1 Nephrology, 2 Haematology and Haemotherapy and 3 Immunology Units, Marqués de Valdecilla Hospital, University of Cantabria, Santander, Spain
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Abstract |
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Methods. The serum pre-/post-transplant profile of anti-HLA Abs was analysed in 71 renal transplant patients by ELISA. The HLA specificity of positive sera was analysed by a different ELISA method. According to the results, patients were classified into two different groups: those who either developed DS-HLA Abs or significantly increased their panel-reactive antibody (PRA) (group A) and those who did not (group B).
Results. Thirteen out of 71 patients showed post-transplant DS-HLA Abs and were included in group A, whereas the remaining 58 were placed in group B. The incidence of acute rejection (AR) was significantly higher in group A than in group B (77 vs 10%). In addition, seven out of eight patients from group A had graft loss secondary to AR, whereas one of nine grafts lost in group B was due to AR. When analysing the clinical outcome according to HLA class specificity, only patients with HLA-I Abs lost their grafts due to vascular AR. The remaining patients with HLA-II Abs who lost their grafts also had HLA-I Abs. In four of the eight patients who lost their grafts, DS HLA-I Abs were detected several days before AR.
Conclusions. The detection of DS HLA-I Abs in the post-transplant period may provide a good marker for AR and graft loss due to immunological origin. Monitorization of these Abs by ELISA may be a useful tool for tailoring immunosuppression after kidney transplantation.
Keywords: acute rejection; anti-HLA antibodies; kidney transplantation; post-transplant
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Introduction |
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The development of kidney graft rejection seems to be more frequent when HLA Abs are donor-specific (DS) [4,5]. A recent study indicated that prevalence of DS HLA Abs detected by ELISA is much lower than reported previously [13]. This study also reported a higher prevalence of HLA-II Abs than HLA-I Abs [3], which is in contrast with previous reports. However, serum HLA Abs were evaluated in this study at the time of rejection and, the authors suggested that HLA Abs binding to damaged tissues may have caused underestimation of its values. Studies showing a temporal evolution of serum HLA Abs during the post-transplant period in kidney transplantation are still lacking. The timing of HLA Abs production should be correlated with the histological and clinical outcome of the kidney grafts in order to evaluate the clinical relevance of HLA Abs monitoring in the daily routine.
In the present study, changes in serum DS HLA-I and HLA-II Abs were analysed by ELISA in kidney transplant recipients, before and after transplantation, by evaluating the production of Abs against new specificities or by increases in per cent of panel-reactive antibody (%PRA). Also studied were correlations between the presence of DS HLA Abs and the development of specific graft pathology and transplant outcome.
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Subjects and methods |
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Screening of HLA Abs and analysis of specificity by ELISA
Sera were screened for the presence of HLA-I and HLA-II IgG Abs by ELISA following manufacturer instructions (LAT-M, One Lambda Inc., CA, USA). The HLA specificity of the positive sera was then analysed by an additional ELISA test (LAT Class I & II, One Lambda Inc.). The ELISA plates were read on an ELX-800 plate reader (Bio-Tek Instruments Inc., VT, USA) and the optical densities obtained were analysed by LATTM software for Windows (One Lambda Inc.). Sera with correlation coefficient (r) of >0.65 were considered specific, and when the donor had the corresponding HLA antigen, the HLA Ab was considered to be DS [6]. A significant increase in reactivity was defined as a change >10% between two consecutive samples [7].
Statistical analysis
Statistical analysis was performed using SPSS software (version 8.0; SPSS, Chicago, IL, USA). Comparisons of quantitative and qualitative variables between groups were performed using MannWhitney and 2 tests, respectively. P values <0.05 were considered significant.
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Results |
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The production of post-transplant HLA Abs was associated with acute rejection and graft loss
Analysis of biopsy-proven AR revealed that 16 of 71 patients (22.5%) experienced AR (Table 2). Importantly, patients that changed antibody status had the highest AR incidence (77% in group A vs 10% in group B, P<0.001). Thus, the sensitivity and specificity of the DS HLA Abs ELISA associated with AR were 63% and 94.6%, respectively.
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Associations of post-transplant HLA-I Abs
As shown in Table 3, 10 out of 13 patients from group A exhibited changes in HLA-I Abs. Seven patients (patients 17) showed HLA-I Abs specific for the antigens of the kidney allograft. In patient 8, the PRA increased by 70% in the post-transplant serum. HLA-I Abs were not DS in patients 9 and 10.
We examined additionally whether there were differences in graft outcome of the 13 patients from group A that developed post-transplant HLA-I Abs compared with patients that had only HLA-II Abs (Tables 3 and 4
). The eight patients (patients 18, Table 3
) with HLA Abs directed against HLA-I lost their grafts and had histological signs of vascular rejection, except for one patient that lost his graft due to relapse of primary disease. Of the remaining five patients from group A, two showed specific HLA-I Abs, but not DS, and this may be related to transfusions during surgery, whereas the other three were negative for HLA-I Abs (Table 3
). These five patients had functioning grafts for 1118 months without experiencing AR episodes (only one case with a borderline AR from the Banff classification, Table 3
).
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Timing of post-transplant HLA Abs production
The temporal relationship between detection of specific HLA-I Abs and graft rejection is shown in Table 5. In three cases (MGF, EAS, RLF) with AR occurring between days 3 and 21, DS HLA Abs were detected later. In one of these cases (EAS) Abs were detected a few days after AR, but not in close proximity to the episode. In the other two patients, samples were not available for study between the last negative sera and the positive sera at 130 days (RLF) and 136 days (MCF) after transplantation. We detected a significant but non-specific increase in PRA at the day of rejection in patient JBP (Table 5
). In the remaining four cases, DS HLA Abs were detected prior to the AR episode (between 1 and 15 days earlier).
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Discussion |
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Our data are in contrast with Supon et al. [3], a group that also used ELISA to examine DS HLA Abs after transplantation. The lower incidence of DS HLA reported by Supon et al. [3] may be due to the binding of Abs to rejected grafts, as sera were analysed at the moment of rejection. In this regard, three of our patients that developed HLA-I Abs failed to show positive titres of Abs near the time of their rejection episodes (Table 5). An increase in PRA was detected in only one patient at the moment of rejection. An additional explanation for the difference in findings could be the different degree of alloimmunization before transplantation. In this regard, Supon et al. [3] reported a higher incidence of post-transplant DS HLA Abs in patients having 350% PRA compared with patients having 0% PRA at the time of transplantation. Nevertheless, the incidence in our patients was still higher than in theirs [3]. An explanation might be the higher prevalence of re-transplant patients in group A from our study, although none of these had pre-transplant DS HLA-I Abs. It is also possible that the higher number of previous kidney transplants in group A induced the production of pre-transplant HLA Abs belonging to the same cross-reactive group post-transplant DS HLA Abs, and that this was independent of %PRA [9]. According to this mechanism, they would induce a more efficient humoral alloimmune response in group A. Nevertheless, most of the sera with pre-transplant HLA Abs were non-specific and may have resulted from polyclonal activation during any inflammatory process [10]. In any case, our clinical policy consists of not repeating previous transplant HLA mismatches, and none of the patients included in the present study had a pre-transplant cross-match positive for either HLA-I or HLA-II.
The incidence of AR in our cohort was equivalent to previously reported values [11]. However, its association with the presence of DS post-transplant HLA Abs, determined by ELISA, differed from other studies [3,12]. Thus, 10 of 16 (62.5%) of patients with AR developed Abs, with most of these having HLA class I. Moreover, all but one of these patients with specific HLA-I Abs lost their grafts as a consequence of AR. This finding suggests that detection of post-transplant specific-HLA-I Abs by ELISA indicates a high-risk factor for graft loss.
Although it is clear that pre-transplant HLA-I Abs are associated with the development of AR and poor graft outcome, its appearance after transplantation has been poorly studied despite early evidence [13,14]. These experiments demonstrated not only appearance of HLA-I Abs using CDC but also its relationship with graft evolution. Although Scornik et al. [15] demonstrated the presence of post-transplant HLA-I Abs by flow cytometry, they could not show a relation with graft rejection [15]. Following the more recent development of ELISA methodology for detecting HLA Abs, several reports [16] showed that patients with pretransplant HLA-I Abs (10% PRA) had greater risk for acute or chronic rejection, or an increase in post-transplant reactivity (also
10% PRA). HLA-I Abs were also related to inefficacy of therapy [12] or graft dysfunction [7].
HLA-II Abs have been examined in more detail mostly because of several technical issues, such as isotypes and subclasses of immunoglobulins. To our knowledge, only one study showed that the presence of HLA-II Abs at the time of transplantation did not correlate with a higher rate of AR [17]. Although other reports have found a negative influence of DS HLA-II Abs by ELISA on graft survival, most of these also detected the presence of DS HLA-I Abs at the same time [5,18]. In the present study, the simultaneous presence of DS HLA-I and HLA-II Abs in most patients led us to question the role of HLA-II Abs in graft outcome. As commented above [17], the production of DS HLA-II Abs (subgroup A1, Table 4) by itself is not deleterious. All patients in subgroup A2 (Table 4
) that had both HLA-II Abs and HLA-I Abs lost their grafts due to AR. Although these findings indicate that the presence of both HLA-I and HLA-II Abs is required for the development of humoral AR, they also demonstrate the importance of DS HLA-I Abs.
By using ELISA, our data are in agreement with others using CDC or flow cytometry in showing an association between DS HLA Abs and AR [2,4,11]. Although we found a higher percentage of DS HLA Abs in patients with AR, this could be attributed to an improved reliability of commercially available tests. The newly developed ELISA techniques are more sensitive, specific and reliable than classic CDC methods. However, as ELISA does not detect HLA IgM Abs, it must be utilized as a complementary test for CDC [4,11]. Nevertheless, the importance of DS HLA-I Abs production for graft outcome indicates that their early detection could play a determinant role in the management of immunosuppressive therapies. In agreement with Piazza et al. [19] using flow cytometry, we detected DS HLA-I Abs by ELISA in half of our cases, between 1 and 15 days before AR diagnosis (Table 5). Altogether, the data point to the need for developing a follow-up protocol to monitor the production of post-transplant HLA Abs, especially in re-transplant or multitransfused recipients. We propose a weekly follow-up during the first month after transplantation, fortnightly in the 2 following months, and then according to each patient's clinical evolution. Such follow-up should be performed by either ELISA or flow cytometry, because of the greater sensitivity, reliability and speed of these techniques. Nevertheless, CDC should be considered as a complementary test because it can detect certain Abs that the other tests cannot. This secondary role must be redefined during the monitoring of post-transplant kidney recipients.
We also studied the association of post-transplant HLA Abs with specific rejection pathology. Most of the AR episodes from group A were classified into grade II and III of the Banff classification, whereas AR in group B did not have a vascular component. Again, the production of HLA-I Abs, but not of HLA-II Abs, was associated with a more aggressive vascular rejection. Moreover, most of the patients that suffered an AR episode in group A lost their grafts during immunosuppresion, but only one patient from group B having AR lost his kidney. This confirms previous findings showing AR episodes within the first month after transplantation in all the patients that developed DS HLA Abs, although most of the graft losses were secondary to thrombosis [4], which differs from our findings. These results suggest a more important role for humoral immune responses in graft rejection than indicated by others [3]. It remains to be determined whether patients from group A would have preserved their kidneys with a more aggressive immunosuppression (e.g. patient 12 in Table 4) or by inhibition of the humoral immune response with immunosuppressive agents that block the production of Abs, such as sirolimus [20]. In other studies, plasmapheresis was performed to remove most of the DS HLA Abs [18]. However, our patients were treated with classical immunosuppression based on corticosteroids, with few receiving anti-lymphocyte antibodies, plasmapheresis, or both, due to high-risk clinical scores arising principally from old age of both recipients and donors.
In summary, our studies suggest that ELISA-detected development of HLA Abs, especially HLA-I Abs, in the post-transplant period may provide a good predictor of AR and of graft survival. Thus, this method should be used to monitor high-risk kidney graft recipients in the early post-transplantation period.
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Notes |
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References |
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