Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: Role of human leukocyte antigen compatibility

Authors


Abstract

The significance of human leukocyte antigen (HLA) compatibility and preformed antibodies in liver transplantation remains unclear. The objectives of this study were to evaluate, in a single-center cohort comprising 896 liver transplants, whether the degree of donor-recipient compatibility and preformed antibodies modified graft survival. Univariate Kaplan-Meier analysis demonstrated that donor-recipient HLA compatibility had a marginal impact on allograft survival. As for compatibility at individual antigen loci, 2 mismatches at HLA-A conferred a survival advantage in retransplanted allografts (P = 0.011). HLA-B and HLA-DR loci did not play a significant role in outcome in any pathology. The concordance of results on preformed antibodies detected by complement-dependent cytotoxicity (CDC) and a multiple bead assay (Luminex® xMAP) showed a strong correlation between both techniques (P < 0.0001). Both CDC-detected and Luminex-detected antibodies were associated with shorter graft survival within the first year post-transplant (P = 0.01 and P = 0.016, respectively). Positive CDC T crossmatches and Luminex-detected HLA class II antibodies played a significant role in decreasing graft survival (P = 0.043 and P = 0.0019 at 1 year, respectively, and P = 0.005 and P = 0.038 at 5 years, respectively). A correlation was also observed between the presence of preformed Luminex-detected class II or Luminex I and II antibodies and allograft rejection (P = 0.001 and P = 0.042, respectively). In conclusion, although HLA typing is not a prerequisite for transplantation, screening of HLA antibodies with Luminex techniques and CDC crossmatch may be useful in the detection of at-risk patients that could benefit from increased surveillance and tailored therapy following transplantation. Liver Transpl, 2008. © 2008 AASLD.

In many end-stage liver diseases, hepatic transplantation often constitutes the only therapeutic option available.1–3 Studies performed in clinical solid organ transplants have endeavored to gain insight into immunological parameters that could be important in altering the course of allograft tolerance, thus modifying the prognosis.

Two main factors, donor-recipient human leukocyte antigen (HLA) compatibility and the absence of preformed, donor-specific anti-HLA cytotoxic antibodies (giving rise to a positive crossmatch), are known to contribute to a more favorable outcome in renal4–6 and heart transplants.7 This has not been the case in liver transplantation. Over the past 2 decades, opinions have been divided, with some groups supporting the detrimental role of HLA incompatibility and presensitization of recipients in the survival of liver grafts8 and others rejecting their impact on prognosis.9, 10 In the largest multicenter cohort of liver transplant patients, Navarro and coworkers11 observed that HLA matching had no clinically significant impact on transplant outcome. Similarly, Neumann and coworkers12 had previously reported in a single-center analysis that HLA compatibility had no effect on graft survival. In contrast, Nikaein and coworkers13 demonstrated increased survival in transplants in which there was a greater degree of HLA donor-recipient compatibility. Markus and coworkers14 showed that diminished allograft survival was associated with HLA compatibility, but in retransplants, a higher incidence of failure due to rejection correlated with a lower degree of HLA compatibility. The authors explained this apparent contradiction by conferring a dualistic effect to HLA compatibility, by which it could reduce rejection yet simultaneously initiate a cascade of immunological mechanisms leading to graft loss, particularly in patients at risk of suffering recurrent autoimmune disease or infection.

The significance of preformed antibodies in the liver recipient also remains controversial. Although not all authors have described this phenomenon,15, 16 some groups have reported that liver transplants can absorb circulating antibodies and protect subsequent kidney grafts transplanted across a positive crossmatch.17, 18 Definitive conclusions, however, have been difficult to establish because published series comprise few recipients with preformed antibodies. More importantly, discrepancies exist when the sensitization status of patients is defined because the different techniques for the detection of antibodies [complement-dependent cytotoxicity (CDC) crossmatch and its variants, flow cytometry crossmatch, panel reactive antibody (PRA), flow cytometry PRA, and enzyme-linked immunosorbent assay] do not provide completely equivalent information. The more recent microsphere-based suspension array technology, Luminex,19 can detect non–donor-specific and donor-specific immunoglobulin G (IgG) anti-HLA antibodies. By exclusion of non-HLA and immunoglobulin M (IgM) antibodies, including most autoantibodies, the specificity of this technique is also higher. Furthermore, non–complement-fixing IgG HLA antibodies that can be clinically relevant can also be detected.20 In kidney transplantation, Luminex-detected anti-HLA antibodies have been shown to maintain a correlation with transplant outcome.21–23 To our knowledge, however, the present study is the first described in the literature to employ Luminex techniques in liver transplantation.

The aims of this study were to investigate the role of HLA mismatching on the outcome of liver transplantation in a large single-center series and to analyze if the degree of histocompatibility in the setting of different base pathologies modified graft outcome. Additionally, we sought to examine the influence of preformed antibodies on liver allograft survival and rejection and to compare results obtained by CDC or by a multiplexed bead based assay, Luminex® xMAP.

Abbreviations

Ab-neg, antibody-negative; Ab-pos, antibody-positive; CDC, complement-dependent cytotoxicity; HBV, hepatitis B virus; HCV, hepatitis C virus; HLA, human leukocyte antigen; IgG, immunoglobulin G; IgM, immunoglobulin M; MM, mismatches; NS, not significant; OLT, orthotopic liver transplantation; PRA, panel reactive antibody; SD, standard deviation.

PATIENTS AND METHODS

This study was approved by the Institutional Review Board of the Doce de Octubre Hospital.

Population

We retrospectively analyzed 896 consecutive orthotopic liver transplants performed at our center for the treatment of end-stage liver disease between March 1986 and May 2006. Selection of allograft recipients was based on various criteria including Child-Turcotte-Pugh score, ABO compatibility, donor-recipient size matching, and time on the waiting list. Although the Model for End-Stage Liver Disease score, based on severity of illness,24, 25 is taken into account at our center, it is not determinant in recipient selection. Graft loss was defined as death of the patient or loss of the graft due to any of the following: vascular complications, bile duct complications, infection, renal failure, respiratory failure, gastrointestinal hemorrhage, malignancy, disease recurrence, rejection, multisystem organ failure, primary graft dysfunction, or other non–transplant-related cause. Patients who died or lost their graft within the first week post–orthotopic liver transplantation were excluded (n = 38).

Immunosuppression

Immunosuppression consisted of double therapy (Prograf® and prednisone) or triple therapy (cyclosporine A, prednisone, and azathioprine) until 1997. Thereafter, because of the increased risk of myelosuppression, azathioprine was substituted by mycophenolate mofetil.

The current treatment protocol includes Prograf® (adults: 0.10–0.20 mg/kg/day, children:0.30 mg/kg/day) plus prednisone (20 mg/day tapering regime until withdrawal 3–6 months post-operation). For patients with an increased risk of rejection, previous rejection episodes, or renal toxicity, the protocol is mycophenolate mofetil (750–1000 mg/12 hours) and prednisone plus/or minus Prograf® (used at a reduced dose or suspended if there is renal toxicity).

Rejection

Rejection episodes were suspected in cases of deranged liver function tests and clinical deterioration of the patient. In all cases, rejection was confirmed by graft biopsy.

Liver biopsy results were available in 753 transplants. Histopathological evaluation was performed following the Banff schema for liver allograft grading.26

HLA Typing

HLA-A, HLA-B, and HLA-DR donor and recipient typing was performed by a standard microlymphocytotoxicity technique using HLA alloantisera from International Histocompatibility Workshops until 1996. Thereafter, commercially available typing trays with anti-HLA monoclonal antibodies (One Lambda, Inc., Canoga Park, CA) were employed.

CDC Crossmatch

Direct antibody cytotoxicity was performed on recipient sera drawn immediately before transplantation and before administration of any blood products. Lymphocyte populations from the spleen or lymph node were obtained by mechanical disruption and density gradient centrifugation. Donor T and B cell lymphocytes were isolated, and the microlymphocytotoxicity assay was performed according to a modified National Institutes of Health standard procedure. A crossmatch was considered positive if cell death above background was ≥20% with T or total lymphocytes or ≥40% with B cell donor lymphocytes.

Antibody Screening by Luminex xMAP Technology

Pretransplant sera were retrospectively analyzed for HLA I and II antibodies by a multiplexed microsphere-based suspension array, Luminex xMAP technology (Luminex Corp., Austin, TX).

Briefly, 2.5 μL of LABScreen® Mixed (One Lambda) color-coded microbeads coated with purified HLA antigens were incubated in the dark for 30 minutes at 20°C to 25°C with 10 μL of pretransplant sera. Any HLA antibodies present in the sera were bound to the LABScreen Mixed surface antigens coating the microbead and were subsequently labeled with R-phycoerythrin conjugated goat anti-human IgG. The microbead fluorescent emission of R-phycoerythrin was then detected and quantified by the LABScan™ 100 flow analyzer (One Lambda).

The determination of positive and negative sera was performed with One Lambda software (LABScreen306) according to the manufacturer's guidelines (One Lambda). Reactivity of sera was assessed by the fluorescent signal for each HLA-coated bead following correction for nonspecific binding to the negative control bead. In the LABScreen® Mixed assay, the normalized fluorescent signal is equal to the value of the class I or II coated bead minus the value of the negative control bead. If any one bead in the mixed assay is positive, the result is considered positive.

Cut-off values were established, as recommended by the manufacturers, based on standards of our laboratory after having confirmed that results of 300 well-characterized sera previously studied by alternative techniques (LAT™ Mixed trays, One Lambda) correlated well with those obtained by the LABScreen® Mixed assay.

Statistical Analysis

Statistical analysis was performed with the SPSS statistical package (SPSS, Inc., Chicago, IL). Actuarial survival was calculated at 1, 3, and 5 years with the life table method, whereas cumulative survival was analyzed with the Kaplan-Meier technique. The log rank test was used to estimate statistical significance, which was established at P ≤ 0.05. The correlation between techniques (CDC versus Luminex) and between allograft rejection and the presence of Luminex-detected preformed antibodies was determined with 2 × 2 tables and the likelihood ratio chi-square test.

RESULTS

Table 1 summarizes the characteristics of the patients included in this study.

Table 1. Patient Characteristics
 n = 896%
  1. Abbreviation: SD, standard deviation.

Age < 15 years  
 Number11513
 Mean ± SD5.5 ± 4.58 
Age > 15 years  
 Number78187
 Mean ± SD49.9 ± 11.5 
Diagnosis  
 Hepatitis C23926.7
 Alcoholic cirrhosis14416.1
 Cryptogenic cirrhosis12313.7
 Carcinoma586.5
 Hepatitis B546.0
 Primary biliary cirrhosis364.0
 Fulminant hepatitis323.6
 Biliary atresia313.5
 Metabolic and congenital323.6
 Others262.9
 Primary sclerosing cholangitis70.8
Retransplants11412.7

Influence of HLA Compatibility on Allograft Survival

Complete donor-recipient HLA-A, HLA-B, and HLA-DR typing was available in 853 transplants.

In order to investigate the influence of HLA compatibility on survival, allografts were categorized into 2 groups [0–3 mismatches (0–3MM) versus 4–6MM], 3 groups (0–4MM, 5MM, or 6MM), or 4 groups (0–3MM, 4MM, 5MM, or 6MM), and Kaplan-Meier estimates of survival were performed initially on the entire cohort and subsequently stratified into adult (n = 741) and pediatric (n = 112) populations. Overall, donor-recipient antigen compatibility had a negligible impact on survival at 5 years when 0–3MM and 4–6MM groups were compared (Fig. 1). This was also observed when the entire population was further categorized into 3 and 4 groups (results not shown). The same trend was observed in adult and pediatric patients when groups were analyzed separately. Because the cohort of liver recipients included patients with distinct base pathologies, we explored whether the impact of HLA compatibility on graft survival was different with respect to the recipients' underlying disease process (Table 1). In the diagnostic subsets, differences in survival stratified by HLA-MM were not significant. Finally, the effect of HLA-A, HLA-B, and HLA-DR compatibility was analyzed separately in retransplants (n = 95). In this group, survival was up to 15% greater in highly mismatched allografts (4–6MM) versus those with 0–3MM. This observation reached statistical significance in children (0% survival in the 0–3MM group versus 82% in the 4–6MM group, P < 0.0001).

Figure 1.

Kaplan-Meier curves of survival in liver allograft recipients based on the number of human leukocyte antigen mismatches (MM) between donor and recipient (P = 0.68, not significant). Abbreviation: OLT, orthotopic liver transplantation.

Influence of Individual HLA Antigen Loci on Survival

As for individual antigen loci, donor-recipient HLA-A typing was available in 886 allografts, HLA-B typing was available in 883, and HLA-DR typing was available in 854. Recipients were stratified into 3 groups of 0MM, 1MM, or 2MM per HLA locus. When analyzing the influence on allograft survival of mismatches within individual antigen loci, we found that actuarial survival at 5 years was virtually the same, regardless of the number of mismatches at the HLA-A, HLA-B, or HLA-DR loci.

A comprehensive analysis investigating the influence of mismatches within different antigen loci was subsequently performed on each etiology. Significant differences in survival were observed at 1, 3, and 5 years in the retransplant and hepatitis B subgroups. In both cases, survival at 5 years in liver allografts with 2MM at the HLA-A locus was greater than in grafts with 1MM or 0MM (P = 0.011 and P = 0.0192 in each subgroup, respectively). Compatibility at HLA-B and HLA-DR loci did not seem to play a role in graft survival in the different diagnostic subsets.

Influence of Preformed Antibodies on Survival

Of the total transplants analyzed (primary and retransplants), 755 (89.5%) tested negative for the presence of anti-donor preformed antibodies by CDC (crossmatch with T and/or B cells), and 89 (10.5%) tested positive. Kaplan-Meier estimates of allograft survival based on crossmatch were lower in patients with a positive crossmatch compared to those with a negative crossmatch. Survival in the CDC-positive group at 1, 3, and 5 years was 72%, 65%, and 56%, whereas in the CDC-negative group, it was 83%, 77%, and 72%, respectively. Overall, we found a highly significant association between anti-donor preformed antibodies detected by CDC and decreased survival at 1, 3, and 5 years (P = 0.01, P = 0.015, and P = 0.004, respectively). Thereafter, the influence on survival of CDC-detected preformed antibodies was studied by stratification of the entire cohort by individual pathologies. The presence of preformed donor-specific antibodies had only a statistically significant effect, decreasing survival in alcoholic cirrhosis recipients at 5 years post–orthotopic liver transplantation (n = 140, P = 0.0011).

A comprehensive analysis was carried out by calculation of survival in separate subgroups of positive and negative crossmatches performed exclusively with T lymphocytes (CDC T), B lymphocytes (CDC B), or both types of cells (CDC T and B; Table 2). Significant differences in survival were associated with CDC T crossmatches, as better graft outcomes were observed in CDC T–negative patients versus CDC T–positive patients at 1, 3, and 5 years (P = 0.043, P = 0.036, and P = 0.005, respectively). However, the antibodies detected by crossmatches performed with B lymphocytes did not seem to play a role in graft outcome.

Table 2. Graft Survival (%) in CDC T and B, Luminex Anti-HLA Class I and II Positive and Negative Cohorts
 Luminex I (n = 810)CDC T (n = 842)Luminex II (n = 810)CDC B (n = 423)Luminex I and II (n = 715)CDC T and B (n = 387)
Ab-pos (n = 115)Ab-neg (n = 695)PAb-pos (n = 57)Ab-neg (n = 785)PAb-pos (n = 118)Ab-neg (n = 692)PAb-pos (n = 59)Ab-neg (n = 364)PAb-pos (n = 69)Ab-neg (n = 646)PAb-pos (n = 27)Ab-neg (n = 360)P
  1. Abbreviations: Ab-neg, antibody-negative; Ab-pos, antibody-positive; CDC, complement-dependent cytotoxicity; HLA, human leukocyte antigen; NS, not significant.

1 year75%83%0.03872%82%0.04371%84%0.001970%78%NS68%83%0.001867%77%NS
3 years70%77%NS65%77%0.03667%77%0.0361%69%NS64%77%0.018956%69%NS
5 years66%71%NS54%71%0.00562%72%0.03853%62%NS61%71%NS44%61%NS

Eight hundred ten sera (90%) of the original group were subsequently analyzed retrospectively for HLA I and II antibodies by a multiplexed bead assay, Luminex xMAP. Of the 810 sera tested, 79.7% (n = 646) were negative and 20.2% (n = 164) were positive for HLA class I and/or HLA class II antibodies. Survival in the Luminex-positive group at 1, 3, and 5 years was 75%, 70%, and 65%, whereas in the Luminex-negative group, it was 83%, 77%, and 71%, respectively. The differences observed between the 2 cohorts were significant at year 1 (P = 0.0158) but not at year 3 or year 5.

An analysis of the influence on survival of preformed antibodies detected by Luminex xMAP was performed by stratification of the entire cohort by individual pathologies. In the alcoholic cirrhosis group, differences in survival were noted between the patients with preformed antibodies and those without antibodies, and these differences were significant at 1, 3, and 5 years (n = 136, P = 0.0009, P = 0.0015, and P = 0.0039, respectively). No other significant differences where observed in the remaining pathologies.

The total group of patients was further categorized into positive and negative for class I antibodies, class II antibodies, and both after analysis of the sera by the Luminex technique, and survival was individually calculated for each group (Table 2). Although the absence of HLA class I antibodies improved survival only at the first year (P = 0.038), HLA class II antibodies significantly reduced graft survival throughout the study (P = 0.0019, P = 0.03, and P = 0.038 at 1, 3, and 5 years, respectively).

To determine the degree of concordance between the 2 techniques, we analyzed how anti-donor antibodies previously detected by CDC correlated with results obtained by Luminex. In all cases, the correlation between CDC and Luminex—CDC T versus Luminex class I (n = 799), CDC B versus Luminex I and Luminex II (n = 400), and CDC T and B versus Luminex class I and II (n = 315)—was statistically significant (P < 0.0001, Table 3).

Table 3. Comparison Between CDC and Luminex xMAP for Anti-HLA Transplant Class I, Class II, and Class I and II Antibodies in Sera from Hepatic TraNSplant Patients
A. Luminex Class I (n = 799)CDC T (n = 799)
Negative (n = 744)Positive (n = 55)Total
  1. The superscripts a-h indicate cohorts of patients in which graft survival was calculated and compared. For CDC T, a crossmatch was performed with isolated T cells or total lymphocytes; for CDC B, a crossmatch was performed with isolated B cells; and for CDC T and B, both crossmatches were performed.

  2. Abbreviations: CDC, complement-dependent cytotoxicity; HLA, human leukocyte antigen.

Luminex-negative (n = 687)665a22687
Luminex-positive (n = 112)79b33112
Total74455799
B. Luminex Class I (n = 400)CDC B (n = 400)
Negative (n = 343)Positive (n = 57)Total
Luminex-negative (n = 339)313c26339
Luminex-positive (n = 61)30d3161
Total34357400
C. Luminex Class II (n = 400)CDC B (n = 400)
Negative (n = 343)Positive (n = 57)Total
Luminex-negative (n = 333)305e28333
Luminex-positive (n = 67)38f2967
Total34357400
D. Luminex I and II (n = 315)CDC T and B (n = 315)
Negative (n = 294)Positive (n = 21)Total
Luminex-negative (n = 290)285g5290
Luminex-positive (n = 25)9h1625
Total29421315

Small numbers of patients were positive for CDC but negative for Luminex (see groups of n = 22, n = 26, n = 28, and n = 5 in Table 3, sections A, B, C, and D). This difference in CDC and Luminex results could be attributed to the presence of IgM antibodies or non-HLA antibodies not detected by the microbead assay. Conversely, groups of 79, 30, 38, and 9 patients were negative for CDC T, CDC B, and CDC T and B, respectively, but showed antibodies detected by the more sensitive Luminex technique.

To explore whether the negative effect of Luminex-detected antibodies on graft outcome was independent of CDC positivity, survival was calculated and compared in cohorts of both Luminex-negative and CDC-negative patients versus Luminex-positive and CDC-negative patients (comparisons of cohort a versus cohort b, cohort c versus cohort d, cohort e versus cohort f, and cohort g versus cohort h in Table 3). In all 4 comparisons, graft survival was lower in Luminex-positive cohorts versus Luminex-negative cohorts. This difference reached statistical significance in cohort g versus cohort h (P = 0.0095). This suggests that antibodies exclusively detected by Luminex decrease graft survival, regardless of results obtained by CDC crossmatch.

Luminex-Detected Preformed Antibodies and Allograft Rejection

Acute or chronic rejection occurred in 355 (47%) of the 753 transplants for which rejection data were available. Of this total, 319 had acute rejection episodes, 18 had chronic rejection, and the remaining 18 had both acute and chronic rejection within the study period.

An analysis of correlation was performed to investigate if there was any relationship between the presence of Luminex-detected antibodies and rejection. In the category of global rejection (acute and/or chronic, n = 355) versus Luminex class I, Luminex class II, and Luminex class I and II detected antibodies, we found that in all cases rejection was more frequent in patients positive for preformed antibodies. This reached statistical significance in the Luminex II–positive patients (P = 0.001) and in the Luminex I and II–positive patients (P = 0.042).

In the category of acute rejection (episodes of acute rejection or both acute and chronic rejection, n = 319 + 18), the same trend was observed, reaching statistical significance in the Luminex class II antibody category (P = 0.002) and the Luminex I and II category (P = 0.037). Finally, in the chronic rejection category (chronic rejection or both chronic and acute rejection, n = 18 + 18), there was no distinct trend observed, and no statistically significant differences were noted.

DISCUSSION

In the analysis of the entire cohort, no significant beneficial or detrimental effects associating the degree of HLA donor-recipient matching and liver graft survival have been observed. These findings are in keeping with results obtained in the 2 largest series published to date: both the single-center analysis of Neumann and coworkers12 at the University of Berlin (n = 924) and the multicenter analysis of the United Network for Organ Sharing database (n = 28,735)11 concluded that HLA matching had no significant impact on liver graft outcome. However, the degree of HLA compatibility seemed to play a role in graft survival of particular cohorts such as hepatitis B virus (HBV) or retransplanted patients. We also observed a trend toward higher survival rates in more incompatible donor-recipient pairs for the autoimmune diseases primary biliary cirrhosis and primary sclerosing cholangitis (data not shown). These results agree with those published by Doran and coworkers,27 who reported beneficial effects of HLA mismatching on graft survival of autoimmune recipients, and with data by Neumann and coworkers,12 who reported a better evolution of primary sclerosing cholangitis patients in the presence of more mismatches. Nonetheless, a larger cohort would be desirable to obtain definitive conclusions.

Our results do not demonstrate an association between HLA compatibility and graft survival in 239 patients transplanted for hepatitis C virus (HCV) infection. This result is in agreement with observations from the German series comprising 119 HCV transplants12, 28 but contrasts with data from Belli and coworkers29 reporting a beneficial effect of HLA matching in 89 HCV transplanted patients. In allografts from the HBV group, however, we noted a positive correlation between survival and highly HLA-B–compatible donor-recipient pairs (not shown). Concurrently with our data, Neumann and coworkers30 observed a similar beneficial role of HLA matching in this population group. The authors noted that increased compatibility at HLA-A or HLA-B had beneficial repercussions on graft survival; this was especially true at the HLA-B locus (5-year graft survival was 79% in patients sharing 1 or more HLA-A antigens with the donor and 94% in patients sharing 1 or more HLA-B antigens with the donor). In addition, the authors reported longer graft survival in patients with HBV reinfection and 1 or 2 HLA-B compatibilities. A possible explanation for the protective role of HLA class I compatibility is that similar HLA class I antigens on the hepatocyte surface could facilitate uninterrupted recognition of viral antigenic peptides by cytotoxic T lymphocytes after transplantation, allowing a more efficient clearance of the virus.

With respect to retransplants, we observed that higher global HLA-A, HLA-B, and HLA-DR mismatching increased liver graft survival, and this effect was mainly attributable to incompatibility at the HLA-A locus. Wong and coworkers31 described how patient survival at retransplantation improved with mismatching not at HLA-A but at the HLA-B and HLA-DR loci. Differences in the linkage disequilibrium and structure of HLA haplotypes in the different populations could help us to understand why a similar effect may be linked to different HLA genes.

In the second part, we aimed at studying the effect of preformed antibodies on graft survival. In our cohort, 10.5% of patients (n = 89) showed anti–donor-specific antibodies in pretransplant sera as detected by positive CDC crossmatch (CDC T and/or B). The frequency is consistent with that commonly reported in the literature.32 When the sera were screened for anti-HLA antibodies (class I and/or II) with the multibead assay, 20.2% (n = 164) yielded a positive result. This increase in frequency can be reasonably attributed to a higher sensitivity of the Luminex technique in detecting donor-specific, non–donor-specific, and some non–complement-fixing anti-HLA antibodies that can be harmful to the graft by other mechanisms.19

We observed a highly significant association between donor-specific preformed antibodies detected by positive CDC, particularly in crossmatches with T or total lymphocytes (Table 2), and decreased graft survival. An analysis of the largest series published thus far failed to demonstrate this effect of positive CDC, although it reported lower early survival rates in the positive crossmatch group (n = 130).33 An absence of association between CDC crossmatch and graft survival was also observed in an analysis of 793 liver transplants with 50 positive crossmatches.34 Even apparently paradoxical results have been obtained by authors reporting fewer early graft losses in patients with positive flow cytometry crossmatch.35 Many studies, however, find a correlation between HLA antibodies and diminished graft survival, and most of them share in common the observation that the deleterious effect is stronger within the first year postransplant.27, 36–39

To investigate whether graft survival was associated with the general status of pretransplant sensitization of patients assessed not by donor-specific crossmatch but by screening techniques, we analyzed our cohort with a Luminex assay and found that preformed antibodies (class I and/or II) affected graft survival negatively. This effect was stronger at year 1 and was maintained all along the observation time for anti-HLA class II antibodies (Table 2). Again, contradictory results are found in the literature. By using PRA, some authors described an association of antibodies with shorter graft survivals,36 whereas others did not observe that correlation.10, 40 In particular, Nikaien and coworkers8 did not find an association between shorter graft survival and PRA, although graft outcome was significantly dependent on positive CDC crossmatch. In contrast, 2 more recent works, using sensitive, solid-phase techniques for the detection of anti-HLA antibodies (enzyme-linked immunosorbent assay and flow cytometry PRA),38, 39 have shown a correlation with poor graft survival.

The association between antibodies and graft survival was observed not in any of the individual pathologies but in the alcoholic cirrhotic recipients. This finding may add further evidence in support of the role of preformed antibodies in graft loss because other factors causing graft deterioration such as recurrent viral infection or autoimmunity are less likely to be acting on the alcoholic cirrhosis cohort.

In addition to the aforementioned epidemiological studies, other data have been published supporting the role of antibodies in liver transplantation outcome. In the past, the presence of donor-specific antibodies to class I antigens was associated with the bile duct loss encountered in chronic rejection.41, 42 Subsequently, the Pittsburgh group reported that in patients with high titers of anti-donor IgG lymphocytotoxic antibodies and persistent positive crossmatch after transplantation, a specific syndrome with low complement activity, increased circulating immune complexes, and refractory trombocytopenia leading to a higher incidence of graft failure was observed.43 More recently, the same center has shown that in a study of 17 transplant recipients withdrawn from any immunosuppression and with stable graft function, all of them lacked donor-specific antibodies.44 Liver biopsies from acute and chronic rejection allografts show immunoglobulin deposition on biliary epithelium and vascular endothelial cells.45 In addition, a cellular-independent, direct injury of graft parenchymal cells by donor reactive alloantibodies has been recently demonstrated in a mouse allogeneic liver transplant model. In this study, the passive transfer of alloantibodies into T-deficient and B-deficient recipients was sufficient to cause rejection of hepatocellular grafts.46 Recent studies from several groups have reported that an important proportion of clinical liver biopsies with specific signs of acute cellular and chronic rejection show deposition of C4d, a marker of immunoglobulin-dependent activation of complement, strongly suggesting a role for a humoral component in liver allograft failure.47–51 All these data are in line with our findings that increased rejection episodes were predominantly observed in allografts positive for Luminex-detected class II antibodies.

On the basis of the results reported here, we conclude that differences in HLA compatibility have an impact not on global liver allograft survival but on selective recipient groups, and donor-recipient HLA typing should not be routinely performed in liver transplantation. On the other hand, screening of HLA antibodies with the Luminex technique and CDC crossmatch may have important implications for graft survival. Information on the sensitization status would allow the detection of at-risk patients requiring closer surveillance and tailored immunosuppressive therapy.

Acknowledgements

We are indebted to Esther Clemente Martinez and Alicia Muñoz Ortiz of the Department of Transplant Surgery for their assistance.

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