Hospital-Acquired Venous Thromboembolism and Invasive Mechanical Ventilation: A Report From the Children's Hospital Acquired Thrombosis Consortium.

OBJECTIVES: To determine if the duration of invasive mechanical ventilation (IMV) was associated with hospital-acquired venous thromboembolism (HA-VTE) among critically ill children. DESIGN: A multicenter, matched case-control study as a secondary analysis of Children's Hospital Acquired Thrombosis (CHAT) Consortium registry. SETTING: PICUs within U.S. CHAT Consortium participating centers. PATIENTS: Children younger than 21 years old admitted to a PICU receiving IMV for greater than or equal to 1 day duration from January 2012 to March 2022 were included for study. Cases with HA-VTE were matched 1:2 to controls without HA-VTE by patient age groups: younger than 1, 1-12, and older than 12 years. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was IMV duration in days. Descriptive data included demographics, anthropometrics, HA-VTE characteristics (i.e., type, location, and timing), central venous catheterization data, thromboprophylaxis practices, and Braden Q mobility scores. Descriptive, comparative, and associative (multivariate conditional logistic regression for HA-VTE) statistics were employed. A total of 152 cases were matched to 304 controls. Cases with HA-VTE were diagnosed at a median of 7 days (interquartile range [IQR], 3-16 d) after IMV. The HA-VTE were limb deep venous thromboses in 130 of 152 (85.5%) and frequently central venous catheterization-related (111/152, 73%). Cases with HA-VTE experienced a longer length of stay (median, 34 d [IQR, 18-62 d] vs. 11.5 d [IQR, 6-21 d]; p < 0.001) and IMV duration (median, 7 d [IQR, 4-15 d] vs. 4 d [IQR, 1-7 d]; p < 0.001) as compared with controls. In a multivariate logistic model, greater IMV duration (adjusted odds ratio, 1.09; 95% CI, 1.01-1.17; p = 0.023) was independently associated with HA-VTE. CONCLUSIONS: Among critically ill children undergoing IMV, HA-VTE was associated with greater IMV duration. If prospectively validated, IMV duration should be included as part of prothrombotic risk stratification and future pediatric thromboprophylaxis trials.

Longitudinal neurocognitive trajectories and risk factors in the first three months following pediatric concussion.

OBJECTIVE: To identify differential trajectories of neurocognitive outcomes following pediatric concussion and investigate predictors associated with patterns of recovery up to 3 months. METHODS: 74 participants aged 8-17 years completed attention/working memory, processing speed, and executive function measures at 2 weeks, 1 month, and 3 months post-injury. We used principal component analysis to generate a composite of information processing. Group-based trajectory modeling identified latent trajectories. Multinominal logistic regression was used to examine associations between risk factors and trajectory groups. RESULTS: We identified three trajectories of neurocognitive outcomes. The medium (54.6%) and high improving groups (35.8%) showed ongoing increase in information processing, while the low persistent group showed limited change 3 months post-injury. This group recorded below average scores on Digit Span Forward and Backward at 3 months. History of pre-injury headache was significantly associated with the persistent low scoring group, relative to the medium improving (p = 0.03) but not the high improving group (p = 0.09). CONCLUSIONS: This study indicates variability in neurocognitive outcomes according to three differential trajectories, with groups partially distinguished by preexisting child factors (history of frequent headaches). Modelling that accounts for heterogeneity in individual outcomes is essential to identify clinically meaningful indices that are indicative of children requiring intervention.

Fatigue recovery and connected factors following paediatric concussion.

OBJECTIVE: Using a biopsychosocial framework and the three-factor fatigue model, we aimed to (1) plot recovery of fatigue over the 3 months following paediatric concussion and (2) explore factors associated with persisting fatigue during the first 3 months postconcussion. METHODS: 240 children and adolescents aged 5-18 years (M=11.64, SD=3.16) completed assessments from time of injury to 3 months postinjury. Separate linear mixed effects models were conducted for child and parent ratings on the PedsQL-Multidimensional Fatigue Scale to plot recovery across domains (General, Cognitive, Sleep/Rest) and Total fatigue, from 1 week to 3 months postinjury. Two-block hierarchical regression analyses were then conducted for parent and child ratings of fatigue at each time point, with age, sex and acute symptoms in block 1 and child and parent mental health variables added to block 2. RESULTS: There was a significant reduction in both child and parent ratings across the 3 months postinjury for all fatigue domains (all p<0.001). For both child and parent fatigue ratings, child mental health was the most significant factor associated with fatigue at all time points. Adding child and parent mental health variables in the second block of the regression substantially increased the variance explained for both child and parent ratings of fatigue. CONCLUSION: Our findings confirm that fatigue improves during the first 3 months postconcussion and highlights the importance of considering child and parent mental health screening when assessing patients with persisting postconcussive symptoms.

What can the plasma proteome tell us about platelets and (vice versa)?

Multi-omics approaches are being used increasingly to study physiological and pathophysiologic processes. Proteomics specifically focuses on the study of proteins as functional elements and key contributors to, and markers of the phenotype, as well as targets for diagnostic and therapeutic approaches. Depending on the condition, the plasma proteome can mirror the platelet proteome, and hence play an important role in elucidating both physiologic and pathologic processes. In fact, both plasma and platelet protein signatures have been shown to be important in the setting of thrombosis-prone disease states such as atherosclerosis and cancer. Plasma and platelet proteomes are increasingly being studied as a part of a single entity, as is the case with patient-centric sample collection approaches such as capillary blood. Future studies should cut across the plasma and platelet proteome silos, taking advantage of the vast knowledge available when they are considered as part of the same studies, rather than studied as distinct entities.

Platelets are key cellular elements of blood with plasma constituting the liquid component. Both platelets and proteins found in plasma rapidly work in unity to prevent/limit blood loss in response to blood vessel damage. Proteomics is the analysis of the entire protein complement of a cell, tissue, or organism under a specific, defined set of conditions. Of note, research to date has shown that platelet and plasma proteomes share many common proteins. In some disease scenarios, plasma proteomes can be used to identify platelet function or dysfunction, while in other scenarios, platelet-specific proteins are needed for physiological assessment. Thus, it may be beneficial to simultaneously study the plasma and platelet proteomes, thereby exploiting the considerable wealth of information provided under such circumstances.

Changes in von Willebrand Factor Multimers, Concentration, and Function During Pediatric Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding. DESIGN: Prospective observational study of pediatric ECMO patients from April 2017 to May 2019. SETTING: The PICU in a large, tertiary referral pediatric ECMO center. PATIENTS: Twenty-five neonates and children (< 18 yr) supported by venoarterial ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children. CONCLUSIONS: Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO.

Current and emerging technologies for the timely screening and diagnosis of neonatal jaundice.

Neonatal jaundice is one of the most common clinical conditions affecting newborns. For most newborns, jaundice is harmless, however, a proportion of newborns develops severe neonatal jaundice requiring therapeutic interventions, accentuating the need to have reliable and accurate screening tools for timely recognition across different health settings. The gold standard method in diagnosing jaundice involves a blood test and requires specialized hospital-based laboratory instruments. Despite technological advancements in point-of-care laboratory medicine, there is limited accessibility of the specialized devices and sample stability in geographically remote areas. Lack of suitable testing options leads to delays in timely diagnosis and treatment of clinically significant jaundice in developed and developing countries alike. There has been an ever-increasing need for a low-cost, simple to use screening technology to improve timely diagnosis and management of neonatal jaundice. Consequently, several point-of-care (POC) devices have been developed to address this concern. This paper aims to review the literature, focusing on emerging technologies in the screening and diagnosing of neonatal jaundice. We report on the challenges associated with the existing screening tools, followed by an overview of emerging sensors currently in pre-clinical development and the emerging POC devices in clinical trials to advance the screening of neonatal jaundice. The benefits offered by emerging POC devices include their ease of use, low cost, and the accessibility of rapid response test results. However, further clinical trials are required to overcome the current limitations of the emerging POC's before their implementation in clinical settings. Hence, the need for a simple to use, low-cost POC jaundice detection technology for newborns remains an unsolved challenge globally.

Platelet Phenotype and Function Changes With Increasing Duration of Extracorporeal Membrane Oxygenation.

OBJECTIVES: To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO). DESIGN: Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019. SETTING: The PICU in a large tertiary referral pediatric ECMO center. PATIENTS: Eighty-seven neonates and children (< 18 yr) supported by ECMO. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020). CONCLUSIONS: The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

Age-related differences in SARS-CoV-2 binding factors: An explanation for reduced susceptibility to severe COVID-19 among children?

CONTEXT: In contrast with other respiratory viruses, children infected with SARS-CoV-2 are largely spared from severe COVID-19. OBJECTIVES: To critically assess age-related differences in three host proteins involved in SARS-CoV-2 cellular entry: angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin. METHODS: We systematically searched Medline, Embase, and PubMed databases for relevant publications. Studies were eligible if they evaluated ACE2, TMPRSS2 or furin expression, methylation, or protein level in children. RESULTS: Sixteen papers were included. Age-dependent differences in membrane-bound and soluble ACE2 were shown in several studies, with ACE2 expression increasing with age. TMPRSS2 and furin are key proteases involved in SARS-CoV-2 spike protein cleavage. TMPRSS2 expression is increased by circulating androgens and is thus low in pre-pubertal children. Furin has not currently been well researched. LIMITATIONS: High levels of study heterogeneity. CONCLUSIONS: Low expression of key host proteins may partially explain the reduced incidence of severe COVID-19 among children, although further research is needed.

Advances and Utility of the Human Plasma Proteome.

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.

Vaccine-induced immune thrombosis and thrombocytopenia syndrome following adenovirus-vectored severe acute respiratory syndrome coronavirus 2 vaccination: a novel hypothesis regarding mechanisms and implications for future vaccine development.

We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus-vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4-heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically. This hypothesis is testable in cell culture and animal models, and potentially in vivo, and if proven correct has significant implications for vaccine development and our understanding of the links between the coagulation and immune systems.

The use of proteomics for blood biomarker research in premature infants: a scoping review.

Over the last decade, the use of proteomics in the setting of prematurity has increased and has enabled researchers to successfully identify biomarkers for an array of associated morbidities. The objective of this scoping review was to identify the existing literature, as well as any knowledge gaps related to proteomic biomarker discoveries in the setting of prematurity. A scoping review was conducted using PubMed, Embase and Medline databases following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines. The study selection process yielded a total of 700 records, of which 13 studies were included in this review. Most studies used a tandem Mass Spectrometry (MS/MS) proteomics approach to identify key biomarkers. The corresponding studies identified proteins associated with retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), necrotising enterocolitis (NEC), late onset sepsis (LOS) and gestational age. This scoping review demonstrates the limited use of proteomics to identify biomarkers associated with severe complications of prematurity. Further research is warranted to identify biomarkers of other important morbidities associated with prematurity, such as intraventricular haemorrhage (IVH) and cerebral palsy, and to investigate the mechanisms associated with these outcomes.

Continuous reference intervals for leukocyte telomere length in children: the method matters.

OBJECTIVES: Children with very short telomeres commonly develop bone marrow failure and other severe diseases. Identifying the individuals with short telomeres can improve outcome of bone marrow transplantation, with accurate diagnosis requiring the use of age-matched reference intervals (RIs). This study aimed to establish RIs for telomere length (TL) in children using three commonly used methods for TL measurement. METHODS: Healthy children aged 30 days to 18 years were recruited for assessment using age as a continuous variable. Venous blood samples were collected and leukocyte TL was measured using terminal restriction fragment (TRF) analysis, quantitative PCR (QPCR) and flow cytometry with fluorescence in situ hybridization (Flow-FISH). Fractional polynomial model and quantile regression were performed to generate continuous RIs. Factors that might contribute to variation in TL, such as gender, were also examined. RESULTS: A total of 212 samples were analyzed. Continuous RIs are presented as functions of age. TRF analysis and QPCR showed significant negative correlation between TL and age (r=-0.28 and r=-0.38, p<0.001). In contrast, Flow-FISH showed no change in TL with age (r=-0.08, p=0.23). Gender did not have significant influence on TL in children. CONCLUSIONS: This study provides three options to assess TL in children by establishing method-specific continuous RIs. Choosing which method to use will depend on several factors such as amount and type of sample available and required sensitivity to age-related change.

Mechanism of bilirubin elimination in urine: insights and prospects for neonatal jaundice.

Despite a century of research, bilirubin metabolism and the transport mechanisms responsible for homeostasis of bilirubin in serum remain controversial. Emerging evidence on the hepatic membrane transporters and inherited disorders of bilirubin metabolism have contributed to a greater understanding of the various steps involved in bilirubin homeostasis and its associated excretory pathways. We discuss these recent research findings on hepatic membrane transporters and evaluate their significance on the newborn bilirubin metabolism and excretion. New insights gained speculate that a proportion of conjugated bilirubin is excreted via the renal system, as an alternative to the intestinal excretion, even in normal physiological jaundice with no associated pathological concerns. Finally, this paper discusses the clinical relevance of targeting the altered renal excretory pathway, as bilirubin in urine may hold diagnostic importance in screening for neonatal jaundice.

Whole blood flow cytometry protocol for the assessment of platelet phenotype, function, and cellular interactions.

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils.

Plasma Proteomic Analysis Reveals Age-Specific Changes in Platelet- and Endothelial Cell-Derived Proteins and Regulators of Plasma Coagulation and Fibrinolysis.

This post hoc study of a plasma proteomic database investigated hemostatic proteins in the context of developmental hemostasis. Twenty-seven hemostatic proteins changed expression with age, and the hemostatic profile of neonates was unique. Appreciating developmental hemostasis through proteomics may lead to more personalized medicine for hospitalized children.

Anticoagulant Heparin Mimetics via RAFT Polymerization.

Heparin, a sulfated polysaccharide derived from animal sources, is the most commonly used parenteral anticoagulant drug, but it suffers from significant safety and supply issues. Herein, we describe the preparation of heparin mimetic homo- and copolymers via the reversible addition-fragmentation chain transfer (RAFT) polymerization in water of commercially available (non-carbohydrate) sulfonated and carboxylated monomers. The anticoagulant activities of the polymers were assessed by activated partial thromboplastin time (APTT), thrombin clotting time (TCT), and for the more promising polymers, thrombin generation, antifactor Xa, and antifactor IIa assays. Sulfonated homopolymers studied herein displayed low cytotoxicity and significant anticoagulant activity in APTT, TCT, and thrombin generation assays. In addition, copolymers of sodium styrenesulfonate and acrylic acid [poly(SSS-co-AA)] displayed unprecedented antifactor IIa activity. This study demonstrates the potential of RAFT polymers as alternative anticoagulants for biomedical applications.

Reference intervals for serum cystatin C in neonates and children 30 days to 18 years old.

BACKGROUND: Serum cystatin C (CysC) is a promising biomarker of kidney function, which has higher accuracy and sensitivity when compared with creatinine. To better utilize serum CysC in clinical practice, this study aimed to establish continuous paediatric reference intervals (RIs) for serum CysC. METHODS: The study subjects consisted of healthy term neonates and children aged 30 days to 18 years. Venous blood samples were collected and serum CysC levels were measured using the immunoturbidimetric measurement principle. Fractional polynomial regression model and quantile regression was applied in the statistical analysis to generate continuous RIs. RESULTS: A total of 378 samples with equal numbers of males and females were analysed for serum CysC. No outliers were found in this analysis. The continuous RIs are presented as equations and graphical scatterplots. CONCLUSIONS: This study established continuous paediatric reference intervals (RIs) for serum CysC in healthy term neonates and children. The continuous RIs generated from this study show age-based dynamic changes as well as blood group and gender-specific differences for serum CysC. Graphical abstract.

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.

Guidelines for panel design, optimization, and performance of whole blood multi-color flow cytometry of platelet surface markers.

Flow cytometry is a valuable tool in determining the phenotype and function of platelets accurately. The emergence of platelet flow cytometry in recent years provides an attractive alternative to other platelet analytical techniques, with advantages such as requiring small volumes and being highly sensitive to minimal changes in receptor function and expression. Here we present a methodical approach encompassing the stages in the development and optimization of platelet flow cytometry panels based on our extensive experience in this area.

Preterm Lung Exhibits Distinct Spatiotemporal Proteome Expression at Initiation of Lung Injury.

The development of regional lung injury in the preterm lung is not well understood. This study aimed to characterize time-dependent and regionally specific injury patterns associated with early ventilation of the preterm lung using a mass spectrometry-based proteomic approach. Preterm lambs delivered at 124-127 days gestation received 15 or 90 minutes of mechanical ventilation (positive end-expiratory pressure = 8 cm H2O, Vt = 6-8 ml/kg) and were compared with unventilated control lambs. At study completion, lung tissue was taken from standardized nondependent and dependent regions, and assessed for lung injury via histology, quantitative PCR, and proteomic analysis using Orbitrap-mass spectrometry. Ingenuity pathway analysis software was used to identify temporal and region-specific enrichments in pathways and functions. Apoptotic cell numbers were ninefold higher in nondependent lung at 15 and 90 minutes compared with controls, whereas proliferative cells were increased fourfold in the dependent lung at 90 minutes. The relative gene expression of lung injury markers was increased at 90 minutes in nondependent lung and unchanged in gravity-dependent lung. Within the proteome, the number of differentially expressed proteins was fourfold higher in the nondependent lung than the dependent lung. The number of differential proteins increased over time in both lung regions. A total of 95% of enriched canonical pathways and 94% of enriched cellular and molecular functions were identified only in nondependent lung tissue from the 90-minute ventilation group. In conclusion, complex injury pathways are initiated within the preterm lung after 15 minutes of ventilation and amplified by continuing ventilation. Injury development is region specific, with greater alterations within the proteome of nondependent lung.