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Ann Thorac Surg 2004;78:557-563
© 2004 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, Centro Cardiologico Monzino IRCCS, Milan, Italy
b Italian Homograft Bank, Centro Cardiologico Monzino IRCCS, Milan, Italy
c Unit of Biostatistics, Centro Cardiologico Monzino IRCCS, Milan, Italy
d Transplant Immunology and Blood Transfusion Center, Ospedale Maggiore Policlinico IRCCS, Milan, Italy
e Department of Cardiac Surgery, Ospedale di Circolo-Fondazione Macchi, Varese, Italy
Accepted for publication February 10, 2004.
* Address reprint requests to Ms Guarino, Banca Italiana Omoinnesti, Centro Cardiologico Monzino IRCCS, Via Parea 4, 20138 Milan, Italy
e-mail: anna.guarino{at}ccfm.it
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Abstract |
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METHODS: Cryopreserved AAVs were obtained from multiorgan HLA-typed donors. Nineteen patients younger than 50 years (mean age, 43.3 ± 8 years) were prospectively studied. After successful surgery, all AAV recipient underwent at 3 and 6 months and each year postoperatively (mean follow-up, 71.9 months) concomitant serum sample collection and two-dimensional transthoracic echocardiography. The presence of anti-HLA antibodies was tested against a panel of lymphocytes obtained from 30 blood donors.
RESULTS: Progressive structural valve deterioration was seen in 6 patients (31.5%) of whom 4 (21%) were reoperated. All pretransplant recipients sera were panel-reactive antibody negative. Seventeen patients (89.4%) demonstrated significant panel-reactive antibody levels, which peaked at 6 months postoperatively, declined from 6 to 24 months, and slowly decreased afterward. In 14 of 19 cases (73.6%) donor-specific HLA antibodies were identified. A strong immunization (6-year persistence of panel-reactive antibody > 70% and peak panel-reactive antibody > 80%) was detected in 31.5% and 36.8% of recipients, respectively. Strong immunization was found to be significantly associated with progressive structural deterioration.
CONCLUSIONS: The immune reaction after cryopreserved AAV implantation is a peculiar long-lasting response occurring in the majority of recipients younger than 50 years of age. An association between a sustained and pronounced immunization and an aggressive AAV degeneration was observed.
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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On the other hand, viable AAVs are known to elicit an immune response after implant because of the presence of alloantigens, which induce specific recipient alloantibodies [6, 7]. Previous studies have demonstrated a specific cellular and humoral [8, 9] immune response directed against HLA determinants of the donor.
Although such an immune response has been postulated as one of the major causes—with surgical technique, mechanical stress, and ischemic or chemical insult—of long-term AAV degeneration, especially in younger patients [3], no clear evidence has been provided to date about its causality with valve durability.
Measurement of the alloreactivity as a result of donor-specific immunoglobulin G antibodies has been serially conducted no longer than 1 year after AAV implant [9, 10]. At later follow-up, prospective data on its magnitude, evolution, and specificity are unknown, although the persistence of antibodies 15 years after the operation in "fresh-fresh" AAV valves has been reported [11].
Moreover, only a few studies have linked immunologic factors to valve function. A concurrent immunologic and functional survey was retrospectively conducted by Smith and coworkers [10, 11] on fresh " homovital" and nonviable antibiotic-stored homograft valves. They were not able to associate both humoral response and degree of HLA mismatch for either class I or II antigens and valve function. Bechtel and colleagues [12] have reported, after a mean follow-up of 15 months, a lack of association between anti-HLA class I antibodies, HLA mismatch, and cryopreserved pulmonary valve function after the Ross procedure. Conversely, Dignan and coworkers [13] have found weak proof of a higher rate of cryopreserved AAV midterm dysfunction in the presence of two HLA class II mismatches.
These conflicting reports point to the need for further prospective studies aimed at characterizing the long-term immune response to AAVs and correlation of results to valve function.
The purpose of this paper was to prospectively serially measure, up to 6 years after AAV implantation, the magnitude and evolution of the recipient anti-HLA class I immune response to cryopreserved AAV valves in patients younger than 50 years of age, and to correlate serologic data with AAV performance by means of a concomitant two-dimensional transthoracic echocardiographic survey.
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Patients and methods |
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Homograft preparation
All AAVs were obtained from HLA-typed multiorgan donors (mean age 38 years, range 17 to 60). Cardiectomy was performed up to 6 hours after cross-clamping, in order to achieve a good valve viability. The entire heart was collected in sterile transport medium (Euro-Collins solution containing glucose, Na, K, Cl; SALF, Italy) at + 4°C and transported from operating room to the laboratory of Italian Homograft Bank at Centro Cardiologico Monzino Foundation IRCCS. Here valves were dissected as soon as possible from a minimum of 1 hour to a maximum of 3 hours after and they were macroscopically examined, cleaned and classified; tissue and medium samples for microbial test were sent to microbiology laboratory. Homograft valves were incubated for 24 hours at 4°C in nutrient medium RPMI 1640 (Roswell Park Memorial Institute 1640, Sigma-Aldrich Co LTD, St. Louis, MO) containing the antibiotics vancomycin 50 µg/mL (Eli Lilly, Indianapolis, IN), polymyxin B sulfate 5 MIoIU 100 g/mL (Biochrom KG, Berlin, Germany), lincomycin 120 µg/mL (Upjohn SARL, Paris, France), and cefoxitin 240 µg/mL (Merck Sharp & Dohme, Chibret, France). After sterilization other samples were taken for microbial tests, and the valve was placed in 100 mL of freezing medium, which was RPMI 1640 containing 10% dimethyl sulfoxide (Sigma-Aldrich Co LTD) and heat sealed inside two hemofreeze bags (NPBI, Emmer-Compascuum, The Netherlands). The packaged valves were then cryopreserved in a controlled-rate freezer (Kryo 10-16 series III; Planer Biomed, Sunbury Middlesex, UK) at a rate of –1°C/min down to –80°C and then were transferred to the vapor phase liquid nitrogen freezer (model 17K, Taylor-Wharton Cryogenics; Harsco Corp, Theodore, AL) at a temperature between –135°C and –185°C. Valves have been stored for a minimum of 4 weeks to a maximum of 12 months before use. At the moment of implantation the valve, in its bag, was sent to the operating room in dry ice, placed in a 37°C saline bath until ice crystals of freezing solution were thawed, aseptically removed from the bag, and had all the dimethyl sulfoxide washed out by sequential immersion in RPMI 1640 medium.
Surgical procedures
All surgical procedures were performed using standard cardiopulmonary bypass and cardioplegia techniques. All AAVs were implanted by the same surgeon with the inclusion cylinder technique [14].
Lymphocytotoxic antibody screening
Serum samples from the 19 patients enrolled in the study were collected before valve replacement and at 3, 6, and 12 months and annually from 1 to 6 years postoperatively. In addition, sera from 10 patients undergoing valve replacement with mechanical valves were studied as control samples to exclude an aspecific sensitizing effect related to open-heart surgery.
All sera were stored at –40°C then analyzed in batch. Panel-reactive antibody (PRA) was determined using a 30-cell selected panel that covered the majority of HLA class I specificities. The panel consisted of T and B lymphocytes obtained from 30 blood donors.
All sera were screened according to the microlymphocytotoxicity assay. Undiluted sera in 1-µl aliquots were added in Terasaki microwells prefilled with light mineral oil containing 1 µL of T-cell and B-cell suspension (at a cell concentration of 2 x 106/L), previously isolated from peripheral blood by Ficoll-Hypaque. After incubation for 60 minutes at 20° to 24°C, 5 µL of noncytotoxic rabbit complement was added for 60 minutes, and then 5 µL of 4% aqueous eosin was added, followed after 5 minutes by 5 µL of formalin to detect nonviable cells. Cytotoxicity was assessed manually in each microwell on a Lambda Scan Plus (One Lambda Inc, Los Angeles, CA), and graded on an increasing scale from 1 (<5% lysis) to 8 (>95% lysis). Sera reacting against at least 5% PRA and with a reaction score of at least 10% of background level were considered positive [11].
Antigen specificity of panel-reactive antibody
All sera found to be PRA positive were studied to evaluate HLA specificities. Reactions of positive sera were interpreted with an automated software (One-Lambda, Inc), and the specificities found were validated manually by two experienced operators in a blind fashion. Positive sera were tested undiluted and after a 1:4 dilution. Subsequently, the specificities identified were compared with donor HLA typing, when available.
HLA-AB typing
All multiorgan donors were typed using microtoxicity with local and commercial trays, according to the standards stated by the European Foundation for Immunogenetics [15]. Fresh lymphocytes were isolated both from peripheral blood samples or lymph nodes by immunomagnetic bead separation. Patients were not HLA-typed, so that AAVs were allocated without considering HLA matching.
Functional assessment of aortic homograft valves
All patients underwent a two-dimensional transthoracic echocardiography immediately after the operation and at the time of PRA assays. Progressive structural deterioration (PSD) was defined as the occurrence of moderate to severe aortic valve incompetence (grade 3 to 4+) or valve stenosis (peak gradient of >40 mm Hg) [13]. A concomitant clinical assessment was obtained.
Statistical analysis
Results are reported as mean ± standard deviations or percentages, unless specified. The association between categorical variables was assessed by 
2 or Fisher's exact test, as appropriate. The time free from PSD was evaluated using Kaplan-Meier survival analysis. The correlation between numeric variables was assessed by the Spearman method. A p value less than 0.05 was considered as significant.
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Results |
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Allograft survival
Mean follow-up was 71.9 ± 15 months. Mortality during the follow-up period was null. Echocardiograms were available for all patients at 3 and 6 months and each year postoperatively until completion of follow-up. Progressive structural allograft valve deterioration was seen in 6 patients (31.5%), of whom 4 (21%) were reoperated on at 24, 41, 51, and 57 months from their first operations. Modes of PSD were isolated insufficiency grade 3 or 4 in 2 patient (10.5%) or insufficiency with associated stenosis (pressure gradient > 40 mm Hg) in 4 patients (21%). The incidence of AAV PSD was 0.2 patients/y.
Incidence, kinetics, and characterization of panel-reactive antibodies
All recipients' sera were PRA negative before operation. Postoperative samples were taken starting at 3 months from AAV implant and continued at 6, 12, 24, 36, 48, 60, and 72 months in all patients. Overall, 17 patients (89.4%) demonstrated positive PRA levels after AAV transplant. Figures 1 and 2, respectively, show the percent of recipients found to be PRA positive and the mean PRA strength of reaction (expressed as percent of activity) during the follow-up period. Briefly, mean levels of PRA peaked at 6 months postoperatively, significantly declined from 6 to 24 months from operation (55.2% versus 31.6%), and slowly decreased afterward (31.5% at 24 months versus 27.7% at 72 months). Conversely, the percentage of patients showing positive antibody production remained quite stable throughout the follow-up period. The 2 patients (10.6%) showing negative anti-HLA reactivity remained negative throughout the entire follow-up period. In control patients receiving a mechanical valve, no PRA activity was detected during the same follow-up period.
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Specificity of antibodies
In sera of the 17 recipients who developed a positive PRA, specificities of HLA antibodies were found in 14 cases (82.4%). After the comparison between these specificities with the donor HLA-I class, we found that anti-HLA antibodies were donor-specific in 13 of 14 cases. In fact, we found that the HLA antibodies corresponded to one or two HLA-1 broad or split antigens belonging to the donor in 10 cases, whereas in the remaining 3 cases only a supertypical reactivity (antiw4 or antiw6) that was donor-related was found (Table 2). Specificities of HLA antibodies were identified in all patients with peak PRA more than 80% and permanent PRA more than 70%.
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= 0.2835; p = 0.0232).
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Comment |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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In this study, attention has been paid to design a prospective model in which selected patients younger than 50 years of age receiving a cryopreserved allograft valve underwent identical processing variables and implant techniques. None of the recipients had prior exposure to allogeneic human tissue and none had sera that were PRA positive before operation. A contemporary yearly immunologic and two-dimensional echocardiographic survey has been prolonged throughout a mean of a 6-year span.
Our results provide evidence that the immune reaction after AVV implant is a peculiar long-lasting response occurring in the great majority of recipients younger than 50 years of age, and, more importantly, suggest that in these patients a strong immunization is likely to be correlated with a progressive midterm valve degeneration.
These findings may add new insights in the controversial debate about the role of chronic rejection in determining durability of allograft valves. Cryopreserved AAVs are considered to have at least 50% leaflet viability (interstitial cells) at the time of valve implantation [16]. According to the available literature, there is no doubt that viable homograft valves can generate an immune response. From a histologic point of view, myocardial rejection after heart transplantation was found to be associated with subendothelial lymphocytic infiltrates and aortic valve edema, suggesting that valve components are a target of an immunologic rejection [17]. Moreover, laboratory studies have shown that AAVs elicit an immune response after implant as a result of the presence of alloantigens that induce specific recipient alloantibodies [6, 7, 18]. The specific cellular [8] and humoral [9] immune response directed against HLA determinants of the donor has been clinically demonstrated. This alloreactivity related to donor-specific immunoglobulin G antibodies was prospectively observed to persist up to 1 year after AAV implant by Smith and colleagues [10].
Nonetheless, antigenicity does not necessarily implicate the occurrence of immune-mediated valve deterioration. Only a few studies have been designed in an attempt to link the immune reaction with AAV function, postulating whether antibody production because of HLA mismatch between donor and recipient has an impact on early and midterm valve deterioration.
A retrospective immunologic survey (with a mean follow-up of 6 years) has been conducted by Smith and coworkers [11] after implant of fresh homovital and nonviable antibiotic-stored homograft valves. They were not able to find a significant correlation between the degree of humoral response and HLA mismatch for either class I or class II antigens with markers of long-term valve function, including patient mortality, reoperation, and progressive valve degeneration. Conversely, Dignan and coworkers [13] have reported by means of an univariate analysis that class II antigen mismatch had a significant association with decrease in cryopreserved aortic valve freedom from structural deterioration. However, they were not able to confirm these results at multivariate analysis, where only young age at operation and short time (<4 hours) between homograft procurement and cryopreservation were associated with an increase in structural failure. More recently, Bechtel and colleagues [19] have reported a lack of association between anti-HLA class I antibodies and homograft function in the first year after the Ross procedure.
In summary, no clear evidence that the immune response against HLA antigens adversely affects valve function has been provided. However, none of the previous studies were prospectively designed to correlate the long-term frequency and magnitude of the immune reaction, in terms of antibody production after AAV implantation, with a concomitant surveillance of valve durability.
The major inferences we have drawn from our results concern the persistence and evolution of the humoral response to cryopreserved AAVs and its implications with valve dysfunction. Approximately 90% of our recipients showed a positive anti-HLA reactivity, which reached its peak after 6 months from implant (mean PRA, 58.3% at 6 months and 50% at 12 months). This is in agreement with previous observations [8–10].
No serial information was available on trend and magnitude of antibody production beyond 1 year from AAV implantation. We have observed that the great majority of patients receiving an AAV presented a long-lasting immune response, which is still significant at 6 years postoperatively. The percentage of our patients showing positive antibodies remained quite stable throughout the 6-year follow-up period, slowly declining from 79% at 6 months to 69% at 72 months postoperatively. Conversely, mean PRA global activity significantly declined in recipients from 6 to 24 months from operation (55.2% versus 31.6%), and slowly decreased afterward (31.5% at 24 months versus 27.7% at 72 months). These data suggest that although the immune response to AAVs reaches its peak within 1 year from implantation, in the majority of AAV recipients a long-lasting immunization persists.
Furthermore, we have better immunologically characterized patients showing a progressive allograft valve structural deterioration, inasmuch as all of them were found presenting at least one of the following two indicators of a broad anti-HLA antibody production: a peak PRA more than 80% and a 6-year persistence of PRA more than 70%. These patients accounted for 36% and 31% of all AAV recipients, respectively. On the other hand, recipients without signs of strong immunization have maintained a perfect freedom from AAV failure up to 6 years postoperatively. Thus, a significant association between progressive valve deterioration within 6 years from implant and strong immunization was statistically calculated.
These findings may partially explain the occurrence of the accelerated and progressive allograft degeneration [20] that is preferably detected in younger patients [3, 13]. In our recipients, the major mode of such an aggressive valve dysfunction was the combination of insufficiency and stenosis (4 of 6 cases), versus the prevalence of pure regurgitations reported in late failure (>10 years) statistics [5, 20]. This may be partially explained with the predominance of the immunologic contribution in premature AAV PSD.
The main limitations of this study were (1) the relatively small number of patients enrolled, which was however at least partially compensated by the frequency and serial nature of serologic and functional measurements, and (2) the lack of HLA mismatch assessment between donor and recipients to exclude that antibodies identified are directed against self antigens. However, the negativity of preoperative PRA and the absence of previous autoimmune diseases of our recipients let us believe that the antibody reaction elicited after AAV implant was caused by anti-HLA alloantibodies.
In conclusion, this study has provided new midterm insight in the determination of frequency, evolution, and magnitude of the humoral immune response after cryopreserved aortic valve implantation in patients younger than 50 years of age. Interestingly, in those recipients showing an accelerated deterioration of the valve the contribution of the immune reaction seems relevant.
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References |
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TopAbstractIntroductionPatients and methodsResultsCommentAcknowledgmentsReferences |
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