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A Practical Guide to Haemostasis


Fibrinogen Assays

Introduction

Fibrinogen defects may be quantitative (hypo- or hyper-fibrinogenaemia) or qualitative (dysfibrinogenaemia). Inherited dysfibrinogenaemia is rare but an acquired defect of Fibrinogen function is more common, especially in liver disease when the Fibrinogen molecule is excessively glycosylated impairing its activity. Fibrinogen levels may also be reduced in liver disease due to reduced synthesis. Elevated levels of fibrin degradation products (FDPs) also impair the conversion of Fibrinogen to fibrin.
Fibrinogen levels are an important part of the investigation of a bleeding tendency or an unexplained prolongation of the APTT or PT. Elevated Fibrinogen levels may correlate with an increased risk of thrombosis in epidemiological studies although the significance in individual patients is unclear.

The structure of Fibrinogen is shown below:


Fibrinogen consists of three pairs of polypeptide chains: two Aα, two Bβ and two γ.  These are linked together by 29 disulphide bonds in such a way that N-terminal regions of the 6-polypeptide chains meet to form a central E-domain. The C-terminal regions [Aα, Bβ and γ] form the D-domain and these are joined by α-helical ropes to the central E-domain to give the characteristic Fibrinogen structure.


Activation of Fibrinogen by Thrombin [Factor IIa] cleaves two short peptides from the N-terminal regions of the Aα and Bβ chains - these peptides are known as Fibrinopeptide A [FpA] and Fibrinopeptide B [FpB] respectively.  Removal of the N-terminal sequences from Aα and Bβ chains chains reveals new N-terminal sequences in the Aα and Bβ chains located within the E domain, known as 'knobs.'  Thrombin cleaves FpA from the Fibrinogen E domain at Arg16-Gly17. These knobs can interact spontaneously with the D-dimer regions to form fibrin polymers.  Under the influence of Factor XIIIa, cross-linking of these fibrin polymers then occurs to form cross-linked fibrin polymers.

Principles

There are a number of assay for measuring Fibrinogen levels in plasma [see References] although in practice most laboratories use the Clauss method.

Fibrinogen Assays

Assays
Clauss Assay A functional assay based upon the time for fibrin clot formation
PT-derived Fibrinogen Assay A functional assay based upon the time for fibrin clot formation
Immunological Assay An immunological method which measures Fibrinogen antigen rather than functional Fibrinogen
Gravimetric Assay A method based upon clot weight [rarely preformed today]
TEG & ROTEM The TEG and ROTEM machines can also be used to provide a qualitative indication of the Fibrinogen concentration.


The diagram below illustrates the Clauss method for measuring Fibrinogen. Cross-linking of Fibrin by Factor XIIIa is not required for the Clauss Fibrinogen assay and so the Fibrinogen levels will be normal in severe Factor XIII deficiency. 

Clauss Fibrinogen Assay Schematic


Method

The methods for measuring Fibrinogen are summarised below:

Assays
Clauss Assay Diluted plasma is clotted with a high concentration of thrombin.

1. The test 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 100 U/ml) ensures that the clotting times are independent of thrombin concentration over a wide range of Fibrinogen levels.

2. The test requires a reference plasma with a known Fibrinogen concentration and that has been calibrated against a known international reference 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.

3. The test platelet poor diluted plasma (diluted 1:10 in buffer) is incubated at 37C, thrombin added (all pre-warmed to 37C). The time taken for the clot to form is compared to the 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.
PT-derived Fibrinogen Assay 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.
Immunological Assay 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.
Gravimetric Assay 1. Clot Weight
Similar to the Clauss method : A Fibrinogen clot is formed by the addition of thrombin and calcium to diluted 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, time consuming and rarely performed in most modern laboratories.

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 fibrin and so the measured protein concentration is taken as equivalent to the Fibrinogen concentration.
TEG & ROTEM The TEG and ROTEM machines can also be used to provide a qualitative indication of Fibrinogen concentration.


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

Assays
Investigation of an inherited bleeding disorder Clauss [& Clottable Protein & Immunoassay]
Investigation of an acquired bleeding disorder Clauss
Suspected dysfibrinogenaemia Clauss & Clottable Protein & Immunoassay
Bleeding disorders affecting factors in addition to Fibrinogen (e.g. DIC) Clauss
Thrombolytic therapy Clauss
Hyperfibrinogenaemia Clauss or Immunoassay


Interpretation

Fibrinogen Level Interpretation
Fibrinogen levels are reduced in: DIC due to 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
Inherited deficiencies e.g. Hypofibrinogenaemia, afibrinogenaemia and dysfibrinogenaemia [the latter is often associated with reduced Fibrinogen levels as well as activity]
Following thrombolytic therapy
In some patients following treatment with asparaginase

Primary hyperfibrinolysis.  This has been reported in individuals with carcinoma of the prostate and other cancers.
Fibrinogen levels are increased in: Increasing age
Female sex, pregnancy, oral contraception
In post-menopausal women
Acute phase reaction
Disseminated malignancy [but may also be decreased if this is associated with DIC]

 

Reference Ranges

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

What Test Next

The Fibrinogen level is usually interpreted in the light of other clotting tests such as the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).
i. If the Clauss Fibrinogen assay is significantly reduced, both the APTT and PT will be prolonged. Although this indicates a hypofibrinogenaemia it does not exclude additional defects in the coagulation cascade such as may be found in Disseminated Intravascular Coagulation [DIC].
ii. 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 with normal plasma may be helpful.
iii. If the Clauss Fibrinogen assay suggests a reduced Fibrinogen concentration 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 to establish if the patient has a hypo- or hypo-dysfibrinogenaemia. 

Any individual with a suspected inherited Fibrinogen deficiency should have molecular studies undertaken to characterise a potential genetic abnormality.