In 1969, a positive complement-dependent cytotoxicity (CDC) crossmatch became a contraindication to kidney transplant, when Patel and Terasaki (1) demonstrated that it represented a major risk for immediate graft rejection. Since then, more sensitive techniques have emerged including the most sensitive one, the flow cytometric crossmatch (FCXM) (2, 3).
Many studies demonstrated an increase in acute rejection episodes, early graft loss, and a decrease in graft survival in kidney transplant recipients transplanted across a positive FCXM, when compared with a negative FCXM (3). However, using FCXM creates a risk of excluding transplant patients who would not experience rejection or graft loss (4, 5). As the significance of antidonor antibodies detected by positive FCXM is still questionable, results of this technique are not currently used to contraindicate kidney transplant.
Twenty years ago, it was demonstrated that transplants performed across a positive crossmatch due to immunoglobulin (Ig)G anti-human leukocyte antigen (HLA) antibodies were associated with graft failure (6–11). The recent development of solid-phase assays using fluorescent beads coated with purified HLA molecules, or single-antigen bead (SAB) assays, has greatly improved the resolution and sensitivity of the identification of anti-HLA donor-specific antibodies (HLA-DSA) in serum. Indeed, antibodies against A, B, and Cw antigens in class I, and DR, DQ, and DP antigens in class II are now almost exhaustively identified, often at the allelic level (12). This approach could be useful to identify positive FCXM associated with poor outcomes after kidney transplantation (13–17).
In this study, historic sera and sera at the time of transplant (day 0 [D0]) of patients transplanted across a positive FCXM were tested with class I and class II Luminex SAB assays, to identify HLA-DSA and to better define the outcome of these patients.
Characteristics and Outcome of Patients According to FCXM
We identified 45 white patients who underwent kidney transplant across a positive historic T-cell or B-cell FCXM (group 1) in our institution from June 2004 to June 2008. Within group 1, 32 patients were transplanted across a positive D0 T-cell or B-cell FCXM. We compared them with a group of 45 patients transplanted across a negative D0 and historic T-cell and B-cell FCXM (group 2), who were matched for donor type (expanded criteria), HLA mismatches (A, B, and DR), gender, and previous transplant. All patients were transplanted across a negative D0 T-cell CDC crossmatch, but two patients in group 1 were transplanted across a positive D0 B-cell CDC crossmatch.
Cold ischemia time and maximal historic values of class I and class II panel-reactive antibody (PRA) values were higher in group 1 than in the group 2 (Table 1). Thirty-one of 32 patients with a positive D0 FCXM received polyclonal intravenous immunoglobulin (IVIG).
Mean follow-up was 30.22±12.9 months. Using Kaplan-Meier analysis, we observed that both graft survival without acute rejection and cumulative graft survival were significantly improved in group 2, when compared with group 1 (P=0.002 and P=0.02, respectively, Fig. 1A and B). At 1-year posttransplantation, mean estimated glomerular filtration rate (e-GFR) was significantly higher in group 2 than group 1 (64.1±20.3 vs. 53.6±22.1 mL/min, P=0.02, data not shown).
In group 1, historic HLA-DSA were identified in 34 recipients (class I for 26 patients and class II for 14 patients). D0 HLA-DSA were identified in 28 patients (class I for 17 patients and class II for 12 patients). There was a poor correlation between the SAB assay mean fluorescence index (MFI) of HLA-DSA and the MFI ratio of the T-cell FCXM or the B-cell FCXM (Fig. 1C and D). In group 2, D0 HLA-DSA were identified in 11 recipients (class I for three patients and class II for eight patients). The D0 HLA-DSA MFI was significantly higher in group 1 (2573±4702, n=28) than in group 2 (509±1484, n=11, P=0.0001, Fig. 1E).
Among the 90 patients, the MFI of D0 HLA-DSA was higher in patients with acute rejection, when compared with patients without acute rejection (2679±3963 vs. 1237±3489, P=0.007). However, high levels of HLA-DSA were observed in patients who did not display acute rejection (Fig. 1F), and reciprocally, low levels of HLA-DSA did not preclude rejection. As well, no correlation was observed between D0 HLA-DSA and 1-year posttransplantation mean e-GFR (r=0.17, P=0.1, data not shown). In summary, no threshold of HLA-DSA identified by the SAB assays was specifically associated with the occurrence of acute rejection or chronic allograft dysfunction.
Risk Factors for Acute Rejection in Patients Transplanted Across a Positive Historic T-Cell or B-Cell FCXM—Group 1 Patients
Patients in group 1 who experienced an episode of acute antibody-mediated rejection (AAMR) had a poorer graft survival compared with patients who did not, or to those who experienced an episode of acute cell-mediated rejection (ACMR) (P=0.05, Fig. 1G). The onset of ACMR or AAMR was associated with a lower e-GFR at 12 months posttransplant (P=0.008 and 0.06, respectively), when compared with the e-GFR of patients who did not experience acute rejection (data not shown).
By univariate analysis, the risk of acute rejection in group 1 was only increased in the presence of a positive D0 T-cell FCXM (odds ratio [OR]=9.04, P=0.002), a positive D0 B-cell FCXM (OR=7.43, P=0.02), and the presence of a D0 HLA-DSA (OR=6.5, P=0.03). We did not observe any significant association between the occurrence of acute rejection and time spent on dialysis, recipient age or sex, history of blood transfusions or pregnancies, number of previous transplants, donor age, cold ischemia time, any treatment, cytomegalovirus infection, delayed graft function, number of HLA mismatches, class I or II PRA, historic positive FCXM, or historic HLA-DSA (data not shown).
The distribution of the patients according to the results of the FCXM and the HLA-DSA analysis is depicted in Figure 2(A). For group 1, when both FCXM and HLA-DSA were positive at D0 (n=21), seven (33%) and six (29%) patients experienced ACMR and AAMR, respectively (Fig. 2B). These patients all had a historic positive FCXM and a historic HLA-DSA. On the contrary, patients with positive historic FCXM and HLA-DSA but negative D0 FCXM with or without D0 HLA-DSA (n=12) and the patients with positive historic and D0 FCXM but no D0 HLA-DSA (n=10) did not experience acute rejection (except for one patient, Fig. 2B). Then, only the combination of positive FCXM and HLA-DSA at D0 was associated with a high risk of acute rejection (sensitivity 87%, specificity 73%, positive predictive value 62%, and negative predictive value 92%). Higher positivity thresholds than 1.2 MFI ratio on T cells, two MFI ratio on B cells for FCXM, and 500 MFI for the class I and class II SAB assays were associated with an increasingly lower prediction sensitivity (data not shown).
Most of the 21 patients transplanted across a positive D0 FCXM with D0 HLA-DSA received tacrolimus and IVIG, but only one of seven patients (14%) who developed ACMR received antithymocyte polyclonal antibodies, when compared with 100% (6/6) of those with AAMR and 63% (5/8) of those without rejection (P=0.007). No significant differences in D0 HLA-DSA MFI and FCXM MFI ratio were observed between patients with ACMR or AAMR and patients without acute rejection. Interestingly, anti-Cw and anti-DP HLA-DSA were found in a significant number of patients displaying acute rejection (Fig. 2B).
Outcome According to the Results of FCXM and Donor-Specific Antibodies
The three previously described subgroups from group 1 were compared with group 2, divided into two subgroups: with HLA-DSA and without HLA-DSA (n=11 and n=34, respectively; Fig. 2A). Using Kaplan-Meier analysis, the rejection-free survival at 30 months of the 21 patients transplanted across a positive D0 FCXM with D0 HLA-DSA was significantly lower than that of the four other groups (P=0.0001, Fig. 3). At 1-year posttransplant, e-GFR of the 21 patients transplanted across a positive D0 FCXM with D0 HLA-DSA was also significantly lower (43±16 mL/min, P=0.0001) than that of the other patients. There was no difference in graft survival between these five groups.
Sensitized patients wait much longer for kidney transplant than nonsensitized patients. The safest strategy for sensitized patients is to perform a graft across a negative FCXM using an acceptable mismatch program, as it gives results similar to those of nonsensitized patients (18, 19). We found that high risk of acute rejection and worse 1-year e-GFR were closely associated with the presence of HLA-DSA at the time of transplant as determined by the two most sensitive techniques, FCXM and SAB. In contrast, the patients transplanted across a negative D0 FCXM in presence of D0 HLA-DSA had a short-term outcome similar to that of patients transplanted without HLA-DSA. On the basis of these results and those from other groups, we propose a decision-making algorithm for transplant-sensitized patients using FCXM and SAB assays (Fig. 4).
Maximal historic values of class I and class II PRA values were higher in group 1 than in group 2. Then, the group 1 patients were more sensitized and were more prioritized at the national level for their access to a donor than the group 2 patients. Consequently, as kidneys for the group 1 came from all the country, the cold ischemia time was higher in group 1 than in group 2.
According to the Banff classification (20), only AAMR is associated with HLA-DSA. However, several groups have observed that ACMR episodes represent 36% to 69% of all acute rejection episodes in patients transplanted across DSA (21, 22). These data suggest that patients with HLA-DSA can also activate donor-reactive, HLA-specific memory T cells that can lead to ACMR (23).
Previous reports have demonstrated that HLA-DSA are associated with subclinical rejection episodes (15, 24), subsequent chronic allograft nephropathy lesions (25–27), and lower graft survival (28). Therefore, our short-term results are not predictive of the long-term outcome of these patients.
Our data do not support previous reports that negative FCXM kidney transplant recipients with pretransplant HLA-DSA identified only by solid phase methods have an increased incidence of AAMR (29). In contrast, we confirmed the findings of other groups that the presence of HLA-DSA detected only by SAB in the context of a negative crossmatch was not associated with increased risk of acute rejection (30, 31). In addition, we confirmed that non-HLA-DSA, detected only by crossmatch but not by the SAB assay, are not relevant (32). Therefore, we question whether FCXM is necessary to predict graft outcome in the absence of HLA-DSA detected with SAB assays.
The poor correlation observed between HLA-DSA MFI and FCXM can be explained by (1) the variation in class I antigen density on SAB (33) and (2) blocking IgM that mask IgG HLA-specific antibody binding to SABs (33, 34). Moreover, a poorer correlation between HLA-C-specific antibodies and FCXM than for A and B antigens was observed (12). For this reason, the identification of anti-Cw antibodies should be considered in future studies.
Many studies report a higher incidence of early kidney graft loss and AAMR in patients with positive CDC T-cell crossmatch (3) and historic HLA-DSA (28, 35). However, we observe in this study that the D0 FCXM is crucial in dissecting failure from success in patients with HLA-DSA. Because graft survival is lower in HLA-DSA transplant recipients who have acute rejection (21, 28), we propose to apply these two sensitive assays at the time of the final prospective crossmatch to better define the rejection risk of sensitized patients presenting historic HLA-DSA. In a recent study, 43% of patients who were DSA positive by both SAB and FCXM assays presented an AMR episode posttransplant (13). We speculate that the prediction of rejection would have been improved if they had analyzed solely the results of D0 DSA.
We found no difference in the prevalence of rejection between class I and class II DSA (16, 28). Interestingly, we also found that circulating anti-Cw and anti-DP antibodies were associated with acute rejection episodes (21, 31) and were detected in a majority of cases. This latter point could be explained by the strategy used for the identification of the HLA antibodies during our clinical practice. Indeed, for class I, we used an enzyme-linked immunosorbent assay (ELISA) single-antigen assay, which was less sensitive than the Luminex SAB assay and which did not analyze the Cw antigens. For class II, we used a Luminex SAB assay, but the panel of the DP antigens was much narrower than with the current test used for the retrospective analysis of the patients. As a whole, our results suggest that donor HLA-Cw and DP typing should be routinely performed.
Thiry-eight percent of recipients with D0 HLA-DSA and a positive FCXM did not develop acute rejection, despite the highest risk for this group. AAMR is not associated with pregraft B-cell or T-cell FCXM MFI values (14, 22) or pregraft HLA-DSA strength values (21, 36). Interestingly, increased HLA-DSA levels posttransplant are associated with acute rejection and could predict them reliably (14, 31). In the future, posttransplantation monitoring with SAB assays should be performed in high-risk patients, as it could help engage early intervention to prevent the complications of acute rejection.
In conclusion, our study demonstrates that the presence of HLA-DSA detected solely by SAB assays in the context of a negative FCXM crossmatch is not associated with increased risk of acute rejection. In addition, the combination of SAB and FCXM assay results to identify HLA-DSA predicts short-term outcome of sensitized kidney transplant recipients, identifying high-risk patients for allograft rejection. Routine donor HLA typing should include information for HLA-Cw and DP to obtain a complete picture of the antigen mismatches capable of triggering a rejection episode.
MATERIALS AND METHODS
Clinically indicated allograft biopsies were performed when serum creatinine deteriorated by more than 20%. All reported rejection episodes were biopsy proven and defined according to the most recent Banff classification (20). We also analyzed graft survival and e-GFR (Cockcroft and Gault). This study was approved by the Institutional Review Board of the Bordeaux Hospital.
Induction treatment included daclizumab or antithymocyte polyclonal antibodies (Thymoglobulin, Genzyme, France). Almost all the patients with a positive current FCXM received polyclonal IVIG (1 g/kg at days 1–2, 20–21, and 41–42). Immunosuppressive treatment included a combination of mycophenolate mofetil and steroids, and tacrolimus or cyclosporine. ACMRs were treated by intravenous methylprednisolone boluses (1000 g/day for 3 days). AAMRs were treated by a combination of intravenous methylprednisolone boluses (500 mg/day for 4 days), plasma exchange for 4 days, IVIG 0.1 g/kg from days 1 to 3, IVIG 1 g/kg from days 4 to 5, and rituximab 375 mg/m2 on day 5.
A serum sample from the day of the transplant was always included and all available historic sera (collected every 3 months since listing). The basic National Institute of Health CDC crossmatch assay was performed on T and B lymphocytes from lymph node or spleen. The lymphocytes were incubated with the sera, treated or not with dithiothreitol at 22°C for 45 min, and then with complement at 22°C for 90 min. The FCXM was also performed prospectively on T and B lymphocytes, looking for IgG and IgM antibodies, with a four-color staining. An autologous crossmatch was systematically performed with the recipient's cells to identify autoreactive nonspecific antibodies. When the autologous crossmatch was positive at a similar level to that of the allogenic crossmatch, the crossmatch was considered negative, as the positive autologous crossmatch demonstrated the presence of autoreactive nonspecific antibodies. The threshold for positivity for CDC was set at 20% of dead cells, and for flow cytometry at a MFI ratio between the patient's serum and the negative control serum (pool from AB group normal donors) of 1.2 for T cells and 2 for B cells (37), which were equivalent to a shift of 20 and 70 fluorescence channels, respectively.
Antibody Screening and Identification
Our routine practice was the following: anti-HLA class I and class II antibody detection and panel identification were performed routinely with the flow bead panel assays (LabScreen, One Lambda, Canoga Park, CA) on a Luminex platform (luminex BV, Oosterhout, The Netherlands), every 6 months since patient listing. Identification was pursued with a high-definition single-antigen ELISA (LAT-1HD, One Lambda) for class I over the whole period and with the SAB assay for class II (LabScreen single-antigen LS2A01, One Lambda) from October 2005, when the kit became commercially available. All HLAs recognized by the identified antibodies were considered unacceptable. In the absence of HLA-DSA detected by ELISA for class I, and SAB for class II, transplants were performed across a positive FCXM, particularly when the patients were waiting a transplant for a long time.
For the purpose of the study, D0 serum and the peak serum (the strongest at the FCXM, mean age=41.6±60 months) were retested for class I and class II antibodies using SAB assays (Luminex platform), encompassing the A, B, Cw, DR, DQ, and DP antigens (LabScreen single-antigen LS1A04 and LS2A01, One Lambda) in the group of patients transplanted across a positive FCXM. Only the D0 serum was retested in the group 2 using the SAB assay. The positivity threshold for the beads MFI was set at 500 after removal of the background according to the “baseline” formula, as reported previously (38, 21). For any given antigen, the MFI was the average of all beads harboring the different alleles of the same antigen. For any given patient, the serum MFI was the sum of all positive antigens (>500). DNA typing for the Cw and DP loci was additionally performed for donors and recipients when the recipient was sensitized against Cw or DP antigens.
We performed univariate analysis for the variables potentially associated with the occurrence of acute rejection. Kaplan-Meier analysis was used to construct graft survival curves. Comparisons were made using the log-rank test. Analyses were performed with Statview software (Abacus Concepts, Berkeley, CA).
The authors thank Catherine Rio, transplant coordinator nurse, for her valued assistance. They also thank the technicians of the immunology laboratory, who carried out the anti-HLA antibodies analysis and crossmatches.
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