BACKGROUND: A gap exists between in vivo and ex vivo coagulation when investigated by use of the coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). The thrombin generation assay (TGA) has been developed to fill this gap.

CONTENT: TGA evaluates thrombin generation (resulting from the action of the procoagulant driver) and decay (resulting from the action of the anticoagulant driver), thus assessing the balance between the two. Coagulation of the test plasma (platelet poor or platelet rich) is activated by small amounts of tissue factor and phospholipids, and the reaction of thrombin generation is continuously monitored by means of a thrombin-specific fluorogenic substrate. Among the parameters derived from the thrombin-generation curve, the most important is the endogenous thrombin potential, defined as the net amount of thrombin that test plasmas can generate on the basis of the relative strength of the pro- and anticoagulant drivers. TGA is therefore the candidate assay to investigate hypo- or hypercoagulability.

SUMMARY: From my analysis of the literature, I draw the following conclusions. There is strong evidence that TGA is helpful to elucidate coagulation mechanisms in various clinical conditions that until recently were poorly understood (chronic liver disease; diabetes; inflammatory bowel disease, myeloproliferative neoplasms, nonalcoholic fatty liver disease). TGA is a promising laboratory tool for investigating hemorrhagic coagulopathies and monitoring replacement therapy in hemophiliacs, predicting the risk of recurrent venous thromboembolism after a first event, and monitoring patients on parenteral or oral anticoagulants. These applications require clinical trials in which TGA results are combined with specific clinical end points.

/pdf/thrombin-generation-assay-and-its-application-in-the-39nknyhovj.pdf

Thrombin Generation Assay and Its Application in the Clinical Laboratory

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jth.13743

Proposal for standardized preanalytical and analytical conditions for measuring thrombin generation in hemophilia: communication from the SSC of the ISTH

The existing techniques to evaluate hemostasis in clinical laboratories are not sensitive enough to detect hypercoagulable and mild hypocoagulable states. Under different experimental conditions, the thrombin generation test may meet these requirements. This technique evaluates the overall balance between procoagulant and anticoagulant forces and has provided new insights in our understanding of the coagulation cascade, as well as of the diagnosis of hypocoagulability and hypercoagulability conditions. Thrombin generated in the thrombin generation test can be quantified as platelet-rich or platelet-poor plasma using the calibrated automated thrombogram method, which monitors the cleavage of a fluorogenic substrate that is simultaneously compared to the known thrombin activity in a non-clotting plasma sample. The calibrated automated thrombogram method is an open system, in which different antibodies, proteins, enzymes and peptides can be introduced to answer specific questions regarding hemostatic processes. The thrombin generation test has great clinical potential, such as in monitoring patients taking anticoagulants and antiplatelet drugs, screening for genetic or acquired thrombotic disorders, and evaluating bleeding risk control in patients with hemophilia using bypass agents or replacement therapy. Different to conventional coagulation tests, the thrombin generation test can be used for an overall evaluation of hemostasis, the results of which can then be used to evaluate specific characteristics of hemostasis, such as prothrombin time, activated partial thromboplastin time, and levels of fibrinogen and other coagulation factors. The introduction of this method will contribute to a better understanding and evaluation of overall hemostatic processes; however, this method still requires standardization and clinical validation.

https://www.scielo.br/pdf/rbhh/v39n3/1516-8484-rbhh-39-03-0259.pdf

Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations

Currently there are three commercially available thrombin generation methods. These methods help detect the levels of thrombin generated in patient samples by the use of chromogenic or fluorogenic substrates in plasma or whole blood. Determining the rate of thrombin generation can help indicate if patients are at risk of clotting or bleeding. This review discusses two fluorogenic and one chromogenic method and focuses on similarities and differences of these three methods. The review specifically focuses on the accuracy of commercial substrates used in thrombin generation, and interference that can occur by various plasma proteins, as well as on evaluating the advantages and disadvantages of each method. The commercial chromogenic assay and both fluorogenic assays are able to monitor the rate of thrombin generation and can give indications towards potential coagulation abnormalities. Overall, the main differences between the thrombin generation methods are based on the type of substrate used, sample preparation, and data processing. Despite advancement in this field there are still technical challenges that preclude the widespread use of thrombin generation in clinical applications.

/pdf/a-review-of-commercially-available-thrombin-generation-5rq95fndw2.pdf

A review of commercially available thrombin generation assays

Assessment of plasma concentration/effect of edoxaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. It was our aim to determine coagulation tests useful for the assessment of edoxaban's pharmacodynamics and provide recommendations for the interpretation of haemostasis diagnostic tests. Edoxaban was spiked at concentrations ranging from 0 to 1,000 ng/ml in platelet-poor plasma which covers the on-therapy range (from ± 25 ng/ml at Ctrough to ± 170 ng/ml at Cmax). aPTT, PT, dRVVT, chromogenic anti-Xa assays, TGA and a large panel of haemostasis diagnostic tests were performed using several reagents. A concentration-dependent prolongation of aPTT, PT and dRVVT was observed. The effect was dependent on the reagents. FXa chromogenic assays showed high sensitivity and a linear correlation depending on the methodology. TGA may be useful to assess the pharmacodynamics of edoxaban but its turnaround time and the lack of standardisation are limitations. Edoxaban impairs the assessment of lupus anticoagulant, protein S (clotting method), APC-R, antithrombin (FXa-based assay) and measurement of clotting factor activity. Immunological assays and assays acting below the FXa are not influenced by edoxaban. In conclusion, some PT reagents could be used to estimate edoxaban activity. Chromogenic anti-Xa assays are required to assess the plasma concentration. TGA may be useful but requires standardisation. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake and the sampling is mandatory to provide a proper assessment of the result.

/pdf/edoxaban-impact-on-routine-and-specific-coagulation-assays-a-3m7wdngoyb.pdf

Edoxaban: Impact on routine and specific coagulation assays. A practical laboratory guide.

https://archive-ouverte.unige.ch/unige:79661/ATTACHMENT01

Large external quality assessment survey on thrombin generation with CAT: further evidence for the usefulness of normalisation with an external reference plasma

Fanny Mingant

Papers

Large external quality assessment survey on thrombin generation with CAT: further evidence for the usefulness of normalisation with an external reference plasma