title: Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis.
abstract: BACKGROUND
  Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations.
  
  
  METHODS
  Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10 min, 180 × g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10 min, 2570 × g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60 kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60 kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179 W/cm(2)) or 2-MHz transcranial Doppler (TCD) (0.457 W/cm(2)).
  
  
  RESULTS
  With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7 ± 7.8% (rt-PA), 40.8 ± 8.6% (rt-PA+TCCS), and 40.4 ± 8.3% (rt-PA+TCD).
  
  
  METHODS
  These systemic investigations were conducted for the first time.
  
  
  CONCLUSIONS
  This protocol should be used for standardized sonothrombolysis investigations.
authors.affiliation: Department of Neurology, University Hospital Gießen, Klinikstraße 33, 35385 Gießen, Germany. Electronic address: florian.roessler@neuro.med.uni-giessen.de.
authors.forename: Florian C
authors.initials: FC
authors.lastname: Roessler
authors.affiliation: Department of Neurology University Hospital of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Teichert
authors.affiliation: Department of Neurology University Hospital of Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Ohlrich
authors.affiliation: Oncology Practice Am Marien-Krankenhaus, Parade 5, 23552 Lübeck, Germany.
authors.forename: Jan H
authors.initials: JH
authors.lastname: Marxsen
authors.affiliation: Research Centre Borstel, Clinical and Experimental Pathology, Parkallee 3a, 23845 Borstel, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Stellmacher
authors.affiliation: Department of Neurology, University Hospital Gießen, Klinikstraße 33, 35385 Gießen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Tanislav
authors.affiliation: Asklepios Klinik Nord, Tangstedter Landstraße 400, 22417 Hamburg, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Seidel
mesh_terms: D001772:Blood Cell Count; D001777:Blood Coagulation / Q000187:drug effects; D063285:Fibrin Clot Lysis Time; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015233:Models, Statistical; D012107:Research Design / Q000592:standards*; D013018:Sound Spectrography / Q000379:methods* / Q000592:standards*; D013927:Thrombosis / Q000628:therapy*; D010959:Tissue Plasminogen Activator / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; D010959:Tissue Plasminogen Activator
keywords: Clot formation protocol; Histological examination; In vitro model; Physiological flow; Sonothrombolysis