Fibrinogen Assays

Fibrinogen defects may be quantitative (hypo- or hyper-fibrinogenaemia) or qualitative (dysfibrinogenaemia). Inherited dysfibrinogenaemia is rare with only 250-300 patients reported worldwide but an acquired defect of fibrinogen function is more common, especially in liver disease when the fibrinogen molecule is excessively glycosylated impairing its activity. Elevated levels of fibrin degradation products (FDPs) also impair the action of fibrinogen.

Fibrinogen levels are a useful as part of the investigation of a bleeding tendency or an unexplained prolongation of the APTT or PT. Elevated levels may correlate with increased risk of thrombosis in epidemiological studies although the significance in individual patients is unclear.

 

Principles

There are four methods for measuring fibrinogen levels although in practice most laboratories use the Clauss method.

1. Clauss method A functional assay based upon the time for fibrin clot formation
2. PT-based derived fibrinogen A derived fibrinogen based upon the prothrombin time
3. Immunological An immunological method which measures fibrinogen antigen rather than functional fibrinogen
4. Gravimetric assays A method based upon clot weight

The diagram illustrates the Clauss and PT-derived fibrinogen assays.

 

Method

The four methods for measuring fibrinogen are summarised below:

1. Clauss Method

 Diluted plasma is clotted with a high concentration of thrombin. The plasma is diluted (usually 1:10 but this may vary if the fibrinogen concentration is very low or very high) to minimise the effect of 'inhibitory substances' within the plasma e.g. heparin, elevated levels of FDPs.

The use of a high concentration of thrombin (typically 100U/ml) ensures that the clotting times are independent of thrombin concentration over a wide range of fibrinogen levels.

The test requires a reference plasma with a known level of fibrinogen calibrated against a known international standard. A calibration curve is constructed using this reference plasma by preparing a series of dilutions (1:5 –1:40) in buffer to give a range of fibrinogen concentrations. The clotting time of each of these dilutions is established (using duplicate samples) and the results (clotting time(s)/fibrinogen concentration (g/L) are plotted on log-log graph paper. The 1:10 concentration is considered to be 100% i.e. normal. There should be a linear correlation between clotting times in the region of 10-50s.

The test platelet poor diluted plasma (diluted 1:10 in buffer) is incubated at 37°C, phospholipid and thrombin are added followed by calcium (all pre-warmed to 37°C). On the addition of the calcium timing begins. The time taken for the clot to form is compared to a calibration curve and the fibrinogen concentration deduced.

Most laboratories use an automated method in which clot formation is deemed to have occurred when the optical density of the mixture has exceeded a certain threshold.

2. PT-based Derived Fibrinogen Assays

The PT is determined by optical density change for a range of plasma dilutions with known fibrinogen levels. The optical change for each different fibrinogen level is plotted as a calibration curve. A PT is performed on the patient’s platelet poor plasma and the fibrinogen derived from the change in optical density compared to the calibration curve.

The derived fibrinogen is a simple and inexpensive test and is widely used. However, the test can give misleading results in some disorders and is not recommended for routine laboratory use.

3. Immunological Fibrinogen Assays

 Assays based on enzyme linked immunoabsorbant assays (ELISA), radial immunodiffusion and electrophoresis are the most commonly employed.
Immunological assays measure protein concentration rather than functional activity. 

They are of value in the investigation of congenital dysfibrinogenaemias where there is a discrepancy between functional activity and antigen level.

4. Gravimetric Assays

1. Clot Weight
Similar to the Clauss method a fibrinogen clot is formed by the addition of thrombin excess and calcium to dilute patient plasma. However, instead of using the time to clot formation to derive the fibrinogen the clot is compressed (to extrude plasma and unused reagents), washed, dried then weighed. This assay is technically difficult and time consuming.

2. Clottable protein
Thrombin is added to plasma without calcium and the clot formed is washed then dissolved in an alkaline reagent then spectrophotometry is performed (e.g. typically absorbance at 282nm). The clot is almost all fibrinogen and so the measured protein concentration is taken as equivalent to the fibrinogen concentration.

 

 

Interpretation

1. Fibrinogen levels are reduced in:

  • DIC due the the consumption of clotting factors

  • Liver disease due to decreased synthesis. An abnormal fibrinogen may be also be found in patients with liver disease due to an abnormal (increased) sialic acid content

  • Massive transfusion leading to a dilutional coagulopathy

  • Hypofibrinogenaemia, afibrinogenaemia and dysfibrinogenaemia

  • Following thrombolytic therapy

  • In some patients following treatment with asparaginase

2. Fibrinogen levels are increased in:

  • Increasing age

  • Female sex, pregnancy, oral contraception

  • In post-menopausal women

  • Acute phase reaction

  • Disseminated malignancy

 

 

Reference Ranges

The reference range for fibrinogen is generally between 1.5-4.0g/L.

 

Comments

  1. If an optical density method is used to monitor clot formation, falsely low readings may occur if the patient’s serum is turbid (e.g. hyperbilirubinaemia, hyperlipidaemia).

  2. Where possible assays should not be performed on samples collected with 4 hours of the administration of therapeutic doses of unfractionated heparin or on samples collected from heparin-contaminated venous or arterial l

  3. High concentrations of unfractionated heparin (>0.8 IU/ml) may lead lead to an underestimate of the true fibrinogen level.

  4. When both immunological and functional fibrinogen assays are performed the same standard should be used to calibrate them to ensure comparability of results.

  5. The derived fibrinogen based upon the PT is relatively easy and inexpensive to perform, and therefore, this technique is popular. However, it is highly dependent on the reagents and analyser used and so results are not readily comparable between laboratories or over time if a laboratory upgrades its instruments or changes reagents. The literature suggests that this method may  also overestimate fibrinogen levels in certain circumstances such as liver failure or DIC. Current UK guidelines recommend that it is not used for fibrinogen estimation in routine haematology practice.

  6. The fibrinogen titre is a rarely used test in which plasma (and therefore fibrinogen is serially diluted in buffer before the addition of thrombin. The titre is reported as the last dilution to display a clot after a given time. The test is time consuming and inaccurate and not recommended.

 

The currently recommended choices of fibrinogen assay for different clinical circumstances are shown below:

Investigation of bleeding

Clauss

Suspected dysfibrinogenaemia

Clauss and clottable protein and immunoassay

Bleeding disorders affecting factors other than the fibrinogen (e.g. DIC)

Clauss

Thrombolytic therapy

Clauss

Very high fibrinogen levels

Clauss or immunoassay

 

What Test Next?

  1. The fibrinogen level is usually interpreted in the light of other clotting tests such as the prothrombin time (PT) and activated thromboplastin time (APTT). If the clotting based fibrinogen assay is significantly reduced both APTT and PT will be prolonged, however, although this indicates hypofibrinogenaemia it does not exclude additional defects in the coagulation cascade such as may be found in disseminated intravascular coagulation [DIC].

  2. Conversely, if the APTT and PT are prolonged but the clotting based fibrinogen assay is normal it suggests a defect higher up the clotting cascade and individual factor assays or a 50:50 mix may be helpful.

  3. If the clotting based fibrinogen assay suggests reduced fibrinogen but there is no obvious reason for this and there is an appropriate clinical context (e.g. family history of bleeding diathesis, poor wound healing, umbilical stump bleeding) it may be useful to perform an immunological fibrinogen assay.

 

Useful Links & References

1. Inherited abnormalities of Fibrinogen:
www.emedicine.com/ped/topic3042.htm.

2. Guidelines on Fibrinogen assays
Mackie et al. Guidelines on fibrinogen assays. Br J Haematol 2003: 121 (3):396-404 or www.bcshguidelines.com/pdf/fibrinogenassays0503.pdf.

3. Hill et al. Diagnosis, clinical features and molecular assessment of the dysfibrinogenaemias. Haemophilia 2008;14:889-897.

4. Hanss, M. and F. Biot, A database for human fibrinogen variants. Ann N Y Acad Sci, 2001. 936: p. 89-90.

5. Bolton-Maggs, P.H., et al., The rare coagulation disorders--review with guidelines for management from the United Kingdom Haemophilia Centre Doctors' Organisation. Haemophilia, 2004. 10(5): p. 593-628.

6. Peyvandi, F., et al., Rare coagulation deficiencies. Haemophilia, 2002. 8(3): p. 308-21.

7. Gandrille, S., et al., A study of fibrinogen and fibrinolysis in 10 adults with nephrotic syndrome. Thromb Haemost, 1988. 59(3): p. 445-50.

8. Henschen, A.H., Human fibrinogen--structural variants and functional sites. Thromb Haemost, 1993. 70(1): p. 42-7.

9. Toulon, P., et al., Fibrin polymerization defect in HIV-infected patients--evidence for a critical role of albumin in the prolongation of thrombin and reptilase clotting times. Thromb Haemost, 1995. 73(3): p. 349-55.

10. Krammer, B., et al., Screening of dysfibrinogenaemia using the fibrinogen function versus antigen concentration ratio. Thromb Res, 1994. 76(6): p. 577-9.

 

Data Interpretation

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