Structural Integrity and Cellular Viability of Cryopreserved Allograft Heart Valves in Right Ventricular Outflow Tract Reconstruction: Correlation of Histopathological Changes with Donor Characteristics and Preservation Times

Abstract

Introduction: Cryopreserved allograft heart valves (CAHV) show longer event-free survival compared to other types of protheses. However, all patients develop early and/or late allograft failure. Negative predictors are clinical, and there is a lack of evidence whether they correspond with the microscopic structure of CAHV. We assessed histopathological signs of structural degeneration, degree of cellular preservation, and presence of antigen-presenting cells (APC) in CAHV and correlated the changes with donor clinical characteristics, cryopreservation times, and CAHV types and diameters.

Methods: Fifty-seven CAHV (48 pulmonary, nine aortic) used for transplantation between November/2017 and May/2019 were included. Donor variables were age, gender, blood group, height, weight, and body surface area (BSA). Types and diameters of CAHV, cold ischemia time, period from decontamination to cryopreservation, and cryopreservation time were recorded. During surgery, arterial wall (n=56) and valvar cusp (n=20) samples were obtained from the CAHV and subjected to microscopy. Microscopic structure was assessed using basic staining methods and immunohistochemistry (IHC).

Results: Most of the samples showed signs of degeneration, usually of mild degree, and markedly reduced cellular preservation, more pronounced in aortic CAHV, correlating with arterial APC counts in both basic staining and IHC. There was also a correlation between the degree of degeneration of arterial samples and age, height, weight, and BSA of the donors. These findings were independent of preservation times.

Conclusion: CAHV show markedly reduced cellular preservation negatively correlating with the numbers of APC. More preserved CAHV may be therefore prone to stronger immune rejection.

Keywords: Allografts; Antigen Presenting Cells; Cryopreservation; Degeneration; Heart Valve; Histopathology; Tissue Donors.

Fig. 1

Bar plots showing individual components of structural degeneration in arterial (A) and valvar (B) samples (only the non-zero variables are shown). X-axis shows grades of severity for each individual variable, y-axis provides number of samples in each category (different colors of the bars were used only for a better understandability of the picture). ELA=elastic fiber; MEMA=mucoid extracellular matrix accumulations.

Fig. 2

Bar plots showing individual components of decreased cellular viability in arterial (A) and valvar (B) samples (only the non-zero variables are shown). X-axis shows grades of severity for each individual variable, y-axis provides number of samples in each category (different colors of the bars were used only for a better understandability of the picture). HE=hematoxylin and eosin.

Fig. 3

Photomicrographs of pulmonary trunk wall. A) Mild reduction of cellular preservation showing shrunken and pyknotic nuclei (arrow), hematoxylin and eosin stain, magnification 100×; B) mild fragmentation and loss of elastic fibers (arrow), Weigert’s resorcin fuchsin stain, magnification 100×; C) mild focal intralamellar mucoid extracellular matrix accumulations (arrow), Alcian blue/periodic acid-Schiff stain, magnification 100×; D) anti-vimentin immunohistochemical stain demonstrating well preserved mesenchymal cells (bar), magnification 100×; E) anti-h-caldesmon immunohistochemical stain demonstrating mild focal loss of smooth muscle cells (arrow), magnification 100×; F) anti-CD34 immunohistochemical stain demonstrating complete loss of endothelial cells (arrow), magnification 200×; G) anti-S100β immunohistochemical stain demonstrating a few positive antigen-presenting dendritic cells (circle), magnification 400×.

Fig. 4

Photomicrographs of aortic wall. A) Marked reduction of cellular preservation showing shrunken and pyknotic nuclei (arrow), hematoxylin and eosin stain, magnification 100×; B) mild fragmentation elastic fibers (arrow) and mild intimal fibrosis (asterisk), Weigert’s resorcin fuchsin stain, magnification 100×; C) mild intralamellar mucoid extracellular matrix accumulations (arrow), Alcian blue/periodic acid-Schiff stain, magnification 100×; D) anti-vimentin immunohistochemical stain demonstrating marked reduction of mesenchymal cells (arrow), magnification 200×; E) anti-h-caldesmon immunohistochemical stain demonstrating marked reduction of smooth muscle cells (arrow), magnification 200×.