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Thrombin calibrator

Manufactured by Diagnostica Stago
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The Thrombin Calibrator is a laboratory instrument used to measure thrombin levels in biological samples. It provides a standardized reference for calibrating thrombin assays, ensuring accurate and reliable results. The Thrombin Calibrator is designed to meet the needs of clinical laboratories and research organizations focused on hemostasis and thrombosis analysis.

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Lab products found in correlation

Fixaβ, by Enzyme Research (6 mentions) α thrombin, by Enzyme Research (6 mentions) Fviia, by Enzyme Research (3 mentions) Human fxia, by Enzyme Research (3 mentions) Hirudin, by Diapharma (1 mentions) Innovin, by Siemens (1 mentions) Enhanced chemifluorescence reagent, by GE Healthcare (1 mentions) Phosphatidylserine, by Avanti Polar Lipids (1 mentions) Phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions) Phosphatidylcholine pc, by Avanti Polar Lipids (1 mentions) Fluorogenic substrate, by Diagnostica Stago (1 mentions) Calibrated automated thrombinoscope, by Diagnostica Stago (1 mentions) Flucakit, by Diagnostica Stago (1 mentions) Immulon 2hb, by Thermo Fisher Scientific (1 mentions) Thrombinoscope, by Stago (1 mentions) Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Fluorogenic thrombin substrate, by Diagnostica Stago (1 mentions) Ge typhoon fla 9000 imager, by GE Healthcare (1 mentions) Prostaglandin i2, by Cayman Chemical (1 mentions)

7 protocols using thrombin calibrator

1

Recombinant FVIII Coagulation Assay

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Recombinant FVIII (Kogenate™) was a generous gift from Dr. Lisa Regan of Bayer Corporation (Berkeley, CA). Dioleoyl phospholipids [Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS)] were purchased from Avanti Polar Lipids (Alabaster, AL). The reagents α-thrombin, FVIIa, FIXaβ, FX, and FXa (Enzyme Research Laboratories, South Bend, IN), hirudin (DiaPharma, West Chester, OH), the chromogenic FXa substrate, Pefachrome Xa (Pefa-5523, CH3OCO-D-CHA-Gly-Arg-pNA·AcOH; Centerchem Inc. Norwalk CT), Enhanced Chemifluorescence reagent (GE Healthcare Bioscience, Piscataway, NJ), recombinant human tissue factor (rTF: Innovin, Dade Behring, Deerfield, IL), flourogenic substrate (Z-Gly-Gly-Arg-AMC: Calbiochem, San Diego, CA), and thrombin calibrator (Diagnostica Stago, Parsippany, NJ) were purchased from the indicated vendors.
Wakabayashi H., Wintermute J.M, & Fay P.J. (2014). Combining mutations that modulate inter-subunit interactions and proteolytic inactivation enhance the stability of factor VIIIa. Thrombosis and haemostasis, 112(1), 43-52.
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2

Plasma Thrombin Generation Assay

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Blood was collected through vena puncture in the antecubital vein and drawn into 0.105 M (3.2%) vacuum citrate tubes. From these tubes, plasma was aliquoted after centrifugation during 10 min at 2500G at 18 °C. Within 4 h after vena puncture, plasma was stored at -80 °C. Ex vivo thrombin generation was measured using the CAT assay (Calibrated Automated Thrombography®; Diagnostica Stago, Asnieres, France), which is a fluorimetric method [14 (link)]. Prior to analyses, plasma was centrifuged again at 10.000 × g for 10 min as described [15 (link)]. Measurements were performed based on protocols of Thrombinoscope BV (Maastricht, the Netherlands). Coagulation was initiated by 5 pM tissue factor (Innovin, Siemens Healthineers, The Hague, The Netherlands) in the presence of 4 mM phospholipid vesicles (PS/PC/PE, 20/40/40, Avanti Polar Lipids, Alabaster, Al, USA), and soluble thrombomodulin (10 mM, Synapse B.V., Maastricht, The Netherlands). A thrombin calibrator and fluorogenic substrate from Diagnostica Stago were used. F1 + 2, TAT, and FPA were measured using commercially available enzyme-linked immunosorbent assays: F1 + 2 and TAT from Siemens Healthcare Diagnostics (The Hague, The Netherlands) and FPA from Bio-Techne (Abingdon, United Kingdom). All measurements were performed according to the manufacturer’s instructions.
Touw C.E., Nemeth B., van Adrichem R.A., van Hylckama Vlieg A., Nelissen R.G., Lisman T, & Cannegieter S.C. (2023). Enhanced in vivo and ex vivo thrombin generation after lower-leg trauma, but not after knee arthroscopy. Thrombosis Journal, 21, 49.
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3

Thrombin Generation Assay Protocol

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Thrombin generation was measured using a calibrated automated thrombinoscope (Diagnostica Stago) in accordance with the manufacturer’s instructions. All reagents including PLT-poor plasma high and low, FluCa-kit, and thrombin calibrator were obtained from Diagnostica Stago. Platelet-poor plasma was prepared by twice centrifuging an aliquot of WB at 2500 G for 10 minutes at room temperature. The plasma was collected and spun at 10,000 G for an additional 10 minutes, transferred to a clean microfuge tube, and stored at between −70°C and −80°C. Plasma samples were thawed for exactly 10 minutes in a water bath at 37°C. A sample of thawed plasma (80 μL) was added to the PLT-poor plasma high and low reagents (20-pmol/L tissue factor and 1-pmol/L tissue factor, respectively) or thrombin calibrator (20 μL) in an Immulon 2HB transparent U-bottom 96-well plate (ThermoFischer Scientific, St. Louis, MO) and incubated for 10 minutes at 37°C. Each sample was run in triplicate. Measurements were started by the addition of Fluo-substrate buffer (40 μL) into each well and were recorded every 20 seconds for 1 hour. The endogenous thrombin potential (ETP) is reported for each sample.
Remy K.E., Yazer M.H., Saini A., Mehanovic-Varmaz A., Rogers S.R., Cap A.P, & Spinella P.C. (2018). Effects of platelet-sparing leukocyte reduction and agitation methods on in vitro measures of hemostatic function in cold-stored whole blood. The journal of trauma and acute care surgery, 84(6), S104-S114.
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4

Thrombin Generation Assay Protocol

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Thrombin generation was measured using a Thrombinoscope (Stago) and performed in Immulon 2HB, round-bottom 96-well plates (Thermo Fisher Scientific). In brief, 60 μL of each plasma sample was spiked with 2.39 to 581 nM PKa in 20 μL with 10 μM PLs. Samples containing 20 μL of thrombin calibrator (Diagnostica Stago) were run in parallel with each cycle of test sample. Thrombin generation was triggered by the addition of PKa, followed by 20 μL of calcium–fluorogenic substrate reagent (16.7 mM calcium and 0.42 mM substrate final reaction concentrations). In some experiments, CTI (1.6 μM) was added to the plasma samples to block FXII activity. All concentrations are final. Thrombograms were produced by monitoring fluorescence at 37 °C at 20-s intervals for 120 min. Each set of conditions was tested in triplicate.
Kearney K.J., Butler J., Posada O.M., Wilson C., Heal S., Ali M., Hardy L., Ahnström J., Gailani D., Foster R., Hethershaw E., Longstaff C, & Philippou H. (2021). Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI. Proceedings of the National Academy of Sciences of the United States of America, 118(3), e2014810118.
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5

Monoclonal Antibody-based Coagulation Assay

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2D2 anti-A3 monoclonal antibody was a gift from Dr. Lisa Regan of Bayer Corporation (Berkeley, CA). Dioleoyl phospholipids phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (PSPCPE) were purchased from Avanti Polar Lipids (Alabaster, AL) and prepared as described [26 (link)]. The reagents FVIII antibody R8B12 ((GMA012) Green Mountain Antibodies, Burlington, VT), α-thrombin, FVIIa, FIXaβ, FX, and FXa (Enzyme Research Laboratories, South Bend, IN), hirudin and chromogenic FXa substrate, Chromogenix S-2765, N-a-Z-D-Arg-Gly-Arg-pNA·2HCl (DiaPharma Group, Inc, West Chester, OH), Enhanced Chemifluorescence (ECF) reagent (GE Healthcare Bioscience, Piscataway, NJ), recombinant human tissue factor (rTF), thrombin fluorogenic substrate, Z-Gly-Gly-Arg-AMC (Calbiochem, San Diego, CA), and thrombin calibrator (Diagnostica Stago, Parsippany, NJ) were purchased from indicated vendors.
Monaghan M., Wakabayashi H., Griffiths A, & Fay P. (2016). Stabilizing Interactions between D666-S1787 and T657-Y1792 at the A2-A3 Interface Support Factor VIIIa Stability in the Blood Clotting Pathway. Journal of thrombosis and haemostasis : JTH, 14(5), 1021-1030.
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6

Coagulation Factor XIII Activation Assay

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Sigmacote® was from Sigma-Aldrich (St. Louis, MO). Prostaglandin-I2 was from Cayman Chemical (Ann Arbor, MI). Lipidated tissue factor (TF, Innovin) was from Siemens (Munich, Germany). Non-reducing 6X sample buffer containing sodium dodecyl sulfate (SDS) was from Boston Bioproducts (Ashland, MA). β-mercaptoethanol was from Fisher Scientific (Hampton, NH). Tris-glycine polyacrylamide gels (10%) were from Bio-Rad (Hercules, CA). Odyssey Blocking Buffer was from LI-COR Biosciences (Lincoln, NE). Anti-human FXIII-A polyclonal antibody (SAF13A-AP) was from Enzyme Research Laboratories (South Bend, IN). FXIII-deficient plasma was from Affinity Biologicals (Ancaster, Ontario). Anti-human fibrinogen polyclonal antibody (A0080) was from Dako (Glostrup, Denmark). Alexa Fluor®−488 anti-rabbit and anti-sheep secondary antibodies were from Jackson Immunoresearch (West Grove, PA). Polyvinylidene difluoride membranes were from Invitrogen (Carlsbad, CA) and scanned on a GE Typhoon FLA-9000 Imager (GE Healthcare, Pittsburgh, PA). Calibrated automated thrombography reagents (fluorogenic thrombin substrate, TF/Lipid Reagents [PPP-Low], and thrombin calibrator) were from Diagnostica Stago (Parsippany, NJ).
Kattula S., Bagoly Z., Tóth N.K., Muszbek L, & Wolberg A.S. (2020). The factor XIII-A Val34Leu polymorphism decreases whole blood clot mass at high fibrinogen concentrations. Journal of thrombosis and haemostasis : JTH, 18(4), 885-894.
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7

Plasma-based Coagulation Assay

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Factor-deficient plasma from patients with severe HA or HB was from George King Bio-Medical (Overland Park, KS, USA), and fresh frozen platelet-poor normal human plasma (referred to as 'control plasma') was from Precision Biologic (Cryocheck; Dartmouth, NS, Canada). Human FXIa was from Enzyme Research Laboratories (South Bend, IN, USA). rFVIII, N8-GP, rFIX and N9-GP were manufactured at Novo Nordisk A/S (Måløv, Denmark). Thrombin calibrator, FluCa substrate and buffer, PPPreagent-LOW and MP reagent were from Diagnostica Stago (Parsippany, NJ, USA).
Waters E.K., Hilden I., Sørensen B.B., Ezban M, & Holm P.K. (2015). Thrombin generation assay using factor XIa to measure factors VIII and IX and their glycoPEGylated derivatives is robust and sensitive. Journal of thrombosis and haemostasis : JTH, 13(11).
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