Relationship of donor-specific class-I anti-HLA antibodies detected by ELISA after kidney transplantation on the development of acute rejection and graft survival

Gema Fernández-Fresnedo1, Jose Manuel Pastor2, Marcos López-Hoyos3, Juan Carlos Ruiz1, Juan Antonio Zubimendi1, Julio Gonzalez-Cotorruelo1, Emilio Rodrigo1, Angel L. M. De Francisco1 and Manuel Arias1,

1 Nephrology, 2 Haematology and Haemotherapy and 3 Immunology Units, ‘Marqués de Valdecilla’ Hospital, University of Cantabria, Santander, Spain



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The objective of this study was to evaluate the role of post-transplant donor-specific anti-HLA antibodies (DS-HLA Abs) detected by an ELISA method on long-term graft survival.

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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Several studies in the past few years have evaluated the prevalence of HLA-specific antibodies (HLA Abs) after renal transplantation and their clinical relevance in terms of acute or chronic rejection risk (reviewed in McKenna et al. [1]). The majority of these studies found a prevalence ~25% positive post-transplant sera for HLA Abs [1]. The presence of HLA Abs was associated with a higher incidence of acute rejection (AR) [1]. Most of these studies were performed with the microlymphocytotoxicity (CDC) or flow cytometry tests, but very few with ELISA testing. This raises the question of sensitivity and specificity of the newly developed ELISA techniques [1,2]. Different studies have reported variable associations between post-transplant HLA Abs and episodes of AR [1]. In addition, the very recent possibility of detecting the HLA-II specifities of Abs [3] has created a new debate focusing on usage of HLA Abs while monitoring post-transplant patients.

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.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and samples
We evaluated all the patients that received kidney transplants at our institution from November 1999 to January 2001. This created a total of 71 patients. Clinical outcomes of these patients were followed-up until August 2002. Hypersensitized patients (PRA >50%) were not included in this study. Analysis of pre-transplant sera and cross-match were performed using the classic microlymphocytotoxicity test with either total blood lymphocytes (HLA-I) or isolated B lymphocytes (HLA-II). In accord with the policy at our centre, all pre-transplant cross-match tests were negative. In each patient, the presence of serum HLA Abs by ELISA was studied in one sample at the closest time before transplantation and in serial post-transplant samples when available.

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 Mann–Whitney and {chi}2 tests, respectively. P values <0.05 were considered significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Kidney transplants and blood transfusions in patients with post-transplant HLA Abs
We placed into group A 13 of 71 patients (18.3%) that had newly developed specific HLA-I and HLA-II Abs or had a significant increase in reactivity (>40%) in the post-transplant period. This group changed its combined antibody status in current and post-transplant sera. The remaining 58 patients (82%) with no qualitative or quantitative change in HLA Abs response were placed into group B. Table 1Go shows pre-transplant characteristics of both groups.


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Table 1.  Pre-transplant recipient characteristics

 
Importantly, 77.6% of patients from group A were receiving second or third kidney grafts compared with 10% in group B (P<0.001). In addition, group A had received more blood transfusions than group B. However, there were no important differences in HLA missmatches or cold ischaemia time.

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 2Go). 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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Table 2.  DS HLA Abs detected by ELISA after kidney transplantation

 
In addition, the Banff-97 classification [8] indicated that the majority of AR episodes in group A were of the vascular type, specially type III with transmural arterial changes (Table 2Go). Likewise, the clinical evolution of transplants after AR, assessed by serum creatinine, oliguria or dialysis therapy, was worse in group A than in group B (data not shown). Moreover, seven of the eight graft losses in group A (87%) were secondary to AR during immunosuppression, whereas only one of nine (11%) from group B lost their grafts due to AR (Tables 2Go and 3Go).


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Table 3.  Specificities of HLA Abs by ELISA and clinical outcome in patients from group A

 

Associations of post-transplant HLA-I Abs
As shown in Table 3Go, 10 out of 13 patients from group A exhibited changes in HLA-I Abs. Seven patients (patients 1–7) 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 3Go and 4Go). The eight patients (patients 1–8, Table 3Go) 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 3Go). These five patients had functioning grafts for 11–18 months without experiencing AR episodes (only one case with a borderline AR from the Banff classification, Table 3Go).


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Table 4.  Specificities of HLA-II Abs by ELISA and clinical outcome

 
It was difficult to analyse the clinical significance of HLA-II Abs due to the close correlation between the production of HLA-I Abs and the graft outcome (Table 4Go). To this end, we divided the 13 patients that developed post-transplant HLA Abs into three subgroups. Of these patients, six (group A1, Table 4Go) developed new post-transplant DS HLA-II Abs, and one of these, who also developed DS HLA-I Abs and lost his graft. The situation was more difficult to understand in group A2 (Table 4Go). The five patients in this group had DS HLA-II Abs before transplantation and retained their specificities or increased their PRA after transplantation. All of these five patients from group A2 lost their grafts and showed signs of vascular rejection. Remarkably, these five also showed post-transplant DS HLA-I Abs. Finally, two patients that did not have HLA-II Abs but developed post-transplant DS HLA-I Abs also lost their grafts (group A3, Table 4Go). However, one of these lost his graft due to relapse of primary disease.

Timing of post-transplant HLA Abs production
The temporal relationship between detection of specific HLA-I Abs and graft rejection is shown in Table 5Go. 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 5Go). 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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Table 5.  Timing of the post-transplant sera positive for HLA-I Abs

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study, the ELISA method detected a prevalence of post-transplant HLA Abs that was similar to the reports using flow cytometry or microlymphocytotoxicity tests [1]. Despite the small number of patients studied, our most important finding was the high percentage of patient sera with HLA Abs directed against donor specifities, and the strong relationship with the development of AR and graft loss, especially for HLA-I Abs.

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 5Go). 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 3–50% 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 4Go) by itself is not deleterious. All patients in subgroup A2 (Table 4Go) 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 5Go). 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 4Go) 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.



   Notes
 
Correspondence and offprint requests to: Dr Manuel Arias, Servicio de Nefrologia, Hospital Universitario Marqués de Valdecilla, E-39008 Santander, Spain. Email: nefarm{at}humv.es Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 7. 8.02
Accepted in revised form: 13.11.02