Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Surgical repair of a coronary artery aneurysm associated with coronary artery fistula - a case report.

Coronary artery aneurysm is a relatively rare disorder that is usually discovered as a secondary finding in patients undergoing coronary artery angiography. Coronary artery fistulas are relatively more frequent than rare aneurysms and are often associated with other cardiac abnormalities. The etiology of aneurysms is mostly atherosclerotic, and they are less frequently associated with other acquired or congenital diseases, such as Kawasaki disease, connective tissue diseases, septic emboli, arteritis, and iatrogenic disease. We report a 70-year-old woman with a rare combination of a coronary artery aneurysm associated with a coronary artery fistula, which drained into the pulmonary artery. The diagnosis of our patient was made by selective coronary angiography and confirmed by computed tomography angiography. The patient was treated surgically because of the symptomatic course of the disease.

Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

OBJECTIVES AND DESIGN: At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS: Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS: Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION: Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

Comparison of Different Thawing Protocols in Human Cryopreserved Venous Grafts.

BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

Current vascular allograft procurement, cryopreservation and transplantation techniques in the Czech Republic.

BACKGROUND: Vascular allotransplantations are performed worldwide in selected patients suffering from vascular prosthesis infection or critical limb ischemia. Either fresh or cryopreserved vascular allograft may be used. OBJECTIVES: In various points, we address several aspects (allograft procurement, cryopreservation and transplantation technique) of the program of vascular allotransplantations in the Czech Republic. MATERIAL AND METHODS: Vascular grafts retrieval has been done within multiorgan harvests using no-touch technique. Very short time of cold ischemia is achieved due to close cooperation with Tissue Establishment where the following processing of cryopreservation is performed. Meeting all necessary quality criteria is a prerequisity for releasing grafts for clinical application. Standardized thawing protocol and surgical handling aims to minimize microfractures before implantation. RESULTS: Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013 in the Czech Republic. The developement of storage of vascular tissue in banks was stimulated in 2000-2010 by the issue of EU directives and national harmonized norms, aimed at assurance of high quality and safety of cells and tissues used for transplantations in humans. CONCLUSIONS: There are several crucial moments affecting final quality, including graft retrieval within a multiorgan harvest, short ischemic time, cryopreservation and thawing technique used. The recommended surgical handling during implantation may also affect results and graft-related complications.

Air Embolism into Superior Mesenteric Artery Following Replacement of Ascending Aorta for Aortic Dissection - A Rare and Fatal Case.

We report an unusual case of fatal air embolism into the superior mesenteric artery in a patient, who underwent replacement of the ascending aorta for aortic dissection type A. CT performed twice on the first postoperative day showed abundant air in the superior mesenteric artery and its branches (but not in the portal-venous system) indicating air embolism with no signs of bowel necrosis. On the second postoperative day, the patient underwent extensive bowel resection due to bowel ischemia and died on the third postoperative day on MODS/SIRS.

Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats.

OBJECTIVES AND DESIGN: The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. MATERIALS AND METHODS: Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. RESULTS: The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, p < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, p < 0.001, anti-MHC class II 80.9±2.7%). CONCLUSION: Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol.

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.

A rationale for early extracorporeal membrane oxygenation in patients with postinfarction ventricular septal rupture complicated by cardiogenic shock.

AIMS: Ventricular septal rupture (VSR) became a rare mechanical complication of myocardial infarction in the era of percutaneous coronary interventions but is associated with extreme mortality in patients who present with cardiogenic shock (CS). Promising outcomes have been reported with the use of circulatory support allowing haemodynamic stabilization, followed by delayed repair. Therefore, we analysed our experience with an early use of Veno-Arterial Extracorporeal Membrane Oxygenation (V-A ECMO) for postinfarction VSR. METHODS AND RESULTS: We conducted a retrospective search of institutional database for patients presenting with postinfarction VSR from January 2007 to June 2016. Data from 31 consecutive patients (mean age 69.5 ± 9.1 years) who were admitted to hospital were analysed. Seven out of 31 patients with VSR who were in refractory CS received V-A ECMO support preoperatively. ECMO improved end-organ perfusion with decreased lactate levels 24 hours after implantation (7.9 mmol/L vs. 1.6 mmol/L, p = 0.01), normalized arterial pH (7.25 vs. 7.40, p < 0.04), improved mean arterial pressure (64 mmHg vs. 83 mmHg, p < 0.01) and lowered heart rate (115/min vs. 68/min, p < 0.01). Mean duration of ECMO support was 12 days, 5 out of 7 patients underwent surgical repair, 4 were weaned from ECMO, 3 survived 30 days and 2 survived more than 1 year. The most frequent complication (5 patients) and the cause of death (3 patients) was bleeding. CONCLUSIONS: Our experience suggests that early V-A ECMO in patients with VSR and refractory CS might prevent irreversible multiorgan failure by improved end-organ perfusion. Bleeding complications remain an important limitation of this approach.

Juxtarenal Modular Aortic Stent Graft Infection Caused by Staphylococcus aureus.

Introduction. We are presenting a case report of an infected modular abdominal stent graft. Case Presentation. A 67-year-old male patient three years after Cook's modular abdominal aortic aneurysm (AAA) graft implantation for juxtarenal AAA with an implantation of a stent extension into the right common iliac artery for type Ib endoleak. The patient was admitted into our center in severe condition with suspected retroperitoneal bleeding. Computed tomography angiography (CTAG) confirmed retroperitoneal bleeding in the right common iliac artery. An urgent surgical revision was indicated; destructed arterial wall around the stent extension in the right common iliac artery was discovered. Due to the severe state of health of the patient, a resection of the infected stent and affected arterial wall was performed, followed by an iliac-femoral crossover bypass. The patient was transported to the intensive care unit with hepatic and renal failure, with maximal catecholamine support. Combined antibiotic treatment was started. The patient died five hours after the procedure. The cause of death was multiorgan failure caused by sepsis. Hemocultures and perioperative microbiological cultures showed the infection agent to be Staphylococcus aureus methicillin sensitive. Conclusion. Stent graft infection is a rare complication. Treatment is associated with high mortality and morbidity.

Differential impact on acute kidney injury incidence between on- and off pump coronary artery bypass grafting in octogenarians.

INTRODUCTION: Acute kidney injury (AKI) following surgical myocardial revascularization is associated with high mortality and morbidity. The aim of this study was to evaluate the risk of acute kidney injury in a population of very old patients following different surgical techniques. PATIENTS AND METHODS: A retrospective study of 310 consecutive patients aged 78 to 93 years, mean 80.5±2.2, who underwent surgery at one cardiac surgery centre. Based on the surgical technique used the patients were divided into: Group I. CABG (n=134) - surgical myocardial revascularization using extracorporeal circulation and arterial and venous grafts. Group II. OPCABG (n=55) - surgical revascularization without extracorporeal circulation but using arterial and venous grafts. Group III. NOTOUCH (n=121) - no handling with the ascending aorta was performed at all. RESULTS: A statistically insignificant renoprotective trend was found in patients who underwent surgery without extracorporeal circulation regardless of technique. Comparing groups II and III vs. group I, a significantly poorer renal functioning (median difference in creatinine was 10.0 (32.9) vs 17.5 (35.0), P=0.05) was shown for patients in group I. CONCLUSION: Surgical myocardial revascularization without extracorporeal circulation in very old patients is safe. The results of this study show a renoprotective trend.

Prediction of compartment syndrome based on analysis of biochemical parameters.

BACKGROUND: The possibility of predicting severe compartment syndrome using simple biochemical parameters was evaluated in a single-center study of 55 patients who presented with acute femoral embolism and who were treated with open surgical embolectomy. METHODS: Parameters related to tissue damage and oxidative metabolism (i.e., lactate, bilirubin, myoglobin, uric acid, glucose, and fibrinogen) were monitored in ipsilateral femoral vein blood. RESULTS: Several statistically significant predictors of relevant compartment syndrome after surgical reperfusion were found, including lactate, uric acid, transcutaneous oxygen pressure, bilirubin, intrafascial pressure, and serum myoglobin. Glycemia and serum albumin did not significantly change over time. CONCLUSIONS: The lactate concentration in femoral vein blood sampled during surgical embolectomy can be used for the stratification of additional postoperative risk of clinically significant compartment syndrome complicating reperfusion after acute embolism of the femoral artery.