ORIGINAL RESEARCH Novel methodology to replicate clot analogs with diverse composition in acute ischemic stroke Sharon Duffy, 1,2 Michael Farrell, 3 Kevin McArdle, 2 John Thornton, 3 David Vale, 2 Eleanor Rainsford, 1 Liam Morris, 1 David S Liebeskind, 4 Eugene MacCarthy, 1 Michael Gilvarry 2 1 Mechanical and Industrial Engineering Department, Galway Medical Technologies Centre, Galway-Mayo Institute of Technology, Galway, Ireland 2 Neuravi Ltd, Galway, Ireland 3 Neuropathology Department, Beaumont Hospital, Dublin, Ireland 4 Neurovascular Imaging Research Core and UCLA Stroke Centre, Los Angeles, California, USA Correspondence to S Duffy, Mechanical and Industrial Engineering Department, Galway Medical Technologies Centre, Galway-Mayo Institute of Technology, Old Dublin Rd, Galway, Ireland; sharon.duffy@research.gmit.ie Received 26 January 2016 Revised 5 April 2016 Accepted 8 April 2016 To cite: Duffy S, Farrell M, McArdle K, et al. J NeuroIntervent Surg Published Online First: [ please include Day Month Year] doi:10.1136/ neurintsurg-2016-012308 ABSTRACT Background Translational research on clot composition may be advanced by the use of clot analogs for the preclinical evaluation of mechanical thrombectomy devices. This work describes a novel set of clot analogs to represent a diverse range of fibrin and red blood cell (RBC) compositions for use in acute ischemic stroke (AIS) occlusion models. Method Fresh whole blood obtained from ovine species was used to create seven different clot analog types. Five replicates were formed for each clot type. Varying amounts of whole blood constituents were mixed with thrombotic factors to create clots of varying compositions. Following histological processing, five sections from each clot were stained with H&E and Martius Scarlet Blue. Fibrin, RBC and white blood cell compositions were quantified. Results Histological examination demonstrated that the clot types had a distinct RBC and fibrin composition. No significant difference in composition was shown between replicates (p>0.05), indicating that the method of clot formation was reproducible. Percentage fibrin composition of the clot types was 1%, 8%, 31%, 38%, 64%, 79%, and 100%. A significant difference in fibrin and RBC composition between clot types was observed ( p<0.05). Conclusions Seven different clot types were developed to replicate common AIS thrombi. These clot analogs may be beneficial for the preclinical evaluation of endovascular therapies, and may be applied to interventional technique training. INTRODUCTION Recanalization and the restoration of downstream blood flow in the territory of a proximal cerebral artery has been established as the definitive treat- ment for acute ischemic stroke (AIS). 1 Timely and effective recanalization of occlusive thrombi or clots in such arteries may be influenced by a number of underlying factors, including clot com- position 2 and type of occluded vessel. 3 Mechanical thrombectomy (MT) has recently been established as an effective revascularization therapy for the treatment of AIS. 45 Clinically relevant clot analogs are valuable for the pre-evaluation of MT devices, to assess the effect of various clot compositions on device performance. A common clot analog model is the Gralla model of thrombin induced clots pre- pared with radiopaque substances 67 for clot visual- ization. This model is a red blood cell (RBC) rich clot, 7 similar in composition to that formed by spontaneous coagulation. 89 Another experimental approach is the preparation of a clot by sedimenta- tion, with a multiple layered structure, consisting of a fibrin rich and RBC rich component. 10 In order to simulate the physiological blood flow environ- ment, Chandler 11 proposed a closed loop tech- nique to form a clot containing a large fibrin component in dynamic conditions. 11–13 There are significant limitations associated with previously described clot models. Homogenous RBC dominant clots tend to be unstable and are sus- ceptible to fragmentation during the MT evaluation procedure. 2 Some clot models are prepared using radiopaque substances, such as barium sulfate, 267 which significantly affects the clot’s mechanical properties by decreasing elasticity. 7 In addition, the histological characteristics are inconsistent with thrombi retrieved from AIS patients. 14–18 A diverse composition of human AIS thrombi is documented in the literature, 14–18 highlighting a clear need for clot analogs that cover a range of fibrin and RBC compositions. Based on a modifica- tion of existing 11 13 19–21 and novel methodologies, this work describes the formation of seven clot analog types with varying fibrin and RBC composi- tions, intended to represent commonly retrieved AIS thrombi. The clot analogs may be used to expand our knowledge about the effect of throm- bus composition on MT device performance, and may facilitate the development of new and refined techniques in the treatment of AIS. They may also be used for in vitro occlusion models for training in interventional techniques, and may facilitate the development of diagnostic imaging technologies to visualize clot composition in vivo. MATERIALS AND METHODS Blood collection Blood was obtained from the jugular vein of ovine species (males and females; age range 1–7 years; weight range 70–120 kg) at a licensed facility (Ash Stream Ltd). Ovine blood was chosen because it has been demonstrated to be most suitable for coagula- tion studies, 22 and venous blood was obtained to minimize stress to the animal. Seven different clot analog types were created, with five clot replicates formed for each type, as described below and illu- strated in figure 1A. For clot types B–G, blood was anticoagulated in 3.2% sodium citrate solution in a 9:1 ratio immediately after collection (mixing ratios Duffy S, et al. J NeuroIntervent Surg 2016;0:1–7. doi:10.1136/neurintsurg-2016-012308 1 Basic science group.bmj.com on July 20, 2016 - Published by http://jnis.bmj.com/ Downloaded from