Quantitative Analysis of Clot Deposition on Extracorporeal Life Support Membrane Oxygenators Using Digital and Scanning Electron Microscopy Imaging Techniques.

Device-induced thrombosis remains a major complication of extracorporeal life support (ECLS). To more thoroughly understand how blood components interact with the artificial surfaces of ECLS circuit components, assessment of clot deposition on these surfaces following clinical use is urgently needed. Scanning electron microscopy (SEM), which produces high-resolution images at nanoscale level, allows visualization and characterization of thrombotic deposits on ECLS circuitry. However, methodologies to increase the quantifiability of SEM analysis of ECLS circuit components have yet to be applied clinically. To address these issues, we developed a protocol to quantify clot deposition on ECLS membrane oxygenator gas transfer fiber sheets through digital and SEM imaging techniques. In this study, ECLS membrane oxygenator fiber sheets were obtained, fixed, and imaged after use. Following a standardized process, the percentage of clot deposition on both digital images and SEM images was quantified using ImageJ through blind reviews. The interrater reliability of quantitative analysis among reviewers was evaluated. Although this protocol focused on the analysis of ECLS membrane oxygenators, it is also adaptable to other components of the ECLS circuits such as catheters and tubing. Key features • Quantitative analysis of clot deposition using digital and scanning electron microscopy (SEM) techniques • High-resolution images at nanoscale level • Extracorporeal life support (ECLS) devices • Membrane oxygenators • Blood-contacting surfaces Graphical overview.

K+ontrol rapidly and efficiently reduces potassium in donor blood during ex vivo circulation.

BACKGROUND: Patients with kidney failure are at risk for lethal complications from hyperkalemia. Resuscitation, medications, and hemodialysis are used to mitigate increased potassium (K+) levels in circulating blood; however, these approaches may not always be readily available or effective, especially in a resource limited environment. We tested a sorbent cartridge (KC, K+ontrol CytoSorbents Medical Inc., Monmouth Junction, New Jersey) which contains a resin adsorber for K+. The objective of this study was to test the utility of KC in an ex vivo circulation system. We hypothesized that KC reduces K+ levels in extracorporeal circulation of donor swine whole blood infused with KCl. METHODS: A six-hour circulation study was carried out using KC, a NxStage (NxStage Medical, Inc., Lawrence, MA) membrane, blood bag containing heparinized whole blood with KCl infusion, 3/16-inch ID tubing, a peristaltic pump, and flow sensors. The NxStage permeate line was connected back to the main circuit in the Control group (n = 6), creating a recirculation loop. For KC group (n = 6), KC was added to the recirculation loop, and a continuous infusion of KCl at 10 mEq/hour was administered for two hours. Blood samples were acquired at baseline and every hour for 6 h. RESULTS: In the control group, K+ levels remained at ∼9 mmol/L; 9.1 ± 0.4 mmol/L at 6 h. In the KC group, significant decreases in K+ at hour 1 (4.3 ± 0.3 mmol/L) and were sustained for the experiment duration equilibrating at 4.6 ± 0.4 mmol/L after 6 h (p = 0.042). Main loop blood flow was maintained under 400 mL/min; recirculation loop flow varied between 60 and 70 mL/min in the control group and 45-55 mL/min in the KC group. Decreases in recirculation loop flow in KC group required 7% increase of pump RPM. CONCLUSIONS: During ex-vivo extracorporeal circulation using donor swine blood, KC removed approximately 50% of K+, normalizing circulating levels.

Expeditionary Mechanical Ventilation in Conjunction With Extracorporeal Life Support During Ground Transport.

BACKGROUND: We assessed the use of an FDA-cleared transport ventilator with limited functions and settings during ground transport in a swine model of ground evacuation. We hypothesized that when used as an adjunct to extracorporeal life support (ECLS), the device would enable safe mobile ventilatory support during ground evacuation. METHODS: Female Yorkshire swine (n = 11; mean, 52.4 ± 1.3 kg) were sedated and anesthetized and received mechanical ventilation (MV) with a standard intensive care unit (ICU) ventilator and were transitioned to the Simplified Automated Ventilator II (SAVe II; AutoMedx) during ground transport. MV served as an adjunct to ECLS in all animals. Ventilator performance was assessed in the uninjured state on day 1 and after bilateral pulmonary contusion on day 2. Data were collected pre- and post-transport on both days. RESULTS: During 33 transports, the SAVe II provided similar ventilation support as the ICU ventilator. Mean total transport time was 38.8 ± 2.1 minutes. The peak inspiratory pressure (PIP) limit was the only variable to show consistent differences pre- and post-transport and between ventilators. No adverse events occurred. CONCLUSION: As an adjunctive supportive device during ground transport, the SAVe II performed adequately without failure or degradation in subject status. Further testing is warranted to elucidate the clinical limits of this device during standalone use.

Dynamics of appearance and decay of gaseous microemboli during in vitro extracorporeal circulation.

INTRODUCTION: Extracorporeal life support (ECLS) patients are at risk for complications caused by gaseous microemboli (GME). GMEs can cause hypoxia, inflammation, coagulation, and end-organ damage. The objective of this in vitro study was to assess dynamics of GME formation during circulation of whole blood or a glycerol blood surrogate. We hypothesized that there is no difference in GME counts and sizes between whole blood and the glycerol blood surrogate and that the membrane lung reduces GME counts over time. METHODS: A circulation platform was developed using the Cardiohelp ECLS system to run either donor blood or glycerol solution. We conducted 10 repetitions consisting of three phases of ultrasound GME detection using the EDAC™ Quantifier (Luna Innovations, Charlottesville, VA, USA) for each group. Phases were 3-minute recordings at the initiation of 2 L/min flow (Phase 1), post-injection of a GME suspension (Phase 2), and 10 minutes after injection (Phase 3). The number and size of GME pre- and post-ML were recorded separately and binned based on diameter ranges. RESULTS: In Phase 1, GME count in blood was higher than in glycerol. In Phase 2, there was a large increase in GME counts; however, most GME were reduced post-membrane in both groups. In Phase 3, there was a significant decrease in GME counts compared to Phase 2. GME > 100 µm in glycerol decreased post membrane. CONCLUSIONS: We demonstrated GME formation and decay dynamics during in vitro circulation in an ECLS system with blood and glycerol. GME counts were higher in blood, likely due to varying rheological properties. There were decreases in GME levels post membrane in both groups after GME injection, with the membrane lung effectively trapping the GME, and additional reduction 10 minutes after GME injection.

Tethered Liquid Perfluorocarbon Coating for 72 Hour Heparin-Free Extracorporeal Life Support.

Coagulopathic complications during extracorporeal life support (ECLS) result from two parallel processes: 1) foreign surface contact and shear stress during blood circulation and 2) administration of anticoagulant drugs to prevent circuit thrombosis. To address these problems, biocompatible surfaces are developed to prevent foreign surface-induced coagulopathy, reducing or eliminating the need for anticoagulants. Tethered liquid perfluorocarbon (TLP) is a nonadhesive coating that prevents adsorption of plasma proteins and thrombus deposition. We examined application of TLP to complete ECLS circuits (membranes, tubing, pumps, and catheters) during 72 hours of ECLS in healthy swine (n = 5/group). We compared TLP-coated circuits used without systemic anticoagulation to standard of care: heparin-coated circuits with continuous heparin infusion. Coagulopathic complications, device performance, and systemic effects were assessed. We hypothesized that TLP reduces circuit thrombosis and iatrogenic bleeding, without impeding gas exchange performance or causing untoward effects. No difference in bleeding or thrombotic complication rate was observed; however, circuit occlusion occurred in both groups (TLP = 2/5; CTRL = 1/5). TLP required elevated sweep gas rate to maintain normocapnia during ECLS versus CTRL (10-20 vs. 5 L/min; p = 0.047), suggesting impaired gas exchange. Thrombus deposition and protein adhesion on explanted membranes were comparable, and TLP did not preserve platelet or blood cell counts relative to controls. We conclude that neither TLP nor standard of care is an efficacious solution to prevent coagulation disturbances during ECLS. Further testing of promising biomaterials for ECLS utilizing the model outlined here is warranted.

Expression of High Mobility Group Box 1 Protein in a Polytrauma Model During Ground Transport and Simulated High-Altitude Evacuation.

BACKGROUND: We investigated the expression of high mobility group box 1 (HMGB1) protein in a combat-relevant polytrauma/ acute respiratory distress syndrome (ARDS) model. We hypothesized that systemic HMGB1 expression is increased after injury and during aeromedical evacuation (AE) at altitude. METHODS: Female Yorkshire swine (n =15) were anesthetized and cannulated with a 23Fr dual-lumen catheter. Venovenous extracorporeal life support (VV ECLS) was initiated via the right jugular vein and carried out with animals uninjured on day 1 and injured by bilateral pulmonary contusion on day 2. On both days, animals underwent transport and simulated AE. Systemic HMGB1 expression was measured in plasma by ELISA. Plasma-free Hb (pfHb) was measured with the use of spectrophotometric methods. RESULTS: Plasma HMGB1 on day 1 was transiently higher at arrival to the AE chambers, increased significantly after injury, reaching highest values at 8,000 ft on day 2, after which levels decreased but remained elevated versus baseline at each time point. pfHb decreased on day 1 at 30,000 ft and significantly increased on day 2 at 8,000 ft and postflight. CONCLUSIONS: Systemic HMGB1 demonstrated sustained elevation after trauma and altitude transport and may provide a useful monitoring capability during en route care.

Heparin-Free Extracorporeal Life Support Using Tethered Liquid Perfluorocarbon: A Feasibility and Efficacy Study.

Coagulation management is the leading challenge during extracorporeal life support (ECLS) due to shear stress and foreign-surface-induced coagulation disturbance during circulation. A nonadhesive, liquid-infused coating called tethered liquid perfluorocarbon (TLP) was developed to prevent adhesion of blood on medical materials. We investigated the novel application of TLP to commercial ECLS circuits compared with standard heparin-coated circuits in vivo in anesthetized swine for 6 hours veno-venous ECLS (1 L/min blood flow) without systemic anticoagulation (n = 3/group). We hypothesized that TLP coating permits heparin-free circulation without untoward effects while reducing thrombus deposition compared with controls. Vital signs, respiration, gas transfer, coagulation, and histology were assessed. Scanning electron microscopy (SEM), elemental mapping, and digital imaging were used to assess thrombus deposition after circulation. There were no group differences in vitals, gas exchange, coagulation, and histology. In both groups, ECLS enabled a decrease in minute volume and end-tidal CO2, with concomitant increase in pH (p < 0.05). Scanning electron microscopy and digital imaging revealed significant thrombus on heparin-coated membranes, which was reduced or absent on TLP-coated materials. Tethered liquid perfluorocarbon permitted heparin-free ECLS without altering device performance and prevented thrombus deposition versus immobilized heparin. Pending multiday in vivo testing, TLP is a promising biomaterial solution to eliminate anticoagulation requirements during ECLS.

Effects of adjunct treatments on end-organ damage and histological injury severity in acute respiratory distress syndrome and multiorgan failure caused by smoke inhalation injury and burns.

BACKGROUND: We investigated effects of mesenchymal stem cells (MSC) or low-flow extracorporeal life support (ECLS) as adjunctive treatments for acute respiratory distress syndrome (ARDS) due to inhalation injury and burns. We hypothesized that these interventions decrease histological end-organ damage. METHODS: Anesthetized female swine underwent smoke inhalation injury and 40% TBSA burns, then critical care for 72h. The following groups were studied: CTR (no injury, n = 4), ICTR (injured untreated, n = 10), Allo (injured treated with allogenic MSC, n = 10), Auto (injured treated with autologous MSC, n = 10), Hemo (injured and treated with the Hemolung low flow ECLS system, n = 9), and Nova (injured and treated with the NovaLung low flow ECLS system, n = 8). Histology scores from lung, kidneys, liver, and jejunum were calculated. Data are presented as means±SEM. RESULTS: Survival at 72h was 100% in CTR; 40% in ICTR; 50% in Allo; 90% in Auto; 33% in Hemo; 63% in Nova. ARDS developed in 0/10 CTR; 10/10 ICTR; 8/9 Hemo; 5/8 Nova; 9/10 Allo; 6/10 Auto. Diffuse alveolar damage (DAD) was present in all injured groups. MSC groups had significantly lower DAD scores than ICTR animals (Allo 26.6 ± 3.4 and Auto 18.9 ± 1.5 vs. ICTR 46.8 ± 2.1, p < 0.001). MSC groups also had lower DAD scores than ECLS animals (Allo vs. Nova, p < 0.05, Allo vs. Hemo p < 0.001, Auto vs. Nova p < 0.001, Auto vs. Hemo, p < 0.001). Kidney injury ICTR (p < 0.05) and Hemo (p < 0.01) were higher than in CTR. By logistic regression, a PaO2-to-FiO2 ratio (PFR) < 300 was a function of the DAD score: logit (PFR < 300) = 0.84 + 0.072*DAD Score, odds ratio 1.074 (1.007, 1.147, p < 0.05) with a ROC AUC of 0.76, p < 0.001. CONCLUSION: Treatment with Auto MSC followed by Allo and then Nova were most effective in mitigating ARDS and MOF severity in this model. Further studies will elucidate the role of combination therapies of MSC and ECLS as comprehensive treatments for ARDS and MOF.

Thromboelastography on-the-go: Evaluation of the TEG 6s device during ground and high-altitude Aeromedical Evacuation with extracorporeal life support.

BACKGROUND: Coagulation monitoring capabilities during transport are limited. Thromboelastography (TEG) is a whole-blood clotting test measuring clot formation, stabilization, and fibrinolysis and is traditionally performed in a laboratory. We evaluated a new point-of-care TEG analyzer, TEG 6s (Haemonetics, Braintree, MA), in a large animal model of combat-relevant trauma managed with extracorporeal life support during ground and high-altitude aeromedical evacuation. The objective was to compare TEG 6s used during transport versus the predicate device, TEG 5000, used in the laboratory. We hypothesized that TEG 6s would be comparable with TEG 5000 during dynamically changing transport conditions. METHODS: Thromboelastography parameters (R, K, angle, MA, LY30) derived by TEG 6s and TEG 5000 were compared during transport of 8 swine. TEG 6s was transported with animals during ground transport and flight. TEG 5000 was stationary in an adjacent building. TEG 6s activated clotting time (ACT) was compared with a Hemochron Junior ACT analyzer (Accriva Diagnostics, San Diego, CA). Statistics were performed using SAS 9.4 with Deming regressions, Spearman correlations, and average differences compared. RESULTS: Correlation between devices was stronger at sea-level (R, r = 0.7413; K, r = 0.7115; angle, r = 0.7192; MA, r = 0.8386; LY30, r = 0.9099) than during high-altitude transport (R, r = 0.4787; K, r = 0.4007; angle, r = 0.3706; MA, r = 0.6573; LY30, r = 0.8481). Method agreement was comparable during stationary operation (R, r = 0.7978; K, r = 0.7974; angle, r = 0.7574; MA, r = 0.7841; LY30, r = 0.9140) versus ground transport (R, r = 0.7927; K, r = 0.6246; angle, r = 0.6967; MA, r = 0.9163; LY30, r = 0.8603). TEG 6s ACT trended higher than Hemochron ACT when subjects were heparinized (average difference, 1,442 ± 1,703 seconds) without a methodological difference by Deming regression. CONCLUSION: Mobile TEG 6s during ground and altitude transport is feasible and provides unprecedented information to guide coagulation management. Future studies should assess the precision and accuracy of TEG 6s during transport of critically ill.

Automatic proximal airway volume segmentation using optical coherence tomography for assessment of inhalation injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury with a mortality rate of up to 40%. Early management of ARDS has been difficult due to the lack of sensitive imaging tools and robust analysis software. We previously designed an optical coherence tomography (OCT) system to evaluate mucosa thickness (MT) after smoke inhalation, but the analysis relied on manual segmentation. The aim of this study is to assess in vivo proximal airway volume (PAV) after inhalation injury using automated OCT segmentation and correlate the PAV to lung function for rapid indication of ARDS. METHODS: Anesthetized female Yorkshire pigs (n = 14) received smoke inhalation injury (SII) and 40% total body surface area thermal burns. Measurements of PaO2-to-FiO2 ratio (PFR), peak inspiratory pressure (PIP), dynamic compliance, airway resistance, and OCT bronchoscopy were performed at baseline, postinjury, 24 hours, 48 hours, 72 hours after injury. A tissue segmentation algorithm based on graph theory was used to reconstruct a three-dimensional (3D) model of lower respiratory tract and estimate PAV. Proximal airway volume was correlated with PFR, PIP, compliance, resistance, and MT measurement using a linear regression model. RESULTS: Proximal airway volume decreased after the SII: the group mean of proximal airway volume at baseline, postinjury, 24 hours, 48 hours, 72 hours were 20.86 cm (±1.39 cm), 17.61 cm (±0.99 cm), 14.83 cm (±1.20 cm), 14.88 cm (±1.21 cm), and 13.11 cm (±1.59 cm), respectively. The decrease in the PAV was more prominent in the animals that developed ARDS after 24 hours after the injury. PAV was significantly correlated with PIP (r = 0.48, p < 0.001), compliance (r = 0.55, p < 0.001), resistance (r = 0.35, p < 0.01), MT (r = 0.60, p < 0.001), and PFR (r = 0.34, p < 0.01). CONCLUSION: Optical coherence tomography is a useful tool to quantify changes in MT and PAV after SII and burns, which can be used as predictors of developing ARDS at an early stage. LEVEL OF EVIDENCE: Prognostic, level III.

Point-of-care endoscopic optical coherence tomography detects changes in mucosal thickness in ARDS due to smoke inhalation and burns.

BACKGROUND: The prevalence of acute respiratory distress syndrome (ARDS) in mechanically ventilated burn patients is 33%, with mortality varying from 11-46% depending on ARDS severity. Despite the new Berlin definition for ARDS, prompt bedside diagnosis is lacking. We developed and tested a bedside technique of fiberoptic-bronchoscopy-based optical coherence tomography (OCT) measurement of airway mucosal thickness (MT) for diagnosis of ARDS following smoke inhalation injury (SII) and burns. METHODS: 16 female Yorkshire pigs received SII and 40% thermal burns. OCT MT and PaO2-to-FiO2 ratio (PFR) measurements were taken at baseline, after injury, and at 24, 48, and 72h after injury. RESULTS: Injury led to thickening of MT which was sustained in animals that developed ARDS. Significant correlations were found between MT, PFR, peak inspiratory pressure (PIP), and total infused fluid volume. CONCLUSIONS: OCT is a useful tool to quantify MT changes in the airway following SII and burns. OCT may be effective as a diagnostic tool in the early stages of SII-induced ARDS and should be tested in humans.

Electron Microscopy as a Tool for Assessment of Anticoagulation Strategies During Extracorporeal Life Support: The Proof Is on the Membrane.

Extracorporeal life support (ECLS) is fast becoming more common place for use in adult patients failing mechanical ventilation. Management of coagulation and thrombosis has long been a major complication in the use of ECLS therapies. Scanning electron microscopy (SEM) of membrane oxygenators (MOs) after use in ECLS circuits can offer novel insight into any thrombotic material deposition on the MO. In this pilot study, we analyzed five explanted MOs immediately after use in a sheep model of different acute respiratory distress syndrome (ARDS). We describe our methods of MO dissection, sample preparation, image capture, and results. Of the five MOs analyzed, those that received continuous heparin infusion showed very little thrombosis formation or other clot material, whereas those that were used with only initial heparin bolus showed readily apparent thrombotic material.