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.

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.

Three-layer collagen-based vascular graft designed for low-flow peripheral vascular reconstructions.

INTRODUCTION: The aim of this study was to develop a prototype of an artificial blood vessel which has similar mechanical properties to a human saphenous vein graft and to experimentally verify the function of the prosthesis via ovine carotid bypass implantation. MATERIAL AND METHODS: The prototype of an artificial graft prosthesis for low flow was developed and manufactured from a collagenous matrix and reinforcing polyester mesh. We compared the results of both the pressurisation and the mechanical stress evaluation tests of VSM with four types of hybrid vascular graft. The most similar graft (type II) was chosen for the first ovine model implantation. RESULTS: Dominant behavior e.g. mechanical response of VSM graft in plots of circumferential and axial stress during loading is observed in circumferential direction. Average results of used VSM showed area of ideal mechanical response and the properties of artificial blood vessels were fitted into this area. Developed graft remained patent after 161 days of follow up in ovine model. CONCLUSIONS: The mechanical properties of the graft were designed and adjusted to be similar to the behaviour of human saphenous veins. This approach showed promising results and enhanced the final performance of the prosthesis.

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.

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.

Technical considerations for isolated limb perfusion: A consensus paper.

BACKGROUND: Isolated limb perfusion (ILP) is a well-established surgical procedure for the administration of high dose chemotherapy to a limb for the treatment of advanced extremity malignancy. Although the technique of ILP was first described over 60 years ago, ILP is utilised in relatively few specialist centres, co-located with tertiary or quaternary cancer centres. The combination of high dose cytotoxic chemotherapy and the cytokine tumour necrosis factor alpha (TNFα), mandates leakage monitoring to prevent potentially serious systemic toxicity. Since the procedure is performed at relatively few specialist centres, an ILP working group was formed with the aim of producing technical consensus guidelines for the procedure to streamline practice and to provide guidance for new centres commencing the technique. METHODS: Between October 2021 and October 2023 a series of face to face online and hybrid meetings were held in which a modified Delphi process was used to develop a unified consensus document. After each meeting the document was modified and recirculated and then rediscussed at subsequent meeting until a greater than 90% consensus was achieved in all recommendations. RESULTS: The completed consensus document comprised 23 topics in which greater than 90% consensus was achieved, with 83% of recommendations having 100% consensus across all members of the working group. The consensus recommendations covered all areas of the surgical procedure including pre-operative assessment, drug dosing and administration, perfusion parameters, hyperthermia, leakage monitoring and theatre logistics, practical surgical strategies and also post-operative care, response evaluation and staff training. CONCLUSION: We present the first joint expert-based consensus statement with respect to the technical aspects of ILP that can serve as a reference point for both existing and new centres in providing ILP.

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.

Cardiogenic shock in patient with posterior postinfarction septal rupture--successful treatment with extracorporeal membrane oxygenation (ECMO) as a ventricular assist device.

We present the case of a 62-year-old female patient admitted to our center for cardiogenic shock due to large inferior myocardial infarct. Echocardiography revealed dysfunction of left ventricle, dilation of right ventricle, mitral valve insufficiency, and a large posterior ventricular septal defect (VSD). Coronary angiography showed occlusion of the right coronary artery. An attempt of percutaneous coronary intervention (PCI) of right coronary and posterior descending artery was not successful due to old thrombi. Despite inotropes and intraaortic balloon pump (IABP) there was severe hemodynamic instability. Therefore, we commenced veno-arterial extracorporeal membrane oxygenation (ECMO) as a ventricular assist device (VAD). Immediately we obtained the stabilization of the patient and the improvement of the clinical conditions. The third day after implantation, the closure of the defect, mitral valve plasty, and bypass to posterior descending artery were performed. The patient was discharged from the hospital 59 days after the operation. Six months after the operation, the patient was in good condition.

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.

In Vivo Evaluation of Short-Term Performance of New Three-Layer Collagen-Based Vascular Graft Designed for Low-Flow Peripheral Vascular Reconstructions.

AIM: The aim of this study was to evaluate short-term patency of the new prosthetic graft and its structural changes after explantation. METHODS: The study team developed a three-layer conduit composed of a scaffold made from polyester coated with collagen from the inner and outer side with an internal diameter of 6 mm. The conduit was implanted as a bilateral bypass to the carotid artery in 7 sheep and stenosis was created in selected animals. After a period of 161 days, the explants were evaluated as gross and microscopic specimens. RESULTS: The initial flow rate (median ± IQR) in grafts with and without artificial stenosis was 120 ± 79 ml/min and 255 ± 255 ml/min, respectively. Graft occlusion occurred after 99 days in one of 13 conduits (patency rate: 92%). Wall-adherent thrombi occurred only in sharp curvatures in two grafts. Microscopic evaluation showed good engraftment and preserved structure in seven conduits; inflammatory changes with foci of bleeding, necrosis, and disintegration in four conduits; and narrowing of the graft due to thickening of the wall with multifocal separation of the outer layer in two conduits. CONCLUSIONS: This study demonstrates good short-term patency rates of a newly designed three-layer vascular graft even in low-flow conditions in a sheep model.

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.

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.

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.

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.

Early pericardial valve deterioration as a result of adhesions with native mitral valve.

We report an unusual case of early pericardial tissue valve prosthesis deterioration that required replacement. Four years after mitral valve replacement, 1 of the 3 leaflets of the valve was thickened and retracted in a fixed open position, which resulted in severe mitral insufficiency. The cause of this was adhesion of the leaflet and the patient's own retained posterior mitral valve leaflet. The finding was confirmed at operation and by histologic examination.

Surgical extirpation of glomus tumor from rare localization on the upper extremity.

Objective. To report on a very rare case of a glomus tumor manifested on the upper arm in a healthy young male patient. Case Presentation and Intervention. A 22-year-old male patient presented with bluish multifocal venous malformation on the left upper arm and was admitted for venous malformation excision. Pain, discomfort, and upper arm paraesthesia had been present for almost 6 years. Ultrasonography revealed septet tumor without blood flow in the subcutaneous region of anterior aspect of the upper arm. A multifocal venous malformation approximately 5-10 mm in diameter was excised. Histological examination showed dilated vascular area with proliferated glomus cells with round nucleus in the wall of dilated vascular structures. Based on histological examination, the final diagnosis was made as "glomangioma." Conclusion. Histological examination is the only method that can establish final diagnosis. Currently, the only available treatment for this type of tumor is surgical excision.

Robot-assisted aortoiliac reconstruction: A review of 30 cases.

OBJECTIVE: The feasibility of laparoscopic aortic surgery with robotic assistance has been sufficiently demonstrated. Reported is the clinical experience of robot-assisted aortoiliac reconstruction for occlusive disease and aneurysm performed using the da Vinci system. METHODS: Between November 2005 and June 2006, 30 robot-assisted laparoscopic aortoiliac procedures were performed. Twenty-seven patients were prospectively evaluated for occlusive disease, two patients for abdominal aortic aneurysm, and one for common iliac artery aneurysm. Dissections of the aorta and iliac arteries were performed laparoscopically using a transperitoneal direct approach technique, a modification of the Stádler method. The robotic system was used to construct anastomoses, to perform thromboendarterectomies and, in most of the cases, for posterior peritoneal suturing. RESULTS: Robot-assisted procedures were successfully performed in all patients. The robot was used to perform both the abdominal aortic and common iliac artery aneurysm anastomoses, the aortoiliac reconstruction with patch, and to complete the central, end-to-side anastomosis in another operation. Median operating time was 236 minutes (range, 180 to 360 minutes), with a median clamp time of 54 minutes (range, 40 to 120 minutes). Operative time is defined as the time elapsed from the initial incision to final skin closure. Median anastomosis time was 27 minutes (range, 20 to 60 minutes), and median blood loss was 320 mL (range, 100 to 1500 mL). No conversion was necessary, 30-day survival was 100%, median intensive care unit stay was 1.8 days, and median hospital stay was 5.3 days. A regular oral diet was resumed after a mean time of 2.5 days. CONCLUSION: Robot-assisted laparoscopic surgery is a feasible technique for aortoiliac surgery. The da Vinci robotic system facilitated the creation of the aortic anastomosis and shortened aortic clamp time in comparison with our laparoscopic techniques.

Diagnosis of vascular prosthesis infection with FDG-PET/CT.

Fluorodeoxyglucose positron emission tomography (FDG-PET) is on the verge of becoming an established imaging tool in the fields of clinical oncology, cardiology, and neurology. Because of the high glucose uptake of inflammatory cells, FDG scanning is an appropriate tool for use in tracing suspected inflammation or to evaluate infection. PET, although highly sensitive, often lacks the ability to define the precise anatomic location of abnormal FDG accumulation. The new PET/computed tomography (CT) technology provides precise registration of metabolic and structural imaging data in a single session. We report positive FDG-PET/CT findings in an infected vascular prosthesis 6 months after grafting. Our experience and a few available case reports support the hypothesis that FDG-PET/CT may have a promising role in future noninvasive diagnosis of infected vascular grafts.