Rare Congenital Fibrinogen Deficiencies
This section describes diagnostic tests, including sensitivities, limitations and interpretation of currently used assays, the importance of identifying molecular defects, and the use of newer assays for thrombin generation and fibrin clot structure which may aid in a better understanding of the bleeding/thrombotic phenotype to predict clinical events and use of replacement therapy.
Levels Associated with Severity of Bleeding
The normal plasma fibrinogen concentration is approximately 150-350 mg/dL with a half-life of about 3.5 days.41 Fibrinogen assay methods are based on one of the following principles:
- A functional activity assay, which measures clottable protein on the basis of either coagulation time or velocity (Clauss or thrombin clotting method)42
- A radial-immunodiffusion assay, utilized by most laboratories, to measure antigen level.43
In general, functional fibrinogen clotting assays are less sensitive and the threshold of detection can be as high as 50 mg/dL. With more sensitive ELISA methods, levels as low as 0.4 mg/dL can be detected.41 Functional fibrinogen levels of ≥100 mg/dL are generally hemostatic unless a large quantity of nonfunctional fibrinogen, which may interfere with normal fibrinogen clotting, is present.
The key diagnostic criterion for afibrinogenemia is the absence of immunoreactive fibrinogen. All coagulation tests that depend on fibrin as the endpoint, including prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT) and reptilase time are infinitely prolonged.
Fibrinogen is undetectable by functional (Clauss) assays in persons with afibrinogenemia. Sensitive ELISA assays may determine trace amounts of fibrinogen (<10 mg/dL). On coagulation screening panels, a normal platelet count and negative D-dimer are useful to discriminate afibrinogenemia from disseminated intravascular coagulation and severe liver disease.
Since hypofibrinogenemia is a proportional decrease of functional and immunoreactive fibrinogen, most fibrin-based coagulation tests are variably prolonged; the most sensitive test is the thrombin time, which is usually prolonged at fibrinogen activity <100 mg/dL. Both total clot-based and immunologic fibrinogen levels are reduced to similar levels. The diagnosis of hypofibrinogenemia using clinical laboratory assays alone may be difficult as heterozygous carriers of afibrinogenemia may have fibrinogen activity within the high-normal range, and patients with confirmed heterozygous fibrinogen gene mutations may have levels of fibrinogen activity that overlap the normal range. Finally, different family members with the same fibrinogen gene mutation can exhibit varying levels of fibrinogen. from a moderate deficiency to normal levels.
Tests should be interpreted with regard to the possibility of acquired hypofibrinogenemia, including consumptive coagulopathy, hepatic failure, and L-asparaginase therapy. Normal platelet count and negative D-dimer, as well as family studies, may be helpful in differentiating acquired fibrinogen deficiency from CFD.
Both the thrombin time and reptilase time are sensitive screening tests. A prolonged reptilase time in the presence of a normal functional fibrinogen provides strong evidence of dysfibrinogenemia. A normal or increased antigen with a lowered functional level (Clauss method) resulting in a low functional-antigen ratio (most commonly 1:2) is usually diagnostic.44
The sensitivity of coagulation tests to dysfibrinogenemia are dependent on the specific mutation, as well as the laboratory reagents and specific testing techniques used.45 A definitive diagnosis can be established by demonstrating the molecular defect. However, as these disorders are dominantly inherited, family studies may be helpful to differentiate congenital from acquired dysfibrinogenemia. In the small percentage of patients that present with thrombosis, a thrombophilia work-up to exclude other co-existing prothrombotic defects may be useful. Results from the prospective study on congenital fibrinogen deficiency (proRBDD project) has confirmed that the correlation between fibrinogen levels and severity of bleeding is strong (β = -0.19, P < 0.05).46
Afibrinogenemia is an autosomal recessive disorder, while hypofibrinogenemia and dysfibrinogenemia are autosomal dominant disorders. Hence, individuals with a family history, especially those with a history of consanguinity, should be appropriately counseled as to the risks of transmission of a congenital fibrinogen disorder to a child. If the mutation is known, genetic testing can be planned during pregnancy using amniotic fluid or maternally-derived fetal fibroblasts to aid in planning an appropriate and safe delivery for an affected child.
In infants born to parents who are either known or suspected carriers, cord blood testing for fibrinogen activity and antigen should be offered to facilitate prompt and accurate diagnosis and management of neonatal bleeding. Avoidance of arterial punctures, intramuscular injections, circumcision and other invasive interventions is recommended. Routine screening for intracranial hemorrhage in neonates is currently under debate, as ultrasound is insensitive to parenchymal bleeding and computed tomography (CT) is associated with radiation exposure; however, the use of magnetic resonance imaging (MRI) in neonates without the need for sedation is increasing in tertiary care hospitals with large neonatal services.
As far as possible, all CFDs should be genotyped. Genotyping provides information regarding the location of the mutation in the fibrinogen molecule, which may aid in classification and understanding of the disease mechanism. Correct classification in cases of hypofibrinogenemia or dysfibrinogenemia may have clinical implications. Deleterious effects of new mutations should be based on “Sorting Intolerant From Tolerant” (SIFT) analysis47 and proper validation.
Patients with afibrinogenemia are homozygous or compound heterozygous for a causative mutation, while hypofibrinogenemia patients are usually heterozygous carriers of afibrinogenemia mutations.
Dysfibrinogenemia is usually associated with an autosomal dominant inheritance caused by heterozygosity for missense mutations, although deletions, frameshift mutations, insertions and intronic mutations have also been reported.48 The two ‘hotspot mutations’ are located in exon 2 of FGA (p.Arg35) and in exon 8 of FGG (p.Arg301) which represent up to 75% of all dysfibrinogens identified in European49-52 and Chinese patients.53 As reported in a cohort of 101 patients, these mutations were neither significantly associated with thrombosis nor with major bleeding.52
However, for both quantitative and qualitative disorders some genotypes are predictive of a specific phenotype, and therefore genotype identification has a direct clinical implication. A distinct group of six mutations clustered in exons 8 and 9 of FGG leads to a specific disorder known as ‘fibrinogen storage disease’.54 This disease is characterized by different degrees of hypofibrinogenemia and hepatic inclusions due to impaired release of mutant fibrinogen that accumulates and aggregates in the hepatocellular reticulum.55 The severity of liver disease is highly variable, from fibrosis to cirrhosis, with an incomplete familial segregation.26 Several mutations in the C-terminal region of the Aa chain are associated with a particular form of hereditary amyloidosis.56 In these cases, fibrinogen variants are functional (fibrinogen activity is not decreased) but unstable and prone to form systemic amyloid fibrils leading to organ damage, especially in the kidneys.57
Given the thrombotic risk in afibrinogenemia and dysfibrinogenemia, both with and without fibrinogen replacement therapy (FRT), a role may exist for monitoring hypercoagulability with global assays, including thrombin-antithrombin complexes for either condition and potentially thromboelastography (TEG or ROTEM) for dysfibrinogenemia.58 Kalina et al. observed that ROTEM parameters FIBTEM and EXTEM provided a consistent, more predictable response to fibrinogen administered in vitro to patients with afibrinogenemia, hypofibrinogenemia or dysfibrinogenemia than assessment of fibrinogen concentrate by the Clauss or ELISA methods.58 More recently, Trelinski et al. conducted a study that demonstrated that ROTEM can help identify dysfibrinogenemia patients at the highest risk of thrombosis.59 An important consideration is that individuals without detectable fibrinogen or with fibrinolytic defects are unable to generate D-dimers or other fibrin degradation products; therefore, these assays are insensitive markers of hypercoagulability in such individuals with CFD.
The structure of the fibrin clot has been identified as a key factor for mechanical thrombus properties (eg, resistance to fibrinolysis),60 and clinical studies have revealed altered fibrin clot structure in a variety of bleeding or thrombotic diseases.61-63 Some specialized laboratories analyze fibrin clot structure with various assays that are not currently considered routine; nonetheless, recent data have confirmed the important role of clot mechanical properties in hemostatic balance.64 Mutant fibrinogens identified in dysfibrinogenemia may provide a distinctive model for understanding the fibrin network and associated properties.65 Most functional studies on dysfibrinogenemic families have described how fibrinogen variants can impact the fibrin clot in both purified fibrinogen and in plasma conditions using turbidity, permeability, and compaction assays, as well as employing various imaging techniques such as scanning or transmission electron microscopy and confocal microscopy.66-68 The comparison of results from different studies is difficult due to the differences in specimen preparation and test conditions.48 In a large series of patients (n = 38), Sugo et al.69 showed that the fibrin clot network could be assigned to one of five classes with a possible correlation to phenotype. Furthermore, abnormal fibrin clot properties have been reported in five dysfibrinogenemic patients suffering from chronic thromboembolic pulmonary hypertension.70 Another retrospective study on 24 dysfibrinogenemic patients suggested that clot permeability and lysis times could distinguish bleeding and thrombotic phenotypes, respectively.71 In addition, more sophisticated research techniques such as atomic force microscopy and magnetic tweezers can also permit analysis of fibrin structure and viscoelastic properties at the level of a single molecule.65