Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft? Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, we report an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at approximately 4 degrees C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85% glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchanged mechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 degrees C, or cryopreservation with 10% Me(2)SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85% glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time.

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.

Histopathologic comparison of a pulmonary autograft and pulmonary homograft in a patient 17 months after a Ross procedure: an autopsy study.

BACKGROUND AND AIM OF THE STUDY: The Ross operation has become very popular during the last decade. However little is known about the cellular behaviour of a normally functioning pulmonary autograft. METHODS: This case report deals with a 14-year-old female who died from a non-valve-related cause 17 months after a Ross-Konno operation using a cryop-reserved viable pulmonary homograft for the right outflow tract. Comparison is made between the homologous and autologous pulmonary valves by macroscopic description, histology and immunohistochemistry. RESULTS: The autograft kept its cellular population-except for the dendritic cells which have disappeared, and developed a jet lesion on the ventricular aspect of one cusp as a likely adaptation to a transvalvular gradient. The homograft was extensively devitalized, its cusps being partially covered with a fibrous sheath of recipient origin; few inflammatory cells, consisting of macrophages and rare T lymphocytes were present. CONCLUSIONS: The most puzzling observation, which needs confirmation, is the selective disappearance of the dendritic cells from the viable autograft. It is disappointing that a viable cryopreserved homograft valve has devitalized in the midterm. This phenomenon seems to result from a clinically silent immune reactions.

Identification of host and donor cells in porcine homograft heart valve explants by fluorescence in situ hybridization.

The pathogenesis of the primary tissue degeneration that limits the life-span of aortic and pulmonary homografts has still not been revealed. Histopathological studies on homograft explants have not given definitive insight into the eventual fate of donor cells, nor have they demonstrated the assumed importance of host cell ingrowth into the graft tissue. In this experimental study, fluorescence in situ hybridization (FISH) is introduced as a new approach to examine the distribution of host and donor cells in homograft explants. Aortic valve replacement was performed with a cryopreserved porcine aortic homograft in three pigs; donor and recipient were of opposite sex. After 4 months, the grafts were explanted and examined by FISH using a biotinylated porcine Y-chromosome-specific library probe. Following probe detection with FITC-conjugated avidin, a clear distinction could be made between cells of host and donor origin without distorting the histological integrity of the explants. There was ingrowth of donor cells into the graft aortic wall and into the valve leaflet, to some extent. In all explants, remaining donor cells were present, though decreased in number. The introduction of FISH in homograft heart valve research provides a powerful tool to study the fate of recipient and donor cellular elements in situ, and may therefore contribute to a better understanding of the histopathological processes that take place in transplanted homograft valves.

Morphologic Study of Homograft Valves before and after Cryopreservation and after Short-Term Implantation in Patients.

Cryopreserved heart valve homografts have been implanted in patients for the past 15 years, but controversies still exist on the survival of donor cells, matrix maintenance, and possible rejection by the host. Therefore a full morphologic study (histology, immunohistochemistry, transmission electron microscopy, and cuprolinic blue-TEM for glycosaminoglycans [GAG]) of short-term implanted uninfected grafts was done using unimplanted valves as the reference. Unimplanted tissues consisted of 5 fresh and 11 cryopreserved valves. Eight implants were recovered at reoperation [4] or autopsy [4], 4 from the right and 4 from the left ventricular outflow tract. The implantation time was 2 hours to 30 days. For unimplanted valves we found a partial preservation of the endothelium, the presence of dendritic Langerhans cells (Lc) and macrophages, and no significant damage to fibroblasts, collagen framework, and GAG pattern, except when the tissues had been ischemic for a long time. Explanted cusps exhibited (i) early disappearance of endothelium and Lc; (ii) nonspecific low-grade inflammatory cell infiltration, mostly of monocytoid type; (iii) viable degree of devitalization of fibroblasts with persistence of viable cells in some areas in most cusps; and (iv) fair preservation of collagen framework and GAGs. It is likely that, in view of the good graft preservation at implantation, humoral rejection is responsible for the earlier destruction of the endothelium and dendritic cells and the delayed devitalization of the fibroblasts and that preservation of the collagen framework and other intercellular matrix components (glycosaminoglycans) should guarantee longterm graft function.

In situ hybridization: a new technique to determine the origin of fibroblasts in cryopreserved aortic homograft valve explants.

Tissue degeneration reduces the durability of cryopreserved homografts. Earlier studies indicated that the presence of fibroblasts in homograft leaflets may contribute to increased valve longevity. These fibroblasts may be of recipient origin or represent surviving donor cells. We developed a method, based on in situ hybridization, to determine the origin of fibroblasts in homograft explants. In young pigs we performed aortic valve replacement with a cryopreserved porcine aortic homograft. A male homograft was implanted in a female pig, whereas two male recipients received a female homograft. After 3 to 4 months the homografts were explanted. Frozen sections were made and alternately examined with hematoxylin-eosin staining and in situ hybridization. With a biotinylated porcine Y chromosome-specific deoxyribonucleic acid probe, male fibroblasts could be clearly distinguished from female fibroblasts. In all leaflets we observed both donor and recipient fibroblasts. The distribution of these populations was marked in schematic drawings. Recipient fibroblasts mostly spread onto the leaflet surface but also penetrated the leaflet tissue. Remaining donor fibroblasts did not show morphologic signs of decreased viability on hematoxylin-eosin staining. In situ hybridization may become a useful technique in homograft research. In this porcine model, the fibroblasts in the aortic homograft explants were of both donor and recipient origin.

The value of the stentless biovalve prosthesis. An experimental study.

Clinical use of the stentless bioprosthesis has not yet been accepted world-wide. Experimental studies are scarce. In a study in growing pigs, 23 aortic valve replacements were performed (7 stented bioprostheses (STB), 10 stentless bioprostheses (SLB) and 6 cryopreserved homografts (CAH)). Valves were studied macroscopically and microscopically after explantation. Five animals died between 1.5 and 4 months after implantation. Two STBs showed extreme calcific degeneration. A third STB animal died suddenly: this STB showed only minimal leaflet calcification. One SLB was stenosed with a large fibrin deposit in one cusp, a second SLB showed slight cusp calcification and three were paracommissural tears in one cusp. In all other animals the valves were explanted after 5 to 6.5 months. All STBs showed severe calcific degeneration. Five SLBs showed mild calcific degeneration, while three others were unaltered after 6 months. All CAHs were free of calcific degeneration, three were perfect, two had a tear in a commisure and another was prolapsed with a fibrin degeneration. The speed and extent of valve degeneration were less than in the STB group. The stentless design is an important contribution which may result in a higher durability of bioprosthetic valves.

Amyloid proteins and amyloidoses: complexity updated.

Amyloid is a beta-pleated fibrillar protein principally constituted of light chains of immunoglobulins (kappa or lambda) in primary or myeloma-associated amyloidosis, of AA proteins in secondary amyloidosis and familial. Mediterranean fever, and of variants of prealbumin - now called transthyretin - in senile amyloidosis and in familial polyneuropathies. Other identified amyloidogenic proteins involve APUD protein derivatives (calcitonin), beta 2 microglobulin in chronic hemodialysis-related amyloidosis and beta protein in Alzheimer disease. After a short review of experimental findings and theories concerning the pathogenesis of amyloid deposition, the clinical aspects of amyloidosis are discussed stressing their great diversity. The diagnostic approach is also examined, with particular emphasis on rectal and kidney biopsy and subcutaneous adipose tissue aspirates. Finally, some comments on the treatment of amyloidosis (role of colchicine and DMSO) are made.

Porcine aortic versus bovine pericardial valves: a comparative study of unimplanted and from patient explanted bioprostheses.

This extensive morphological study (macroscopy, x-ray, histology, histochemistry and electron microscopy) compares two types of bioprosthetic valves, porcine aortic (PAV) and bovine pericardial (BPV) of various models, both unimplanted (five) and explanted (229). There were 197 PAV and 32 BPV explanted from the mitral, aortic and tricuspid positions, with a mean duration of implantation of 70.3 and 13.5 months, respectively. Within that material, a smaller, rather homogeneous, series of 11 Carpentier-Edwards PAV (CE) and 11 Ionescu-Shiley BPV (IS) explants (mean implantation period 53 and 49, mean patient-age 45 and 47 years) made the comparison of clinical and macroscopic features more valid. In the total series, the leading causes of failure were cuspal tear/perforation with calcification in the PAV group (64 per cent); non-calcified leaflet rupture (27 per cent) and infective endocarditis (27 per cent) in the BPV group. In the small series of CE PAV and IS PAV, the characteristic modes of failure were calcified juxta-commissural cusp rupture for CE and non-calcified leaflet rupture at the suture for IS. The most characteristic x-ray features were calcification of fibrous cords irradiating from the commissures and calcific nodules in the centre in PAV and large plaques extending from the commissures and leaflet base in all directions in BPV. The main microscopic features of leaflet degradation were: the soaked sponge phenomenon (loosening and plasma and fat insudation) and the nodular, protein-rich calcification, both centred in the spongiosa, in PAV explants; important macrophagic activity and destruction of collagenous structures at the outflow layer and along the suture of the leaflets, with preservation of the middle layer, and the intrinsic calcification of the deep collagenous bundles, in BPV explants. Those alterations and other features are discussed with reference to leaflet structure and design, haemodynamics and possible causal mechanisms.

Histotopographic evidence that amyloid deposits in sclerocalcific heart valves and other chronic lesions of the cardiovascular system are related to old thrombotic material.

Deposition of amyloid in human sclero-calcific heart valves has been reported recently as a localized age-independent and dystrophic form of amyloidosis. Histochemical studies have shown that the deposits are permanganate resistant, contain tryptophan and P component and are immunologically unrelated to any known type of amyloid fibril protein. In this study histological observations from a series of four selected sclerotic heart valves show amyloid deposition in old thrombotic material covering fusing commissures or appositional collagen on the body of the leaflets. Similar cases from extravalvular sites have been added to the series: a partly hyalinized thrombus of the left atrium, a thrombotic aneurysm of the left ventricle, 2 thrombotic atherosclerotic aneurysms of the aorta and popliteal artery respectively, and an encapsulated haematoma of the scalp. The deposits are Congo red positive with typical green dichroism in polarized light, permanganate resistant and contain tryptophan. Electron microscopy of 3 cases displays small fibrils which are typical of amyloid. No patient showed evidence of systemic amyloidosis. The natural history of sclero-calcific valvulopathies and present observations favour the following pathogenesis: first, recurrent thrombotic deposition on thickened and fibrotic endocardium; second, degradation of a coagulation-related protein with beta potential during the aging of the clot with transformation into amyloid fibrils; finally, inclusion of the amyloid in sclerotic replacement tissue.

Amyloidosis of the joints: evidence that human hip capsules have a unique predisposition for amyloid of the senile systemic type.

Amyloid-containing tissues were sampled from 19 capsules and 10 cartilages from hip, knee and sternoclavicular joints of 16 elderly patients. Heart ventricles from 3 of these patients were also studied. Histochemistry and immunofluorescence were performed and amyloid fibrils extracted from one hip capsule. The results indicate that 9 of 13 hip capsules contained prealbumin-like amyloid fibril protein ASc1, a finding characteristic of senile systemic amyloidosis. The amyloid of 16 out of 17 other joint structures showed no reaction with anti-ASc1 or antisera to 3 other systemic forms of amyloid, indicating that they represent localized forms of amyloid.

Normally and abnormally functioning left-sided porcine bioprosthetic valves after long-term implantation in patients: distinct spectra of histologic and histochemical changes.

This morphologic study (X-ray examination of gross specimens, histologic study and histochemical staining) compares two groups of explanted left-sided bioprosthetic valves: group I, 6 valves with normal cusp function and group II, 10 valves with significant dysfunction. Implantation periods ranged from 26 to 79 months. A computerized descriptive statistical method (principal component analysis) is used to analyze the qualitative results. Although qualitatively identical alterations are observed in both groups, the findings in the deep layers of the cusps of severe collagen breakdown, intensive fibrin penetration and various degrees of calcification are restricted to group II. Other findings of interest in both groups include amyloid deposits (four cases) and layering of fusiform host cells on the cusp surface (three cases). The computerized study shows that individuals of one clinical group are morphologically different from those of the other. Mechanical stress may contribute to surface alterations early after implantation, while further collagen breakdown and macrophagic activity result in deep penetration of plasma components and fibrin. Subsequent calcification is likely to be dystrophic rather than metabolic. Colonization of the cuspal surface by endothelial cells after long-term implantation of bioprosthetic valves expresses a new type of relation between host and bioprosthesis.

Amyloid deposits in bioprosthetic cardiac valves after long-term implantation in man. A new localization of amyloidosis.

Congo red staining with microscopic examination under polarized light was performed in 30 porcine bioprosthetic cardiac valves and one autologous fascia lata valve explanted from 31 patients in order to detect the presence of amyloid. Microdeposits of amyloid were present in the sewing ring of the fascia lata valve and in 10 porcine bioprostheses, and this finding was confirmed by transmission electron microscopy in 3 porcine bioprostheses. All amyloid-laden porcine valves had been implanted for at least 33 months before removal, and all except two showed dysfunction and/or severe degeneration of cuspal tissue. Statistical analyses failed to establish any correlation between the presence of amyloid and patient-related factors. In a majority of porcine bioprostheses amyloid was permanganate-sensitive and tryptophan-positive. The pathogenesis of this new form of heart valve amyloidosis might consist in penetration of human macrophages in deteriorated bioprosthetic cusps and their interaction with blood-borne amyloid precursors.

Histopathology of the aortic valve in patients with a previous history of acute rheumatic fever. An analysis of 63 surgical specimens.

In a series of 63 surgical patients with a positive history of rheumatic fever and whose aortic valve was removed either singly or in combination with other valves, the histological examination of the aortic valves showed functional lesions in 22 cases (35%). Organic lesions of inflammatory origin, destruction of the architecture with scarring and presence of hypertrophic vessels, were observed in 19 valves (30%). The other cases were doubtful. This ratio was identical in uni- and plurivalvular involvement. Anatomic aspects and significance of microscopic lesions of the aortic valves were discussed. The role of microthrombi and turbulent flow in the pathogenesis of the functional lesions is stressed. The authors conclude that there is no correlation between the anatomic aspects of the aortic valve deformity and the presence of histologically proven organic lesions of inflammatory origin. This provides support to the current opinion that rheumatic carditis is mainly a disease of the mitral valve.

Microdeposits of amyloid in sclerocalcific heart valves: a histochemical and immunofluorescence study.

Amyloid associated with seven sclerotic and two normal aortic and mitral valves was studied. The sclerotic valve amyloid contained microfibrils with typical random orientation and a fibril width of 9.5-12.5 nm. The amyloid deposits demonstrated permanganate-resistant Congophilia and contained the amino acid tryptophan. Immunofluorescence studies showed P-component in amyloid deposits of 6 of 7 valves, but none of the sclerotic valves contained amyloid fibril proteins of the AL (primary), AA (secondary), AEt (medullary thyroid carcinoma) or ASc1 (senile cardiac) types. Two non-sclerotic valves, removed from a patient with systemic amyloidosis, showed permanganate-sensitive Congophilic amyloid deposits which contained amyloid fibril protein AA.