Thrombin generation was measured in PPP using the Calibrated Automated Thrombogram (CAT) (Diagnostica Stago, Asnière, France) with the Thrombinoscope software (Thrombinoscope BV®, Maastricht, The Netherlands) [24 (link), 25 (link)]. PPP, supplemented with the three different reversal agents in three different concentrations, were run in triplicates. The thrombin generation parameters lag time, peak of maximum thrombin concentration, velocity index and the total amount of thrombin generated, i.e. endogenous thrombin potential (ETP), were recorded. The PPP reagent containing 5 pM TF and 4 μM phospholipids was used to initiate thrombin generation.
Calibrated automated thrombogram
Manufactured by Diagnostica Stago
Sourced in France, United States
The Calibrated Automated Thrombogram is a laboratory instrument designed to measure the thrombin generation potential in a sample. It provides quantitative data on the kinetics of thrombin generation, offering insights into the hemostatic and thrombotic status of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Tissue factor, by Diagnostica Stago (1 mentions)
Ppp reagent low, by Diagnostica Stago (1 mentions)
Fluoroskan ascent plate reader, by Thermo Fisher Scientific (1 mentions)
Fluoroskan ascent fluorometer, by Thermo Fisher Scientific (1 mentions)
Acl top 700 analyzer, by Werfen (1 mentions)
Thrombinoscope software, by Diagnostica Stago (1 mentions)
Bcs analyzer, by Siemens (1 mentions)
Vwf antigen, by Siemens (1 mentions)
8 protocols using calibrated automated thrombogram
1
Thrombin Generation Assay with Reversal Agents
2
Thrombogram Analysis of Platelet-Poor Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TG was measured in platelet‐poor plasma (PPP) using the Calibrated Automated Thrombogram (CAT; (Diagnostica Stago, Asnière, France) with Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands).14 , 15 PPP was prepared by double centrifugation, first for 15 minutes at 2500 g, thereafter for 10 minutes at 10 000 g. For the youngest patients (patients 1 and 4), a smaller amount of blood was collected than for the adult patients, and only a single centrifugation was performed. TG in PPP containing the different BPAs in different concentrations, including one unspiked sample, were run in triplicates. The PPP reagent “low,” containing 1 pM of tissue factor (TF) and 4 µM phospholipids (Diagnostica Stago, Asnière, France), was used to initiate TG.
3
Thrombin Generation Assay in Platelet-Poor Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CAT is a micro‐well assay performed in 96‐well plates that measures thrombin generation in platelet poor plasma (PPP). Primary metrics include lag time which reflects time to initial thrombin production, peak height which reflects the maximum amount of thrombin produced, time to peak which reflects the time to peak height, and endogenous thrombin potential which reflects the total amount of thrombin produced.
To prepare PPP, whole blood was collected through approved IRB protocol HSC‐MS‐09‐0314 from eight healthy volunteers into 4.5 ml vacutainers containing 3.2% sodium citrate. Samples were centrifuged for 10 minutes at 200g and platelet rich plasma supernatant was centrifuged again for 10 minutes at 2,000g to obtain PPP. Samples from each donor were pooled and aliquoted. CAT was performed using a Calibrated Automated Thrombogram (Diagnostica Stago, Parsippany NJ). In a 96‐well plate, 20 μl of phospholipid reagent was added to 72 μl of PPP and either 8 μl of CCTs suspended in PBS at concentration 106 cells per milliliter or PBS vehicle. Thrombin generation was initiated with 20 μl of 0.1 M calcium chloride and fluorogenic substrate solution. Samples were performed in duplicate. Reactions were measured using a Fluoroskan Ascent plate reader (ThermoFisher, Waltham, MA).
To prepare PPP, whole blood was collected through approved IRB protocol HSC‐MS‐09‐0314 from eight healthy volunteers into 4.5 ml vacutainers containing 3.2% sodium citrate. Samples were centrifuged for 10 minutes at 200g and platelet rich plasma supernatant was centrifuged again for 10 minutes at 2,000g to obtain PPP. Samples from each donor were pooled and aliquoted. CAT was performed using a Calibrated Automated Thrombogram (Diagnostica Stago, Parsippany NJ). In a 96‐well plate, 20 μl of phospholipid reagent was added to 72 μl of PPP and either 8 μl of CCTs suspended in PBS at concentration 106 cells per milliliter or PBS vehicle. Thrombin generation was initiated with 20 μl of 0.1 M calcium chloride and fluorogenic substrate solution. Samples were performed in duplicate. Reactions were measured using a Fluoroskan Ascent plate reader (ThermoFisher, Waltham, MA).
4
Thrombin Generation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The thrombin generation assay (TGA; calibrated automated thrombogram; all reagents from Diagnostica Stago) was conducted with a validated method [9 (link)], in which thrombin generation was triggered in 80 µL of citrated platelet-poor plasma (PPP) by addition of a PPP reagent solution containing TF (final concentration 5 µM) and phospholipids (final concentration 4 µM). After 5-min incubation time at 37 °C, 20 µL of the Flu-Ca reagent was added. The activity of the thrombin generated resulted in conversion of a fluorogenic substrate. Fluorescence was measured continuously for 90 min with a MTP fluorometer (Diagnostica Stago), which relates the fluorescence to a thrombin calibrator. TGA parameters (lag time, time to peak and peak height) were derived with Thrombinoscope software (Thrombinoscope, Maastricht, The Netherlands).
5
Apolipoprotein and Coagulation Factors Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Levels of apolipoprotein (apo) A-I, A-II, A-IV, (a), B-100, B-total, C-I, C-II, C-III and E were measured on unthawed serum samples. To determine the apolipoprotein profile, a mass spectrometric method was developed for multiplexed quantification of apolipoproteins [14 (link),19 (link)].
All coagulation-related laboratory measurements (FVII, FVIII, FIX, FXI, vWF:Ag) were analyzed on the ACL-Top 700 Analyzer (Instrumentation Laboratory). FVIII, FIX, and FXI levels were measured using modified activated partial thromboplastin time assays with immunodepleted plasmas. Similarly, FVII was determined using a modified prothrombin time assay. vWF antigen was measured using an automated latex–enhanced immunoassay with the HemosIL vWF:Ag Reagent Kit [10 (link)].
The thrombin generation potential was assessed by the Calibrated Automated Thrombogram (Diagnostica Stago) according to the specifications of the manufacturer [20 (link)]. The fluorescent signal representing generated thrombin was monitored in a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific), and the parameters were calculated with the Thrombinoscope software (Thrombinoscope BV). Endogenous thrombin potential (ETP) was the parameter determined through the thrombin generation assay [11 (link),15 (link)].
All coagulation-related laboratory measurements (FVII, FVIII, FIX, FXI, vWF:Ag) were analyzed on the ACL-Top 700 Analyzer (Instrumentation Laboratory). FVIII, FIX, and FXI levels were measured using modified activated partial thromboplastin time assays with immunodepleted plasmas. Similarly, FVII was determined using a modified prothrombin time assay. vWF antigen was measured using an automated latex–enhanced immunoassay with the HemosIL vWF:Ag Reagent Kit [10 (link)].
The thrombin generation potential was assessed by the Calibrated Automated Thrombogram (Diagnostica Stago) according to the specifications of the manufacturer [20 (link)]. The fluorescent signal representing generated thrombin was monitored in a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific), and the parameters were calculated with the Thrombinoscope software (Thrombinoscope BV). Endogenous thrombin potential (ETP) was the parameter determined through the thrombin generation assay [11 (link),15 (link)].
6
Calibrated Automated Thrombogram Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TF‐TG was measured using a Calibrated Automated Thrombogram (Diagnostica Stago, Inc, Parsippany, NJ, USA) per the manufacturer’s recommendations using the PPP‐reagent (5 pM TF/4 µM phospholipids). TF‐TG profiles were analyzed using Thrombinoscope software (Diagnostica Stago) with five parameters: ETP, Peak, lag time, time‐to‐peak, and velocity index. The results were reported as mean ± standard deviation.
7
Comprehensive Blood Coagulation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were collected into 3 mL tubes (Sarstedt) containing 3.2% citrate. The samples were processed within 1 hour after the blood collection, centrifuged at 2000 g for 20 minutes to prepare platelet-poor plasma, aliquoted, and immediately stored at −80 °C. The aliquots were thawed directly before the analysis. Anti-Xa activity was determined with a chromogenic substrate and a bovine FXa (Hyphen BioMed), and a chromogenic anti-IIa assay with human thrombin (Hyphen BioMed) was used to measure anti-IIa activity. The FVIII activity was assessed by a one-stage clotting test (Siemens Healthineers). In addition, activities of D-dimer, prothrombin fragments, VWF antigen, and VWF (all purchased from Siemens Healthineers) were measured on a BCS analyzer (Siemens Healthineers). The TG assay was carried out on a Fluoroscan Ascent (Fisher Scientific) at a 390/460-nm wavelength using a calibrated automated thrombogram (Diagnostica Stago) activated with 5-pM tissue factor.
8
Thrombin Generation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TGA determinations were made using standardized methods on a dedicated Thrombinoscope platform (also known as the Calibrated Automated Thrombogram) as recommended by the manufacturer (Diagnostica Stago Inc., Parsippany, NJ, USA). Briefly, frozen platelet poor plasma was thawed in a 37 °C water bath for exactly 10 minutes, followed by pipetting 80 μL of each sample plasma into each of 6 wells of a 96-well Immulon 2HB plate. To 3 of 6 wells, 20 μL of platelet poor plasma Reagent HIGH was added [31] , along with 20 μL of Thrombin Calibrator to the other 3 wells of each 6-well grouping. The plates were then inserted into a Fluoroskan Ascent instrument equipped with 1 liquid dispenser unit set to a nominal temperature of 37 °C for 10 minutes, followed by dispensing of 20 μL of FluCa reagent into each well. Fluorescence measurements were collected for a period of 60 minutes, followed by determination of ETP, peak height, time-to-max peak height, lag time, and velocity index by the Thrombinoscope software. The method used was consistent with previously published methods [32, 33] .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!