The Clauss assay is the most commonly used assay for measuring fibrinogen function in the clinic (Clauss, 1957; Besser & McDonald, 2016). Other traditional functional assays from which fibrinogen function is derived include prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and reptilase time (RT).
The Clauss assay, also known as the von Clauss assay, is the most commonly used assay for measuring fibrinogen function in the clinic, with a turnaround time of 30 to 60 minutes (Huissoud et al., 2009; Solomon et al., 2011). Plasma is typically diluted to 1:10 and mixed with a high concentration of thrombin (usually 100 U/mL), phospholipid and calcium, all at body temperature (37°C). Plasma dilution reduces the effect of plasma factors that negatively impact on clotting efficiency during the reaction. The use of a high thrombin concentration ensures that thrombin does not become a limiting factor in the reaction.
Clot formation is measured by the time it takes for the reaction to achieve either:
The latter is measured through loss of contact between a steel ball and a magnetic sensor, the result of their incorporation into the developing fibrin network (Schlimp et al., 2015). To correlate clotting time to fibrinogen levels, the clotting time is placed on to a calibration curve, created using a dilution range of plasma or standard with known fibrinogen concentrations, measured in g/L, plotted against clotting time. Importantly, fibrinogen concentration is inversely proportional to clotting time.
There are limitations to the Clauss assay. Optical density measurements may give false reads when fibrin monomer polymerisation is slow, however the assay is good for detecting weak fibrin formation. The presence of factors such as bile pigment and free haemoglobin can also impact on optical density readings (Mackie et al., 2003). Mechanical end points are sensitive at a low fibrinogen concentration; however, read-outs can be influenced by the presence of heparin, an anticoagulant drug that may have been administered to patients experiencing cardiac complications.
The prothrombin time-derived fibrinogen assay, or PT-derived fibrinogen assay, is an indirect measure of fibrinogen concentration (Mackie et al., 2003; Undas et al., 2016). The prothrombin time, which has a normal range of 11 to 13.5 seconds (or international normalised ratio, INR, of 0.8–1.1), is used to estimate fibrinogen levels (Chernecky & Berger, 2013). Note that this normal range is dependent on the presence of vitamin K antagonists such as warfarin and phenprocoumon, which if present, produces an average INR range of 2–3. Like the Clauss assay, a calibration curve is generated using a standard plasma of known fibrinogen concentration measured by optical density. The key difference in this assay is the addition of thromboplastin to the reaction instead of thrombin. Thromboplastin converts prothrombin into thrombin which in turn impacts on fibrinogen conversion to fibrin. This assay is therefore an indirect measure of fibrinogen activity and a key limitation is that the results could be due to either low fibrinogen levels/function, or reduced levels of prothrombin.
Similar to the PT-derived fibrinogen assay, the aPTT-derived fibrinogen assay is an indirect measure of fibrinogen activity (Sobas et al., 2002). For the aPTT assay, platelet poor plasma that has undergone chelation to remove calcium is incubated at 37°C with phospholipid (cephalin), a contact activator (such as kaolin or micronised silica), and calcium in molar excess. Clotting time is measured from the addition of calcium and the aPTT is the time taken to form a fibrin clot with a normal range of 30–40 seconds, measured using optical density. The patient aPTT is used against a fibrinogen standard for estimating fibrinogen levels.
Other methods that can give an indication of fibrinogen activity include thrombin time (TT) and reptilase time (RT). Similar to the PT-derived fibrinogen assay, thrombin time is the time it takes for a plasma sample to create a clot when thrombin is added to the reaction, with a normal range of between 12 to 14 seconds (Hoffbrand, 2002; Lillicrap et al., 2009). Prolonged clotting time equates to either a quantitative or qualitative defect in fibrinogen (Hatton, 2008). This assay is particularly sensitive to the presence of heparin. If heparin is present, reptilase is instead added to the reaction in the place of thrombin, effectively giving a readout of reptilase time (RT), the normal range for which is 15 to 20 seconds (Lillicrap et al., 2009).
It is important to note that tests which focus solely on measuring fibrinogen function provide only a fraction of information on the overall haemostatic condition of a patient. To get an overall picture of coagulopathy in the clinic, particularly for cases of acquired fibrinogen deficiency, multiple tests may be needed in conjunction with fibrinogen testing. Decisions on which tests are needed should be influenced by the individual patient circumstance and their comorbidities.
Learn about point-of-care viscoelastic devices such as rotational thromboelastometry (ROTEM) and thromboeslastography (TEG) that can provide a better overview of coagulation, including fibrinogen function and fibrinolysis.
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