Prediction of Biliary Complications After Living-Donor Liver Transplantation Based on Serum Cytokine Profile J.M. Kim, J.H. Kim, S.-Y. Lee, J.B. Park, C.H. David Kwon, S.J. Kim, J.-W. Joh, S.-K. Lee, and E.-S. Kang ABSTRACT Biliary tract complications are the main concern associated with living-donor liver transplantation (LDLT). Many laboratory parameters have been studied for the early detection of post-LDLT complications, including various cytokines. To explore immunologic activation status and its clinical significance, the cytokine secretion patterns of LDLT patients who developed biliary complications were analyzed. Serum samples from LDLT recipients were collected 1 day before and 3, 7, 14, and 30 days after transplantation. Each sample was tested for interleukin (IL) 2, IL-4, IL-6, IL-8, IL-10, IL-12, interferon-a, and tumor necrosis factor a with the use of multiplex bead flow cytometry. Fifteen patients without any complications and 6 patients with biliary complications showed differential serum cytokine profiles. The biliary complication group (4 biliary stricture and 2 biliary obstruction patients) displayed significantly increased concentrations of IL-2 and IL-12 on posttransplantation days 3 and 7 and of IL-4 on post-transplantation day 7. Profiling cytokine secretion in the serum of patients in the first month of LDLT may be helpful for the prediction and diagnosis of biliary complications within 1 year.
B
ILIARY COMPLICATIONS (BC), including anastomotic strictures, anastomotic leaks, intrahepatic strictures, biliary lithiasis, and cholangitis, are one of the main concerns associated with living-donor liver transplantation (LDLT) and cause postoperative morbidity and mortality [1]. The overall incidence of BC has been reported to range from 9% to 67% and mostly appears within the 1st year after LDLT [2]. Early diagnosis and prompt treatment of BC reduce unnecessary procedures and improve outcomes related to LDLT. Although confirmative diagnosis of BC relies on invasive techniques, such as radiographic imaging and cholangiography, elevated liver enzymes are an initial biochemical diagnostic clue, despite being measured with the use of a nonspecific test. For the early detection of complications after transplantation, many laboratory parameters have been studied. Cytokines are known to be crucial mediators of the immune response, and changes in cytokine levels precede post-transplantation complications [3,4]. Increased T-helper (TH) 2 cytokines have been suggested as an important factor preceding organ rejection, although some controversy exists [5]. Only a few studies have addressed cytokine levels associated with biliary pathogenesis [4,6]. The development of the multiplex assay has enabled the detection of soluble analytes such as cytokines and the rapid ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 861e864 (2014)
and accurate performance of dozens of assays in a single sample [7]. The present study evaluated the pattern of cytokine secretion with the use of multiplex bead flow cytometry in LDLT recipients with post-transplantation biliary complications in an effort to explain patient immunologic activation status and its clinical significance. MATERIALS AND METHODS Twenty-one patients who underwent their first LDLT from 2007 to 2009 at Samsung Medical Center, Seoul, Korea, were enrolled in
From the Department of Surgery (J.M.K., J.B.P., C.H.D.K., S.J.K., J.-W.J., S.-K.L.) and Department of Laboratory Medicine and Genetics (S.-Y.L., E.-S.K.), Samsung Medical Center; and Department of Laboratory Medicine (J.H.K.), Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea. The first 2 authors contributed equally to this work. Funding: Samsung Medical Center Clinical Research Development Program grant #CRS110-31-2. Address reprint requests to Eun-Suk Kang, MD, PhD, Associate Professor, Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, #50 Irwon-Dong, Gangnam-Gu, Seoul 135710, Korea. E-mail: [email protected] 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.038 861
862
KIM, KIM, LEE ET AL Table 1. Baseline Characteristics Biliary complications
Male Age (y) Diagnosis HBV HBV, alcoholic HBV, HCC HCC, non-B, non-C HCV, HCC History Hypertension Diabetes CTP A B C MELD score Intensive care unit stay (d) Hospitalization (d)
No (n ¼ 15)
Yes (n ¼ 6)
P value
13 (86.7%) 53 (20e62)
6 (100%) 49 (43e71)
NS NS NS
4 0 10 1 0
(26.7%) (0%) (66.7%) (6.7%) (0%)
4 (26.7%) 2 (13.3%) 5 7 3 11 8 23
(33.3%) (46.7%) (20.0%) (7e33) (6e12) (21e36)
1 1 3 0 1
(16.7%) (16.7%) (50.0%) (0%) (16.7%)
0 (0%) 1 (16.7%) 0 2 4 16 8 32
(0%) (33.3%) (66.7%) (14e35) (5e12) (21e45)
NS NS .007
NS NS .029
Abbreviations: NS, not significant; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; CTP, Child-Turcotte-Pugh; MELD, Model for End-Stage Liver Disease.
this study. The sera in these patients were serially collected and stored for analysis. Informed and written consent was obtained from each patient, and the study protocol was approved by the hospital’s
Fig 1. Serial liver function tests in the patient group with biliary complications (BC; n ¼ 6) and the group with no complications (N; n ¼ 15) at baseline before transplantation and 3, 7, 14, and 30 days after transplantation. (A) Aspartate transaminase (AST), (B) alanine transaminase (ALT), (C) total bilirubin, and (D) alkaline phosphatase (ALP).
Institutional Review Board. The presence of BC was identified through review of the medical records. BC types were classified as biliary leakage, strictures, or obstruction. The diagnosis of BC was based on clinical symptoms, liver function tests, and radiologic examination, including computerized tomography, ultrasonography, endoscopic retrograde cholangiopancreaticography, or diisopropyl iminodiacetic acid scan. Serum samples from LDLT recipients were collected 1 day before and 3, 7, 14, and 30 days after transplantation. The aliquots of serum were stored at 80 C before being analyzed. The concentrations of serum TH1 (interferon [IFN] g, interleukin [IL] 2, IL12, and tumor necrosis factor [TNF] a), TH2 (IL-4 and IL-10), and proinflammatory (IL-6 and IL-8) cytokines were measured with the use of multiplex bead flow cytometry (Flowcytomix; Bender Medsystems, Vienna, Austria). Briefly, patient serum was added to bead mixtures coated with antibodies to human cytokines. A biotinconjugated mixture was added to the serum antibody-bead mixture complex. Following incubation, a streptavidin-phycoerythrin (PE) solution was added to bind the biotin-conjugate. Following incubation, unbound streptavidin-PE was removed during a subsequent wash step, and the analyte was coupled with bead mixtures, biotinylated antibody, and streptavidin-PE emitting fluorescent signals. A standard curve was prepared from serial dilutions of a standard mixture. The concentrations of human cytokines were analyzed with the use of FACScanto flow cytometry (Becton Dickinson Biosciences, San Jose, California, USA) and Flowcytomix Pro 2.2 software (Bender Medsystems). Statistical analyses were performed with the use of SPSS 21.0 software (SPSS, Chicago, Illinois, USA) for Windows. Continuous
19.7 14.8 0
variables are presented as median and range and were compared with the use of the Mann-Whitney U test. Categoric variables were compared with the use of the Fisher exact test, as appropriate. A P value of
B
ILIARY COMPLICATIONS (BC), including anastomotic strictures, anastomotic leaks, intrahepatic strictures, biliary lithiasis, and cholangitis, are one of the main concerns associated with living-donor liver transplantation (LDLT) and cause postoperative morbidity and mortality [1]. The overall incidence of BC has been reported to range from 9% to 67% and mostly appears within the 1st year after LDLT [2]. Early diagnosis and prompt treatment of BC reduce unnecessary procedures and improve outcomes related to LDLT. Although confirmative diagnosis of BC relies on invasive techniques, such as radiographic imaging and cholangiography, elevated liver enzymes are an initial biochemical diagnostic clue, despite being measured with the use of a nonspecific test. For the early detection of complications after transplantation, many laboratory parameters have been studied. Cytokines are known to be crucial mediators of the immune response, and changes in cytokine levels precede post-transplantation complications [3,4]. Increased T-helper (TH) 2 cytokines have been suggested as an important factor preceding organ rejection, although some controversy exists [5]. Only a few studies have addressed cytokine levels associated with biliary pathogenesis [4,6]. The development of the multiplex assay has enabled the detection of soluble analytes such as cytokines and the rapid ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 46, 861e864 (2014)
and accurate performance of dozens of assays in a single sample [7]. The present study evaluated the pattern of cytokine secretion with the use of multiplex bead flow cytometry in LDLT recipients with post-transplantation biliary complications in an effort to explain patient immunologic activation status and its clinical significance. MATERIALS AND METHODS Twenty-one patients who underwent their first LDLT from 2007 to 2009 at Samsung Medical Center, Seoul, Korea, were enrolled in
From the Department of Surgery (J.M.K., J.B.P., C.H.D.K., S.J.K., J.-W.J., S.-K.L.) and Department of Laboratory Medicine and Genetics (S.-Y.L., E.-S.K.), Samsung Medical Center; and Department of Laboratory Medicine (J.H.K.), Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea. The first 2 authors contributed equally to this work. Funding: Samsung Medical Center Clinical Research Development Program grant #CRS110-31-2. Address reprint requests to Eun-Suk Kang, MD, PhD, Associate Professor, Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, #50 Irwon-Dong, Gangnam-Gu, Seoul 135710, Korea. E-mail: [email protected] 0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.038 861
862
KIM, KIM, LEE ET AL Table 1. Baseline Characteristics Biliary complications
Male Age (y) Diagnosis HBV HBV, alcoholic HBV, HCC HCC, non-B, non-C HCV, HCC History Hypertension Diabetes CTP A B C MELD score Intensive care unit stay (d) Hospitalization (d)
No (n ¼ 15)
Yes (n ¼ 6)
P value
13 (86.7%) 53 (20e62)
6 (100%) 49 (43e71)
NS NS NS
4 0 10 1 0
(26.7%) (0%) (66.7%) (6.7%) (0%)
4 (26.7%) 2 (13.3%) 5 7 3 11 8 23
(33.3%) (46.7%) (20.0%) (7e33) (6e12) (21e36)
1 1 3 0 1
(16.7%) (16.7%) (50.0%) (0%) (16.7%)
0 (0%) 1 (16.7%) 0 2 4 16 8 32
(0%) (33.3%) (66.7%) (14e35) (5e12) (21e45)
NS NS .007
NS NS .029
Abbreviations: NS, not significant; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; CTP, Child-Turcotte-Pugh; MELD, Model for End-Stage Liver Disease.
this study. The sera in these patients were serially collected and stored for analysis. Informed and written consent was obtained from each patient, and the study protocol was approved by the hospital’s
Fig 1. Serial liver function tests in the patient group with biliary complications (BC; n ¼ 6) and the group with no complications (N; n ¼ 15) at baseline before transplantation and 3, 7, 14, and 30 days after transplantation. (A) Aspartate transaminase (AST), (B) alanine transaminase (ALT), (C) total bilirubin, and (D) alkaline phosphatase (ALP).
Institutional Review Board. The presence of BC was identified through review of the medical records. BC types were classified as biliary leakage, strictures, or obstruction. The diagnosis of BC was based on clinical symptoms, liver function tests, and radiologic examination, including computerized tomography, ultrasonography, endoscopic retrograde cholangiopancreaticography, or diisopropyl iminodiacetic acid scan. Serum samples from LDLT recipients were collected 1 day before and 3, 7, 14, and 30 days after transplantation. The aliquots of serum were stored at 80 C before being analyzed. The concentrations of serum TH1 (interferon [IFN] g, interleukin [IL] 2, IL12, and tumor necrosis factor [TNF] a), TH2 (IL-4 and IL-10), and proinflammatory (IL-6 and IL-8) cytokines were measured with the use of multiplex bead flow cytometry (Flowcytomix; Bender Medsystems, Vienna, Austria). Briefly, patient serum was added to bead mixtures coated with antibodies to human cytokines. A biotinconjugated mixture was added to the serum antibody-bead mixture complex. Following incubation, a streptavidin-phycoerythrin (PE) solution was added to bind the biotin-conjugate. Following incubation, unbound streptavidin-PE was removed during a subsequent wash step, and the analyte was coupled with bead mixtures, biotinylated antibody, and streptavidin-PE emitting fluorescent signals. A standard curve was prepared from serial dilutions of a standard mixture. The concentrations of human cytokines were analyzed with the use of FACScanto flow cytometry (Becton Dickinson Biosciences, San Jose, California, USA) and Flowcytomix Pro 2.2 software (Bender Medsystems). Statistical analyses were performed with the use of SPSS 21.0 software (SPSS, Chicago, Illinois, USA) for Windows. Continuous
19.7 14.8 0
variables are presented as median and range and were compared with the use of the Mann-Whitney U test. Categoric variables were compared with the use of the Fisher exact test, as appropriate. A P value of
