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a Division of Cardiac Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland
b Division of Cardiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland
Accepted for publication May 29, 2007.
* Address correspondence to Dr Conte, Division of Cardiac Surgery, The Johns Hopkins Medical Institutions, 600 N Wolfe St, Blalock 618, Baltimore, MD 21287 (Email: jconte{at}csurg.jhmi.jhu.edu).
Presented at the Fifty-third Annual Meeting of the Southern Thoracic Surgical Association, Tucson, AZ, Nov 8–11, 2006.
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Abstract |
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Methods: A retrospective cohort study of using data reported to the United Network for Organ Sharing /Organ Procurement and Transplantation Network (UNOS/OPTN) registry from January 1, 2000, to December 31, 2004, was performed. The association between PRA at transplant and primary end points, allograft and patient survival, as well as a secondary end point, rejection within 1 year, was analyzed.
Results: Pretransplant PRA was reported for 8,160 (79.4%) of the 10,279 first heart transplant recipients during the study period. Panel-reactive antibody was 0% in 6,481 (79.4%) patients (group 1), 1% to 10% in 930 (11.4%) patients (group 2), 11% to 25% in 309 (3.8%) patients (group 3), and greater than 25% in 440 (5.4%) patients (group 4). Actuarial survival was significantly different among the four groups by Kaplan–Meier method (p < 0.001). Furthermore, using PRA cutoffs of 0%, 10%, or 25%, the group with lower PRA had significantly better patient and allograft survival. Cox proportional hazard modeling revealed increasing PRA as a significant predictor of mortality (p < 0.001). However, when each group (2, 3, and 4) was compared with group 1 (PRA 0%), only group 4 (PRA > 25%) had worse survival on multivariate analysis. Patients with PRA greater than 25% confirmed by the flow cytometric technique had the worst overall survival. Rejection rate within 1 year after transplantation also significantly increased with increasing PRA. Propensity-matched patients demonstrated similar results.
Conclusions: This large series of patients from the United Network for Organ Sharing database has demonstrated that elevated PRA remains a significant risk factor in a recent cohort of heart transplant recipients. Patients with PRA greater than 25% are at a particularly high risk.
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Introduction |
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The PRA result is usually presented as the percent of panel reactivity (ie, the number of wells with positive reactivity divided by the total number of wells tested x 100). What value constitutes a significantly elevated PRA is variably defined, and may prompt a prospective cross-match, that is, direct testing of the recipient’s sera against the donor’s lymphocytes. Generally, a positive donor-specific cross-match is considered a contraindication to solid organ transplantation. Although current standards in kidney transplantation mandate a routine cross-match between donor–recipient pairs before transplantation, constraints of prolonged ischemic time do not allow for routine prospective cross-matches in heart transplantation (HTx). Cross-matching in HTx is generally performed for recipients with an elevated pretransplant PRA. A recent survey of adult heart transplant programs showed that there is considerable variation among centers about the timing of PRA determination, importance of PRA values, threshold for treatment or prospective cross-match, and the clinical implications of sensitization [4]. According to the survey, the most common criterion used for elevated PRA to be considered positive was a value of 10% or more, although there is considerable variation. Furthermore, different methods are used at different centers for PRA determination. The complement-dependent cytotoxicity assay is the older and most commonly used method. During recent years, many laboratories have applied more-sensitive serum screening and cross-match techniques. The anti-human globulin–augmented lymphocytotoxicity test, the complement-independent enzyme-linked immunosorbent assay, and the flow cytometric (FCM) assay are prime examples of these more sensitive techniques. Some advocate that the use of FCM technique is more predictable of outcomes [5, 6]. Only a few laboratories use more than one technique to assess PRA.
The development of sensitization may occur as a result of previous blood transfusions, previous pregnancies, and more recently, the use of a ventricular assist device (VAD) as a bridge to heart transplantation. Some studies have suggested that although VAD support leads to increased sensitization this does not translate into worse outcomes [7, 8]. Thus, the acceptable threshold and the overall significance of elevated pretransplant PRA in HTx has not been clearly defined. To evaluate the effect of pretransplant PRA on outcomes in a large and recent cohort of heart transplant recipients, we analyzed the influence of different PRA cutoff levels, 0%, 10%, and 25%, using data reported to the United Network for Organ Sharing /Organ Procurement and Transplantation Network (UNOS/OPTN) registry, between 2000 and 2004.
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Patients and Methods |
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Statistical Analyses and Survival
The STAR files from the UNOS/OPTN registry were imported into Stata version 9.0 (StataCorp. College Station, TX), which was used for statistical computations. For all statistical tests, probability values less than 0.05 were considered significant. Continuous variables are summarized as median with interquartile range when they are not normally distributed, and categorical variables are expressed in frequencies and percentages. Bivariate comparison of continuous variables was performed using a Student’s t test if values were normally distributed; if not, a Wilcoxon rank sum test was used. Categorical variables were tested using Pearson’s 
2 test. Survival estimates were based on the Kaplan–Meier method, and statistical differences between survival curves were assessed by the log-rank (Mantel-Cox) test. Multivariate analyses were performed by multiple logistic and Cox proportional hazards regression. During the analyses for model selection, only variables with more than two thirds of available data were considered. The only exception made was VAD support because only about half of the patients have information available. To more accurately estimate confidence intervals, we made the assumption that the missing data were missing at random and performed imputation of missing data using the single regression method. Our findings proved to be robust across models that included both imputed and nonimputed (ie, casewise deletion of observations with missing data points) data. Our final model was chosen to reflect the effect of clinically relevant potential cofounders, namely recipient’s age, sex, body mass index, donor’s age, ischemia time, transfusions between listing and transplantation, life support (which includes any of the following: VAD support, ventilator support, intraaortic balloon pump, and extracorporeal membrane oxygenation), HLA mismatch, and transplant year. Another model was also analyzed in which VAD support was considered as a cofactor separately from other forms of life support only after imputation of missing data, as fewer than two thirds of the patients had data available about VAD support.
In addition, to reduce bias and increase precision, propensity scores were generated by developing a model that estimates the probability of being in any of the patient groups based on clinically relevant variables listed above. Propensity scores were analyzed using the regression adjustment technique. That is, both the generated propensity scores and comparative PRA groups were included in a Cox regression model. Subsequently, we also obtained an additional dataset from UNOS that contains information about the techniques used for PRA determination and cross-match results and incorporated these data in our analyses.
Rejection Comparisons
A subgroup analysis was performed on recipients with at least 365 days of follow-up to evaluate the association between the development of rejection and PRA level. Patients were excluded from this analysis if they had fewer than 365 days of follow-up as determined by subtracting the date of transplant from the date that the data were captured in the dataset. A total of 1,684 patients (20.6%) were excluded with follow-up of fewer than 365 days. The chosen variable from the database was "treatment for rejection within 1 year," which was used to define the rejection events. Other variables in the database that define rejection such as acute rejection episodes had too many missing data points (more than one third) and were not uniformly reported; thus, those were not used for analysis. Adjusted probabilities of rejection within 1 year after HTx accounted for the same clinically relevant cofounders used for survival analysis.
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Results |
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Techniques Used for Panel-Reactive Antibody Determination
We further requested an additional dataset from UNOS containing information about the technique used for PRA determination and results of retrospective cross-match. These data were merged into the original STAR files. Of the 8,160 patients, 7,003 (86%) patients had information about the technique used for the determination of the most recent pretransplant PRA. The complement-dependent cytotoxicity technique was used in 2,576 patients (37%), the anti-human globulin–augmented lymphotoxicity test was used in 2,761 patients (39%), and the FCM technique was used in 1,664 patients (24%). The enzyme-linked immunosorbent assay technique was reported in only 2 patients and thus was not included in these analyses. Among the patients with PRA greater than 25%, the anti-human globulin–augmented lymphotoxicity test or FCM technique was the method used for PRA determination in 70% of the patients.
All the above multivariate analyses were repeated controlling for techniques used for PRA determination, and only PRA greater than 25% still emerged as a significant predictor of worse survival. Additional subanalyses of the techniques revealed that the patients with PRA greater than 25% determined by the FCM technique had the worst overall survival. Furthermore, when all the patients with positive retrospective cross-match were excluded (350 patients), PRA greater than 25% remained a significant predictor of worse survival only among those patients for whom PRA was determined by the FCM technique.
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Comment |
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The standards of the American Society of Histocompatibility and Immunogenetics and UNOS recommend that pretransplant cross-matches be performed only when the PRA activity of potential cardiac allograft recipients is 10% or greater [9, 10]. Even though PRA activity does not necessarily predict that a donor-specific cross-match will be positive or that the heart will be lost to hyperacute rejection if transplanted without a prospective cross-match, several studies report that a PRA of 10% or greater affects the duration and incidence of rejections after transplant, as well as patient survival [1–3]. Loh and colleagues [1] evaluated 125 heart transplant recipients and reported that an elevated PRA value of 25% or greater at the time of HTx may be a risk factor for decreased long-term survival. A trend toward an increased risk of death caused by rejection was also observed. Lavee and associates [2] demonstrated in a cohort of 463 heart transplant recipients that PRA greater than 10% is a risk factor for rejection-related events, and a negative lymphocytotoxic cross-match in patients with an elevated PRA does not reduce the risk of death resulting from acute or chronic rejection. These findings were supported by the report of 311 patients from Kobashigawa and colleagues [3], who also found that patients with PRA of 11% or greater at the time of transplant appear to have earlier and more severe rejection with significantly lower survival after transplant surgery, despite negative donor-specific cross-match.
One of the major issues about determining truly sensitized patients is the fact that different approaches are used in different laboratories for PRA screening [4]. Historically, PRA analysis to detect HLA antibodies has been performed using cell-based complement-dependent cytotoxicity techniques and more recently the anti-human globulin–augmented lymphocytotoxicity test. Enzyme-linked immunosorbent assay and FCM assay were introduced as an alternative approach to detect HLA antibodies. The latter seem to overcome the drawbacks of complement-dependent cytotoxicity as a more sensitive technique and the ability to detect class II antigens. Flow-cytometric technique–detectable pretransplant antibodies have been shown to better differentiate sensitized versus nonsensitized patients in terms of outcomes after HTx [5, 6]. With these developments it appears logical to evaluate the influence of PRA in a recent cohort of patients to see whether they are comparable to previous reports when only the complement-dependent cytotoxicity technique was used, although some major centers started using FCM techniques in earlier years.
Furthermore, the proportion of these so-called sensitized patients has been rising with the increased use of VAD as a bridge to HTx [7, 11]. A single center report and another report using the International Society for Heart and Lung Transplantation (ISHLT) registry have suggested that although VAD use does increase sensitization, these patients have comparable outcomes to nonbridged patients [7, 8]. This raises the important and persistently controversial question regarding the overall influence of elevated PRA on outcomes in HTx. A potential explanation for the lack of influence on outcome in VAD patients maybe the finding from some studies, which have shown a temporal pattern of HLA sensitization during VAD support [12]. This temporal pattern consisted of a rapid increase followed by a rapid progressive decrease, and several mechanisms have been proposed [12]. One may rationalize from these findings that although VAD support increases PRA, the level may be normalized at the time of transplantation. This is also suggested by our findings that poorer survival among patients with PRA greater than 25% on multivariate analysis is no longer significant when "most recent PRA at transplant" is replaced with "peak PRA at transplant." Thus, the reports that elevated PRA in patients supported with VAD does not influence HTx outcomes may not necessarily translate to other patients with elevated PRA.
Modern improvements and better outcomes in HTx have emboldened more programs to offer HTx to patients with elevated PRA. We hypothesized that despite advancements in HTx, humoral sensitization as reflected by PRA is still a significant predictor of mortality and rejection in the recent era. We explored our hypothesis using data available on first HTx recipients reported to UNOS from 2000 to 2004 (all retransplantations were excluded). Unlike several reports in the literature that use a 10% cutoff to determine positive PRA result, we analyzed PRA as a continuous value from 0% to 100%. We used all available information about PRA in the database and selected "most recent PRA at transplant" as the most complete and reliably reported variable. Furthermore, we also divided the patients in four clinically relevant groups: group 1 is PRA 0% (which is also used as the reference group in most of the analyses), group 2 is PRA 1% to 10%, group 3 is PRA 11% to 25%, and group 4 is PRA greater than 25%. This allowed for evaluation of outcomes in these separate groups. Additionally the entire patient cohort was divided into two groups using PRA level cutoff points of 0% or 10% or 25%.
Pertinent clinical characteristics that were more prevalent in sensitized patients included younger age (may be related to enhanced immune responsiveness), female recipient (likely associated with pregnancies), transfusions between listing and transplantation (presence of HLA antigens on blood cells), more likely to be on VAD support (possibly related to interactions between the ventricular assist device surface and host immune system), and having spent longer time on the waiting list (could be related to duration of immunomodulatory therapy). These findings are in agreement with predictors of sensitization reported in the literature [12–14]. Some other findings are the higher prevalence of congenital heart disease and fewer patients in UNOS class 2 status. Our analyses clearly demonstrated that elevated PRA (as a continuous value greater than 0%) was a predictor of mortality by Kaplan–Meier unadjusted estimates as well as in an adjusted multivariate Cox regression model. When the cohort was analyzed at different cutoff levels described above, the group with lower PRA always had better survival by log rank test. Comparison of each of the other three groups (2, 3, and 4) to reference group 1 (PRA 0%) showed that group 4 (PRA>25%) remained a significant predictor of mortality in a multivariate model. Similar results were obtained when the above analyses were also performed for allograft survival. Notably, these effects of PRA on allograft and patient survival were no longer present when only patients who survived more than 30 days were analyzed. Likewise, PRA also emerged as a significant predictor of 30-day mortality. These findings suggest that the influence of pretransplant PRA on outcomes in HTx is an early phenomenon.
The most reliably reported variable about rejection in the database is "treatment for rejection within 1 year after transplantation." Sensitized patients with PRA greater than 25% demonstrated statistically significant higher rejection rates compared with PRA 0%, in an unadjusted and adjusted model. Additionally, patients were propensity-matched according to baseline characteristics, and the same findings were noted for all the outcome variables, namely, allograft and patient survival as well as rejection. Comparable results were also obtained when "peak PRA at transplant" was substituted for "most recent PRA at transplant"; however, none of the groups remained significant on multivariate analyses when compared with PRA 0%. This would suggest that "most recent PRA at transplant" is a more important predictor of outcome, because some patients with high "peak PRA at transplant" may have their PRA level normalized or reduced at the time of HTx.
The current study has the inherent limitations of the review of a multiinstitutional voluntary registry. Analysis of data may be compromised by lack of uniformity of reporting, nonstandardized method of PRA screening, and other pertinent variations such as organ preservation, surgical techniques, and postoperative and immunosuppressive management. Only first heart transplant recipients were included to help achieve a more uniform cohort of patients; moreover, retransplantation may significantly increase sensitization. The 2000 to 2004 era was also selected to reflect the advancements that have recently evolved in HTx. We also do not have enough information to evaluate why these patients died; it could be that they were overimmunosuppressed because of high PRA, which could lead to higher incidence of infection.
One of the major disadvantages of database analyses is the large amount of missing data in the reporting of some important variables, such as acute rejection episodes, previous pregnancies, and primary cause of graft failure or death. We compensated for these issues by analyzing only variables with more than two thirds available data except for VAD support as described above; however, statistical imputation of missing data showed that the results of our analyses were robust. Poorer outcome in the sensitized group may also be a reflection of other variables that were not accounted for.
Our analysis of the UNOS database from a recent era showed that PRA is still a significant risk factor in the outcomes of HTx, and its effect appears to be an early phenomenon. These data suggest that patients with PRA greater than 0% have worse posttransplant outcomes than those with PRA of 0% and should still receive careful evaluation before undergoing HTx. Patients with PRA greater than 25% are at a particularly high risk, and pretransplant lymphocyte cross-match should be considered, especially if confirmed by the FCM technique. Further advancements in HTx will better define the future role of pretransplant PRA.
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Discussion |
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Use of these national databases opens an opportunity for quite a bit of clinical research in the field of transplantation, similar to what the STS database does, and a lot to be gleaned from these. Unfortunately, as with the STS database, one of the major problems with the UNOS (United Network for Organ Sharing) database is the lack of complete data in many areas, and this may have hampered some of the analyses for you. I have got several questions for you.
First of all, you have shown that PRA (panel-reactive antibody) does have an impact, largely for high PRA, within the first 30 days. Where you did utilize life support as one of your variables, were you able to tease out ventricular assist devices (VADs) separate from other forms of life support such as balloon pump and ventilator status at the time of transplant and able to distinguish that as an independent variable in these patients, because I suspect it will affect the outcomes? In other words, how much of the high PRA is related to the use of VADs, which actually have been shown to have a higher early mortality rate than non-VAD patients in the UNOS registry?
The second question I have is, PRA is an evolving concept. Historically it has been the cytotoxic antibodies using the classic AHG (anti-human globulin–augmented lymphotoxicity test) wash or NIH (National Institutes of Health) protocol well that you describe where it is a percentage of reaction and cell lysis to a panel of something less than 100 random donors that are supposed to represent the population. As we have moved through the ELISA (enzyme-linked immunosorbent assay) and the flow cytometry and more recently to the use of the bead—movement x bead technology—we have been able to distinguish class I from class II antibodies a little bit better, and more specifically, to separate out donor-specific antibodies. Now, I know the UNOS registry doesn’t collect a lot of this data currently, but the question I have is related to a study that we recently presented at the American Transplant Congress whereby we were able to show that PRA was, or the outcomes were, related to not the nondonor-specific antibodies but purely to the presence of donor-specific antibodies in the population. So that PRA, per se, may not be the critical point here. It may be the presence of donor-specific antibodies in a population where you are not doing prospective cross-matches. And the follow-up of this question is, I was a little surprised that you did not use either prospective or retrospective cross-match as one of the variables in your analysis, because it may be that it is not the PRA per se that is the problem but it is the presence of a positive donor-specific cross-match that is contributing to the early poor outcomes.
Thank you.
DR NWAKANMA: Thank you, Dr Ring, for your comments. Your first question was to separate VAD support from life support. We did think about that. As you acknowledged, one of the disadvantages of using the UNOS database is incomplete data. One of the ways we have been able to make reliable conclusions using the database is to use only variables where at least two thirds of the patients have data available, because whenever we do imputation of missing data, if two thirds of the data are available, then the results are usually similar. Unfortunately, VAD support happens to be one of those variables where less than two thirds of the data were available. However, we did still analyze it and did find, like you would expect, that patients with higher PRA or patients that were on VAD support had higher PRA, and it did not affect the survival. But one needs to be careful to interpret these data because, again, the data were incomplete for that variable.
Your next question was about the technique used. We do appreciate the fact that with the new flow cytometric method you are more able to detect class II antigens from class I. Unfortunately again, the UNOS database does not specify the type of technique that was used. We just expect by using a recent cohort of patients we may be able to capture more patients where the new technique is used as opposed to the CDC (complement-dependent cytotoxicity) technique.
The next question is about the presence of donor-specific antibodies. That was a very, very important question that we went back to address with UNOS, because the standard transplant research files which they provide whenever one asks for data from the UNOS provides a variable called "cross-match done." It does not tell you whether the cross-match was done prospectively or retrospectively or when it was done. It just tells you yes or no. So we are going to go back to have them reload the information and give us exactly when it was done, prospectively or retrospectively; then we will be able to analyze whether it was the donor-specific antibodies that affected survival.
So thank you for those comments. We do acknowledge those limitations and we will try our best to include more information in our future analyses as we have them available.
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Acknowledgments |
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Doctor Nwakanma is a Hugh R. Sharp Cardiac Surgery Research Fellow. Doctors Williams and Weiss are Irene Piccinini Cardiac Surgery Research Fellows.
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25% versus panel-reactive antibody > 25%.