- Email: [email protected]
Prevalence and Impact of Reformed and De Novo Anti-HLA Donor-Specific Antibodies in Liver Transplantation
Prevalence and Impact of Reformed and De Novo Anti-HLA Donor-Specific Antibodies in Liver Transplantation M. Papachristoua, A. Fylaktoua,*, M. Daoudakib, E. Cholongitasc, T. Karampatakisa, A. Anastasioua, G. Chatzikaa, F. Makrovasilia, L. Vagiotasd, K. Karakasid, and I. Fouzasd a National Peripheral Histocompatibility Center, Hippokration General Hospital, Thessaloniki, Greece; bBiochemistry Laboratory, Medical School Aristotle University of Thessaloniki, Thessaloniki, Greece; cFirst Department of Internal Medicine, Medical School of National & Kapodistrian University of Athens, Athens, Greece; and dDivision of Transplantation, Department of Surgery, Hippokration General Hospital, Thessaloniki, Greece
ABSTRACT Introduction. The prevalence and impact of pre-existing and de novo anti-HLA donorspecific antibodies (DSAs) after orthotopic liver transplantation (OLT) is still controversial. We investigated the prevalence of DSAs and their implication in the development of allograft dysfunction after OLT. Patients and Methods. A total of 65 liver transplant patients were tested for anti-HLA antibodies, with single antigen bead technology, before, 1, 3, 6, and 12 months after transplantation, and thereafter annually, along with other risk factors. Sixteen out of 65 patients (24.6%) had circulating pre-existing anti-HLA antibodies, and 4 of them (25%) had DSAs. All patients positive for anti-HLA antibodies (100%) presented allograft dysfunction. Fourteen out of 65 patients (21.5%) had circulating de novo DSAs, and 12 out of 14 (85.7%) presented allograft dysfunction. The investigated risk factors for allograft dysfunction were: recipient and donor age, time on the waiting list, cold ischemia time, cytomegalovirus infection, immunosuppression regimen, de novo DSAs, Model for End-Stage Liver Disease, aspartate aminotransferase, alanine aminotransferase, gammaglutamyl transpeptidase (GGT), direct bilirubin and total bilirubin peak post-transplant, and alkaline phosphatase. The multivariate analysis showed that de novo DSAs and time on the waiting list were independent risk factors for allograft dysfunction. Conclusion. Our results show that de novo DSAs are an independent risk factor for allograft dysfunction, along with time on the waiting list.
T
HE PATHOGENIC role of pre-existing and de novo anti-HLA donor-specific antibodies (DSAs) after orthotopic liver transplantation (OLT) is still controversial [1]. Humoral rejection in clinical ABO-compatible OLT is considered clinically insignificant because liver is an immunologically privileged organ resistant to HLA alloantibodies [2]. Based on literature data, DSA emergence is associated with post-OLT complications including ductopenia, de novo autoimmune hepatitis, biliary strictures, accelerated fibrosis associated with recurrent liver disease, refractory thrombocytopenia and acute liver injury that can evolve into combined acute antibody-mediated rejection (AMR), and T-cell-mediated rejection [3,4]. Inadequately treated, the end result can be chronic or ductopenic rejection post-OLT [5].
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The latter is developed more frequently in patients with detected DSAs. In fact, there is an association of high-mean fluorescence intensity (MFI) DSAs with chronic rejection after OLT [6], and chronic rejection is considered the result of pre-existing or postemerging OLT-DSAs with MFI >10,000 [7]. De novo post-OLT DSAs usually are against HLA class II antigens, and the majority of them are against
*Address correspondence to Asimina Fylaktou, MD, PhD, National Peripheral Histocompatibility Center, Immunology Laboratory, Hippokration General Hospital, Kostantinoupoleos 49, 54642 Thessaloniki, Greece. Tel: þ003 2310 312 000; Fax: þ003 2310 818 254. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 424e428 (2019)
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Table 1. Main Demographic Data of the Study
Recipients Donors Mean recipient age SD Mean donor age SD
Male
Female
Total
48 (73.8%) 34 (52.3%) 53.5 9.7 44.8 14.0
17 (26.2%) 31 (47.7%) 48.9 11.8 49.2 13.6
65 (100%) 65 (100%) 52.3 10.4 46.9 13.9
Abbreviation: SD, standard deviation.
DQ antigens [6]. In general, recent reports indicate that the development of de novo DSAs is associated with lower liver graft survival [7,8]. A low immunosuppression load increases the risk of DSA development, so screening of anti-HLA antibodies is needed for early identification of acute or chronic AMR in order to develop therapeutic strategies [9,10]. The aim of this study is to define the frequency of preformed and de novo DSAs after OLT and to assign whether they act as risk factors for biliary complications and allograft dysfunction, potentially affecting graft survival. METHODS Seventy-five OLT patients (January 2010-December 2017) were evaluated. Ten patients were excluded due to death <3 months post-transplantation. Table 1 shows the demographic data of patients (n ¼ 65) enrolled in the study.
HLA-A, B, C, DR, DQ, DP loci by polymerase chain reaction (PCR) sequencing specific probes and PCR sequencing specific oligonucleotides were performed for all donor-recipient pairs. Anti-HLA antibodies of all OLT patients were tested by Luminex assay, before, 1, 3, 6, and 12 months post-transplantation and thereafter annually. Antibodies detected with a cut-off value of MFI 1000 were further classified into DSA and non-DSA. Demographic and clinical variables for each patient at the time of baseline visit are as follows: age at OLT, sex, indication for transplantation (including the presence of hepatocellular carcinoma before OLT based on radiological and/or histological findings and confirmed in the explant), concomitant diseases (e.g. diabetes mellitus, arterial hypertension, hyperlipidemia, coronary artery disease, and IgM positive cytomegalovirus [CMV] infection), cold ischemia time, time on the waiting list, and Model for End-Stage Liver Disease (MELD) score at the time of OLT. The laboratory variables recorded pre-transplantation and 1, 3, 6, and 12 months post-transplantation were serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), direct bilirubin, and total bilirubin (TBIL). In addition, immunosuppression regimen including the use or not of mammalian target of rapamycin inhibitors (mTORi) was also recorded. The patients received calcineurin inhibitor (CNI)-based (mainly cyclosporine) or everolimus-based immunosuppression regimen with or without mycophenolate mofetil. Everolimus was given instead of CNIs in patients with hepatocellular carcinoma before OLT or development of renal dysfunction (defined as serum creatinine 1.5 mg/dL and/or Modification of Diet in Renal
Table 2. Patients Characteristics in the Study Groups With Respect to De Novo DSAs Continuous Variable (mean SD)
Recipient’s age (years) Donor’s age (years) Time on waiting list (months) CIT (hours) MELD AST pre-transplantation (U/L) AST peak value (U/L) post-OLT‡ ALT peak value (U/L) post-OLT‡ GGT pre-transplantation (U/L) GGT peak value (U/L) post-OLT‡ ALP pre-transplantation (U/L) ALP peak value (U/L) post-OLT‡ DBIL peak value (mg/dL) post-OLT TBIL peak value (mg/dL) post-OLT‡ Categorical Variables Donor’s sex: female/male [n (%)] Recipient’s sex: female/male [n (%)] Viral disease: yes/no [n (%)] mTORi: yes/no [n (%)] Recipient’s CMV pre-transplantation: yes/no [n (%)] Recipient’s CMV post-transplantation: yes/no [n (%)] Donor’s CMV: yes/no [n (%)] Allograft dysfunction: no/yes [n (%)]
NoneDe Novo Anti-HLA
53.1 45.8 14.2 9.0 14.4 63.3 48.5 78.7 56.6 113.7 134.2 156.4 0.8 1.9
10.0 14.4 19.6 1.8 4.6 52.0 33.6 72.1 46.5 129.7 69.7 155.3 1.0 3.2
17 (42.5)/23 (57.5) 9 (22.5)/31 (77.5) 20 (50)/20 (50) 21 (52.5)/19 (47.5) 2 (5.6)/34 (94.4) 5 (16.7)/25 (83.3) 1 (2.6)/37 (97.4) 19 (47.5)/21 (52.5)
De Novo DSA
48.1 47.9 10.7 8.6 19.1 170.8 54.6 71.1 60.5 184.4 199.6 218.4 1.3 4.6
12.9 12.7 10.6 1.5 5.2 233.3 42.7 71.8 42.2 230.2 110.2 165.0 1.2 4.3
9 (64.3)/5 (35.7) 4 (28.6)/10 (71.4) 8 (57.1)/6 (42.9) 8 (57.1)/6 (42.9) 1 (8.3)/11 (91.7) 5 (45.5)/6 (54.5) 0 (0)/12 (100) 12 (85.7)/2 (14.3)
De Novo Anti-HLA, Non-DSA
P Value*
P Value†
7.6 14.4 14.4 2.0 1.9 50.5 29.0 29.7 103.2 48.9 159.6 61.4 1.1 3.3
.143 .805 .789 .476 .002 .002 .499 .952 .558 .414 .059 .059 .057 .040
.684 .536 .705 .985 .029 .026 .443 .500 .061 .781 .056 .191 .450 .590
(45.5)/6 (54.5) (36.4)/7 (63.6) (54.5)/5 (45.5) (54.5)/5 (45.5) 0 (0)/9 (100) 1 (12.5)/7 (87.5) 0 (0)/11 (100) 8 (72.7)/3 (27.3)
.160 .647 .645 .764 .731 .057 .570 .013
.861 .350 .789 .904 .469 .774 .587 .138
54.5 49.4 14.4 9.0 16.5 93.0 52.6 69.0 106.7 64.0 222.8 156.3 1.0 2.6 5 4 6 6
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; OLT, orthotopic liver transplantation; SD, standard deviation; TBIL, total bilirubin. *Comparison between nonede novo anti-HLA and de novo DSA. † Comparison between nonede novo anti-HLA and de novo anti-HLA, non-DSA. ‡ The peak post-transplant values were recorded at the 30th postoperative day.
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PAPACHRISTOU, FYLAKTOU, DAOUDAKI ET AL Table 3. Patients and Donor Characteristics With Respect to Allograft Dysfunction Continuous Variables (mean SD)
No Allograft Dysfunction (n ¼ 26)
Recipient’s age (years) Donor’s age (years) Time on waiting list (months) CIT (hours) MELD score AST pre-transplantation (U/L) AST peak value (U/L) post-OLT* ALT peak value (U/L) post-OLT* GGT pre-transplantation (U/L) GGT peak value (U/L) post-OLT* ALP pre-transplantation (U/L) ALP peak value (U/L) post-OLT* TBIL peak value (mg/dL) post-OLT* DBIL peak value (mg/dL) post-OLT Categorical Variables Donor’s sex: female/male [n (%)] Recipient’s sex: female/male [n (%)] Viral disease: yes/no [n (%)] mTORi: yes/no [n (%)] Recipient’s IgM positive CMV infection pre-transplantation: yes/no [n (%)] Recipient’s IgM positive CMV infection post-transplantation: yes/no [n (%)] Donor’s IgM positive CMV infection: yes/no [n (%)] De novo DSAs: yes/no [n (%)]
52.10.4 47.3 13.1 8.9 9.2 8.7 1.5 14.3 4.1 63.4 55.1 30.1 14.2 38.0 33.0 66.4 53.7 32.6 23.4 140.5 75.0 103.7 51.9 2.8 4.9 0.6 0.8 12 (46.2)/14 (53.8) 7 (26.9)/19 (73.1) 14 (53.8)/12 (46.2) 12 (46.2)/14 (53.8) 1 (4.2)/23 (95.8)
Allograft Dysfunction (n ¼ 39)
P Value
.722 .778 .051 .169 .045 .013 <.001 <.001 .543 <.001 .227 <.001 .104 .006
52.4 46.6 16.5 9.0 16.6 111.4 62.5 97.8 65.8 175.5 179.1 209.8 2.6 1.2 19 10 20 23 2
10.5 14.6 20.3 1.9 5.0 151.0 37.8 72.0 66.1 172.2 119.1 171.7 2.7 1.2
(48.7)/20 (51.3) (25.6)/29 (74.4) (51.3)/19 (48.7) (59.0)/16 (41.0) (6.1)/31 (93.9)
.839 .908 .839 .310 .752
2 (9.1)/20 (90.9)
9 (33.3)/18 (66.7)
.043
0 (0)/25 (100) 2 (7.7)/24 (92.3)
1 (2.8)/35 (97.2) 12 (30.8)/27 (69.2)
.401 .027
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; OLT, orthotopic liver transplantation; SD, standard deviation; TBIL, total bilirubin. *The peak post-transplant values were recorded at the 30th postoperative day.
Diseaseebased glomerular filtration rate <60 mL/min) during their follow-up after OLT. Informed consent from all patients and approval from local ethics committee were obtained. During the follow-up period, development of biliary complications and/or graft dysfunction including biliary strictures, ductopenia, and liver fibrosis were recorded. Allograft dysfunction was defined by increased international normalized ratio, bilirubin, and lactate levels after the third postoperative day. Diagnosis of complications was based on ultrasound, magnetic resonance imaging, and histological liver biopsies. The latter were performed whenever clinically indicated.
Statistical Analysis Categorical variables were compared using c2and Fisher’s exact test. Continuous variables for two groups were tested using Student t test and Mann-Whitney U test for parametric and nonparametric tests, respectively. Variables for more groups were tested using Kruskal-Wallis for nonparametric tests. The predictive factors for allograft dysfunction were determined by binary logistic regression. Statistically significant factors were included in multivariate logistic regression analysis to highlight the independent risk factors for allograft dysfunction. A P value of < .05 was considered statistically significant. Statistical analysis was performed with SPSS version 22 (IBM, Armonk, NY, United States).
RESULTS
Patients’ demographic data and transplant characteristics are listed on Table 1 and Table 2, respectively. Sixteen out
of 65 patients (24.6%) had circulating pre-existing antiHLA antibodies. Four of them (25%) had DSAs. One out of 4 patients (25%) with both class I and II DSAs kept the DSAs on follow-up. All 4 patients (100%) presented allograft dysfunction with biliary strictures (50%) and ductopenia (50%). Fourteen out of 65 patients (21.5%) had circulating de novo DSAs, and eleven out of 65 (17.2%) presented de novo anti-HLA antibodies but not DSAs. The total amount of de novo DSAs detected were 20, and 18 of them (90%) belonged to class II while two (10%) belonged to class I. Most of de novo class II DSAs (44.4%) belonged to DQ locus, and all 8 patients presenting them had allograft dysfunction (3 with biliary strictures, 2 with liver fibrosis, 3 with ductopenia). Moreover, the 2 patients with class I de novo DSAs had allograft dysfunction, as well. The median MFI for de novo class I DSAs was 2646.5 (1140e4153) and for class II DSAs 3774 (1048e21,205). Especially for DQ, the median MFI was 6499 (1048e21,205). In univariate analysis, patients with de novo DSAs (n ¼ 14) displayed, compared to patients without de novo anti-HLA (n ¼ 40), higher values of MELD score at the time of OLT (19.1 5.2 vs 14.4 4.6, respectively, P ¼ .002), higher levels of AST pre-transplantation (170.8 233.3 vs 63.3 52.0, respectively, P ¼ .002), and higher levels of TBIL peak value (4.6 4.3 vs 1.9 3.2 respectively, P ¼ .004). The most
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Table 4. Binary Logistic Regression Analysis for Allograft Dysfunction Outcome
Table 5. Multivariate Logistic Regression Analysis for Allograft Dysfunction Outcome
Variable
OR
95% CI
P Value
Variables
OR
95% CI
P
Recipient’s age Donor’s age Time on waiting list CIT MELD score AST pre-transplantation AST peak value post-OLT ALT peak value post-OLT GGT pre-transplantation GGT peak value post-OLT ALP pre-transplantation ALP peak value post-OLT TBIL peak value post-OLT DBIL peak value post-OLT Donor’s sex Recipient’s sex Viral disease mTORi Recipient’s IgM positive CMV infection pre-transplantation Recipient’s IgM positive CMV infection post-transplantation De novo DSAs
1.002 0.996 1.042 1.116 1.121 1.009 1.063 1.033 1.000 1.039 1.004 1.015 1.600 2.258 0.902 1.068 0.902 1.677 1.484
0.955e1.051 0.960e1.033 1.093e1.106 0.836e1.491 0.993e1.265 0.999e1.020 1.024e1.104 1.011e1.055 0.992e1.008 1.014e1.065 0.998e1.010 1.004e1.026 1.002e2.553 0.967e5.274 0.334e2.439 0.347e3.294 0.334e2.439 0.616e4.562 0.127e17.374
.938 .823 .002 .456 .064 .091 .002 .003 .971 .002 .157 .008 .049 .060 .839 .908 .839 .311 .753
De novo DSAs Time on waiting list
6.91 1.074
1.198e39.861 1.012e1.141
.031 .019
5.000
0.951e26.276
.057
5.333
1.082e26.278
.022
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; TBIL, total bilirubin; OLT, orthotopic liver transplantation; OR, odds ratio.
crucial finding was that these patients displayed a higher percentage of allograft dysfunction (85.7% vs 47.5%, respectively, P ¼ .013) (Table 2). Especially, 12 out of 14 patients with de novo DSA had graft dysfunction: 6 patients presented biliary strictures, 4 ductopenia, and 2 liver fibrosis. In univariate analysis with respect to allograft dysfunction, patients with allograft dysfunction (n ¼ 39) presented, compared to patients without allograft dysfunction (n ¼ 26), higher percentages of de novo DSAs (P ¼ .027) and recipient’s CMV IgM positive infection (P ¼ .043), confirmed by PCR, and received antiviral therapy. Moreover, these patients displayed higher values of MELD score at the time of OLT (P ¼ .045) and serum levels of AST pretransplantation (P ¼ .013), as well as higher peak values of AST (P < .001), ALT (P < .001), GGT (P < .001), ALP (P < .001), and direct bilirubin (P¼.006) posttransplantation (Table 3). Binary logistic regression revealed that de novo DSAs (P ¼ .022), time on the waiting list (P ¼ .002), serum peak values of AST (P ¼ .002), ALT (P ¼ .003), GGT (P ¼ .002), ALP (P ¼ .008), and TBIL (P ¼ .049) post-transplantation were significant predictive factors for allograft dysfunction (Table 4). Most of all, multivariate logistic regression analysis highlighted de novo DSA (P ¼ .031) and time on the waiting list (P ¼ .019) as independent risk factors for allograft dysfunction (Table 5).
Abbreviations: CI, confidence interval; DSA, donor-specific antibody; OR, odds ratio.
DISCUSSION
Graft-liver dysfunction with the concomitant presence of DSAs in serum is one of the criteria introduced by the Banff Working Group [2,11,12]. Our study shows that despite initial tolerance of the liver transplant to pre-existing anti-HLA DSAs and despite escape from AMR, pre-existing and de novo anti-HLA DSAs are most likely related to chronic graft dysfunction. This finding is in agreement with the recently published data by Del Bello et al [13]. Acute AMR is rare, but chronic rejection is more common, is usually presented with graft fibrosis and biliary strictures, and is associated with the presence of anti-HLA DSAs [1,6,12,14]. O’Leary et al found that de novo DSAs are associated with chronic rejection [6]. Musat et al reported that patients who present DSAs may develop ductopenic rejection [2]. In our study, de novo DSAs are the most significant factor for biliary strictures and graft failure. The pathogenic effects of anti-HLA DSAs are related to the antibody class, the specificity, and the strength reflected by MFI [14e16]. Most studies, including ours, have used an MFI 1000 cut-off value for positive anti-HLA antibodies [12]. It has also been reported, in agreement with our results, that allograft dysfunction is associated with the presence of class II DSAs, especially anti-DQ DSAs [17]. A long time on the waiting list is a significant risk factor for allograft dysfunction and failure [18,19], as has also been confirmed in our study. Higher MELD score is present in HLA-DSA positive recipients [16] and is associated with early allograft dysfunction [20]. According to our results, patients with allograft dysfunction had significantly higher MELD score, compared to patients with no allograft dysfunction. The diagnosis of acute and chronic liver rejection can be proved only with liver biopsy, usually has few clinical symptoms, and is based on a level increase of ALT, AST, GGT, ALT, and TBIL, irrespective of DSA presence [21], which is consistent with our results. There are several studies on the correlation between DSAs and immunosuppression and the different immunosuppression regimens used in OLT [22]. The liver recipients in our Transplantation Center are routinely treated with steroids, mycophenolate mofetil, and CNIs; depending on the case, they may be treated with mTORi. Our study did not reveal any statistical significant correlation between the frequency of de novo DSAs development or the MFI levels with mTORi patients’ treatment, as reported in other studies [23]. CMV infection is associated with AMR and chronic
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rejection, as revealed by other projects [24,25]. Our findings revealed that CMV infection is associated with graft dysfunction. A limitation of our study is the small number of patients with pre-existing DSAs and the inadequate follow-up period, due to the performance of most OLTs during the 2016e2017 period. In conclusion, our results show that DSAs are independent risk factor for allograft dysfunction along with time on the waiting list. REFERENCES [1] Del Bello A, Congy-Jolivet N, Muscari F, Lavayssière L, Esposito L, Cardeau-Desangles I, et al. Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients. Am J Transplant 2014;14:867e75. [2] Musat AI, Agni RM, Wai PY, Pirsch JD, Lorentzen DF, Powell A, et al. The significance of donor-specific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation. Am J Transplant 2011;11:500e10. [3] O’Leary JG, Shiller SM, Bellamy C, Nalesnik MA, Kaneku H, Jennings LW, et al. Acute liver allograft antibodymediated rejection: an inter-institutional study of significant histopathological features. Liver Transplant 2014;20:1244e55. [4] O’Leary JG, Demetris AJ, Friedman LS, Gebel HM, Halloran PF, Kirk AD, et al. The role of donor-specific HLA alloantibodies in liver transplantation. Am. J. Transplant 2014;14: 779e87. [5] Iacob S, Cicinnati VR, Lindemann M, Heinemann FM, Radtke A, Kaiser GM, et al. Donor-specific anti-HLA antibodies and endothelial C4d deposition-association with chronic liver allograft failure. Transplantation 2015;99:1869e75. [6] O’Leary JG, Kaneku H, Susskind BM, Jennings LW, Neri MA, Davis GL, et al. High mean fluorescence intensity donorspecific anti-HLA antibodies associated with chronic rejection postliver transplant. Am J Transplant 2011;11:1868e76. [7] Kaneku H, O’Leary JG, Banuelos N, Jennings LW, Susskind BM, Klintmalm GB, et al. De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients. Am J Transplant 2013;13:1541e8. [8] Watson R, Kozlowski T, Nickeleit V, Woosley JT, Schmitz JL, Zacks SL, et al. Isolated donor specific alloantibodymediated rejection after ABO compatible liver transplantation. Am J Transplant 2006;6:3022e9. [9] Chan KM, Lee CS, Wu TJ, Lee CF, Chen TC, Lee WC. Clinical perspective of acute humoral rejection after blood typecompatible liver transplantation. Transplantation 2011;91:e29e30. [10] Iacob S, Cicinnati VR, Dechêne A, Lindemann M, Heinemann FM, Rebmann V, et al. Genetic, immunological and
PAPACHRISTOU, FYLAKTOU, DAOUDAKI ET AL clinical risk factors for biliary strictures following liver transplantation. Liver Int 2012;32:1253e61. [11] Demetris AJ, Bellamy C, Hübscher SG, O’Leary J, Randhawa PS, Feng S, et al. 2016 comprehensive update of the Banff working group on liver allograft pathology: Introduction of antibody-mediated rejection. Am J Transplant 2016;16:2816e35. [12] Del Bello A, Congy-Jolivet N, Danjoux M, Muscari F, Kamar N. Donor-specific antibodies and liver transplantation. Hum Immunol 2016;77:1063e70. [13] Del Bello A, Congy-Jolivet N, Danjoux M, Muscari F, Lavayssière L, Esposito L, et al. De novo donor-specific anti-HLA antibodies mediated rejection in liver-transplant patients. Transpl Int 2015;28:1371e82. [14] Au KP, Chan SC, Chok KSH, Sharr WW, Dai WC, Sin SL, et al. Clinical factors affecting rejection rates in liver transplantation. Hepatobiliary Pancreat Dis Int 2015;14:367e73. [15] Ali S, Ormsby A, Shah V, Segovia MC, Kantz KL, Skorupski S, et al. Significance of complement split product C4d in ABO-compatible liver allograft: diagnosing utility in acute antibody mediated rejection. Transpl Immunol 2012;26:62e9. [16] Musat AI, Pigott CM, Ellis TM, Agni RM, Leverson GE, Powell AJ, et al. Pretransplant donor-specific anti-HLA antibodies as predictors of early allograft rejection in ABO-compatible liver transplantation. Liver Transplant 2013;19:1132e41. [17] Wozniak LJ, Hickey MJ, Venick RS, Vargas JH, Farmer DG, Busuttil RW, et al. Donor-specific HLA antibodies are associated with late allograft dysfunction after pediatric liver transplantation. Transplantation 2015;99:1416e22. [18] Howard D. The impact of waiting time on liver transplant outcomes. Health Serv Res 2000;35:1117e34. [19] Gheorghe L, Popescu I, Iacob R, Iacob S, Gheorghe C. Predictors of death on the waiting list for liver transplantation characterized by a long waiting time. Transpl Int 2005;18: 572e6. [20] Croome KP, Hernandez-Alejandro R, Chandok N. Early allograft dysfunction is associated with excess resource utilization after liver transplantation. Transplant Proc 2013;45:259e64. [21] Adams DH, Sanchez-Fueyo A, Samuel D. From immunosuppression to tolerance. J Hepatol 2015;62:S170e85. [22] Demetris AJ, Zeevi A, O’Leary JG. ABO-compatible liver allograft antibody-mediated rejection: an update HHS public access. Curr Opin Organ Transpl 2015;20:314e24. [23] Willuweit K, Heinold A, Rashidi-Alavijeh J, Heinemann FM, Horn PA, Paul A, et al. Immunosuppression with mTOR inhibitors prevents the development of donor-specific antibodies after liver transplant. Clin Transplant 2017;3:e12974. [24] Razonable RR. Cytomegalovirus infection after liver transplantation: current concepts and challenges. World J Gastroenterol 2008;14:4849e60. [25] Weigand K, Schnitzler P, Schmidt J, Chahoud F, Gotthardt D, Schemmer P, et al. Cytomegalovirus infection after liver transplantation incidence, risks, and benefits of prophylaxis. Transplant Proc 2010;42:2634e41.
ABSTRACT Introduction. The prevalence and impact of pre-existing and de novo anti-HLA donorspecific antibodies (DSAs) after orthotopic liver transplantation (OLT) is still controversial. We investigated the prevalence of DSAs and their implication in the development of allograft dysfunction after OLT. Patients and Methods. A total of 65 liver transplant patients were tested for anti-HLA antibodies, with single antigen bead technology, before, 1, 3, 6, and 12 months after transplantation, and thereafter annually, along with other risk factors. Sixteen out of 65 patients (24.6%) had circulating pre-existing anti-HLA antibodies, and 4 of them (25%) had DSAs. All patients positive for anti-HLA antibodies (100%) presented allograft dysfunction. Fourteen out of 65 patients (21.5%) had circulating de novo DSAs, and 12 out of 14 (85.7%) presented allograft dysfunction. The investigated risk factors for allograft dysfunction were: recipient and donor age, time on the waiting list, cold ischemia time, cytomegalovirus infection, immunosuppression regimen, de novo DSAs, Model for End-Stage Liver Disease, aspartate aminotransferase, alanine aminotransferase, gammaglutamyl transpeptidase (GGT), direct bilirubin and total bilirubin peak post-transplant, and alkaline phosphatase. The multivariate analysis showed that de novo DSAs and time on the waiting list were independent risk factors for allograft dysfunction. Conclusion. Our results show that de novo DSAs are an independent risk factor for allograft dysfunction, along with time on the waiting list.
T
HE PATHOGENIC role of pre-existing and de novo anti-HLA donor-specific antibodies (DSAs) after orthotopic liver transplantation (OLT) is still controversial [1]. Humoral rejection in clinical ABO-compatible OLT is considered clinically insignificant because liver is an immunologically privileged organ resistant to HLA alloantibodies [2]. Based on literature data, DSA emergence is associated with post-OLT complications including ductopenia, de novo autoimmune hepatitis, biliary strictures, accelerated fibrosis associated with recurrent liver disease, refractory thrombocytopenia and acute liver injury that can evolve into combined acute antibody-mediated rejection (AMR), and T-cell-mediated rejection [3,4]. Inadequately treated, the end result can be chronic or ductopenic rejection post-OLT [5].
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The latter is developed more frequently in patients with detected DSAs. In fact, there is an association of high-mean fluorescence intensity (MFI) DSAs with chronic rejection after OLT [6], and chronic rejection is considered the result of pre-existing or postemerging OLT-DSAs with MFI >10,000 [7]. De novo post-OLT DSAs usually are against HLA class II antigens, and the majority of them are against
*Address correspondence to Asimina Fylaktou, MD, PhD, National Peripheral Histocompatibility Center, Immunology Laboratory, Hippokration General Hospital, Kostantinoupoleos 49, 54642 Thessaloniki, Greece. Tel: þ003 2310 312 000; Fax: þ003 2310 818 254. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 424e428 (2019)
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Table 1. Main Demographic Data of the Study
Recipients Donors Mean recipient age SD Mean donor age SD
Male
Female
Total
48 (73.8%) 34 (52.3%) 53.5 9.7 44.8 14.0
17 (26.2%) 31 (47.7%) 48.9 11.8 49.2 13.6
65 (100%) 65 (100%) 52.3 10.4 46.9 13.9
Abbreviation: SD, standard deviation.
DQ antigens [6]. In general, recent reports indicate that the development of de novo DSAs is associated with lower liver graft survival [7,8]. A low immunosuppression load increases the risk of DSA development, so screening of anti-HLA antibodies is needed for early identification of acute or chronic AMR in order to develop therapeutic strategies [9,10]. The aim of this study is to define the frequency of preformed and de novo DSAs after OLT and to assign whether they act as risk factors for biliary complications and allograft dysfunction, potentially affecting graft survival. METHODS Seventy-five OLT patients (January 2010-December 2017) were evaluated. Ten patients were excluded due to death <3 months post-transplantation. Table 1 shows the demographic data of patients (n ¼ 65) enrolled in the study.
HLA-A, B, C, DR, DQ, DP loci by polymerase chain reaction (PCR) sequencing specific probes and PCR sequencing specific oligonucleotides were performed for all donor-recipient pairs. Anti-HLA antibodies of all OLT patients were tested by Luminex assay, before, 1, 3, 6, and 12 months post-transplantation and thereafter annually. Antibodies detected with a cut-off value of MFI 1000 were further classified into DSA and non-DSA. Demographic and clinical variables for each patient at the time of baseline visit are as follows: age at OLT, sex, indication for transplantation (including the presence of hepatocellular carcinoma before OLT based on radiological and/or histological findings and confirmed in the explant), concomitant diseases (e.g. diabetes mellitus, arterial hypertension, hyperlipidemia, coronary artery disease, and IgM positive cytomegalovirus [CMV] infection), cold ischemia time, time on the waiting list, and Model for End-Stage Liver Disease (MELD) score at the time of OLT. The laboratory variables recorded pre-transplantation and 1, 3, 6, and 12 months post-transplantation were serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), direct bilirubin, and total bilirubin (TBIL). In addition, immunosuppression regimen including the use or not of mammalian target of rapamycin inhibitors (mTORi) was also recorded. The patients received calcineurin inhibitor (CNI)-based (mainly cyclosporine) or everolimus-based immunosuppression regimen with or without mycophenolate mofetil. Everolimus was given instead of CNIs in patients with hepatocellular carcinoma before OLT or development of renal dysfunction (defined as serum creatinine 1.5 mg/dL and/or Modification of Diet in Renal
Table 2. Patients Characteristics in the Study Groups With Respect to De Novo DSAs Continuous Variable (mean SD)
Recipient’s age (years) Donor’s age (years) Time on waiting list (months) CIT (hours) MELD AST pre-transplantation (U/L) AST peak value (U/L) post-OLT‡ ALT peak value (U/L) post-OLT‡ GGT pre-transplantation (U/L) GGT peak value (U/L) post-OLT‡ ALP pre-transplantation (U/L) ALP peak value (U/L) post-OLT‡ DBIL peak value (mg/dL) post-OLT TBIL peak value (mg/dL) post-OLT‡ Categorical Variables Donor’s sex: female/male [n (%)] Recipient’s sex: female/male [n (%)] Viral disease: yes/no [n (%)] mTORi: yes/no [n (%)] Recipient’s CMV pre-transplantation: yes/no [n (%)] Recipient’s CMV post-transplantation: yes/no [n (%)] Donor’s CMV: yes/no [n (%)] Allograft dysfunction: no/yes [n (%)]
NoneDe Novo Anti-HLA
53.1 45.8 14.2 9.0 14.4 63.3 48.5 78.7 56.6 113.7 134.2 156.4 0.8 1.9
10.0 14.4 19.6 1.8 4.6 52.0 33.6 72.1 46.5 129.7 69.7 155.3 1.0 3.2
17 (42.5)/23 (57.5) 9 (22.5)/31 (77.5) 20 (50)/20 (50) 21 (52.5)/19 (47.5) 2 (5.6)/34 (94.4) 5 (16.7)/25 (83.3) 1 (2.6)/37 (97.4) 19 (47.5)/21 (52.5)
De Novo DSA
48.1 47.9 10.7 8.6 19.1 170.8 54.6 71.1 60.5 184.4 199.6 218.4 1.3 4.6
12.9 12.7 10.6 1.5 5.2 233.3 42.7 71.8 42.2 230.2 110.2 165.0 1.2 4.3
9 (64.3)/5 (35.7) 4 (28.6)/10 (71.4) 8 (57.1)/6 (42.9) 8 (57.1)/6 (42.9) 1 (8.3)/11 (91.7) 5 (45.5)/6 (54.5) 0 (0)/12 (100) 12 (85.7)/2 (14.3)
De Novo Anti-HLA, Non-DSA
P Value*
P Value†
7.6 14.4 14.4 2.0 1.9 50.5 29.0 29.7 103.2 48.9 159.6 61.4 1.1 3.3
.143 .805 .789 .476 .002 .002 .499 .952 .558 .414 .059 .059 .057 .040
.684 .536 .705 .985 .029 .026 .443 .500 .061 .781 .056 .191 .450 .590
(45.5)/6 (54.5) (36.4)/7 (63.6) (54.5)/5 (45.5) (54.5)/5 (45.5) 0 (0)/9 (100) 1 (12.5)/7 (87.5) 0 (0)/11 (100) 8 (72.7)/3 (27.3)
.160 .647 .645 .764 .731 .057 .570 .013
.861 .350 .789 .904 .469 .774 .587 .138
54.5 49.4 14.4 9.0 16.5 93.0 52.6 69.0 106.7 64.0 222.8 156.3 1.0 2.6 5 4 6 6
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; OLT, orthotopic liver transplantation; SD, standard deviation; TBIL, total bilirubin. *Comparison between nonede novo anti-HLA and de novo DSA. † Comparison between nonede novo anti-HLA and de novo anti-HLA, non-DSA. ‡ The peak post-transplant values were recorded at the 30th postoperative day.
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PAPACHRISTOU, FYLAKTOU, DAOUDAKI ET AL Table 3. Patients and Donor Characteristics With Respect to Allograft Dysfunction Continuous Variables (mean SD)
No Allograft Dysfunction (n ¼ 26)
Recipient’s age (years) Donor’s age (years) Time on waiting list (months) CIT (hours) MELD score AST pre-transplantation (U/L) AST peak value (U/L) post-OLT* ALT peak value (U/L) post-OLT* GGT pre-transplantation (U/L) GGT peak value (U/L) post-OLT* ALP pre-transplantation (U/L) ALP peak value (U/L) post-OLT* TBIL peak value (mg/dL) post-OLT* DBIL peak value (mg/dL) post-OLT Categorical Variables Donor’s sex: female/male [n (%)] Recipient’s sex: female/male [n (%)] Viral disease: yes/no [n (%)] mTORi: yes/no [n (%)] Recipient’s IgM positive CMV infection pre-transplantation: yes/no [n (%)] Recipient’s IgM positive CMV infection post-transplantation: yes/no [n (%)] Donor’s IgM positive CMV infection: yes/no [n (%)] De novo DSAs: yes/no [n (%)]
52.10.4 47.3 13.1 8.9 9.2 8.7 1.5 14.3 4.1 63.4 55.1 30.1 14.2 38.0 33.0 66.4 53.7 32.6 23.4 140.5 75.0 103.7 51.9 2.8 4.9 0.6 0.8 12 (46.2)/14 (53.8) 7 (26.9)/19 (73.1) 14 (53.8)/12 (46.2) 12 (46.2)/14 (53.8) 1 (4.2)/23 (95.8)
Allograft Dysfunction (n ¼ 39)
P Value
.722 .778 .051 .169 .045 .013 <.001 <.001 .543 <.001 .227 <.001 .104 .006
52.4 46.6 16.5 9.0 16.6 111.4 62.5 97.8 65.8 175.5 179.1 209.8 2.6 1.2 19 10 20 23 2
10.5 14.6 20.3 1.9 5.0 151.0 37.8 72.0 66.1 172.2 119.1 171.7 2.7 1.2
(48.7)/20 (51.3) (25.6)/29 (74.4) (51.3)/19 (48.7) (59.0)/16 (41.0) (6.1)/31 (93.9)
.839 .908 .839 .310 .752
2 (9.1)/20 (90.9)
9 (33.3)/18 (66.7)
.043
0 (0)/25 (100) 2 (7.7)/24 (92.3)
1 (2.8)/35 (97.2) 12 (30.8)/27 (69.2)
.401 .027
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; OLT, orthotopic liver transplantation; SD, standard deviation; TBIL, total bilirubin. *The peak post-transplant values were recorded at the 30th postoperative day.
Diseaseebased glomerular filtration rate <60 mL/min) during their follow-up after OLT. Informed consent from all patients and approval from local ethics committee were obtained. During the follow-up period, development of biliary complications and/or graft dysfunction including biliary strictures, ductopenia, and liver fibrosis were recorded. Allograft dysfunction was defined by increased international normalized ratio, bilirubin, and lactate levels after the third postoperative day. Diagnosis of complications was based on ultrasound, magnetic resonance imaging, and histological liver biopsies. The latter were performed whenever clinically indicated.
Statistical Analysis Categorical variables were compared using c2and Fisher’s exact test. Continuous variables for two groups were tested using Student t test and Mann-Whitney U test for parametric and nonparametric tests, respectively. Variables for more groups were tested using Kruskal-Wallis for nonparametric tests. The predictive factors for allograft dysfunction were determined by binary logistic regression. Statistically significant factors were included in multivariate logistic regression analysis to highlight the independent risk factors for allograft dysfunction. A P value of < .05 was considered statistically significant. Statistical analysis was performed with SPSS version 22 (IBM, Armonk, NY, United States).
RESULTS
Patients’ demographic data and transplant characteristics are listed on Table 1 and Table 2, respectively. Sixteen out
of 65 patients (24.6%) had circulating pre-existing antiHLA antibodies. Four of them (25%) had DSAs. One out of 4 patients (25%) with both class I and II DSAs kept the DSAs on follow-up. All 4 patients (100%) presented allograft dysfunction with biliary strictures (50%) and ductopenia (50%). Fourteen out of 65 patients (21.5%) had circulating de novo DSAs, and eleven out of 65 (17.2%) presented de novo anti-HLA antibodies but not DSAs. The total amount of de novo DSAs detected were 20, and 18 of them (90%) belonged to class II while two (10%) belonged to class I. Most of de novo class II DSAs (44.4%) belonged to DQ locus, and all 8 patients presenting them had allograft dysfunction (3 with biliary strictures, 2 with liver fibrosis, 3 with ductopenia). Moreover, the 2 patients with class I de novo DSAs had allograft dysfunction, as well. The median MFI for de novo class I DSAs was 2646.5 (1140e4153) and for class II DSAs 3774 (1048e21,205). Especially for DQ, the median MFI was 6499 (1048e21,205). In univariate analysis, patients with de novo DSAs (n ¼ 14) displayed, compared to patients without de novo anti-HLA (n ¼ 40), higher values of MELD score at the time of OLT (19.1 5.2 vs 14.4 4.6, respectively, P ¼ .002), higher levels of AST pre-transplantation (170.8 233.3 vs 63.3 52.0, respectively, P ¼ .002), and higher levels of TBIL peak value (4.6 4.3 vs 1.9 3.2 respectively, P ¼ .004). The most
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Table 4. Binary Logistic Regression Analysis for Allograft Dysfunction Outcome
Table 5. Multivariate Logistic Regression Analysis for Allograft Dysfunction Outcome
Variable
OR
95% CI
P Value
Variables
OR
95% CI
P
Recipient’s age Donor’s age Time on waiting list CIT MELD score AST pre-transplantation AST peak value post-OLT ALT peak value post-OLT GGT pre-transplantation GGT peak value post-OLT ALP pre-transplantation ALP peak value post-OLT TBIL peak value post-OLT DBIL peak value post-OLT Donor’s sex Recipient’s sex Viral disease mTORi Recipient’s IgM positive CMV infection pre-transplantation Recipient’s IgM positive CMV infection post-transplantation De novo DSAs
1.002 0.996 1.042 1.116 1.121 1.009 1.063 1.033 1.000 1.039 1.004 1.015 1.600 2.258 0.902 1.068 0.902 1.677 1.484
0.955e1.051 0.960e1.033 1.093e1.106 0.836e1.491 0.993e1.265 0.999e1.020 1.024e1.104 1.011e1.055 0.992e1.008 1.014e1.065 0.998e1.010 1.004e1.026 1.002e2.553 0.967e5.274 0.334e2.439 0.347e3.294 0.334e2.439 0.616e4.562 0.127e17.374
.938 .823 .002 .456 .064 .091 .002 .003 .971 .002 .157 .008 .049 .060 .839 .908 .839 .311 .753
De novo DSAs Time on waiting list
6.91 1.074
1.198e39.861 1.012e1.141
.031 .019
5.000
0.951e26.276
.057
5.333
1.082e26.278
.022
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; CIT, cold ischemia time; CMV, cytomegalovirus; DBIL, direct bilirubin; DSA, donor-specific antibody; GGT, gamma-glutamyl transpeptidase; MELD, Model for End-Stage Liver Disease; mTORi, mammalian target of rapamycin inhibitors; TBIL, total bilirubin; OLT, orthotopic liver transplantation; OR, odds ratio.
crucial finding was that these patients displayed a higher percentage of allograft dysfunction (85.7% vs 47.5%, respectively, P ¼ .013) (Table 2). Especially, 12 out of 14 patients with de novo DSA had graft dysfunction: 6 patients presented biliary strictures, 4 ductopenia, and 2 liver fibrosis. In univariate analysis with respect to allograft dysfunction, patients with allograft dysfunction (n ¼ 39) presented, compared to patients without allograft dysfunction (n ¼ 26), higher percentages of de novo DSAs (P ¼ .027) and recipient’s CMV IgM positive infection (P ¼ .043), confirmed by PCR, and received antiviral therapy. Moreover, these patients displayed higher values of MELD score at the time of OLT (P ¼ .045) and serum levels of AST pretransplantation (P ¼ .013), as well as higher peak values of AST (P < .001), ALT (P < .001), GGT (P < .001), ALP (P < .001), and direct bilirubin (P¼.006) posttransplantation (Table 3). Binary logistic regression revealed that de novo DSAs (P ¼ .022), time on the waiting list (P ¼ .002), serum peak values of AST (P ¼ .002), ALT (P ¼ .003), GGT (P ¼ .002), ALP (P ¼ .008), and TBIL (P ¼ .049) post-transplantation were significant predictive factors for allograft dysfunction (Table 4). Most of all, multivariate logistic regression analysis highlighted de novo DSA (P ¼ .031) and time on the waiting list (P ¼ .019) as independent risk factors for allograft dysfunction (Table 5).
Abbreviations: CI, confidence interval; DSA, donor-specific antibody; OR, odds ratio.
DISCUSSION
Graft-liver dysfunction with the concomitant presence of DSAs in serum is one of the criteria introduced by the Banff Working Group [2,11,12]. Our study shows that despite initial tolerance of the liver transplant to pre-existing anti-HLA DSAs and despite escape from AMR, pre-existing and de novo anti-HLA DSAs are most likely related to chronic graft dysfunction. This finding is in agreement with the recently published data by Del Bello et al [13]. Acute AMR is rare, but chronic rejection is more common, is usually presented with graft fibrosis and biliary strictures, and is associated with the presence of anti-HLA DSAs [1,6,12,14]. O’Leary et al found that de novo DSAs are associated with chronic rejection [6]. Musat et al reported that patients who present DSAs may develop ductopenic rejection [2]. In our study, de novo DSAs are the most significant factor for biliary strictures and graft failure. The pathogenic effects of anti-HLA DSAs are related to the antibody class, the specificity, and the strength reflected by MFI [14e16]. Most studies, including ours, have used an MFI 1000 cut-off value for positive anti-HLA antibodies [12]. It has also been reported, in agreement with our results, that allograft dysfunction is associated with the presence of class II DSAs, especially anti-DQ DSAs [17]. A long time on the waiting list is a significant risk factor for allograft dysfunction and failure [18,19], as has also been confirmed in our study. Higher MELD score is present in HLA-DSA positive recipients [16] and is associated with early allograft dysfunction [20]. According to our results, patients with allograft dysfunction had significantly higher MELD score, compared to patients with no allograft dysfunction. The diagnosis of acute and chronic liver rejection can be proved only with liver biopsy, usually has few clinical symptoms, and is based on a level increase of ALT, AST, GGT, ALT, and TBIL, irrespective of DSA presence [21], which is consistent with our results. There are several studies on the correlation between DSAs and immunosuppression and the different immunosuppression regimens used in OLT [22]. The liver recipients in our Transplantation Center are routinely treated with steroids, mycophenolate mofetil, and CNIs; depending on the case, they may be treated with mTORi. Our study did not reveal any statistical significant correlation between the frequency of de novo DSAs development or the MFI levels with mTORi patients’ treatment, as reported in other studies [23]. CMV infection is associated with AMR and chronic
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rejection, as revealed by other projects [24,25]. Our findings revealed that CMV infection is associated with graft dysfunction. A limitation of our study is the small number of patients with pre-existing DSAs and the inadequate follow-up period, due to the performance of most OLTs during the 2016e2017 period. In conclusion, our results show that DSAs are independent risk factor for allograft dysfunction along with time on the waiting list. REFERENCES [1] Del Bello A, Congy-Jolivet N, Muscari F, Lavayssière L, Esposito L, Cardeau-Desangles I, et al. Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients. Am J Transplant 2014;14:867e75. [2] Musat AI, Agni RM, Wai PY, Pirsch JD, Lorentzen DF, Powell A, et al. The significance of donor-specific HLA antibodies in rejection and ductopenia development in ABO compatible liver transplantation. Am J Transplant 2011;11:500e10. [3] O’Leary JG, Shiller SM, Bellamy C, Nalesnik MA, Kaneku H, Jennings LW, et al. Acute liver allograft antibodymediated rejection: an inter-institutional study of significant histopathological features. Liver Transplant 2014;20:1244e55. [4] O’Leary JG, Demetris AJ, Friedman LS, Gebel HM, Halloran PF, Kirk AD, et al. The role of donor-specific HLA alloantibodies in liver transplantation. Am. J. Transplant 2014;14: 779e87. [5] Iacob S, Cicinnati VR, Lindemann M, Heinemann FM, Radtke A, Kaiser GM, et al. Donor-specific anti-HLA antibodies and endothelial C4d deposition-association with chronic liver allograft failure. Transplantation 2015;99:1869e75. [6] O’Leary JG, Kaneku H, Susskind BM, Jennings LW, Neri MA, Davis GL, et al. High mean fluorescence intensity donorspecific anti-HLA antibodies associated with chronic rejection postliver transplant. Am J Transplant 2011;11:1868e76. [7] Kaneku H, O’Leary JG, Banuelos N, Jennings LW, Susskind BM, Klintmalm GB, et al. De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients. Am J Transplant 2013;13:1541e8. [8] Watson R, Kozlowski T, Nickeleit V, Woosley JT, Schmitz JL, Zacks SL, et al. Isolated donor specific alloantibodymediated rejection after ABO compatible liver transplantation. Am J Transplant 2006;6:3022e9. [9] Chan KM, Lee CS, Wu TJ, Lee CF, Chen TC, Lee WC. Clinical perspective of acute humoral rejection after blood typecompatible liver transplantation. Transplantation 2011;91:e29e30. [10] Iacob S, Cicinnati VR, Dechêne A, Lindemann M, Heinemann FM, Rebmann V, et al. Genetic, immunological and
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