Whole blood coagulation in an ex vivo thrombus is sufficient to induce clot neutrophils to adopt a myeloid-derived suppressor cell signature and shed soluble Lox-1.

BACKGROUND: Blood clots are living tissues that release inflammatory mediators including IL-8/CXCL8 and MCP-1/CCL2. A deeper understanding of blood clots is needed to develop new therapies for prothrombotic disease states and regenerative medicine. OBJECTIVES: To identify a common transcriptional shift in cultured blood clot leukocytes. METHODS: Differential gene expression of whole blood and cultured clots (4 hours at 37 °C) was assessed by RNA sequencing (RNAseq), reverse transcriptase-polymerase chain reaction, proteomics, and histology (23 diverse healthy human donors). Cultured clot serum bioactivity was tested in endothelial barrier functional assays. RESULTS: All cultured clots developed a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) signature, including up-regulation of OLR1 (mRNA encoding lectin-like oxidized low-density lipoprotein receptor 1 [Lox-1]), IL-8/CXCL8, CXCL2, CCL2, IL10, IL1A, SPP1, TREM1, and DUSP4/MKP. Lipopolysaccharide enhanced PMN-MDSC gene expression and specifically induced a type II interferon response with IL-6 production. Lox-1 was specifically expressed by cultured clot CD15+ neutrophils. Cultured clot neutrophils, but not activated platelets, shed copious amounts of soluble Lox-1 (sLox-1) with a donor-dependent amplitude. sLox-1 shedding was enhanced by phorbol ester and suppressed by heparin and by beta-glycerol phosphate, a phosphatase inhibitor. Cultured clot serum significantly enhanced endothelial cell monolayer barrier function, consistent with a proresolving bioactivity. CONCLUSION: This study suggests that PMN-MDSC activation is part of the innate immune response to coagulation which may have a protective role in inflammation. The cultured blood clot is an innovative thrombus model that can be used to study both sterile and nonsterile inflammatory states and could be used as a personalized medicine tool for drug screening.

Optimizing RBC Transfusion Outcomes in Patients with Acute Illness and in the Chronic Transfusion Setting.

Red blood cell (RBC) transfusion is a common clinical intervention used to treat patients with acute and chronic anemia. The decision to transfuse RBCs in the acute setting is based on several factors but current clinical studies informing optimal RBC transfusion decision making (TDM) are largely based upon hemoglobin (Hb) level. In contrast to transfusion in acute settings, chronic RBC transfusion therapy has several different purposes and is associated with distinct transfusion risks such as iron overload and RBC alloimmunization. Consequently, RBC TDM in the chronic setting requires optimizing the survival of transfused RBCs in order to reduce transfusion exposure over the lifespan of an individual and the associated transfusion complications mentioned. This review summarizes the current medical literature addressing optimal RBC-TDM in the acute and chronic transfusion settings and discusses the current gaps in knowledge which need to be prioritized in future national and international research initiatives.

Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles.

Introduction: Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. Methods: RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation. Results: Our results showed that treating RBCs at 6% hematocrit with 10 µM yoda1 for 30 min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. Conclusion: This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated in vivo under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.

Hyperferritinemic sepsis, macrophage activation syndrome, and mortality in a pediatric research network: a causal inference analysis.

BACKGROUND: One of five global deaths are attributable to sepsis. Hyperferritinemic sepsis (> 500 ng/mL) is associated with increased mortality in single-center studies. Our pediatric research network's objective was to obtain rationale for designing anti-inflammatory clinical trials targeting hyperferritinemic sepsis. METHODS: We assessed differences in 32 cytokines, immune depression (low whole blood ex vivo TNF response to endotoxin) and thrombotic microangiopathy (low ADAMTS13 activity) biomarkers, seven viral DNAemias, and macrophage activation syndrome (MAS) defined by combined hepatobiliary dysfunction and disseminated intravascular coagulation, and mortality in 117 children with hyperferritinemic sepsis (ferritin level > 500 ng/mL) compared to 280 children with sepsis without hyperferritinemia. Causal inference analysis of these 41 variables, MAS, and mortality was performed. RESULTS: Mortality was increased in children with hyperferritinemic sepsis (27/117, 23% vs 16/280, 5.7%; Odds Ratio = 4.85, 95% CI [2.55-9.60]; z = 4.728; P-value < 0.0001). Hyperferritinemic sepsis had higher C-reactive protein, sCD163, IL-22, IL-18, IL-18 binding protein, MIG/CXCL9, IL-1ß, IL-6, IL-8, IL-10, IL-17a, IFN-γ, IP10/CXCL10, MCP-1/CCL2, MIP-1α, MIP-1ß, TNF, MCP-3, IL-2RA (sCD25), IL-16, M-CSF, and SCF levels; lower ADAMTS13 activity, sFasL, whole blood ex vivo TNF response to endotoxin, and TRAIL levels; more Adenovirus, BK virus, and multiple virus DNAemias; and more MAS (P-value < 0.05). Among these variables, only MCP-1/CCL2 (the monocyte chemoattractant protein), MAS, and ferritin levels were directly causally associated with mortality. MCP-1/CCL2 and hyperferritinemia showed direct causal association with depressed ex vivo whole blood TNF response to endotoxin. MCP-1/CCL2 was a mediator of MAS. MCP-1/CCL2 and MAS were mediators of hyperferritinemia. CONCLUSIONS: These findings establish hyperferritinemic sepsis as a high-risk condition characterized by increased cytokinemia, viral DNAemia, thrombotic microangiopathy, immune depression, macrophage activation syndrome, and death. The causal analysis provides rationale for designing anti-inflammatory trials that reduce macrophage activation to improve survival and enhance infection clearance in pediatric hyperferritinemic sepsis.

Early, Persistent Lymphopenia Is Associated With Prolonged Multiple Organ Failure and Mortality in Septic Children.

OBJECTIVES: Sepsis-associated immune suppression correlates with poor outcomes. Adult trials are evaluating immune support therapies. Limited data exist to support consideration of immunomodulation in pediatric sepsis. We tested the hypothesis that early, persistent lymphopenia predicts worse outcomes in pediatric severe sepsis. DESIGN: Observational cohort comparing children with severe sepsis and early, persistent lymphopenia (absolute lymphocyte count < 1,000 cells/µL on 2 d between study days 0-5) to children without. The composite outcome was prolonged multiple organ dysfunction syndrome (MODS, organ dysfunction beyond day 7) or PICU mortality. SETTING: Nine PICUs in the National Institutes of Health Collaborative Pediatric Critical Care Research Network between 2015 and 2017. PATIENTS: Children with severe sepsis and indwelling arterial and/or central venous catheters. INTERVENTIONS: Blood sampling and clinical data analysis. MEASUREMENTS AND MAIN RESULTS: Among 401 pediatric patients with severe sepsis, 152 (38%) had persistent lymphopenia. These patients were older, had higher illness severity, and were more likely to have underlying comorbidities including solid organ transplant or malignancy. Persistent lymphopenia was associated with the composite outcome prolonged MODS or PICU mortality (66/152, 43% vs 45/249, 18%; p < 0.01) and its components prolonged MODS (59/152 [39%] vs 43/249 [17%]), and PICU mortality (32/152, 21% vs 12/249, 5%; p < 0.01) versus children without. After adjusting for baseline factors at enrollment, the presence of persistent lymphopenia was associated with an odds ratio of 2.98 (95% CI [1.85-4.02]; p < 0.01) for the composite outcome. Lymphocyte count trajectories showed that patients with persistent lymphopenia generally did not recover lymphocyte counts during the study, had lower nadir whole blood tumor necrosis factor-α response to lipopolysaccharide stimulation, and higher maximal inflammatory markers (C-reactive protein and ferritin) during days 0-3 ( p < 0.01). CONCLUSIONS: Children with severe sepsis and persistent lymphopenia are at risk of prolonged MODS or PICU mortality. This evidence supports testing therapies for pediatric severe sepsis patients risk-stratified by early, persistent lymphopenia.

Capacity Building in Pediatric Critical Care-Global Health Research and Education: The Blantyre Experience.

Pediatric critical care medicine (PCCM), as it is practiced in high-income countries, is focused on specialized medical care for the most vulnerable pediatric patient populations. However, best practices for provision of that care globally are lacking. Thus, PCCM research and education programming can potentially fill significant knowledge gaps by facilitating the development of evidence-based clinical guidelines that reduce child mortality on a global scale. Malaria remains a leading cause of pediatric mortality worldwide. The Blantyre Malaria Project (BMP) is a research and clinical care collaborative that has focused on reducing the public health burden of pediatric cerebral malaria in Malawi since 1986. In 2017, the requirements of a new research study led to the creation of PCCM services in Blantyre, creating the opportunity to establish a PCCM-Global Health Research Fellowship by BMP in collaboration with the University of Maryland School of Medicine. In this perspective piece, we reflect on the evolution of the PCCM-Global Health research fellowship. Although the specifics of this fellowship are out of the scope of this perspective, we discuss the context allowing for the development of this program and explore some early lessons learned to consider for future capacity-building efforts in the future of PCCM-Global Health research.

Acute Disorders of Consciousness in Pediatric Severe Sepsis and Organ Failure: Secondary Analysis of the Multicenter Phenotyping Sepsis-Induced Multiple Organ Failure Study.

OBJECTIVES: Acute disorders of consciousness (DoC) in pediatric severe sepsis are associated with increased risk of morbidity and mortality. We sought to examine the frequency of and factors associated with DoC in children with sepsis-induced organ failure. DESIGN: Secondary analysis of the multicenter Phenotyping Sepsis-Induced Multiple Organ Failure Study (PHENOMS). SETTING: Nine tertiary care PICUs in the United States. PATIENTS: Children less than 18 years old admitted to a PICU with severe sepsis and at least one organ failure during a PICU stay. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was frequency of DoC, defined as Glasgow Coma Scale (GCS) less than 12 in the absence of sedatives during an ICU stay, among children with severe sepsis and the following: single organ failure, nonphenotypeable multiple organ failure (MOF), MOF with one of the PHENOMS phenotypes (immunoparalysis-associated MOF [IPMOF], sequential liver failure-associated MOF, thrombocytopenia-associated MOF), or MOF with multiple phenotypes. A multivariable logistic regression analysis was performed to evaluate the association between clinical variables and organ failure groups with DoC. Of 401 children studied, 71 (18%) presented with DoC. Children presenting with DoC were older (median 8 vs 5 yr; p = 0.023), had increased hospital mortality (21% vs 10%; p = 0.011), and more frequently presented with both any MOF (93% vs 71%; p < 0.001) and macrophage activation syndrome (14% vs 4%; p = 0.004). Among children with any MOF, those presenting with DoC most frequently had nonphenotypeable MOF and IPMOF (52% and 34%, respectively). In the multivariable analysis, older age (odds ratio, 1.07; 95% CI, 1.01-1.12) and any MOF (3.22 [1.19-8.70]) were associated with DoC. CONCLUSIONS: One of every five children with severe sepsis and organ failure experienced acute DoC during their PICU stay. Preliminary findings suggest the need for prospective evaluation of DoC in children with sepsis and MOF.

Factors influencing pediatric transfusion: A complex decision impacting quality of care.

BACKGROUND: The risks of red blood cell transfusion may outweigh the benefits for many patients in pediatric intensive care units (PICUs), but guidelines from the Transfusion and Anemia eXpertise Initiative (TAXI) have not been consistently adopted. We sought to identify factors that influenced transfusion decision-making in PICUs to explore potential barriers and facilitators to implementing the guidelines. STUDY DESIGN AND METHODS: A total of 50 ICU providers working in eight US ICUs of different types (non-cardiac PICUs, cardiovascular ICUs, combined units) and variable sizes (11-32 beds) completed semi-structured interviews. Providers included ICU attendings and trainees, nurse practitioners, nurses, and subspecialty physicians. Interviews examined factors that influenced transfusion decisions, transfusion practices, and provider beliefs. Qualitative analysis utilized a Framework Approach. Summarized data was compared between provider roles and units with consideration to identify patterns and unique informative statements. RESULTS: Providers cited clinical, physiologic, anatomic, and logistic factors they considered in making transfusion decisions. Improving oxygen carrying capacity, hemodynamics and perfusion, respiratory function, volume deficits, and correcting laboratory values were among the reasons given for transfusion. Other sought-after benefits included alleviating symptoms of anemia, improving ICU throughput, and decreasing blood waste. Providers in different roles approached transfusion decisions differently, with the largest differences noted between nurses and subspecialists as compared with other ICU providers. While ICU attendings most often made the decision to transfuse, all providers influenced the decision-making. DISCUSSION: Implementation of transfusion guidelines requires multi-professional approaches that emphasize the known risks of transfusion, its limited benefits, and highlight evidence around the safety and benefit of restrictive approaches.

The Impact of Restrictive Transfusion Practices on Hemodynamically Stable Critically Ill Children Without Heart Disease: A Secondary Analysis of the Age of Blood in Children in the PICU Trial.

OBJECTIVES: Guidelines recommend against RBC transfusion in hemodynamically stable (HDS) children without cardiac disease, if hemoglobin is greater than or equal to 7 g/dL. We sought to assess the clinical and economic impact of compliance with RBC transfusion guidelines. DESIGN: A nonprespecified secondary analysis of noncardiac, HDS patients in the randomized trial Age of Blood in Children (NCT01977547) in PICUs. Costs analyzed included ICU stay and physician fees. Stabilized inverse propensity for treatment weighting was used to create a cohort balanced with respect to potential confounding variables. Weighted regression models were fit to evaluate outcomes based on guideline compliance. SETTING: Fifty international tertiary care centers. PATIENTS: Critically ill children 3 days to 16 years old transfused RBCs at less than or equal to 7 days of ICU admission. Six-hundred eighty-seven subjects who met eligibility criteria were included in the analysis. INTERVENTIONS: Initial RBC transfusions administered when hemoglobin was less than 7 g/dL were considered "compliant" or "non-compliant" if hemoglobin was greater than or equal to 7 g/dL. MEASUREMENTS AND MAIN RESULTS: Frequency of new or progressive multiple organ system dysfunction (NPMODS), ICU survival, and associated costs. The hypothesis was formulated after data collection but exposure groups were masked until completion of planned analyses. Forty-nine percent of patients (338/687) received a noncompliant initial transfusion. Weighted cohorts were balanced with respect to confounding variables (absolute standardized differences < 0.1). No differences were noted in NPMODS frequency (relative risk, 0.86; 95% CI, 0.61-1.22; p = 0.4). Patients receiving compliant transfusions had more ICU-free days (mean difference, 1.73; 95% CI, 0.57-2.88; p = 0.003). Compliance reduced mean costs in ICU by $38,845 U.S. dollars per patient (95% CI, $65,048-$12,641). CONCLUSIONS: Deferring transfusion until hemoglobin is less than 7 g/dL is not associated with increased organ dysfunction in this population but is independently associated with increased likelihood of live ICU discharge and lower ICU costs.

Anticoagulation practices associated with bleeding and thrombosis in pediatric extracorporeal membrane oxygenation; a multi-center secondary analysis.

To determine associations between anticoagulation practices and bleeding and thrombosis during pediatric extracorporeal membrane oxygenation (ECMO), we performed a secondary analysis of prospectively collected data which included 481 children (<19 years), between January 2012 and September 2014. The primary outcome was bleeding or thrombotic events. Bleeding events included a blood product transfusion >80 ml/kg on any day, pulmonary hemorrhage, or intracranial bleeding, Thrombotic events included pulmonary emboli, intracranial clot, limb ischemia, cardiac clot, and arterial cannula or entire circuit change. Bleeding occurred in 42% of patients. Five percent of subjects thrombosed, of which 89% also bled. Daily bleeding odds were independently associated with day prior activated clotting time (ACT) (OR 1.03, 95% CI= 1.00, 1.05, p=0.047) and fibrinogen levels (OR 0.90, 95% CI 0.84, 0.96, p <0.001). Thrombosis odds decreased with increased day prior heparin dose (OR 0.88, 95% CI 0.81, 0.97, p=0.006). Lower ACT values and increased fibrinogen levels may be considered to decrease the odds of bleeding. Use of this single measure, however, may not be sufficient alone to guide optimal anticoagulation practice during ECMO.

COVID-19 impairs oxygen delivery by altering red blood cell hematological, hemorheological, and oxygen transport properties.

Introduction: Coronavirus disease 2019 (COVID-19) is characterized by impaired oxygen (O2) homeostasis, including O2 sensing, uptake, transport/delivery, and consumption. Red blood cells (RBCs) are central to maintaining O2 homeostasis and undergo direct exposure to coronavirus in vivo. We thus hypothesized that COVID-19 alters RBC properties relevant to O2 homeostasis, including the hematological profile, Hb O2 transport characteristics, rheology, and the hypoxic vasodilatory (HVD) reflex. Methods: RBCs from 18 hospitalized COVID-19 subjects and 20 healthy controls were analyzed as follows: (i) clinical hematological parameters (complete blood count; hematology analyzer); (ii) O2 dissociation curves (p50, Hill number, and Bohr plot; Hemox-Analyzer); (iii) rheological properties (osmotic fragility, deformability, and aggregation; laser-assisted optical rotational cell analyzer (LORRCA) ektacytometry); and (iv) vasoactivity (the RBC HVD; vascular ring bioassay). Results: Compared to age- and gender-matched healthy controls, COVID-19 subjects demonstrated 1) significant hematological differences (increased WBC count-with a higher percentage of neutrophils); RBC distribution width (RDW); and reduced hematocrit (HCT), Hb concentration, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC); 2) impaired O2-carrying capacity and O2 capacitance (resulting from anemia) without difference in p50 or Hb-O2 cooperativity; 3) compromised regulation of RBC volume (altered osmotic fragility); 4) reduced RBC deformability; 5) accelerated RBC aggregation kinetics; and (6) no change in the RBC HVD reflex. Discussion: When considered collectively, homeostatic compensation for these RBC impairments requires that the cardiac output in the COVID cohort would need to increase by ∼135% to maintain O2 delivery similar to that in the control cohort. Additionally, the COVID-19 disease RBC properties were found to be exaggerated in blood-type O hospitalized COVID-19 subjects compared to blood-type A. These data indicate that altered RBC features in hospitalized COVID-19 subjects burden the cardiovascular system to maintain O2 delivery homeostasis, which appears exaggerated by blood type (more pronounced with blood-type O) and likely plays a role in disease pathogenesis.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma.

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Mortality Risk in Pediatric Sepsis Based on C-reactive Protein and Ferritin Levels.

OBJECTIVES: Interest in using bedside C-reactive protein (CRP) and ferritin levels to identify patients with hyperinflammatory sepsis who might benefit from anti-inflammatory therapies has piqued with the COVID-19 pandemic experience. Our first objective was to identify patterns in CRP and ferritin trajectory among critically ill pediatric sepsis patients. We then examined the association between these different groups of patients in their inflammatory cytokine responses, systemic inflammation, and mortality risks. DATA SOURCES: A prospective, observational cohort study. STUDY SELECTION: Children with sepsis and organ failure in nine pediatric intensive care units in the United States. DATA EXTRACTION: Two hundred and fifty-five children were enrolled. Five distinct clinical multi-trajectory groups were identified. Plasma CRP (mg/dL), ferritin (ng/mL), and 31 cytokine levels were measured at two timepoints during sepsis (median Day 2 and Day 5). Group-based multi-trajectory models (GBMTM) identified groups of children with distinct patterns of CRP and ferritin. DATA SYNTHESIS: Group 1 had normal CRP and ferritin levels ( n = 8; 0% mortality); Group 2 had high CRP levels that became normal, with normal ferritin levels throughout ( n = 80; 5% mortality); Group 3 had high ferritin levels alone ( n = 16; 6% mortality); Group 4 had very high CRP levels, and high ferritin levels ( n = 121; 11% mortality); and Group 5 had very high CRP and very high ferritin levels ( n = 30; 40% mortality). Cytokine responses differed across the five groups, with ferritin levels correlated with macrophage inflammatory protein 1α levels and CRP levels reflective of many cytokines. CONCLUSIONS: Bedside CRP and ferritin levels can be used together to distinguish groups of children with sepsis who have different systemic inflammation cytokine responses and mortality risks. These data suggest future potential value in personalized clinical trials with specific targets for anti-inflammatory therapies.

Etiology, Pathophysiology and Mortality of Shock in Children in Low (Middle) Income Countries: A Systematic Review.

OBJECTIVES: Shock is a life-threatening condition in children in low- and middle-income countries (LMIC), with several controversies. This systematic review summarizes the etiology, pathophysiology and mortality of shock in children in LMIC. METHODS: We searched for studies reporting on children with shock in LMIC in PubMed, Embase and through snowballing (up to 1 October 2019). Studies conducted in LMIC that reported on shock in children (1 month-18 years) were included. We excluded studies only containing data on neonates, cardiac surgery patients or iatrogenic causes. We presented prevalence data, pooled mortality estimates and conducted subgroup analyses per definition, region and disease. Etiology and pathophysiology data were systematically collected. RESULTS: We identified 959 studies and included 59 studies of which six primarily studied shock. Definitions used for shock were classified into five groups. Prevalence of shock ranged from 1.5% in a pediatric hospital population to 44.3% in critically ill children. Pooled mortality estimates ranged between 3.9-33.3% for the five definition groups. Important etiologies included gastroenteritis, sepsis, malaria and severe anemia, which often coincided. The pathophysiology was poorly studied but suggests that in addition to hypovolemia, dissociative and cardiogenic shock are common in LMIC. CONCLUSIONS: Shock is associated with high mortality in hospitalized children in LMIC. Despite the importance few studies investigated shock and as a consequence limited data on etiology and pathophysiology of shock is available. A uniform bedside definition may help boost future studies unravelling shock etiology and pathophysiology in LMIC.

Machine learning derivation of four computable 24-h pediatric sepsis phenotypes to facilitate enrollment in early personalized anti-inflammatory clinical trials.

BACKGROUND: Thrombotic microangiopathy-induced thrombocytopenia-associated multiple organ failure and hyperinflammatory macrophage activation syndrome are important causes of late pediatric sepsis mortality that are often missed or have delayed diagnosis. The National Institutes of General Medical Science sepsis research working group recommendations call for application of new research approaches in extant clinical data sets to improve efficiency of early trials of new sepsis therapies. Our objective is to apply machine learning approaches to derive computable 24-h sepsis phenotypes to facilitate personalized enrollment in early anti-inflammatory trials targeting these conditions. METHODS: We applied consensus, k-means clustering analysis to our extant PHENOtyping sepsis-induced Multiple organ failure Study (PHENOMS) dataset of 404 children. 24-hour computable phenotypes are derived using 25 available bedside variables including C-reactive protein and ferritin. RESULTS: Four computable phenotypes (PedSep-A, B, C, and D) are derived. Compared to all other phenotypes, PedSep-A patients (n = 135; 2% mortality) were younger and previously healthy, with the lowest C-reactive protein and ferritin levels, the highest lymphocyte and platelet counts, highest heart rate, and lowest creatinine (p < 0.05); PedSep-B patients (n = 102; 12% mortality) were most likely to be intubated and had the lowest Glasgow Coma Scale Score (p < 0.05); PedSep-C patients (n = 110; mortality 10%) had the highest temperature and Glasgow Coma Scale Score, least pulmonary failure, and lowest lymphocyte counts (p < 0.05); and PedSep-D patients (n = 56, 34% mortality) had the highest creatinine and number of organ failures, including renal, hepatic, and hematologic organ failure, with the lowest platelet counts (p < 0.05). PedSep-D had the highest likelihood of developing thrombocytopenia-associated multiple organ failure (Adj OR 47.51 95% CI [18.83-136.83], p < 0.0001) and macrophage activation syndrome (Adj OR 38.63 95% CI [13.26-137.75], p < 0.0001). CONCLUSIONS: Four computable phenotypes are derived, with PedSep-D being optimal for enrollment in early personalized anti-inflammatory trials targeting thrombocytopenia-associated multiple organ failure and macrophage activation syndrome in pediatric sepsis. A computer tool for identification of individual patient membership ( www.pedsepsis.pitt.edu ) is provided. Reproducibility will be assessed at completion of two ongoing pediatric sepsis studies.

Prevalence of Pathogenic and Potentially Pathogenic Inborn Error of Immunity Associated Variants in Children with Severe Sepsis.

PURPOSE: Our understanding of inborn errors of immunity is increasing; however, their contribution to pediatric sepsis is unknown. METHODS: We used whole-exome sequencing (WES) to characterize variants in genes related to monogenic immunologic disorders in 330 children admitted to intensive care for severe sepsis. We defined candidate variants as rare variants classified as pathogenic or potentially pathogenic in QIAGEN's Human Gene Mutation Database or novel null variants in a disease-consistent inheritance pattern. We investigated variant correlation with infection and inflammatory phenotype. RESULTS: More than one in two children overall and three of four African American children had immunodeficiency-associated variants. Children with variants had increased odds of isolating a blood or urinary pathogen (blood: OR 2.82, 95% CI: 1.12-7.10, p = 0.023, urine: OR: 8.23, 95% CI: 1.06-64.11, p = 0.016) and demonstrating increased inflammation with hyperferritinemia (ferritin [Formula: see text] ng/mL, OR: 2.16, 95% CI: 1.28-3.66, p = 0.004), lymphopenia (lymphocyte count < 1000/µL, OR: 1.66, 95% CI: 1.06 - 2.60, p = 0.027), thrombocytopenia (platelet count < 150,000/µL, OR: 1.76, 95% CI: 1.12-2.76, p = 0.013), and CRP greater than 10 mg/dl (OR: 1.71, 95% CI: 1.10-2.68, p = 0.017). They also had increased odds of requiring extracorporeal membrane oxygenation (ECMO, OR: 4.19, 95% CI: 1.21-14.5, p = 0.019). CONCLUSION: Herein, we describe the genetic findings in this severe pediatric sepsis cohort and their microbiologic and immunologic significance, providing evidence for the phenotypic effect of these variants and rationale for screening children with life-threatening infections for potential inborn errors of immunity.

Outcomes Associated With Early RBC Transfusion in Pediatric Severe Sepsis: A Propensity-Adjusted Multicenter Cohort Study.

BACKGROUND: Little is known about the epidemiology of and outcomes related to red blood cell (RBC) transfusion in septic children across multiple centers. We performed propensity-adjusted secondary analyses of the Biomarker Phenotyping of Pediatric Sepsis and Multiple Organ Failure (PHENOMS) study to test the hypothesis that early RBC transfusion is associated with fewer organ failure-free days in pediatric severe sepsis. METHODS: Four hundred one children were enrolled in the parent study. Children were excluded from these analyses if they received extracorporeal membrane oxygenation (n = 22) or died (n = 1) before sepsis day 2. Propensity-adjusted analyses compared children who received RBC transfusion on or before sepsis day 2 (early RBC transfusion) with those who did not. Logistic regression was used to model the propensity to receive early RBC transfusion. A weighted cohort was constructed using stabilized inverse probability of treatment weights. Variables in the weighted cohort with absolute standardized differences >0.15 were added to final multivariable models. RESULTS: Fifty percent of children received at least one RBC transfusion. The majority (68%) of first transfusions were on or before sepsis day 2. Early RBC transfusion was not independently associated with organ failure-free (-0.34 [95%CI: -2, 1.3] days) or PICU-free days (-0.63 [-2.3, 1.1]), but was associated with the secondary outcome of higher mortality (aOR 2.9 [1.1, 7.9]). CONCLUSIONS: RBC transfusion is common in pediatric severe sepsis and may be associated with adverse outcomes. Future studies are needed to clarify these associations, to understand patient-specific transfusion risks, and to develop more precise transfusion strategies.

Pediatric Organ Dysfunction Information Update Mandate (PODIUM) Contemporary Organ Dysfunction Criteria: Executive Summary.

Prior criteria for organ dysfunction in critically ill children were based mainly on expert opinion. We convened the Pediatric Organ Dysfunction Information Update Mandate (PODIUM) expert panel to summarize data characterizing single and multiple organ dysfunction and to derive contemporary criteria for pediatric organ dysfunction. The panel was composed of 88 members representing 47 institutions and 7 countries. We conducted systematic reviews of the literature to derive evidence-based criteria for single organ dysfunction for neurologic, cardiovascular, respiratory, gastrointestinal, acute liver, renal, hematologic, coagulation, endocrine, endothelial, and immune system dysfunction. We searched PubMed and Embase from January 1992 to January 2020. Study identification was accomplished using a combination of medical subject headings terms and keywords related to concepts of pediatric organ dysfunction. Electronic searches were performed by medical librarians. Studies were eligible for inclusion if the authors reported original data collected in critically ill children; evaluated performance characteristics of scoring tools or clinical assessments for organ dysfunction; and assessed a patient-centered, clinically meaningful outcome. Data were abstracted from each included study into an electronic data extraction form. Risk of bias was assessed using the Quality in Prognosis Studies tool. Consensus was achieved for a final set of 43 criteria for pediatric organ dysfunction through iterative voting and discussion. Although the PODIUM criteria for organ dysfunction were limited by available evidence and will require validation, they provide a contemporary foundation for researchers to identify and study single and multiple organ dysfunction in critically ill children.

Hematologic Dysfunction Criteria in Critically Ill Children: The PODIUM Consensus Conference.

CONTEXT: Studies of organ dysfunction in children are limited by a lack of consensus around organ dysfunction criteria. OBJECTIVES: To derive evidence-informed, consensus-based criteria for hematologic dysfunction in critically ill children. DATA SOURCES: Data sources included PubMed and Embase from January 1992 to January 2020. STUDY SELECTION: Studies were included if they evaluated assessment/scoring tools to screen for hematologic dysfunction and assessed outcomes of mortality, functional status, organ-specific outcomes, or other patient-centered outcomes. Studies of adults or premature infants, animal studies, reviews/commentaries, small case series, and non-English language studies with inability to determine eligibility were excluded. DATA EXTRACTION: Data were abstracted from each eligible study into a standard data extraction form along with risk of bias assessment. RESULTS: Twenty-nine studies were included. The systematic review supports the following criteria for hematologic dysfunction: thrombocytopenia (platelet count <100000 cells/µL in patients without hematologic or oncologic diagnosis, platelet count <30000 cells/µL in patients with hematologic or oncologic diagnoses, or platelet count decreased ≥50% from baseline; or leukocyte count <3000 cells/µL; or hemoglobin concentration between 5 and 7 g/dL (nonsevere) or <5 g/dL (severe). LIMITATIONS: Most studies evaluated pre-specified thresholds of cytopenias. No studies addressed associations between the etiology or progression of cytopenias overtime with outcomes, and no studies evaluated cellular function. CONCLUSIONS: Hematologic dysfunction, as defined by cytopenia, is a risk factor for poor outcome in critically ill children, although specific threshold values associated with increased mortality are poorly defined by the current literature.