Notice: Wiley Online Library will be unavailable on Saturday 27th February from 09:00-14:00 GMT / 04:00-09:00 EST / 17:00-22:00 SGT for essential maintenance. Apologies for the inconvenience.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
Human leukocyte antigen (HLA) crossmatching is currently accepted as a test that is mandatory for kidney, heart, and lung transplants. Only for liver transplants (LTs) does this test seem to be used sporadically. The first experience of Starzl et al.1, 2 indicated that crossmatching was not necessary. However, as more experience accrued, emerging evidence showed that a positive crossmatch test was associated with early graft failure.3, 4 Since that time, accumulating evidence has shown that crossmatches do have an early lasting effect.5-10 In 1995, an extensive study from London of 158 consecutive LTs showed that there was no effect of a positive crossmatch on overall graft survival by the usual cytotoxicity test or by flow cytometry.11 In 1996, a retrospective study from Pittsburgh on 1520 LTs was published; it showed that the early failure rate in positive crossmatch cases disappeared by the second year.12 More recently, in 2005, Muro et al.13 showed in 268 LTs that a very strong effect on graft survival was obtained with complement-dependent cytotoxicity (CDC) crossmatching: there was a 5-year survival rate of 21% in crossmatch-positive transplants versus a rate of 60% in crossmatch-negative transplants (P = 0.0016). Also in 2005, significantly poorer survival was noted with positive T cell crossmatches in 104 living donor LTs by Suehiro et al.14 In 2008, Castillo-Rama et al.15 showed that antibodies detected by either CDC or Luminex assays were associated with significantly lower graft survival among 896 transplants. These significant correlations with survival were confirmed by the collaborative studies of Opelz et al. in 2009.16 Thus, although historically there has been a tendency for many centers to skip this test, recent evidence seems to indicate that possibly with better technology, the crossmatch test is of importance in predicting graft survival for primary LTs.
There has, however, not been any study to date that has focused on the role of preformed HLA antibodies in second liver allograft survival. We therefore sought to determine the influence of preformed HLA antibodies on graft survival in liver retransplantation.
Ab, antibody; CDC, complement-dependent cytotoxicity; DSA, donor-specific antibody; HBV, hepatitis B virus; HCV, hepatitis C virus; HLA, human leukocyte antigen; LT, liver transplant; MHC, major histocompatibility complex; NS, not significant.
PATIENTS AND METHODS
We retrospectively analyzed sera from 139 patients who had undergone transplantation at 9 different LT centers that take part in the North Italy Transplant Program. The only inclusion criterion was that all patients must have received at least 2 LTs. Criteria for liver allocation included the patient's Model for End-Stage Liver Disease score, the ABO blood group matching, the donor-recipient age and size matching, and the balance of organs procured and transplanted by each center. A positive pretransplant crossmatch was not an absolute contraindication for a retransplant. The outcome of the second LT was then analyzed according to the age groups of the patients [ie, adults (≥18 years old) and pediatrics (<18 years old)].
Each patient had his or her serum tested once before the second LT for preformed HLA antibodies. These sera were sent to the Terasaki Foundation Laboratory (Los Angeles, CA) between November 2008 and March 2009, and they were tested for HLA class I and II immunoglobulin G antibodies with single antigen beads (One Lambda, Inc., Canoga Park, CA) according to the manufacturer's recommended protocol. Both serological and allelic HLA specificities were reported for HLA antibodies. The pretransplant CDC crossmatch was performed in Milan with unseparated donor lymphocytes. HLA typing of all donors, also performed in Milan, was by either sequence-specific primer or sequence-specific oligonucleotide probe hybridization, and only the HLA serological specificity was reported. Recipient HLA typing was not done.
Each patient had at least 6 months of follow-up from the second LT, which was deemed to have failed if the patient had a third LT or died. Second liver allograft survival was taken as the interval from the second LT to the third LT or death, whichever occurred earlier. The date of last follow-up for this study was February 28, 2009.
All patients involved gave informed consent. Ethical approval for the study was granted on the condition that the collected samples were screened for the purpose of assessing patients for transplantation.
Single Antigen Bead Assays
Screening tests for HLA-specific immunoglobulin G antibodies were performed with LABScreen single antigen class I (lot 5) and II (lot 6) beads (One Lambda). The LABScreen assay was performed according to the manufacturer's protocol. In brief, 20 μL of test serum was incubated with the beads for 30 minutes at room temperature in the dark. All samples were diluted 1:3 in 1× phosphate-buffered saline. Next, the samples were washed, and 100 μL of 1:100 anti-human immunoglobulin G-phycoerythrin was added. After a second incubation step, samples were washed twice and read on the LABScreen 100 Luminex machine (One Lambda). Raw trimmed mean fluorescence intensity values were obtained from the output file generated by the flow analyzer and normalized with the following formula:
A normalized trimmed mean fluorescence intensity greater than 1000 was taken as the cutoff for a positive value.
Frequency tables were used to summarize the counts of each variable by group. Graphs were plotted with Excel spread sheets (Microsoft, Redmond, WA). Statistical analyses were performed with Stata software, version 10.1 (StataCorp, College Station, TX). Graft survival was estimated by the Kaplan-Meier method and tested for statistical significance with the log-rank test. Categorical variables were compared via the chi-square test, and continuous variables were compared via the Student t test. Two-sided P values less than 0.05 were considered significant.
Table 1 summarizes the characteristics of our study population, which consisted of 118 adults and 21 pediatric patients. There were significantly more males among the adult patients (73.7%), and there were more female patients in our pediatric cohort (66.7%, P < 0.001). Significantly more adults were positive for hepatitis C (43.2% versus 4.8% in adult and pediatric patients, respectively, P < 0.001). The indications for liver retransplantation are listed in Table 2.
Table 1. Characteristics of the Study Population
Entire Study Population (n = 139)
Adults (n = 118, 84.9%)
Children (n = 21, 15.1%)
Values are reported as means and standard deviations.
Interval from serum collection to transplant (months)*
2.4 ± 7.8
2.4 ± 8.4
2.0 ± 3.1
Current patient status [n (%)]
Table 2. Causes of First Graft Failure
Cause of Liver Failure
Adults [n (%)]
Children [n (%)]
Hepatic artery thrombosis
One hundred three adults (87.3%) had HLA antibodies present; 24 (20.3%) had class I antibodies only, 23 (19.5%) had class II antibodies only, and 56 (47.5%) had both class I and II antibodies. With the exclusion of 8 patients whose donor HLA typing was unknown, 27 adult patients (24.5%) had donor-specific antibodies (DSAs) detected by single antigen beads (Fig. 1). In the pediatric cohort, 15 patients (71.4%) had HLA antibodies; 2 (9.5%) had class I antibodies only, 2 (9.5%) had class II antibodies only, and 11 (52.4%) had both class I and II antibodies. Six pediatric patients (28.6%) had DSAs. No significant differences were noted in the major histocompatibility complex (MHC) class distribution of HLA antibodies between adult and pediatric patients (data not shown). As donor HLA typing was reported on the basis of serological specificity, the recipient DSA status was assigned on the basis of the serological specificity of detected antibodies. The occurrence of HLA antibodies was not associated with the etiology of first graft failure or hepatitis C serostatus.
Class I antibodies were associated with significantly poorer regraft survival throughout the study in adults (P = 0.046, P = 0.042, and P = 0.033 at 1, 3, and 5 years, respectively; Fig. 2), but not in pediatric patients (P = not significant at 1, 3, and 5 years). Of the 80 adults with class I antibodies present, 28 (35.0%) and 37 (46.3%) lost their grafts within 3 months and 1 year of retransplantation, respectively; this highlighted the rapid rate of second liver graft loss in the first year after retransplantation. When adults with and without class I antibodies were compared, the survival difference at 1 year was 21.3%. Although graft loss beyond the first year occurred at a much slower rate, significant survival differences of 22.1% and 23.7% were still seen at 3 and 5 years, respectively. The time-dependent Cox proportional hazard model also showed class I antibodies to be a significant predictor of second liver allograft survival in adults (hazard ratio = 2.0, 95% confidence interval = 1.0-3.8, P = 0.028).
To eliminate the confounding effect of other HLA antibodies on second allograft survival, further analysis was performed according to the presence of class II antibodies, overall DSAs, class I DSAs, class II DSAs, and anti-HLA antibodies to each class II HLA locus (ie, DR, DP, and DQ). None of the other HLA antibodies showed a significant association, positive or negative, with graft survival in either the adult cohort or the pediatric cohort. Among adults with and without class I HLA antibodies, the patient characteristics, causes of primary liver failure, and causes of first graft failure were similar between those with a failed second LT and those with a functioning second LT (Table 3).
Table 3. Comparison of Patient Characteristics by Graft Status in Adults
The present study demonstrates a deleterious effect of preformed class I HLA antibodies on second liver allograft survival in adults. This effect was seen as early as 1 year after the second LT and persisted up to 5 years after retransplantation. Indeed, several groups recently have reported that T cell–positive crossmatches appear to be associated with poorer graft survival rates,8, 17, 18 and a recent study by Muro et al.13 further showed that the majority of patients with positive crossmatches lost their grafts during the first 3 months after transplantation. Opelz et al.16 also showed that class I antibodies resulting in a positive T cell crossmatch are harmful to grafts. The results of our study corroborate these findings; that is, mainly class I antibodies that are detrimental to graft survival.
The high proportion of patients in our study with preformed HLA antibodies (87.3% and 71.4% in adults and children, respectively) before the second LT was likely due to exposure to allo-antigens during the first LT. We speculate that patients who have required at least 2 LTs would also have had previous exposure to blood products resulting in further allo-immunization.
Although the multivariate analysis by Evrard et al.19 involving 500 consecutive pediatric recipients of primary liver allografts showed that a positive T cell crossmatch was one of the variables that had a negative impact on acute rejection–free graft survival, acute rejection per se did not significantly influence overall patient or graft survival in their study. The absence of a significant deleterious effect of class I antibodies on second liver graft survival in the pediatric population in our study supports this finding.
Earlier publications have identified various nonimmunological factors that influence long-term liver regraft survival, such as donor age, interval to retransplantation, need for preoperative mechanical ventilation, severity of graft failure, and total number of grafts.20-22 Rosen et al.23 created a model to predict liver retransplantation outcomes based on recipient preoperative factors, but they did not take into account the role of preformed HLA antibodies.
It is well known that liver grafts are more resistant to injury by HLA antibodies in comparison with kidney, heart, and lung transplants. The type of graft injury mediated by these antibodies, namely, direct hepatocyte injury or vascular injury mediated through antibody binding to hepatic sinusoidal endothelial cells, remains largely unclear. Both MHC class I and II molecules are expressed on hepatic sinusoidal endothelial cells, whereas resting hepatocytes and biliary epithelial cells, which constitute 30% of nonparenchymal liver cells, express only MHC class I molecules, and this allows them to act as antigen-presenting cells only for MHC class I–restricted T cells.24, 25 In contrast, MHC class II molecules are not constitutively expressed on hepatocytes or biliary epithelial cells and become up-regulated only after inflammation. Class I antibodies may thus mediate graft injury via direct binding to class I antigens on hepatocytes and biliary epithelial cells and indirectly through vascular injury after binding of the antibody to its antigen on hepatic sinusoidal endothelial cells. The former would give rise to a clinical picture of cellular rejection, whereas the latter would result in a clinical picture of vascular rejection. Class II antibodies, however, may not play a key role in early liver graft rejection.
Early reports on the role of HLA antibodies in graft survival were based on direct lymphocytotoxic crossmatching of donor and recipient sera, and a positive crossmatch was reported to have a negative influence on graft survival. Although it was surprising that preformed DSAs were not significantly associated with second liver allograft survival in this study, this could have been due to the false-positive labeling of a recipient's DSA status. Because the serological specificity of an HLA antigen could be shared by several allelic specificities, an antibody labeled as DSA by serological specificity could have a different allelic specificity than the donor's HLA antigen. This point should be further explored, especially in light of evidence suggesting that class I antibodies are negatively associated with graft survival. To accurately investigate the effect of DSAs on liver regraft survival, pretransplant HLA typing of all donors by allelic specificity as well as testing of recipients' sera with sensitive solid-phase assays would be necessary.
In summary, this study shows that HLA class I antibodies have a definite deleterious effect on short-term and long-term liver regraft survival in adults. After undergoing regrafting, adults with class I antibodies have significantly lower early and late graft survival. Sufficient attention needs to be paid to preformed HLA antibodies when the second transplant is being performed. Crossmatching of donors and recipients should be performed prior to liver retransplantation to optimize short-term and long-term graft survival.
The authors thank the 9 liver transplantation centers in the North Italy Transplant Program for their kind assistance with serum collection and for the provision of the relevant clinical information.