Comprehensive Characterization of Surface-Bound Proteins and Measurement of Fibrin Fiber Thickness on Extracorporeal Membrane Oxygenation Circuits Collected From Patients.

OBJECTIVE: To characterize surface-bound proteins and to measure the thickness of fibrin fibers bound to extracorporeal membrane oxygenation (ECMO) circuits used in children. DESIGN: Single-center observational prospective study, April to November 2021. SETTING: PICU, Royal Children's Hospital, Melbourne, Australia. PATIENTS: Patients aged less than 18 years on venoarterial ECMO and without preexisting disorder. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: ECMO circuits were collected from six patients. Circuit samples were collected from five different sites, and subsequently processed for proteomic and scanning electron microscopy (SEM) studies. The concentration of proteins bound to ECMO circuit samples was measured using a bicinchoninic acid protein assay, whereas characterization of the bound proteome was performed using data-independent acquisition mass spectrometry. The Reactome Over-representation Pathway Analyses tool was used to identify functional pathways related to bound proteins. For the SEM studies, ECMO circuit samples were prepared and imaged, and the thickness of bound fibrin fibers was measured using the Fiji ImageJ software, version 1.53c (https://imagej.net/software/fiji/). Protein binding to ECMO circuit samples and fibrin networks showed significant intra-circuit and interpatient variation. The median (range) total protein concentration was 19.0 (0-76.9) µg/mL, and the median total number of proteins was 2011 (1435-2777). A total of 933 proteins were commonly bound to ECMO circuit samples from all patients and were functionally involved in 212 pathways, with signal transduction, cell cycle, and metabolism of proteins being the top three pathway categories. The median intra-circuit fibrin fiber thickness was 0.20 (0.15-0.24) µm, whereas the median interpatient fibrin fiber thickness was 0.18 (0.15-0.21) µm. CONCLUSIONS: In this report, we have characterized proteins and fiber fibrin thickness bound to ECMO circuits in six children. The techniques and approaches may be useful for investigating interactions between blood, coagulation, and the ECMO circuit and have the potential for circuit design.

Neurocognitive Dysfunction and Smaller Brain Volumes in Adolescents and Adults With a Fontan Circulation.

BACKGROUND: Neurocognitive outcomes beyond childhood in people with a Fontan circulation are not well defined. This study aimed to investigate neurocognitive functioning in adolescents and adults with a Fontan circulation and associations with structural brain injury, brain volumetry, and postnatal clinical factors. METHODS: In a binational study, participants with a Fontan circulation without a preexisting major neurological disability were prospectively recruited from the Australia and New Zealand Fontan Registry. Neurocognitive function was assessed by using Cogstate software in 107 participants with a Fontan circulation and compared with control groups with transposition of the great arteries (n=50) and a normal circulation (n=41). Brain MRI with volumetric analysis was performed in the participants with a Fontan circulation and compared with healthy control data from the ABIDE I and II (Autism Brain Imaging Data Exchange) and PING (Pediatric Imaging, Neurocognition, and Genetics) data repositories. Clinical data were retrospectively collected. RESULTS: Of the participants with a Fontan circulation who had a neurocognitive assessment, 55% were male and the mean age was 22.6 years (SD 7.8). Participants with a Fontan circulation performed worse in several areas of neurocognitive function compared with those with transposition of the great arteries and healthy controls (P<0.05). Clinical factors associated with worse neurocognitive outcomes included more inpatient days during childhood, younger age at Fontan surgery, and longer time since Fontan procedure (P<0.05). Adults with a Fontan circulation had more marked neurocognitive dysfunction than adolescents with a Fontan circulation in 2 domains (psychomotor function, P=0.01 and working memory, P=0.02). Structural brain injury was present in the entire Fontan cohort; the presence of white matter injury was associated with worse paired associate learning (P<0.001), but neither the presence nor severity of infarct, subcortical gray matter injury, and microhemorrhage was associated with neurocognitive outcomes. Compared with healthy controls, people with a Fontan circulation had smaller global brain volumes (P<0.001 in all regions) and smaller regional brain volumes in most cerebral cortical regions (P<0.05). Smaller global brain volumes were associated with worse neurocognitive functioning in several domains (P<0.05). A significant positive association was also identified between global brain volumes and resting oxygen saturations (P≤0.04). CONCLUSIONS: Neurocognitive impairment is common in adolescents and adults with a Fontan circulation and is associated with smaller gray and white matter brain volume. Understanding modifiable factors that contribute to brain injury to optimize neurocognitive function is paramount.

Recombinant Factor VIIa in Pediatric Cardiac Surgery.

OBJECTIVES: Recombinant activated factor VIIa (rVIIa) is used off-label for refractory bleeding after cardiac surgery. This study reviewed the indications, usage rates, and complications of rVIIa. DESIGN: A retrospective case-control observational study. SETTING: A single quaternary pediatric hospital. PARTICIPANTS: All children undergoing cardiac surgery with cardiopulmonary bypass over a three-year period. INTERVENTIONS: Administration of rVIIa as rescue therapy for refractory bleeding after weaning from cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS: Onethousand, five hundred fifteen cardiopulmonary bypass procedures were reviewed. Patients receiving rVIIa were each matched to two control patients by age, procedure type, and bypass time. Data collected included weight, crossclamp time, anticoagulant and antifibrinolytic dose, return to the operating room for bleeding, thrombotic events, and extracorporeal membrane oxygenation (ECMO) circuit interventions. Forty-two patients received rVIIa (2.8%). Major systemic thrombotic complications were observed in 19% (controls 12.5%) of patients; 80% of recombinant factor VIIa patients requiring postoperative ECMO had interventions for circuit thrombosis (controls 31.25%); 4.76% of rVIIa recipients required reexploration for intractable bleeding (controls 1.39%). CONCLUSIONS: This study added to understanding regarding the use of recombinant factor VIIa in pediatric cardiac surgery and reported increased thrombotic complications, especially for children who progress to ECMO. Prospective studies to better understand the pathophysiology of coagulopathy and hemorrhage in pediatric cardiac surgery and the role of hemostatic agents, such as rVIIa, are required.

Development and Validation of the Warfarin-Aspirin Bleeding Assessment Tool (WA-BAT) in Children.

Bleeding assessment tools (BATs) aim to screen and estimate bleeding risk in patients with inherited bleeding disorders. However, the use of BAT as a standardized measure for comparing bleeding in patients on long-term thromboprophylaxis has not yet been validated. We developed a self-administrable BAT to assess bleeding in patients undergoing long-term thromboprophylaxis with aspirin or warfarin. Eligible participants were invited to complete the warfarin-aspirin -BAT (WA-BAT) online. The WA-BAT was readministered a number of weeks later to determine intrarater reliability. The WA-BAT showed substantial intrarater reliability and assesses major and minor bleeding associated with long-term warfarin or aspirin use.

Platelets from children are hyper-responsive to activation by thrombin receptor activator peptide and adenosine diphosphate compared to platelets from adults.

Platelets are crucial subcellular elements of haemostasis at sites of vascular injury and are also known to be immune mediators in pathological thrombosis. Despite the integral role of platelets in many disease processes, there is very little information available on platelet function and response to agonists in healthy children. We recently reported important differences in the interaction of platelets with monocytes in the circulation, including increased formation of monocyte-platelet aggregates (MPAs) without concomitant increase in P-selectin expression. Our current study investigates parameters of platelet activation (PAC-1 binding and P-selectin expression) and MPA formation in response to a range of physiologically relevant platelet agonists in healthy children compared to healthy adults. All parameters were significantly higher in children in response to sub-maximal concentrations of thrombin receptor activator peptide and adenosine diphosphate, reflecting an age-specific difference in agonist-stimulated platelet reactivity in children. The results of our study challenge the general assumption that platelet reactivity in children is similar to adults. This finding is fundamental to investigating the role of platelets in diseases of childhood and pathogenesis of adult-based diseases that have their origins in childhood. Our findings underscore the need for age-specific reference ranges for platelet function in children rather than extrapolation from adult data.

Continuous reference intervals for holotranscobalamin, homocysteine and folate in a healthy paediatric cohort.

BACKGROUND: The detection of deficiencies in B12 and folate children is important. However, despite the availability of various markers to assess B12 and folate metabolism, there are limited studies describing the reference intervals (RIs) and changes during growth and development for these markers in healthy children. METHODS: Using samples collected from 378 children aged 30 days-< 18 years, we derived continuous RIs for holotranscobalamin, homocysteine and red cell folate. RESULTS: The lower RI for holotranscobalamin was lowest at birth, rising during early childhood and then declining following ages 4-6 years whereas red cell folate was highest early in life and then declined steadily towards adulthood. Total homocysteine, reflective of both B12 and folate status was elevated early in life, reaching a nadir at age 2 and then increasing towards adulthood. CONCLUSIONS: Continuous central 95th percentile RI for holotranscobalamin, homocysteine and red cell folate for children ages 30 days to <18 years were established. Each marker shows dynamic changes throughout childhood and adolescence which will assist clinicians in more appropriately assessing B12 and folate status in this population.

Immunofluorescence Protocol for Characterization of Platelet and Leukocyte Binding in Extracorporeal Membrane Oxygenation (ECMO) Circuits.

The continuous contact between blood and the foreign surface of the extracorporeal membrane oxygenation (ECMO) circuit contributes to hemostatic, inflammatory, and other physiological disturbances observed during ECMO. Although previous studies have extensively investigated blood samples from patients on ECMO, cell adsorption to the ECMO circuit as an additional factor that could potentially influence clinical outcomes, has largely been overlooked. Here we provide a detailed immunofluorescence (IF) protocol designed to characterize cellular binding on ECMO circuits collected from patients. Extracorporeal membrane oxygenation circuits were collected from three pediatric patients and an albumin primed-only ECMO circuit was used as control. Circuit samples from five different sites within each ECMO circuit were collected and processed for the IF protocol. CD14 and CD42a antibodies were used to identify platelets and leukocytes bound to each ECMO circuit sample and images captured using inverted fluorescence microscopy. The protocol enables the comprehensive characterization of platelet and leukocyte binding to ECMO circuits collected from patients, which could in turn extend our knowledge of the characteristics of circuit binding and may provide guidance for improved ECMO circuit design.

Capillary blood protein markers of posttraumatic headache in children after concussion.

OBJECTIVE: Posttraumatic headache (PTH) represents the most common acute and persistent symptom in children after concussion, yet there is no blood protein signature to stratify the risk of PTH after concussion to facilitate early intervention. This discovery study aimed to identify capillary blood protein markers, at emergency department (ED) presentation within 48 hours of concussion, to predict children at risk of persisting PTH at 2 weeks postinjury. METHODS: Capillary blood was collected using the Mitra Clamshell device from children aged 8-17 years who presented to the ED of the Royal Children's Hospital, Melbourne, Australia, within 48 hours of sustaining a concussion. Participants were followed up at 2 weeks postinjury to determine PTH status. PTH was defined per clinical guidelines as a new or worsened headache compared with preinjury. An untargeted proteomics analysis using data-independent acquisition (DIA) was performed. Principal component analysis and hierarchical clustering were used to reduce the dimensionality of the protein dataset. RESULTS: A total of 907 proteins were reproducibly identified from 82 children within 48 hours of concussion. The mean participant age was 12.78 years (SD 2.54 years, range 8-17 years); 70% of patients were male. Eighty percent met criteria for acute PTH in the ED, while one-third of participants with follow-up experienced PTH at 2 weeks postinjury (range 8-16 days). Hemoglobin subunit zeta (HBZ), cystatin B (CSTB), beta-ala-his dipeptidase (CNDP1), hemoglobin subunit gamma-1 (HBG1), and zyxin (ZYX) were weakly associated with PTH at 2 weeks postinjury based on up to a 7% increase in the PTH group despite nonsignificant Benjamini-Hochberg adjusted p values. CONCLUSIONS: This discovery study determined that no capillary blood protein markers, measured at ED presentation within 48 hours of concussion, can predict children at risk of persisting PTH at 2 weeks postinjury. While HBZ, CSTB, CNDP1, HBG1, and ZYX were weakly associated with PTH at 2 weeks postinjury, there was no specific blood protein signature predictor of PTH in children after concussion. There is an urgent need to discover new blood biomarkers associated with PTH to facilitate risk stratification and improve clinical management of pediatric concussion.

Blood Collection Processing and Handling for Plasma and Serum Proteomics.

The plasma and serum proteome has enormous potential as a tool for understanding the health of a number of physiological systems. Despite this potential, the use of plasma and serum proteomics clinically and for research is limited, and there are no strict guidelines on how samples should be collected and prepared for proteomic analysis. Given the sensitivity of proteomic analysis, there are a number of pre-analytical variables that should be considered and determined prior to undertaking proteomics-based methodologies.In this chapter, we provide an example of a blood processing protocol and highlight major considerations for pre-analytical variables involving the collection, processing, and handling of blood samples for plasma and serum proteomics. We provide comprehensive notes on aspects of the protocol that must be considered before commencing sample collections for a proteomic study as well as a thorough checklist to be used when designing new proteomic studies.

Proteomic Applications and Considerations: From Research to Patient Care.

Despite technological advancements in the field of proteomics, the rate at which serum and plasma biomarkers identified using proteomic approaches are translated into clinical use remains extremely low. In this chapter, we describe recent technological advancements and analytical strategies in proteomic methods. We also describe the progress of proteomic blood-based biomarkers to date and discuss what the future of proteomics might entail with the use of multi-omic approaches and implementing machine learning on large proteomic datasets. Lastly, we provide several key considerations for biomarker studies, ranging from sample type to the use of reference samples, in order to achieve progress from bench to bedside, ultimately improving patient diagnosis, disease, and/or therapeutic monitoring and care.

Protocol for the Investigation of Plasma and Whole Blood Clot Property of Fibrin Fiber Thickness Using Scanning Electron Microscopy.

Blood clot formation represents a key component of the coagulation process for preventing excessive hemorrhage. The structural characteristics of blood clots are associated with their strength and susceptibility to fibrinolysis. Scanning electron microscopy is a technique that allows for state-of-the-art image capture of blood clots, providing visualization of topography, fibrin thickness, fibrin network density, and blood cell involvement and morphology. In this chapter, we provide a detailed protocol for characterization of plasma and whole blood clot structure using SEM, covering the spectrum from blood collection, in vitro clot formation, sample preparation for SEM, imaging, and image analysis, specifically focusing on the measurement of fibrin fiber thickness.

Factors XI and XII in extracorporeal membrane oxygenation: longitudinal profile in children.

Background: Extracorporeal membrane oxygenation (ECMO) is used in children with cardiopulmonary failure. While the majority of ECMO centers use unfractionated heparin, other anticoagulants, including factor XI and factor XII inhibitors are emerging, which may prove suitable for ECMO patients. However, before these anticoagulants can be applied in these patients, baseline data of FXI and FXII changes need to be acquired. Objectives: This study aimed to describe the longitudinal profile of FXI and FXII antigenic levels and function before, during, and after ECMO in children. Methods: This is a prospective observational study in neonatal and pediatric patients with ECMO (<18 years). All patients with venoarterial ECMO and with sufficient plasma volume collected before ECMO, on day 1 and day 3, and 24 hours postdecannulation were included. Antigenic levels and functional activity of FXI and FXII were determined in these samples. Longitudinal profiles of these values were created using a linear mixed model. Results: Sixteen patients were included in this study. Mean FXI and FXII antigenic levels (U/mL) changed from 7.9 and 53.2 before ECMO to 6.0 and 34.5 on day 3 and they recovered to 8.8 and 39.4, respectively, after stopping ECMO. Function (%) of FXI and FXII decreased from 59.1 and 59.0 to 49.0 and 50.7 on day 3 and recovered to 66.0 and 54.4, respectively. Conclusion: This study provides the first insights into changes of the contact pathway in children undergoing ECMO. FXI and FXII antigen and function change during ECMO. Results from this study can be used as starting point for future contact pathway anticoagulant studies in pediatric patients with ECMO.

Increased Risk for Thromboembolism After Fontan Surgery: Considerations for Thromboprophylaxis.

The Fontan circulation introduces an increased risk of thromboembolism which is associated with substantial mortality and morbidity. Adverse outcomes of thromboembolic complications post-Fontan surgery vary in both nature and severity, ranging from local tissue infarction and pulmonary embolism to Fontan failure and ischemic stroke. Furthermore, recent studies have identified that subclinical stroke is common yet underdiagnosed in Fontan patients. Fontan patients are commonly treated with antiplatelet agents and/or anticoagulants as primary thromboprophylaxis. Optimal thromboprophylaxis management in the Fontan population is still unclear, and clinical consensus remains elusive despite the growing literature on the subject. This perspective will describe the nature of thromboembolism post-Fontan surgery and provide evidence for the use of both current and emerging thromboprophylaxis options for children and adults living with Fontan circulation.

Proteomics in Thrombosis and Hemostasis.

Proteomics, the simultaneous study of all proteins in a given cell, tissue or organism, is an innovative approach used to identify novel markers for diagnosis, prognosis and the pathophysiological mechanisms associated with diseases. Proteomic methodologies have been used in a variety of contexts such as investigating changes in protein abundance that may occur with disease presence, the response to therapeutic treatments as well as the impacts of age on the plasma proteome.Over the last decade, significant technological advancements in proteomic techniques have resulted in an increase in the use of proteomics in thrombosis and hemostasis research, particularly in order to identify relevant and novel clinical markers associated with bleeding and thrombosis. This mini-review explores the use of proteomics in the setting of thrombosis and hemostasis from 2010-2020, across five main domains (platelets, blood clot composition, stroke, venous thromboembolism, and therapeutics), as well as provides insights into key considerations for conducting proteomic studies.

Pathophysiological pathway differences in children who present with COVID-19 ARDS compared to COVID -19 induced MIS-C.

COVID-19 has infected more than 275 million worldwide (at the beginning of 2022). Children appear less susceptible to COVID-19 and present with milder symptoms. Cases of children with COVID-19 developing clinical features of Kawasaki-disease have been described. Here we utilise Mass Spectrometry proteomics to determine the plasma proteins expressed in healthy children pre-pandemic, children with multisystem inflammatory syndrome (MIS-C) and children with COVID-19 induced ARDS. Pathway analyses were performed to determine the affected pathways. 76 proteins are differentially expressed across the groups, with 85 and 52 proteins specific to MIS-C and COVID-19 ARDS, respectively. Complement and coagulation activation are implicated in these clinical phenotypes, however there was significant contribution of FcGR and BCR activation in MIS-C and scavenging of haem and retinoid metabolism in COVID-19 ARDS. We show global proteomic differences in MIS-C and COVID-ARDS, although both show complement and coagulation dysregulation. The results contribute to our understanding of MIS-C and COVID-19 ARDS in children.

Fibrin clot characteristics and anticoagulant response in a SARS-CoV-2-infected endothelial model.

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma (p = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation.

Investigating potential protein markers of cardiovascular disease in children with type 1 diabetes mellitus.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a metabolic disease characterized by dysglycaemia. Cardiovascular disease (CVD) is a major complication among T1DM patients and the leading cause of mortality later in life. METHODS: The study subjects consisted of T1DM children with poor glycemic control (HbA1c > 7.5%) and healthy age and gender matched controls. Venous blood samples were collected and tested by utilizing a novel immunoassay panel with 96 protein biomarkers. Data were analyzed using non-linear regression analysis and the expression of biomarkers was compared between T1DM and healthy control groups using an unpaired student's t-test. Dynamic principal component analysis (PCA) was operated based on the differentially expressed proteins. RESULTS: Ten T1DM children and 10 healthy controls were analyzed. Twelve CVD markers show significant differential expression between T1DM patients and healthy controls (p < 0.05). Dynamic PCA clustering based on differentially expressed proteins demonstrated an obvious clustering between the two populations. CONCLUSIONS: This preliminary study reveals the feasibility of utilizing a novel immunoassay panel to investigate potential biomarkers for predicting incipient CVD in children with T1DM. In future, longitudinal studies are required to track the relationships between measurements of the selected protein markers and the development of CVD in T1DM patients.

Cross-sectional assessment of haemostatic profile and hepatic dysfunction in Fontan patients.

BACKGROUND: Fontan-associated liver disease is accompanied by a hypercoagulable state. While hepatic dysfunction in Fontan patients is common, its relationship with haemostatic changes and clinical outcomes in this patient population remains unclear. OBJECTIVE: To correlate liver dysfunction and haemostatic profiles with clinical outcomes in the Fontan population. PATIENTS/METHODS: Patients were enrolled in a multicentre, cross-sectional study in Australia and New Zealand. Hepatic structure and function were assessed using serum-based calculations (Fibrotest and model for end-stage liver disease excluding international normalised ratio scores). Haemostatic profiles were assessed by Thrombin Generation. Platelet function was assessed via Platelet Factor 4 (PF4) and P-selectin (P-SEL). Clinical outcomes were obtained from the Australian and New Zealand Fontan Registry. RESULTS: Seventy-three patients participated in the study (mean age 18.9±8.5 years with a mean of 13.5±6.9 years post-Fontan). The Endogenous Thrombin Potential (ETP) for patients who suffered thrombotic events (TE) (1366.4±66.2 nM/min) was higher compared with patients with major bleeding events (1011.1±138.4 nM/min) (p=0.03). Except for a negative correlation between Fibrotest-score and PF4 (p=0.045), PF4 and P-SEL concentrations did not correlate with markers of hepatic dysfunction or structural abnormality. CONCLUSIONS: Increased ETP is associated with TE during clinical follow-up after Fontan. This study reinforces that hepatic dysfunction may contribute to the derangement of coagulation factors, impacting the individual risk of haemostatic complications for the Fontan population.