Guidelines on fibrinogen assays

Abstract

Haematology departments in the UK have traditionally performed fibrinogen assays to detect decreased levels and abnormalities of fibrinogen, and to assess haemorrhagic risk. It has also been shown that elevated fibrinogen levels are a predictor of a variety of arterial cardiovascular events, and fibrinogen assays are sometimes recommended with this in mind. The Clauss fibrinogen assay (based on the thrombin clotting time) is the most popular technique in UK hospital laboratories, although many other methods are also in use. There appears to be great variability in both the source of reagents and the exact method used for the Clauss assay. Most laboratories are now equipped with automated coagulation analysers, and many of these perform a fibrinogen estimation derived from the degree of change of light scatter or optical density during the prothrombin time (PT-Fg). A number of problems have been described in the use of the PT-Fg method; it generally gives higher values than the Clauss technique, but the exact degree of discrepancy seems to depend on a number of different variables. International and National standards are available for fibrinogen, but do not appear to be universally used. These guidelines have been prepared against this background and recommend which methods should be used in various clinical settings, as well as highlighting a variety of problems with fibrinogen assays. Fibrinogen is a major plasma protein (normal concentration 1·5–4·0 g/l), which is synthesized in the hepatocyte. It comprises two each of three different polypeptide chains (Aα, Bβ and γ) linked by disulphide bridges. Thrombin cleaves the Aα and Bβ chains to release fibrinopeptides A and B, respectively, from the amino-terminal ends. After the fibrinopeptides are released, the resulting fibrin monomers undergo polymerization to form an insoluble fibrin clot (Hantgan et al, 1994). Fibrinogen also shows heterogeneity due to cleavage in the circulation, at the carboxyl termini of the polypeptide chains, giving rise to a series of molecules of varying size (de Maat et al, 1999). Fibrinogen and fibrin can be degraded to progressively smaller fragments by proteolytic enzymes, including plasmin and neutrophil elastase. Fibrinogen is an acute phase reactant (Whicher & Dieppe, 1985; Hantgan et al, 1994) and levels may be raised in relation to a variety of physiological variables and inflammatory conditions (Table I). The variation of fibrinogen levels within the same normal subject has been observed to be approximately 1·00 g/l in a study of 14 normal subjects over a 3-year period, although in some of the subjects fibrinogen levels varied by more than 2·5 g/l (Thompson et al, 1987). Such variation must be taken into account when fibrinogen is being considered as a risk factor for ischaemic heart disease. One further pre-analytical variable that has been reported to affect fibrinogen measurements, which is relevant to epidemiological studies, is the season of the year; fibrinogen levels are considerably higher in winter (van der Bom et al, 1997). A seasonal difference of 0·34 g/l (95% CI 0·29–0·30 g/l) was present in subjects aged 55–75 years which was more pronounced at age > 75 years. A polymorphsim of the beta fibrinogen gene promoter (G-455A) has been associated with increased levels of fibrinogen (Humphries et al, 1999). Carriers of the A-allele (about 20% of the population) have 7–10% higher fibrinogen levels than those of the GG genotype. Reduced fibrinogen levels may occur as a result of inherited gene defects, resulting in abnormal, reduced or absent fibrinogen protein, or as a result of liver disease or consumptive coagulopathies. Traditionally, fibrinogen assays have been required in the investigation of haemorrhagic states or in laboratory testing for haemorrhagic disorders (Table II). Some centres use a fibrinogen assay along with the prothrombin time (PT) and activated partial thromboplastin time (APTT), as part of a general haemostatic screen. In this situation, the fibrinogen level replaces the use of a thrombin clotting time, but the information provided may differ depending on the type of assay used. Whereas a sensitive thrombin time will detect low fibrinogen levels, decreased fibrin monomer polymerization, abnormal fibrinopeptide release, and may be influenced by acquired dysfibrinogens, heparin or fibrin/ fibrinogen degradation products (FDP's), different fibrinogen assays show varying sensitivity to each of these factors. The rare conditions of congenital afibrinogenaemia, hypofibrinogenaemia and dysfibrinogenaemia are due to gene defects, resulting in reduced fibrinogen synthesis and release and/or abnormal molecular species. In afibrinogenaemia, there is a grossly decreased hepatic synthesis of fibrinogen, with very low or undetectable plasma fibrinogen, which results in a haemorrhagic diathesis, with prolonged clotting times and abnormal platelet function. In hypofibrinogenaemia, circulating fibrinogen levels show a mild-to-moderate reduction, and patients may be asymptomatic or have haemorrhagic problems. Dysfibrinogenaemia is characterized by abnormal fibrinogen function, therefore, immunoassays tend to give disproportionately higher levels than clottable protein measurements. In some patients, hypodysfibrinogenaemia is present (reduced levels of fibrinogen protein and abnormal function). Approximately 250 patients with dysfibrinogenaemia have been reported in the literature, 55% of whom were asymptomatic, 25% of whom had a haemorrhagic tendency and 20% of whom had thrombophilia (Haverkate & Samama, 1995). The precise incidence of inherited dysfibrinogenaemia is unclear, as patients are usually detected because of a prolonged thrombin clotting time and obviously this will only be sensitive to certain types of fibrinogen abnormality. Acquired dysfibrinogens are usually due to excessive glycosylation of the fibrinogen molecule and typically occur in liver disease. In addition, in hepatocellular failure, fibrinogen levels may be markedly reduced and fibrinogen function may be influenced by the presence of elevated levels of FDPs. In disseminated intravascular coagulation (DIC), there is gross consumption of fibrinogen due to generalized fibrin formation throughout the microvasculature, and the presence of a variety of degraded forms of fibrinogen and FDPs. Massive blood transfusion or the use of colloids may also result in decreased fibrinogen levels as a result of haemodilution. Systemic thrombolytic therapy results in a gross reduction in fibrinogen levels and there is a high risk of haemorrhage. Prior to starting a prolonged infusion (over more than 1 d) of a plasminogen activator, and serially after starting therapy, the fibrinogen level may be monitored as an indication of appropriate therapy (target 0·5–1·0 g/l; Ludlam et al, 1995). In some patients receiving thrombolytic therapy, a fall in fibrinogen does not occur and an adjustment of therapy is necessary to prevent venous thrombotic extension (Marder & Sherry, 1988). Elevated fibrinogen levels are also clinically relevant. Fibrinogen is a major plasma protein and, therefore, a small elevation in fibrinogen level will have a significant impact on plasma viscosity, and thereby modify blood rheology. Increased plasma viscosity (e.g. in hyperviscosity syndrome) has been associated with an increased risk of thromboembolism. In recent years, a number of large prospective studies have shown that the fibrinogen level is a predictor of a variety of cardiovascular events, including stroke, myocardial infarction, leg ischaemia (in arterial disease) and postsurgical arterial re-occlusion (Lowe & Rumley, 1999). This statistical association has remained significant regardless of the type of fibrinogen assay employed, although some authors have suggested that immunological assays may be better predictors of cardiovascular disease than functional assays (Cremer et al, 1994; Sweetnam et al, 1996, 1998). However, the interpretation of a fibrinogen value at the upper end of the normal range, or of an elevated fibrinogen measurement, in an individual patient is often difficult because of the genetic, physiological and other factors outlined above (Machin & Mackie, 1993). It is not known whether all types of fibrinogen assay are equally sensitive to the increased fibrinogen levels resulting from these different conditions. The various fibrinogen assays may have a quite different sensitivity and clinical utility when used on an individual patient basis. A variety of different tests and assays of fibrinogen have been used by clinical laboratories (for review see de Maat et al, 1999). The assays vary in the degree of expertise and time required, and the equipment available. Some tests are designed for emergency situations, where an exact fibrinogen level may not be required but merely an estimate of whether the levels are normal or grossly decreased. The expanded range of coagulation automation and reagents has had a varying impact on the performance of laboratory tests and their precision. Automated coagulometers utilize different technologies to detect fibrin clot end-points. Some employ mechanical principles involving the movement of a metal hook or clot impedance of a ball bearing in a magnetic field; others use photo-optical devices to measure changes in light transmission or light scatter through 90°. Coagulation analysers that use a photo-optical detection principle continuously monitor the change in optical density or light scatter and the degree of change is proportional to the fibrinogen concentration: hence the derived fibrinogen assay. The most reliable derived fibrinogen methods are based on the prothrombin time (PT-Fg), where there is rapid thrombin generation and fibrin formation. A high concentration of thrombin (ranging from 35 to 200 U/ml, but typically about 100 U/ml) is added to dilute test plasma and the clotting time is measured (Clauss, 1957). The test result is compared with a calibration curve prepared by clotting a series of dilutions of a reference plasma sample of known fibrinogen concentration, and a result in g/l is obtained. The technique is relatively time consuming and requires a high degree of technical expertise if performed by manual methods, as the end-point is very difficult to determine at the higher dilutions of standard plasma, in test samples with low fibrinogen content, or in samples forming friable clots. Mechanical end-point techniques depend on the tensile strength of the clot and, although this makes them sensitive at low fibrinogen concentrations, they are particularly affected by heparin therapy. Photo-optical systems depend on the change in optical density resulting from fibrin formation, which decreases light transmission and increases scattered light. These methods are usually suitable for detecting weak fibrin formation, but may give erroneous values when fibrin monomer polymerization is slow. They are often affected by turbid or lipaemic plasmas, while the light transmission may be affected by bile pigment and free haemoglobin, depending on the wavelength used by the optical system. Most laboratories now use commercial reagents and an automated method, and a multiplicity of products are available, varying in thrombin strength, buffer of of heparin and calibration method and The reagents in use in the UK have been et al, and only small in fibrinogen levels were There were significant in Clauss depending on the but these are to have a clinical impact A degree of was with normal plasma of variation typically by optical methods and by The performance of Clauss fibrinogen assays by automated coagulometers in clinical laboratories may the for samples only and and many laboratories only perform them when there is a There is also the on some analysers for as the Clauss assay a high concentration of which has the of to and may tests (e.g. resulting in a of clotting The standard plasma or must be as very turbid reference may on photo-optical coagulometers and with dilutions of test 1999). This may to particularly for samples with low fibrinogen levels, when assays are Some commercial reference designed for use in Clauss appear to be when compared with the International This may the calibration method used by the or the use of other than the International there is generally impact of or on the Clauss certain clinical (e.g. thrombolytic may show as a result of the thrombin concentration and buffer will the impact of heparin and assays should not be performed on samples within of of of or on samples from venous or arterial This may be a during or and in these it may be necessary to the heparin suitable or The must only be used during of and should be taken that changes do not occur as a result of the to the for prolonged calibration are used for the of the that the is for the samples should be used to for most Clauss reagents high of appropriate methods should be on the of the to fibrinogen standard is to of on some A suitable range must be used will vary depending on the and and the calibration curve must be requires The calibration curve must at three dilutions of the plasma, and clotting times are samples should be if they are the range of the assay. a type of standard and within the as the results may not be Fibrinogen derived from the prothrombin time have been used in recent years et al, & et al, et al, 1985; et al, de & & et al, 1988). The is by a prothrombin time on a plasma series of plasma of known fibrinogen concentration and by a of the optical change against fibrinogen The optical change in each test sample is to a fibrinogen the technique is an of fibrinogen, the of or standard is very The of the PT-Fg method is that it a fibrinogen value at than that of the and it is used in approximately of UK Haematology laboratories National for although there is the for clinical use. PT-Fg assays are a of associated methods that vary depending on the method of type of and and optical of and test plasmas, as well as the of methods recommended by they may or and the use of normal plasma of optical or plasma with a calibration It is difficult to calibration in some PT-Fg methods if the reference plasma has a high fibrinogen level or 1999). and test with high tend to show an increased discrepancy PT-Fg and Clauss assays et al, if not PT-Fg methods give higher values than the Clauss technique et al, et al, 1994; et al, et al, et al, et al, This has been observed in samples from patients with liver high fibrinogen levels and in those receiving or thrombolytic therapy. In clinical the discrepancy is not over the range of observed fibrinogen values and may depend on the of the clinical as well as the thromboplastin used. Some laboratories have normal fibrinogen levels by but reduced results by the Clauss assay in patients with dysfibrinogenaemia or those receiving thrombolytic therapy. In these it is which method with in have a Clauss and types of PT-Fg assays et al, de & the that was in PT-Fg assays if the was prolonged (e.g. as a result of or the fibrinogen level was studies et al, have shown that PT-Fg results vary to the and different from the same A higher level of typically for normal was compared with Clauss particularly if turbid reagents and reference were used. In thrombolytic therapy, fibrinogen degradation products were in of fibrin in the Clauss but not in fibrinogen molecules may fibrin polymerization by of the fibrin as they at fibrinopeptide A & 1999). The resulting clot formation and clot detection affect the Clauss technique, while a large change giving a higher PT-Fg This be as the fibrinogen level and by the in Clauss assays. and a in Clauss fibrinogen and an in PT-Fg & 1997). turbid fibrin are at thrombin (de & 1998). the thrombin concentration in the PT-Fg method is a than that typically used in the Clauss while the fibrinogen concentration is the fibrin by the PT-Fg method will be more This may particularly the results conditions of abnormal fibrin PT-Fg results on clinical particularly those with abnormal fibrinogen molecules and high or low fibrinogen levels, show a with Clauss and other functional assays et al, et al, The of the curve depending on the PT-Fg thromboplastin the upper of detection by as as g/l Clauss values of It is not known whether the upper vary all reagents or whether there is a significant This the use of to the PT-Fg results to Clauss PT-Fg assays are not recommended for general use in Haematology both the and PT-Fg each must be as a assay The results from different or thromboplastin reagents should not be and reference are PT-Fg assays are only suitable for samples with optical where a normal fibrinogen value can be as large epidemiological studies of normal where an of normal or elevated fibrinogen not be In this as well as it is that from different PT-Fg assays are not PT-Fg assays have been there may be in the interpretation of fibrinogen results from patient as the results are not laboratories or because of the and The reference plasma must be for optical and calibration values must be available in relation to the International or plasma should be against the International in a Clauss assay and the observed value should be used in the PT-Fg calibration These assays are very and have been used as reference assays for Thrombin is added to plasma, in the of the clot is and in or other by a protein assay or estimation & & & & the of the protein in the clot will be the protein concentration will be approximately to the fibrinogen One method & is to measure the at and to the fibrinogen concentration the protein assays are time and them for use in However, they can be of use in the investigation of congenital fibrinogen A variety of immunological assays are assays and many to the giving the and precision. Some clinical analysers (e.g. have a for fibrinogen assay available immunological assays have the of protein concentration than functional may be in some methods where degraded forms of fibrinogen are as these have a different and may at different in and These are to the fibrinogen and Clauss or PT-Fg results observed in certain clinical Some commercial assays utilize against of the fibrinogen these the detection of fibrinogen or various types of degraded fibrinogen Such assays are sometimes in studies or of individual and assays give results although the has a high degree of The International for fibrinogen has been by functional than immunological a standard available, the same standard should be used for both types of fibrinogen assay and It is that the same standard be used for different fibrinogen assays within an individual to Fibrinogen is suitable for studies fibrinogen as a risk factor for cardiovascular disease (Cremer et al, 1994; Sweetnam et al, 1996, where normal or elevated fibrinogen levels are It also has utility in the investigation and of congenital fibrinogen (Table The fibrinogen is an emergency test where plasma therefore, is in different types of the of thrombin & The is reported as the to a clot after a The test can be performed in the presence of various as to and to heparin and However, the results are very and the test is time consuming and does not to to the of automated analysers, the test has from clinical laboratories and it is not In assays & fibrinogen is from dilute plasma, a thrombin and The clot is to and in and and on an number of available on the obviously The assay a high degree of technical to results and is time as the clot must be It is therefore, of clinical utility when fibrinogen assays are required The method does not to large of be automated and is not recommended for use. techniques & have been but are in some clinical in patients with acute phase and are not used in hospital The variables fibrinogen assays are in The recommended for fibrinogen assay is at a strength of part as used for other blood coagulation tests 1998). of the this and to than the blood concentration requires adjustment and are available which the of and blood to be 1998). It is that blood is by with and as as et al, 1998). Prior to fibrinogen blood samples should be by for the presence of clots. with of or should be should be prepared that are a of by at a of for this can be performed at lipaemic or samples may in some photo-optical methods of fibrinogen can be at to fibrinogen for at and more prolonged is required, is a suitable for Clauss fibrinogen and at for to not affect assay results in study et al, 1998). can be for et al, at where it is for at et al, of samples from patients with or those receiving thrombolytic therapy may to in of fibrinogen suitable are in the plasma is to it should be to a for at and by to or partial can to the formation of which fibrinogen, and may affect the assay of fibrinogen in products and other fibrinogen a at is necessary by to the This will a than normal plasma in to a concentration that can be by the Clauss reference may be prepared by from at normal and against National for Coagulation or International International for These should be in at and at use. commercial reference are they should be some have shown significant from the International in their et al, commercial are it is to with a optical In to reference for is required to prevent the of should not be as this problems with the calibration of photo-optical coagulometers for Clauss and PT-Fg assays. fibrinogen may be with PT-Fg when certain analysers and fibrinogen is generally in the of standard for assay in clinical laboratories, as after there are often problems with degradation and optical should be in each at normal a age range and both samples from patients should not be if they are free from major as it is that they will be or may have an acute phase which the range of It is recommended that all laboratories in an and of problems. in it may be to to of the assay as reference plasma or source of and these should be taken into account when a with a normal fibrinogen should be with each of fibrinogen at of for A sample with reduced or raised fibrinogen may be depending on the for which fibrinogen assays are being or if results are from test A of results should be to the time of and It is to a as a because this can in which that the method is of the range of the a result provided by as a patient testing must be the has been and of the laboratory and the clinical utility of fibrinogen assays should be into The variability of laboratory involving both reagents and end-point detection should be by in an is more and should the value of a fibrinogen level in the investigation of acquired and inherited and also the value of levels in the risk of thrombotic cardiovascular disease. the investigation of a of fibrinogen levels with the thrombin time, the variation during thrombolytic therapy and the with therapy fibrinogen should be levels of fibrinogen should be for further as part of clinical the levels transfusion or acute when therapy must be of arterial is more but levels of risk in myocardial to be with fibrinogen levels into of the normal These be with the relevant that patients may be as to their as and the of high levels from an acute phase with other acute phase particularly protein and factor can be The Clauss fibrinogen assay appears to be the most reliable method for general use in clinical In the most clinical with fibrinogen levels in to the Clauss assay and most is also based on this The PT-Fg method and immunological techniques may be in large studies where elevated fibrinogen is but may not fibrinogen measurements. the investigation of congenital both functional or clottable protein and immunological fibrinogen assays should be performed (Haverkate & Samama, to discrepancy PT-Fg levels appear to show a elevation in a variety of clinical and, if the assay is it must be by a different functional assay or are In light of the available literature, which shows that the PT-Fg is influenced by the of and the test is not recommended for clinical use. The authors and to the and on this by of the for the and information in these guidelines is to be and at the time of to the authors the can for or that may have been