SDC, Patients and Methods
Complement-dependent lymphocytotoxic crossmatch test (CDC-CXM)
Serum samples from recipients were obtained within 48 hours before transplantation. Pre-transplant donor-specific CXM was performed by the standard CDC technique. In brief, the donor’s B and T lymphocytes were isolated from peripheral blood, the recipient’s serum was added, and the mixture was incubated at room temperature or 37°C for 60 minutes. This was followed by a 120-minute incubation with rabbit complement in order to detect any cytotoxic antibody activity against donor cells. Target cell lysis was determined by trypan blue exclusion. The CXM test was interpreted as positive when more than 20% of donor lymphocytes were lysed in excess of the baseline rate. If the test was positive, the recipient’s serum was treated with dithiothreitol (DTT) for 30 minutes to act as a reducing agent to inactivate immunoglobulin M (IgM) antibodies, and the test was subsequently repeated (8). A negative CXM after DTT reduction was indicative of the presence of IgM antibodies only, whereas a positive CXM in spite of DTT treatment indicated the presence of immunoglobulin G (IgG) antibodies.
Immunosuppressive regimen
Before 2001, the immunosuppression protocol consisted of standard triple-drug therapy with cyclosporine, mycophenolate, and methylprednisolone. However, since 2001, a four-drug routine immunosuppressive protocol was established which included basiliximab (Simulect®, Novartis), a calcineurin inhibitor (tacrolimus or cyclosporine), mycophenolate, and methylprednisolone. Basiliximab, an IL-2R antibody, was given for immune induction during the intraoperative period and then on postoperative day four. Tacrolimus (Prograft®, Astellas) was started at a dose of 0.5 mg twice daily on postoperative day three. Trough concentrations of tacrolimus were adjusted to 10-12 ng/mL for the first month, and then tapered over 6 months to a maintenance level of 5-8 ng/mL. Cyclosporine (Sandimmune®, Novartis) was given at a dose of 2 mg/kg/day orally, and subsequently adjusted to maintain blood levels of 200-300 ng/mL for the first month. After the first month, target levels were set at 100-150 ng/mL. A total of 1.5 g (two doses of 750 mg) of mycophenolate (CellCept®, Roche) was provided the day following liver transplantation. The mycophenolate dose was adjusted according to the occurrence of hematologic or gastrointestinal side effects.
A total of 500 mg of methylprednisolone was injected in all patients during the anhepatic period, followed by 500 mg on the first postoperative day. Thereafter, the drug was gradually tapered to 32 mg/day over a period of 10 days, and discontinued by 12 months after LT. None of the patients received immunosuppression therapy with anti-lymphocyte antibodies.
SDC, Table S1. Comparison of liver function test values for aspartate aminotransferase and alanine aminotransferase between positive and negative lymphocytotoxic crossmatch recipients
Posttransplant day Aspartate aminotransferase (U/L) Alanine aminotransferase (U/L) CDC-CXM
Negative
CDC-CXM
True positive P-value CDC-CXM Negative
CDC-CXM
True positive P-value
0 (immediate) 259.9 256.1 0.793 161.7 148.4 0.149
0 (6 hr later) 448.1 335.1 0.023 342.2 311.0 0.014
1 597.1 694.5 0.015 440.6 528.8 0.025
2 502.8 729.5 0.021 461.8 577.5 0.107
3 281.9 341.7 0.017 367.8 419.2 0.093
4 143.5 156.3 0.051 263.8 299.3 0.163
5 87.2 88.3 0.117 197.5 209.4 0.179
6 72.5 62.4 0.497 167.3 159.6 0.578
7 83.1 48.5 0.471 162.2 131.6 0.602
8 90.3 50.4 0.227 172.9 127.1 0.191
9 79.6 48.0 0.010 189.5 127.4 0.013
10 135.7 47.0 0.008 216.2 128.5 0.003
11 93.7 51.8 0.177 221.9 135.8 0.009
12 75.1 54.0 0.347 218.4 151.6 0.098
13 67.3 77.0 0.577 211.3 165.3 0.145
14 62.0 59.6 0.558 207.7 176.9 0.179
1 month 44.4 40.4 0.169 99.4 83.8 0.320
3 months 36.2 38.1 0.915 49.7 55.0 0.883
6 months 41.9 35.7 0.430 48.4 44.0 0.826
12 months 41.8 46.3 0.369 47.7 52.7 0.096
24 months 43.9 90.8 0.566 46.3 53.6 0.154
36 months 40.9 37.3 0.684 43.0 36.0 0.831
48 months 41.5 35.8 0.935 41.1 33.6 0.948
60 months 37.6 29.7 0.439 38.2 26.2 0.178
*All values are expressed as means
SDC, Table S2. Effect of true positive lymphocytotoxic crossmatch and false positive lymphocytotoxic crossmatch (IgM autoantibodies) on liver transplant outcome
Negative CXM Positive CXM
aAb* (–) aAb* (+) P-value
Final CXM result
True negative (A)
True positive (B)
False positive
(C) A+B† A+C‡ Total B vs. A B vs.
A+C C vs. A C vs.
A+B B vs. C A vs. B v
Number of case 1011 80 42 1091 1053 1133 C
Allograft rejection 188 18 13 206 201 219 0.390 0.456 0.068 0.052 0.308 0.105
(18.6) (22.5) (31.0) (18.9) (19.1) (19.3)
Biliary complication 274 27 13 301 287 314 0.200 0.211 0.583 0.633 0.754 0.394
(27.1) (33.8) (31.0) (27.6) (27.3) (27.7)
Vascular complication
Hepatic artery 48 4 2 52 50 54 0.788 0.788 1.000 1.000 1.000 0.995
(4.7) (5.0) (4.8) (4.8) (4.7) (4.8)
Hepatic vein 28 3 2 31 30 33 0.491 0.502 0.338 0.348 1.000 0.677
(2.8) (3.8) (4.8) (2.8) (2.8) (2.9)
Portal vein 55 5 1 60 56 61 0.797 0.613 0.721 0.723 0.663 0.648
(5.4) (6.3) (2.4) (5.5) (5.3) (5.4)
Viral disease recurrence
Hepatitis B virus 79 6 2 85 81 79 0.920 0.950 0.765 0.766 0.713 0.766
(7.8) (7.5) (4.8) (7.8) (7.7) (7.0)
Hepatitis C virus 35 7 0 42 35 42 0.176 0.171 0.395 0.400 0.094 0.171
(3.5) (8.8) (0.0) (3.8) (3.3) (3.7)
CMV§ infection 325 30 9 355 334 364 0.325 0.286 0.144 0.130 0.071 0.196
(32.1) (37.5) (21.4) (32.5) (31.7) (32.1)
de novo malignancy 43 8 7 51 50 58 0.061 0.058 0.003 0.004 0.287 0.000
(4.3) (10.0) (16.7) (4.7) (4.7) (5.1)
Re-transplantation 37 4 1 41 38 42 0.535 0.532 1.000 1.000 0.659 0.745
(3.7) (5.0) (2.4) (3.8) (3.6) (3.7)
Graft loss (GL) 237 24 11 261 248 272 0.186 0.193 0.681 0.736 0.658 0.394
(23.4) (30.0) (26.2) (23.9) (23.6) (24.0)
Liver-specific GL 110 17 5 127 115 132 0.005 0.006 0.800 1.000 0.202 0.021
(10.9) (21.3) (11.9) (11.6) (10.9) (11.7)
Patient death 223 22 11 245 234 256 0.261 0.277 0.528 0.570 0.877 0.454
(22.1) (27.5) (26.2) (22.5) (22.2) (22.6)
Values are expressed as number (%); CXM, crossmatch; *aAb, IgM autoantibod
†A+B, absence of IgM autoantibody in recipient sera, ‡A+C, finally reported negative CDC-CXM result; §CMV, Cytomegalovir
SDC, Table S3. Multivariate analysis to identify risk factors for liver-specific graft survival
Variables in the equation B SE Wald df p-value Exp(B) 95.0% CI for Exp(B) Lower Upper Type of donor (living vs. deceased) -0.154 0.277 0.308 1 0.579 0.857 0.498 1.476 Type of recipient (adult vs. pediatric) 0.190 0.323 0.347 1 0.556 1.210 0.642 2.279 Relationship with the donor -0.268 0.227 1.387 1 0.239 0.765 0.490 1.195
Donor gender -0.390 0.180 4.679 1 0.031 0.677 0.475 0.964
Recipient gender -0.037 0.208 0.032 1 0.859 0.964 0.641 1.449 Allograft rejection -0.230 0.200 1.321 1 0.250 0.795 0.537 1.176 Biliary complication -0.129 0.196 0.434 1 0.510 0.879 0.598 1.291 Hepatic artery complication -1.489 0.256 33.852 1 0.000 0.226 0.137 0.372 Hepatic vein complication -0.686 0.350 3.838 1 0.049 0.504 0.254 1.000 Portal vein complication -0.381 0.316 1.450 1 0.228 0.683 0.368 1.270 Hepatitis B viral disease recurrence -0.463 0.261 3.149 1 0.076 0.629 0.378 1.050 Hepatitis C viral disease recurrence -0.569 0.360 2.495 1 0.114 0.566 0.280 1.147 Cytomegalovirus infection 0.303 0.201 2.266 1 0.132 1.354 0.913 2.009 de novo malignancy 0.843 0.517 2.660 1 0.103 2.324 0.844 6.399 True positive crossmatch -0.622 0.267 5.421 1 0.020 0.537 0.318 0.906 IgM autoantibody in recipient sera 0.201 0.464 0.187 1 0.665 1.222 0.492 3.037
