Point-of-care functional assays

Viscoelastic devices such as rotational thromboelastometry (ROTEM) and thromboeslastography (TEG) measure overall coagulation ability by indicating clotting time, time to maximum clot strength and clot degradation (fibrinolysis) (Peng et al., 2018). They also provide a sensitive readout for the diagnosis of hyperfibrinolysis compared to other techniques such as immunochemical techniques that lack total biomarker specificity.

Since whole bloods can be tested quickly and in real-time with TEG and ROTEM devices, they are increasingly being used as point-of-care tools at the bedside for directing treatment choices made during surgical procedures (Peng et al., 2018). A study on perioperative samples taken from major paediatric surgery patients indicated no significant difference in readings for ROTEM testing carried out at the bedside versus in a laboratory setting, however bedside testing saved an average of 11 minutes compared to laboratory-based ROTEM testing (p<0.001) (Haas et al., 2012). Further, various studies covering cardiac surgery, trauma, postpartum haemorrhage (PPH) and liver transplantation have indicated that point-of-care coagulation testing leads to a reduced need for transfusions, reduced mortality and increased cost-effectiveness (Görlinger et al., 2011; Schöchl et al., 2011; Weber et al., 2012; Kirchner et al., 2014; Spahn et al., 2014; Leon-Justel et al., 2015; Mallaiah et al., 2015; Nardi et al., 2015; Roullet et al., 2015; Whiting et al., 2015; Solomon et al., 2016).

Despite recognition of the potential for the use of viscoelastic devices in the clinic, earlier limitations included ease of use as well as variability and accuracy concerns that were based on limited clinical data (Chitlur et al., 2011, Spahn et al., 2013). Full automation as well as more recent studies indicating improved reliability means that these devices, particularly ROTEM, are increasingly used to guide treatment decisions during medical procedures involving cardiac surgery, liver transplantation and PPH. To reflect this progress, the European Society of Anaesthesiology recommends viscoelastic assays for monitoring coagulation status in cases of peri-operative bleeding (Kozek-Langenecker et al., 2017). The 2019 European guidelines on management of major bleeding and coagulopathy following trauma recommends that resuscitation measures be continued using a goal-directed strategy, guided by the standard laboratory coagulation values and/or viscoelastic methods (Spahn et al., 2019).

Rotational thromboelastometry (ROTEM)

Rotational thromboelastometry (ROTEM) plots multiple aspects of the clotting cascade on to a trace in real-time. Initial indications of clot formation can be read as soon as 7 to 10 minutes following sampling, with full qualitative results available within 20 minutes (Collins et al., 2014). For the reaction, blood is mixed with reagents in a disposable container that has an oscillating sensor pin which detects changes in resistance as the reaction progresses. Greater resistance correlates with clot firmness. The changes in resistance are plotted on to a trace, the shape of which is used to determine readouts for both clot formation and degradation (Figure 2). ROTEM offers multiple channels for reactions that analyse different coagulation components (Crochemore et al., 2017):  

  1. INTEM: activation of intrinsic pathway (contact phase) using ellagic acid (evaluates factors XII, XI, IX, VIII, X, V, II, I and von Willebrand)
  2. EXTEM: activation of extrinsic pathway using thromboplastin or tissue factor (evaluates factors II, VII, IX, X)
  3. HEPTEM: like INTEM but with neutralisation of heparin, using heparinase
  4. FIBTEM (fibrin-based extrinsically activated test): like EXTEM but with addition of cytochalasin D to inhibit platelet function, allowing for the analysis of fibrinogen on clot formation
  5. APTEM: activation like EXTEM but with addition of aprotinin to inhibit fibrinolysis. APTEM relative to EXTEM gives a true indication of hyperfibrinolysis

The resulting ROTEM trace provides the following values (Figure 2):

  • CT (clotting time): time from mixing reagents to clot initiation detected as 2 mm amplitude
  • CFT (clot formation time): time from CT to clot firmness of 20 mm amplitude
  • alpha angle (α-angle): indication of how fast clot is forming
  • A5/A10/A20: amplitude 5/10/20 minutes following CT
  • MCF (maximum clot firmness): the maximum amplitude measured on trace
  • LI30/LI60: clot lysis index 30/60 minutes following CT
  • ML (maximum lysis): fibrinolysis is measured by ML > 15%
Features of plots for rotational thromboelastometry, ROTEM (top half) and thromboelastography, TEG (bottom half)

Figure 11. Features of plots for rotational thromboelastometry, ROTEM (top half) and thromboelastography, TEG (bottom half) (Adapted from Winearls et al., 2016).
For ROTEM: CT, clotting time; CFT, clot formation time; MCF, maximum clot firmness; LI60, lysis index 60 minutes following CT; ML, maximum lysis. For TEG: R, reaction time; K, clot formation time; MA, maximum amplitude; LY30, percentage lysis 30 minutes following maximum amplitude.

The FIBTEM and the EXTEM/APTEM ratio give an indication of fibrinogen deficiency and hyperfibrinolysis, respectively (Figure 3). The FIBTEM protocol assesses the impact of fibrinogen on clot formation with a readout of maximum clot firmness (MCF). In this reaction, a platelet inhibitor (cytocholasin D) is added to the reaction to remove the effect of platelet variation on the MCF readout (Bolliger et al., 2012). Correlation studies indicate that MCF readouts correlate positively with Clauss measurements for fibrinogen levels during cardiac surgery (Mace et al., 2016; Tirotta et al., 2019; ) liver transplantation (Roullet et al., 2010; Song et al., 2014; Hashir et al., 2019) and postpartum haemorrhage (Huissoud et al., 2009; Solomon et al., 2012; van Rheenan-Flach et al., 2013; Collins et al., 2014; Gillisen et al., 2019).

ROTEM and TEG traces for normal patient, patient with fibrinogen deficiency or fibrinolysis

Figure 12. ROTEM and TEG traces for normal patient, patient with fibrinogen deficiency or fibrinolysis. (Adapted from Zostautiene et al., 2017; ROTEM, 2019).

The ROTEM EXTEM and APTEM protocols were found to be comparable to the standard euglobulin lysis time in accurately indicating ongoing hyperfibrinolysis in less than 15 minutes (Levrat et al., 2008). The APTEM reaction uses aprotinin to inhibit fibrinolysis whereas the EXTEM reaction does not. EXTEM viewed in the context of APTEM allows for accurate assessment of changes in EXTEM that can be attributed to hyperfibrinolysis. Roullet et al concluded that ROTEM EXTEM was useful for understanding overall coagulation during liver transplantation, and that the A10 value could guide platelet and fibrinogen transfusion (Roullet et al., 2010).

Despite the promise of these devices, some studies have highlighted both variability and accuracy issues in some contexts (Hunt et al., 2015; Seo et al. 2015; Blasi et al., 2017). For instance, Blasi et al indicate that FIBTEM MCF is not a good indicator of plasma fibrinogen after graft reperfusion during liver transplantation (Blasi et al., 2017). Whilst there have been recent improvements in the devices, such as full automation to remove user variability, more studies are required to understand the full potential for ROTEM in assessing fibrinogen and hyperfibrinolysis in specific clinical contexts.

Thromboelastography (TEG)

Similar to ROTEM, a thromboelastograph (TEG) depicts clot time, clot formation, clot stability and clot degradation, effectively plotting the interaction between fibrinogen, platelets and other clotting factors (Curry & Pierce, 2007; Hunt et al., 2015) (Figure 2). The functional fibrinogen (FF) assay of TEG measures the stability of polymerised fibrin. Platelets also contribute to clot strength and is a factor that varies depending on the individual patient situations. For this reason, the FF reaction is treated with the glycoprotein IIb/IIIa platelet inhibitor abciximab to eliminate the effect of platelet variation on the FF readout.

ROTEM is based on TEG, however unlike ROTEM which uses an optical detector, a torsion wire is used to measure clot formation in TEG. TEG also initiates movement through rotation of the cuvette, rather than the pin. The interpretation for ROTEM and TEG results is similar but they are not interchangeable, and there is evidence that they can yield differing results, particularly for fibrin clotting ability (Solomon et al., 2012; Rizoli et al., 2016) (Table 1). The outcome for TEG is a trace that is directly related to clot strength and allows for the assessment of different coagulation states; however, the nomenclature is somewhat different to that of ROTEM.

  • R value (reaction time): The time taken from start until TEG amplitude reaches 2 mm, which corresponds to fibrin formation. Note that this time is prolonged in severe hypofibrinogenaemia.
  • K value (clot formation time): The time taken from R-time until TEG amplitude reaches 20 mm, which corresponds to clot firmness.  Note that this time is prolonged in cases of hypofibrinogenaemia.
  • α-angle: Angle formed between the middle of the trace and a line drawn between the R and K-values. This is a function of the rate of fibrin polymerisation and is decreased in hypofibrinogenaemia.
  • MA (maximum amplitude): Highest amplitude achieved which is representative of maximum clot strength. This is decreased in the case of hyperfibrinolysis.
  • LY30/A30: a measurement of the percentage lysis 30 minutes following the maximum amplitude.
Reference values for rotational thromboelastometry, ROTEM and thromboelastography, TEG

Table 1: Reference values for rotational thromboelastometry, ROTEM and thromboelastography, TEG (adapted from Walsh et al., 2011; ROTEM, 2019). 

TEG MA readings for fibrinogen have been positively correlated to fibrinogen levels measured using the standard Clauss assay in a trauma (Harr et al., 2013; Kornblith et al., 2014; Meyer et al., 2014; Meyer et al., 2015; Peng et al., 2018) and liver transplantation setting (Yang et al., 2014). For cardiac surgery patients, the correlation is less clear. Some studies indicate a moderate correlation between TEG MA and Clauss fibrinogen levels (Fluger et al., 2011; Fluger et al., 2012; Gautam et al., 2017) whereas at least one study indicated that there is no correlation (Agarwal et al., 2015).

Whilst functional fibrinogen assays provide an indication of how much active fibrinogen is present in a patient sample, quantitative fibrinogen assays provide a better indication of the levels of fibrinogen present. 

Other point-of-care devices

The latest point-of-care devices are being designed with the emergency setting in mind. One such device is the HemoSonics Quantra® which uses novel patented ultrasound technology (sonic estimation of elasticity via resonance, SEER) to measure clot stiffness. This new technology means that readings supposedly remain accurate even in the presence of vibration. The device is small, performs automatic quality checks every 8 hours, and uses a blood collection cartridge that negates the need for sample handling. New devices such as the HemoSonics Quantra® also aim to minimise decision-making and simplify display, both time-saving measures.

Learn about traditional functional assays such as the Clauss assay and the PT-derived assay here. For more on quantitative fibrinogen assays, such as ELISA and clottable protein assay that are used to confirm congenital fibrinogen deficiencies, click here.

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