Viability of Human Arterial Grafts Monitored by Comet Assay.

An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy.

The identification of patient-derived, tumor-reactive T cell receptors (TCRs) as a basis for personalized transgenic T cell therapies remains a time- and cost-intensive endeavor. Current approaches to identify tumor-reactive TCRs analyze tumor mutations to predict T cell activating (neo)antigens and use these to either enrich tumor infiltrating lymphocyte (TIL) cultures or validate individual TCRs for transgenic autologous therapies. Here we combined high-throughput TCR cloning and reactivity validation to train predicTCR, a machine learning classifier that identifies individual tumor-reactive TILs in an antigen-agnostic manner based on single-TIL RNA sequencing. PredicTCR identifies tumor-reactive TCRs in TILs from diverse cancers better than previous gene set enrichment-based approaches, increasing specificity and sensitivity (geometric mean) from 0.38 to 0.74. By predicting tumor-reactive TCRs in a matter of days, TCR clonotypes can be prioritized to accelerate the manufacture of personalized T cell therapies.

Reconstruction of Angstrom resolution exit-waves by the application of drift-corrected phase-shifting off-axis electron holography.

Phase-shifting electron holography is an excellent method to reveal electron wave phase information with very high phase sensitivity over a large range of spatial frequencies. It circumvents the limiting trade-off between fringe spacing and visibility of standard off-axis holography. Previous implementations have been limited by the independent drift of biprism and sample. We demonstrate here an advanced drift correction scheme for the hologram series that exploits the presence of an interface of the TEM specimen to the vacuum area in the hologram. It allows to obtain reliable phase information up to 2π/452 at the 1 Å information limit of the Titan 80-300 kV environmental transmission electron microscope used, by applying a moderate voltage of 250 V to a single biprism for a fringe spacing of 1 Å. The obtained phase and amplitude information is validated at a thin Pt sample by use of multislice image simulation with the frozen lattice approximation and shows excellent agreement. The presented method is applicable in any TEM equipped with at least one electron biprism and thus enables achieving high resolution off-axis holography in various instruments including those for in-situ applications. A software implementation for the acquisition, calibration and reconstruction is provided.

Langmuir analysis of electron beam induced plasma in environmental TEM.

The characterisation of the gas state under electron impact is of high importance for the understanding of materials in-situ environmental transmission electron microscopy (ETEM) experiments. We report on the formation of a dilute plasma state of Ar and He based on the development of a Langmuir probe as a plasma diagnostic tool for the differentially pumped volume at the TEM sample position in the octagon. In order to keep stray magnetic field influences of the objective lens small, and thus the results readily interpretable within existing theory, the experiments are performed in Lorentz mode. The applicability of Langmuir type analysis for the identification of plasma parameters, such as ion density, plasma temperature, sheath extension and electron energy distribution is examined. The systematic study as a function of gas pressure and beam current reveals cold plasma characteristics with electron temperatures of around 0.4 eV and ion and electron densities that are orders of magnitude below the expected values from ionisation cross sections. The loss of charged particles appears to be due to surface collisions at the electrically conducting pole piece surfaces as well as at the conductive parts of the TEM holder.

Partial resection of native and prosthetic arteriovenous fistula as a treatment of late infections complications - series of case reports.

INTRODUCTION: Infection of arteriovenous fistula (AV) used for hemodialysis (HD) is associated with massive bleeding, sepsis development, formation of metastatic infectious foci, and a high risk of AV loss. Urgent management of an infected AV is crucial for successful treatment and AV salvage. CASE REPORTS: We present the use of partial resection as a successful method of dealing with late AV infection in two cases. In case 1, the resection was performed due to an infection of the native arteriovenous fistula (AVF) with two defects above the drainage vein aneurysms. In case 2, partial resection and replacement of the prosthetic arteriovenous fistula (AVG) were done due to an infection of HD puncture site. The AVs remained patent in both cases, with no further signs of infection postoperatively and, most importantly, without the need to use a temporary HD catheter. CONCLUSION: The establishment of a new AV is limited by the quality of the venous and arterial systems. All surgical, interventional and non-surgical means should be used to safely maintain the created AV patent. Provided that the requirements of an early indication are met, partial resection of the AV is the method of choice for AV infections and allows us to avoid using a permanent dialysis catheter in our patients.

Increased Number of Mast Cells in Epicardial Adipose Tissue of Cardiac Surgery Patients With Coronary Artery Disease.

Chronic inflammation of adipose tissue is associated with the pathogenesis of cardiovascular diseases. Mast cells represent an important component of the innate defense system of the organism. In our work, we quantified mast cell number in epicardial adipose tissue (EAT), subcutaneous adipose tissue (SAT), and right atrial myocardium (RA) in patients undergoing open heart surgery (n=57). Bioptic samples of EAT (n=44), SAT (n=42) and RA (n=17) were fixed by 4 % paraformaldehyde and embedded into paraffin. An anti-mast cell tryptase antibody was used for immunohistochemical detection and quantification of mast cells. We also demonstrated immunohistochemically the expression of CD117 and chymase markers. In EAT of patients with coronary artery disease (CAD), higher incidence of mast cells has been found compared to patients without CAD (3.7±2.6 vs. 2.1±1.2 cells/mm(2)). In SAT and RA, there was no difference in the number of mast cells in CAD and non-CAD patients. Mast cells in SAT, EAT and RA expressed CD117 and chymase. An increased incidence of mast cells in EAT of CAD patients may indicate the specific role of these inflammatory cells in relation to EAT and coronary arteries affected by atherosclerosis.

Long-term experience with implantable treprostinil pumps in pulmonary arterial hypertension.

OBJECTIVES: To compare clinical parameters and quality of life in patients with pulmonary arterial hypertension (PAH) at the time of diagnosis, at the time of LenusPro pump implantation and during intravenous treptostinil treatment. METHODS: Seven patients with severe PAH treated with intravenous treptostinil via implantable LenusPro pumps were evaluated, including NYHA classification, six­minute walking test, BNP and quality of life assessment using the EQ-5D-5L questionnaire before and after pump implantation. RESULTS: No significant changes were observed in NYHA class and six­minute walking distance test. There was however a significant improvement in the quality of life and a decrease in BNP levels. The mean EQ-5D-5L index assessed during subcutaneous treptostinil treatment was significantly worse when compared to that assessed during its intravenous application (0.39 ± 0.24 vs 0.78 ± 0.28, p ˂ 0.05); the same is true about the pain/discomfort dimension. Complications occurred, namely one nonfatal pneumothorax, one nonfatal hemothorax, and one event of nonfatal treptostinil intoxication after refilling. CONCLUSIONS: In patients who do not tolerate subcutaneous treptostinil treatment, the use of the LenusPro implantable pump results in a significant improvement in quality of life with an acceptable safety profile (Tab. 2, Fig. 2, Ref. 19).

Predictors of survival in patients with pulmonary hypertension and acute right heart failure.

OBJECTIVES: The aim of this study was to analyse survival of patients with pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) hospitalized due to an acute right heart failure (ARHF) with emphasis on risk factors and effectiveness of treatment following current guidelines. METHODS: We retrospectively analysed 117 hospitalizations of 70 patients (59 PAH patients; 11 CTEPH patients, mean age 53.1 ± 16.77 years, 54 % females) between 2004 and 2013. RESULTS: 96 cases were hospitalized at cardiology wards (CW) while 21 at intensive care unit (ICU). The overall hospital mortality was 12.8 %, CW mortality was 4 %, and ICU mortality was 52.4 %. Higher risk of in-hospital mortality was associated with younger age, lower sodium levels, severe forms of PAH (heritable PAH, CTD-PAH) and need of PAH combination treatment. The one-year survival from the first ARHF hospitalization was 67.6 % (95 % CI 57.1-80 %), the two-year survival was 41.9 % (95 % CI 30.8-56.9 %). The presence of ascites was a predictor of long-term mortality. CONCLUSIONS: Mortality in patients with PH and ARHF remains very high. Identification of its risk factors could be used as basis of risk-adapted therapy (Tab. 5, Fig. 2, Ref. 14).

Sourdough bacterial dynamics revealed by metagenomic analysis in Brazil.

This study dealt with the influence of the temperature on the bacterial dynamics of two spontaneously fermented wheat sourdoughs, propagated at 21 ±â€¯1 °C (SD1) and 30 ±â€¯1 °C (SD2), during nine backslopping steps (BS1 to BS9). Proteobacteria was the only phylum found in flour. Escherichia hermannii was predominant, followed by Kosakonia cowanii, besides species belonging to the genera Pantoea and Pseudomonas. After one step of propagation, Clostridium and Bacillus cereus group became predominant. Lactobacillus curvatus was found at low relative abundance. For the second backslopping step, Clostridium was flanked by L. curvatus and Lactobacillus farciminis. From BS4 (6th day) onward, lactic acid bacteria (LAB) became predominant. L. farciminis overcame L. curvatus and remained dominant until the end of propagations for both sourdoughs. At 21 °C, Bacillus, Clostridium, Pseudomonas, and Enterobacteriaceae were gradually inhibited. At the end of propagation, SD1 harbored only LAB. Otherwise, the temperature of 30 °C favored the persistence of atypical bacteria in SD2, as Pseudomonas and Enterobacteriaceae. Therefore, the temperature of 21 °C was more suitable for sourdough propagation in Brazil. This study enhanced the knowledge of temperature's influence on microbial assembly and contributed to the elucidation of sourdough microbial communities in Brazil.

Ascending aorta replacement vs. total aortic arch replacement in the treatment of acute type A dissection: a meta-analysis.

OBJECTIVE: Acute type A aortic dissection (ATAAD) is a severe, rapidly progressing disease which typically requires patients to undergo emergency surgical intervention. Despite advancements in surgical procedures, still, ATAAD remains a surgical emergency associated with high mortality. The aim of this systematic review and meta-analysis was to compare whether either ascending aorta replacement (AR) or total aortic arch replacement (TR) leads to improved short- and long-term clinical outcomes. MATERIALS AND METHODS: A search of PubMed, Embase, Science Direct, Web of Science, SciELO, BIOSIS, and China National Knowledge Infrastructure (CNKI) databases were supplemented by searching through bibliographies of key articles. Thereafter, data on early and late prognostic factors were extracted. A systematic review and meta-analysis of 15 studies were performed to compare whether either AR or TR leads to a reduction in the risk of in-hospital and short-term mortality, postoperative complications, re-operation rate, and long-term mortality. RESULTS: A total of 15 cohort studies (n = 2822 patients with ATAAD; AR with HA, partial arch = 1911, TR = 911) were deemed eligible and included in the meta-analysis. Compared with TR, AR led to a significantly lower risk of in-hospital mortality (RR = 0.77; 95% CI: 0.61-0.96), shorter cardiopulmonary bypass time (CPB, mean difference = -53.09; 95% CI: -56.68--49.50), circulatory arrest time (CA, mean difference = -8.09; 95% CI: -9.04-7.15), and antegrade cerebral perfusion (ACP, mean difference = -28.62; 95% CI: -30.23--27.00). Differences in the incidence rates of neurological dysfunctions and renal dialysis were not significant. The pooled rate of aortic re-operation was lower in TR group (AR 7.6% vs. TR 5.3%), albeit not significantly (risk ratio = 1.39; 95% CI: 0.94-2.07; p = 0.10). CONCLUSIONS: These findings demonstrate that AR is associated with a lower early mortality rate and shorter operative times overall. Nevertheless, the incidence of postoperative complications in patients undergoing AR is comparable to that of patients undergoing TR. Further prospective follow-up data needs to be collected and analyzed to discern whether there are statistically significant differences in the risks of re-operation and long-term mortality between AR and TR procedures.

Development of vascular substitutes for low-flow peripheral bypass grafting - a review.

The development of a low-flow vascular prosthesis is a very topical issue. The authors present a pathway for the development of a prosthesis with optimal properties based on the idea of mimicking the characteristics of a biological model (saphenous vein graft) and programming these properties in the model of the prosthetic substitute. The vascular prosthesis presented consists of three layers - a non-absorbable scaffold representing vascular “media”, and two absorbable collagen layers - pseudointima and pseudoadventitia. The basic methods of physical testing are presented - the single axis stretch test and inflation-extension test, as well as other procedures that affect the final properties. These include collagen curing, antithrombotic treatment of the inner layer and the use of sterilization methods. The designed new graft was successfully implanted in an ovine model.

The influence of purification of carp collagen used in a novel composite graft with sandwich construction of the wall on its biological properties and graft patency rates.

We compared graft outcome between two types of a novel composite three-layer carp-collagen-coated vascular graft in low-flow conditions in a sheep model. Collagen in group A underwent more cycles of purification than in group B in order to increase the ratio between collagen and residual fat. The grafts were implanted end-to-side in both carotid arteries in sheep (14 grafts in 7 sheep in group A, 18 grafts in 9 sheep in group B) and artificially stenosed on the right side. The flow in the grafts in group A decreased from 297±118 ml/min to 158±159 ml/min (p=0.041) after placement of the artificial stenosis in group A, and from 330±164ml/min to 97±29 ml/min (p=0.0052) in group B (p=0.27 between the groups). From the five surviving animals in group A, both grafts occluded in one animal 3 and 14 days after implantation. In group B, from the six surviving animals, only one graft on the left side remained patent (p=0.0017). Histology showed degradation of the intimal layer in the center with endothelization from the periphery in group A and formation of thick fibrous intimal layer in group B. We conclude that the ratio between collagen and lipid content in the novel three-layer graft plays a critical role in its patency and structural changes in vivo.

Neoadjuvant Hypertermic Isolated Limb Perfusion in Treatment of Undifferentiated Spindle Cell Sarcoma of Lower Limb with Achieved Complete Pathologic Response.

BACKGROUND: Hyperthermic isolated limb perfusion uses therapeutic effect of hyperthermia in the bounded compartment of the limb together with increased concentration of chemotherapy effect than what would be achieved in systemic application. Gold standard was melphalan (Alkeran) in combination with tasonermin (Beromun, tumor necrosis factor alpha). The efficacy of this combination has been demonstrated in limb soft tissue sarcomas and in patients with limb isolated bulky disease of malignant melanoma. CASE: We describe a case of a 65-year-old female patient with undifferentiated spindle-cell sarcoma treated by a multidisciplinary team at the 2nd Surgical Clinic of Cardiovascular Surgery and Clinic of Oncology General University Hospital in Prague and at the Department of Orthopaedics Na Bulovce Hospital with the aim of preserving the limb despite the advanced disease. The patient underwent hyperthermic isolated limb perfusion with tasonermin and melphalan with partial response on magnetic resonance imaging. Subsequent wide resection was done with complete pathological remission according to histological examination maintaining a fully functional limb. The patient is followed without signs of recurrence. CONCLUSION: Hyperthermic isolated limb perfusion with tasonermin and melphalan is an important part of a multimodal approach in the treatment of extremity sarcomas with a high percentage of responses that increase the percentage of limbs retaining resections. Multidisciplinary team should consider this option in patients with localized limb sarcomas and should be performed in specialized centers with experience in this procedure. This work was supported by project Progres Q28-LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Flat Bands, Indirect Gaps, and Unconventional Spin-Wave Behavior Induced by a Periodic Dzyaloshinskii-Moriya Interaction.

Periodically patterned metamaterials are known for exhibiting wave properties similar to the ones observed in electronic band structures in crystal lattices. In particular, periodic ferromagnetic materials are characterized by the presence of bands and band gaps in their spin-wave spectrum at tunable GHz frequencies. Recently, the fabrication of magnets hosting Dzyaloshinskii-Moriya interactions has been pursued with high interest since properties, such as the stabilization of chiral spin textures and nonreciprocal spin-wave propagation, emerge from this antisymmetric exchange coupling. In this context, to further engineer the magnon band structure, we propose the implementation of magnonic crystals with periodic Dzyaloshinskii-Moriya interactions, which can be obtained, for instance, via patterning of periodic arrays of heavy metal wires on top of an ultrathin magnetic film. We demonstrate through theoretical calculations and micromagnetic simulations that such systems show an unusual evolution of the standing spin waves around the gaps. We also predict the emergence of indirect gaps and flat bands, effects that depend on the strength of the Dzyaloshinskii-Moriya interaction. Such phenomena, which have been previously observed in different systems, are observed here simultaneously, opening new routes towards engineered metamaterials for spin-wave-based devices.

Concomitant embolism in the subclavian and pulmonary artery.

INTRODUCTION: Pulmonary embolism is a life-threatening condition that causes obstruction of the pulmonary arteries by an embolus, most often originating from the venous system of the lower limbs or pelvic veins. Depending on the extent of the embolism, an acute right-sided heart failure may result, with subsequent death. Paradoxical embolism is a condition in which a venous thrombus is embolized into the systemic circulation arteries by a right-to-left heart shunt. This condition most commonly occurs in the interatrial septum. The aim of treatment for pulmonary embolism is to remove the obstruction of the lung. Depending on the extent of the embolism and the patients hemodynamic status, thrombolytic or surgical treatment can be chosen. Thrombolytic therapy has become the basis of treatment for all types of acute pulmonary embolism. Currently, open surgery is indicated in hemodynamically unstable patients with massive pulmonary embolism, and increasingly frequently in submassive embolism where thrombolytic therapy is not effective or contraindicated. CASE REPORT: The case report describes a less common condition of pulmonary and concurrently systemic embolization in the subclavian artery. Thrombolytic therapy was contraindicated in this case due to the high risk of secondary embolism to the brain. Surgical embolectomy was performed from the pulmonary arteries simultaneously with direct embolectomy from the subclavian and brachiocephalic artery. The surgical management of acute pulmonary embolism is a suitable method of treatment for both massive pulmonary embolism where the procedure is performed as a life-saving indication, and submassive embolism where less invasive treatment would not be effective. CONCLUSION: Direct embolectomy of subclavian and brachiocephalic artery is associated with minimal risk of iatrogenic embolism into the arteries supplying the brain compared to indirect embolectomy performed from the cubital or brachial artery. Key words: embolism paradoxical embolization - embolectomy foramen ovale stroke.

Changes in omentin levels and its mRNA expression in epicardial adipose tissue in patients undergoing elective cardiac surgery: the influence of type 2 diabetes and coronary heart disease.

Omentin is a protein produced by numerous tissues including adipose tissue. Its concentrations are decreased in patients with obesity, type 2 diabetes mellitus (DM) and coronary artery disease (CAD). Experimental studies suggest that omentin may have anti-inflammatory and insulin-sensitizing properties. In the present study, we measured circulating omentin levels and its mRNA expression in epicardial and subcutaneous fat, intercostal and heart muscle before and after elective cardiac surgery in patients with CAD (CAD+, DM-, n=18), combination of CAD and DM (CAD+, DM+, n=9) or with none of these conditions (CAD-, DM-, n=11). The groups did not differ in baseline anthropometric and biochemical characteristics with the exception of higher blood glucose and HBA(1c) in CAD+, DM+ group. Baseline circulating omentin levels tended to be lower in CAD+, DM- and CAD+, DM+ groups as compared to CAD-, DM- group and cardiac surgery increased its concentration only in CAD-, DM- group. The change in serum omentin levels during surgery inversely correlated with epicardial fat thickness. While baseline omentin mRNA expression did not differ among the groups in any of the studied tissues, its increase after surgery was present only in subcutaneous fat in CAD-, DM- and CAD+, DM- groups, but not in CAD+, DM+ group. Intercostal muscle omentin mRNA expression increased after surgery only in CAD-, DM- group. In conclusion, cardiac surgery differentially affects omentin levels and subcutaneous fat and skeletal muscle mRNA expression in patients without coronary artery disease and diabetes as compared to patients with these conditions.

Branched pedal bypass in the treatment of critical limb ischemia - a single center experience.

INTRODUCTION: Pedal bypass is a therapeutic option for limb salvage in critical limb ischemia. METHOD: Retrospective analysis of 20 patients who underwent either simple (14 patients) or branched (6 patients) pedal bypass in a single center and had at least one postoperative follow-up. In patients with branched pedal bypass, the main trunk was connected to the pedal artery of better quality and the side branch to an artery on the opposite side of the foot. The main trunk was constructed as a reverse saphenous bypass in all patients with branched bypass. From patients with simple bypass, nine had reverse saphenous graft, three had an allograft, one patient had in situ saphenous graft, and one PTFE prosthesis. RESULTS: The difference in the operation time was not significant. One patient with simple bypass required reoperation for wound bleeding and there was one case of perioperative mortality in the same group. The difference between the groups in the primary or secondary patency rates, limb salvage and overall survival was not significant. CONCLUSIONS: We did not find any significant difference in patency rates, limb salvage, or perioperative complications between patients with simple and branched pedal bypass in our cohort. We believe that the anticipated benefits of constructing an additional branch are masked by the disadvantageous presence of an additional anastomosis. Adoption of a branched pedal bypass may therefore require further improvements - perhaps the utilization of a branched saphenous graft. Key words:  critical limb ischemia - peripheral arterial disease - bypass - revascularization - amputation.

Dunbar syndrome - single-center experience with surgical treatment.

INTRODUCTION: Dunbar syndrome is caused by compression of the truncus coeliacus (TC), most commonly by the median arcuate ligament. Chronic irritation of the TC during breathing leads to fibrous changes of the arterial wall and formation of fixed stenosis. This compression syndrome is often associated with specific complaints including weight loss and early postprandial epigastric pain. In this study, we summarize our experience with a group of 14 patients from a single institution. METHODS: In 14 patients who were diagnosed with Dunbar syndrome and who were referred for surgery, we performed an invasive measurement of systemic pressure in a. radialis during the operation and compared it with invasively measured pressure in a. gastrica sinistra before and after the release of TC. In patients with significant stenosis (pressure gradient above 15 mmHg), a bypass was performed. RESULTS: The initial pressure gradient of 56±19 mmHg decreased after the release of TC to 39±16 mmHg (p.

Spin caloritronic nano-oscillator.

Energy loss due to ohmic heating is a major bottleneck limiting down-scaling and speed of nano-electronic devices, and harvesting ohmic heat for signal processing is a major challenge in modern electronics. Here, we demonstrate that thermal gradients arising from ohmic heating can be utilized for excitation of coherent auto-oscillations of magnetization and for generation of tunable microwave signals. The heat-driven dynamics is observed in Y3Fe5O12/Pt bilayer nanowires where ohmic heating of the Pt layer results in injection of pure spin current into the Y3Fe5O12 layer. This leads to excitation of auto-oscillations of the Y3Fe5O12 magnetization and generation of coherent microwave radiation. Our work paves the way towards spin caloritronic devices for microwave and magnonic applications.Harvesting ohmic heat for signal processing is one of major challenges in modern electronics and spin caloritronics, but not yet well accomplished. Here the authors demonstrate a spin torque oscillator device driven by pure spin current arising from thermal gradient across an Y3Fe5O12/Pt interface.

[Isolated Perfusion of the Upper Extremity with TNF-α - Double Venous Cannulation]. / Izolovaná perfuze horní koncetiny s TNF-α - dvojitá kanylace zilního systému.

BACKGROUND: The authors present a technical variation of the standard cannulation for cardiopulmonary bypass perfusion during hyperthermic isolated limb perfusion (ILP) procedures in selected patients with unresectable soft tissue sarcoma or malignant melanoma. PATIENTS: Of 55 ILP procedures performed at our institution since the procedure was established in 2009, nine were performed at the upper extremity. Standard single venous cannulation was used in five cases, and extended, double venous cannulation in the last four. The standard technique for brachial vein cannulation in a small compartment of the upper extremity entails a problematic and longer perfusion of the upper extremity. This is due to the lower flow rate in the venous system and relatively large surface area with respect to weight. We present a simple technique based on a "Y" cannulation of the venous system via the deep brachial vein and superficial venous system via the basilic vein, delivering a 20% increase in flow rate in the extracorporeal circulation. Faster heating of the upper extremity and a stable thermal environment throughout upper-extremity ILP are essential for successful treatment. CONCLUSION: Extended technique of venous cannulation for extracorporeal circulation setting, due to their advantages, became standard in the upper limb ILP procedure at our institution.Key words: isolated limb perfusion - malignant melanoma - soft tissue sarcoma - upper limb - extracorporeal circulation The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 8. 1. 2017Accepted: 15. 1. 2017.