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Fibrinogen Deficiency
Fibrinogen Deficiency in Bleeding


Read time: 15 mins
Last updated:31st Oct 2023
Published:29th Jan 2020

The outcome is a reduced ability to form blood clots which can lead to excessive bleeding if left untreated. Indeed, low fibrinogen levels have been correlated to excessive blood loss during trauma, surgery and to the severity of postpartum haemorrhage, both of which present an increased risk of mortality (Frith et al., 2010; Rainer et al., 2011; Cortet et al., 2012; Gielen et al., 2014; Walden et al., 2014; Liu et al., 2018). Fibrinogen replenishment has been shown to improve outcome and therefore early assessment of fibrinogen levels and activity should be a key consideration for perioperative management (Mallaiah et al., 2015; Matsunaga et al., 2017; Li et al., 2018).

Fibrinogen replenishment may prevent excessive bleeding and save lives. Early assessment of fibrinogen deficiency using reliable and rapid diagnostic tests should therefore be at the forefront of perioperative management.


Figure 10. Quantitative and functional assays for measuring fibrinogen levels and activity. ELISA, enzyme-linked immunosorbent assay; FF, functional fibrinogen; FIBTEM, fibrin-based extrinsically activated test; PT, prothrombin time; ROTEM, rotational thromboelastometry; TEG, thromboelastography.

In this section, we discuss and compare the available diagnostic tools for measuring fibrinogen levels (quantitative assays) and quality (functional assays) (Figure 1). We also highlight recent data on the increasing use and reliability of point-of-care viscoelastic functional assays such as rotational thromboelastometry (ROTEM) and thromboelastography (TEG).

Traditional functional assays

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).

Clauss Assay

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:

  1. an optical density threshold, or
  2. a mechanical end point that measures the tensile strength of the clot

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.

Prothrombin time (PT)-derived fibrinogen assay

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.

Activated partial thromboplastin time (aPTT)-derived fibrinogen assay

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.   

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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%


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).


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

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Quantitative fibrinogen assays

Quantitative measurement of fibrinogen can be carried out using various immunological assays and the clottable protein assay (Mackie et al., 2003). It is important to note that whilst these assays give an indication of how much fibrinogen is present, they do not give an indication of the functional activity of fibrinogen. They are therefore used alongside functional assays to determine fibrinogen levels and activity, and are most often used to confirm suspected cases of congenital fibrinogen deficiency alongside genetic analysis.

Immunological assays

Immunological assays allow for quantitative measurements since they detect antigen, however it is not possible to distinguish between functional and non-functional protein. They are also time consuming and can take many hours to complete. They are therefore not regularly used to diagnose fibrinogen deficiency in acute settings; however, they are often used to confirm congenital fibrinogen deficiency. The enzyme-linked immunosorbent assay (ELISA) is the most accurate and widely used immunological assay compared to other techniques such as electrophoretic techniques, radial immunodiffusion, rapid latex agglutination, immune turbidimetry and nephelometry (Mackie et al., 2003; Chen et al., 2010). Polyclonal antibodies are generally utilised for these assays to ensure full coverage of fibrinogen protein; however, monoclonal antibodies that are specific to non-proteolysed fibrinogen are also available. The latter allows for the identification of fibrinogen that has not been processed through plasmin digestion and gives a better indication of functional fibrinogen levels.

Clottable protein assay

A clottable protein assay can be used to determine clot weight. In this assay, thrombin is added to patient sample plasma in the absence of calcium ions. The resulting clot is washed and treated with alkaline urea for protein measurement by spectrophotometry (Ratnoff & Menzie, 1951; Jacobsson et al., 1955, Blomback & Blomback, 1956). Since fibrin is the only protein in the clot structure, the protein concentration achieved is related directly to the amount of fibrin present in clots. This diagnostic test is therefore highly accurate and is often used to confirm congenital fibrinogen deficiencies; however, it is not used as a standard diagnostic test since it is a time-consuming and laborious technique (Mackie et al., 2003).

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