To prepare PRP, citrated whole blood collected from the inferior vena cava was centrifuged at 150g for 5 min without brakes. The supernatant was collected, and platelet counts were measured on a scil Vet abc Plus hematology analyzer (scil animal care company, Ontario, Canada). Platelet counts were adjusted to 600 × 103 platelets/μl using platelet-poor plasma prepared from the same whole-blood sample using a second spin at 800g for 8 min. In vitro thrombin generation assays were conducted using a modified Technothrombin Thrombin Generation Assay (catalog no. 5006010, DiaPharma Group, West Chester Township, OH). Thrombin generation was initiated by the addition of recombinant TF (1.0pM) (RecombiPlasTin 2G, Instrumentation Laboratory, Bedford, MA). Fluorescence was immediately measured with a SpectraMax Gemini EM fluorimeter and SoftMax Pro software (Molecular Devices, San Jose, CA) at 355 nm/460 nm (excitation/emission) for 2 hours at 37°C with measurements collected at 1-min intervals. Raw fluorescence data were analyzed using Technothrombin TGA evaluation software.
Technothrombin thrombin generation assay
Manufactured by Diapharma
Sourced in United States
The Technothrombin Thrombin Generation Assay is a laboratory equipment used to measure the generation of thrombin, a key enzyme involved in blood clotting. The assay provides quantitative information about the overall coagulation potential of a sample.
Automatically generated - may contain errors
2 protocols using technothrombin thrombin generation assay
1
Platelet-Rich Plasma Preparation and Thrombin Generation Assay
2
Measuring Thrombin Generation in Plasma Microparticles
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Thrombin generation capacity (TGC) was measured at baseline (t0) and post-intervention (t24) in citrated platelet-poor plasma (PPP), which was further treated to ensure only microparticles remained in the plasma (no platelets or large platelet fragments). In this way, the TGC related to plasma microparticle load was measured. Aliquots of 25 μL were stored frozen at − 80 °C for up to 1 month before analysis. Analyses were carried out using the Technothrombin® Thrombin Generation Assay (Diapharma Group Inc., West Chester, Ohio, US), using the protocol specified by the manufacturer but with some modification to sample preparation: platelet-poor plasma was generated by double centrifugation directly from citrated whole blood, not sequentially after generation of platelet-rich plasma. The final concentrations of tissue factor (TF) used were 1 pmol/L. Microparticle-free plasma and microparticle-high plasma (Diapharma Group Inc., West Chester, Ohio, US) were used as negative and positive controls.
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