Decellularized Cryopreserved Allografts as Off-the-Shelf Allogeneic Alternative for Heart Valve Replacement: In Vitro Assessment Before Clinical Translation.

Cryopreserved allogeneic conduits are the elective biocompatible choice among currently available substitutes for surgical replacement in end-stage valvulopathy. However, degeneration occurs in 15 years in adults or faster in children, due to recipient's immunological reactions to donor's antigens. Here, human aortic valves were decellularized by TRICOL, based on Triton X-100 and sodium cholate, and submitted to standard cryopreservation (TRICOL-human aortic valves (hAVs)). Tissue samples were analyzed to study the effects of the combined procedure on original valve architecture and donor's cell removal. Residual amounts of nucleic acids, pathological microorganisms, and detergents were also investigated. TRICOL-hAVs proved to be efficaciously decellularized with removal of donor's cell components and preservation of valve scaffolding. Trivial traces of detergents, no cytotoxicity, and abrogated bioburden were documented. TRICOL-hAVs may represent off-the-shelf alternatives for both aortic and pulmonary valve replacements in pediatric and grown-up with congenital heart disease patients.

[Which indications and access routes for transcatheter aortic valve implantation? Health technology assessment from HAS]. / Quelles indications et quelles voies d'abord pour l'implantation des valves aortiques percutanées ? Évaluation technologique menée par la Haute Autorité de santé (HAS).

The transcatheter aortic valve implantation should be restricted to patients with severe symptomatic aortic stenosis with a contraindication for a surgical replacement (taking into account surgical risk scores, comorbidities, anatomical conditions, life expectancy and frailty). Patient eligibility should be performed by a heart team with the involvement of a cardiac surgeon, an interventional cardiologist, a clinical cardiologist and an anaesthetist/resuscitation specialist. The advice of a geriatrician is strongly recommended. The long-term efficacy remains unknown. The French National Authority for Health (Haute Autorité de santé [HAS]) reminds that contraindications in the CE mark should be strictly respected (i.e.instructions for use). Extension of current indications should be conditional to the presentation of clinical evidence. Thus, implantation in patients at lower surgical risk or the use of direct transaortic route are not eligible for reimbursement given the current state of knowledge.

Development of a growing rat model for the in vivo assessment of engineered aortic conduits.

BACKGROUND: Numerous limitations of aortic valve grafts currently used in pediatric patients cause the need for alternative prostheses. For the purpose of in vivo evaluation of novel engineered aortic conduit grafts, we aimed at downsizing a previously described model to create a growing rodent model. MATERIALS AND METHODS: U-shaped aortic conduits were sutured to the infrarenal aorta of young Wistar rats (70-80 g, n = 10) in an end-to-side manner. Functional assessment was performed by Doppler sonography and high resolution rodent MRI. Histology and immunohistochemistry followed after 8 wk. RESULTS: Postoperative recovery rate was 80%. Conforming to clinical observations, postoperative MRI (d 5) and Doppler sonography (wk 8) revealed unimpaired conduit perfusion. Explanted implants were luminally completely covered by an endothelial cell layer with local hyperplasia and accumulation of α-smooth muscle actin (+) cells. Moreover microcalcification of the decellularized scaffolds was observed. CONCLUSIONS: Our downsized model of aortic conduit transplantation enables overall characterization with detailed analysis of maturation of engineered aortic grafts in a growing organism.

NMR assessment of Me(2)SO in decellularized cryopreserved aortic valve conduits.

INTRODUCTION: Decellularized cryopreserved allograft vascular tissue may provide a nonimmunogenic scaffold that is suitable for repopulation by cells from a variety of sources, conferring the potential for growth and repair. Although dimethyl sulfoxide (Me(2)SO) is generally regarded as a safe cryoprotectant, even low levels may alter function of repopulating cells. We investigated the residual concentration of Me(2)SO in the aqueous compartment of cryopreserved ovine aortic valve conduits following decellularization. MATERIALS AND METHODS: Aortic valve conduits from Suffolk sheep were cryopreserved in 1.1 M (7.5% vol/vol) Me(2)SO according to the protocol of our local tissue bank. Three aortic valve conduits were decellularized in a series of hypotonic and hypertonic Tris buffers. Tissue samples were taken at regular time intervals throughout the decellularization process and equilibrated in double distilled, deionized H(2)O for 28 days. Quantitative proton nuclear magnetic resonance spectroscopy was used to determine the residual Me(2)SO concentration in the equilibration solutions from which Me(2)SO tissue concentrations were calculated. RESULTS: After thawing, the mean Me(2)SO concentration in the valve conduit was 0.302 +/- 0.081 M. The decellularization process resulted in a stepwise reduction in the Me(2)SO concentration to less than 8.56 x 10(-5) +/- 9 x 10(-5) M (P = 0.02). The diffusion coefficient was 2.5 x 10(-6) cm(2)/s. CONCLUSIONS: Our study demonstrates that Me(2)SO is effectively washed out of the aortic valve conduit during decellularization, resulting in a final concentration that is several orders of magnitude less than Me(2)SO concentrations reported to alter cell function.

Echocardiographic assessment and preliminary clinical results after aortic valve replacement with the Medtronic Mosaic bioprosthesis.

BACKGROUND AND AIM OF THE STUDY: The study aim was to examine prospectively the clinical performance and durability of the Medtronic Mosaic bioprosthesis, a stented porcine aortic valve that combines improvements in tissue preservation, notably net zero differential pressure fixation of the leaflets, with antimineralization treatment using 2-amino-oleic acid (AOA). METHODS: A total of 158 Mosaic valves was implanted; 152 in patients aged over 70 years, and six in patients aged <70 years with contraindications to anticoagulant therapy. Mean age was 73.7 years. All valves were implanted in the supraannular position. Thirty-two patients (20%) required concomitant procedures, including coronary revascularization, ascending aorta replacement and/or mitral annuloplasty. Postoperative anticoagulation (heparin) was prescribed for ten days, followed by antiplatelet therapy. No long-term oral anticoagulants were prescribed, except in some patients with atrial fibrillation. The follow up included routine clinical and blood work-up, and echocardiography at six months and one year after surgery. RESULTS: There were seven early (0-30 days) and five late deaths (>30 days). One death was caused by a hemorrhagic stroke at three months in a patient without anticoagulant or antiplatelet therapy. No thromboembolic complications or structural valve deterioration were observed during follow up. At two years, freedom from endocarditis and reoperation was each 99.6%. NYHA class was excellent, with 98% of patients in class I or II at one year. Patient survival was 92% at two years. Hemodynamically, the valve was performing well, with mean systolic gradients of 13.6, 13.2, 12.6 and 9.6 mmHg for the 21, 23, 25 and 27 mm valves, respectively. There was no evidence of structural valve deterioration. CONCLUSION: Long-term evaluations are mandatory to confirm the durability of any new bioprosthetic valve. Satisfactory early clinical and hemodynamic results with the new Mosaic bioprosthesis warrant its continued implantation in the aortic position for patients over the age of 70 years.

Banking of cryopreserved heart valves in Europe: assessment of a 10-year operation in the European Homograft Bank (EHB).

BACKGROUND AND AIM OF THE STUDY: The preparation, banking and distribution of cryopreserved heart valves has been carried out at the European Homograft Bank (EHB) in Brussels without interruption since January 1989. We present an assessment of the Bank's activities during this 10-year period. METHODS: Heart valve donors aged <62 years form three categories: multiorgan donors with non-transplantable hearts; recipients of cardiac transplantation; and non-beating heart cadavers with a warm ischemia time of less than 6 h. Past history and biology are checked for transmissible diseases. Dissection, incubation in antibiotics and cryopreservation in 10% dimethylsulfoxide with storage in liquid nitrogen vapors (about -150 degrees C), and quality control are according to the standards of the Belgian Ministry of Health. Cryopreserved valves are shipped to the implantation centers in a dry shipper at about -150 degrees C. RESULTS: Between January 30th 1989 and December 31st 1998, 1,817 non-transplantable hearts and 12 excised semilunar valves were obtained. In total, 2,077 valves (1,032 pulmonary, 931 aortic and 13 mitral) were decontaminated, cryopreserved and stored in liquid nitrogen vapor (six more valves were refrigerated). In total, 1,515 valves were discarded at different stages of the protocol, the main causes of rejection being significant macroscopic lesions (68.2% aortic and 26.67% pulmonary). Inadequate excision at procurement (10.37% pulmonary), persistent contamination after antibiotics (5.6%) and positive serology for hepatitis B and C and Q fever (5.4%) were other frequent causes for rejection. Among the 2,117 accepted valves, 1,398 were graded first and 719 second choice, mainly on the basis of morphology. In total, 2,090 cryopreserved valves and one refrigerated valve were implanted in 39 institutions between May 1989 and December 1998. Of requests, 10.02% could not be satisfied. In total, 967 pulmonary valves were implanted in the right ventricular outflow tract (RVOT); 424 during a Ross procedure, and 76 in the left ventricular outflow tract (LVOT). Of the aortic valves, 732 were implanted in the LVOT and 266 in the RVOT. Mitral homografts were used for tricuspid valve replacement in two cases, and in the mitral position in seven. Complications at distribution and thawing included 10 bag ruptures and 16 transversal conduit wall fractures. Of the valves shipped, 317 (13.16%) were not used and were returned safely in the dry shipper. Comparison of distribution rates in the first 5.5 and last 4.5 years of EHB activity shows: (i) a significant increase in pulmonary valve implantations in the RVOT (from 71.95% to 81.95%); and (ii) a marked increase (265%) in pulmonary homograft implantations as part of a Ross operation, and a significant decrease (28%) in aortic homograft implantation in the LVOT. CONCLUSION: While macroscopic lesions of procured aortic valves remain the most frequent and unavoidable cause of homograft rejection during quality control, the high percentage of inadequate surgical heart valve excision should be corrected. The rates of bacterial contamination and positive serology seem acceptable. Storage and shipping of cryopreserved homografts in liquid nitrogen vapor permits them to be spared very efficiently. The increasing use of pulmonary valves for RVOT reconstruction either in congenital heart disease or as part of the Ross procedure compensates for the limited availability of good quality aortic valves.

Histologic and genetic assessment of explanted allograft valves.

A possible way of analyzing the immune response triggered by the allograft and the cellular viability is to compare immunocompetent and immunosuppressed patients, such as those having valve replacement and heart transplantation, respectively. These groups differ in immunosuppression therapy, preparation methods, valve hemodynamics. In the present study, we investigated polymerase chain reaction-amplified DNA flanking hypervariable (CA)n regions obtained from valve leaflets taken from patients having valve replacement or heart transplantation and performed a histologic analyses of the cells. In addition, we assessed an autograft valve to compare the hemodynamic effects on the cellular composition of the valve leaflet. We conclude that leaflet cellularity of the heart transplantation and autograft patients is superior to that of the valve replacement patients. These differences were consistent with the occurrence of an immune response in the valve replacement group, which was prevented or abrogated by immunosuppressive therapy administered to the heart transplantation group. However, it cannot be excluded that preparation procedures have a long-term effect on the extracellular matrix, leading to deterioration of cell adhesion and homing conditions.

Perioperative assessment of aortic homograft, Toronto stentless valve, and stented valve in the aortic position.

We investigated aortic valve hemodynamic performance and perioperative left ventricular function in 50 patients (mean [+/- SD] age, 64 +/- 9 years; 34 men, 16 women) undergoing elective aortic valve replacement, using an aortic homograft (n = 20), a Toronto stentless porcine valve (n = 20), or a stented bioprosthesis (n = 10), by transesophageal echocardiography combined with high-fidelity cavity pressure recordings and thermodilution cardiac output measurements. Thirty-nine patients had aortic stenosis; 11 had predominant regurgitation. Thirteen patients with concomitant coronary artery stenosis underwent grafting. Left ventricular mass index in all patients was 280 +/- 110 g/m2. The transvalvular pressure drop and energy consumption were significantly higher with stented than stentless valves (5 with aortic homograft and 11 with Toronto valve, with matched age and valve size; 20 +/- 12 versus 3 +/- 9 mm Hg; 21% +/- 13% versus 8% +/- 8%, both p < 0.01). However, there was no difference in these variables between the Toronto valve and the aortic homograft (3 +/- 12 versus 2 +/- 10 mm Hg; 5% +/- 14% versus 2% +/- 12%, both p > 0.05), although the Toronto valves (normalized to body surface area) were larger than the aortic homografts (14.4 +/- 1.9 versus 12.6 +/- 1.8 mm/m2, p < 0.01). There was no significant difference in left ventricular stroke volume index or stroke work index in the systemic circulation, either between stentless and stented valves or between aortic homografts and Toronto valves, although the cross-clamp time required to insert a stentless valve was 20 minutes longer than that for a stented valve.(ABSTRACT TRUNCATED AT 250 WORDS)

[Non-invasive and invasive assessment of the function of aortic valve homografts in infancy and childhood]. / Homograft aorta billentyü conduit funkciójának noninvazív és invazív vizsgálata csecsemö- és gyermekkorban.

Study population involved 21 pts with complex congenital heart disease after corrective surgery using homografts between the years 1986 and 1992. Diagnoses included double outlet right ventricle, tetralogy of Fallot, transposition of great arteries, truncus arteriosus, pulmonary atresia with VSD, corrected transposition with pulmonary stenosis, and absent pulmonary valve. Pts age at surgery ranged from 18 days--to 15 yrs mean, 6.7 yrs. Time interval between surgery and diagnostic procedures was 8 days--6 yrs (mean 11 months). All pts were studied by Echo/Doppler. 2 pts by TEE and 3 pts by MRI. 6 pts had cardiac catheterisation. Distal (5) or proximal (1) stenosis was present in 6 cases. The severity and the type of stenosis was correctly identified by noninvasive technique compared to invasive findings. Homograft valve regurgitation was mild (4) or moderate (3). In two pts severe insufficiency was associated to homograft endocarditis. Postoperative residual shunts were found in 8 pts. Homograft stenosis can correctly be diagnosed using Echo/Doppler technique. Distal stenosis was more frequent than proximal. Homograft tends to become insufficient, but severe incompetence did not occur except in endocarditis.

The allograft valve in heart transplantation and valve replacement. Genetic assessment of the origin of the cells by means of deoxyribonucleic acid profiles.

Assessment of the cellular origin of allograft valves is essential in comprehending their biologic behavior and in improving preparation methods. In this study we retrospectively analyzed 10 allografts obtained from patients who underwent valve replacement or heart transplantation. Histologic evaluation and deoxyribonucleic acid amplification by polymerase chain reaction technology with fluorescence labeled primers was performed on different parts of the valve leaflets. Automated analyses of the obtained amplimers showed in the heart transplantation group the presence of receptor cells interspersed with native donor cells in three cases. Preliminary results for the valve replacement group are inconclusive as yet.

Comparative assessment of bioprosthesis durability in the aortic position.

Published durability results of porcine and pericardial bioprostheses in the aortic position have been reviewed for the purpose of a qualitative comparison between these valve types, and also with previously reviewed series of homograft valves. Porcine bioprostheses exhibit a wide range of failure curves, with a characteristic Weibull-shaped pattern. Patient characteristics must account for the majority of differences seen among the series. Newer series generally contain older patients and have better results. A second generation pericardial valve has distinctly better durability than a previously discontinued pericardial model, and appears to be as durable as that of the best porcine valve series. Homograft valves using cold antibiotic storage have durability results approximately equal to porcine valves, but with younger patients on the average. The results with cryopreserved valves may be comparable, but insufficient long term information is available. To make these qualitative comparisons quantitative would require a formal analysis which takes age and other patient-related characteristics into account.

[Quantitative assessment of fibroblast viability in cryopreserved aortic valve allografts].

The purpose of this study is to clarify whether serum (fetal bovine serum or human serum) is necessary or not for cryopreservation of aortic valve allografts. The protective effects of fetal bovine serum compared with human serum were evaluated by means of quantitative assessment of fibroblast viability. Porcine aortic valves were excised and rinsed immediately after death, followed by treatment with low concentration antibiotics. Valves were gradually frozen at a control-rate of -1 degree C/min and then stored in liquid nitrogen vapor-phase. The samples were classified into three groups by nutrient medium as follows. Group A (n = 5); Dulbecco's Modified Eagle Medium (DMEM), Group B (n = 5); DMEM containing 10% fetal bovine serum, Group C (n = 5); DMEM containing 20% human serum. At 1 week, 1 month, and 3 months after initiation of storage, the valves were thawed rapidly and examined for fibroblast viability, which was assessed quantitatively by means of autoradiography with tritiated proline. After 1 week storage, the fibroblast viability rate reduced from 98% to 80.7% in group A, 79.4% in group B and 79.2% in group C respectively, but there were no significant differences among three groups. The viability rate decreased with the passage of time, and remained around 72% in all groups after 3 months storage. The histological examination did not show any changes in the structure of the valves in all samples up to 3 months of storage. The results demonstrate that the fibroblasts are still viable after 3 months storage by cryopreservation. The addition of fetal bovine serum or human serum does not improve fibroblast viability.(ABSTRACT TRUNCATED AT 250 WORDS)

Donor heart valves: electron microscopic and morphometric assessment of cellular injury induced by warm ischemia.

Cryopreserved allograft valves are increasingly being used as valvular replacements. Leaflet fibroblast viability has been suggested to influence clinical durability. The warm ischemic time is thought to be a critical determinant of this cell viability. The purpose of this study was to apply quantitative morphometric methods to characterize, by transmission electron microscopy, valvular cellular injury resulting from progressive warm ischemic time. Porcine aortic valves were harvested with a spectrum of warm ischemic times (40 minutes and 2, 6, 12, 24, and 36 hours; five valves per warm ischemic time; n = 30) and processed by standard electron microscopic methods. To ensure randomized tissue selection within each warm ischemic time interval, we randomly selected one thin section from each leaflet. The first ten cells in each thin section were photographed and cellular injury was assessed (cell disruption, dilation of endoplasmic reticulum, cytoplasmic edema, nuclear and mitochondrial changes). Nine hundred micrographs have been analyzed by Cochran-Mantel-Haenszel statistics to determine if a significant association between warm ischemic time and cellular injury exists. Our findings indicate a significant association between reversible cell injury through 24 hours of warm ischemic injury (p less than 0.0001). Furthermore, a significant association between irreversible cell injury and progressive warm ischemia through 36 hours was also found. These findings indicate that the ischemic interval after donor death is associated with progressive leaflet cell injury. Cellular damage begins shortly after donor death and continues incrementally throughout 36 hours. After 2 hours of warm ischemic injury 37% of the cells had morphologic evidence of injury. After 6 hours of warm ischemic injury the number of injured cells increased to 73%. By 36 hours 22% of the cells appeared normal. Irreversible cell injury increases with prolonged ischemia and becomes quantitatively impressive at 24 hours, by which time 26% of cells are so affected. Conversely, some cells are resistant to irreversible injury for a prolonged ischemic interval.

Assessment of different preservation and storage conditions on aortic valves: introduction of a quantitative measuring method of the integrity of endothelial cells.

The fate of human allogeneic aortic valves depends mainly on their histological and immunological condition at the time of transplantation. A screening test making novel use of Alcian Blue was used to determine the integrity of endothelial cells as a prerequisite to their function. The dye uptake into the nucleus was measured quantitatively. The test was used to compare the effect of different storage mediums and temperatures (+4 degrees C, -30 degrees C, -80 degrees C, DMSO, FCS, RPMI, antibiotic solution) on aortic valves of rats. The cell integrity decreased with increasing storage time and higher storage temperature. The cryoprotective agent DMSO had no essential effect on the maintenance of cell integrity.

Doppler echocardiography in the assessment of the homograft aortic valve.

To determine the utility of Doppler echocardiography in the evaluation of the homograft valve in the aortic position, 27 patients with normally functioning valves (group 1) and 30 patients with suspected malfunctioning valves (group 2) were examined. Simultaneous cardiac catheterization and Doppler echocardiography were performed in 23 group 2 patients. Doppler and surgical findings were compared in 7 patients too ill for invasive studies. In group 1 patients, the maximal velocity (+/- standard deviation) was 1.8 +/- 0.37 m/s, the mean pressure gradient was 7.1 +/- 3.07 mm Hg and the mean aortic valve area was 2.2 +/- 0.79 cm2. The maximal velocity in group 2 patients with aortic regurgitation (AR) classified as moderate or greater was 2.5 +/- 0.55 m/s, compared with 1.8 +/- 0.44 m/s in patients with mild AR or less (p less than 0.01). In the quantitation of AR, pulsed-wave mapping and angiographic grades were identical in 18 patients and differed by 1 grade in 5. Seven patients too ill for catheterization had severe destruction of valve leaflets at cardiac surgery. In 6 patients, both Doppler grading methods suggested severe AR. In a seventh patient, who had an obstructed Starr-Edwards valve in the mitral position, AR was graded as mild by pulsed-wave mapping. Only 1 patient had homograft valve stenosis, with a withdrawal gradient at catheterization of 34 mm Hg and a Doppler maximal gradient of 36 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemically determined mineral content of explanted porcine aortic valve bioprostheses: correlation with radiographic assessment of calcification and clinical data.

Bioprosthetic valve calcification is usually assessed pathologically by gross inspection, radiographic studies, and histologic examination. Quantitation of mineral content by chemical assay has not been reported for failed clinical valves removed from adults. In this study, calcium determination by atomic absorption spectroscopy was done on 52 removed porcine valves after routine pathologic examination, including specimen radiography done by a standard technique. Specimens included 31 valves with calcific primary tissue failure, two calcified (but not overtly dysfunctional) valves removed simultaneously with failed valves, 14 nondeteriorated valves obtained at reoperation or autopsy after long-term implantation, and five valves removed within 1 month after insertion. Chemically determined mineral content varied widely among patients and duration of function. Valves with calcific failure had 113 +/- 68 micrograms/mg calcium overall (mean +/- SD) after 36 to 156 months (mean 87) of function. Almost all dysfunctional porcine valves with radiographically demonstrated calcific deposits had greater than 34 and 67 micrograms/mg calcium for mitral and aortic valves, respectively. Nondeteriorated valves (implanted 8 to 145 months, mean 57) had 5 +/- 6 micrograms/mg calcium. Failed aortic valves had more calcium than failed mitral valves and valves with calcific stenosis more than valves with regurgitation caused by calcification with tearing. Correlation of semiquantitative radiographic grading with chemically determined valve mineral was good, indicating that radiographic assessment of calcification may be used reliably for clinical comparisons between valves.

Histological assessment of orthotopic aortic valve leaflet allografts: its role in selecting graft pre-treatment.

Histopathological studies of human cardiac valve grafts recovered at autopsy or re-operation, together with long-term clinical follow-up of valve graft recipients, have indicated that the success of grafts is largely dependent upon the extent to which they are replaced by host fibrous connective tissue. To find the valve preparation technique with least inhibitory effect on tissue ingrowth after grafting, various sterilizing and storage procedures were evaluated using a series of aortic valve leaflet allografts in dogs. To facilitate evaluation, a method for rapidly assaying relative degrees of colonization of grafts was first devised. Application of this method has unequivocally identified a newly-formulated antibiotic solution as the pre-treatment most compatible with host tissue ingrowth.

Clinical and hemodynamic assessment of the Angell-Shiley porcine xenograft.

Valve replacement with an Angell-Shiley bioprosthesis was accomplished in 449 patients. To evaluate the bioprostheses from this total series, 344 patients who did not undergo associated operation, had no previous operations, or had no other valve substitutes were selected. Hospital mortality was 2.6% for aortic (4 out of 156), 7.2% for mitral (9 out of 125), and 12.7% for multiple-valve replacements (8 out of 63). The 323 patients discharged from the hospital were followed for 6 to 36 months. There were 15 late deaths. Hepatitis, bleeding, thromboembolism, endocarditis, and residual valvular incompetence, always periprosthetic, were the major complications. Forty-five patients with single-valve replacement (16 mitral and 29 aortic) without clinical valve dysfunction were electively recatheterized to assess hemodynamic performance. Measurements were recorded at rest and during exercise on a bicycle ergometer. Functional aortic valve orifice averaged 1.23 +/- 0.33 cm2 and the mean systolic gradient was 21.51 +/- 6.68 mm Hg at rest. During exercise, aortic gradient increased to 26.60 +/- 7.54 mm Hg and mean functional area to 1.51 +/- 0.34 cm2. In the mitral position, the mean diastolic gradient at rest was 8.44 +/- 3.17 mm Hg and the functional orifice area averaged 1.67 +/- 0.51 cm2. Exercise increased the mean gradient to 11.92 +/- 3.8 mm Hg and the mean orifice area to 2.05 +/- 0.57 cm2.

An 11-year assessment of 93 flash-frozen homograft valves in the aortic position.

Flash-frozen homograft aortic valves were used for isolated aortic valve replacement at the National Heart Hospital between 1968 and 1971. This study of 93 patients with an average follow-up of 8.5 years complements a previous report on 30 patients but shows a higher incidence of degeneration. There was an early mortality of 4.8% and a late mortality of 15.9% over 11 years. Only five of the 15 late deaths were directly attributable to valve malfunction. There were no episodes of thromboembolism during the 11 year follow-up and only three cases of infective endocarditis. Despite the late onset of valve degeneration there is a 77% probability of patient survival at 11 years. The use of frozen valves delayed the time of onset of degenerative changes when compared over an 11-year period with the earlier use of freeze-dried valves. The evaluation of replacement cardiac valves is thus seen to be time-dependent and needs a long follow-up and an adequate number of patients in the sample for a meaningful assessment. There was no significant difference in the long-term performance between the frozen valves sterilized in ethylene oxide or sterilized with gamma irradiation.