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.

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.

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.

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.

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.

A clickable probe for versatile characterization of S-nitrosothiols.

S-nitrosation of cysteine thiols (SNOs), commonly referred to as S-nitrosylation, is a cysteine oxoform that plays an important role in cellular signaling and impacts protein function and stability. Direct labeling of SNOs in cells with the flexibility to perform a wide range of cellular and biochemical assays remains a bottleneck as all SNO-targeted probes to date employ a single analytical modality such as biotin or a specific fluorophore. We therefore developed a clickable, alkyne-containing SNO probe 'PBZyn' based on the o-phosphino-benzoyl group warhead that enables multi-modal analysis via click conjugation. We demonstrate the utility of PBZyn to assay SNOs using in situ cellular imaging, protein blotting and affinity purification, as well as mass spectrometry. The flexible PBZyn probe will greatly facilitate investigation into the regulation of SNOs.

Red Blood Cell Dysfunction in Critical Illness.

Oxygen (O2) delivery, which is fundamental to supporting patients with critical illness, is a function of blood O2 content and flow. This article reviews red blood cell (RBC) physiology and dysfunction relevant to disordered O2 delivery in the critically ill. Flow is the focus of O2 delivery regulation: O2 content is relatively fixed, whereas flow fluctuates greatly. Thus, blood flow volume and distribution vary to maintain coupling between O2 delivery and demand. This article reviews conventional RBC physiology influencing O2 delivery and introduces a paradigm for O2 delivery homeostasis based on coordinated gas transport and vascular signaling by RBCs.

Effects of blood storage age on immune, coagulation, and nitric oxide parameters in transfused patients undergoing cardiac surgery.

BACKGROUND: Retrospective studies suggested that storage age of RBCs is associated with inflammation and thromboembolism. The Red Cell Storage Duration Study (RECESS) trial randomized subjects undergoing complex cardiac surgery to receive RBCs stored for shorter versus longer periods, and no difference was seen in the primary outcome of change in multiple organ dysfunction score. STUDY DESIGN AND METHODS: In the current study, 90 subjects from the RECESS trial were studied intensively using a range of hemostasis, immunologic, and nitric oxide parameters. Samples were collected before transfusion and on Days 2, 6, 28, and 180 after transfusion. RESULTS: Of 71 parameters tested, only 4 showed a significant difference after transfusion between study arms: CD8+ T-cell interferon-γ secretion and the concentration of extracellular vesicles bearing the B-cell marker CD19 were higher, and plasma endothelial growth factor levels were lower in recipients of fresh versus aged RBCs. Plasma interleukin-6 was higher at Day 2 and lower at Days 6 and 28 in recipients of fresh versus aged RBCs. Multiple parameters showed significant modulation after surgery and transfusion. Most analytes that changed after surgery did not differ based on transfusion status. Several extracellular vesicle markers, including two associated with platelets (CD41a and CD62P), decreased in transfused patients more than in those who underwent surgery without transfusion. CONCLUSIONS: Transfusion of fresh versus aged RBCs does not result in substantial changes in hemostasis, immune, or nitric oxide parameters. It is possible that transfusion modulates the level of platelet-derived extracellular vesicles, which will require study of patients randomly assigned to receipt of transfusion to define.

Consensus Recommendations for RBC Transfusion Practice in Critically Ill Children From the Pediatric Critical Care Transfusion and Anemia Expertise Initiative.

OBJECTIVES: To date, there are no published guidelines to direct RBC transfusion decision-making specifically for critically ill children. We present the recommendations from the Pediatric Critical Care Transfusion and Anemia Expertise Initiative. DESIGN: Consensus conference series of multidisciplinary, international experts in RBC transfusion management of critically ill children. SETTING: Not applicable. INTERVENTION: None. SUBJECTS: Children with, or children at risk for, critical illness who receive or are at risk for receiving a RBC transfusion. METHODS: A panel of 38 content and four methodology experts met over the course of 2 years to develop evidence-based, and when evidence lacking, expert consensus-based recommendations regarding decision-making for RBC transfusion management and research priorities for transfusion in critically ill children. The experts focused on nine specific populations of critically ill children: general, respiratory failure, nonhemorrhagic shock, nonlife-threatening bleeding or hemorrhagic shock, acute brain injury, acquired/congenital heart disease, sickle cell/oncology/transplant, extracorporeal membrane oxygenation/ventricular assist/ renal replacement support, and alternative processing. Data to formulate evidence-based and expert consensus recommendations were selected based on searches of PubMed, EMBASE, and Cochrane Library from 1980 to May 2017. Agreement was obtained using the Research and Development/UCLA Appropriateness Method. Results were summarized using the Grading of Recommendations Assessment, Development, and Evaluation method. MEASUREMENTS AND RESULTS: The Transfusion and Anemia Expertise Initiative consensus conference developed and reached consensus on a total of 102 recommendations (57 clinical [20 evidence based, 37 expert consensus], 45 research recommendations). All final recommendations met agreement, defined a priori as greater than 80%. A decision tree to aid clinicians was created based on the clinical recommendations. CONCLUSIONS: The Transfusion and Anemia Expertise Initiative recommendations provide important clinical guidance and applicable tools to avoid unnecessary RBC transfusions. Research recommendations identify areas of focus for future investigation to improve outcomes and safety for RBC transfusion.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Viral DNAemia and Immune Suppression in Pediatric Sepsis.

OBJECTIVES: Demonstrate that DNA viremia is common in pediatric sepsis and quantitate its associations with host immune function and secondary infection risk. DESIGN: Retrospective analysis of a prospective cohort study. PATIENTS: Seventy-three children admitted with sepsis-induced organ failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: This study was performed as an ancillary investigation to a single-center prospective study of children with severe sepsis. Longitudinally collected, batched, frozen plasma was examined using real time-polymerase chain reaction for the presence of cytomegalovirus, Epstein-Barr virus, herpes simplex virus, human herpes virus-6, torque teno virus, and adenovirus DNA. Innate immune function was also measured longitudinally via quantification of ex vivo lipopolysaccharide -induced tumor necrosis factor-α production capacity. Viral DNAemia with a virus other than torque teno virus was detected in 28 of 73 subjects (38%) and included cytomegalovirus 5%, Epstein-Barr virus 11%, herpes simplex virus 4%, human herpes virus-6 8%, and adenovirus 26%. In addition, torque teno virus was detected in 89%. Epstein-Barr virus DNAemia was associated with preexisting immune suppression (p = 0.007) Viral DNAemia was associated with preexisting immune suppression and high risk for the subsequent development of secondary infection (p < 0.05 for both). Subjects with viral DNAemia had lower innate immune function over time compared with those who were virus negative (p < 0.05). CONCLUSIONS: DNAemia from multiple viruses can be detected in septic children and is strongly associated with preexisting immune suppression and secondary infection risk. The role of DNA viruses in the perpetuation of impaired host defense in this setting should be the subject of prospective study.

Controlling Phlebotomy Volume Diminishes PICU Transfusion: Implementation Processes and Impact.

BACKGROUND AND OBJECTIVES: Phlebotomy excess contributes to anemia in PICU patients and increases the likelihood of red blood cell transfusion, which is associated with risk of adverse outcomes. Excessive phlebotomy reduction (EPR) strategies may reduce the need for transfusion, but have not been evaluated in a PICU population. We hypothesized that EPR strategies, facilitated by implementation science methods, would decrease excess blood drawn and reduce transfusion frequency. METHODS: Quantitative and qualitative methods were used. Patient and blood draw data were collected with survey and focus group data to evaluate knowledge and attitudes before and after EPR intervention. The Consolidated Framework for Implementation Research was used to interpret qualitative data. Multivariate regression was employed to adjust for potential confounders for blood overdraw volume and transfusion incidence. RESULTS: Populations were similar pre- and postintervention. EPR strategies decreased blood overdraw volumes 62% from 5.5 mL (interquartile range 1-23) preintervention to 2.1 mL (interquartile range 0-7.9 mL) postintervention (P < .001). Fewer patients received red blood cell transfusions postintervention (32.1% preintervention versus 20.7% postintervention, P = .04). Regression analyses showed that EPR strategies reduced blood overdraw volume (P < .001) and lowered transfusion frequency (P = .05). Postintervention surveys reflected a high degree of satisfaction (93%) with EPR strategies, and 97% agreed EPR was a priority postintervention. CONCLUSIONS: Implementation science methods aided in the selection of EPR strategies and enhanced acceptance which, in this cohort, reduced excessive overdraw volumes and transfusion frequency. Larger trials are needed to determine if this approach can be applied in broader PICU populations.

Recommendations for utilization of the paracorporeal lung assist device in neonates and young children with pulmonary hypertension.

The management of decompensating critically ill children with severe PH is extremely challenging and requires a multidisciplinary approach. Unfortunately, even with optimal care, these children might continue to deteriorate and develop inadequate systemic perfusion and at times cardiac arrest secondary to a pulmonary hypertensive crisis. Tools to support these children are limited, and at times, the team should proceed with offering extracorporeal support, especially in newly diagnosed patients who have not benefitted from medical therapy prior to their acute deterioration, in patients with severe pulmonary venous disease and in patients with alveolar capillary dysplasia. Currently, the only approved mode for extracorporeal support in pediatric patients with PH eligible for lung transplantation is ECMO. To decrease the risks associated with ECMO, and offer potential for increased duration of support, extubation, and rehabilitation, we transitioned four small children with refractory PH from ECMO to a device comprising an oxygenator interposed between the PA and LA. This work describes in great detail our experience with this mode of support with emphasis on exclusion criteria, the implantation procedure, and the post-implantation management.

Pediatric intensive care outcomes: development of new morbidities during pediatric critical care.

OBJECTIVE: To investigate significant new morbidities associated with pediatric critical care. DESIGN: Randomly selected, prospective cohort. SETTING: PICU patients from eight medical and cardiac PICUs. PATIENTS: This was a randomly selected, prospective cohort of PICU patients from eight medical and cardiac PICUs. MEASUREMENTS AND MAIN RESULTS: The main outcomes measures were hospital discharge functional status measured by Functional Status Scale scores and new morbidity defined as an increase in the Functional Status Scale of more than or equal to 3. Of the 5,017 patients, there were 242 new morbidities (4.8%), 99 PICU deaths (2.0%), and 120 hospital deaths (2.4%). Both morbidity and mortality rates differed (p < 0.001) among the sites. The worst functional status profile was on PICU discharge and improved on hospital discharge. On hospital discharge, the good category decreased from a baseline of 72% to 63%, mild abnormality increased from 10% to 15%, moderate abnormality status increased from 13% to 14%, severe status increased from 4% to 5%, and very severe was unchanged at 1%. The highest new morbidity rates were in the neurological diagnoses (7.3%), acquired cardiovascular disease (5.9%), cancer (5.3%), and congenital cardiovascular disease (4.9%). New morbidities occurred in all ages with more in those under 12 months. New morbidities involved all Functional Status Scale domains with the highest proportions involving respiratory, motor, and feeding dysfunction. CONCLUSIONS: The prevalence of new morbidity was 4.8%, twice the mortality rate, and occurred in essentially all types of patients, in relatively equal proportions, and involved all aspects of function. Compared with historical data, it is possible that pediatric critical care has exchanged improved mortality rates for increased morbidity rates.

Citrate anticoagulation during continuous renal replacement therapy in pediatric critical care.

OBJECTIVE: To provide the pediatric intensivist an in-depth understanding of citrate as regional anticoagulant during continuous renal replacement therapy. DATA SOURCES AND DATA SELECTION: We searched the PubMed.gov database using the initial key words: citrate anticoagulation [title] AND continuous; citrate [title] AND pediatric AND continuous; prospective pediatric renal replacement AND citrate; and regional citrate anticoagulation. Additional searchers were performed using EMBASE, CINAHL, and SCOPUS with similar keywords and limits. Further articles were gathered from bibliographic references of relevant studies and reviews. Only articles published in English were reviewed. DATA EXTRACTION AND DATA SYNTHESIS: In the pediatric population, there are no prospective interventional or randomized studies comparing regional versus systemic anticoagulation. However, there are 11 (retrospective and prospective observational studies) in the pediatric population using citrate anticoagulation. These studies have shown that regional citrate anticoagulation in the pediatric population can be effective, provide equivalent circuit survival, and decrease bleeding compared with heparin anticoagulation. In the adult population, there are six prospective randomized controlled trials comparing the efficacy of regional citrate anticoagulation versus heparin. Two systematic reviews with meta-analysis of these six trials have been performed. The adult data on the use of regional citrate anticoagulation during continuous renal replacement therapy show a decreased risk of bleeding and at the least equivalent circuit survival as compared to heparin. Current pediatric and adult studies support regional citrate anticoagulation as an effective alternative to systemic heparin anticoagulation in most patient populations. CONCLUSIONS: Continuous renal replacement therapy is the most common modality of renal replacement in the critical care setting. Regional anticoagulation is an ideal option in a critically ill child after recent surgery or with coagulopathy. Therefore, regional citrate anticoagulation in the pediatric critical care population requiring renal replacement therapy is commonly employed. Complications of citrate anticoagulation can be avoided with a greater understanding of the properties and clearance of citrate. Continued reporting of observational data and the development of prospective multicenter trials using citrate anticoagulation are needed to ensure safe and standardized care in the pediatric population.

Analysis of S-nitrosothiols via copper cysteine (2C) and copper cysteine-carbon monoxide (3C) methods.

This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C)±carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO detection sensitivity of ozone based chemiluminescence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions.

Nitric oxide transport in blood: a third gas in the respiratory cycle.

The trapping, processing, and delivery of nitric oxide (NO) bioactivity by red blood cells (RBCs) have emerged as a conserved mechanism through which regional blood flow is linked to biochemical cues of perfusion sufficiency. We present here an expanded paradigm for the human respiratory cycle based on the coordinated transport of three gases: NO, O2, and CO2. By linking O2 and NO flux, RBCs couple vessel caliber (and thus blood flow) to O2 availability in the lung and to O2 need in the periphery. The elements required for regulated O2-based signal transduction via controlled NO processing within RBCs are presented herein, including S-nitrosothiol (SNO) synthesis by hemoglobin and O2-regulated delivery of NO bioactivity (capture, activation, and delivery of NO groups at sites remote from NO synthesis by NO synthase). The role of NO transport in the respiratory cycle at molecular, microcirculatory, and system levels is reviewed. We elucidate the mechanism through which regulated NO transport in blood supports O2 homeostasis, not only through adaptive regulation of regional systemic blood flow but also by optimizing ventilation-perfusion matching in the lung. Furthermore, we discuss the role of NO transport in the central control of breathing and in baroreceptor control of blood pressure, which subserve O2 supply to tissue. Additionally, malfunctions of this transport and signaling system that are implicated in a wide array of human pathophysiologies are described. Understanding the (dys)function of NO processing in blood is a prerequisite for the development of novel therapies that target the vasoactive capacities of RBCs.

Direct regulation of striated muscle myosins by nitric oxide and endogenous nitrosothiols.

BACKGROUND: Nitric oxide (NO) has long been recognized to affect muscle contraction, both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins. PRINCIPAL FINDINGS: Exposure of skeletal and cardiac myosins to physiological concentrations of nitrogen oxides, including the endogenous nitrosothiol S-nitroso-L-cysteine, reduced the velocity of actin filaments over myosin in a dose-dependent and oxygen-dependent manner, caused a doubling of force as measured in a laser trap transducer, and caused S-nitrosylation of cysteines in the myosin heavy chain. These biomechanical effects were not observed in response to S-nitroso-D-cysteine, demonstrating specificity for the naturally occurring isomer. Both myosin heavy chain isoforms in rats and cardiac myosin heavy chain from human were S-nitrosylated in vivo. SIGNIFICANCE: These data show that nitrosylation signaling acts as a molecular "gear shift" for myosin--an altogether novel mechanism by which striated muscle and cellular biomechanics may be regulated.

Evolution of adverse changes in stored RBCs.

Recent studies have underscored questions about the balance of risk and benefit of RBC transfusion. A better understanding of the nature and timing of molecular and functional changes in stored RBCs may provide strategies to improve the balance of benefit and risk of RBC transfusion. We analyzed changes occurring during RBC storage focusing on RBC deformability, RBC-dependent vasoregulatory function, and S-nitrosohemoglobin (SNO-Hb), through which hemoglobin (Hb) O(2) desaturation is coupled to regional increases in blood flow in vivo (hypoxic vasodilation). Five hundred ml of blood from each of 15 healthy volunteers was processed into leukofiltered, additive solution 3-exposed RBCs and stored at 1-6 degrees C according to AABB standards. Blood was subjected to 26 assays at 0, 3, 8, 24 and 96 h, and at 1, 2, 3, 4, and 6 weeks. RBC SNO-Hb decreased rapidly (1.2 x 10(-4) at 3 h vs. 6.5 x 10(-4) (fresh) mol S-nitrosothiol (SNO)/mol Hb tetramer (P = 0.032, mercuric-displaced photolysis-chemiluminescence assay), and remained low over the 42-day period. The decline was corroborated by using the carbon monoxide-saturated copper-cysteine assay [3.0 x 10(-5) at 3 h vs. 9.0 x 10(-5) (fresh) mol SNO/mol Hb]. In parallel, vasodilation by stored RBCs was significantly depressed. RBC deformability assayed at a physiological shear stress decreased gradually over the 42-day period (P < 0.001). Time courses vary for several storage-induced defects that might account for recent observations linking blood transfusion with adverse outcomes. Of clinical concern is that SNO levels, and their physiological correlate, RBC-dependent vasodilation, become depressed soon after collection, suggesting that even "fresh" blood may have developed adverse biological characteristics.