Human Cryopreserved Homografts: Electron Microscopic Analysis of Cellular Injury Donald G. Crescenzo, MD, Stephen L. Hilbert, PhD, Robert H. Messier, Jr, MD, Patrick W. Domkowski, MS, Mary Kay Barrick, BS, Perry L. Lange, BS, Victor J. Ferrans, MD, PhD, Robert B. Wallace, MD, and Richard A. Hopkins, MD Georeetown Universitv School of Medicine, WashinHon, DC, and Center for Devices and Radiological Health, Food and Drug Admkstration, RockGille, Maryland

I

Twenty-five human cryopreserved valves with harvest- related warm ischemic times (WITs) ranging from 0 to 20 hours were studied using transmission electron micros- copy to characterize the effects of harvesting and preser- vation on leaflet matrix cells. The valves were divided into seven groups on the basis of WIT and processed using standard transmission electron microscopic meth- ods. Each cell (528 micrographs) was graded for revers- ible and irreversible cellular injury and subjected to a Cochran-Mantel-Haenszel trend analysis. Our results demonstrated a progression in cellular injury with in- creasing WIT. During the first 12 hours of warm isch- emia, reversible cellular injury predominated (O.O%, 30.0%, 51.2%, 31.3%,35.1%, 45.1%, and40.0% at WITsof 0, 1, 2, 8, 12, 16, and 20 hours, respectively). A positive correlation (p < 0.OOOl) between increasing WIT and reversible cellular injury through the first 12 hours was

urrently, there are three categories of prostheses C available, with the selection of a specific replace- ment heart valve design being dictated by clinical indica- tions, patient factors, and surgeons’ preference. Although the ideal cardiac valve replacement has yet to be identi- fied, the human cryopreserved homograft offers numer- ous clinical advantages for ventricular outflow reconstruc- tion over both mechanical and xenograft valves: (1) relative resistance to endocarditis; (2) optimal hydraulic performance especially in the small aortic root size of children; (3) low incidence of thromboembolic complica- tions; and (4) freedom from long-term anticoagulation and superior performance in the right ventricular outflow tract position [l]. Long-term durability of the homograft, al- though uncertain, seems to be influenced by the preim- plantation processing method chosen (Fig 1). Historically,

Presented in part at the Twenty-eighth Annual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Feb S 5 , 1992.

The opinions or assertions contained herein are the private views of the authors and are not to be construed as conveying either an official endorsement or criticism by the US Department of Health and Human Services, the Food and Drug Administration; LifeNet, Inc; the National Institutes of Health; The American Heart Association; or The National Research Council.

Address reprint requests to Dr Hopkins, Department of SurgeryAPHC, Georgetown University, 3800 Reservoir Road, NW, Washington, DC 20007.

observed. Minimal morphologic evidence of irreversible injury was noted in valves harvested with less than 12 hours of warm ischemia; however, after 12 hours there was a marked increase (O.O%, O.O%, 4.7%, 2.4%, 2.7%, 31.4%, and 40.0% at WITs of 0, 1, 2, 8, 12, 16, and 20 hours, respectively) in irreversible cellular injury ( p < 0.001 between 12 and 20 hours WIT). These data demon- strate a progression in cellular injury with increasing WIT. There was virtually no morphologic injury in valves with harvest-related WITs less than 2 hours and minimal irreversible cellular injury observed in valves exposed to 12 hours or less of warm ischemia. If cellular viability is critical to homograft durability then harvest- related warm ischemia may need to be restricted to 12 hours.

(Ann Tliorac Surx 1993;55:25-31)

the spectrum of these methods ranges from cold storage in nutrient media to treatment with aldehydes, antibiot- ics, or ppropiolactonc to ethylene oxide and irradiation [3,6]. Harsh chemical disinfection (ie, high concentrations of antibiotics, aldehydes, or ppropiolactone) and irradia- tion have been shown to reduce allograft durability [4, 61. Homografts prepared by various gentle methods (ie, low-dose antibiotics) appear to have durability exceeding porcine, but perhaps less than mechanical valves [7-91. Currently, homografts are disinfected using lower con- centrations of antibiotics followed either by cryopreserva- tion or storage of the valve in cold (4°C) nutrient media. There remains a paucity of data assessing the effects of procurement and processing on the morphologic integrity of the cryopreserved valve leaflets.

Current harvesting protocols are based on the premise that cellular viability enhances allograft durability [2, 10-161. All valves harvested have an obligatory period of warm ischemic time from the death of the donor to immersion of the valve in cold storage solution. This obligatory warm ischemic time has been shown to affect matrix cell viability, but the time-dependent injury re- sponse has not been well characterized. Historically, valves have been harvested up to 72 hours after donor death. Most centers limit the warm ischemic time to less

0 1993 by The Society of Thoracic Surgeons

26 CRESCENZO ET AL HUMAN CRYOPRESERVED HOMOGRAFTS

Ann Thorac Surg 19!33;55:25-31

90

E RO t": 70 .e 60 e e

u m 50 W P C 0 40 o m

a 20 10

E" 30 tlanh C h e m i c y Preservation

0 - 0 4 5 6 8 10 12 I 4 I 6

Years

Fig I . Comparison of the durability of homografts processed by cryo- preservation (OBrien and associates [21), with storage in 4°C nutri- ent media (Barratt-Boyes and colleagues [31), and using harsh chemi- cal preservation methods (Duly and co-u)orkers 141). Also included is the expected durability of the xenograft rialz~es (Spampinato and associ- ates f.51).

than 24 hours, whereas others severely restrict harvest to only virtual beating heart donors.

The purpose of this study was to quantitatively charac- terize the combined consequences of variable warm isch- emic injury, antibiotic disinfection, cryopreservation, and thawing on human leaflet matrix cell morphology using transmission electron microscopy.

Material and Methods Study Design Twenty-five human cryopreserved aortic and pulmonic valves with warm ischemic times ranging from 0 minutes to 20 hours were obtained (LifeNet Transplant Services, Virginia Beach, VA) and studied using transmission elec- tron microscopy to characterize the effects of harvesting, antibiotic disinfection, and cryopreservation on leaflet matrix cell ultrastructure. Valves were acquired by means of a standard clinical procurement and cryopreservation protocol [17]. Homograft valves selected for study were rejected for transplantation for the following reasons: (1) positive bacterial cultures (18/25 valves); (2) thickened semilunar cusps or atheromatous plaques (3/25 valves); (3) technical error during valve harvest (3/25 valves); and (4) annular hematoma (1/25 valves). The valves were divided into seven groups on the basis of warm ischemic time: group 1, 0 minutes; group 2, 0 to 1 hour; group 3, 1 to 2 hours; group 4,2 to 8 hours; group 5,8 to 12 hours; group 6, 12 to 16 hours; and group 7, 16 to 20 hours. Two leaflets from each valve were randomly selected for study using standard transmission electron microscopic methods.

Valve Procurement and Preservation All the valves were aseptically procured in an operating room and cryopreserved by LifeNet. The warm ischemic time from each valve was recorded and the valve trans- ported (4°C) in either RPMI 1640 nutrient media, 0.9% saline solution, or lactated Ringer's to the tissue bank for processing [17]. Each homograft was cultured, excess myocardium and fat were removed, and the internal valve

diameter was determined. Subsequently, the homograft was disinfected in RPMI 1640 nutrient media-antibiotic solution (cefoxitin, 240 pg/mL medium; lincomycin, 120 @mL medium; polymyxin B, 100 pg/mL medium; and vancomycin, 50 pg/mL medium) at 4°C for 24 hours. The homografts were then cryopreserved (1°C per minute, 10% dimethyl sulfoxide) and stored in vapor liquid nitrogen (-150°C to -190OC). The homografts were initially thawed by immersion of the storage packet in a 40°C water bath for 10 minutes with subsequent stepwise dilution of the dimethyl sulfoxide cryoprotectant using increasing volumes of fresh RPMI 1640 nutrient media (dimethyl sulfoxide concentrations, 7.5%, 5.0%, 2.5%, and 0.0%). Two semilunar cusps from each valve were fixed using 1% glutaraldehyde + 4% formaldehyde in 0.1 mol/L phosphate buffer (pH 7.4).

Transmission Electron Microscopy After fucation, each cusp was cut from the free edge to the base and embedded in epoxy resin. To ensure that the cells observed were representative of the population of cells from each leaflet, multiple thin sections from each tissue block were cut, and one thin section from each valve leaflet was randomly selected for morphologic ob- servation. Transmission electron microscopic studies (80 kV; JEOL 100 CX transmission electron microscope, To- kyo, Japan) were conducted using standard methods [18]. The first ten cells observed in each thin section were photographed (20 photomicrographs per valve), and the extent of cellular injury was assessed using the following criteria: (1) reversible injury (cytoplasmic edema, dilation of the endoplasmic reticulum, mitochondrial swelling) and (2) irreversible injury (mitochondrial flocculent den- sities, karyolysis, and disruption of the plasma mem- brane).

Statistical Analysis For this study, 528 photomicrographs were analyzed. A spreadsheet containing the valve identification and the results of our analysis was constructed (Quattro Pro 3.0, Borland, Santa Cruz, CA). These data were cross- tabulated, and the resulting contingency table was ana- lyzed for a possible linear association between time and the number (percentage) of damaged cells. A Cochran- Mantel-Haenszel ,$ test (PC-SAS PROC FREQ; SAS Insti- tute, Cary, NC) was used to identify a linear association between the rows and columns of the contingency table.

Results Fibroblasts and myofibroblasts are the predominant cell components of the leaflet matrix [19]. Endothelial cells were occasionally observed; however, they appear to be more susceptible to the effects of warm ischemia (Fig 2). Quantitative studies of leaflet collagen bundle crimp [20] and proteoglycans were not addressed in this study. A gradual increase in the number of matrix cells demonstrat- ing morphological evidence of cellular injury with increas- ing duration of warm ischemia was observed (Table 1; Fig 3). After 12 hours of warm ischemia virtually two-thirds of

Ann Thorac Surg 1993;55W1

CRESCENZO CT Al. HUMAN CRYOPRESERVED HOMOGRAbTS

27

i

Fig 2 . lrreversibly injured endothelial cell observed in a homograft exposed to 6 hours of warm ischemia, illustrating the sensitivity of this cell type to ischemic inju y . Note the presence of ka yolysis (short arrow) and loss of plasma membrane integrity (long arrow) (compare with Fig 4). (Uranyl acetate and lead citrate stain; ~ 5 , 4 0 0 before 35% reduction.)

the leaflet matrix cells were morphologically unaltered (Fig 4). However, 20 hours of warm ischemia resulted in 80% of the cell population demonstrating evidence of cellular injury (Figs 5, 6). For the first 12 hours, injury was predominantly reversible with a positive correlation be- tween reversible cellular injury and increasing warm ischemic time (p < 0.0001). Thereafter, irreversible injury was noted in an increasing proportion of cells (ie, 2.7% at 12 hours up to 40% at 20 hours). No irreversible injury was observed in any cells exposed to 1 hour or less of warm ischemia (ie, beating heart donors) followed by standard cryopreservation processing. There was a grad- ual increase in the number of irreversibly injured cells between 1 and 12 hours of warm ischemia; however, greater than 95% of cells were free of irreversible injury. A rapid increase in the number of irreversibly injured cells was noted when harvest-related warm ischemic times increased from 12 to 20 hours (p < 0.001). After 20 hours of warm ischemia 80% of the matrix cells demonstrated evidence of cellular injury (40% reversible, 40% irrevers- ible). The alteration of matrix cell ultrastructural morphol-

I Normal = Reversiblelnjury Irreversible Injury 1 Fig 3. Percentage of cells observed in each category of cellular i n j u y . X-axis groups represent the various warm ischemic intervals. Using a Cochran-Mantel-Haenszel trend analysis a positive correlation exists (*p < 0.0001) for reversible injury through the first 12 hours of warm ischemic time with a positive correlation (**p < 0.001) twtween 12 and 20 hours of warm ischemia for irreversible cellular inju y .

ogy was observed in the following sequence: (1) dilation of endoplasmic reticulum; (2) cytoplasmic edema; (3) mitochondrial swelling; (4) presence of mitochondrial flocculent densities (ie, small, irregularly shaped electron- dense particles); (5) karyolysis; and (6) plasma membrane disruption.

Comment The results of this study quantitatively describe the ultra- structural changes associated with variable warm ischemic injury followed by antibiotic disinfection and cryopreserva- tion to leaflet matrix cells of human cryopreserved ho- mograft valves. Our findings demonstrate a gradual in- crease in reversible cellular injury through the first 12 hours of warm ischemia; however, after 20 hours of warm ischemia, 80% of the matrix cells demonstrated morpho- logic evidence for either reversible or irreversible cell injury. Remarkably, these data further demonstrate little irreversible damage to the leaflet matrix cells when pro- cessing is preceded by less than 12 hours of harvest-

Table 1. Percentage of Reversible and Irreversible Cellular Injury in Cryopreserved Human Values IJrocessed for Use as Homografts Result 0 Minutes 0-1 Hour 1-2 Hours 2-8 Hours 8-12 Hours 12-16 Hours 16-20 Hours

Normal 100.0% 70.0% 44.1% 66.3% 62.2% 23.5% 20.0w

Reversible injury 0.0%” 30.0%” 51.2%” 31.3%” 35.1%“ 45.1% 40.0% (20120) (14120) (38186) (1101166) (46174) (241102) (1360)

(0120) (6120) (M86) (52/166) (26174) (461102) (2460) Irreversible injury 0.0% 0.0% 4.7% 2.4% 2.7% 31.4%h 40.0%h

(0120) (0120) (41%) (4166) (374) (32/102) (24160)

A positive correlation exists between increasing warm ischemia and reversible cellular injury ( p < 0.0001 by Cochran-Mantel-Haenszel trend analysis). ” A positive correlation exists between increasing warm ischemic time and irreversible cellular injury (17 < 0.001 by Cochran-Mantel-Haenszel trend analysis).

28 CRf:;Cl:N/( 1 F.T Al. IiIJMAN C KYOPRESERVED HOMOCRAFTS

Ann Thorac Surg 1993;55:Wl

Fig 4 . Representative micrograph depicts the ultrastructural features of an uninjured leaflet fibroblast after 6 hours of warm ischemia. Note the appearance of the nucleus (euchromatin, heterochromatin), mito- chondria (long arrow), endoplasmic reticulum (short arrow), and cytoplasmic density. (Uranyl acetate and lead citrate stain; x4,350 before 35% reduction.)

related ischemia; however, an additional 8 hours of warm ischemia greatly increases the number of cells that under- went irreversible cellular injury. Valves harvested with relatively short warm ischemic times (12 hours or less) contain a population of matrix cells in which the majority are morphologically unaltered and thus presumably will be viable after transplantation. The exact role of these fibroblasts in promoting homograft valve durability is currently speculative; it has been suggested that viable fibroblasts may facilitate the active renewal of extracellular components depleted as a consequence of harvesting, processing, valve implantation, and the dynamic leaflet motion during the cardiac cycle. Alternatively, the pres- ence of viable leaflet fibroblasts may simply reflect gentle

Fig 5 . Severdy injured leaflet fibroblast after 20 hours of warm isch- emia, demonstrating extensive dilation of endoplasmic reticulum and mitochondria1 flocculent densities. (Uranyl acetate und lead citrate stain; ~ 5 , 4 0 0 before 35'70 reduction.)

Fig 6 . Higher magnification Vipw of the cell depicted in Figure 4, demonstrating flocculent densities (short arrow) and dilation of endo- plasmic reticulum (long arrow) after 20 hours of warm ischemia. (Uranyl acetate and lead citrate stain; X10,500 before 35% reduc- tion.)

preservation techniques and have little direct impact on the long-term durability of homografts.

Current harvesting and cryopreservation protocols are based on the premise that leaflet fibroblast viability en- hances valve durability [2, 10-161. Although this hypoth- esis has yet to be proved, this premise has gained accep- tance by comparison with studies demonstrating the reduced durability of homografts processed by methods known to be toxic to leaflet matrix cells [4, 61. Early attempts at increasing the shelf-life of banked homografts involved harsh preservation methods such as lyophiliza- tion, irradiation, and aldehyde pretreatment. The dura- bility of homografts processed by these harsh methods is clearly inferior to that attained with present-day tech- niques, because 62% and 95% of patients required reop- eration within 10 and 20 years of implantation, respec- tively [4]. In comparison, a 95% actuarial freedom from valve failure at 4 years and 92% at 10 years have been reported for cryopreserved homograft valves placed in the aortic position in adults [2]. Cold wet-stored homografts as reported by Barratt-Boyes and others (ie, a gentle technique with presumably low cellular viability) attain an actuarial freedom from valve replacement of 95%, 78%, and 42% after 5, 10, and 14 years of implantation, respec- tively [3]. The Brisbane experience with cold wet-stored homografts is similar, with 58% freedom from reoperation at 15 years [12]. Cryopreserved valves demonstrate a moderate advantage in terns of durability compared with cold wet-stored valves with a moderate increase in dura- bility compared with porcine aortic valve xenografts in similar patient populations [7]. All homograft valves, regardless of processing and storage methods, demon- strate increased durability and performance in the right ventricular outflow tract as compared with xenograft valves [7, 21-23].

Harvest-associated warm ischemia may be a critical

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CRESCENZO ET AL 29 HUMAN CRYOPRESERVED HOMOGRAFTS

determinant of fibroblast viability. Previous laboratory studies in which porcine aortic valves were harvested and exposed to variable periods of warm ischemia demon- strated a resilience by leaflet fibroblasts to the effects of warm ischemic injury for more than 24 hours [19] when studied before antibiotic disinfection and cryopreserva- tion (ie, analogous to the time of cadaveric harvest-related ischemia). However, after 24 hours of warm ischemia a marked increase in the number of irreversibly injured cells was observed. A positive correlation between increasing warm ischemic time and irreversible cellular injury was demonstrated through 36 hours of warm ischemia. In the present study of human cryopreserved valves, our find- ings indicate the window of minimal warm ischemic injury is shorter (ie, 12 hours) before there is a marked increase in the number of irreversibly injured cells in leaflet tissue that has been harvested, processed, cryopre- served, and thawed in a manner analogous to current clinical protocols. This observation suggests that an addi- tional reduction in the number of viable leaflet fibroblasts occurs when cadaveric harvest ischemia is followed by antibiotic disinfection, cryopreservation, and thawing. The animal studies are an important complement to understanding these human results, because the limited availability of donor tissue mandates full processing and clinical use of all human valves except for those found to be nontransplantable. Thus a control series for human valves that have only experienced cadaveric harvest isch- emia is not available in this country.

The mechanism that renders the matrix cells susceptible to destruction may be related to depletion of high-energy phosphate intermediates during the warm ischemic inter- val. In an analysis of human cryopreserved valves pro- cessed for use as homografts, adenosine triphosphate levels decreased from 0.127 nmol/mg protein after 2 hours of warm ischemia to complete depletion of adenosine triphosphate after 13 hours of warm ischemia [24]. Signif- icant reductions in adenosine triphosphate levels have also been observed in porcine aortic valves after brief periods of warm ischemia, antibiotic disinfection, and cryopreservation [25]. Furthermore, as the warm ischemic interval is increased, reductions in the levels of other adenine nucleotides (ie, adenosine diphosphate and ade- nosine monophosphate) occurs [26]. Accumulation of lactate in valvular tissue has been measured after 2 hours of warm ischemia, suggesting that around this time, ischemic leaflet cells have converted from aerobic to anaerobic metabolism [27]. Teleologically, cellular injury should progressively increase as metabolic reserves are further depleted during subsequent processing steps (ie, antibiotic disinfection, cryopreservation, and thawing) even if these steps are by themselves not toxic. Our morphologic observations in clinical human cryopre- served aortic homografts support these biochemical find- ings. Within relatively short periods of warm ischemia (1 to 12 hours), we observed evidence of reversible cellular injury (ie, cytoplasmic and mitochondrial edema, dilation of the endoplasmic reticulum). However, marked mor- phological evidence of irreversible cellular injury (ie, mitochondrial flocculent densities, karyolysis, plasma

membrane disruption) was not observed until after 12 hours of warm ischemia. These findings suggest that periods of warm ischemia less than 12 hours may repre- sent the optimal window for valve procurement.

This study demonstrates that after 20 hours of warm ischemia and subsequent disinfection and cryopreserva- tion 40% of the matrix cells in cryopreserved valves have been irreversibly injured. Morphologic criteria for revers- ible cellular injury (ie, cytoplasmic edema, dilated endo- plasmic reticulum, swelling of the mitochondria) repre- sent observations consistent with an inability of the cells to maintain osmotic gradients. With restoration of the metabolic reserves it is expected that these findings may regress. The markers of irreversible cellular injury (ie, mitochondrial flocculent densities, karyolysis, plasma membrane disruption) represent extensive cellular de- struction with no chance of salvage. Mitochondria1 floc- culent densities, which are electron-dense irregularly shaped particles composed of calcium and phosphate found within the mitochondria matrix, correspond to the biochemical uncoupling of oxidative phosphorylation. Their presence may signal irreversible mitochondrial damage and severe depletion of intracellular adenosine triphosphate.

Results of this morphologic study demonstrate a marked increase in matrix cell irreversible injury after harvest-related warm ischemia greater than 12 hours in human allografts processed as clinical quality homograft valves. Previously, worldwide harvesting protocols al- lowed widely variable cadaveric harvests up to 72 hours postmortem, but currently, most restrict harvest to within 24 hours. If fibroblast viability is a significant determinant of homograft durability and performance, then warm ischemic time associated with valve procurement should be restricted to 12 hours or less unless modifications of processing methods can be implemented that reverse the progression of injury. Limiting harvests to beating heart donors is probably too restrictive and is not supported by our data. Unlike myocardial cells, fibroblasts are remark- ably resilient and do not die within minutes after the onset of ischemia. Minimal cellular damage observed after short periods of cadaveric ischemia, cold antibiotic disin- fection, and cryopreservation is quite impressive. Intu- itively, one would expect negative effects of such proc- essing; cryopreservation, as currently performed, is a standardized method extraordinarily effective in preserv- ing the viability of previously uninjured valve leaflet cells. Cardiac surgeons should understand the implications of preprocessing warm ischemia and be aware of that time interval when selecting a cryopreserved homograft for transplantation, especially when prolonged durability is :n issue for a specific patient. Currently our findings support three (conceptual) categories of cryopreserved homograft valves: (1) beating heart or virtual beating heart donors (less than 2 hours of warm ischemia); (2) short cadaveric harvest ischemia (2 to 12 hours); and (3) pro- longed cadaveric ischemia (>12 hours but 524 hours). The matrix cell population in valves harvested more than 24 hours after donor death are almost certainly either nonviable or irreversibly injured, especially once exposed

30 CRESCENZO ET AL HUMAN CRYOPRESERVED HOMOGRAFTS

Ann Thorac Surg 1993;552531

to the rigors of preimplantation processing and transplan- tation.

Grant support was provided by The American Heart Association (Capital Affiliate), and LifeNet, Inc. Research funding for Dr Crescenzo was supported by a National Research Council Fel- lowship. The cryopreserved valves were provided by LifeNet, Inc.

References 1.

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Hopkins RA. Historical development of the use of homograft valves. In: Hopkins RA, ed. Cardiac reconstruction with allograft valves. New York: Springer-Verlag, 1989:9. OBrien MF, Stafford EG, Gardner MA, Pohlner PG, McGif- fin DC. A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies. J Thorac Cardiovasc Surg 1987;94: 812-23. Barratt-Boyes BG, Roche MB, Subramanyan R, Pemberton JR, Whitlock RM. Long-term follow-up of patients with the antibiotic-sterilized aortic homograft valve inserted freehand in the aortic position. Circulation 1987;75:768-77. Daly RC, Orszulak TA, Schaff HV, McGovern E, Wallace RB. Long-term results of aortic valve replacement with nonviable homografts. Circulation 1991;84(Suppl 3):81-8. Spampinato N, Stassano P, Cammarota A, et al. Bioprosthe- ses at twelve years. J Cardiac Surg 1988;3(Suppl):383-90. Moodie DS, Mair DD, Fulton RE, Wallace RB, Danielson GK, McGoon DC. Aortic homograft obstruction. J Thorac Cardio- vasc Surg 1976;72:553-61. Kirklin JW, Blackstone EH, Maehara T, et al. Intermediate- term fate of cryopreserved allograft and xenograft valved conduits. Ann Thorac Surg 1987;44:59%606. Borkon AM, Soule LM, Baughman KL, et al. Comparative analysis of mechanical and bioprosthetic valves after aortic valve replacement. J Thorac Cardiovasc Surg 1987;9420-33. Hammond GL, Geha AS, Kopf GS, Hashim SW. Biological versus mechanical valves: analysis of 1,116 valves inserted in 1,012 adult patients with a 4,818 patient-year and a 5,327 valve-year follow-up. J Thorac Cardiovasc Surg 1987;93:

Angell WW, Oury JH, Lamberti JJ, Koziol J. Durability of the viable aortic allograft. J Thorac Cardiovasc Surg 1989;98: 48-56. OBrien MF, Stafford EG, Gardner MAH, et al. The viable cryopreserved allograft aortic valve. J Cardiac Surg 1987;

McGiffin DC, O'Brien MF, Stafford EG, Gardner MA, Pohl- ner PG. Long-term results of the viable cryopreserved al-

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lograft aortic valve: continuing evidence for superior valve durability. J Cardiac Surg 1988;3(Suppl):289-96. OBrien MF, Johnston N, Stafford G, et al. A study of cells in the explanted viable cryopreserved allograft valve. J Car- diothorac Surg 1988;3(Suppl):279-87. Penta A, Qureshi S, Radley-Smith R, Yacoub MH. Patient status 10 or more years after "fresh" homograft replacement of the aortic valve. Circulation 1984;7O(Suppl 1):182-6. Angell WW, Angell JD, Oury JH, et al. Long-term follow-up of viable frozen aortic homografts: a viable homograft valve bank. J Thorac Cardiovasc Surg 1987;93:815-22. Al-Janabi N, Ross DN. Enhanced viability of fresh aortic homografts stored in nutrient media. Cardiovasc Res 1973;7

Hopkins RA. Allograft valve banking: techniques and tech- nology. In: Hopkins RA, ed. Cardiac reconstruction with allograft valves. New York: Springer-Verlag, 1989:3743. Ferrans VJ, Spray TL, Billingham MG, Roberts WC. Struc- tural changes in glutaraldehyde-treated porcine heterografts used as substitute cardiac valves. Transmission and scanning electron microscopic observations in 12 patients. Am J Car- diol 1978;41:1159-84. Crescenzo DG, Hilbert SL, Barrick MK, et al. Donor heart valves: electron microscopic and morphometric assessment of cellular injury induced by warm ischemia. J Cardiothorac Surg 1992;103:253-8. Hilbert SL, Barrick MK, Ferrans VJ. Porcine aortic valve bioprosthesis: a morphologic comparison of the effects of fixation pressure. J Biomed Mat Res 1990;24:773-87. Hopkins RA. Right ventricular outflow tract reconstructions. The role of valves in the viable allograft era. Ann Thorac Surg 1988;45:593-4. Fontan F, Choussat A, Deville C, Doutremepuich C, Coupil- laud J, Vosa C. Aortic valve homografts in the surgical treatment of complex cardiac malformations. J Thorac Car- diovasc Surg 1984;87649-57. Kay PH, Ross DN. Fifteen years' experience with the aortic homograft: the conduit of choice for right ventricular outflow tract reconstruction. Ann Thorac Surg 1985;40:36&4. Messier RH, Domkowski PW, Abd-Elfattah AS, et al. Anal- ysis of the total adenine nucleotide pool in 25 cryopreserved human cardiac valves. Circulation 1991;4(Suppl 2):640. Domkowski PW, Messier RH, Crescenzo DG, et al. Preim- plantation alteration of adenine nucleotides in cryopreserved heart valves. Ann Thorac Surg (in press). Messier RH, Domkowski PW, Aly H, et al. High energy phosphate depletion in leaflet matrix cells during processing of cryopreserved cardiac valves. J Surg Res (in press). St. Louis J, Corcoran P, Rajan S, et al. Characterization of the effects of warm ischemia following harvesting of allograft cardiac valves by magnetic resonance spectroscopic analysis of ATP, phosphorus, lactate and electron microscopy. Eur J Cardiothorac Surg 1991;5:458-65.

817-22.

DISCUSSION

MR ENDRE BODNAR (Pinner, United Kingdom): 1 would like to congratulate Dr Crescenzo and associates. I hope that this excellent report will be published and quoted in relevant publi- cations for many years to come because it clarifies a number of problematic issues. I would like to make a few comments.

First of all, this report seemed to prove what we have been believing for some time, that all of those homografts that have been implanted over the past 25 years were nonviable because they were harvested from cadavers 24 to 72 hours after death. One may conclude this from Crescenzo and associates' data.

The second thing I would like to comment on is that it may be a mistake to equate cryopreservation with viability. The Mark OBrien series that you have used here as an example was a

mortuary series. Therefore, in light of your results, they were cryopreserved but nonviable valves. We presented to this Society a couple of years ago results obtained in London over a 20-year period in which there was no difference in clinical performance whether the homografts were believed to be viable or not.

As far as fibroblast viability is concerned, I would like to note that it does not mean that the valve is normal. First of all, a normal valve has endothelium, and fibroblasts do not produce endothelium. Neither do they produce elastin or collagen. They produce only procollagen, and a normal extracellular environ- ment is necessary to turn it into collagen. The so-called viable valves are not normal, and there is a question as to whether the life of a fibroblast will extend the life of the homograft.

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Finally, with a view to this, there may be a new evaluation of the truly viable valves. They may be much more antigenic than those homografts that we have been working with over the past 25 years, as acute rejection was a totally unknown complication of homograft insertion until these truly and highly viable valves arrived. So when you are proposing that the best valve should be given to the best patient, I would raise the question, which is the best valve? I am not sure that the most viable is the best.

DR CRESCENZO Thank you very much for your comments. I would like to address several points in response to your remarks.

First, endothelial cells have never been shown to be important in terms of valve durability. In fact, preservation of endothelial cells may reduce valve durability by an immunologic mediated response. It has been shown that the immunologic reaction is a result of class I antigens expressed on the surface of the endo- thelial cells.

As far as our statements concerning the different categories of cryopreserved valves, we still have not been able to fully eluci- date the exact role of the fibroblast in promoting valve durability. We do know that cryopreservation has been shown to preserve cellular viability; however, what may be more important in terms of valve durability is that cryopreservation causes less damage to the acellular components, and this is the reason that cryopre- served valves perform better than the harsh preservation series published by the Mayo Clinic.

DR PETER K. SMITH (Durham, NC): How would you handle the bias that is introduced into your data by the fact that such a large proportion of the valves were rejected because of positive cultures? We all understand that they have been warm ischemic in a medium that is conducive to this. To what extent do your data support your conclusions even in the remaining 8 or so that were not infected?

DR CRESCENZO Dr Smith, thank you very much for your comments as well. This very question has been raised by mem- bers of our research team. The morphologic observations re- ported in this study were performed on human cryopreserved valves that were processed on line in a commercial distribution center. These valves were later rejected for transplantation be- cause of technical reasons such as positive bacterial cultures. These were "technical" infections by virtue of positive surveil- lance culture-not invasive infections causing thickened cusps, for example. If we assume a worse-case scenario, our data would be overreporting cellular injury, and I think you would agree that valves with warm ischemic times of less than 12 hours had remarkably little injury.

DR FLAVIAN M. LUPINElTI (Ann Arbor, MI): I enjoyed your report very much. 1 think it is very important to attempt to quantitate the quality of "viability," which is sometimes referred to rather loosely. But I wonder whether your methods really determine fibroblast viability as opposed to cell death; they may not be opposite sides of the same coin. Do you have any data that relate your histologic grading to proline uptake or autoradiogra- phy or some other more affirmative line of evidence that would indicate cellular viability?

DR CRESCENZO The proline and inulin uptake studies have been done at Old Dominion University by Dr Wolfinbarger. Our morphologic observations correlate with his findings. Recently, our laboratory has been able to culture fibroblasts in porcine heart valves with warm ischemic time as long as 48 hours. We are currently looking at whether these fibroblasts can secrete colla- gen in a normal fashion. Our preliminary results demonstrate that fibroblasts will proliferate in cell culture, but we still do not know if these cells will function normally.