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.

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.

Platelet function in neonates and children.

Platelets are major regulators of haemostasis and coagulation. The primary role of platelets in coagulation is to form a stable clot and stop bleeding. Studies of platelet phenotype and function in neonates and children have been restricted by the large volumes required for many common platelet function tests such as platelet aggregometry. Developmental changes in platelets have not been as well described as developmental changes in plasma coagulation proteins, and overall, platelet phenotype and function in neonates and children has been understudied when compared to adults. Recent developments in more sensitive platelet function testing methods requiring smaller blood volumes such as flow cytometry has enabled recent studies to further investigate platelet phenotype and function in neonates and children. In this review we will provide an overview of recent advances from the past five years in platelets in the context of developmental haemostasis, as well as the role of platelets in neonatal paediatric disease.

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.

The Evolution of Extracorporeal Membrane Oxygenation Circuitry and Impact on Clinical Outcomes in Children: A Systematic Review.

This systematic review summarizes the major developments in extracorporeal membrane oxygenation (ECMO) circuitry in pediatrics over the past 20 years and demonstrates the impacts of those developments on clinical outcomes. This systematic review followed structured Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A total of 1987 studies were retrieved, of which 82 were included in the final analysis. Over the past 20 years, ECMO pumps have shifted from roller pumps to centrifugal pumps. Silicone and polypropylene hollow fiber membrane oxygenators were initially used but have been replaced by polymethylpentene hollow fiber membrane oxygenators, with other ECMO components poorly reported. Considerable variability in mortality was found across studies and there was no statistical difference in mortality rates across different periods. The duration of ECMO and other outcome measures were inconsistently reported across studies. This systematic review demonstrated technological developments in pumps and oxygenators over the last two decades, although patient mortality rates remained unchanged. This could be because of ECMO support applied to patients in more critical conditions over the years. We also highlighted the limitations of methodology information disclosure and outcome measures in current ECMO studies, showing the need of reporting standardization for future ECMO studies.

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.

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.

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.

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.

Coagulation in pediatric extracorporeal membrane oxygenation: A systematic review of studies shows lack of standardized reporting.

Objectives: Extracorporeal membrane oxygenation (ECMO) involves complex coagulation management and frequent hemostatic complications. ECMO practice between centers is variable. To compare results between coagulation studies, standardized definitions and clear documentation of ECMO practice is essential. We assessed how study population, outcome definitions, and ECMO-, coagulation-, and transfusion-related parameters were described in pediatric ECMO studies. Data sources: Embase, Medline, Web of Science, Cochrane Library and Google Scholar. Study selection: English original studies of pediatric ECMO patients describing hemostatic tests or outcome. Data extraction: Eligibility was assessed following PRISMA guidelines. Study population, outcome and ECMO-, coagulation, and transfusion parameters were summarized. Data synthesis: A total of 107 of 1312 records were included. Study population parameters most frequently included (gestational) age (79%), gender (60%), and (birth) weight (59%). Outcomes, including definitions of bleeding (29%), thrombosis (15%), and survival (43%), were described using various definitions. Description of pump type, oxygenator and cannulation mode occurred in 49%, 45%, and 36% of studies, respectively. The main coagulation test (53%), its reference ranges (49%), and frequency of testing (24%) were the most prevalent reported coagulation parameters. The transfusion thresholds for platelets, red blood cells, and fibrinogen were described in 27%, 18%, and 18% of studies, respectively. Conclusions: This systematic review demonstrates a widespread lack of detail or standardization of several parameters in coagulation research of pediatric ECMO patients. We suggest several parameters that might be included in future coagulation studies. We encourage the ECMO community to adopt and refine this list of parameters and to use standardized definitions in future research.

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.

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.

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.

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.

Adsorption of Blood Components to Extracorporeal Membrane Oxygenation (ECMO) Surfaces in Humans: A Systematic Review.

The accumulation of blood proteins and cells on extracorporeal membrane oxygenation (ECMO) circuits has been proposed as a contributing factor to the coagulopathic state of many patients. This systematic review aims to summarize and discuss the existing knowledge of blood components binding to the ECMO circuits in human patients. A systematic review was conducted using the Medline, PubMed and Embase databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Seven studies were included in this review. Three studies identified a leukocyte adhesion, three studies observed von Willebrand factor accumulation and four studies identified bound platelets on the surface of the circuits. Other identified components included fibrin, albumin, hemoglobin, erythrocytes, progenitor cells, fibronectin and IgG. This systematic review demonstrates the limited state of knowledge when it comes to adsorption to the ECMO circuits in humans. Most of the studies lacked insight or detail into the mechanisms of binding and the interactions between different components bound to the ECMO circuits. Further research is required to comprehensively characterize surface adsorption to ECMO circuits in humans and to define the specific mechanisms of binding, enabling improvements that increase biocompatibility between the blood-circuit interface in this important clinical setting.

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.

Platelet Phenotype and Function in the Setting of Pediatric Extracorporeal Membrane Oxygenation (ECMO): A Systematic Review.

Background: Despite increasing technical improvement and extracorporeal membrane oxygenation (ECMO)-related knowledge over the past three decades, morbidity and mortality associated with bleeding and clotting complications remain high in pediatric patients undergoing ECMO. Platelets, a key element of the coagulation system, have been proposed to be the main cause of coagulopathy in the setting of ECMO. This systematic review aims to summarize and discuss the existing knowledge of platelet phenotype and function in the pediatric ECMO population. Methods: A systematic review was conducted for the Embase, Medline, and PubMed databases following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Results: The detailed study selection process yielded a total of 765 studies and only 3 studies that fulfilled the selection criteria were included in this review. Techniques used to assess platelet function in the three existing studies included platelet aggregometry, flow cytometry, and thromboelastography-platelet mapping. The finding that is common to the three studies is reduced platelet function in pediatric patients during ECMO either compared to before the initiation of ECMO or in non-survivors compared to survivors. Two studies demonstrated reduced platelet aggregation that are irreversible by platelet transfusion during ECMO. Two studies reported bleeding events and mortality in children on ECMO and none of the studies investigated thrombotic events. Conclusions: This systematic review demonstrates the extremely limited information available for platelet phenotype and function in the pediatric ECMO population. Evidence from the existing literature suggests reduced platelet aggregation and increased platelet activation in children during ECMO. However, this needs to be interpreted with care due to the limitations associated with the techniques used for platelet function testing. Furthermore, the association between platelet dysfunction and clinical outcomes in the pediatric ECMO population remains elusive. Multiple research gaps have been identified when it comes to the knowledge of platelet phenotype and function of children on ECMO, highlighting the need for robust, well-designed studies in this setting.