (Table 2). Patient 6 received an HLA-A28 and HLA-B27 mis-
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TABLE 1 Patients positive with FlowPRA-immunoglobulin M
Patient No. Posttransplant patients 1 2 3 4 5 6 7 8 9 10 11 Nontransplanted patients 12 13
Class I beads positivity A (%)
Control beads positivity B (%)
Test C (%)a
Negative control D (%)b
IgM class I positivity E (%)c
28.6 31.5 64 37.1 10.9 75.9 32.5 17.6 81.7 36.5 13.3
15.7 4.74 21.1 6.67 2.17 1.90 2.66 9.58 0.21 21.5 4.51
12.9 26.76 42.9 30.43 8.73 74 29.84 8.02 81.49 15 8.79
6.91 3.58 3.58 3.58 3.58 4.93 3.58 3.28 6.61 3.58 6.61
5.99 23.18 39.32d 26.85d 5.15d 69.07d 26.26d 4.74 74.88d 11.42d 2.18d
73.5 35.2
0.88 2.14
76.62 33.06
8.63 4.93
67.99d 28.13
A ⫺ B ⫽ C. D ⫽ class I bead positivity of negative control serum– control bead positivity of negative control serum. c C ⫺ D ⫽ E. d Immunoglobulin M antibody specificities determined (see Table 2). a
b
matched renal graft. Posttransplant sera had been positive by QS using combined IgG/A/M conjugate but negative by QID with the IgG conjugate. Using FlowPRAIgM we identified IgM donor–specific antibodies to HLAA28 and HLA-B27. HLA-B27 shares an epitope with HLA-B13, -B40, -B22, -B48, -B41, and -B42 to which antibodies were also detected and defined (Table 2). Patient 7 received a cadaver HLA-A31 mismatch kidney transplant and received anti-thymocyte globulin treatment in the postoperative period. Patient 7 had been positive by CDC, but we were unable to define a specificity. HLA-A31 is a split of the broad antigen HLAA19. The positive CDC result may have been due to autoantibodies or possibly the ATG treatment, which
can cause false-positive CDC results. However using FlowPRA-IgM, IgM antibodies to HLA-A30 and HLAA32 were defined (Table 2). These antigens are also the splits of the broad HLA-A19. A 1 month posttransplant serum from patient 10 had reacted with 90% of the panel when tested by CDC, but no HLA antibody specificity had been defined. The QID test was negative. Using FlowPRA-IgM, IgM antibodies to the A1 CREG were found (Table 2). These did not relate to the HLA-B7 transplant mismatch but may have resulted from blood transfusions. Patient 9 received an HLA-B7 and HLA-B60 mismatched renal transplant. CDC had defined donor-specific antibodies in posttransplant serum. When tested by
TABLE 2 IgM HLA class I–specific antibodies as determined by Flow PRA-IgM Patient no.
HLA antibody defined by CDC
HLA IgG antibody specificity
3
B7, B22, B27, B41, B60, B61, B81
Negative
4 5 6
A1, A3, B5, B12, B15, B21, B22 B12 No specificity defined
Negative B12 Negative
7 9 10 11 12
No specificity defined B7, B55, B56, B60, B61, Bw6 No specificity defined B40 A1, A3, A10, A11, A19
Negative Not tested Negative Not tested Not tested
HLA IgM antibody specificity B7, B8, B13, B27, B41, B42, B44, B45, B48, B55, B60, B61 A1, A3, A11, A23, A24, A36 A30, A31 A28, B7, B13, B27, B22, B40, B41, B42 A30, A32 B27, B44, B45 A3, A9, A10, A29, A30, A31, A32 B40 A1, A3, A10, A11, A19
Abbreviations: IgM ⫽ immunoglobulin; HLA ⫽ human leukocyte antigen; CDC ⫽ complement-dependent cytotoxicity.
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FlowPRA-IgM, we identified additional antibodies to HLA-B27, ⫺44 and ⫺45. For patients 11 and 12, FlowPRA-IgM identified the specificities defined by CDC (Table 2). For patient 11, reduction in panel reactivity by DTT suggested the presence of an IgM antibody, which was confirmed by FlowPRA-IgM.
DISCUSSION This study has shown that the FlowPRA-IgM assay is a more sensitive technique for the detection and definition of IgM antibodies than is CDC. FlowPRA-IgM detected and defined IgM antibodies in three patient samples when the CDC assays had proved unsuccessful and QID was negative for IgG antibodies. In another CDC-positive patient, the antibody defined by CDC was confirmed as IgG HLA-B12 by the QID test. When this sample was tested by FlowPRA-IgM, IgM-HLA-A30 and HLAA31 antibodies were also detected. CDC had failed to detect and define these IgM specificities. In one patient the CDC assay had defined HLA class I–specific antibodies but the patient was negative for IgG alloantibodies by QID. When tested using FlowPRA-IgM, the CDC-defined specificities were confirmed, and further IgM specificities were also defined. There were no particular specificities missed by FlowPRA-IgM, and there was only one patient for whom CDC-defined specificities were not confirmed as either IgG by QID or IgM by FlowPRA-IgM. The role of IgM alloantibodies in graft survival is controversial. Previous studies have shown their association with early graft loss and their relevance in retransplant patients. One study showed that 8.4% of an IgM autoantibody–positive population had IgM alloantibodies in the absence of IgG alloantibodies [7]. In addition they reported a case study suggesting that IgM donor–specific antibodies may contribute to early failure of a graft [3]. In contrast there have also been reports of cases of successful renal transplantation in the presence of HLA donor–specific IgM antibody [5]. The low affinity and limited intravascular distribution of IgM may make IgM alloantibodies relatively nonhazardous. We found donor-specific IgM HLA class I antibodies in three patients with failed transplants. These antibodies may have contributed to failure of the grafts, which may suggest that specificity rather than isotype is of key importance. One reason why IgM antibodies have generally not been considered to be graft damaging is because, usually, after relevant tests, they have been defined as autoantibodies. In this study we have established the existence of IgM alloantibodies in the presence of IgM autoantibody.
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Thirty percent of posttransplant patients with existing IgM autoantibodies selected for this study have been shown to have IgM HLA class I–specific antibodies. Whether these antibodies are deleterious for the transplant or not and whether a patient should be denied a transplant because of these antibodies in current or historic sera is debatable. The policy employed at our center is to take both IgG and IgM HLA–specific antibodies into account. IgM HLA class I antigens to which a patient is sensitized are avoided when selecting a kidney donor. Transplanting across these mismatch antigens could induce the risk of isotype switching from IgM to IgG HLA–specific antibodies, which could prove to be detrimental to the graft. We therefore consider it important that routine screening in the laboratory detects and defines these IgM HLA class I antibodies. In our current screening strategy, QS positive sera are investigated for IgG alloantibodies by QID. As a result of this study, If the QID test were negative, the sera would now be screened for the presence of IgM HLA class I antibodies using FlowPRA panel and specificity beads consecutively. CONCLUSION We have demonstrated the presence of IgM HLA–specific antibodies in patients with IgM autoantibodies. Studies have shown the relevance of IgM alloantibodies in graft loss; therefore, we take into account the presence of these antibodies in patient sera when selecting a kidney donor. This study has demonstrated the value of FlowPRA-IgM for the simultaneous detection and definition of IgM antibodies. REFERENCES 1. Scornik JC, Brunson ME, Howard RJ, Pfaff WW: Alloimmunisation, memory and the interpretation of crossmatch results for renal transplantation. Transplantation 54:389, 1992. 2. Taylor CJ, Ting A, Morris PJ: Production and characterisation of human monoclonal lymphocytotoxic autoantibodies from a renal dialysis patient. Tissue Antigens 37: 112, 1991. 3. Worthington JE, Thomas AA, Dyer PA, Martin S: Detection of HLA-specific antibodies by PRA-STAT and their association with transplant outcome. Transplantation 65:12, 1998. 4. Smith JD, Danskine AJ, Rose ML, Yacoub MH: Successful renal transplantation in the presence of donor specific HLA IgM antibodies. Transpl Proc 27:664, 1992. 5. Tardif GN, McCalmon RT Jr: Successful renal transplantation in the presence of donor specific HLA IgM antibodies. Transpl Proc 27:664, 1995.
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6. Sumethkul V, Mongkolsuk T, Sujirachato K, Chiewsilp P: Immunoglobulin class switch of posttransplant panel reactive antibody and the impact on kidney allograft outcome. Transpl Proc 30:1167, 1998. 7. Worthington JE: Investigation of the incidence of IgM alloantibodies in renal patients with IgM autoantibodies. Manchester, UK: Transplantation Laboratory, Manchester Royal Infirmary, 1997. 8. Chapman JR, Taylor CJ, Ting A: Immunological class and specificity of antibodies causing positive T-cell cross-
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matches: relationship to renal transplant outcome. Transplantation 42:608, 1986. 9. Worthington JE, Robson AJ, Sheldon S, Langton A: A comparison of enzyme-linked immunoabsorbent assays and flow cytometry for the detection of HLA specific antibodies. Hum Immunol 62:1178, 2001. 10. Robson AJ, Langton A, Worthington JE, Martin S: A comparison of flow cytometry screening methods. Eur J Immunogen 26:47, 1999.