Objectives: Although previous studies have investigated the effect of human leukocyte antigen matching on long-term outcomes after heart transplants, its role in the prognosis after a heart transplant remains unclear, particularly with respect to short-term survival.

Materials and Methods: We evaluated the human leukocyte antigen mismatch on in-hospital mortality of 158 consecutive patients who had undergone a heart transplant between 2000 and 2008. Human leukocyte antigens-A, -B, and -DR were determined by means of serologic and molecular techniques. Univariate analysis and a multiple logistic regression models evaluated the effect of human leukocyte antigen variants on mortality, independent of clinical variables.

Results: In-hospital mortality was 11.4%. Higher prevalence of acute kidney injury (50.0% vs 12.9%), higher levels of troponins 48 hours after transplant (15.6 ± 12.0 ng/mL vs 9.7 ± 9.4 ng/mL), prolonged ischemia (188.2 ± 32.5 min vs 162.6 ± 40.7 min), higher frequency of reoperation (61.1% vs 17.9%), and higher human leukocyte antigen-DR mismatch (1.61 ± 0.5 vs 1.30 ± 0.6) were found in patients who died. By logistic regression analysis, humanleukocyte antigen-DR mismatch is associated with in-hospital mortality (OR=5.159, 95% CI=1.348-19.754), independent of the effect of covariates such as recipient age, mismatch sex, mismatch human leukocyte antigen-A, human leukocyte antigen-B, acute kidney injury, reoperation, ischemia duration, and levels of troponins.

Conclusions: Human leukocyte antigen-DR mismatch is associated with in-hospital mortality in heart transplant.

Key words : Immunological compatibility, Human leukocyte antigen, HLA-DR mismatch, Heart transplant, In-hospital mortality

Introduction

Differently from kidney transplant, in which the role of immunogenetics is well established,^1,2 in heart transplants, the relation between outcome and immunogenetic compatibility remains an open question, although during the last 10 years, there have been many improvements in understanding the role of the immune system in patients’ recovery after a heart transplant.^3-5 Indeed, current methods for screening suitable donor hearts do not avoid a high number of early graft failures. Therefore, we must increase the specificity of biomarkers that predict the success or failure of a heart transplant.

Human leukocyte antigen (HLA) compatibility significantly influences graft survival after a heart transplant, independent of donor and recipient age, cold ischemic time, sex, type of underlying disease, and prophylaxis with antilymphocyte globulins.⁶ Indeed, 2 HLA-A, -B, or -DR mismatches between donor organ and recipient were associated with a 25% increased risk of graft failure within 3 years when compared with transplants having 0 or 1 mismatch.⁶ More recently, Kaczmarek and associates showed that the number of HLA-DR mismatches affects graft failure, and patients survival within 5 years of a heart transplant.⁷ Furthermore, several single-center studies, such as the studies of UNOS/ISHLT registry (Thoracic and International Registry for Thoracic Organ Transplantation) have revealed significant benefits of HLA-compatible heart transplants.^8,9 Significant evidence has demonstrated a notable proportion of allograft rejection episodes are not caused only by T-cell response, but also by specific alloantibodies against the donor graft HLA class I and class II molecules.¹⁰

Other observations, however, have not demonstrated statistically significant differences in the 1-year survival rate of HLA-DR–matched versus mismatched cardiac grafts, although a trend for higher survival rates has been found in HLA-DR–matched patients, and HLA-DR–matched hearts had a lower frequency of acute cellular rejection after a heart transplant.¹¹ Also, conflicting with the relevance of HLA-matching in heart transplant success were other data, which do not demonstrate any positive effect of HLA-A, -B, and -DR matching on survival and incidence of complications, such as rejection episodes or infections during a 1-year follow-up or more.12 Aside from the debate on the relevance of HLA matching in the long term, a possible role of HLA-A, -B, and -DR with respect to in-hospital outcomes after heart transplants remains poorly investigated. Thus, this study aimed to analyze the possible prognostic effect of donor-recipient HLA genotype compatibility on short-term survival (30 days after a heart transplant) in a single center using heart-transplanted patients from 2000 to 2008.

Materials and Methods

Patients
After the approval of the Local Ethical Committee, we enrolled 158 consecutively heart-transplanted patients between 2000 to 2008 from the division of heart transplant of Monaldi Hospital of Naples, Italy. The study was performed in accordance of the Helsinki Declaration of 1975, revised in 2000. Written, informed consent was provided by all patients. The study population consisted of 158 consecutively heart transplanted patients, mostly male (79.1%), with a mean age of 48.9 ± 13.1 years, and known HLA typing of both donors and recipients (Table 1).

Data from all patients were prospectively entered into a dedicated database containing all the recipient’s preoperative data (UNOS status, data from the right chamber catheter, and comorbidities), variables related to the donor (cause of death, the number and dosage of inotropic drugs, echocardiography parameters, and comorbidities), the data for the donor-recipient matching (genetic typing of donor and recipient, HLA mismatch, body size mismatch, and sex mismatch), data relating to the procedures (ischemic time, use of blood products, and technical notes), all given treatments. Patients were analyzed retrospectively with a focus on the effect of donor-recipient HLA genotype profiles on in-hospital mortality, HLA-A, -B, and -DR were determined by serologic and molecular techniques (Complement Dependent Cytotoxicity and polymerase chain reaction [PCR] amplification with sequence-specific primers) before being placed on the waiting list at the Regional Laboratory of Transplantation Immunology of the Second University of Naples.

Specimen characteristics
Graft surgical care and protection
Donor and recipients were matched for ABO blood type compatibility and size of the body (within 20% difference in body weight). Prospective HLA matching was not used with the exception of recipients with high levels of anti-HLA antibodies who had undergone a prospective cross-match by following instructions for the Regional Laboratory of Transplantation Immunology of the Second University of Naples. Evaluation of the donor heart was performed according to the rules of the National Quality Control Centre (Italian National Transplant Centre).¹³ Donors positive for human immunodeficiency virus or cancer were excluded. Donor heart procurement was performed by standard techniques, and the grafts were protected via infusion of 2 L of cold Celsior solution (4°C to 8°C) in the aortic root and topical cold saline slush.13 Principles of intraoperative and postoperative care, along with details on the immunosuppressive regimen, are reported extensively elsewhere.¹³ Notably, during the early postoperative phase, immunosuppressive therapy with calcineurin inhibitors was withheld until complete normalization of renal function. Patients were maintained on induction therapy and steroids during this time.

Cardiopulmonary bypass weaning and perioperative inotropic support
In the early postoperative phase, transplanted heart allografts were reperfused and circulation was supported to maintain hemodynamic stability while the allografts gradually restored their normal function. Usually, at our Institution, the length of reperfusion (without inotropes) is approximately 15 minutes for every hour of cold ischemic time. After reperfusion, the patient is always provided with an active hemodynamic management and support. Generally, a dosage of dobutamine 5 to 15 mcg/kg/min and isoprenaline 5 to 15 ng/kg/min was sufficient for optimal hemodynamic performance. The length of inotropic support was determined by several factors including the metabolic status of the myocardial tissue, and the quality of preservation of the donor heart. Number and dosage of inotropes were decided, based on hemodynamic monitoring directly measuring arterial pressure, right atrial pressure, pulmonary arterial pressure, cardiac output, and continuous sVO₂ (Vigilance Indwelling Catheter; Baxter Healthcare Corporation, Deerfield, IL, USA).

Event definition
Definitions of graft failure, multiple organ failure, graft rejection, and infection strictly adhere to current released guidelines and are reported elsewhere.13 In-hospital mortality is defined as mortality occurring within 30 days after a heart transplant.

Assay methods and study design
Analyzed variables
Analyses included variables of demographics (recipient age, donor age, recipient sex, donor sex, and mismatch sex), mismatch weight, anemia, and acute kidney injury (AKI), which was assessed by means of RIFLE criteria¹⁴ (3 grades of increasing severity of acute renal disease as risk, injury, and failure). Pulmonary vascular resistance (PVR), ischemic duration, troponin I, and reoperation, HLA-A, -B, and -DR also were analyzed. The number of HLA mismatches was calculated, with grading from 0 to 6, where 0 is full match among HLA loci of donors and recipients, and 6 is the complete mismatch of the same loci.

Human leukocyte antigen typing: serologic and molecular techniques
Serologic HLA typing was performed using a standard microcytotoxicity assay,¹⁵ and molecular HLA typing was performed by PCR amplification with sequence-specific primer methodology. The discrimination between the different alleles took place during PCR. This shortens and simplifies the postamplification step to a simple gel electrophoresis detection step. The sequence-specific primer test results are either positive or negative, which abolishes the need for complicated interpretation of results.

Statistical analyses
Data are expressed as means ± SD, and median and interquartiles range for continuous variables, and as percentages and 95% confidence intervals for categorical variables. Normal distribution of scalar parameters was assessed by Kolmogorov-Smirnov test. All parameters were normally distributed, except PVR and troponin I. Log-transformation improved troponins normality, and slightly improves that of PVR. Bivariate analyses (using the chi-square statistic for categoric variables and the t test for continuous variables) were used to identify significant factors associated with in-hospital death. Multiple logistic regression using a stepwise-backward selection algorithm was used to evaluate the predictive role of HLA-DR mismatch on mortality independent of the effect of recipient age, mismatch HLA-A and HLA-B, AKI (RIFLE injury and failure),¹⁴ reoperation, ischemia duration, and levels of troponins.

To balance between the test of a full model including all measured variables, and a more parsimonious reduced model, model building and variables selection was achieved by subject-matter knowledge and data driven model selection. Age is regarded as a confounder (and as such, it is forced in the model irrespective of the statistical significance). All others variables were considered effect modifiers, and were tested before inclusion in the final model, except the 3 HLA variables, which were tested in the model, independent of their statistical univariate significance, since they were the main target of the study. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 17.0, IBM Corporation, Armonk, NY, USA). A P value < .05 was considered statistically significant.

Results

Data
The allelic distribution was homogeneous. The more-common HLA-A alleles in heart transplant recipients were 36 for HLA-A1 (11.4%), 68 for HLA-A2 (21.5%), 53 for HLA-A3 (16.8%), and 43 for HLA-A24 (13.6%). We found in the donor population 40 HLA-A1 alleles (12.7%), 64 HLA-A2 (20.3%), 38 HLA-A3 (12.0%), and 36 HLA-A24 (11.4%). Also, for HLA-B alleles, there is a certain level of correspondence between recipients and donors. In fact, in the recipient group, there were 32 HLA-B18 (10.1%), 64 HLA-B35 (20.3%), 35 HLA-B44 (11.1%), and 30 HLA-B51 (9.5%). Donor HLA-B alleles were distributed as follows: 32 cases of HLA-B18 (10.1%), 53 of HLA-B35 (16.8%), and 28 of HLA-B44 (8.9%). Finally, the most frequent recipient HLA-DR alleles were 110 for HLA-DR11 (34.8%), 30 for HLA-DR13 (9.5%), and 40 for HLA-DR7 (12.7%). Conversely the most common alleles in the donor group were 75 for HLA-DR11 (23.7%), 27 for HLA-DR13 (8.5%), 38 for HLA-DR7 (12.0%), 26 for HLA-DR15 (8.2%), and 26 for HLA-DR1 (8.2%).

Analysis and presentation
Of the 18 patients who died (11.4%), 11 received reoperation heart surgery (61.1%) compared with 25 in the survivor group (17.9%) (P < .001). A higher prevalence of AKI (50.0% vs 12.9%; P < .001), higher levels of troponins 48 hours after transplant (15.6 ± 12.0 vs 9.7 ± 9.4; P = .017), longer ischemia duration (188.2 ± 32.5 vs 162.6 ± 40.7; P = .011), and higher HLA-DR mismatch (1.61 ± 0.5 vs 1.30 ± 0.6; P = .046) were found in patients who died (Table 2 and Figure 1).

No differences were found between the dead and survivor groups when we analyzed the following variables: recipient and donor ages, recipient and donor sex, weight mismatch, HLA-A mismatch, HLA-B mismatch, and total HLA mismatch. Also, clinical conditions such as anemia, diabetes, and pulmonary vascular resistance, were not statistically different between the dead and the survivors (Table 2).

Logistic regression analysis demonstrated the HLA-DR mismatch predicts hospital mortality (OR=5.159, 95% CI=1.348-19.754; P = .017). Obviously, this effect is independent from the effect of the remaining variables included in the model (recipient age, mismatch HLA-A and HLA-B, AKI, reoperation, ischemia duration, and levels of troponin I; Table 3).

Immunosuppressive therapy was not introduced in the model because immunosuppression of the 18 patients that died in the hospital differed from the survivors owing to a more complex clinical situation because of a higher frequency of acute kidney failure, longer ischemic times, and in turn—more extensive organ dysfunction. Additionally, for 9 patients, immunosuppressive therapy was not initiated, and no differences were found in dosage and type of immunosuppressant therapy between the survivors and the other 9 patients who died in the hospital (cyclosporine 100% in deceased vs 90% in survived, P = NS; mycophenolate mofetil 66.7% in the deceased patients vs 64.3% in those who survived; P = NS).

Discussion

This study shows that the presence of HLA-DR mismatch predicts in-hospital (30-day follow-up) mortality after a heart transplant, as well as the presence of reoperation. High plasma levels of troponins, measured 48 hours after the transplant, were associated with higher in-hospital mortality.

Recently, the predictive role of HLA-B and -DR mismatch in acute rejection after a lung transplant has been shown.¹⁶ Whereas, the relevance of HLA matching on a renal transplant has been accepted¹⁷ and consecutively transferred to heart transplants,⁷ its benefits remain unclear for the heart.^3,5 The number of clinical publications on HLA matching for heart transplants is rather scant owing to the fact that HLA matching is not usually taken into account when selecting an organ for a specific recipient.^3,5 Furthermore, patients with heart failure require an urgent transplant and cannot wait for an organ with a high degree of HLA matching to become available. These factors, together with the elevated level of polymorphism of the HLA system, make the actual probability of a high degree of donor-recipient HLA matching quite low.^3,5

Therefore, the relevance of HLA matching for the outcome of heart transplants is a particularly critical issue because the cost-benefit balance of a better immunocompatibility and longer waiting before transplant may determine a patient’s survival. Our study demonstrates that in a consecutive series of heart-transplanted patients, that HLA-DR mismatch predicts in-hospital mortality, independent of the effect of clinical variables, previously recognized as risk factors for short-term mortality. These results reinforce the concept of the importance of HLA-DR mismatch not only for the long term, but also for immediate outcome such as in-hospital mortality.

In this study, there is a good correlation between donor and recipient allelic frequencies; nevertheless, the degree of mismatch within the single donor-recipient couple is high, ranging from 4.4 to 6. A similarly high level of HLA mismatch is seen in several US populations, but in no other regions close to the geographic origin of the study population, ie, the Campania Region in southern Italy, thus indicating a remarkable level of heterogeneity at the HLA locus.

It is significant to emphasize the observed inverse relation between HLA-DR mismatching and 30-day survival after a heart transplant is independent of relevant clinical factors, such as the 48-hour posttransplant plasma levels of troponins. In fact, the latter provides an important indication of graft function within the recipient’s body¹⁸; therefore, independence from this important marker is a further indication of the strength of the observed HLA-DR role in the outcomes of a heart transplant.

In recent years, better management of immunosuppressive therapy has reduced immunologic insults in heart transplants. The purpose of a beneficial immunosuppressive therapy is to prevent graft rejection, while minimizing negative adverse events.^3,5 Nevertheless, rejection remains the leading cause of transplanted heart loss.^3,5,19 Indeed, the significant degree of HLA mismatch and its correlation with in-hospital death, as observed in the present study, tend to support the idea that current immunosuppressive therapy is not optimal. To improve immunosuppressive therapy, particularly in cases where there is donor-recipient HLA-DR mismatch, should be a primary aim of research.

Based on the available scientific data as well as the results of this study, it is possible to claim short-term and long-term benefits of HLA-DR matching for heart-transplanted patient survival. Caution should be exercised, considering our results even when statistical significance is clearly defined, and even more so, in presence of borderline statistical significance, given the small sample size of the population studied.

However, at least 2 issues awaits further action: firstly, larger, multicenter, prospective studies should provide conclusive evidence about the importance of introducing HLA-DR matching into allocation policies to improve the outcome of heart transplant; and secondly, but certainly not the least, the same studies should reveal what the best combination is between host-versus-graft immunocompatibility and wait time for transplant, which will improve survival after heart transplant. We conclude that HLA-DR mismatch is associated with in-hospital mortality independent of reoperation and 48-hour levels of troponins after a heart transplant.

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