Clinical relevance of cell-free DNA during venovenous extracorporeal membrane oxygenation.

BACKGROUND: Thrombosis remains a critical complication during venovenous extracorporeal membrane oxygenation (VV ECMO). The involvement of neutrophil extracellular traps (NETs) in thrombogenesis has to be discussed. The aim was to verify NETs in the form of cell-free DNA (cfDNA) in the plasma of patients during ECMO. METHODS: A fluorescent DNA-binding dye (QuantifFluor®, Promega) was used to detect cell-free DNA in plasma samples. cfDNA concentrations from volunteers (n = 21) and patients (n = 9) were compared and correlated with clinical/technical data before/during support, ECMO end and time of a system exchange. RESULTS: Before ECMO, patients with a median (IQR) age of 59 (51/63) years, SOFA score of 11 (10/15), and ECMO run time of 9.0 (7.0/19.5) days presented significantly higher levels of cfDNA compared to volunteers (6.4 (5.8/7.9) ng/µL vs. 5.9 (5.4/6.3) ng/µL; p = 0.044). Within 2 days after ECMO start, cfDNA, inflammatory, and hemolysis parameters remained unchanged, while platelets decreased (p = 0.005). After ECMO removal at the end of therapy, cfDNA, inflammation, and coagulation data (except antithrombin III) remained unchanged. The renewal of a system resulted in known alterations in fibrinogen, d-dimers, and platelets, while cfDNA remained unchanged. CONCLUSION: Detection of cfDNA in plasma of ECMO patients was not an indicator of acute and circuit-induced thrombogenesis.

A solution for global hygienic challenges regarding the application of heater-cooler systems in cardiac surgery.

71.759 surgical procedures were performed in 2019 with the aid of cardiopulmonary bypass in Germany. To adjust the patient's body temperature on extracorporeal circulation, the application of a heater-cooler unit (HCU) is mandatory. However, in case of insufficient sanitisation of HCU, life-threatening infections can be transmitted by the device to the patients, including Legionella bacteria, Mycobacterium chimaera, Pseudomonas aeruginosa. To avoid disease transmission, as a requirement for safe medical practice established by regulatory authorities, HCUs must be regularly disinfected by hazardous chemicals posing a danger for both handling humans and the environment. Therefore, to comply with regulations, HCU manufacturers have introduced both timely and financially extensive sanitisation procedures. Our paper describes a novel, effective and easy to handle disinfection method for the above problematics without utilising hazardous chemicals. The method's technical principle is electrolysis, resulting in drinking water quality regarding the analysed germs in the worldwide most commonly utilised heater-cooler device. The main aim of the study was to prove the efficacy and reliability of the device cleansing process. Furthermore, the economic impact of the novel method was evaluated. Therefore, we have undertaken 60 microbiological sampling series between December 2019 and November 2020 from a conventional HCU (3T LivaNova, Germany). During the total investigational period, no contamination with Pseudomonas aeruginosa or Legionellae could have been demonstrated in the HCU. The extreme slow-growing nontuberculous M. chimaera was detected only in one sample obtained from diamond electrode cleansed HCU water, and source of contamination was promptly eliminated by a simple technical modification of the device test-site. Additionally, the diamond electrode application is beneficial for eliminating potentially hazardous cleansing material from the process, which may affect otherwise both patients operated on cardiopulmonary bypass and the perfusionists.

Coagulation abnormalities in patients with COVID-19 on venovenous ECLS increased risk for technical complications and support times but had no impact on survival.

BACKGROUND: Patients with severe coronavirus disease-19 (COVID-19)-associated acute respiratory distress on venovenous extracorporeal lung support (V-V ECLS) showed a high incidence of vascular as well as ECLS-related thrombotic complications. The latter may influence the outcome of the patients. METHODS: This is a retrospective monocentric study on prospectively collected data of technical complications including 69 adult COVID-19 patients on V-V ECLS (ECLS Registry, March 2020 until April 2021) without and with system exchanges. Alterations in ECLS-specific data, hemolysis, coagulation, and hemostasis parameters were analyzed. RESULTS: Every second COVID-19 patient on V-V ECLS developed technical complications. Optimized ECLS management at our ECLS center reduced cases of acute clot formation (pump head thrombosis, acute oxygenator thrombosis) (17%), and allowed early identification of progressive clotting processes (worsened gas transfer, coagulation disorder) (14%, 54%) with a significant overhang of hyperfibrinolysis (37%). Although COVID-19 disease and technical complications caused the prolonged length of stay at the intensive care unit and ECLS support times, the proportion of successful weaning and survival rates were comparable with patients without system exchange. CONCLUSION: The survival of ECLS patients with COVID-19 was independent of the requirement for system exchange due to technical-induced coagulation disorders. Close monitoring for circuit clotting is mandatory in COVID-19 patients and is one prerequisite for successful organ support in these difficult patients.

Introduction of a New ex vivo Porcine Coronary Artery Model: Evaluation of the Direct Vascular Injury after Stent Implantation with and without Dogbone Effect.

INTRODUCTION: Neointimal hyperplasia after percutaneous coronary intervention remains a major determinant of in-stent restenosis (ISR). The extent of mechanical vessel injury correlates with ISR. A new ex vivo porcine stent model was introduced and evaluated comparing different stent designs. METHODS: Coronary arteries were prepared from pig hearts from the slaughterhouse and used for ex vivo implantations of coronary stents. One basic stent design in two configurations (dogbone, DB; nondogbone, NDB) was used. Vascular injury was determined according to a modified injury score (IS). RESULTS: Standardized experimental conditions ensured comparable vessel dimensions and overstretch data. DB stents caused more severe IS compared to NDB stents. The mean IS and the IS at the distal end of all stents were significantly reduced for NDB stents (ISMean, DB, 1.16 ± 0.12; NDB, 1.02 ± 0.12; p = 0.018; ISDist, DB, 1.39 ± 0.28; NDB, 1.13 ± 0.24; p = 0.03). DISCUSSION/CONCLUSION: The introduced ex vivo model allowed the evaluation of different stent designs, which exclude unfavorable stent designs.

Anti-oxidative effect of the tyrosine kinase inhibitor nintedanib: a potential therapy for chronic lung allograft dysfunction?

Background: The long-term survival after lung transplantation (LTx) is often limited by the development of chronic lung allograft dysfunction (CLAD). Increased oxidative stress has been found to occur in chronic lung allograft dysfunction because of several risk factors, e.g. immunological factors or drug related factors. The aim of this study was to investigate the anti-oxidative effect of the receptor tyrosine kinase (RTK) inhibitor nintedanib on immunologically induced oxidative stress and on drug induced oxidative stress.Methods: In-vivo studies were used for investigation of immunologically induced oxidative stress: Immunohistochemistry of transglutaminase-2 (TGM-2) was used to figure out a potential anti-oxidative effect of receptor tyrosine kinase inhibitor nintedanib in a rat model of allogeneic left LTx. In-vitro studies were used for investigation of drug induced oxidative stress: Cell viability assay, 2'7'-dichlorodihydrofluorescein diacetate (DCFDA) and immunofluorescence of transglutaminase-2 were disposed to examine the potential impact of nintedanib on cyclosporin A (CsA) treated lung fibroblasts of the rat.Results: In-vivo studies: Allogeneic transplanted animals without drug interaction showed severe chronic rejection and an excessive expression of TGM-2, whereas the application of nintedanib significantly decreased the number of TGM-2 positive cells. In-vitro studies: Concentrations of CsA ranging from 250 ng/ml to 500 ng/ml demonstrated oxidative stress caused by an increased production of reactive oxygen species (ROS) and an overexpression of TGM-2 without inducing apoptosis in cells. Concentrations of more than 1000 ng/ml led to a considerable decrease of cellularity. 30 min-pre-incubation with nintedanib at a concentration between 25 and 100 nM reduced generation of intracellular ROS and expression of TGM-2.Conclusion: These results demonstrate a downregulation of ROS and TGM-2 by pretreatment with the receptor tyrosine kinase inhibitor nintedanib and present its potential anti-oxidative and immunomodulatory effect in the treatment of chronic lung allograft dysfunction.

Accumulations of von Willebrand factor within ECMO oxygenators: Potential indicator of coagulation abnormalities in critically ill patients?

Clot formation within membrane oxygenators (MOs) remains a critical problem during extracorporeal membrane oxygenation (ECMO). The composition of the clots-in particular, the presence of von Willebrand factor (vWF)-may be an indicator for prevalent nonphysiological flow conditions, foreign body reactions, or coagulation abnormalities in critically ill patients. Mats of interwoven gas exchange fibers from randomly collected MOs (PLS, Maquet, Rastatt, Germany) of 21 patients were stained with antibodies (anti-vWF and anti-P-selectin) and counterstained with 4',6-diamidino-2-phenylindole. The extent of vWF-loading was correlated with patient and technical data. While 12 MOs showed low vWF-loadings, 9 MOs showed high vWF-loading with highest accumulations close to crossing points of adjacent gas fibers. The presence and the extent of vWF-fibers/"cobwebs," leukocytes, platelet-leukocyte aggregates (PLAs), and P-selectin-positive platelet aggregates were independent of the extent of vWF-loading. However, the highly loaded MOs were obtained from patients with a significantly elevated SOFA score, severe thrombocytopenia, and persistent liver dysfunction. The coagulation abnormalities of these critically ill patients may cause an accumulation of the highly thrombogenic and elongated high-molecular-weight vWF multimers in the plasma which will be trapped in the MOs during the ECMO therapy.

Quantitative assessment of peripheral limb perfusion using a modified distal arterial cannula in venoarterial ECMO settings.

In cases of severe cardiopulmonary deterioration, quick establishment of venoarterial extracorporeal membrane oxygenation (ECMO) represents a support modality. After successful arterial peripheral cannulation, a certain grade of peripheral limb malperfusion is a fairly common phenomenon. Detection of peripheral malperfusion is vital, since it can result in compartment syndrome or even loss of the affected limb. To prevent or resolve emerging lower limb ischaemia, a newly designed perfusion catheter is placed into the superficial femoral artery, distal to the arterial cannula via ECMO. The aim of our study was to evaluate flow and haemodynamic characteristics of this novel distal limb perfusion cannula for ECMO therapy and present these important findings for the first time. The distal perfusion cannula blood flow increases in linear correlation with ECMO blood flow The variability of distal perfusion cannula blood flow with a 15 Fr cannula ranges between 160 ± 0.40 mL min-1 at 1.5 L min-1 ECMO flow rate and 480 ± 80 mL min-1 at 5.0 L min-1 ECMO blood flow, respectively. Comparatively, the 17-Fr-sized cannula performs on a scale of 140 ± 20 to 390 ± 60 mL distal perfusion cannula blood flow at 1.5-5.0 L min-1 ECMO blood flow, respectively. The quantitative assessment of the distal perfusion cannula blood flow has revealed that distal perfusion cannula blood flow can measure up to 10% of the ECMO blood flow. Furthermore, it has been also well demonstrated that the novel distal perfusion cannula is sufficient to compensate peripheral limb ischaemia.

The activity of nintedanib in an animal model of allogenic left lung transplantation resembling aspects of allograft rejection.

AIM OF THE STUDY: The prevention and treatment of chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx) remain unsatisfactory. Growth factors may play an important role in the development of CLAD. This study evaluated the effects of nintedanib, a receptor tyrosine kinase inhibitor, in the treatment of CLAD after experimental LTx. MATERIALS AND METHODS: A rat model of left lung allo-transplantation (Fisher 344 to Wistar Kyoto) was used to evaluate the effect of nintedanib (50 mg/kg per day) on the development of CLAD. Therapy with nintedanib began 2 days before LTx and ended on postoperative day (POD) 20 (n = 6) or 60 (n = 6). Nontreated animals who underwent LTx (n = 12) were used as controls, whereas naïve lungs (n = 24) served as reference for physiological healthy organs without transplantation damage or medical effects. Acute and chronic rejection were evaluated on POD 20 and 60, respectively. RESULTS: Immunohistologic analysis showed a decrease in growth factors/receptors on POD 60 (nintedanib-treated vs. nontreated controls: platelet-derived growth factor (PDGF) A: [P ≤ 0.001]; PDGF receptor-α: [P ≤ 0.001]; vascular endothelial growth factor (VEGF) A: [P ≤ 0.001]; VEGF receptor-2: [P ≤ 0.001]). However, no reductions in fibrotic changes were observed in nintedanib-treated allografts compared with nontreated allografts. Although nintedanib treatment started before LTx none of the animals showed impaired wound healing. No dehiscence of the sutures of the bronchus, vessels or skin, or stenosis of the bronchus was found. CONCLUSION: In conclusion, while nintedanib reduced the expression of growth factors/receptors in a rat LTx model, a reduction in fibrotic alterations was not observed at POD 60.

Hemostatic Changes During Extracorporeal Membrane Oxygenation: A Prospective Randomized Clinical Trial Comparing Three Different Extracorporeal Membrane Oxygenation Systems.

OBJECTIVE: Extracorporeal membrane oxygenation is a rescue therapy for patients with severe lung failure. Major complications caused by extracorporeal membrane oxygenation are bleeding, thrombosis, and hemolysis. The aim of this study was to compare the impact of different extracorporeal membrane oxygenation systems on blood hemostasis in adults during veno-venous extracorporeal membrane oxygenation therapy. DESIGN: Single center prospective randomized study. SETTING: University Hospital Regensburg, Germany. PATIENTS: Adult patients with severe acute respiratory distress syndrome requiring veno-venous extracorporeal membrane oxygenation therapy. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Three different extracorporeal membrane oxygenation systems: the Cardiohelp system (Maquet Cardiopulmonary AG), the Dideco ECC.O5 (Sorin Group), and the Deltastream system with Hilite 7000 LT + DP3 pumphead (Medos Medizintechnik AG) were compared. Therefore hemostasis, anticoagulation, hemolysis, and inflammatory parameters were monitored. Of the 54 patients included in the study, 18 patients each were randomly assigned to the three different extracorporeal membrane oxygenation systems. Exclusion criteria were acute renal failure, trauma, and surgery within 2 days. The median time on veno-venous extracorporeal membrane oxygenation support was 13.5 days (4-70 d). Median platelet count had dropped from 220.5 G/L before extracorporeal membrane oxygenation therapy to a minimum of 133 G/L by the last day of extracorporeal membrane oxygenation support. During the first 5 days of extracorporeal membrane oxygenation therapy, prothrombin fragment 1.2 (F1.2) (1.36-2.4 µM), thrombin-antithrombin complex (14.5-50 µg/L), and D-dimers (6.00-27.0 mg/L) increased, whereas fibrinogen values dropped from 5.8 to 4.1 g/L. The three different extracorporeal membrane oxygenation systems did not show any differences with regard to hemostasis, anticoagulation, hemolysis, and inflammatory parameters within the first 5 days of extracorporeal membrane oxygenation therapy. CONCLUSIONS: Over time, miniaturized veno-venous extracorporeal membrane oxygenation therapy increasingly activates coagulation. The different types of membrane oxygenators and pumps did not significantly alter hemostasis.

Existence of circulating anti-endothelial cell antibodies after heart transplantation is associated with post-transplant acute allograft rejection.

Anti-endothelial cell antibodies (AECA) may be involved in the development of heart allograft rejection. Its detection might be a cheap and noninvasive method to identify high-risk patients. An indirect immunofluorescence method on human umbilical vein endothelial cells was used to investigate the presence of AECAs in 260 pre- and post-transplant serum samples sequentially collected from 34 patients within the first year after heart transplantation (HTX). The presence of AECAs before (23.5 %) and early after HTX (14.7 %) was associated with a significantly increased risk of early acute rejection (75 and 60 %, respectively) compared to 33 % in AECA-negative patients (p = 0.049). Moreover, rejections from AECA-positive patients were more severe (p = 0.057) with a significantly increased incidence of multiple (p = 0.025). The mean number of the sum of rejection episodes was significantly higher in AECA-positive patients (p ≤ 0.05). Patients free of AECAs mainly received mycophenolate mofetil as primary immunosuppression (p = 0.067). Nevertheless, the presence of AECAs did not affect long-term outcome and mortality of HTX patients. Despite a low number of patient samples, the detection of AECAs before and early after HTX could be used as a biomarker for an increased risk of early acute rejection in high-risk patients. This easy method might be a valuable tool to support screening procedures to improve individualized immunosuppressive therapy.

Accumulation of Multipotent Progenitor Cells on Polymethylpentene Membranes During Extracorporeal Membrane Oxygenation.

Multipotent progenitor cells were mobilized during pediatric extracorporeal membrane oxygenation (ECMO). We hypothesize that these cells also adhered onto polymethylpentene (PMP) fibers within the membrane oxygenator (MO) during adult ECMO support. Mononuclear cells were removed from the surface of explanted PMP-MOs (n = 16). Endothelial-like outgrowth and mesenchymal-like cells were characterized by flow cytometric analysis using different surface markers. Spindle-shaped attaching cells were identified early, but without proliferative activity. After long-term cultivation palisading type or cobblestone-type outgrowth cells with high proliferative activity appeared and were characterized as (i) leukocytoid CD45+/CD31+ (CD133+/VEGFR-II+/CD90+/CD14+/CD146dim/CD105dim); (ii) endothelial-like CD45-/CD31+ (VEGF-RII+/CD146+/CD105+/CD133-/CD14-/CD90-); and (iii) mesenchymal-like cells CD45-/CD31- (CD105+/CD90+/CD133dim/VEGFR-II-/CD146-/CD14-). The distribution of the cell populations depended on the MO and cultivation time. Endothelial-like cells formed capillary-like structures and did uptake Dil-acetylated low-density lipoprotein. Endothelial- and mesenchymal-like cells adhered on the surface of PMP-MOs. Further research is needed to identify the clinical relevance of these cells.

D-dimers Are a Predictor of Clot Volume Inside Membrane Oxygenators During Extracorporeal Membrane Oxygenation.

Thrombosis inside the membrane oxygenator (MO) is a critical complication during venovenous extracorporeal membrane oxygenation (ECMO). The aim of this study was to prove if thrombotic clots manifest within the MO when D-dimer levels are elevated over a long-term period. Heparin-coated polymethylpentene MOs (n = 13) were exchanged due to high plasma D-dimer levels. Clot volume was calculated using multidetector computed tomography (MDCT). Coagulation parameters and MO function were analyzed before and after MO exchange. Before MO exchange, D-dimer levels increased significantly in each patient (11.5 [6.5-15.5] mg/L to 35.0 [34-35] mg/L, P ≤ 0.001). High levels of D-dimers were tolerated for 1 to 6 days. Additionally, fibrinogen concentration (n = 8) and platelet count decreased (n = 8). Within 48 h after exchange, D-dimer levels decreased significantly (n = 11, 12 [8-16] mg/L, P = 0.004). Fibrinogen concentration and platelet counts increased. Clots were found in all MOs in the inlet part of the device. Clot volume (16-106 cm(3) ) did not correlate with MO support time but increased significantly when high D-dimer levels were accepted for >2 days. An increase or high levels of D-dimers in absence of other explaining pathology during ECMO therapy reflected coagulation activity within the MO. Evidence of clots within the MO at high D-dimer levels and decrease after exchange underline the relevance of D-dimer testing during ECMO treatment. Besides, surveillance of MOs during ongoing ECMO therapy will help to predict clot formation, and to avoid system-induced coagulation disorders as well as critical situations.

Flow dynamics of different adult ECMO systems: a clinical evaluation.

Membrane oxygenator (MO) failure is a known hazard during venovenous extracorporeal membrane oxygenation (v-v ECMO) therapy. Knowledge about technical and performance details of different ECMO systems (Maquet, Rastatt, Germany; Medos, Stolberg, Germany; Sorin, Modena, Italy) licensed for adults with acute lung failure might improve their handling. This retrospective study comprises 186 adult patients (Regensburg ECMO Registry) treated with v-v ECMO. Flow dynamic data were used to analyze the performance of different blood pumps, cannula types, and MOs to maintain an adequate blood flow (1-5 L/min). Usage of the Medos ECMO system in critically ill patients required a higher pump speed and generated a higher pressure drop across the MO (dpMO), however, without an increase in free plasma hemoglobin. The dpMO depended on the type of MO and increased with blood flow as expected. Type-specific normal values are reported. A distinct increase in dpMO above normal values within 1 day required an immediate MO exchange. This was an infrequent technical complication (3%). Finally, pressure-flow performance of single dual-lumen cannulas (27 Fr) was comparable with small single-lumen cannulas (15 Fr), without an increased risk of technical-induced hemolysis. Despite different performances, all current commercially available adult v-v ECMO systems produce adequate blood flow without an increased risk in technical-induced hemolysis. Familiarity with the specific properties of individual systems allows early detection of technical complications. Additionally, the choice of an adequate cannula requires a closer consideration of the individual patient situation.

Computer based visualization of clot structures in extracorporeal membrane oxygenation and histological clot investigations for understanding thrombosis in membrane lungs.

Extracorporeal membrane oxygenation (ECMO) was established as a treatment for severe cardiac or respiratory disease. Intra-device clot formation is a common risk. This is based on complex coagulation phenomena which are not yet sufficiently understood. The objective was the development and validation of a methodology to capture the key properties of clots deposed in membrane lungs (MLs), such as clot size, distribution, burden, and composition. One end-of-therapy PLS ML was examined. Clot detection was performed using multidetector computed tomography (MDCT), microcomputed tomography (µCT), and photography of fiber mats (fiber mat imaging, FMI). Histological staining was conducted for von Willebrand factor (vWF), platelets (CD42b, CD62P), fibrin, and nucleated cells (4', 6-diamidino-2-phenylindole, DAPI). The three imaging methods showed similar clot distribution inside the ML. Independent of the imaging method, clot loading was detected predominantly in the inlet chamber of the ML. The µCT had the highest accuracy. However, it was more expensive and time consuming than MDCT or FMI. The MDCT detected the clots with low scanning time. Due to its lower resolution, it only showed clotted areas but not the exact shape of clot structures. FMI represented the simplest variant, requiring little effort and resources. FMI allowed clot localization and calculation of clot volume. Histological evaluation indicated omnipresent immunological deposits throughout the ML. Visually clot-free areas were covered with leukocytes and platelets forming platelet-leukocyte aggregates (PLAs). Cells were embedded in vWF cobwebs, while vWF fibers were negligible. In conclusion, the presented methodology allowed adequate clot identification and histological classification of possible thrombosis markers such as PLAs.

Neutrophil extracellular traps - a potential trigger for the development of thrombocytopenia during extracorporeal membrane oxygenation.

Neutrophil extracellular traps (NETs) have recently emerged as a potential link between inflammation, immunity, and thrombosis, as well as other coagulation disorders which present a major challenge in the context of extracorporeal membrane oxygenation (ECMO). By examining blood from ECMO patients for NETs and their precursors and correlating them with clinical and laboratory biomarkers of coagulation and inflammation, this study aims to evaluate the association between the presence of NETs in the bloodstream of ECMO patients and the development of potentially severe coagulation disorders during ECMO therapy. Therefore, blood samples were collected from healthy volunteers (n=13) and patients receiving veno-venous (VV) ECMO therapy (n=10). To identify NETs and their precursors, DNA and myeloperoxidase as well as granulocyte marker CD66b were visualized simultaneously by immunofluorescence staining in serial blood smears. Differentiation of DNA-containing objects and identification of NETs and their precursors was performed semiautomatically by a specific algorithm using the shape and size of DNA staining and the intensity of MPO and CD66b signal. Neutrophil extracellular traps and their precursors could be detected in blood smears from patients requiring VV ECMO. Compared to volunteers, ECMO patients presented significantly higher rates of NETs and NET precursors as well as an increased proportion of neutrophil granulocytes in all detected nucleated cells. A high NET rate prior to the initiation of ECMO therapy was associated with both increased IL-6 and TNF-α levels as an expression of a high cytokine burden. These patients with increased NET release also presented an earlier and significantly more pronounced decrease in platelet counts and ATIII activity following initiation of therapy compared with patients with less elevated NETs. These findings provide further indications for the development of immune-mediated acquired thrombocytopenia in ECMO patients.

Extended Cellular Deposits on Gas Exchange Capillaries Are Not an Indicator of Clot Formation: Analysis of Different Membrane Oxygenators.

Antithrombogenic coatings of artificial surfaces within extracorporeal membrane oxygenation (ECMO) circuits improved its bio- and hemocompatibility. However, there is still a risk of thrombus formation in particular within the membrane oxygenator (MO). Since inflammatory cells are essential components within clots, the aim was to identify the extent of cellular accumulations on gas exchange capillaries from different ECMO systems. Thirty-four MOs (PLS, n = 27, Getinge; Hilite 7000 LT, n = 7, Fresenius Medical Care, Germany) were collected from adult patients. The extent of cellular deposits on gas exchange capillaries was classified using nuclear 4',6-diamidino-2-phenylindole staining and fluorescence microscopy. All Hilite oxygenators exhibited small cellular deposits. In contrast, the cellular distribution was heterogeneous on capillaries from PLS oxygenators: small deposits (34%), clusters (44%) and membrane-spanning cell structures (pseudomembranes) (22%). Overall, the median fluorescence intensity was significantly higher in the PLS group. Nevertheless, within 3 days before MO removal, there was no alteration in critical parameters ( d -dimer and fibrinogen levels, platelet counts, and pressure drop across the MO). In conclusion, despite the histological differences on the gas capillaries from different types of oxygenators, there was no further evidence of increased inflammation and coagulation parameters that indicate clot formation within oxygenators.

Prevalence and outcomes of patients developing heparin-induced thrombocytopenia during extracorporeal membrane oxygenation.

OBJECTIVES: Unfractionated heparin (UFH) is the commonly used anticoagulant to prevent clotting of the ECMO circuit and thrombosis of the cannulated vessels. A side effect of UFH is heparin-induced thrombocytopenia (HIT). Little is known about HIT during ECMO and the impact of changing anticoagulation in ECMO patients with newly diagnosed HIT. The aim of the study was to determine the prevalence, complications, impact of switching anticoagulation to argatroban and outcomes of patients developing heparin-induced thrombocytopenia (HIT) during either veno-venous (VV) or veno-arterial (VA) ECMO. METHODS: Retrospective observational single centre study of prospectively collected data of consecutive patients receiving VV ECMO therapy for severe respiratory failure and VA ECMO for circulatory failure from January 2006 to December 2016 of the Medical intensive care unit (ICU) of the University Hospital of Regensburg. Treatment of HIT on ECMO was done with argatroban. RESULTS: 507 patients requiring ECMO were included. Further HIT-diagnostic was conducted if HIT-4T-score was ≥4. The HIT-confirmed group had positive HIT-enzyme-linked-immunosorbent-assay (ELISA) and positive heparin-induced-platelet-activation (HIPA) test, the HIT-suspicion group a positive HIT-ELISA and missing HIPA but remained on alternative anticoagulation until discharge and the HIT-excluded group a negative or positive HIT-ELISA, however negative HIPA. These were compared to group ECMO-control without any HIT suspicion. The prevalence of HIT-confirmed was 3.2%, of HIT-suspicion 2.0% and HIT-excluded 10.8%. Confirmed HIT was trendwise more frequent in VV than in VA (3.9 vs. 1.7% p = 0.173). Compared to the ECMO control group, patients with confirmed HIT were longer on ECMO (median 13 vs. 8 days, p = 0.002). Different types of complications were higher in the HIT-confirmed than in the ECMO-control group, but in-hospital mortality was not different (31% vs. 41%, p = 0.804). CONCLUSION: HIT is rare on ECMO, should be suspected, if platelets are decreasing, but seems not to increase mortality if treated promptly.

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks.

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 µm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.

Risk of circuit infection in septic patients on extracorporeal membrane oxygenation: a preliminary study.

Extracorporeal membrane oxygenation (ECMO) is the ultimate treatment option to improve gas exchange and decrease the aggressiveness of mechanical ventilation in septic patients with uncontrolled severe lung failure. However, potential microbiological colonization of the artificial surfaces of membrane oxygenator (MO) remains a critical issue in patients with bacteremia. The current study investigates the risk of MO infection in 10 consecutive septic patients on long-term treatment with ECMO. The flushing fluids of all investigated MOs were sterile. After incubation with nutrient solution for 14 days in one MO Enterococci spp. were isolated. In the patient concerned, a diffuse, unaccountable bleeding diathesis had developed, which stopped after exchange of the MO. Analysis of clinical parameters showed that D dimers had increased and fibrinogen levels had decreased before exchange of this MO, but standard markers of infection had remained unremarkable. In conclusion, circuit infection may be a potential cause for unexplained clinical deterioration of patients on ECMO, which therefore should be considered as an indication for exchange of the device.