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8 protocols using hemosil protein c

1

Comprehensive Coagulation Profile Analysis

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Coagulation tests, including PT, aPTT, and factor assays, were performed on an automated coagulation analyzer, ACL TOP (Instrumentation Laboratory, Lexington, MA, USA). PT was measured using the HemosIL RecombiPlasTin reagent (Instrumentation Laboratory,), and aPTT was measured using the SynthASil reagent (Instrumentation Laboratory). Fibrinogen was measured using the HemosIL Fibrinogen-C XL reagent (Instrumentation Laboratory) based on the Clauss method. Coagulation factors were tested using a PT-based clotting assay with the HemosIL RecombiPlasTin reagent (for FV) and an aPTT-based clotting assay using the SynthASil reagent (for FVIII, FXI, and FXII). Antithrombin and protein C activity were determined with chromogenic assays (HemosIL liquid antithrombin and HemosIL Protein C; Instrumentation Laboratory) and free protein S antigen with an immunoassay (HemosIL Free Protein S, Instrumentation Laboratory).
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2

Coagulation Biomarker Measurement Protocol

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Plasma APC levels were analyzed by the OLIGOBIND APC activity assay (Sekisui Diagnostics GmbH, Pfungstadt, Germany) according to the manufacturer’s instructions. Plasma sTM levels were measured by ELISA using monoclonal antibodies against thrombomodulin. The plasma sTM levels determined by this technique were well correlated with a functional assay [15 (link)]. Plasma protein C levels were measured by a synthetic chromogenic substrate method using HemosIL Protein C (Instrumentation Laboratory, Bedford, MA). Plasma TAT levels were analyzed using STACIA CLEIA TAT (LSI Medience, Tokyo, Japan) according to the manufacturer’s instructions. Plasma F1 + 2 levels were analyzed using Enzygnost F1 + 2 (Siemens Healthcare Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions.
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3

Coagulation Factor Assays in Clinic

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PT and aPTT were assayed by using the RecombiPlasTin (Instrumentation Laboratory, Milan, Italy) and SynthASil (Instrumentation Laboratory) on an ACL 3000 (Beckman Coulter Inc., Fullerton, CA, USA). Procoagulant factor II, VII, and X levels were measured by a one-stage PT-based clotting assay. Procoagulant factor VIII, IX, XI, and XII levels were measured by an aPTT-based clotting assay on the ACL 3000. Antithrombin, protein C, and protein S activity was determined by using HemosIL liquid antithrombin, HemosIL Protein C, and HemosIL Free Protein S (Instrumentation Laboratory).
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4

Coagulation Factors and Inhibitors Measurement

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Levels of factor (F) VIII, II, antithrombin (AT) and Protein C were measured on an automated coagulation analyzer (ACL 300 TOP) with reagents and protocols from the manufacturer (Recombiplastin 2G and FII depleted plasma for FII, Hemosil (R) SynthASil and FVIII depleted plasma for FVIII, Liquid Antithrombin reagent for AT, and Hemosil Protein C for Protein C measurements; Instrumentation Laboratory, Breda, the Netherlands).
Plasma levels of Tissue Factor Pathway Inhibitor (TFPI) were determined with an in-house ELISA as previously described
18 (link).
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5

Coagulation Assays: A Comprehensive Approach

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Both PT and aPTT were assayed using the RecombiPlasTin (Instrumentation Laboratory, Milan, Italy) and SynthASil (Instrumentation Laboratory) on an ACL 3000 (Instrumentation Laboratory). Fibrinogen was measured using the HemosIL Fibrinogen-C XL reagent (Instrumentation Laboratory SpA). Coagulation factors were assayed using a PT-based clotting assay with the HemosIL RecombiPlasTin reagent (for FII, FV, FVII, and FX) and an aPTT-based clotting assay using the SynthASil reagent (for FVIII, FXI, FXI, and FXII). d-dimers were measured using an immunoturbidimetric method on ACL 3000. Antithrombin and protein C activity was determined using chromogenic assays (HemosIL liquid antithrombin and HemosIL protein C, respectively; Instrumentation Laboratory SpA). The tissue factor pathway inhibitor (TFPI) was measured using a TFPI Human ELISA Kit (Abcam (Cambridge, MA)) according to the manufacturer’s protocol.
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6

Coagulation Factors and Activity Assays

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Prothrombin time, fibrinogen and antithrombin were analysed on the ACL-TOP 750 (HemosIL ReadiPlasTin, HemosIL Q.F.A Thrombin, HemosIL Liquid Antithrombin, Werfen Barcelona, Spain). Von Willebrand antigen and activity were determined by immunoturbidimetric latex based assays (HemosIL von Willebrand Factor Antigen, HemosIL von Willebrand Ristocetin Cofactor assay; Werfen) on the ACL-TOP 700. Protein C was analyzed on ACL-TOP 700 (HemosIL Protein C, Werfen). ADAMTS13 was determined with the Technozym® ADAMTS-13 Activity ELISA kit (Technoclone, Wien, Austria). Von Willebrand antigen and activity are expressed in %. This is equivalent to IU/dL since these tests are calibrated against HemosIL Calibration Plasma (Werfen), which is traceable to the current International Standards.
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7

Hemostasis Biomarkers Measurement Protocol

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The determination of fibrinogen was based on the Clauss method (Fibrinogen-C, Werfen) by using excess thrombin to convert fibrinogen into fibrin and results were expressed in mg/dL.
Protein C was measured by an assay based on a synthetic chromogenic substrate, after incubation of plasma with snake venom as activator (HemosIL protein C, Werfen). Antithrombin was measured by chromogenic assay containing bovine FXa and excess heparin (HemosIL liquid antithrombin, Werfen). Results of both tests were reported as percentage activity in comparison to a pooled normal plasma with an assigned arbitrary activity of 100%. All of the above tests were performed on automated coagulometer (ACLTOP, Werfen).
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8

Automated Coagulation Assay Protocol

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Plasma levels of PC (HemosIL Protein C, Werfen Group) and Free-protein S (HemosIL Free Protein S, Werfen Group) were measured on an automated coagulometer analyzer (ACL TOP500, Werfen Group).
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