Single HLA flow-beads (SAFB) analysis has emerged as an ideal tool to define the specificity of HLA-antibodies. By comparison of the donor’s HLA-typing with the HLA-antibody specificities of the recipient, the presence or absence of donor-specific HLA-antibodies (HLA-DSA) can be determined “virtually” without performing a cell-based crossmatch (i.e., virtual crossmatch). Since November 2004, our renal transplant center uses this approach for pretransplant risk assessment and subsequent adaptation of the immunosuppressive regimen (1). Although a negative virtual crossmatch has been associated with a low risk for early antibody-mediated rejection (AMR) due to HLA-DSA, it became questionable whether all HLA-antibodies detected by SAFB are in fact clinically relevant (1, 2). The virtual crossmatch approach has also been successfully used to allocate suitable renal allograft to sensitized patients (3). Therefore, many organ procurement organizations consider implementing such a strategy for efficient organ allocation. Because both applications of the virtual crossmatch approach (i.e., pretransplant risk assessment and organ allocation) rely on SAFB analysis, defining the clinical relevance of HLA-DSA detected by SAFB as an individual test is of major importance.
How can the clinical relevance of HLA-DSA detected by SAFB be determined? Basically, it can only be studied in transplantations performed in the presence of HLA-DSA defined by SAFB, but without applying an immunosuppressive regimen that can potentially modulate the development of AMR (i.e., polyclonal anti–T-lymphocyte globulin [ATG], intravenous immunoglobulin, anti-CD20 antibody [rituximab]). Because such prospective studies will unlikely be performed, retrospective analyses are necessary. So far, three retrospective studies investigating the clinical relevance of HLA-DSA detected by SAFB have been published with conflicting results (4–6). Notably, all these studies had important limitations. First, small patient populations were analyzed limiting interpretation of clinical outcomes because of insufficient statistical power (4–6). Second, outcomes might have been confounded by induction therapies, which can modulate the development of AMR (4). Third, AMR as a separate entity of rejection was not specifically assessed (5, 6). Finally, protocol biopsies were not performed to prove presence or absence of subclinical AMR (4–6). Therefore, it is currently unknown to which extent HLA-DSA detected by SAFB represent a risk for development of AMR and allograft failure.
In addition, it would be helpful to identify the parameters of HLA-DSA (e.g., number, class, strength, and prior sensitizing event) that are predictive for a good or poor outcome. This information could be used to assign an individual risk for specific HLA-DSA. Currently, the strength of HLA-DSA at the time of transplantation is regarded as a risk factor for the occurrence of early AMR and allograft failure, whereas other parameters have not been studied in detail (7, 8).
The aims of this retrospective study were (1) to investigate the clinical relevance of preformed HLA-DSA detected by SAFB at the time of transplantation in a large unselected cohort of patients, who have been transplanted without an induction therapy potentially modulating the development of AMR, and (2) to define HLA-DSA characteristics that are predictive for the occurrence of AMR.
PATIENTS AND METHODS
All 372 consecutive patients transplanted at the University Hospital Basel between January 1999 and November 2004 were investigated. Thirty-eight patients were excluded (no pretransplant serum available [n=13]; induction therapy with ATG [n=25]) leaving 334 patients (90%) for the final analysis. Induction therapy with ATG was given to patients considered at high risk for rejection based on a current or remote positive B-cell complement-dependent cytotoxicity crossmatch (CDC-XM), current CDC panel reactive antibody more than 20%, or prior allograft loss due to rejection within the first 6 months posttransplant. Of the 334 patients included in the analysis, only two had a weak remote positive B-cell CDC-XM, whereas all other patients had current and remote negative T-cell and B-cell CDC-XM. No prospective flow-cytometric crossmatches (FC-XM) were performed. On the basis of the CDC-XM results, these 334 patients were considered as low risk for rejection. Immunosuppressive regimens were changing over time and are detailed in the patient characteristics (Table 1). Presensitizing events, prior HLA- mismatches, clinical, and histologic data were obtained by carefully reviewing the patient charts. All retrospective analyses were performed with approval of the local Institutional Review Board.
Diagnosis of Rejection and Definition of AMR
All reported rejection episodes were biopsy proven. Clinically indicated allograft biopsies were preformed when serum creatinine deteriorated by more than 20%. Since January 2001, protocol biopsies scheduled at month 3 and 6 posttransplant were part of the clinical routine and were performed in 186 of 209 possible patients (89%). Biopsy specimens (two cores obtained with a 16-gauge needle) were evaluated by light microscopy and immunofluorescence for C4d as previously reported (9, 10). Findings were graded according to the Banff 2007 classification (11). Positive C4d-staining was defined as focal or diffuse detection of C4d in peritubular capillaries (PTC) by immunofluorescence. AMR was defined as C4d positivity in PTC alone or with transplant glomerulitis, or peritubular capillaritis, or arteritis, or thrombotic microangiopathy in glomeruli. AMR was also assumed in the presence of at least moderate transplant glomerulitis and peritubular capillaritis without C4d positivity in PTC. If AMR occurred together with acute T-cell mediated rejection, it was classified as AMR.
Detection of HLA-Antibodies and Assignment as HLA-DSA
All day-of-transplant sera were tested for class I (i.e., HLA-A/B/Cw) and class II (i.e., HLA-DR/DQ/DP) HLA-antibodies using SAFB on a Luminex platform (LabScreen single antigen LS1A04 Lot 002 and LS2A01 Lot 005; OneLambda Inc., Canoga Park, CA). A positive result was defined as a baseline normalized mean fluorescence intensity (MFI) more than 500. Quantiplex beads (OneLambda) were used to calculate standard fluorescence intensities for better comparison between different laboratories, but the definition of a positive result was based on MFI as described earlier. Preliminary assignment as HLA-DSA was performed on a serologic level by virtual crossmatching (i.e., comparison of the HLA-typing of the donor with the HLA-antibody specificities of the recipient). If only one of several different HLA-alleles of a serologically defined HLA-antigen on the SAFB was positive, high resolution typing of the donor was performed to determine true donor specificity. For example, one patient had a positive result for the B*2708 bead, whereas the B*2705 bead was negative. He received an allograft, which was typed as B27 by serology suggesting a possible HLA-DSA. However, high resolution typing revealed a B*2705, finally demonstrating that in fact no HLA-DSA was present. High resolution typing of the donor was also performed when the HLA-allele(s) in the used SAFB panel covered less than 90% of the expected alleles of a serologically defined HLA-antigen in our almost exclusively Caucasian population. The final assignment as HLA-DSA was thus determined on a HLA-allele level with more than or equal to 90% confidence. HLA-Cw and HLA-DP typing of the donor was performed if an isolated HLA-Cw or HLA-DP-DSA was potentially present. If a serologic equivalent of a HLA-antigen was represented by several alleles on the SAFB and high resolution typing of the donor was not performed, the MFI value of the most likely allele in Caucasians was selected (e.g., if SAFB A*0201, A*0203, and A*0206 were positive, the MFI value of A*0201 was selected). Because many HLA-DQβ alleles on the SAFB are paired with different DQ α-chains, the bead with the most likely DQ α-chain in Caucasians was selected to assess the strength of HLA-DSA against HLA-DQ antigens. Therefore for every individual HLA-DSA, the strength was based on the MFI of one SAFB. In case of several HLA-DSA against different HLA-antigens, the cumulative strength was calculated by adding the individual MFI values.
T and B cells were isolated using immunomagnetic beads (Dynabeads, Dynal Biotech, Oslo, Norway). One microliter of donor T and B cells were incubated with 1 μL of recipient sera for 30 and 40 min, respectively. Five microliters of rabbit complement and staining solution was added and incubated for 45 min. T and B cell CDC-XM were considered positive when the observed cell death exceeded 10% above background.
Typing of HLA-Antigens
HLA-A/B/DR antigens were determined by serology (Biotest, Rockaway, NJ) and sequence-specific primer (SSP) DNA-typing (Protrans, Indianapolis, IN). HLA-DQ antigens were primarily inferred from the HLA-DR antigens, which are in strong linkage disequilibrium (12). If a potential donor-specific HLA-DQ antibody was present, HLA-DQβ antigens of the donor were verified by SSP DNA-typing (Protrans). HLA-C antigens were assigned by sequence-specific oligonucleotide DNA-typing (OneLambda) on a Luminex platform, HLA-DPβ by SSP DNA-typing. High resolution HLA-typing was performed by sequence-specific oligonucleotide (OneLambda) on a Luminex platform or by SSP (Genovision, West Chester, PA).
Primary outcomes were development of clinical or subclinical AMR up to day 200 posttransplant and death-censored graft survival by the end of the year 2007. The cutoff was set at 200 days posttransplant, because we expected that AMR because of preformed DSA will mostly become apparent within this time frame.
We used JMP software version 7.0 (SAS Institute Inc., Cary, NC) for statistical analysis. For categorical data, Fisher’s exact test or Pearson’s chi-square test were used. Parametric continuous data were analyzed by Student’s t tests. For nonparametric continuous data, the Wilcoxon rank sum test was used. Survival analysis was performed by the Kaplan-Meier method, and groups were compared using the log-rank test. A P value less than 0.05 was considered to indicate statistical significance.
Patient characteristics are detailed in Table 1. In the whole population, presensitizing events were observed in 163 of 334 patients (49%) including 53 patients with prior renal transplants (16%). As expected, presensitizing events and female gender were more common in the group of patients with HLA-DSA than in the group without HLA-DSA (P≤0.002). There were no statistical differences between patients with/without HLA-DSA regarding donor type, donor age, HLA-mismatches, and baseline immunosuppression.
Assignment of HLA-DSA and Phenotypes of AMR
Initially, 73 of 334 patients were considered to have HLA-DSA defined by virtual crossmatching on a serologic level. After accomplishment of additional HLA-typings of the donors prompted as described earlier, 67 of 334 patients (20%) were finally assigned to have HLA-DSA on an allele level.
In total, 54 of 334 patients (16%) experienced AMR within the first 200 days posttransplant. Thirty-seven of 54 patients (69%) had acute clinical AMR, 7 patients (13%) showed subclinical AMR, and 10 patients (18%) demonstrated C4d deposition without morphologic evidence of active AMR. Twelve of 44 patients (27%) with histologic features of AMR (i.e., glomerulitis and peritubular capillaritis) had negative C4d staining in PTC. All these C4d-negative cases presented as acute clinical AMR and mostly occurred within the first 7 days posttransplant.
Correlation of HLA-DSA With AMR and Allograft Survival
The overall incidence of clinical/subclinical rejection (i.e., AMR and acute T-cell mediated rejection) at day 200 posttransplant was significantly higher in patients with HLA-DSA (48/67; 71%) than in patients without HLA-DSA (94/267; 35%) (P<0.0001; Fig. 1A). After exclusion of patients with subclinical rejection episodes the incidence was 63% for patients with HLA-DSA and 26% for patients without HLA-DSA (P<0.0001).
The cumulative incidence of clinical/subclinical AMR at day 200 posttransplant was significantly higher in patients with HLA-DSA (37/67; 55%) than in patients without HLA-DSA (17/267; 6%) (P<0.0001; Fig. 1B). By contrast, pure acute clinical/subclinical T-cell mediated rejection episodes were observed more often in patients without HLA-DSA (77/267; 29%) than in patients with HLA-DSA (11/67; 16%) (P=0.05).
Notably, 30 of 67 patients (45%) with HLA-DSA did not experience AMR until day 200 posttransplant. Therefore, the long-term impact of HLA-DSA was studied separately for patients, who experienced AMR and those who did not. Death-censored allograft survival at 5 years was equal in patient without HLA-DSA (89%) and patients with HLA-DSA but no AMR (87%) (P=0.95). However, patients with HLA-DSA and AMR had significantly lower death-censored allograft survival (68%; P=0.002; Fig. 2). Patient survival at 5 years was equal in all three groups (HLA-DSA and AMR: 87%; HLA-DSA, but no AMR: 92%; no HLA-DSA: 93%; P=0.69).
Correlation of HLA-DSA Characteristics and Immunosuppression With the Occurrence of AMR
The number, cumulative strength, and class of HLA-DSA were not different between patients with HLA-DSA and AMR and patients with HLA-DSA but no AMR (P≥0.08) (Table 2). Blood transfusions were more often noticed in patients with HLA-DSA but no AMR, whereas other sensitizing events were equally distributed. Interestingly, even a repeated HLA-mismatch with a previous renal transplant and detectable HLA-DSA against it led to AMR in only 5/9 patients (Table 2). The association of individual HLA-DSA with the occurrence of AMR is summarized in Table 3. Although the low number of individual HLA-DSA precludes any statistical analysis, some specific SAFB seem to be less often associated with AMR. Induction therapy and baseline immunosuppression were not different between the two patient groups (P≥0.14) (Table 2). In addition, donor type, donor age, recipient age, and gender were statistically not different (P≥0.15) (data not shown).
Details of Patients With HLA-DSA but Without AMR
Individual details of these 30 patients are summarized in Table 4. Twenty-seven of 30 patients (90%) with HLA-DSA but no AMR had at least one allograft biopsy within the first 200 days posttransplant. None demonstrated glomerulitis or peritubular capillaritis, and all C4d stainings were negative. However, 11/30 patients (36%) experienced T-cell mediated rejection. Six of 30 patients (20%) had an unstable clinical course (four allograft failed, two had deteriorating function). The other 24/30 patients (80%) had a stable clinical course during a median follow-up time of 7.2 years (range 3.2–9.0) (creatinine at 6 months: 1.6 mg/dL [range 0.8–2.4]; creatinine at last follow-up: 1.4 mg/dL [range 0.7–2.5]; P=0.35 by matched pairs analysis). HLA-DSA strength measured by MFI was not different between patients with stable and unstable clinical courses (P=0.50).
Defining the clinical relevance of HLA-DSA detected by SAFB is important as these assays are increasingly used for pretransplant risk assessment and organ allocation. The main observation in this study was that 37/67 patients (55%) with HLA-DSA detected by SAFB at the time of transplantation experienced clinical/subclinical AMR, which was associated with a 20% lower death-censored allograft survival 5 years posttransplant. However, 30/67 patients (45%) with HLA-DSA were devoid of AMR and showed an equal allograft survival as patients without HLA-DSA. Interestingly, readily available characteristics of HLA-DSA (e.g., number, class, strength, sensitizing events) were not predictive for the occurrence of AMR. These results indicate that more than half of HLA-DSA defined by SAFB are clinically relevant and emphasize the difficulty to predict their impact based on readily available parameters.
All patients in our study had negative current T-cell and B-cell CDC-XM indicating that the levels of HLA-DSA were low. Within this arbitrary category of HLA-DSA level, we observed a wide spectrum of clinical effects ranging from acute clinical AMR to no obvious detrimental impact. While cases with acute AMR and detectable HLA-DSA can be regarded as conclusive, the presence of HLA-DSA without pathologic evidence of AMR is a diagnostic challenge and offers two major explanations.
First, the detected antibodies bind exclusively to the HLA-molecules on SAFB, but not to the ones of the donor in vivo. During the production process of SAFB, HLA-molecules may have been denatured exposing novel epitopes that are not present on properly configured HLA-molecules of the donor in vivo. Furthermore, the detected HLA-DSA might be directed against epitopes of the HLA-molecules, which are accessible on the SAFB but not in vivo, and thus are likely not pathogenic (i.e., epitopes in the proximity of the cell membrane ). Clearly, FC-XM would have been of major interest to resolve these two particular confounding factors, but unfortunately we only had sufficient donor cells in approximately 15% of patients precluding a meaningful interpretation.
Second, SAFB have detected antibodies that truly bind to HLA-molecules of the donor in vivo, but which are of limited clinical relevance because of intrinsic characteristic of the HLA-DSA and protective factors of the donor endothelial cells. In our patient population, the number, class, and cumulative strength of HLA-DSA measured by MFI and known sensitizing events were not predictive for the occurrence of AMR. This suggests that these readily available characteristics are not sufficient to determine the clinical impact of HLA-DSA. These results are consistent with Burns et al. (14), who found that pretransplant HLA-DSA levels determined by FC-XM and the route of sensitization did not correlate with the occurrence and severity of AMR in patients transplanted across positive FC-XM with an intensified immunosuppressive regimen. By contrast, Lefaucheur et al. (8) found in a population with negative CDC-XM that the strength of HLA-DSA measured by ELISA was associated with a higher risk of AMR, whereas the class of HLA-DSA was also not predictive.
Another often considered pathogenic characteristic of HLA-DSA is the capability to activate complement dependent on the IgG subclasses. However, complement fixing IgG1/3 seem to be predominant and often coexist with noncomplement fixing IgG2/4 complicating a detailed analysis in this regard (15, 16). Maybe the most decisive characteristic of an antibody for its clinical impact is the binding strength to the recognized epitope, which is not easy to assess (17). In fact, in addition to the identification of the specific epitope, functional studies of the binding features of the HLA-DSA would also be required (13, 18).
With respect to protective factors on endothelial cells, several studies indicate that they can modulate the clinical impact of HLA-DSA potentially leading to a state of accommodation (19, 20). Indeed, in our study, 24/30 patients (80%) with HLA-DSA but no histologic evidence of AMR within the first 200 days posttransplant demonstrated stable clinical courses over a median follow-up time of 7.2 years (range 3.2–9.0 years). However, the long-term impact of these HLA-DSA cannot yet be conclusively assessed, because the detrimental effect of HLA-DSA might take years to become clinically apparent and only additional allograft biopsies can assure absence of late subclinical or chronic AMR (21).
How can the results of this study be used for clinical management of renal allograft recipients? Our data suggest that readily available characteristics of HLA-DSA (i.e., number, class, strength, sensitizing events) were not predictive for the occurrence of AMR. The presence of HLA-DSA was, however, associated with a 55% risk of early AMR and a 20% lower death-censored allograft survival 5 years posttransplant. Therefore, regarding all HLA-DSA as a potential risk for AMR is a safe strategy, until more predictive parameters exist. Notably, some SAFB were found to be less often associated with AMR suggesting that they might be prone to give “technically” false-positive results or that these specific HLA-DSA might in fact be of limited clinical relevance (see Table 3). This preliminary observation is intriguing and clearly requires further analysis in a larger patient population.
The overall rejection rate at 6 months posttransplant in our patient population transplanted between 1999 and 2004 was high (i.e., 32%), but comparable (i.e., 11%–35%) with a recent large multicenter study performed between 2002 and 2004 using similar immunosuppressive regimens (22). The rate of rejection episodes observed in patients with HLA-DSA was driven by AMR, whereas it was a rare event in patients without HLA-DSA at the time of transplantation. These few AMR cases occurring in the early phase after transplantation might be due to non-HLA-DSA, remote HLA-DSA, or HLA-DSA that were not detected by the used SAFB panel, whereas beyond the first 3 months they might also indicate de novo HLA-DSA (2). This confirms that the absence of HLA-DSA detected by SAFB is associated with a low risk for early AMR (1).
Our study had several advantages to determine the clinical relevance of HLA-DSA detected by SAFB. First, a large unselected patient population was investigated, which did not receive an induction therapy potentially modulating the development of AMR. Second, HLA-DSA were defined at the allele level with more than or equal to 90% confidence. Third, the presence/absence of HLA-DSA was correlated with clinical/subclinical AMR as a separate entity of rejection.
However, this study has also important limitations. As mentioned earlier, no FC-XM could be performed and it is thus impossible to ascertain whether FC-XM alone or in combination with SAFB results would have improved prediction of AMR. Recently, two small studies suggested that SAFB have a higher sensitivity than FC-XM to detect clinically relevant HLA-DSA, whereas the specificity of SAFB compared with the FC-XM is still a matter of debate (1, 4, 23–25). We would like to emphasize here that the aim of this study was not to compare SAFB with FC-XM results, but to determine the predictive value of SAFB as an individual test. Another limitation of our study is that protocol biopsies were only obtained in 56% of all patients, which might confound the true incidence of subclinical AMR in the different groups. However, protocol biopsies were equally often performed in patients with/without HLA-DSA likely leading to a small balanced underestimation. Finally, as donor specificity of HLA-DSA was not determined at the allele level in every case (i.e., when the likelihood was ≥90%) and HLA-antibodies against DQα could not conclusively assessed due to lack of DQα typing, rare false assignments as HLA-DSA can not be excluded.
In conclusion, the absence of HLA-DSA detected by SAFB was associated with a low risk for early clinical/subclinical AMR. By contrast, patients with HLA-DSA had a 55% risk of AMR, which was associated with a 20% lower death-censored allograft survival at 5 years posttransplant. This supports the utility of SAFB for pretransplant risk assessment and organ allocation. However, further improvement of the positive predictive value of HLA-DSA detected by SAFB will require an enhanced definition of pathogenic factors of HLA-DSA.
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