- Email: [email protected]
Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant
Human Immunology 70 (2009) 580 –583
Contents lists available at ScienceDirect
Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant Vincent Aubert a,*, Jean-Pierre Venetz b, Giuseppe Pantaleo a, Manuel Pascual b a b
Service of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland Organ Transplant Center, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
A R T I C L E
I N F O
Article history: Received 10 March 2009 Accepted 9 April 2009 Available online 16 April 2009
Keywords: DSA Luminex Solid phase assay HLA-antibodies Antibody-mediated rejection
A B S T R A C T
Since new technologies based on solid phase assays (SPA) have been routinely incorporated in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules has generally been considered as a risk factor for acute rejection (AR) and, in particular, for acute humoral rejection (AHR). We retrospectively studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell complement-dependent cytotoxicity (CDC) crossmatches at the time of transplant. Pretransplantation sera were screened for the presence of circulating anti-HLA antibody and DSA by using highly sensitive and HLA-specific Luminex assay, and the results were correlated with AR and AHR posttransplantation. We found that approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation. Of 113 patients, 11 (9.7%) had HLA-DSA. Of 11 rejection episodes post-transplant, only two patients had pretransplantation DSA, of whom one had a severe AHR (C4d positive). One-year allograft survival was similar between the pretransplantation DSA-positive and -negative groups. Number, class, and intensity of pretransplantation DSA, as well as presensitizing events, could not predict AR. We conclude that, based on the presence of pretransplantation DSA, post-transplantation acute rejections episodes could not have been predicted. The only AHR episode occurred in a recipient with pretransplantation DSA. More work should be performed to better delineate the precise clinical significance of detecting low titers of DSA before transplantation. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction An increasing number of clinical studies have demonstrated adverse allograft survival in patients who have anti– human leukocyte antigen (HLA) antibodies, whether preformed pretransplantation or developed de novo post-transplantation [1–5]. Recently, since new technologies based on solid phase assays (SPA) have been routinely used in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against HLA antigens has generally been considered as a risk factor for acute rejection (AR) and/or acute humoral rejection (AHR) [6]. However, the precise clinical relevance of anti-HLA antibodies detected by very high analytical sensitivity assays, as well as the exact importance of the presence of pretransplantation DSA (either anti– class I HLA or anti– class II HLA or both) is not completely established. Moreover, the significance of DSA detectable only by SPA but not by cytotoxicity assays is still controversial. So far, retrospective studies investigating the clinical relevance of DSA detected by multiplex technology SPA have demonstrated different results [7–9]. Among the various limitations detected in these studies, perhaps
* Corresponding author. E-mail address: [email protected] (V. Aubert).
the most important is the lack of standardization of the different immunosuppressive regimens that were administered. Moreover, protocol biopsies are not performed in all studies to determine the presence or absence of subclinical AR or humoral rejection. At our institution, since April 2003, we have used the same IS regimen for all kidney transplant recipients [10]. The aim of this retrospective study was to determine the clinical relevance of preformed DSA detected by multiplex technology SPA at the time of kidney transplantation in patients receiving a common immunosuppressive protocol drug regimen. 2. Subjects and methods 2.1. Patients and sera All consecutive patients (n ⫽ 113) undergoing transplantation at the Centre Hospitalier Universitaire Vaudois in Lausanne, Switzerland, between April 2003 and April 2007 were investigated. The patient characteristics are shown in Table 1. All patients had current and peak negative T-cell and B-cell CDC crossmatches. No prospective flow-cytometric or Luminex crossmatches were performed. All retrospective data were obtained by analyzing the clinical records and electronic databases of the Transplant Center.
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.04.011
V. Aubert et al. / Human Immunology 70 (2009) 580 –583
Table 1 Patient characteristics All
Patients, n 113 Male, n (%) 79 (70) Age, mean ⫾ SD, at tx 46.2 ⫾ 15.1 Age, median (range) at tx 49 (3–78) Kidney disease, n (%) Diabetes 8 (7) Hypertension 9 (8) Glomerulonephritis 33 (29) Genetic disease 25 (22) Others 38 (34) Sensitizing events, n (%) Prior tx 21 (19) Blood transfusions 63 (56) Pregnancies 20 (18) Living donor, n (%) 45 (40) HLA mismatches, n (%) 0 9 (8) 1 2 (2) 2 12 (11) 3 24 (21) 4 35 (31) 5 18 (16) 6 13 (11) HLA A/B mismatches, n (%) 0 9 (8) 1 7 (6) 2 38 (34) 3 34 (30) 4 25 (22) HLA DR mismatches, n (%) 0 27 (24) 1 51 (45) 2 35 (31) Current PRA I at tx, n (%) 0% 102 (90) ⬍10% 4 (4) 10–49% 4 (4) 50–79% 1 (1) ⬎80% 2 (2)
DSA negative
DSA positive
p Value (DSA negative vs DSA positive)
102 11 76 (74) 3 (27) ⬍0.005 46.6 ⫾ 15.5 42.8 ⫾ 10.2 NS 50 (3–78) 39 (31–65) 8 (8) 9 (9) 28 (27) 24 (24) 33 (32)
0 0 5 (45) 1 (10) 5 (45)
NS NS NS NS NS
16 (16) 56 (55) 13 (13) 41 (40)
5 (45) 7 (64) 7 (64) 4 (36)
⬍0.01 NS ⬍0.0001 NS
9 (9) 2 (2) 11 (11) 21 (20) 32 (31) 15 (15) 12 (12)
0 0 1 (9) 3 (27) 3 (27) 3 (27) 1 (9)
NS NS NS NS NS NS NS
9 (9) 7 (7) 34 (33) 30 (29) 22 (21)
0 0 4 (36) 4 (36) 3 (27)
NS NS NS NS NS
23 (22) 49 (48) 30 (29)
4 (36) 2 (18) 5 (45)
NS ⬍0.03 NS
92 (90) 4 (4) 4 (4) 1 (1) 1 (1)
10 (91) 0 0 0 1 (9)
NS NS NS NS ⬍0.03
DSA ⫽ donor-specific antibody; tx ⫽ transplantation.
Immunosuppresive regimens were induction therapy with basiliximab for first kidney transplants (Table 2). In the event of retransplant or if panel-reactive antibody antibody was ⬎50%, thymoglobulin at 1.5 mg/kg/day for 4 days was given. Maintenance therapy consisted in tacrolimus (targeted plasma levels 8 –10 mg/ l), steroids (daily bolus of 500 mg, 250 mg, and 125 mg methylprednisolone for the first 3 days post-transplantation, followed by tapered steroids to a dose of 5 mg prednisone per day at 3 months post-transplantation), and MMF (initially 2 g/day). In hepatitis C virus–infected patients, cyclosporine was given instead of tacrolimus. 2.2. Detection of anti-HLA antibodies and definition of DSA Stored sera from consecutive patients undergoing transplantation between April 2003 and April 2007 who had negative pretransplantation CDC crossmatches with T and B cells were retested for the presence of anti-HLA antibodies using the multiplex technology SPA. Class I (i.e., HLA-A/B) and class II (i.e., HLA-DR/DP/DQ) HLA antibodies were tested using LabScreen LS1A01 Lot 007 and LS2A01 Lot 004 (OneLambda, Canoga Park, CA) commercialized in Switzerland by Ingen. Briefly, 20 l of serum samples were incubated with HLA class I– coated and HLA class II– coated microspheres, respectively, for 30 minutes in the dark under gentle agitation. The specimens were then washed five times before being incubated with anti-human IgG-conjugated phyco-erythrine in the same condi-
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tions as in the first incubation. The Labscan 100 flow analyzer (Luminex, Austin, TX) was used for beads and data acquisition. Data were then exported to HLA Visual software (One Lambda) for analysis. The cut-off level was defined as a baseline normalized ⬎500 mean fluorescence intensity units (MFI). The presence of DSA was assigned by comparing the various HLA specificities proposed by the software analysis with the HLA typing of the donor for all transplanted patients. Luminex results did not influence transplant management, as this was a retrospective analysis. 2.3. Definitions and management of rejection In cases of acute renal dysfunction, renal biopsy was performed. Acute rejection was defined by the Banff 03 working classification of renal allograft pathology. Since 2003, C4d staining has been performed on all renal allograft biopsy samples and correlated with the presence of posttransplantation donor-specific antibody in recipient’s serum. Treatment for acute rejection consisted of 3 days of high-dose methylprednisolone (500 mg daily boluses), followed by a prednisone taper. In addition, steroid-resistant patients were treated with thymoglobulin for 4 –7 days. 2.4. Statistical analysis Differences between groups were assessed by the 2 test and Fisher’s exact test for categoric variables. Parametric continuous data were analyzed by Student t test. Values of p ⬍ 0.05 were considered statistically significant. 3. Results 3.1. Patient characteristics Baseline characteristics of the 113 patients (all pretransplantation DSA-positive or -negative recipients) enrolled in the study are shown in Table 1. Of the patients, 70% were male and 30% female, with a mean age of 46.2 years at the time of transplantation. A history of sensitizing event was documented in 64% of the overall study patients, with 19% having prior transplants, 56% blood transfusions, and 18% pregnancies. Kidney transplants were from a living donor in 40% of cases. Presensitizing events (prior transplants, pregnancies, and female gender) were significantly more common in the group of patients with DSA than in the group without DSA. No statistical differences between patients with or without DSA were found regarding age, donor type, HLA mismatches, and current percentage PRA at transplantation. Detailed analysis showed that
Table 2 Baseline immunosuppression, acute rejection, and survival
Patients, n Induction Basiliximab, n (%) Thymoglobulin, n (%) Basiliximab ⫹ Thymo, n (%) Immunosuppression Tac, n (%) CsA, n (%) Rejection All acute rejection, n (%) Acute cellular rejection, n (%) Borderline, n (%) Ia, n (%) Acute humoral rejection, n (%) Clinically suspected Graft survival at 1 year, n (%) Patient survival at 1 year, n (%) NS ⫽ not significant.
All
DSA negative
DSA positive
113
102
11
P Value (DSA negative vs DSA positive)
⬍0.03 ⬍0.005 NS
81 (72) 25 (22) 7 (6)
76 (75) 19 (19) 7 (6)
5 (45) 6 (55) 0
108 (96) 5 (4)
97 (95) 5 (5)
11 (100) 0
NS NS
8 (7) 6 (5) 2 (2) 4 (3) 1 (1) 1 (1) 112 (99) 113 (100)
7 (7) 6 (6) 1 (1) 5 (5) 0 1 (1) 101 (99) 102 (100)
1 (9) 0 0 0 1 (9) 0 11 (100) 11 (100)
NS NS NS NS ⬍0.005 NS NS NS
582
V. Aubert et al. / Human Immunology 70 (2009) 580 –583
there were no differences in any PRA category between patients with or without DSA, except in the ⬎80% PRA category. In the DSA-positive group, significantly more patients (6/11) received thymoglobulin induction as compared with DSA-negative patients (19/102). 3.2. HLA antibodies and correlation of DSA with AR Approximately half of our patient population (55/113, 48.7%) had pretransplantation circulating anti-HLA antibodies. Of 113 patients, 11 (9.7%) had pretransplantation DSA (DSA-positive group). The antibodies identified were anti– class I DSA only in six patients, anti– class II DSA only in four patients. One patient had both anti– class I and anti– class II DSA. Of 11 AR episodes post-transplantation, six patients were negative for anti-HLA antibodies and five were positive for antiHLA, but only two patients had pretransplantation DSA, of whom one developed AHR (C4d positive) at day 7 post-transplantation. Of note, nine of 11 recipients had pretransplantation DSA and did not present any post-transplantation rejection episodes. Moreover, differences in the number, cumulative strength, and class of DSA were not statistically significant between DSA-positive patients with or without AR (data not shown). One AHR episode occurred, in a recipient with pretransplantation DSA. 4. Discussion With the advent of highly sensitive antibody screening tests using SPA and multiplex technology, it has become more important to identify whether the presence of pretransplantation anti-HLA antibodies or DSA may be associated with higher risk of AR, AHR, or worse clinical outcomes. To investigate the clinical relevance of pretransplantation DSA, we studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell CDC crossmatches at the time of transplantation. We retrospectively screened pretransplantation sera for the presence of circulating anti-HLA antibody and DSA on serum samples that were taken immediately before transplantation by using highly sensitive and HLA-specific Luminex assays. Approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation by Luminex testing. Interestingly, only seven of 113 of these recipients (6%) would have been considered as immunized before transplantation when using a traditional PRA-CDC test (PRA ⬎10%). This discrepancy is probably caused by the high sensitivity of the SPA, which also identifies HLA class I and class II noncytolytic antibodies [11]. This high sensitivity of the SPA was also confirmed by the analysis of DSA. Indeed, 10 of 11 patients (91%) who were DSA positive before transplantation had a PRA level of 0%. This high sensitivity of SPA may be related in part to the cut-off level fixed at 500 MFI according to the manufacturer’s criteria. Indeed recent studies have reported a higher cut-off value [12–14]. In our study, no hyperacute rejection occurred, and patient and graft survival were excellent in anti-HLA or DSA-positive and -negative groups. Similar good results have been previously reported by Gibney et al., who observed an AR of 3% in patients with anti-HLA antibodies in the absence of DSA [15]. Of 11 rejection episodes post-transplantation, only two patients had pretransplantation DSA, of whom one had AHR (C4d positive) at 1 week post-transplantation. This latter patient had become highly sensitized (PRA ⬎80%) after transplantectomy 15 years after her first kidney transplant. Patients waiting for a second or third transplant are generally considered at higher immunologic risk, as they have an increased likelihood of having DSA, especially if they have undergone transplantectomy [12]. Thus our patient who had AHR experienced the cumulative effect of the risk factors of being female, having a previous transplantectomy, waiting for a second transplant, and being hyperimmunized with post-transplantation DSA. Of note, nine of 11 recipients had pretransplantation DSA
without any post-transplantation rejection episodes after transplantation. Thus, regardless of the analysis, the development of AR was not associated with the presence of pretransplantation DSA. Biopsies were not performed in these nine patients because these patients had normal graft function at 1 year post-transplantation (mean estimated glomerular filtration rate [GFR] by modified diet in renal disease [MDRD], 57 ⫾ 7 ml/min/1.73 m2; mean creatinine level, 116 ⫾ 19 mol/l) with no significant proteinuria (⬍0.5 g/24 h). Our data are in contradiction to some studies showing that the presence of pretransplantation DSA had a significant impact on AR rates and graft survival [9,16]. One possible explanation of our low AR rates in our DSA-positive group was the predominant use of a potent induction regimen with thymoglobulin because of prior transplants or high PRA at transplantation. When we analyzed the rate of AR between the groups with and without HLA antibodies, we found no significant differences. In this series, we also observed a lack of significant differences for the presence of AR between the positive and negative DSA patients. Therefore, our study did not show any usefulness in differentiating between patients with only class I or only class II pretransplantation DSA compared with patients with both or more in predicting outcomes as previously described [16,17]. Indeed, the patient with AHR had only class I DSA, whereas the only patient in this series with class I and class II DSA pretransplantation had no AR at all (data not shown). Similarly, our data do not support previous findings that suggest that only anti-class II DSA pretransplantation, even if this is a relatively uncommon finding, could be associated with a high rate of humoral rejection [16]. In this experience, if we had taken into consideration the presence of pretransplantation DSA as an absolute contraindication to transplantation, 10% (11/113) of the patients would not have received a kidney transplant. Recently, Akalin et al. reported, in an analysis of 35 kidney transplant recipients with pretransplantation DSA, that the addition of plasmapheresis to high-dose intravenous Ig decreased the incidence of acute rejection [18]. The original point of their study was to stratify kidney transplant recipients according to the mean fluorescence indices of Luminex (MFI), a measure which may reflect the amount of circulating antibodies [18,19]. Their results indicated that patients with “strong DSA” were at higher risk for developing AHR and needed more intensive desensitization protocols. By reviewing our own data, the pretransplantation DSA-MFI values of the two kidney transplant recipients with acute rejection were at 1004 (patient 1, anti-DR11), 1008 and 2659 (patient 2, anti-A26 and anti-A11, respectively), that is, values relatively low according to the stratification proposed by Akalin et al. On the other hand, the pretransplantation DSA-MFI values of the nine kidney transplant recipients without any rejection episodes ranged from 694 to 5717 (mean, 2758 ⫾ 1938). Thus, based on these pretransplantation DSA-MFI values alone, we could not have predicted the post-transplantation acute rejection episodes. Altogether, our data confirm the high sensitivity of solid phase assays (SPA) of the Luminex type to detect circulating DSA antibodies. However, they also suggest that the positive predictive value of low-levels pretransplantation DSA (e.g., MFI values ⬍6000, such as those found in this series) remains incompletely understood. More studies are needed to define which pretransplantation DSA are pathogenic post-transplantation and/or to decide on the most suitable cut-off level for optimal use of determining DSA in routine transplantation. Finally, it may be that potent immunosuppressive induction protocols (e.g., with thymoglobulin) may protect against AR in patients with low levels of DSA pretransplantation. In summary, the presence of pretransplantation anti-HLA or DSA were correlated with pre-existing sensitizing events before transplantation but were not correlated with high risk of AR or worse clinical outcomes. Pretransplantation DSA (especially at low levels) detected by SPA multiplex technology should not be used
V. Aubert et al. / Human Immunology 70 (2009) 580 –583
alone to discourage transplantation, as most patients with DSA had good outcomes. Detecting DSA pretransplantation by using new SPA will likely become very useful in the near future, in particular for retransplantation or sensitized recipients at risk for AR, as it is the case in the exerience of Akalin et al. and others [18,20]. However, as a result of our recent experience, we have now changed the estimation of the positive cut-off level, and we take into consideration only DSA with ⬎2000 MFI values. Overall, the high sensitivity of SPA brings new knowledge to the field of solid organ transplantation that is valuable but still quite complex to understand. More work, therefore, should continue to be performed to better delineate the precise clinical significance of detecting circulating DSA before and after transplantation, to fully validate the routine implementation of these new techniques in allocation policies as well as in the clinic. Acknowledgments The authors acknowledge the Ingen Company for help in providing anti-HLA kits and MichÉle Estoppey for excellent technical assistance. References [1] Terasaki PI. Humoral theory of transplantation. Am J Transplant 2003;3: 665–73. [2] Cai J, Terasaki PI. Humoral theory of transplantation—mechanism, prevention, and treatment. Hum Immunol 2005;66:334 – 42. [3] Terasaki PI, Cai J. Humoral theory of transplantation: Further evidence. Curr Opin Immunol 2005;17:541–5. [4] Akalin E, Pascual M. Sensitization after Kidney Transplantation. Clin J Am Soc Nephrol 2006;1:433– 40. [5] Mao Q, Terasaki PI, Cai J, Briley K, Catrou P, Haisch C, et al. Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study. Am J Transplant 2007;7:864 –71. [6] Lefaucheur C, Superbielle-Boissel C, Hill GS, Nochy D, Andrade J, Antoine C, et al. Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation. Am J Transplant 2008;8:324 –31. [7] Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, Thuraisingham RC. Pretransplant donor-specific antibodies in cytotoxic negative crossmatch kidney transplants: Are they relevant? Transplantation 2008;85:1200 – 4.
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[8] van den Berg-Loonen EM, Billen EV, Voorter CE, van Heurn LW, Claas FH, van Hoof JP, et al. Clinical relevance of pretransplant donor-directed antibodies detected by single antigen beads in highly sensitized renal transplant patients. Transplantation 2008;85:1086 –90. [9] Patel AM, Pancoska C, Mulgaonkar S, Wenig FL. Renal transplantation in patients with pretransplantation donor-specific antibodies and negative flow cytometry crossmatches. Am J Transplant 2007;7:2371–7. [10] Manuel O, Venetz JP, Fellay J, Wasserfallen JB, Sturzenegger N, Fontana M, et al. Efficacy and safety of universal valganciclovir prophylaxis combined with a tacrolimus/mycophenolate-based regimen in kidney transplantation. Swiss Med Weekly 2007;137:669 –76. [11] Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: Contraindication vs. risk. Am J Transplant 2003;3:1488 –1500. [12] Billen EVA, Christiaans MHL, Lee J, van den Berg-Loonen EM. Donor-directed HLA antibodies before and after transplantectomy detected by the Luminex single antigen assay. Transplantation 2009;87:563–9. [13] Higgins R, Hathaway M, Lowe D, Lam F, Kashi H, Tan LC, et al. Blood levels of donor-specific human leukocyte antigen antibodies after renal transplantation: Resolution of rejection in the presence of circulating donor-specific antibody. Transplantation 2007;84:876 – 84. [14] Billen EVA, Voorter CEM, Christiaans MHL, van den Berg-Loonen E. Luminex donor-specific crossmatches. Tissue Antigens 2008;71:507–13. [15] Gibney EM, Cagle LR, Freed B, Warnell SE, Chan L, Wiseman AC. Detection of donor-specific antibodies using HLA-coated microspheres: Another tool for kidney transplant risk stratification. Nephrol Dial Transplant 2006;21: 2625–9. [16] Pollinger HS, Stegall MD, Gloor JM, Moore SB, Degoey SR, Ploeger NA, et al. Kidney transplantation in patients with antibodies against donor HLA class II. Am J Transplant 2007;7:857– 63. [17] Su¨sal C, Opelz G. Good kidney transplant outcome in recipients with presensitization against HLA class II but not HLA class I. Hum Immunol 2004; 65:810 – 6. [18] Akalin E, Dinavahi R, Friedlander R, Ames S, de Boccardo G, Sehgal V, et al. Addition of plasmapheresis decreases the incidence of acute antibody-mediated rejection in sensitized patients with strong donor-specific antibodies. Clin J Am Soc Nephrol 2008;3:1160 –7. [19] Mizutani K, Terasaki P, Hamdani E, Esquenazi V, Rosen A, Miller J, et al. The importance of anti-HLA-specific antibody strength in monitoring kidney transplant patients. Am J Transplant 2007;7:1027–31. [20] Mai ML, Ahsan N, Wadei HM, Genco PV, Geiger XJ, Willingham DL, et al. Excellent renal allograft survival in donor-specific antibody positive transplant patients—role of intravenous immunoglobulin and rabbit antithymocyte globulin. Transplantation 2009;87:227–32.
Contents lists available at ScienceDirect
Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant Vincent Aubert a,*, Jean-Pierre Venetz b, Giuseppe Pantaleo a, Manuel Pascual b a b
Service of Immunology and Allergy, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland Organ Transplant Center, Centre Hospitalier Universitaire Vaudois (CHUV), Lausanne, Switzerland
A R T I C L E
I N F O
Article history: Received 10 March 2009 Accepted 9 April 2009 Available online 16 April 2009
Keywords: DSA Luminex Solid phase assay HLA-antibodies Antibody-mediated rejection
A B S T R A C T
Since new technologies based on solid phase assays (SPA) have been routinely incorporated in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules has generally been considered as a risk factor for acute rejection (AR) and, in particular, for acute humoral rejection (AHR). We retrospectively studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell complement-dependent cytotoxicity (CDC) crossmatches at the time of transplant. Pretransplantation sera were screened for the presence of circulating anti-HLA antibody and DSA by using highly sensitive and HLA-specific Luminex assay, and the results were correlated with AR and AHR posttransplantation. We found that approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation. Of 113 patients, 11 (9.7%) had HLA-DSA. Of 11 rejection episodes post-transplant, only two patients had pretransplantation DSA, of whom one had a severe AHR (C4d positive). One-year allograft survival was similar between the pretransplantation DSA-positive and -negative groups. Number, class, and intensity of pretransplantation DSA, as well as presensitizing events, could not predict AR. We conclude that, based on the presence of pretransplantation DSA, post-transplantation acute rejections episodes could not have been predicted. The only AHR episode occurred in a recipient with pretransplantation DSA. More work should be performed to better delineate the precise clinical significance of detecting low titers of DSA before transplantation. 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction An increasing number of clinical studies have demonstrated adverse allograft survival in patients who have anti– human leukocyte antigen (HLA) antibodies, whether preformed pretransplantation or developed de novo post-transplantation [1–5]. Recently, since new technologies based on solid phase assays (SPA) have been routinely used in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against HLA antigens has generally been considered as a risk factor for acute rejection (AR) and/or acute humoral rejection (AHR) [6]. However, the precise clinical relevance of anti-HLA antibodies detected by very high analytical sensitivity assays, as well as the exact importance of the presence of pretransplantation DSA (either anti– class I HLA or anti– class II HLA or both) is not completely established. Moreover, the significance of DSA detectable only by SPA but not by cytotoxicity assays is still controversial. So far, retrospective studies investigating the clinical relevance of DSA detected by multiplex technology SPA have demonstrated different results [7–9]. Among the various limitations detected in these studies, perhaps
* Corresponding author. E-mail address: [email protected] (V. Aubert).
the most important is the lack of standardization of the different immunosuppressive regimens that were administered. Moreover, protocol biopsies are not performed in all studies to determine the presence or absence of subclinical AR or humoral rejection. At our institution, since April 2003, we have used the same IS regimen for all kidney transplant recipients [10]. The aim of this retrospective study was to determine the clinical relevance of preformed DSA detected by multiplex technology SPA at the time of kidney transplantation in patients receiving a common immunosuppressive protocol drug regimen. 2. Subjects and methods 2.1. Patients and sera All consecutive patients (n ⫽ 113) undergoing transplantation at the Centre Hospitalier Universitaire Vaudois in Lausanne, Switzerland, between April 2003 and April 2007 were investigated. The patient characteristics are shown in Table 1. All patients had current and peak negative T-cell and B-cell CDC crossmatches. No prospective flow-cytometric or Luminex crossmatches were performed. All retrospective data were obtained by analyzing the clinical records and electronic databases of the Transplant Center.
0198-8859/09/$32.00 - see front matter 䉷 2009 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2009.04.011
V. Aubert et al. / Human Immunology 70 (2009) 580 –583
Table 1 Patient characteristics All
Patients, n 113 Male, n (%) 79 (70) Age, mean ⫾ SD, at tx 46.2 ⫾ 15.1 Age, median (range) at tx 49 (3–78) Kidney disease, n (%) Diabetes 8 (7) Hypertension 9 (8) Glomerulonephritis 33 (29) Genetic disease 25 (22) Others 38 (34) Sensitizing events, n (%) Prior tx 21 (19) Blood transfusions 63 (56) Pregnancies 20 (18) Living donor, n (%) 45 (40) HLA mismatches, n (%) 0 9 (8) 1 2 (2) 2 12 (11) 3 24 (21) 4 35 (31) 5 18 (16) 6 13 (11) HLA A/B mismatches, n (%) 0 9 (8) 1 7 (6) 2 38 (34) 3 34 (30) 4 25 (22) HLA DR mismatches, n (%) 0 27 (24) 1 51 (45) 2 35 (31) Current PRA I at tx, n (%) 0% 102 (90) ⬍10% 4 (4) 10–49% 4 (4) 50–79% 1 (1) ⬎80% 2 (2)
DSA negative
DSA positive
p Value (DSA negative vs DSA positive)
102 11 76 (74) 3 (27) ⬍0.005 46.6 ⫾ 15.5 42.8 ⫾ 10.2 NS 50 (3–78) 39 (31–65) 8 (8) 9 (9) 28 (27) 24 (24) 33 (32)
0 0 5 (45) 1 (10) 5 (45)
NS NS NS NS NS
16 (16) 56 (55) 13 (13) 41 (40)
5 (45) 7 (64) 7 (64) 4 (36)
⬍0.01 NS ⬍0.0001 NS
9 (9) 2 (2) 11 (11) 21 (20) 32 (31) 15 (15) 12 (12)
0 0 1 (9) 3 (27) 3 (27) 3 (27) 1 (9)
NS NS NS NS NS NS NS
9 (9) 7 (7) 34 (33) 30 (29) 22 (21)
0 0 4 (36) 4 (36) 3 (27)
NS NS NS NS NS
23 (22) 49 (48) 30 (29)
4 (36) 2 (18) 5 (45)
NS ⬍0.03 NS
92 (90) 4 (4) 4 (4) 1 (1) 1 (1)
10 (91) 0 0 0 1 (9)
NS NS NS NS ⬍0.03
DSA ⫽ donor-specific antibody; tx ⫽ transplantation.
Immunosuppresive regimens were induction therapy with basiliximab for first kidney transplants (Table 2). In the event of retransplant or if panel-reactive antibody antibody was ⬎50%, thymoglobulin at 1.5 mg/kg/day for 4 days was given. Maintenance therapy consisted in tacrolimus (targeted plasma levels 8 –10 mg/ l), steroids (daily bolus of 500 mg, 250 mg, and 125 mg methylprednisolone for the first 3 days post-transplantation, followed by tapered steroids to a dose of 5 mg prednisone per day at 3 months post-transplantation), and MMF (initially 2 g/day). In hepatitis C virus–infected patients, cyclosporine was given instead of tacrolimus. 2.2. Detection of anti-HLA antibodies and definition of DSA Stored sera from consecutive patients undergoing transplantation between April 2003 and April 2007 who had negative pretransplantation CDC crossmatches with T and B cells were retested for the presence of anti-HLA antibodies using the multiplex technology SPA. Class I (i.e., HLA-A/B) and class II (i.e., HLA-DR/DP/DQ) HLA antibodies were tested using LabScreen LS1A01 Lot 007 and LS2A01 Lot 004 (OneLambda, Canoga Park, CA) commercialized in Switzerland by Ingen. Briefly, 20 l of serum samples were incubated with HLA class I– coated and HLA class II– coated microspheres, respectively, for 30 minutes in the dark under gentle agitation. The specimens were then washed five times before being incubated with anti-human IgG-conjugated phyco-erythrine in the same condi-
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tions as in the first incubation. The Labscan 100 flow analyzer (Luminex, Austin, TX) was used for beads and data acquisition. Data were then exported to HLA Visual software (One Lambda) for analysis. The cut-off level was defined as a baseline normalized ⬎500 mean fluorescence intensity units (MFI). The presence of DSA was assigned by comparing the various HLA specificities proposed by the software analysis with the HLA typing of the donor for all transplanted patients. Luminex results did not influence transplant management, as this was a retrospective analysis. 2.3. Definitions and management of rejection In cases of acute renal dysfunction, renal biopsy was performed. Acute rejection was defined by the Banff 03 working classification of renal allograft pathology. Since 2003, C4d staining has been performed on all renal allograft biopsy samples and correlated with the presence of posttransplantation donor-specific antibody in recipient’s serum. Treatment for acute rejection consisted of 3 days of high-dose methylprednisolone (500 mg daily boluses), followed by a prednisone taper. In addition, steroid-resistant patients were treated with thymoglobulin for 4 –7 days. 2.4. Statistical analysis Differences between groups were assessed by the 2 test and Fisher’s exact test for categoric variables. Parametric continuous data were analyzed by Student t test. Values of p ⬍ 0.05 were considered statistically significant. 3. Results 3.1. Patient characteristics Baseline characteristics of the 113 patients (all pretransplantation DSA-positive or -negative recipients) enrolled in the study are shown in Table 1. Of the patients, 70% were male and 30% female, with a mean age of 46.2 years at the time of transplantation. A history of sensitizing event was documented in 64% of the overall study patients, with 19% having prior transplants, 56% blood transfusions, and 18% pregnancies. Kidney transplants were from a living donor in 40% of cases. Presensitizing events (prior transplants, pregnancies, and female gender) were significantly more common in the group of patients with DSA than in the group without DSA. No statistical differences between patients with or without DSA were found regarding age, donor type, HLA mismatches, and current percentage PRA at transplantation. Detailed analysis showed that
Table 2 Baseline immunosuppression, acute rejection, and survival
Patients, n Induction Basiliximab, n (%) Thymoglobulin, n (%) Basiliximab ⫹ Thymo, n (%) Immunosuppression Tac, n (%) CsA, n (%) Rejection All acute rejection, n (%) Acute cellular rejection, n (%) Borderline, n (%) Ia, n (%) Acute humoral rejection, n (%) Clinically suspected Graft survival at 1 year, n (%) Patient survival at 1 year, n (%) NS ⫽ not significant.
All
DSA negative
DSA positive
113
102
11
P Value (DSA negative vs DSA positive)
⬍0.03 ⬍0.005 NS
81 (72) 25 (22) 7 (6)
76 (75) 19 (19) 7 (6)
5 (45) 6 (55) 0
108 (96) 5 (4)
97 (95) 5 (5)
11 (100) 0
NS NS
8 (7) 6 (5) 2 (2) 4 (3) 1 (1) 1 (1) 112 (99) 113 (100)
7 (7) 6 (6) 1 (1) 5 (5) 0 1 (1) 101 (99) 102 (100)
1 (9) 0 0 0 1 (9) 0 11 (100) 11 (100)
NS NS NS NS ⬍0.005 NS NS NS
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there were no differences in any PRA category between patients with or without DSA, except in the ⬎80% PRA category. In the DSA-positive group, significantly more patients (6/11) received thymoglobulin induction as compared with DSA-negative patients (19/102). 3.2. HLA antibodies and correlation of DSA with AR Approximately half of our patient population (55/113, 48.7%) had pretransplantation circulating anti-HLA antibodies. Of 113 patients, 11 (9.7%) had pretransplantation DSA (DSA-positive group). The antibodies identified were anti– class I DSA only in six patients, anti– class II DSA only in four patients. One patient had both anti– class I and anti– class II DSA. Of 11 AR episodes post-transplantation, six patients were negative for anti-HLA antibodies and five were positive for antiHLA, but only two patients had pretransplantation DSA, of whom one developed AHR (C4d positive) at day 7 post-transplantation. Of note, nine of 11 recipients had pretransplantation DSA and did not present any post-transplantation rejection episodes. Moreover, differences in the number, cumulative strength, and class of DSA were not statistically significant between DSA-positive patients with or without AR (data not shown). One AHR episode occurred, in a recipient with pretransplantation DSA. 4. Discussion With the advent of highly sensitive antibody screening tests using SPA and multiplex technology, it has become more important to identify whether the presence of pretransplantation anti-HLA antibodies or DSA may be associated with higher risk of AR, AHR, or worse clinical outcomes. To investigate the clinical relevance of pretransplantation DSA, we studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell CDC crossmatches at the time of transplantation. We retrospectively screened pretransplantation sera for the presence of circulating anti-HLA antibody and DSA on serum samples that were taken immediately before transplantation by using highly sensitive and HLA-specific Luminex assays. Approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation by Luminex testing. Interestingly, only seven of 113 of these recipients (6%) would have been considered as immunized before transplantation when using a traditional PRA-CDC test (PRA ⬎10%). This discrepancy is probably caused by the high sensitivity of the SPA, which also identifies HLA class I and class II noncytolytic antibodies [11]. This high sensitivity of the SPA was also confirmed by the analysis of DSA. Indeed, 10 of 11 patients (91%) who were DSA positive before transplantation had a PRA level of 0%. This high sensitivity of SPA may be related in part to the cut-off level fixed at 500 MFI according to the manufacturer’s criteria. Indeed recent studies have reported a higher cut-off value [12–14]. In our study, no hyperacute rejection occurred, and patient and graft survival were excellent in anti-HLA or DSA-positive and -negative groups. Similar good results have been previously reported by Gibney et al., who observed an AR of 3% in patients with anti-HLA antibodies in the absence of DSA [15]. Of 11 rejection episodes post-transplantation, only two patients had pretransplantation DSA, of whom one had AHR (C4d positive) at 1 week post-transplantation. This latter patient had become highly sensitized (PRA ⬎80%) after transplantectomy 15 years after her first kidney transplant. Patients waiting for a second or third transplant are generally considered at higher immunologic risk, as they have an increased likelihood of having DSA, especially if they have undergone transplantectomy [12]. Thus our patient who had AHR experienced the cumulative effect of the risk factors of being female, having a previous transplantectomy, waiting for a second transplant, and being hyperimmunized with post-transplantation DSA. Of note, nine of 11 recipients had pretransplantation DSA
without any post-transplantation rejection episodes after transplantation. Thus, regardless of the analysis, the development of AR was not associated with the presence of pretransplantation DSA. Biopsies were not performed in these nine patients because these patients had normal graft function at 1 year post-transplantation (mean estimated glomerular filtration rate [GFR] by modified diet in renal disease [MDRD], 57 ⫾ 7 ml/min/1.73 m2; mean creatinine level, 116 ⫾ 19 mol/l) with no significant proteinuria (⬍0.5 g/24 h). Our data are in contradiction to some studies showing that the presence of pretransplantation DSA had a significant impact on AR rates and graft survival [9,16]. One possible explanation of our low AR rates in our DSA-positive group was the predominant use of a potent induction regimen with thymoglobulin because of prior transplants or high PRA at transplantation. When we analyzed the rate of AR between the groups with and without HLA antibodies, we found no significant differences. In this series, we also observed a lack of significant differences for the presence of AR between the positive and negative DSA patients. Therefore, our study did not show any usefulness in differentiating between patients with only class I or only class II pretransplantation DSA compared with patients with both or more in predicting outcomes as previously described [16,17]. Indeed, the patient with AHR had only class I DSA, whereas the only patient in this series with class I and class II DSA pretransplantation had no AR at all (data not shown). Similarly, our data do not support previous findings that suggest that only anti-class II DSA pretransplantation, even if this is a relatively uncommon finding, could be associated with a high rate of humoral rejection [16]. In this experience, if we had taken into consideration the presence of pretransplantation DSA as an absolute contraindication to transplantation, 10% (11/113) of the patients would not have received a kidney transplant. Recently, Akalin et al. reported, in an analysis of 35 kidney transplant recipients with pretransplantation DSA, that the addition of plasmapheresis to high-dose intravenous Ig decreased the incidence of acute rejection [18]. The original point of their study was to stratify kidney transplant recipients according to the mean fluorescence indices of Luminex (MFI), a measure which may reflect the amount of circulating antibodies [18,19]. Their results indicated that patients with “strong DSA” were at higher risk for developing AHR and needed more intensive desensitization protocols. By reviewing our own data, the pretransplantation DSA-MFI values of the two kidney transplant recipients with acute rejection were at 1004 (patient 1, anti-DR11), 1008 and 2659 (patient 2, anti-A26 and anti-A11, respectively), that is, values relatively low according to the stratification proposed by Akalin et al. On the other hand, the pretransplantation DSA-MFI values of the nine kidney transplant recipients without any rejection episodes ranged from 694 to 5717 (mean, 2758 ⫾ 1938). Thus, based on these pretransplantation DSA-MFI values alone, we could not have predicted the post-transplantation acute rejection episodes. Altogether, our data confirm the high sensitivity of solid phase assays (SPA) of the Luminex type to detect circulating DSA antibodies. However, they also suggest that the positive predictive value of low-levels pretransplantation DSA (e.g., MFI values ⬍6000, such as those found in this series) remains incompletely understood. More studies are needed to define which pretransplantation DSA are pathogenic post-transplantation and/or to decide on the most suitable cut-off level for optimal use of determining DSA in routine transplantation. Finally, it may be that potent immunosuppressive induction protocols (e.g., with thymoglobulin) may protect against AR in patients with low levels of DSA pretransplantation. In summary, the presence of pretransplantation anti-HLA or DSA were correlated with pre-existing sensitizing events before transplantation but were not correlated with high risk of AR or worse clinical outcomes. Pretransplantation DSA (especially at low levels) detected by SPA multiplex technology should not be used
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alone to discourage transplantation, as most patients with DSA had good outcomes. Detecting DSA pretransplantation by using new SPA will likely become very useful in the near future, in particular for retransplantation or sensitized recipients at risk for AR, as it is the case in the exerience of Akalin et al. and others [18,20]. However, as a result of our recent experience, we have now changed the estimation of the positive cut-off level, and we take into consideration only DSA with ⬎2000 MFI values. Overall, the high sensitivity of SPA brings new knowledge to the field of solid organ transplantation that is valuable but still quite complex to understand. More work, therefore, should continue to be performed to better delineate the precise clinical significance of detecting circulating DSA before and after transplantation, to fully validate the routine implementation of these new techniques in allocation policies as well as in the clinic. Acknowledgments The authors acknowledge the Ingen Company for help in providing anti-HLA kits and MichÉle Estoppey for excellent technical assistance. References [1] Terasaki PI. Humoral theory of transplantation. Am J Transplant 2003;3: 665–73. [2] Cai J, Terasaki PI. Humoral theory of transplantation—mechanism, prevention, and treatment. Hum Immunol 2005;66:334 – 42. [3] Terasaki PI, Cai J. Humoral theory of transplantation: Further evidence. Curr Opin Immunol 2005;17:541–5. [4] Akalin E, Pascual M. Sensitization after Kidney Transplantation. Clin J Am Soc Nephrol 2006;1:433– 40. [5] Mao Q, Terasaki PI, Cai J, Briley K, Catrou P, Haisch C, et al. Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study. Am J Transplant 2007;7:864 –71. [6] Lefaucheur C, Superbielle-Boissel C, Hill GS, Nochy D, Andrade J, Antoine C, et al. Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation. Am J Transplant 2008;8:324 –31. [7] Gupta A, Iveson V, Varagunam M, Bodger S, Sinnott P, Thuraisingham RC. Pretransplant donor-specific antibodies in cytotoxic negative crossmatch kidney transplants: Are they relevant? Transplantation 2008;85:1200 – 4.
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[8] van den Berg-Loonen EM, Billen EV, Voorter CE, van Heurn LW, Claas FH, van Hoof JP, et al. Clinical relevance of pretransplant donor-directed antibodies detected by single antigen beads in highly sensitized renal transplant patients. Transplantation 2008;85:1086 –90. [9] Patel AM, Pancoska C, Mulgaonkar S, Wenig FL. Renal transplantation in patients with pretransplantation donor-specific antibodies and negative flow cytometry crossmatches. Am J Transplant 2007;7:2371–7. [10] Manuel O, Venetz JP, Fellay J, Wasserfallen JB, Sturzenegger N, Fontana M, et al. Efficacy and safety of universal valganciclovir prophylaxis combined with a tacrolimus/mycophenolate-based regimen in kidney transplantation. Swiss Med Weekly 2007;137:669 –76. [11] Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: Contraindication vs. risk. Am J Transplant 2003;3:1488 –1500. [12] Billen EVA, Christiaans MHL, Lee J, van den Berg-Loonen EM. Donor-directed HLA antibodies before and after transplantectomy detected by the Luminex single antigen assay. Transplantation 2009;87:563–9. [13] Higgins R, Hathaway M, Lowe D, Lam F, Kashi H, Tan LC, et al. Blood levels of donor-specific human leukocyte antigen antibodies after renal transplantation: Resolution of rejection in the presence of circulating donor-specific antibody. Transplantation 2007;84:876 – 84. [14] Billen EVA, Voorter CEM, Christiaans MHL, van den Berg-Loonen E. Luminex donor-specific crossmatches. Tissue Antigens 2008;71:507–13. [15] Gibney EM, Cagle LR, Freed B, Warnell SE, Chan L, Wiseman AC. Detection of donor-specific antibodies using HLA-coated microspheres: Another tool for kidney transplant risk stratification. Nephrol Dial Transplant 2006;21: 2625–9. [16] Pollinger HS, Stegall MD, Gloor JM, Moore SB, Degoey SR, Ploeger NA, et al. Kidney transplantation in patients with antibodies against donor HLA class II. Am J Transplant 2007;7:857– 63. [17] Su¨sal C, Opelz G. Good kidney transplant outcome in recipients with presensitization against HLA class II but not HLA class I. Hum Immunol 2004; 65:810 – 6. [18] Akalin E, Dinavahi R, Friedlander R, Ames S, de Boccardo G, Sehgal V, et al. Addition of plasmapheresis decreases the incidence of acute antibody-mediated rejection in sensitized patients with strong donor-specific antibodies. Clin J Am Soc Nephrol 2008;3:1160 –7. [19] Mizutani K, Terasaki P, Hamdani E, Esquenazi V, Rosen A, Miller J, et al. The importance of anti-HLA-specific antibody strength in monitoring kidney transplant patients. Am J Transplant 2007;7:1027–31. [20] Mai ML, Ahsan N, Wadei HM, Genco PV, Geiger XJ, Willingham DL, et al. Excellent renal allograft survival in donor-specific antibody positive transplant patients—role of intravenous immunoglobulin and rabbit antithymocyte globulin. Transplantation 2009;87:227–32.