The holy grail of coagulation testing should mimic in vivo clot formation—the interplay between blood cells, coagulation factors, and related proteins, platelets, endothelium, blood flow, pH, and temperature. Additionally, it should be easy-to-use, accurate, reliable, and ultimately predict bleeding or thrombosis. For now, global coagulation tests only fulfill some of these criteria. While viscoelastic tests are best known, there is more than just ROTEM and TEG.

Viscoelastic Testing

Viscoelastometry has evolved over 70 years into the leading global coagulation test in human and veterinary medicine. A pin-and-cup model measures the changing viscous and elastic properties of activated whole blood to produce a graphical representation of coagulation factor activation, thrombin generation, platelet aggregation, and fibrinolysis as a function of time. Additional algorithms offer validated measurements of clot strength. TEG and ROTEM are most widely available compared with the Sonoclot analyser, and the more recent VCM point-of-care machine has also been validated for veterinary use.

Performance

A large multi-centre diagnostic study evaluated identical samples in 10 academic institutions and found considerable variation within and between centres for most results produced by both TEG and ROTEM, reflecting similar findings in human hospitals. This highlights the importance of the PROVETS guidelines—evidence-based recommendations on viscoelastic testing aiming to standardise the use of these devices and improve their performance in clinical and research settings. This includes the importance of establishing institutional reference intervals (RI) following published guidelines.

Clinical Use

Studies can be broadly categorised into healthy animal studies, observational studies in sick animals, and exploratory or interventional studies evaluating hypo- or hypercoagulability in sick animals, with or without control populations. Thus far, these studies have explored viscoelastic testing to measure the effect of fluids on coagulation, to describe coagulation profiles in infectious diseases, and to identify hypercoagulable and hyperfibrinolytic patients in disease states such as hypoproteinaemia and trauma. Ongoing research must evaluate further its utility in hypercoagulable and hyperfibrinolytic patients.

Sonorheometry

Researchers developed this point-of-care viscoelastic device to address some of the shortfalls of existing devices, including operator variability, large shear forces that may alter viscoelastic properties of nascent clots, and the limitations of relying on clot amplitude to measure viscoelastic clot strength. Upon connecting a syringe of whole blood, a fully automated system warms the sample, distributes to the operating channels, adds reagents, administers ultrasound pulses, and records reflected sound waves to create shear modulus curves all within 15 minutes. This has recently been FDA approved.

Turbidimetric Assays

Turbidimetric assays measure the change in light absorbance (or transmission) over time during clot formation. The resulting waveforms provide information on initiation of clot formation, rate and strength of clot formation, and fibrinolysis (i.e. global coagulation). Currently, these devices demand platelet-poor plasma (PPP) samples with minimal turbidity to maximise photo-optic performance. The contribution of the endothelium, flow, and blood cells are not evaluated.

There are several variations. Overall hemostatic potential (OHP) and clot formation and lysis (CloFAL) assays use standard assay microplates filled with reagents plus control or patient samples, from which light optics are measured using standard spectrophotometers. Samples are activated by thrombin, tissue plasminogen activator (tPA), or tissue factor. For OHP, thrombin or thrombin plus tPA are added to buffered PPP samples to produce absorbance curves; calculating the area under the curve equals overall coagulation potential or OHP, respectively. Subsequently, overall fibrinolysis potential can be calculated (OFP = [OCP – OHP]/OCP). Clot lysis time, fibrin lag time, maximum slope, and maximum/minimum optical density can also be calculated.

For clot waveform analysis (CWA), certain PT/aPTT devices that measure the change over time in light absorbance/transmittance can produce clot waveforms: change in light optics represents clot strength; the first derivative of this curve represents clot formation velocity, including the maximum velocity; and the second derivative represents clot formation acceleration, including maximum acceleration. These results are described in CWA studies as pre-coagulation, coagulation, and post-coagulation phases. Although, theoretically, any raw data output from PT/aPTT testing can be used to produce these measurements, there are very few devices for which capable software/algorithms are available.

Performance

There are limited veterinary studies involving these devices. One study created OHP RIs in healthy dogs, starting with human protocols. Modifications were required and resulted in assay coefficients of variation >10% for several parameters, poor correlation with aPTT, strong negative correlation with PT, and strong correlation with fibrinogen levels. All recent veterinary CWA studies used the ACL-TOP turbidimetric analyser to produce single institution RIs, but no intra- and interassay variability data. These authors noted that abnormal sample turbidity (such as lipaemia or haemolysis) altered curves but not the end results.

Clinical Use

One small study in 10 privately-owned dogs with thrombosis showed significant changes in OHP parameters compared with healthy dogs, although some values remained within normal. In a retrospective study in dogs with DIC (excluding dogs exposed to antithrombotics or those with icteric or lipaemic samples or unreadable waveforms), CWA demonstrated significant differences between bleeders and non-bleeders compared with traditional PT/aPTT results. But most values remained within normal and there was large overlap between groups.

Simultaneous Thrombin and Plasmin Generation Assays

Also using assay microplates and spectrophotometry, simultaneous thrombin and plasmin generation assays (STP) track fluorescence markers incorporated into the substrates added to microplate wells containing PPP, TF, phospholipids, tPA, and either of the fluorescing substrates specific for thrombin or plasmin. Increasing fluorescence indicates increasing thrombin or plasmin generation, represented graphically to allow measurement of thrombin generation lag time, peak thrombin quantity and time, plasmin peak, fibrinolysis time, and AUC values. A newer variant performs the thrombin and plasmin measurements from the same well. Recordings are taken for up to 4 hours. No veterinary studies have evaluated this technology; however, fibrinolysis has been evaluated using a similar microplate assay.

Performance

The first STP study created paediatric and adult RIs—paediatric values were significantly different, veering towards a more hypocoagulable profile. Like turbidimetric assays, thrombin lag time had unacceptable inter-assay variability (CV>10%), as did velocity measurements, but the remainder were acceptable.

Clinical Use

An ex vivo study found significant differences in all STP assays in samples taken from human patients with factor deficiencies; those with major bleeding diatheses also exhibited significantly lower thrombin and plasmin potential. Authors also reported changes specific to the different bleeding disorders involved.

The Future

Much research focuses on developing a ‘gold standard’ test of global coagulation. Advances within viscoelastic technology include laser speckle rheometry that tracks Brownian motion to measure coagulation, ultrasonic waves used to levitate a drop of blood and quantify viscoelasticity-related shape deformation, and a ROTEM/TEG-like device with prefilled reagents that may be better suited to point-of-care use.

Microfluidics encompasses flow chamber devices that permit controlled blood flow under pressure to multiple channels using very small blood volumes. Different channels within a single cartridge can measure different components of global coagulation by means of altering reagents, coatings, and of course, flow. Some research models aim to create channels that mimic the endothelium. Modelling thrombosis in large vessels is a major limitation.

Additional technologies currently being investigated include video microscopic tracking of clot formation (Thrombodynamics), and high- and low-shear point-of-care devices (global thrombosis test). These are unlikely to be used in veterinary settings in the near future.

Conclusion

A device that measures coagulation under in vivo-like conditions rests in the realm of science fiction. Current devices perform admirably to provide clinicians with meaningful measures of hemostasis but have numerous limitations. Best practice requires a good understanding of their mechanism of action and current literature.

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