Risk Factors for dDSA
Table 1A compares baseline characteristics of recipients that developed dDSA to those who did not. In univariate analysis, risk factors associated with dDSA included AA recipient and donor race, deceased donor transplant, number of HLA mismatches, antithymocyte globulin (ATG) induction, dialysis pretransplant, simultaneous kidney-pancreas (SPK) transplant, and packed red blood cell (PRBC) transfusion during the initial transplant operation or hospitalization. Of note, re-transplant and sensitized recipients were not at greater risk of dDSA. After multivariate analysis, AA recipient race, SPK patients, and an increased number of HLA mismatches remained significant risk factors for dDSA (Table 1B). For every one mismatch at any of the HLA-A, B, DR, or DQ alleles, there was a 19% increased risk of development of a dDSA.
Mycophenolate (MMF) doses, prednisone doses, and tacrolimus levels in AA were compared to non-AA at 1, 3, 6, 12, 18, 24, and 36 months posttransplant. There were no differences in mean tacrolimus level between the two groups. At months 1 and 3, the MMF dose was higher in the AA group (P<0.05), and at months 1, 3, 6, 12, 18, and 24, the prednisone dose was higher in the AA group compared to the non-AA group (P<0.05) (see Table S1, SDC, http://links.lww.com/TP/A900).
Of the entire cohort, 16% (80) developed an AR. Recipients who developed dDSA were more likely to suffer an AR than the No dDSA cohort (35% vs. 10%, P<0.001; Table 2A). In addition, the dDSA group was more likely to suffer an AMR (16% vs. 0.3%, P<0.001), an AR ascribed to noncompliance (8% vs. 2%, P=0.001), and a recurrent AR (6% vs. 1%, P=0.02). Of the 42 recipients with dDSA who developed an AR, 13 (31%) developed dDSA before the AR, 12 (29%) were detected after the AR, and 17 (40%) were detected at the time of AR (Fig. 3). Because of this equal split, it was unclear whether dDSA were a result of AR or whether AR led to dDSA.
To understand the temporal association of dDSA with AR, recipients with dDSA and AR were divided into three groups. Thirteen recipients had dDSA before AR, 10 (77%) of whom suffered an AMR with or without a cellular component. Seventeen recipients developed dDSA and AR concurrently, all with an AMR component. Fully, 12 recipients developed dDSA after AR of which 11 (92%) experienced a purely cellular rejection. Those with dDSA after AR were significantly more likely to have a purely cellular rejection compared to both those with dDSA at the time of AR and those with dDSA before AR (OR 32, 95% CI 4.4, 262; P<0.0001).
Of recipients who developed dDSA, those with dDSA directed against both class I and class II antigens were more likely to be associated with an AR, compared to those with only class I or only class II antigens. In fact, the AR risk among recipients with dDSA against both classes I and II was approximately 10-fold higher than those with only class I or class II dDSA (Fig. 1B).
Mean serum creatinine (SCr) was compared between the dDSA and No dDSA cohorts (Table 2A). At 3, 6, 12, and 24 months posttransplant, the mean SCr of the dDSA group was higher than the No dDSA group, reaching statistical significance at the most recent follow-up (Table 2A). Proteinuria, as estimated by mean protein-to-creatinine ratios, was not significantly different between groups.
Excluding recipients who suffered an AR, there was no difference in mean SCr or protein-to-creatinine ratio (Table 2B).
At a median follow-up of 31 months, death-censored actuarial graft survival for recipients with dDSA was significantly worse than that of the No dDSA cohort (89% vs. 97.5%; P=0.002, Fig. 4A). Causes of graft failure were not statistically different between groups (see Table S2, SDC, http://links.lww.com/TP/A900). In the dDSA group, 28.6% of graft losses were a result of AR, all of which were AMR related; in the No dDSA group, there were no graft losses resulting from AR. Furthermore, graft loss resulting from chronic rejection occurred in 7% of dDSA patients and only 3% of No dDSA patients; these graft losses were diagnosed as chronic rejection based on biopsy findings consistent with chronic cell-mediated rejection (diffuse, mononuclear inflammatory cell infiltrate, marked tubulitis) and chronic AMR (thrombosis of arterial vessels and glomerular capillaries, multifocal transmural necrotizing vasculitis, marked peritubular capillaritis). After excluding recipients who had suffered an AR, there was no difference in death-censored graft survival (P=0.66, log-rank test, Fig. 4B). Moreover, the timing of onset of DSA in relation to AR had no impact on graft survival (Fig. 4C).
Herein the authors describe 503 renal and renal-pancreas transplant recipients prospectively screened for dDSA. Twenty-four percent developed dDSA, the majority of which were directed against class II antigens. In multivariate analysis, AA recipient race, receiving a SPK transplant, and an increase in HLA mismatches were significant risk factors for the development of dDSA. Interestingly, re-transplant and sensitized recipients, traditionally considered those with a high-immune risk, were not found to have an increased risk of dDSA. The median time to dDSA development was 6.1 months posttransplantation, with about 20% of patients developing dDSA within the first year.
Recipients who developed dDSA were more likely to have suffered an AR, AMR, and rejection related to noncompliance compared to recipients without dDSA. With a median follow-up of 31 months, development of a dDSA was associated with an increased incidence of graft loss. Yet, by excluding those recipients who suffered an AR, there was no difference in graft survival. The timing of dDSA in relation to the timing of AR had no impact on graft survival. the authors did however see that those with dDSA after AR were significantly more likely to have a purely cellular rejection. Thus, at an intermediate time point (2–5 years), the detrimental effect of dDSA was limited to those recipients who had also suffered an AR.
A number of studies have similarly examined posttransplant dDSA using prospective monitoring protocols. Everly et al. included a 64% AA demographic, where 25% of recipients developed dDSA, mostly within the first year posttransplant (12). Risk factors for dDSA were younger recipient age, deceased donor transplant, pretransplant sensitization, and HLA-DQ mismatch. The rate and timing of dDSA was similar to our findings, yet they did not find AA recipient race to be a risk factor for dDSA (12).
Another study by Wiebe et al. found 15% of recipients developed dDSA at a mean of 4.6 years (16). HLA mismatch and medication nonadherence were significantly associated with dDSA, while AR trended toward significance. Again, most dDSA were class II antigens, though none developed before 6 months. The study cohort was mostly Caucasian with only 2% AA recipients (16).
Finally, Cooper et al. detected dDSA in 27% of recipients, most of which were class II, and developed by 6 months posttransplant (10). Increasing HLA mismatch was a risk factor for dDSA and development of dDSA was associated with AR. Again, the timing, type, and rate of dDSA were similar to the data presented here. Importantly, they also demonstrated that the detrimental impact of dDSA in the early posttransplant period was limited to those recipients with AR (10).
While not the first of its kind, this is among the largest studies presented to date. Willicombe et al. presented 505 patients who were prospectively monitored for dDSA. They reported an 18.2% rate of dDSA with the majority of patients developing a DQ antibody (54.3%) (17). Findings from their study were similar to a report the authors published on the high rate of de novo DQ DSA and their detrimental effect on graft outcomes. Of note, the report herein expands upon this previously published study of 347 patients (18).
The data are unique in that an ethnically diverse population of AA, Hispanic, and Caucasian recipients was included. Similar studies, noted above, have been restricted to either predominantly low-immune risk Caucasians or mostly AA recipients (10, 12, 16). Additionally, the finding that AA race was a risk factor for dDSA has not been previously reported. The timing and incidence of dDSA is similar to other studies, as are the risk factors of increasing HLA mismatch, deleterious effects of dDSA with AR, and high frequency of posttransplant class II DSA. Comparison of immunosuppression between AA and non-AA recipients suggests that the increased incidence of dDSA in the AA population is more likely an immunologic issue than an issue with medication adherence and dosing.
It was also found that recipients of SPK transplants were at an increased risk for development of dDSA versus kidney-alone. While few studies have commented on this risk, a report of 167 pancreas transplants, of which 152 were SPK recipients, showed a 16% incidence of dDSA. In that study, pancreas transplant recipients with dDSA had an increased risk for AR and worse graft survival (19).
Limitations of this study include that, to date, no universally accepted median florescence index (MFI) cutoff exists to define a positive dDSA using the SAB Luminex platform. In this study, all positive dDSA had a MFI of greater than 2,000, yet other reports have utilized lower levels of 300, 500, and 1,000 MFI (10, 12, 16). It is possible that if a DSA were present pretransplant at an MFI less than 2,000, it would be considered de novo if it occurred more than 1 month posttransplant at an MFI greater than 2,000. However, in this study, the signal-to-noise ratio below 2,000 is not high enough that one can confidently say these are actually dDSA and not background noise and the day-to-day reproducibility of the test is not adequate to definitively call these dDSA. Although this study included a large number of recipients, the follow-up was relatively short, with no grafts followed for more than 5 years. Studies have shown a correlation between subclinical AMR and chronic rejection, renal dysfunction, and graft failure (20–23). Protocol biopsies were not performed in the cohort, thus it is plausible that recipients may have had subclinical AMR that was not identified and it is possible that longer follow-up will reveal a detrimental effect of dDSA even in those without an AR. Antibodies other than HLA class A, B, DR, and DQ were not tested. It is recognized that these antibodies, such as HLA-C and DP, may be clinically significant, and this is a limitation of the study. The authors also did not include recipients with pretransplant DSA; that data is discussed in a separate publication (24).
In summary, utilizing a prospective screening protocol, 24% of renal and renal-pancreas transplant recipients developed dDSA at a median of 6 months posttransplantation, and most dDSA were directed against class II. AA recipient race, the receipt of a SPK transplant, and an increasing number of HLA mismatches were significant risk factors for the development of dDSA. Those who developed dDSA were more likely to have suffered an AR compared to recipients without dDSA. With intermediate follow-up, although the development of a dDSA was associated with an increased incidence of graft loss, the detrimental effect of dDSA was limited to those recipients who had also suffered an AR. Longer follow-up will be necessary to determine the long-term effect of dDSA on allograft outcomes, even in the absence of AR.
MATERIALS AND METHODS
A nested case-control study was performed on all adult kidney or kidney-pancreas recipients from July 2007 through July 2011 at Houston Methodist, Houston, Texas. Cases were defined as dDSA positive and controls were all of the dDSA-negative (No dDSA) patients. The study was approved by the Houston Methodist Institutional Review Board.
HLA Typing Methods
Low-resolution donor and recipient HLA typing was performed by molecular methods according to manufacturers’ directions. All recipients and living donors were typed by a flow bead array using sequence-specific oligonucleotide probes (Labtype SSO, OLI) with positive hybridization detected by a Luminex100 (Luminex, Austin, TX) instrument and data analyzed by Fusion software (One Lambda/OLI, Canoga Park, CA). Deceased donors were typed by PCR-SSP (HLA-A/B/DRB1/DQB1-SSP UniTray; Life Technologies/Invitrogen, Carlsbad, CA) and data analyzed by vendor-supplied software (Unimatch, Life Technologies).
Pretransplant Panel Reactive Antibodies (PRA)
Pretransplant HLA antibodies were determined via Luminex multichannel array using SAB (LabScreen; One Lambda). A “fresh” SAB analysis of each cross-match serum was done at the time of transplant. Previous parallel studies using traditional flow cross-matches and flow antiglobulin cross-matches established a clinical threshold of greater than or equal to 2,000 MFI as positive. In the system used in this study, 4,000 MFI is the approximate lower limit of a positive B-cell flow cross-match (Canto II; Becton-Dickinson, San Jose, CA), while antibodies with greater than or equal to 8,000 MFI are associated with positive CDC cross-matches.
Determination of Posttransplant DSA
Posttransplant sera were screened for class I and II HLA dDSA via SAB according to the manufacturer’s instructions (LABScreen; One Lambda/OLI). Antibodies were detected by a multichannel flow array (Luminex 100; Luminex, Austin, TX) and identified via Fusion software (LABScreen; One Lambda/OLI). Prospective dDSA were determined at 1, 3, 6, 9, and 12 months posttransplant, every 6 months thereafter, and when clinically warranted (presence of graft dysfunction or suspicion of rejection). dDSA were defined as HLA-A, B, DR, or DQ antibodies directed against the donor HLA that were not present pretransplant. All dDSA had a MFI of greater than 2,000, as this is the lowest limit of detection by flow cytometry in the laboratory.
Subjects considered at high risk of AR (AA, re-transplants, and sensitized recipients) received a 3-day course of ATG at a dose of 1.5 mg/kg/day. All other subjects received either 2.0 mg/kg of daclizumab or 20 mg of basiliximab for two doses. Maintenance immunosuppression consisted of tacrolimus, MMF, and prednisone. The dose of tacrolimus was adjusted to maintain a trough level of 8 to 10 ng/mL for the first 3 months posttransplantation and tapered to 5 to 8 ng/mL thereafter. MMF was given at a dose of 1,000 mg twice daily. Methylprednisolone (250 mg) was given on the day of transplantation, tapered to 25 mg by day 5, and then to 5 to 10 mg by 6 months posttransplantation. Forty-nine patients (11%) were withdrawn from prednisone on posttransplant day 6.
Diagnosis of Rejection and Definition of AMR
Renal biopsies were evaluated by light microscopy and immunofluorescence. Electron microscopy examination was performed when glomerular pathology was suspected. Histopathology and classification of rejection was reported according to Banff 2005 and subsequent updates. AMR was defined as the simultaneous presence of diffuse linear C4d deposition in the peritubular capillaries and presence of DSA or morphological evidence, or both, of graft injury. Snap-frozen portions of biopsies were stained by indirect immunofluorescence using a specific monoclonal antibody (Serotec Inc., Raleigh, NC) at a dilution of 1:75. In the rare event that snap-frozen tissue of the kidney biopsy was not available, staining was performed on paraffin sections with a polyclonal antibody (1:50 dilution; American Research Products, Inc., Waltham, MA).
Characteristics of dDSA were compared with No dDSA by chi-square analysis (or Fisher exact testing when at least one cell size was ≤5). Logistic regression was used to analyze continuous covariates and for multivariate analysis with backward elimination. Odds ratios (OR) presented refer to the relative risk associated with dDSA. Graft survival was visualized using Kaplan-Meier methods with significance assessed by log-rank test. P values less than 0.05 were considered statistically significant. Statistical analyses were conducted with STATA version 10 SE (StataCorp, College Station, TX) and SAS 9.3 (SAS Institute Inc., Cary, NC).
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Keywords:© 2014 by Lippincott Williams & Wilkins
Donor-specific antibody; Kidney transplant; Acute rejection