Clinical experience of reoperative right ventricular outflow tract reconstruction with valved conduits: risk factors for conduit failure in long-term follow-up.

Reconstruction of right ventricular outflow tract in patients with congenital heart disease in various age groups remains a controversial issue. Currently, a little is known about the fate of secondary and subsequent conduit. The aim of the study was to determine risk factors of conduit failure, evaluate long-term conduit survival, find out which type of conduit should be preferred in case of reoperations. We performed a retrospective analysis of a total of 249 records of valved conduit secondary and subsequent replacement in right ventricular outflow tract in 197 patients. Median follow-up was 5.7 years. The study endpoints were defined as conduit explants; balloon dilatation of the graft (excluding balloon dilatation of left/right pulmonary artery), transcatheter pulmonary valve implantation; heart transplantation or death of the patient. There were total of 21 deaths (11% mortality) among 197 patients during the follow-up, 2 patients underwent heart transplant, in 23 implanted conduits pulmonary angioplasty or/including transcatheter pulmonary valve implantation was afterwards performed due to graft failure, conduit had to be explanted in 46 cases. After 28 years follow-up, freedom from graft failure after 5 years was 77%, 48% after 10 years and 21% after 15 years. Reoperative right ventricular outflow tract reconstruction demonstrates good mid-term and acceptable long-term outcomes regardless of the type of conduit implanted. Worse long-term graft survival of secondary and further conduits is associated with younger age of the recipient at implantation, small size of the conduit, younger age of donor and male donor in case of allograft implantation.

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.

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.

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.

The time has come to extend the expiration limit of cryopreserved allograft heart valves.

Despite the wide choice of commercial heart valve prostheses, cryopreserved semilunar allograft heart valves (C-AHV) are required, and successfully transplanted in selected groups of patients. The expiration limit (EL) criteria have not been defined yet. Most Tissue Establishments (TE) use the EL of 5 years. From physiological, functional, and surgical point of view, the morphology and mechanical properties of aortic and pulmonary roots represent basic features limiting the EL of C-AHV. The aim of this work was to review methods of AHV tissue structural analysis and mechanical testing from the perspective of suitability for EL validation studies. Microscopic structure analysis of great arterial wall and semilunar leaflets tissue should clearly demonstrate cells as well as the extracellular matrix components by highly reproducible and specific histological staining procedures. Quantitative morphometry using stereological grids has proved to be effective, as the exact statistics was feasible. From mechanical testing methods, tensile test was the most suitable. Young's moduli of elasticity, ultimate stress and strain were shown to represent most important AHV tissue mechanical characteristics, suitable for exact statistical analysis. C-AHV are prepared by many different protocols, so as each TE has to work out own EL for C-AHV.

Mechanical and structural properties of human aortic and pulmonary allografts do not deteriorate in the first 10 years of cryopreservation and storage in nitrogen.

The aortic and pulmonary allograft heart valves (AHV) are used in the cardiac surgery for replacing the impaired semilunar valves. They are harvested from donor hearts and cryostored in tissue banks. The expiration period was set to 5 years arbitrarily. We hypothesized that their mechanical and structural properties do not deteriorate after this period. A total of 64 human AHV (31 aortic and 33 pulmonary) of different length of cryopreservation (fresh, 0-5, 5-10, over 10 years) were sampled to different tissue strips (artery, leaflet, ventriculo-arterial junction) and tested by tensile test with loading velocity 10 mm/min until tissue rupture. Neighbouring regions of tissue were processed histologically and evaluated for elastin and collagen area fraction. The results were evaluated statistically. In aortic AHV, the physical deformation response of wall samples to stress did not changed significantly neither during the process of cryopreservation nor during the first 10 years of storage. In pulmonary AHV, the ultimate strain dropped after 5 years of cryopreservation indicating that pulmonary artery was significantly less deformable at the time of rupture. On the other hand, the ultimate stress was equal during the first 10 years of cryostorage. The changes in collagen and elastin amount in the tissue samples were not associated with mechanical impairment. Neither elasticity, stiffness and solidity nor morphology of aortic and pulmonary AHV did not change reasonably with cryopreservation and in the first 10 years of cryostorage. This evidence suggests that the expiration period might be extended in the future.

Antimicrobial efficiency and stability of two decontamination solutions.

Two decontamination solutions, commercially produced BASE•128 and laboratory decontamination solution (LDS), with analogous content of antibiotic and antimycotic agents, were compared in their antimicrobial efficiency and stability (pH and osmolarity). Both solutions were compared immediately after thawing aliquots frozen for 1, 3 or 6 months. Agar well diffusion method was used to test their antimicrobial efficiency against five human pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Enterococcus faecalis. The difference in the inhibition of growth between the two decontamination solutions was mostly not statistically significant, with few exceptions. The most pronounced difference between the LDS and BASE•128 was observed in their decontamination efficacy against E. coli and E. faecalis, where the LDS showed to be more efficient than BASE•128. The osmolarity value of LDS decreased with cold-storage, the osmolarity values of the BASE•128 could not be measured as they were below the range of the osmometer. Slight changes were found in pH of the less stable LDS solution, whose pH increased from initial value 7.36 ± 0.07 to 7.72 ± 0.19 after 6 m-storage. We verified that BASE•128 and LDS are similarly efficient in elimination of possible placental bacterial contaminants and may be used for decontamination of various tissues.

Structural changes arising from different thawing protocols on cryopreserved human allograft's aortic valve leaflets.

BACKGROUND: The aim of our experimental work was to assess the impact and morphological changes that arise during different thawing protocols on human aortic valve (AV) leaflets resected from cryopreserved aortic root allografts (CARAs). OBJECTIVES: Two thawing protocols were tested: 1. CARAs were thawed at a room temperature (23°C); 2. CARAs were placed directly into a water bath at a temperature of 37°C. After all the samples were thawed, non-coronary AV leaflets were sampled from each specimen and fixed in a 4% formaldehyde solution before they were sent for morphological analysis. MATERIAL AND METHODS: All the samples were washed in distilled water for 5 min and dehydrated in a graded ethanol series (70%, 85%, 95%, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane (HMDS) for 10 min, and then air-dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs and coated with gold. Histological analysis was performed with the use of an electron microscope on a scanning mode operating at 25 kV - BS 301. RESULTS: Thawing protocol 1 (room temperature at 23°C): 6 (100%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. Thawing protocol 2 (water bath at 37°C): 5 (83%) samples showed loss of the endothelial covering of the basal membrane with no damage to the basal lamina. One (17%) sample showed loss of the endothelial covering the basal membrane with significant damage to the basal membrane. CONCLUSIONS: Based on our experimental work, we can clearly conclude that cryopreserved AV leaflet allografts show identical structural changes at different rates of thawing.

Organization model for allotransplantations of cryopreserved vascular grafts in Czech Republic.

The transplantation of fresh or cryopreserved vascular allografts in patients with a prosthetic graft infection or critical limb ischemia is necessary for their limb salvage and, in many cases, represents a lifesaving procedure. While transplantation of fresh allografts has a long history in the Czech Republic, the standard use of cryopreserved vascular allografts was introduced into the clinical practice in 2011 as a result of the implementation of EU Directive 2004/23/EC into national legislation (Human Cell and Tissue Act No. 296/2008 Coll.). The authors present an organizational model based on cooperation between the majority of Czech Transplant Centers with a tissue establishment licensed by the national competent authority. In various points, we are addressing individual aspects of experimental and clinical studies which affect clinical practice. Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013. The growing number of centers participating in this programme led to a growing number of patients who underwent transplantation of vascular allografts. In 2015 the numbers of transplanted fresh versus cryopreserved allografts in the Czech Republic were almost equal. Cooperation of the participating centers in the Czech Republic with the licensed Tissue Establishment made it possible to achieve a full compliance with the European Union Directives, and harmonized national legal norms and assured a high quality of cryopreserved vascular allografts.

Cryopreserved human aortic root allografts arterial wall: Structural changes occurring during thawing.

BACKGROUND: The aim of our experimental work was to assess morphological changes of arterial wall that arise during different thawing protocols of a cryopreserved human aortic root allograft (CHARA) arterial wall. METHODS: The experiment was performed on CHARAs. Two thawing protocols were tested: 1, CHARAs were thawed at a room temperature at +23°C; 2, CHARAs were placed directly into a water bath at +37°C. MICROSCOPIC SAMPLES PREPARATION: After fixation, all samples were washed in distilled water for 5 min, and dehydrated in a graded ethanol series (70, 85, 95, and 100%) for 5 min at each level. The tissue samples were then immersed in 100% hexamethyldisilazane for 10 minutes and air dried in an exhaust hood at room temperature. Processed samples were mounted on stainless steel stubs, coated with gold. RESULTS: Thawing protocol 1: All 6 (100%) samples showed loss of the endothelium and damage to the subendothelial layers with randomly dispersed circular defects and micro-fractures without smooth muscle cells contractions in the tunica media. Thawing protocol 2: All 6 (100%) samples showed loss of endothelium from the luminal surface, longitudinal corrugations in the direction of blood flow caused by smooth muscle cells contractions in the tunica media with frequent fractures in the subendothelial layer. CONCLUSION: All the samples thawed at the room temperature showed smaller structural damage to the CHARA arterial wall with no smooth muscle cell contraction in tunica media when compared to the samples thawed in a water bath.

The composition and biomechanical properties of human cryopreserved aortas, pulmonary trunks, and aortic and pulmonary cusps.

Human cryopreserved allografts of pulmonary and aortic heart valves, aortas and pulmonary trunks are used for valve replacement. However, it is unknown how the composition of these allografts relate to their mechanical properties. Our aims were to correlate the histological compositions and passive mechanical properties of aortic and pulmonary valves and to observe the microcracks of aortas and pulmonary trunks. The following parameters were quantified: ultimate stress; ultimate strain; Young's modulus of elasticity; valve cusp wall thickness; pulmonary and aortic intima-media thickness; area fraction of elastin, collagen and calcification; and length density of elastic fibres. The propagation of experimentally induced microcracks avoided elastic fibres. Ultimate strain was negatively correlated with the area fraction of calcification (r=-0.4) in aortas. Ultimate stress (r=0.27) and Young's modulus in small deformation (r=0.29) and in large deformation (r=0.32) correlated with wall thickness in valve cusps. Young's modulus (r=0.34) and ultimate strain (r=0.31) correlated with intima-media thickness. Ultimate strain correlated with the area fraction of elastin (r=-0.40) and collagen in the arteries (r=0.31). As conventional histology does not fully explain the mechanical properties of cryopreserved grafts, both morphological and biomechanical tests should be used complementarily when characterizing the ageing of the grafts.

Myocardial iron content and mitochondrial function in human heart failure: a direct tissue analysis.

AIMS: Iron replacement improves clinical status in iron-deficient patients with heart failure (HF), but the pathophysiology is poorly understood. Iron is essential not only for erythropoiesis, but also for cellular bioenergetics. The impact of myocardial iron deficiency (MID) on mitochondrial function, measured directly in the failing human heart, is unknown. METHODS AND RESULTS: Left ventricular samples were obtained from 91 consecutive HF patients undergoing transplantation and 38 HF-free organ donors (controls). Total myocardial iron content, mitochondrial respiration, citric acid cycle and respiratory chain enzyme activities, respiratory chain components (complex I-V), and protein content of reactive oxygen species (ROS)-protective enzymes were measured in tissue homogenates to quantify mitochondrial function. Myocardial iron content was lower in HF compared with controls (156 ± 41 vs. 200 ± 38 µg·g-1 dry weight, P < 0.001), independently of anaemia. MID (the lowest iron tercile in HF) was associated with more extensive coronary disease and less beta-blocker usage compared with non-MID HF patients. Compared with controls, HF patients displayed reduced myocardial oxygen2 respiration and reduced activity of all examined mitochondrial enzymes (all P < 0.001). MID in HF was associated with preserved activity of respiratory chain enzymes but reduced activity of aconitase and citrate synthase (by -26% and -15%, P < 0.05) and reduced expression of catalase, glutathione peroxidase, and superoxide dismutase 2. CONCLUSION: Myocardial iron content is decreased and mitochondrial functions are impaired in advanced HF. MID in HF is associated with diminished citric acid cycle enzyme activities and decreased ROS-protecting enzymes. MID may contribute to altered myocardial substrate use and to worsening of mitochondrial dysfunction that exists in HF.

Coronéo extra-aortic annuloplasty ring could stabilize even the pulmonary autograft annulus in a Ross operation.

The Ross procedure is an alternative technique to mechanical prosthesis and recent evidence suggests that it is associated with improved long-term survival and quality of life. However, late pulmonary autograft failure can occur more frequently in patients with significant dilation of the aortic annulus. Here, we present a technical modification of the Ross procedure--a case report of a patient who underwent a Ross procedure in which the gross dilation of the aortic annulus was reduced by implantation of an extra-aortic Coronéo annuloplasty ring. Before discharge, we performed a multidetector computed tomography (MDCT) to verify the effect of extra-aortic annuloplasty on the geometry of the neoaortic root and particularly on the dimension of the aortic annulus. The result of the MDCT demonstrated that the ring sits in the optimal position at the level of aortoventricular base and that aortic annulus is significantly reduced.

Malignant Wegener's granulomatosis with fibrosing mediastinitis and vena cava superior syndrome.

A 47-year-old man was admitted to hospital for migratory joint pain, fatigue, and cough with bloody sputum and proteinuria with increased serum creatinine level. Diagnosis of Wegener's granulomatosis was established. During follow-up, the vena cava superior syndrome developed. The patient died of respiratory failure after 12 years of follow-up. The autopsy revealed rigid, whitish, 12 mm thick tissue, which embedded and compressed the large vessels upwards from their origin in the heart, thus causing vena cava superior syndrome. This tissue was composed of fibrous material without inflammatory cellulization. We consider this fibrous tissue as a manifestation of fibrosing mediastinitis that may or may not share pathogenesis with Wegener's granulomatosis.

Effect of donor/recipient body weight ratio, donor weight, recipient weight and donor age on kidney graft function in children.

BACKGROUND: We hypothesized that supplementing a higher mass of renal parenchyma from adult donors, and their younger age, would improve graft function in paediatric recipients. METHODS: We calculated estimated glomerular filtration rate (eGFR; Schwartz formula) and absolute glomerular filtration rate (absGFR) in 57 renal-grafted children (1995-2007) aged 3.1-17.9 years, weighing 12.9-85.0 kg, on discharge from the hospital after transplantation (TPL), 1 year after TPL and at the last follow-up (1.5-11.7 years after TPL). We correlated their eGFR with the individual ratio between the donor and the recipient body weight at the time of TPL (donor/recipient body weight ratio; D/R BWR), and we evaluated the effect of the donor and the actual recipient body weight on the eGFR and absGFR. RESULTS: The D/R BWR varied from 0.65 to 5.23. We found a significant positive correlation between D/R BWR and eGFR at discharge from the hospital (P < 0.001), 1-year post-TPL (P < 0.001) and at the last follow-up (P < 0.05). Using multiple linear regression analyses, we found that both eGFR and absGFR values were much more determined by the actual recipient weight than by the donor weight (27/6% and 43/4% at discharge, by 24/4% and 57/0% 1 year after TPL, and 0/0% and 20/0% at the end of the follow-up). A tendency for lower eGFR with increasing age of donors was apparent at discharge and 1 year after TPL, but it reached statistical significance only at the last follow-up (r = 0.4254, P < 0.01). CONCLUSION: In paediatric renal transplants, the value of D/R BWR directly correlated with eGFR in the early and late posttransplant periods. However, this correlation was mainly influenced by the recipient weight, while the donor weight played only a minor or negligible role.

Profiling proteinuria in children after renal transplantation.

Proteinuria is a common complication after renal transplantation (RTx). In adults, tubular proteinuria prevails and is associated with impaired graft survival. In the absence of studies on proteinuria profiling in transplanted children, we aimed at analyzing the types of proteinuria in transplanted children. Fifty-three children (11.8 years) were analyzed in a cross-sectional study. Morning urine was tested for total protein (PROT), albumin (ALB) and alpha-1-microglobulin (AMG). The type of proteinuria was assessed by the alpha-1-microglobulin/albumin algorithm (AAA): [AAA = AMG x 100/(AMG+ALB]. Median PROT, ALB, and AMG (in mg/mmol creatinine) were 20.0, 3.8, and 4.9, respectively. Pathological total proteinuria (>22 mg protein/mmol creatinine) was found in 47% of children (25/53). Only 20% of patients with pathological total proteinuria (5/25) had glomerular proteinuria, whereas 80% (20/25) had tubular proteinuria. Three of five children with glomerular proteinuria had chronic allograft nephropathy. Both AMG and albuminuria negatively correlated with the estimated glomerular filtration rate (eGFR) (p = 0.021 and 0.003, respectively). In conclusion, tubular proteinuria was present in 80% of children post-RTx and may be associated with impaired graft function; glomerular proteinuria is associated mainly with chronic allograft nephropathy.

Urinary tract infections in pediatric renal transplant recipients--a two center risk factors study.

UTI are common in renal Tx recipients and may significantly impact on the graft function. The aim of our study was to evaluate the prevalence, risk factors, and significance of UTI in Tx children. We performed a retrospective cross-sectional study of 76 Tx patients, median age at Tx was 13.4 yr. Twenty-one of 76 (28%) patients developed at least one UTI during the mean follow-up time of 3.3 +/- 2.0 yr post-Tx. The first UTI occurred at a median of 160 days post-Tx. The RR of having UTI was significantly higher in patients with the primary diagnosis of obstructive uropathy (RR = 2.6, 95th CI = 1.1-6.0, p = 0.032), history of PN pre Tx (RR = 2.7, 95th CI = 1.3-5.4, p = 0.009) and pre Tx VUR (RR = 2.2, 95th CI = 1.1-4.5, p = 0.045). These three factors also significantly decreased the infection-free survival time to the first UTI. Most UTI caused reversible acute allograft dysfunction, but the long-term graft function could not be reliably assessed with SCr. In conclusion, UTI occurred in 28% of pediatric Tx recipients, mostly during the first year post-Tx despite antibiotic prophylaxis. The diagnosis of obstructive uropathy, history of UTI and VUR prior to Tx were significant risk factors.

Kirschner wire migration from the right sternoclavicular joint to the heart: a case report.

Presented here is a rare case in which Kirschner wires migrated from the right sternoclavicular joint to the heart. A 29-year-old man suffering from sternoclavicular instability due to a motorcycle accident received surgical fixation with Kirschner wires. Six months after the surgery, the chest x-ray showed migration of the 3 broken wires to the anterior mediastinum and to the right hemithorax. The patient was asymptomatic and was scheduled for elective surgical extraction of the migrating wires because of the potential danger of injuring the mediastinal organs.During the surgery, the intracardiac location of all wires was discovered,and the wires were successfully extracted from the extracorporeal circulation. This rare and potentially lethal complication is discussed.

Extension of pulmonary adenocarcinoma into the left atrium.

A 62-year-old man presented with pulmonary adenocarcinoma that penetrated through the pulmonary vein into the left atrium. The tumor in the left atrium was removed via a right lower lobectomy under cardiopulmonary bypass. In selected cases, radical removal of a tumor in patients without mediastinal lymph node involvement may improve the prognosis. The use of cardiopulmonary bypass extends the possibilities of radical tumor removal.

Impact of processing on surface structure of human cardiac valve allografts.

Methods of processing and cryopreservation are believed to be the most important factors of long term clinical performance of biological heart valve prostheses. That is why we decided to cooperate in evaluating the impact of current AHV (allograft heartvalve) bank protocol on valve tissue morphology. AHV harvested from "heart-beating" cadaveric donors, considered as a fresh tissue, were compared with valve samples from non-heart beating donors, samples stored in saline, samples treated with antibiotic solution, and finally with cryopreserved valves, stored in liquid nitrogen for months. All samples were dissected, dried with hexamethyldisilazane (HMDS) method, gold-coated, studied and photographed in scanning electron microscope Tesla BS 301. Different superficial patterns were found on ventricular and vascular surfaces of "fresh" semilunar valves. We were able to detect early changes of endothelium after harvesting, denudation of endothelial covering during preservation with and without freezing. Our alternative method of drying samples by HMDS method proved to be suitable for thin membranes of human semilunar valves. Scanning electron microscopy seems to be helpful for morphological control of processing, cryopreservation and liquid nitrogen storage of AHV. We believe that further confrontation of morphological investigation with other methods helps us to develop more suitable protocol of handling AHV in heart valve banking.