(258e) Microfluidic Flow Assays for Diagnosing Bleeding and Thrombotic Disorders

Bleeding and thrombotic disorders are characterized by significant phenotypic variability. Over the last decade important advances have been made in the understanding of the molecular basis of these disorders; however a large portion of the observed variability remains unknown. Current assays for the diagnosis of bleeding disorders have limitations, particularly for the identification of mild to moderate bleeding and the discrimination of common variations in the hemostatic system. Also, quantifying clinical bleeding is extremely difficult. Therefore, a method that shows high sensitivity and specificity is highly desirable.

We have developed a microfluidic flow assay (MFA) to quantify platelet function and coagulation under physiologic flow conditions [1].  The MFA consists of a series of microfluidic channels (250 um X 50 um) in which whole blood is perfused over micropatterned prothrombotic proteins over a range of wall shear rates (Fig 1).  The micropatterned proteins include type 1 collagen, the primary adhesive ligand of platelets, and lipidated tissue factor, the initiator of extrinsic pathway of coagulation.  We have shown that the focal "injury" in the MFA correlates will with the animal models of thrombosis and can be used to detect dysfunction in platelet function [2,3].

In this talk I will describe the application of the MFA to diagnosing bleeding disorders.  We are currently in the middle of a large clinical evaluation of the MFA in collaboration the Hemophilia and Thrombosis Center at the University of Colorado, Denver in which we are screening 500 individuals with bleeding disorders and 1000 controls.  Preliminary results with type 1 von Willebrand disease patients show strong correlation between platelet accumulation in the MFA and clinical phenotypes. 

[1]  K.B. Neeves, S.F. Maloney, K.P. Fong, A.A. Schmaier, M.L. Kahn, L.F. Brass, and S. L. Diamond.  Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates.  Journal of Thrombosis and Haemostasis, 6 (2008), 2193-2201.

[2] L. Zhu, T.J. Stalker, K.P. Fong, H. Jiang, A. Tran, I. Crichton, E.K. Lee, K.B. Neeves, S.F. Maloney, H. Kikutani, A. Kumanogoh, E. Pure, S.L. Diamond, and L.F. Brass.  Disruption of SEMA4D ameliorates platelet hypersensitivity in dyslipidemia and confers protection against the development of atherosclerosis.  Arteriosclerosis, Thrombosis, and Vascular Biology, 29 (2009), 1039-1045.

[3] A.A. Schmaier, Z. Zou, A. Kazlauskas, L. Emert-Sedlak, K.P. Fong, K.B. Neeves, S.F. Maloney, S.L. Diamond, S.P. Kunapuli, J. Ware, L.F. Brass, T.E. Smithgall, K. Saksela, M.L. Kahn.  Molecular priming of Lyn by GPVI enables an immune receptor to adopt a hemostatic role.  Proceedings of the National Academy of Sciences of the USA, 106 (2009), 21167-21172.