HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Online Discussion Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Claus Bartels Claudia Schmidtke Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bechtel, J.F. M. Articles by Sievers, H.-H. Search for Related Content PubMed PubMed Citation Articles by Bechtel, J.F. M. Articles by Sievers, H.-H. Related Collections Molecular biology Valve disease

Ann Thorac Surg 2001;71:2003-2007
© 2001 The Society of Thoracic Surgeons

---

Original article: cardiovascular

Anti-HLA class I antibodies and pulmonary homograft function after the Ross procedure

^a Clinic for Cardiac Surgery, Medical University of Luebeck, Luebeck, Germany
^b Institute for Immunology and Transfusion Medicine, Medical University of Luebeck, Luebeck, Germany

Accepted for publication February 14, 2001.

Address reprint requests to Dr Sievers, Klinik für Herzchirurgie, Universitätsklinikum Lübeck, Ratzeburger Alle 160, D-23538 Lübeck, Germany
e-mail: sievers{at}medinf.mu-luebeck.de

	Abstract

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. The Ross procedure provides excellent long-term results in the majority of patients. However, degeneration of the pulmonary homograft in some patients remains an unresolved problem that may be related to immunologic factors. Therefore, we studied the prevalence of antihuman leukocyte antigen (HLA) class I antibodies and echocardiographic results of homograft function at rest.

Methods. Forty-seven patients (37 men, 10 women; 47 ± 15 years) were seen for echocardiography 1.1 to 63.9 months (median, 27 months) postoperatively. The presence of anti-HLA antibodies was tested against a panel of lymphocytes of 50 donors.

Results. Twenty-seven (57%) of the patients produced anti-HLA class I antibodies. No difference in the maximal or mean transhomograft pressure gradient, or in the frequency of homograft regurgitation according to the presence or absence of anti-HLA antibodies was found. However, the right ventricle was slightly but significantly larger in antibody-positive patients (26.3 ± 4.2 versus 30.7 ± 3.5 mm; p = 0.001).

Conclusions. In the first years after the Ross procedure, we could not detect significant evidence of an association between anti-HLA class I antibodies and echocardiographic results of homograft function at rest in adults.

	Introduction

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The Ross procedure for treatment of aortic valve disease offers excellent long-term results in the majority of children [1] and adults [2]. In some patients, however, degeneration of the homograft used for reconstruction of the right ventricular outflow tract occurs. With current preservation protocols, homograft valves can be expected to contain at least some viable cells at the time of implantation [3] resulting in a specific cellular [4] and humoral immune response [5] directed against human leukocyte antigen (HLA) determinants of the donor. To date, however, there is no direct evidence of a deleterious effect of this immunologic response. Studies on homograft valves explanted at autopsy or reoperation gave conflicting results as to whether there is rejection of homograft valves [6, 7]. Valve function in vivo may be a more sensitive marker for damage to the homograft than freedom from reoperation and can be studied appropriately by echocardiography. There are two studies on aortic homograft function and immunologic factors: Smith and coworkers [8] found no significant correlation between the degree of HLA mismatch for either class I or II antigens with markers for long-term valve function. Dignan and colleagues [9] reported a significantly higher rate of valve dysfunction in the presence of two HLA class II mismatches, but this association was not significant in the multivariate analysis. There are no studies linking homograft function and immunologic factors after the Ross procedure. We, therefore, studied the prevalence of anti-HLA antibodies in adult patients who had undergone a Ross procedure and their relevance to results of pulmonary homograft function.

	Material and methods

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
A random sample of 47 patients (of a total of 113 patients) of at least 1-month follow-up was chosen to undergo anti-HLA antibody determination in addition to our regular echocardiographic examination. Of the patients, 37 were men, 10 were women; mean weight and height were 78 ± 12 kg and 173 ± 8 cm, respectively. Follow-up ranged from 1.1 to 63.9 months (median, 27 months). The study was approved by the local ethics committee, and informed consent was obtained from the patients.

Operative technique
Median sternotomy and standard cardiopulmonary bypass with a membrane oxygenator (Hollow Fiber Oxygenator; Spiral Gold; Baxter, Anacos, Puerto Rico) at moderate hypothermia (26°C temperature nasopharyngeal) with cold crystalloid cardioplegia (St Thomas’ Hospital solution) for myocardial protection was used. Pulmonary homografts (mean diameter, 26 ± 2 mm) were obtained from five different homograft valve banks and were handled according to the standard thawing protocol of the particular homograft bank. Implantation of the pulmonary homograft into the right ventricular outflow tract was performed with continuous running 4-0 Prolene suture lines (Ethicon, Norderstedt, Germany) proximally and 5-0 Prolene distally. Perioperatively, 15 patients (33%) had different types of allogenic blood transfused.

Screening for anti-HLA antibodies
The blood sample was obtained at the time of echocardiography or within 2 months before or after. Whole blood was centrifuged immediately, and serum samples stored at -80°C until analysis. Lymphocytotoxic antibodies in serum specimen were detected using National Institutes of Health standard and antihuman globulin protocols [10]. Lymphocytes from 50 HLA-typed blood donors were used to determine panel reactive antibodies (PRA). Results are expressed as percentage PRA, that is, the number of positive donors divided by the number of donors tested times 100. A PRA greater than or equal to 6% was considered positive.

Determination of antibody specificity
All patients were serologically typed for HLA-A and HLA–B (Lymphotype HLA-ABC; Biotest, Dreireich, Germany). For determination of antibody specificity, a group of 13 patients had HLA class I typing of the valve donor, which was performed from a small specimen of the homograft myocardium that had been harvested intraoperatively and stored at -80°C. In brief, the DNA was first isolated enzymatically. HLA class I typing was performed using a polymerase chain reaction protocol with sequence-specific primers (Micro SSP Generic HLA Class I DNA Typing Tray; One Lambda Inc, Canoga Park, CA).

Echocardiographic data acquisition and measurements
Resting transthoracic echocardiograms were made with 2.5-MHz ultrasound transducers (Hewlett-Packard Sonos 2500 system; Andover, MA) in standard longitudinal and cross-sectional views during routine follow-up investigation and recorded on VHS videotape. A modified echocardiogram lead I was continuously recorded. The end-systolic right ventricular diameter was measured from the standard parasternal long axis view. Maximum velocities across the pulmonary homograft valve were calculated by continuous wave Doppler imaging transducer. For determination of the pressure gradient the modified Bernoulli equation ( , where p is the pressure gradient and v is the velocity across the valve) was used. To assess pulmonary homograft regurgitation, pulsed wave, continuous wave, and color flow Doppler were performed. Semiquantitative assessment from grade 0 to 3 of pulmonary homograft regurgitation was based on the length and width of the regurgitant jet and the distance it reaches into the right ventricular outflow tract on the parasternal short-axis view.

Statistical analysis
Data are presented as absolute numbers and percentages, or mean ± standard deviation. Relative frequencies were compared using Fisher’s exact test or the Kruskal-Wallis test, and means were compared using the unpaired t test. Correlation between continuous data were assessed using Pearson’s bivariate or Spearman’s rank correlation. When analyzing the pressure gradients, a repeat analysis was also performed in which the transhomograft pressure gradients were first divided by the diameter of the homograft and by the body surface area ( ) to correct for these variables. Multiple regression analysis was performed if univariate analysis indicated a significant effect of the antibody status. All analyses were performed using Minitab, release 12 (Minitab Inc, State College, PA).

	Results

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
All patients presented in good health. Twenty-seven patients (57%) were found to be anti-HLA antibody positive. The median PRA of these patients was 47% (range, 8% to 96%). The specificity for HLA-A and HLA-B antibodies was tested in 13 patients. All but one was found to be donor specific. The 1 patient in whom antibody specificity could not be determined had a PRA of 96% precluding the determination of specificity. There were no significant differences according to sex, height, weight, age at operation, length of follow-up, diameter of the homograft, allogenic blood transfusions (Table 1), or bank of origin of the homograft (p = 0.214). There was no (rank) correlation between PRA and length of follow-up (r = 0.13, p = 0.399). No evidence of a significant influence of follow-up (p = 0.26) on the frequency of a positive antibody determination was found.

View larger version (12K): [in this window] [in a new window]   Fig 1. Scatterplot of the maximal transpulmonary homograft pressure gradient at rest against the length of follow-up. (+ = anti-HLA antibody positive; = anti-HLA antibody-negative patients.)

View larger version (11K): [in this window] [in a new window]   Fig 2. Scatterplot of the mean transpulmonary homograft pressure gradient at rest against the length of follow-up. (+ = anti-HLA antibody positive; = anti-HLA antibody-negative patients.)

Right ventricular diameter
The end-systolic right ventricular diameter was found to be significantly larger (30.7 ± 3.5 mm) in patients with anti-HLA antibodies as compared with those who did not produce antibodies (26.3 ± 4.2 mm; p = 0.001). No correlation could be elucidated between the transhomograft pressures and right ventricular diameter (r = 0.15, p = 0.346, and r = 0.10, p = 0.534 for maximal and mean transpulmonary pressure, respectively). Multiple regression analysis including the presence or absence of anti-HLA antibodies, sex, transfusions, age, follow-up, BSA, and homograft diameter, revealed that the presence of anti-HLA antibodies (coefficient = 3.958, p < 0.001), age at operation (coefficient = 0.128, p = 0.001), and BSA (coefficient = 7.315, p = 0.020) were significantly associated with higher right ventricular diameters.

	Comment

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Our study provides evidence that the implantation of a cryopreserved homograft during the Ross procedure induces a specific humoral immune response. However, we could not detect significant evidence of an association between this immunologic reaction and homograft valve function at rest in adults during the first postoperative years.

Whether an implanted homograft valve suffers immunologic-mediated damage has been controversially debated for decades. In recent years, modern technologies have allowed a donor-specific immunologic response to be detected in vitro and in vivo, but its clinical significance is still unknown. One study on recipients of homovital aortic homograft valves found no significant differences regarding long-term valve function according to anti-HLA class I or II antibody status or the degree of HLA class I or II mismatch, although there tended to be a slight, nonsignificant increase in the frequency of mild aortic homograft stenosis and in the rate of homograft degeneration in antibody positive patients [8]. As compared with our study, the prevalence of anti-HLA antibodies was higher (82% versus 57%). This difference might result from the longer follow-up (6.4 ± 0.6 versus 2.1 ± 1.2 years in our study) or from the fact that we used cryopreserved homograft valves that have been shown to possess reduced antigenicity [11].

Antibodies against both HLA class I and II after cardiac transplantation are associated with decreased survival [12]. In cardiac transplantation, HLA-DR matching has a significant impact on graft survival [13], and the antigenicity of homograft valves can be reduced in vitro by HLA-DR matching [11]. Dignan and coworkers [9] recently reported that recipients of cryopreserved aortic homografts had a significantly lower rate of freedom from structural deterioration if they had two mismatches at the HLA-DR locus. There was no such association for HLA class I mismatches, and the prevalence of anti-HLA antibodies was not reported. Multivariate analysis revealed age of operation less than 25 years and a time from procurement until cryopreservation of less than 4 hours, but not HLA-DR mismatch as independent predictors for structural deterioration [9]. The antigenicity of homograft valves seems to be mainly mediated by endothelial cells that rapidly lose HLA class II expression during storage and antibiotic treatment of valve tissue [14], and no professional antigen-presenting dentritic cells have been identified so far in homograft valves. Therefore, the relevance of HLA class II antigens in any presumed immune-mediated damage of the valve remains uncertain.

In children, indirect evidence from clinical studies points toward an immune-mediated damage of homograft valves [15]. In addition, Rajani and coworkers [7] reported that explanted homografts of infants showed thickened leaflets, and that the leaflets and aortic sleeves contained a hyperplastic intimal layer with numerous spindle cells (similar to those seen in coronary arteries in transplant vasculopathy) and multiple foci of inflammation. In contrast, Mitchell and colleagues [6] found no evidence for immune-mediated valve dysfunction in children and adults. They reported that cryopreserved allografts show early cellular autolysis and exhibit severe degeneration after implantation with disruption of normal architecture and loss of endothelial and interstitial cells, and that inflammatory cell infiltrates are absent or trivial. Noteworthy, Mitchell and associates [6] were able to control for "background" lymphocytic infiltrates because they studied thawed but unimplanted cryopreserved homograft valves, too. However, no explants could be examined (except cases of infective endocarditis) between the 10th postoperative day and 11th month, the period likely to show evidence of immune-mediated damage.

After heart transplantation, the aortic valve shows remarkable structural preservation, and a low tendency for calcification [6, 16]. Whether this difference is due to ABO matching, immunosuppression regimen, the length of the preharvest period of warm ischemia, cryopreservation, or a combination of these factors is unknown. Myocardial rejection is reported to be associated with subendothelial lymphocytic infiltrates and aortic valve edema indicating that valvular components can be targeted by a cellular immune response [16]. However, the fact that homograft valves are immunogenic and that immunosuppressive drugs may modulate the ability to generate an immunologic response does not prove that rejection of cardiac valves (ie, the immune-mediated destruction of valve components) occurs [6]. Our study is one of the first to link immunologic data and homograft valve function in humans, and although it cannot disprove the presence of rejection, it does not provide evidence that the humoral response against HLA class I antigens adversely affect valve function at rest during medium-term follow-up.

Antibiotic pretreatment and cryopreservation may affect the antigenicity of homograft valves [11, 14]. Because slightly different protocols for cryopreservation are used in different homograft banks, we tested whether the prevalence of a positive antibody determination varied according to the homograft bank, but we found no evidence for this to occur, nor did we find evidence that perioperative blood transfusions affected its prevalence.

The pulmonary autograft procedure offers optimal hemodynamics [17], compares at least favorably with aortic homograft implantation [18], and is associated with good long-term results (84% actuarial rate of freedom from reoperation at 20 years for the autograft [2]). The rate of reoperation for pulmonary homograft failure (15% at 20 years [2]) or overt right heart failure appears also to be low. However, most [19, 20], but not all [18], echocardiographic studies report a significant increase of the transpulmonary pressure gradients during follow-up. Given the cross-sectional design of our study, we could not assess temporal changes of the pressure gradient. An unexpected and unexplained finding of our study is the increased diameter of the right ventricle in antibody positive patients seemingly unrelated to the transhomograft pressure gradient. Although without apparent clinical sequela in our patients, this finding clearly warrants further investigation. Serial examinations, measurement of homograft function during exercise, HLA class II typing, longer follow-up, and a larger sample size will be crucial to address the relevance of immunology after transplantation of a homograft valve and to further improve the outcome of the Ross procedure.

	Acknowledgments

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We are indebted to Dr Derek Robinson, Center for Statistics and Stochastic Processes, School of Mathematical Sciences, University of Sussex, Brighton, UK, for expert statistical analysis.

	Footnotes

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This article has been selected for the open discussion forum on the STS Web site: http://www.sts.org/section/atsdiscussion/

	References

Top Footnotes Abstract Introduction Material and methods Results Comment Acknowledgments References

 

1. Jones T.K., Lupinetti F.M. Comparison of Ross procedures and aortic valve allografts in children. Ann Thorac Surg 1998;66:S170-S173.
2. Oury J.H., Hiro S.P., Maxwell J.M., Lamberti J.J., Duran C.M.G. The Ross Procedure: current Registry results. Ann Thorac Surg 1998;66:S162-S165.
3. Salomon R.N., Friedman G.B., Callow A.D., Libby P. Cryopreserved aortic homografts contain viable smooth muscle cells capable of expressing transplantation antigens. J Thorac Cardiovasc Surg 1993;106:1173-1180.[Abstract]
4. Hoekstra F., Knoop C., Vaessen L., et al. Donor-specific cellular immune response against human cardiac valve allografts. J Thorac Cardiovasc Surg 1996;112:281-286.[Abstract/Free Full Text]
5. Hogan P., Duplock L., Green M., et al. Human aortic valve allografts elicit a donor-specific immune response. J Thorac Cardiovasc Surg 1996;112:1260-1267.[Abstract/Free Full Text]
6. Mitchell R.N., Jonas R.A., Schoen F.J. Pathology of explanted cryopreserved allograft heart valves: comparison with aortic valves from orthotopic heart transplants. J Thorac Cardiovasc Surg 1998;115:118-127.[Abstract/Free Full Text]
7. Rajani B., Mee R.B., Ratliff N.B. Evidence for rejection of homograft cardiac valves in infants. J Thorac Cardiovasc Surg 1998;115:111-117.[Abstract/Free Full Text]
8. Smith J.D., Hornick P.I., Rasmi N., Rose M.L., Yacoub M.H. Effect of HLA mismatching and antibody status on "homovital" aortic valve homograft performance. Ann Thorac Surg 1998;66:S212-S215.
9. Dignan R., O’Brien M., Hogan P., et al. Influence of HLA matching and associated factors on aortic valve homograft function. J Heart Valve Dis 2000;9:504-511.[Medline]
10. Mittal K.D., Mickey M.R., Singal D.P., Terasaki P.I. Serotyping for homotransplantation by the microdroplet lymphocyte cytotoxicity test. Transplantation 1968;6:913-916.[Medline]
11. Hoekstra F., Knoop C., Jutte N., et al. Effect of cryopreservation and HLA-DR matching on the cellular immunogenicity of human cardiac valve allografts. J Heart Lung Transplant 1993;13:1095-1098.
12. Suciu-Foca N., Reed E., Marboe C., et al. The role of anti-HLA antibodies in heart transplantation. Transplantation 1991;51:716-724.[Medline]
13. Smith J.D., Rose M.L., Pomerance A., Burke M., Yacoub M.H. Reduction of cellular rejection and increase in longer-term survival after heart transplantation after HLA-DR matching. Lancet 1995;346:1318-1322.[Medline]
14. Johnson D.L., Sloan C., O’Halloran, Yacoub M.H. Effect of antibiotic pretreatment on immunogenicity of human heart valves and component cells. Ann Thorac Surg 1998;66:S221-S224.
15. Baskett R.J., Ross D.B., Nanton M.A., Murphy D.A. Factors in the early failure of cryopreserved homograft pulmonary valves in children: preserved immunogenicity?. J Thorac Cardiovasc Surg 1996;112:1170-1179.[Abstract/Free Full Text]
16. Valente M., Faggian G., Billingham M.E., et al. The aortic valve after heart transplantation. Ann Thorac Surg 1995;60:S135-S140.
17. Schmidtke C., Bechtel J.F.M., Hueppe M., Noetzold A., Sievers H.H. Size and distensibility of the aortic root and aortic valve function after different techniques of the Ross procedure. J Thorac Cardiovasc Surg 2000;119:990-997.[Abstract/Free Full Text]
18. Aklog L., Carr-White G.S., Birks E., Yacoub M.H. Pulmonary autograft versus aortic homograft for aortic valve replacement: interim results from a prospective randomized trial. J Heart Valve Dis 2000;9:176-189.[Medline]
19. Ward K.E., Elkins R.C., Overholt E.D., et al. Evaluation of cryopreserved homografts in the right ventricular outflow tract after the Ross procedure: Intermediate-term follow-up. J Heart Valve Dis 1997;6:130-133.[Medline]
20. Savoye C., Auffray J.L., Hubert E., et al. Echocardiographic follow-up after Ross procedure in 100 patients. Am J Cardiol 2000;85:854-857.[Medline]

This article has been cited by other articles:

				K. J. Zehr, M. Yagubyan, H. M. Connolly, S. M. Nelson, and H. V. Schaff Aortic root replacement with a novel decellularized cryopreserved aortic homograft: Postoperative immunoreactivity and early results J. Thorac. Cardiovasc. Surg., October 1, 2005; 130(4): 1010 - 1015. [Abstract] [Full Text] [PDF]

				J.F. M. Bechtel, J. Gellissen, A. W. Erasmi, M. Petersen, A. Hiob, U. Stierle, and H.-H. Sievers Mid-term findings on echocardiography and computed tomography after RVOT-reconstruction: comparison of decellularized (SynerGraft) and conventional allografts Eur. J. Cardiothorac. Surg., March 1, 2005; 27(3): 410 - 415. [Abstract] [Full Text] [PDF]

				G. Pompilio, G. Polvani, G. Piccolo, A. Guarino, A. Nocco, A. Innocente, M. Porqueddu, L. Dainese, F. Veglia, A. Sala, et al. Six-year monitoring of the donor-specific immune response to cryopreserved aortic allograft valves: Implications with valve dysfunction Ann. Thorac. Surg., August 1, 2004; 78(2): 557 - 563. [Abstract] [Full Text] [PDF]

				W. M. Novick, D. Anic, A. Lora Solf, M. Arboleda Torres, I. Nino De Guzman Leon, R. W. Reid, and T. G. Di Sessa Medtronic freestyle valve for right ventricular reconstruction in pediatric ross operations Ann. Thorac. Surg., May 1, 2004; 77(5): 1711 - 1716. [Abstract] [Full Text] [PDF]

				D. A. Fullerton, J. W. Fredericksen, R. S. Sundaresan, and K. A. Horvath The Ross procedure in adults: intermediate-term results Ann. Thorac. Surg., August 1, 2003; 76(2): 471 - 477. [Abstract] [Full Text] [PDF]

				J.J.M. Takkenberg, K.M.E. Dossche, M.G. Hazekamp, A. Nijveld, E.W.L. Jansen, T.W. Waterbolk, and A.J.J.C. Bogers Report of the Dutch experience with the Ross procedure in 343 patients Eur. J. Cardiothorac. Surg., July 1, 2002; 22(1): 70 - 77. [Abstract] [Full Text] [PDF]

				J.F. M. Bechtel, C. Bartels, C. Schmidtke, W. Skibba, M. Muller-Steinhardt, H. Kluter, and H.-H. Sievers Does Histocompatibility Affect Homograft Valve Function After the Ross Procedure? Circulation, September 18, 2001; 104(90001): I-25 - 28. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Online Discussion Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Claus Bartels Claudia Schmidtke Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bechtel, J.F. M. Articles by Sievers, H.-H. Search for Related Content PubMed PubMed Citation Articles by Bechtel, J.F. M. Articles by Sievers, H.-H. Related Collections Molecular biology Valve disease