Comparison of a New Automated Amidolytic Assay of Thrombin Generation with “ Prothrombin Time” and “Thrombotest”.

1979 ◽  
Author(s):  
J.J.C. Jonker ◽  
L.H.M. van Riel ◽  
M.M.P. Paulssen
Keyword(s):  
Prothrombin Time ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Automated Method

An automated method was developed for the determination of thrombin generation in citrated whole blood during activation by thromboplastin. The thrombin generated was allowed to split the chromogenic substrate Tos-Gly-Pro-Arg-pNA yielding p-Nitroaniline. The comparison with this method and “Prothrombin time” in 50 samples was significant (r = 0,88 P < 0,001) and also in 503 samples with “Thrombotest” (r = 0,73 P < 0,001). This assay may serve as a specific, precise and fast and cheaper alternative for “prothrombin time” and “Thrombotest” in large Thrombosis Services.

Levels of Platelet Factor 3 in Citrate Plasma and in Whole Blood as Determined by A Chromogenic Peptide Substrate Assay

1979 ◽  
Author(s):  
H. Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PCE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PCE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PCE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PCE 1/theophy11ine anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and βtg are released in parallel.

Automated Spectrophotometric Method Using 2,2’-Dithiodipyridine Acid for Determination of Cholinesterase in Whole Blood

1996 ◽  
Vol 79 (3) ◽  
pp. 757-763 ◽  
Author(s):  
Jose J Cerón ◽  
María J Fernandez Del Palacio ◽  
Luis J Bernal ◽  
Cándido Gutierrez
Keyword(s):  
Detection Limit ◽  
Spectrophotometric Method ◽  
Whole Blood ◽  
Cholinesterase Activity ◽  
New Method ◽  
Good Precision ◽  
Throughput Rate ◽  
Nitrobenzoic Acid ◽  
Automated Method

Abstract An automated method using 2,2’-dithiodipyridine (2-PDS) as chromophore for determination of wholeblood cholinesterase activity was developed. Assay procedures, optimal concentrations of chromophore and substrate, detection limit, precision, backgrounds, and sensitivity of the method were compared with those of an earlier automated method based on the Eilman method and using 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) as chromophore. The new method has the advantages of automation (resulting in increase throughput rate and decrease in amount of reagents used) and good precision and sensitivity. Sample dilutions also are reduced in the new method because hemoglobin interference is less.

Effective Reversal of Edoxaban-associated Bleeding with Four-factor Prothrombin Complex Concentrate in a Rabbit Model of Acute Hemorrhage

2015 ◽  
Vol 122 (2) ◽  
pp. 387-398 ◽  
Author(s):  
Eva Herzog ◽  
Franz Kaspereit ◽  
Wilfried Krege ◽  
Baerbel Doerr ◽  
Jochen Mueller-Cohrs ◽  
...  
Keyword(s):  
Blood Loss ◽  
Prothrombin Time ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Blood Clotting ◽  
Prothrombin Complex Concentrate ◽  
Rabbit Model ◽  
Clotting Time ◽  
Blood Clotting Time

Abstract Background: Edoxaban is an oral, selective direct factor Xa inhibitor approved in Japan for venous thromboembolism prevention after orthopedic surgery. Data are lacking regarding reversal strategies for edoxaban; this study assessed whether four-factor prothrombin complex concentrate (Beriplex®/Kcentra®; CSL Behring GmbH, Marburg, Germany) can effectively reverse its effects on hemostasis using a previously described rabbit model. Methods: The study comprised assessments of thrombin generation in vitro, pharmacokinetic parameters, and edoxaban reversal in vivo. In a blinded in vivo stage, a standardized kidney incision was performed in animals (n = 11 per group) randomized to receive vehicle + saline, edoxaban (1,200 μg/kg) + saline, or edoxaban (1,200 μg/kg) + four-factor prothrombin complex concentrate (50 IU/kg). Animals were monitored for treatment impact on hemostasis and coagulation parameters. Data are median (range). Statistical tests were adjusted for multiple testing. Results: Edoxaban administration increased blood loss (30 [2 to 44] ml) and time to hemostasis (23 [8.5 to 30.0] min) compared with the control group (3 [1 to 8] ml and 3 [2.0 to 5.0] min, respectively). Biomarkers of coagulation (prothrombin time, activated partial thromboplastin time, whole blood clotting time) and thrombin generation parameters (e.g., peak thrombin, endogenous thrombin potential, lag time) were also affected by edoxaban. Administration of four-factor prothrombin complex concentrate significantly reduced time to hemostasis (to 8 [6.5 to 14.0] min, observed P &lt; 0.0001) and total blood loss (to 9 [4 to 22] ml, observed P = 0.0050) compared with the edoxaban + saline group. Of the biomarkers tested, prothrombin time, whole blood clotting time, and endogenous thrombin potential correlated best with clinical parameters. Conclusion: In a rabbit model of hemostasis, four-factor prothrombin complex concentrate administration significantly decreased edoxaban-associated hemorrhage.

scholarly journals Measurement of Thrombin Generation in Whole Blood - The Effect of Heparin and Aspirin

1994 ◽  
Vol 72 (01) ◽  
pp. 078-083 ◽  
Author(s):  
Han Kessels ◽  
Suzette Béguin ◽  
Harry Andree ◽  
H Coenraad Hemker
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Lag Time ◽  
Coagulation System ◽  
Common Denominator ◽  
Centrifugal Separation ◽  
Chromogenic Substrate ◽  
Tissue Thromboplastin ◽  
Cellular Components ◽  
Inhibition Of Thrombin

SummaryA technique has been developed to monitor the development of thrombin in freshly collected whole blood in the absence of anticoagulants. It is based on the centrifugal separation of the cellular components from subsamples of blood drawn from non-anticoagulated clotting whole blood which are diluted in buffer containing a chromogenic substrate.It is shown that the burst of thrombin generation after triggering coagulation with trace amounts of tissue thromboplastin occurs sooner in non-anticoagulated whole blood than in citrated whole blood. Heparin is shown to prolong the lag-time of thrombin generation more in native blood than in recalcified citrated blood.It is also demonstrated that intake of 500 mg of aspirin significantly delays and inhibits thrombin generation in non-anticoagulated, thromboplastin triggered whole blood, whereas it has no effect on the coagulation in citrated plasma. The effect of aspirin intake on thrombin generation in blood is roughly equal to that of 0.03 U/ml of unfractionated heparin. This demonstrates that platelet reactions and the coagulation system are closely linked processes. It further lends support to the hypothesis that inhibition of thrombin generation is a common denominator of antithrombotic therapy.

scholarly journals A Chromogenic Test to Determine the Procoagulant Phospholipids in Platelet-rich Plasma and Whole Blood

1994 ◽  
Vol 72 (04) ◽  
pp. 582-587 ◽  
Author(s):  
R J Wagenvoord ◽  
H H Hendrix ◽  
Hu Kai ◽  
H C Hemker
Keyword(s):  
Whole Blood ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Product Formation ◽  
Phosphatidyl Serine ◽  
Linear Increase ◽  
Control Group ◽  
Chromogenic Substrate ◽  
Chromogenic Assay ◽  
Two Parameters

SummaryWe have developed a chromogenic assay to measure the phospholipid-related procoagulant activity (PPA) in whole blood, or platelet-rich plasma. The test is based upon thrombin formation from prothrombin by prothrombinase and is designed in such a way that procoagulant lipids are rate limiting for the prothrombinase activity. In the chromogenic test PPA concentrations equivalent to 0-10 nM phospholipid vesicles containing 75% phosphatidyl choline (PC) and 25% phosphatidyl serine (PS) can be measured.The thrombin, which develops during the test, is measured with a chromogenic substrate. By the action of thrombin on this chromogenic substrate p-nitroaniline is liberated, which causes an increase in absorbance. Thrombin formed in the assay mixture activates the present platelets. This causes a linear increase of the velocity of thrombin generation during the test, i. e. a parabolic increase of product formation. For that reason the thrombin generation in time is characterized by two parameters, the basal PPA (PPA-B) of the original mixture and the increase in PPA due to platelet activation (PPA-A). To determine these figures the absorbency-data were fitted to parabolas. In most cases the contribution of PPA-A to the total amount of formed thrombin becomes considerable already after 30 s.Preliminary tests show that PPA-B activity in whole blood or platelet-rich plasma of patients with a thrombotic disorder is significantly higher than the activity of a control group of the same age.

Multicenter Evaluation of a Chromogenic Substrate Method for Photometric Determination of Prothrombin Time

1987 ◽  
Vol 58 (03) ◽  
pp. 856-865 ◽  
Author(s):  
F Dati ◽  
M Barthels ◽  
J Conard ◽  
J Flückiger ◽  
A Girolami ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Anticoagulant Therapy ◽  
Photometric Determination ◽  
Coagulation Factors ◽  
Stable Phase ◽  
Photometric Method ◽  
Sensitivity Index ◽  
Chromogenic Substrate ◽  
Plasma Samples

SummaryA multicenter study of a chromogenic substrate method for photometric determination of prothrombin time was conducted in order to evaluate its clinical application. Seven laboratories pailicipaled in the study using a total of 742 plasma samples from 417 patients on oral anticoagulant therapy, 261 healthy subjects and 64 patients with different diseases especially of the liver as well as 30 patients with hereditary deficiency of coagulation factors II, V, VII, X. The chromogenic PT method was compared to a standardized coagulometric PT assay which uses the same sensitive human placenta thromboplastin calibrated against international reference preparations. A high correlation of the prothrombin ratio values of the chromogenic and the coagulometric assay was obtained in 402 plasma samples (r = 0.940; y = 1.02x − 0.1). The study showed that the chromogenic PT reagent is sensitive to deficiency of the coagulation factors of the extrinsic pathway but not affected by heparin up to 1 IU/ml because of the heparin antagonist added. The precision (coefficient of variation) of the photometric method ranged between 0.6 and 3% (intraassay CV) and between 1.4 and 5.8 (interassay CV). The International Sensitivity Index (ISI) obtained for the used lot was 1.09. The therapeutical range in percentage activity for patients in a stable phase of an anticoagulant therapy was found to be from 15 to 27 percent of normal. The results of the clinical evaluation proved the good comparability of the new chromogenic PT test with coagulometric methods, its high factor sensitivity, good reproducibility and easy performance.

Levels of platelet factor 3 in citrate plasma and in whole blood as determined by a chromogenic peptide substrate assay

1979 ◽  
Author(s):  
H Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PGE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PGE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PGE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PGE 1/theophylline anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and β-tg are released in parallel.

An automated method for the determination of acetyl and pseudo cholinesterase in hemolyzed whole blood

1992 ◽  
Vol 22 (2) ◽  
pp. 231-241 ◽  
Author(s):  
W. J. A. Meuling ◽  
M. J. M. Jongen ◽  
J. J. Van Hemmen
Keyword(s):  
Whole Blood ◽  
Automated Method

APPLICATIONS OF INTERNATIONAL RECOMMENDATIONS ON THE STANDARDIZATION OF PROTHROMBIN TIME IN ORAL ANTICOAGULANT CONTROL

1987 ◽  
Author(s):  
F Dati ◽  
U Becker ◽  
N Heimburger
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant ◽  
Calibration Procedure ◽  
Sensitivity Index ◽  
Chromogenic Substrate ◽  
Clotting Factors ◽  
Reference Preparation ◽  
Broad Variation ◽  
The Mean

The determination of prothrombin time (PT) in oral anticoagulant control is affected by a broad variation. The responsible factors are: type of thromboplastin incorporated in the PT reagents, procedure for use, clotting factors or heparin inhibitors added to the reagent, method of expression of PT results. Recently, joint recommendations have been issued by International Committees (ICSH/ICTH) taking into account the system of International Thromboplastins and the statistical model for thromboplastin calibration established by WHO. The aim is a standardization of commercial thromboplastins for PT tests in order to allow the use of the international scale of oral anticoagulant intensity (INR: Intern. Normalized Ratio). Following such recommendations we have standardized two new PT tests, based on coagulometric and photometric methods which rely on the same sensitive human placental thromboplastin. The coagulometric PT test (Thromborel®S) is performed with conventional coagulometers. The photometric PT assay (Chromoquick®) uses a new chromogenic substrate specific for thrombin. This method is based on the measurement of the time necessary to reach a fixed increase of absorbance (0.1 A) using a special microprocessor-controlled photometer.The two PT reagents were calibrated either directly against a reference preparation (BCT) or via an intermediate standard thromboplastin in two multicentric studies. The calibration procedure by the WHO method allows to assign the corresponding ISI (Intern. Sensitivity Index) to the PT reagent used and the transformation of the obtained prothrombin ratio (PR) into INR by the equation INR = PRISI. The calculated ISI values were 1.08 for the coagulometric PT reagent (n = 330) and 1.07 for the photometric reagent (n = 365), respectively.The reproducibility of the ISI value for the new human placental thromboplastin for 64 different batches amounts to 3.6 %, the mean ISI value being 1.12.Comparison with the reference thromboplastins in PR values gave a good correlation.A) Coagul. PT assay (x): r = 0.964; y = 1.03x ™ 0.1;B) Photom. PT assay (x): r = 0.940; y = 1.02x ™ 0.1.

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