ICU Clinician Perspectives on Machine Learning and the Implementation of a Mechanical Ventilation Prediction Tool: A Single Center Survey Study

Rationale Although there is considerable interest in machine learning (ML) algorithms to improve patient care, implementation of these algorithms into practice has been limited. Our team developed and validated a deep learning algorithm to predict respiratory failure requiring mechanical ventilation in patients in the intensive care unit (ICU), including those with COVID-19. To help optimize implementation of this tool, we developed and disseminated a survey assessing ICU physician perspectives on the acceptability and feasibility of this tool at our institution. Methods We distributed an 8-item survey to 99 critical care trainees and faculty at our institution via email. The survey consisted of 6 multiple choice and 2 free response questions, with an ordinal scale of 1-5 used in perception-based questions. The survey was designed in accordance with international recommendations for web-based surveys. Our survey was reviewed for completeness by a team of critical care, machine learning, and implementation science experts. Data were collected over a 2- week period in May of 2021. This survey was anonymous and exempt from IRB review. Results Fifty-three critical care physicians (53.5% of providers contacted) started the survey, and of these, 88.7% (47/53) completed the survey. Fifty-nine percent (n=31) of respondents were attendings, 36% (n=19) fellows, and 3.7% (n=2) residents. Baseline knowledge of ML was low (mean= 2.40/5), with only 7.5% (n=4) of respondents rating their knowledge as a 4 or 5. Fifteen percent (n=8) had knowingly used an ML-based tool in their clinical practice. Confidence in predicting the need for mechanical ventilation due to COVID-19 (mean=3.57/5) was slightly lower than for respiratory failure due to all other causes (mean=3.89/5). Overall willingness to utilize an ML-based algorithm was favorable (mean=3.32/5). Factors most likely to increase likelihood of utilization were “high quality evidence that it outperformed trained clinicians” (mean=4.28/5), “transparency of the data utilized” (mean= 4.13/5), and “limited workflow interruption” (mean=4.09/5). Shared concerns from participants included “alarm fatigue” and “workflow interruption.” Conclusion The results suggest that ICU physicians have had limited exposure to ML-based tools, but feel such a tool would be beneficial in the context of predicting need for mechanical ventilation in ICU patients and those with COVID-19. Evidence of the tool's efficacy and data transparency were high priorities for respondents, and there was concern over workflow interruptions. This survey provided a baseline assessment of physician acceptance of a novel ML-based tool, which will be crucial in optimizing its implementation into clinical practice at our institution.

Intravenous Immunoglobulin but Not Dexamethasone Inhibits Neutrophil Pro-Inflammatory Activity While Maintaining Key Antimicrobial Functions

Rationale. Acute respiratory distress syndrome (ARDS) is a heterogeneous clinical disease. ARDS immunopathology due to lung infection involves an array of immune cells and the importance of granulocytes, and in particular neutrophils and neutrophil extracellular trap production (NETosis), has recently come to light. Despite over 20 well run, randomized, controlled trials, no specific therapies for ARDS are available and mortality remains high. Current treatments for ARDS are primarily limited to supportive therapies, including lung protective ventilation, and in certain situations, systemic steroid administration. Recently, clinical studies adding intravenous immunoglobulin (IVIG), an FDA approved drug, to standard ARDS therapy have shown faster recovery with less severe symptoms, suggesting a complementary beneficial effect, but the mechanism(s) remain unknown. Interestingly, previous in vitro studies found that IVIG can impair some inflammatory pathways in neutrophils. Our study assessed effects of IVIG with and without dexamethasone (a key glucocorticoid used in COVID-19 ARDS) in neutrophils ex vivo and in vivo in COVID-19 patients. Methods. Ex vivo treatment of neutrophils with IVIG or dexamethasone was conducted, followed by assessment of NETosis, oxidative burst and phagocytosis. Additionally, cell-free DNA was quantified in the blood of COVID-19 patients before and after treatment with IVIG. Ex vivo NETosis and plasma cell-free DNA was quantified using the QuantiT ™ PicoGreen™ dsDNA Assay Kit (Invitrogen). Oxidative burst was assessed by OxyBURST™ Green H2DCFDA, SE (Invitrogen) and phagocytosis of pHrodo™ Red S. aureus Bioparticles™ (Invitrogen) was quantified. Results. IVIG inhibits crucial neutrophil inflammatory pathways such as NETosis and oxidative burst while concomitantly enhancing phagocytic activity (Figure panels A-C). Notably, dexamethasone does not impact any of these critical pathways. Moreover, COVID-19 patients undergoing standard treatment plus IVIG had decreased cell-free DNA in the circulation 5 days after initiation of a 4 day treatment course, suggesting decreased NETs in circulation (Figure panel D) which possibly reverted at a later timepoint. Conclusion. Our data demonstrate potential targeted beneficial effects of IVIG in the context of neutrophil-mediated immunopathology. We demonstrate an ex vivo inhibitory effect of IVIG on pro-inflammatory pathways in neutrophils, which may lead to diminished immunopathology in disease states worsened by neutrophil-driven destruction. Based on the compelling evidence of the contribution of neutrophils to development and severity in ARDS, our evidence of IVIG impairing key pro-inflammatory functions in neutrophils (where dexamethasone does not) suggests a theoretical potential complementary beneficial effect of adding IVIG to standard treatment for infection induced ARDS although further research is needed.

Circulating neutrophils from COVID-19 patients have elevated rates of phagocytosis

Rationale: Neutrophils are an important component of the immune system and are crucial for the inflammatory response during infection from a foreign pathogen. Phagocytosis is a vital cellular mechanism in which pathogens are engulfed in a phagosome to be eliminated. Once taken inside the cell, the pathogen is subject to a cytotoxic environment inside the phagolysosome. While neutrophils are critical in suppressing the spread of infection, too much activity has been linked to tissue damage. Inflammatory lung conditions such as acute respiratory distress syndrome (ARDS) can occur, often leading to death. Our aim is to determine whether the rate of phagocytosis is associated with disease severity among patients with COVID-19. Methods: Neutrophils were isolated from the peripheral blood of 20 COVID-19 human subjects. Neutrophils were isolated with Polymorphoprep and mixed in a 1:1 ratio with pHrodo TM Red Staphylococcus aureus Bioparticles TM (Invitrogen) in a 96-well plate and centrifuged at 1200 rpm for 6 min. Fluorescence was quantified via plate reader (Infinite M200, TECAN) every 15min for 2 hours to define the rate of phagocytosis, with excitation at 560nm and emission at 585nm. Cells were incubated at 37°C with 5% CO2 in-between reads. Statistical analysis was performed via simple linear regression with detection of significant differences in slopes. Paired two-tailed non-parametric t-tests (Wilcoxon) were conducted for each time point. Results: Rates of phagocytosis were significantly higher in neutrophils from critically ill COVID-19 patients relative to neutrophils from healthy controls (487.5 versus 374.5 at 120min;p<0.05 at 75, 90, 105 and 120 min). Conclusion: Despite the functional role of neutrophils in suppression of foreign pathogens, increased activity of neutrophils can lead to collateral damage and thus exacerbated lung injury. We found that neutrophils from COVID-19 patients have higher rates of phagocytosis than those from healthy subjects. These findings suggest that neutrophils circulating in COVID-19 patients have an elevated activation state and may contribute to increased severity of disease. However, the elevation in phagocytosis may also suggest that neutrophils in critically ill COVID patients are well able to phagocytose and kill pathogens which cause secondary infections in this at-risk cohort. Figure: .

Neutrophils from the circulation of critically ill COVID-19 patients may promote inflammation via increased production of reactive oxygen species

Background: Neutrophils are an important part of the innate immune system and play a vital role in host defense. Neutrophils target invading pathogens through both intracellular and extracellular mechanisms. One of their primary antimicrobial mechanisms is the production of reactive oxygen species (ROS) within phagolysosomes during oxidative burst. ROS production is a marker of the inflammatory/activity state of neutrophils. Although neutrophils are crucial in the defense against microorganisms, recruitment and hyperactivation of neutrophils can also cause collateral damage to the host. Recent studies have shown that neutrophils may be playing a role in acute respiratory distress syndrome (ARDS) in COVID-19. We hypothesized that ROS production would be increased secondary to elevated activation of circulating neutrophils in COVID-19 patients and may be playing a role in immunopathology.Methods: Circulating neutrophils were isolated from the blood of 20 human subjects with COVID-19 and 12 healthy controls. Neutrophils at 2x106 cells/ml were incubated with 10μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for 20min at 37°C. Neutrophils were centrifuged at 500g, resuspended in HBSS-/-, plated at 2x105 in 96-well plates and exposed to 0, 2.5, 25 and 250nM phorbol myristate acetate (PMA;in triplicate). Fluorescence was measured (Infinite M200, TECAN) at 495nm:520nm every 15min for 2hr. ROS production was analyzed using mixed-effects models with Geisser- Greenhouse correction and Sidak's multiple comparisons test.Results: ROS production was increased in neutrophils isolated from the circulation of critically ill COVID-19 patients as compared to healthy controls (2581nm versus 3790nm, respectively, at 120min, p<0.05). Upon stimulation with PMA, ROS production was also increased in COVID-19 patients relative to controls (2.5 PMA: 16597 versus 10549, respectively, <0.001;25 PMA: 20633 versus 11312, respectively, p<0.0001). At the final timepoint, 1.57, 1.82 and 1.85-fold difference in ROS production was noted at 2.5, 25 and 250nM respectively between patient populations. Conclusion: Increased ROS production at baseline and upon stimulation demonstrates that neutrophils in the circulation of critically ill COVID-19 patients with ARDS have an elevated activation state. These patients are known to have recruitment of neutrophils to the lung parenchyma, where the production and release of ROS may cause collateral damage. Further, neutrophils have been implicated in the vascular and systemic immunopathology of COVID-19, which may be driven by release of these damaging and toxic molecules. Neutrophil-associated pathways may serve as therapeutic targets and quantitative markers of disease in COVID-19 patients.

Inhalant use is associated with sleep quality and mental health during the COVID-19 pandemic

RATIONALE: Cigarette smoking and sleep quality are known to impact mental health considerably. As nicotine is a primary component of both cigarettes and electronic (e)-cigarettes, we launched a survey-based study in order to study the effects of e-cigarettes, conventional tobacco, and dual usage on mental health as well as sleep quality. We sent out this survey in the time of COVID-19 in order to assess the impact of nicotine-based inhalant use on sleep quality and mental health. We hypothesized that e-cigarette use impacts mental illnesses and sleep quality, which is exacerbated by the presence of additional stress such as a global pandemic. METHODS: We combined our established UCSD Inhalant Questionnaire with the Pittsburgh Sleep Quality Index (PSQI), Hospital Anxiety and Depression Scale (HADS), and Patient Health Questionnaire (PHQ-9). Participants were recruited through online advertisements posted to social media sites including Facebook, Craigslist, Reddit, and Twitter. Participants (n=554) were recruited in April 2020, and a subset (n=217) retook the survey in June 2020. The survey was broken up into four sections - past and current type of inhalant use (UCSD Inhalant Questionnaire), PSQI, HADS, and PHQ-9. All participants were incentivized via a weekly lottery for a gift card. RESULTS: Inhalant users reported worse sleep quality (PSQI, 6.74) when compared to non-smokers/non-vapers (PSQI, 5.72, p=0.02). Higher anxiety scores were associated with worse sleep quality (linear regression, r2=0.28, p<0.001). E-cigarette users and dual users of both e-cigarettes and conventional tobacco had higher anxiety scores (HADS anxiety;12.08 and 11.37, respectively) than non-smokers/non-vapers (7.94, p=0.028). Dual users were found to have higher depression scores relative to controls as well (HADS depression;7.42 versus 4.68, p=0.017). Sleep quality improved during the COVID pandemic from April 2020 (PSQI, 7.92) to June 2020 (PSQI, 6.10, p<0.0001). Interestingly, anxiety scores increased over the same period of time (HADS anxiety, 7.51 to 8.76, p<0.01). CONCLUSION: Our finding of diminished sleep quality in inhalant users is novel. Based on these data, we speculate that inhalant users may be more susceptible to sleep disturbances in the setting of a stressor, such as a global pandemic. Higher anxiety scores in e-cigarette and dual users, and higher depression scores in dual users, imply a direct relationship between inhalant use and mental disorders. Higher anxiety scores as the pandemic progressed may in theory reflect the lack of having an end in sight, or having loved ones affected by COVID-19.

Using apache longitudinally in ards to assess disease severity

Introduction: The acute physiology, age, chronic Health Evaluation (APACHE) II score has been used to assess risk of mortality in admitted intensive care unit (ICU) patients since 1985. There are few examples of a longitudinal correlation over time. As the scores rises from 0 to 71, risk of mortality increases. For this study, other scores were also considered - SOFA (Sequential Organ Failure Assessment) was excluded due to a lack of variable diversity and small range of values. APACHE III and IV are more complex scores with additional variables, such that calculating daily scores manually was impracticable. APACHE II has consistent calibration compared to III and IV. We assessed whether APACHE III and IV scores had any additional benefit over APACHE II. Methods: APACHE II score was used to determine clinical severity daily of sixteen critically ill COVID-19 patients and compared to daily changes in cytokine levels. If any studies were unavailable for a given day, data was pulled from a maximum of 24 hours before or after the day of interest. Although GCS (Glasgow Coma Scale) is a large part of the score, and difficult to assess in an intubated patient, however it contributed to documenting sedation and intubation state. Results: APACHE II score assessed clinical severity daily in COVID-19 more effectively relative to SOFA. The wide score range allowed comparison of patients without overlap, as compared to SOFA which has a range of 0-24. With a larger range of 0-71, we believe that it increased the sensitivity for detection of small changes in clinical status. We analyzed APACHE II in context of absolute neutrophil count, plasma cytokine levels, as well as neutrophil functional studies. For our study, APACHE II, not SOFA, was helpful in demonstrating changes in severity of illness, which correlated with some of these assessments. For example, APACHE II showed a significant linear correlation with pro-inflammatory cytokine IL-8 in plasma (r2=0.47, p=0.0017;Fig 1). Conclusion: APACHE II was able to define the severity of illness in COVID-19 patients on a daily basis. APACHE II score allowed us to tie immunophenotyping to clinical disease severity over time and was helpful in broadening our approach to data comparison with clinical status. This was a pilot study to assess the ability of APACHE II to track severity of illness, but in the future, we plan to correlate APACHE II in a larger cohort of a variety of ARDS patients.

Neutrophil extracellular trap formation (NETOSIS) increases with severity of disease in COVID-19 patients

Background: Neutrophils are key players in the immune and aid in the defense against microorganisms. Neutrophil extracellular traps (NETs) are extracellular DNA complexes, which are released during NETosis, a programmed form of cell death. Although NETs are crucial in the fight against infectious agents, an overabundance of neutrophils has been implicated in many inflammatory lung conditions. Our aim is to determine whether an overabundance of NETosis is associated with clinical deterioration of patients with COVID-19. Methods: Circulating polymorphonuclear cells (neutrophils) were isolated from human peripheral blood of 20 human subjects with COVID-19. Neutrophils were seeded in 96-well plates and treated with 0, 2.5 nM, 25 nM, and 250 nM of phorbol 12-myrisate 13-acetate (PMA) or 12 uM nigericin for 2 hours to stimulate NET production via canonical and noncanonical pathways, respectively. Following incubation, wells were treated with micrococcal nuclease, supernatants were collected from each well, and extracellular DNA content to quantify NETosis was detected by fluorescent plate reader. We calculated acute physiology and chronic health evaluation (APACHE-II) scores for every human subject. These were calculated at the same time point at which the neutrophils were collected. They were then compared to the degree of NETosis and absolute neutrophil count (ANC). These were analyzed using a simple linear regression model. We also categorized participants based on APACHE-II scores (APACHE-II <15, APACHE-II>15) and compared them to rates of NETosis using a bar graph. Results: APACHE II is a widely used ICU mortality prediction score that is used to risk-stratify patients. We found that participants with higher APACHE-II scores had higher rates of NETosis, both at 0 nM PMA and when stimulated with nigericin (figure 1a-b). This suggests that higher rates of NETosis correlate with increased disease severity. Additionally, we found a positive correlation between ANC and NETosis (Figure 1c-1d), suggesting that ANC itself is a reliable marker of NETosis and disease severity. Conclusion: NETosis is an important player in immune system defense but has also been implicated in various inflammatory lung conditions. We found that in patients with COVID-19, there was a positive correlation between worsening disease state, measure by APACHE II scores, and increased NETosis. This suggests that over-activation of neutrophils may play a role in disease progression. We also found a positive correlation between NETosis and ANC, indicating that the degree of circulating neutrophils is a reliable marker of the functional state of neutrophils, as well as disease severity.

Medical and pharmacy students learning trends in the COVID-19 pandemic

Introduction: The COVID-19 pandemic can be seen as a source of disruption for many people's lives, but it has also served as a stimulus, changing the medical education environment. In response to the pandemic, UC San Diego School of Medicine converted its in-person preclerkship education into an online format using the software combination of the Zoom video communication, Panopto video platform and Canvas learning platform. By using data from the COVID-19 virtual format (CVF) and the in-person format (IPF) in the fall of 2019 extracted from Panopto and Canvas, we compared student lecture viewing trends and analyzed Canvas student usage to determine if student engagement to educational material has differed between the two learning structures. Methods: Student lecture viewing data were collected by using the “Analytics” function built into Panopto which recorded student views, lecture viewing completion, viewing trends over time, and total time spent watching lectures. On Canvas, data on student click-through rates for resources provided to students were extracted comparing this year to last year. Data acquired from Zoom was used to observe student attendance during lectures that were held synchronously. All statistics (t-tests) were calculated using Prism. Results: In the setting of the COVID-19 pandemic, there was a higher proportion of CVF students that watched the recorded live lectures compared to the in-person 2019 class (p=0.011). The CVF class also had a higher recorded lecture completion rate (p=0.003). It was observed that on average, 57.2% of CVF students attended live Zoom lectures if they were held. 56.3% of all CVF students also watched the live lecture recording, which indicates that a small proportion of students are re-watching the lecture after viewing them synchronously. Discussion: CVF students are more likely to watch and have higher complete rates of recorded live lectures compared to the IPF counterpart. These observations are significant as they indicate a change in student learning behavior in this predominantly virtual environment. Students may be more inclined to the flexibility that the virtual format offers. Further studies should be done to assess student performance between the CVF and IPF formats.

Assessing electronic cigarette use and perceptions during the COVID-19 pandemic in the united states

Rationale: As the COVID-19 pandemic changes the social landscape, distinguishing what is important to electronic (e)-cigarette users as well as changing perceptions and behaviors informs research on the current impact that e-cigarettes have on communities. We designed the current study to define how e-cigarette use has been impacted by COVID-19, and how the pandemic has changed e-cigarette user habits and their risk perceptions. Methods: To assess e-cigarette use and perceptions, a nationally distributed 52-item questionnaire assessed e-cigarette use, dependence and perceptions, COVID-19 diagnosis, demographic data, and pre/during COVID-19 vaping habits. E-cigarette usage/dependence was assessed via the validated Penn State Electronic Cigarette Dependence Index. Questions were developed in-house to assess pre/during COVID-19 vaping habits of users and risk perceptions of both non-users and users of e-cigarettes. Results: A total of 565 survey responses were obtained with 393 (69%) reporting no e-cigarette use and 172 (31%) e-cigarette users. Fifty (30%) vapers believed there were no adverse health effects due to vaping e-cigarettes while only 38 (9%) nonusers agreed (P<0.0001). Two hundred eight (76%) non-users believed that e-cigarette use would lead to worse COVID-19 symptoms, compared to 40% of users (P<0.001). Twenty-eight percent of non-users also believed that e-cigarette users were more likely to be infected with SARS-CoV-2, versus only 11% of e-cigarette users. Thirty percent of all vapers reported high dependence on vaping. Fifty-three (33%) users believed that ecigarettes are not a safe alternative to conventional tobacco (P<0.001). Of total respondents, 11 (6%) reported having had COVID-19. Fifty-nine (38%) e-cigarette users reported changing their habits during the pandemic, with 31% reporting increased vaping (P<0.001). Although 109 e-cigarette users (70%) reported vaping in a social setting before the pandemic, 38 (25%) users switched to vaping non-socially during the pandemic (P<0.001). One hundred three (67%) e-cigarette users replied that they would decrease or stop vaping if diagnosed with COVID, while 50 (31%) said they would continue vaping (P<0.001). Conclusions: Our findings show a large difference in risk perception of e-cigarette use between users and non-users, which is consistent with past studies and data. Additionally, we characterize and document the habits of e-cigarette users during the COVID-19 pandemic, finding that e-cigarette users report increased use during the pandemic, show more caution in social settings, and reduced usage if hypothetically diagnosed with COVID-19.

Effect of intravenous immunoglobulin on neutrophil antimicrobial and inflammatory functions-implications in neutrophil-mediated immunopathology

Rationale. Neutrophils are the most abundant circulating leukocytes and the first line of defense against invading pathogens. Known for their function as phagocytic cells, neutrophils also capture and kill pathogens via elaboration of Neutrophil Extracellular Traps (NETosis), and production of reactive oxygen species (ROS), antimicrobial peptides and proteases. Conversely, neutrophils also have been associated with immunopathology. For example, numerous studies have indicated that neutrophils play a role in acute respiratory distress syndrome (ARDS), which is a major cause of death in COVID-19. We found that COVID-19 patients with ARDS have circulating neutrophils with a hyper-activated phenotype: elevated NETosis, ROS production and phagocytosis. While a small prospective clinical trial showed benefit of intravenous immunoglobulin (IVIG) in mitigating respiratory comorbidity in COVID-19, and prior literature showed benefit of IVIG in viral ARDS, the exact mechanisms are not known. Current clinical therapeutic trials have targeted NET clearance following their release, but not aimed at preventing their formation. In concordance with previous findings, we hypothesized that inhibition of systemic activation of neutrophils with IVIG may reduce collateral tissue damage by blocking NETosis. Methods. Neutrophils from healthy controls were treated ex vivo with IVIG and NETosis, ROS production and phagocytosis quantified. Stimulants phorbol myristate acetate (PMA), for the canonical pathway, and nigericin, for the non-canonical pathway, were used. NETosis was quantified with Quant-iT™ PicoGreen™ dsDNA Assay (Invitrogen) and by NET visualization using myeloperoxidase staining;ROS production was assessed by OxyBURST™ Green H2DCFDA (Invitrogen). Phagocytosis of pHrodo™ Red S. aureus Bioparticles™ (Invitrogen) was also quantified.Results. Ex vivo treatment of healthy neutrophils with IVIG was associated with a significant decrease in NETosis at concentrations of 5 and 10 mg/ml of IVIG (p<0.0001;Figure 1A). IVIG suppression of NETosis was associated with a dose effect. ROS production was also diminished in all concentrations of IVIG tested, including as low as 0.2 mg/ml (p<0.0001;Figure 1B). Phagocytosis was not only preserved but was boosted in the setting of IVIG treatment at 10 mg/ml (p<0.05;Figure 1C).Conclusion. These ex vivo data utilizing fresh human neutrophils demonstrate the potential beneficial effect of IVIG, an FDAapproved treatment, in the context of neutrophil-mediated immunopathology. IVIG may be a potential treatment to prevent progression of vascular and lung inflammation and damage in COVID-19, consistent with emerging evidence of its therapeutic benefit in this disorder. Results from an FDA Phase 3 prospective randomized international study that evaluates IVIG in COVID-19 are highly anticipated.

Circulating neutrophils from critically ill covid-19 patients have increased netosis via canonical and non-canonical pathways

Introduction. The pathophysiology of infection with SARS-CoV-2 involves the lower airways and host-launched aggressive inflammatory responses leading to exacerbated lung damage in these vital tissues. Early clinical studies found that COVID-19 patients have higher levels of neutrophils in the circulation. Neutrophils are the most abundant leukocyte in circulation and are known to be highly proinflammatory due to production of neutrophil extracellular traps (NETosis). NETs are web-like chromatin structures coated with histones and proteases that both capture and kill invading pathogens. However, while being an effective countermeasure towards foreign microbes, this process also causes undesirable damage in host tissues. Therefore, we sought to characterize NETosis in circulating neutrophils from COVID-19 patients to determine whether this immunological response might be exacerbating or driving the disease state in COVID-19, rather than mitigating the virus. Methods.Blood was drawn daily from critically ill COVID-19 patients (n=16) after consent was obtained. Healthy controls (n=13) were screened for COVID-19 and gave blood once a week. Blood was drawn into lithium heparin tubes (BD Vacutainer). Neutrophils were isolated using PolymorphprepTM(PROGEN) per manufacturer's instructions. Cells were resuspended at 2x106 cells/ml for functional assays. Neutrophils were stimulated with increasing concentrations of PMA (Phorbol 12-myristate 13-acetate) of 2.5nM, 25nM and 250nM to stimulate NETosis via the canonical pathway, and nigericin at 15uM for the non-canonical pathway. NETosis was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen) and by NET visualization via myeloperoxidase and nuclear staining (using Polyclonal Rabbit Anti-Human Myeloperoxidase by Dako and Hoescht stain by Invitrogen). Results.Functional NETosis assays of circulating neutrophils from COVID-19 patients demonstrate overall increased NETosis determined by increased release of dsDNA. This enhanced NETosis occurred at baseline and after stimulation with PMA when compared to healthy controls (Figure 1A, p <0.0001). Fluorescent microscopy also demonstrated increased NETosis in neutrophils from COVID-19 patients (Figure 1B;MPO-green and nucleus-blue). NETosis via the non-canonical pathway (induction with nigericin) was also increased in COVID-19 patients versus controls (p=0.02). Conclusions.Circulating neutrophils from critically ill COVID-19 patients are more prone to produce NETs than circulating neutrophils from healthy individuals. This is likely to lead to NETmediated tissue injury once neutrophils enter inflamed tissue, where they can potentially drive acute lung injury and acute respiratory distress syndrome, common causes of mortality in COVID-19. The finding of increased production of NETs by both canonical and non-canonical pathways is consistent with an overall hyper-activated state in COVID-19.

Very-low driving pressure ventilation in patients with acute respiratory distress syndrome on extracorporeal membrane oxygenation

Rationale: The ideal ventilator settings in patients with severe acute respiratory distress syndrome (ARDS) while on Extracorporeal Membrane Oxygenation (ECMO) are unknown. ELSO recommends modest driving pressures and PEEP (i.e., “lung rest”) during ECMO to minimize ventilator induced lung injury (VILI) and improve mortality. However, even lower driving pressures (DP) may improve inflammatory and lung injury biomarker profiles, and possibly improve mortality. The focus of this study was to evaluate the feasibility and impact of very-low DP in patients with ARDS on ECMO. Methods: Patients on ECMO due to ARDS from coronavirus disease 2019 had their DP decreased from 10 to 1-5 cmH2O for two hours, as tolerated. The other ventilator settings were unchanged and were consistent with ELSO guidelines;positive end-expiratory pressure (10-15 cmH2O) and a respiratory rate 10 breaths per minute. During the low DP protocol, the transpulmonary pressure was monitored with esophageal manometry. Plasma (for IL6 and sRAGE) and urine were collected before and after the protocol. Results: Thirty patients were enrolled and ultimately 21 patients underwent the low DP protocol. Seventeen were male, aged 51 ± 9 years, median BMI of 31.7 kg/m2 (IQR 27.4-35.2), with a median SOFA score of 10 (IQR 8-12) at ICU admission. There were no complications during the low DP protocol (1-5 cm H2O). The protocol was performed a median of 16 (IQR 13.5-20) hours after ECMO initiation. Average ECMO days were 21 (IQR 12.5-41) with a survival to hospital discharge of 43% (9/21). Baseline average tidal volume (TV) was 3.4±2.6 ml/kg of predicted body weight (PBW), during the low DP protocol the TV decreased to 2.7±3.2 ml/kg of PDW. Two groups were noted-those who had spontaneous breaths vs. those who did not (see Figure 1). Ten patients during the low DP protocol continued to have unchanged TV, 5.2±2.8 to 5.4±2.7 ml/kg of PBW due to spontaneous breathing found on esophageal manometry. Eleven patients had no spontaneous breaths and TV decreased from 1.76±0.57 to 0.2±0.38 ml/kg of PBW during the low DP protocol. The spontaneous vs. nonspontaneous breathing groups has a statistically different TV before, during, and after the low DP protocol (p<0.001). Conclusions: Very-low DP is feasible in patients on ECMO support which may decrease VILI. However, in the absence of neuromuscular blockade patients might spontaneously breathe with the same transpulmonary pressure. Thus, whether low DP offers any advantage in spontaneous breathing vs. non-spontaneous breathing requires further study. (Table Presented).