Global coagulation assays versus differentiated testing: will endogenous thrombin potential replace established thrombophilia screening? Pro. 1

SummaryDevelopmental haemostasis is a concept, now universally accepted, introduced by Andrew et al. in the late 1980’s. However, coagulation analysers and reagents have changed significantly over the past 15 years. Coagulation testing is known to be sensitive to changes in individual reagents and analysers. We hypothesised that the reference ranges developed by Andrew et al. may not be appropriate for use in a modern coagulation laboratory. Our study was designed to determine whethera current day coagulation testing system (STA Compact analyser and Diagnostica Stago reagent system) was sensitive to agerelated changes in coagulation assays. This is the first large scale study since Andrew et al. to determine the age associated numerical changes in coagulation proteins. Our results confirm the concepts of developmental haemostasis elucidated by Andrew et al. However, our results clearly demonstrate that the absolute values of reference ranges for coagulation assays in neonates and children vary with analyser and reagent systems. The results confirm the need for coagulation laboratories to develop age-related reference ranges specific to their own testing systems. Without this, accurate diagnosis and management of neonates and children with suspected bleeding or clotting disorders is not possible. Finally we present age related reference ranges for D-dimers, TFPI, and endogenous thrombin potential, previously not described.

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Endogenous Thrombin Potential (ETP) in Patients with Hereditary Antithrombin (AT) Deficiency or Carriers of the Prothrombin (PT) 20210A Gene Mutation.

Blood ◽

10.1182/blood.v110.11.1623.1623 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1623-1623

Author(s):

Jacqueline Conard ◽

Nicolas Soustelle ◽

Laura Plouy ◽

Marie-Helene Horellou ◽

Meyer-Michel Samama

Keyword(s):

Gene Mutation ◽

Thrombin Generation ◽

Type I ◽

Type Ii ◽

Factor V Leiden Mutation ◽

Endogenous Thrombin Potential ◽

Thrombin Inhibition ◽

Thrombophilia Screening ◽

At Deficiency ◽

Deficiency Type

Abstract Hereditary thrombophilias are associated with an increased risk of venous thromboembolism (VTE) but the magnitude of the risk is not the same: very high in AT deficiency type I or type II RS (Reactive Site) or PE (Pleiotropic Effect), moderate or nul in HBS (Heparin Binding Site) type AT deficiency. According to Butenas S, van’t Veer C, Mann KG. (Blood1999; 94: 2169–78), the dominant factors influencing thrombin generation in a synthetic ‘plasma’ system are prothrombin and AT. Determination of the Endogenous Thrombin Potential (ETP) has been proposed as a valuable tool to detect thrombin generation and hypercoagulability. Aim of the study: Evaluation of ETP in AT deficient patients with different types of deficiency (type I or II) and in carriers of the prothrombin (PT) 20210A gene mutation. Methods The study concerns 35 AT deficient patients: type I or II RS or PE (n=28) or type II HBS (n=7) and 38 carriers of the PT 20210A mutation (24 heterozygous, 14 homozygous). Patients had no antithrombotic or hormonal treatment and women were not pregnant. They have been compared to 59 healthy subjects. ETP was measured in citrated frozen platelet poor plasma by Hemker method initiated by addition of diluted recombinant tissue factor and phospholipids. The following parameters were recorded: ETP (nM/min) as total Thrombin Generation, Peak (nM) as amount of thrombin generated, Start-to-Tail (ST) minus Time-to-Peak (TTP) (min) as an index of thrombin inhibition. Complete thrombophilia screening was also performed. Results: ETP is significantly increased in type I or type II RS or PE AT deficient patients and in patients with heterozygous or homozygous PT 20210A mutations as compared to controls (p<.001), but less increased in HBS type II AT deficiency. n ETP (nM/min) Peak (nM) ST-TTP (min) Controls 59 1484+/−309 296+/−45 15.2+/−1.9 AT HBS 7 1734+/−172 341+/−43 15.2+/−1.1 PT heterozygous 24 2221+/−391 373+/−57 18.3+/−2.7 PT homozygous 14 2606+/−474 385+/−59 21.4+/−3.8 AT I or II RS or PE 28 2655+/−391 359+/−41 25.4+/−5.1 These results demonstrate that ETP, marker of hypercoagulability, is gradually increased in patients with HBS type II AT deficiency, heterozygous and homozygous PT 20210A mutation and in types of AT deficiency other than HBS. The parameter ST-TTP is normal in HBS type II AT deficiency. It reflects the speed of inhibition of the thrombin generated, depending on the amount of thrombin generated and the degree of inhibition. Conclusion: Our findings suggest that ETP might be useful as a first line test for thrombophilia screening to detect hypercoagulability and ST-TTP as a marker of thrombin inhibition. Results of this large series of patients with AT deficiency and homozygous PT 20210A mutation should be compared with the magnitude of the risk of VTE in different thrombophilias, including patients with factor V Leiden mutation or combined thrombophilias.

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Reference values of coagulation assays performed for thrombophilia screening after a first venous thrombosis and their intra-patient associations

Thrombosis Research ◽

10.1016/j.thromres.2022.01.005 ◽

2022 ◽

Author(s):

Jean-Christophe Gris ◽

Éva Cochery-Nouvellon ◽

Chloé Bourguignon ◽

Éric Mercier ◽

Sylvie Bouvier ◽

...

Keyword(s):

Venous Thrombosis ◽

Reference Values ◽

Coagulation Assays ◽

Thrombophilia Screening

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Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature

Thrombosis and Haemostasis ◽

10.1160/th07-07-0448 ◽

2008 ◽

Vol 99 (02) ◽

pp. 416-426 ◽

Cited By ~ 51

Author(s):

Manuel Zürcher ◽

Irmela Sulzer ◽

Gabriela Barizzi ◽

Bernhard Lämmle ◽

Lorenzo Alberio

Keyword(s):

Ambient Temperature ◽

Whole Blood ◽

Repeated Measures ◽

Blood Samples ◽

Coagulation Assays ◽

Specimen Collection ◽

Collection Tube ◽

Thrombophilia Screening ◽

Clinically Significant ◽

The Stability

SummaryMany preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4–6, 8–12, 24–28 and 48–52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C,VWF:RCo,VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24–28 hours, and for PT, aPTT and FVII:C at 48–52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance).The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factorV andVIII coagulant activity, and total PS antigen all investigated parameters can be measured 24–28 hours after specimen collection without observing clinically relevant changes.

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Fibrin Clot Formation and Lysis in Plasma

Methods and Protocols ◽

10.3390/mps3040067 ◽

2020 ◽

Vol 3 (4) ◽

pp. 67

Author(s):

Julie Brogaard Larsen ◽

Anne-Mette Hvas

Keyword(s):

Fibrinolytic Activity ◽

Research Laboratory ◽

Ex Vivo ◽

Fibrin Clot ◽

Clot Formation ◽

Coagulation Assays ◽

Fibrin Formation ◽

Over Time ◽

Routine Coagulation

Disturbance in the balance between fibrin formation and fibrinolysis can lead to either bleeding or thrombosis; however, our current routine coagulation assays are not sensitive to altered fibrinolysis. The clot formation and lysis assay is a dynamic plasma-based analysis that assesses the patient’s capacity for fibrin formation and fibrinolysis by adding an activator of coagulation as well as fibrinolysis to plasma and measuring ex vivo fibrin clot formation and breakdown over time. This assay provides detailed information on the fibrinolytic activity but is currently used for research only, as the assay is prone to inter-laboratory variation and as it demands experienced laboratory technicians as well as specialized personnel to validate and interpret the results. Here, we describe a protocol for the clot formation and lysis assay used at our research laboratory.

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