Using Well-Plate Microfluidic Devices to Conduct Shear-Based Thrombosis Assays

Conant, Carolyn G.; Nevill, J. Tanner; Zhou, Zhou; Dong, Jing; Schwartz, Michael A.; Ionescu-Zanetti, Cristian

doi:10.1016/j.jala.2010.10.005

Cited by 27 publications

(17 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…25 Microfluidic channels were coated for 1 hour at room temperature with a 25 mL volume of PBS containing 20 mg/mL of mVWF or hVWF, washed with PBS, and then blocked with 0.5% BSA in PBS. Whole blood samples were incubated for 30 minutes at 37°C with hPF4 (10 mg/mL) and either a specific mAb (KKO, RTO, or isotype control) at 50 mg/mL or polyclonal hIgG at 1.0 mg/mL.…”

Section: Analysis Of Microfluidic Thrombosismentioning

confidence: 99%

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

Tutwiler

Madeeva

Ahn

et al. 2016

Blood

125

View full text Add to dashboard Cite

• The procoagulant nature of HIT can be simulated in a microfluidic model using human blood and its components.• PF4/glycosaminoglycans/ immunoglobulin G complexes activate monocytes through FcgRIIA to generate TF and thrombin, leading to coated platelets in HIT.Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)gRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcgRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcgRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcgRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk. (Blood. 2016;127(4):464-472) IntroductionHeparin-induced thrombocytopenia (HIT) is an iatrogenic, immunemediated disorder characterized by antibodies that recognize complexes between the platelet chemokine platelet factor 4 (PF4, CXCL4) and heparin or cell surface glycosaminoglycans (GAGs). 1,2 It is estimated that up to 50% of patients with HIT develop thrombosis that might be limb-and/or life-threatening. [3][4][5] Even with early recognition, cessation of heparin, and institution of alternative forms of anticoagulation, recurrent thromboembolic complications may occur and 10% to 20% of patients go on to amputation and/or death. 6 Thus, there is a need for a better understanding of the pathogenesis of HIT and to determine how this information can be used to mitigate the risk of thrombosis.Thrombocytopenia and thrombosis in HIT have been attributed to binding of PF4/heparin/immunoglobulin G (IgG) immune-complexes to the platelets through the IgG fragment crystallizable (Fc) region, which activates platelets through their immunoreceptor tyrosine-based activation motif (ITAM) receptor, FcgRIIA. 7,8 However, monocytes, endothelial cells, and other cell types might also be activated by these immune complexes and contribute to the underlying pathology, 9 but their contribution to the process is less well characterized. Indeed, recent evidence suggests that thrombosis in HIT is initiated by binding of pathogenic antibodies to antigenic complexes of PF4 and GAGs expressed by the endothelium as well as circulating cells, includi...

show abstract

Section: Analysis Of Microfluidic Thrombosismentioning

confidence: 99%

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

Tutwiler

Madeeva

Ahn

et al. 2016

Blood

125

View full text Add to dashboard Cite

show abstract

“…This approach avoids the pitfalls of the standard point of care devices that rely on clot-based endpoints with high variability and low reliability, in addition to measuring only certain portions of the coagulation pathway. Similarly, a microfluidic assay was used to study thrombus formation on collagen and von Willebrand factor matrices, in addition to a dose-response effect of abciximab, demonstrating the ability to obtain a large number of data points per single patient sample using small blood volumes and high throughput approach [51]. Similarly, a recent report describes the development of a low-cost paper-based microfluidic system to help titrate oral anticoagulation doses [52].…”

Section: Current Applications For the Use Of Microfluidics In Clinicamentioning

confidence: 99%

Microfluidic technology as an emerging clinical tool to evaluate thrombosis and hemostasis

Branchford

Neeves

et al. 2015

Thrombosis Research

View full text Add to dashboard Cite

Assessment of platelet function and coagulation under flow conditions can augment traditional static assays used to evaluate patients with suspected hemostatic or thrombotic disorders. Among the available flow-based assays, microfluidic devices require the smallest blood volume and provide multiple output options. These assays are based on the presence of wall shear stress that mimics in vivo interactions between blood components and vessel walls. Microfluidic devices can generate essential information regarding homeostatic regulation of platelet activation and subsequent engagement of the coagulation cascade leading to fibrin deposition and clot formation. Emerging data suggest that microfluidic assays may also reveal consistent patterns of hemostatic or thrombotic pathology, and could aid in assessing and monitoring patient-specific effects of coagulation-modifying therapies.

show abstract

“…Microfluidicbased systems, e.g. the BioFlux system from Fluxion, enable simultaneous analyses of cells in different microfluidic flow channels loaded with solution into well plates acting as input and output reservoirs for these channels 12,13,14 . However, these and other microfluidic systems are not compatible with standard microscope slides and do not allow for recovery of a sufficiently large number of cells for further experiments, such as RT-PCR or Western Blot.…”

Section: The Following Steps Are Critical For the Successful Executiomentioning

confidence: 99%

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Lane

Jantzen

Carlon

et al. 2012

JoVE

View full text Add to dashboard Cite

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses 1 .Our flow chamber design and flow circuit ( Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs 2,3 . This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry ( Fig. 11E), or scanning electron microscopy 5 . The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12) . Video LinkThe video component of this article can be found at https://www.jove.com/video/3349/ Protocol 1. Endothelial progenitor cell isolation 1. Prior to any collection of peripheral human blood, submit your research protocol to your Institutional Review Board (IRB), and after its approval, obtain the volunteer donors' informed consent (peripheral blood collection and EPC isolation had been approved by the Duke University IRB and is in full compliance with U.S. regulatory requirements related to the protection of human research participants). 2. When working with animal-derived EPCs, have your research protocol approved by your Institutional Animal Care and Use Committee (IACUC). All our porcine experiments had been approved by the Duke University IACUC and were conducted in accordance with the highest standards of humane care. 3. For isolation of endothelial progenitor cells, collect 50 ml of peripheral blood via standard phlebotomy technique from a consented volunteer donor into blood collection bags filled with the anticoagulant citrate phosphate dextrose and dilute the solution 1:1 with Hank's buffered salt solution (without CaCl 2 , MgCl 2 , MgSO 4 ) and layer on equal volumes of Histopaque to create well-defined layers. 4. Centrifuge (30 min, 740 g, low break setting) and colle...

show abstract

Using Well-Plate Microfluidic Devices to Conduct Shear-Based Thrombosis Assays

Cited by 27 publications

References 15 publications

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia

Microfluidic technology as an emerging clinical tool to evaluate thrombosis and hemostasis

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Contact Info

Product

Resources

About