Unexpected laboratory diagnosis: Acquired dysfibrinogenemia in a bleeding patient with liver disease

Case report
A 49-year-old African-American female with history of hepatitis C and hypertension was investigated for a possible dysfibrinogenemia when an abnormal thrombin time (TT) and reptilase time (RT) were reported by the laboratory. The patient presented with generalized weakness, abdominal pain, vomiting of blood, and epistaxis. Two years prior, the patient presented with epistaxis as well as excessive bleeding during menstrual cycles. On examination, the patient was awake, conscious, and in no acute distress. She had no signs of jaundice. Findings of elevated total bilirubin and liver enzymes, along with a reduction in albumin level, were in accordance with chronic/active liver disease. Screening coagulation assays revealed a prolonged prothrombin time (PT), prolonged activated partial thromboplastin time (aPTT), and low fibrinogen-activity level. A mixing study was performed which included the use of a TT and RT to check the patient's sample for heparin contamination. Both the TT and RT were prolonged, suggesting an abnormality of fibrinogen. Mixing with normal pooled plasma corrected the PT and aPTT. The clinical team was notified of these findings, and additional laboratory tests were performed, including a TT mixing study, antigenic (immunologic) fibrinogen level, and fibrinogen activity/fibrinogen-antigen ratio. The results confirmed the presence of dysfibrinogenemia. The clinical team expressed surprise at the diagnosis, as the patient's bleeding had been attributed to multiple coagulation-factor deficiencies, given the prolonged PT and aPTT. Given this information, cyroprecipitate, in addition to fresh frozen plasma, could be considered for the treatment of future bleeding episodes. Results of coagulation studies and liver function tests are presented in Table 1.

Discussion
Dysfibrinogenemia is the production by the liver of quantitatively normal — but abnormally functioning — fibrinogen. Dysfibrinogenemia can be inherited, but is more often acquired. Inherited dysfibrinogenemia may be associated with bleeding, thrombosis, or both. Acquired dysfibrinogenemia has been associated with bleeding, but how strong dysfibrinogenemia is as an independent risk factor for bleeding is unknown, as patients with acquired dysfibrinogenemia often have other risk factors for bleeding.1 Most coagulation proteins, including fibrinogen, are synthesized by the liver; therefore, acquired dysfibrinogenemia may be present in chronic liver disease, or liver malignancies such as hepatocellular carcinoma.2,3 Acquired dysfibrinogenemia has also been reported in obstructive biliary disease, and in immune disorders (e.g., multiple myeloma and systemic lupus erythematosus).4,5,6

After injury, regenerating hepatocytes have increased sialyltransferase activity and synthesize hypersialylated proteins (e.g., fibrinogen B and chains) with the increased negative charge, causing a decrease in polymerization rate.7 These anionic sugars cause the fibrinogens to polymerize slowly and abnormally, as the increased negative charge causes repulsion between fibrin monomers.1,8 The alteration of the fibrin monomers renders them to be poor substrates for thrombin, inhibiting the formation of a clot. Accumulation of altered proteins in the plasma of patients with liver disease may occur due to impaired removal by the diseased liver. Normally, asialoglycoproteins are rapidly removed from the circulation by the liver as the result of binding of their terminal galactosyl residues to the hepatocyte membrane.9 Impairment of this clearance mechanism might be responsible for elevated levels of sialic acid in patients with dysfibrinogenemia leading to a prolonged TT. Often, the prolonged TT may be explained clinically as secondary to increased circulating fibrinogen-fibrin degradation products due to delayed clearance, which act as natural anticoagulants; and, thus, the diagnosis of dysfibrinogenemia may be missed.10,11

Discussion of tests and results
The laboratory results observed in this case are consistent with other studies of acquired dysfibrinogenemia, showing prolonged TT and RT, and a decrease in the fibrinogen clotting activity. The diagnosis of dysfibrinogenemia is dependent on laboratory investigation, traditionally using tests of fibrin clot formation; the TT and RT are the screening tests, and the fibrinogen clotting activity-antigen ratio is the confirmatory test.1 The acquired form is diagnosed by demonstrating abnormal liver function tests as well. Acquired dysfibrinogenemia is typically diagnosed by demonstrating: (a) abnormal laboratory tests of hepatocellular or cholestatic function and (b) normal TT and/or RT in family members. The possibility of an inherited dysfibrinogenemia should be considered if fibrinogen dysfunction persists after resolution of the hepatobiliary disease, or when liver function tests are normal.1 The following is a discussion of the common laboratory tests used to diagnose dysfibrinogenemia:

Thrombin time: The TT, which is an initial screening test, detects the rate of fibrin clot formation after a fixed concentration of thrombin is added to a sample, thus reflecting the amount and the quality of fibrinogen present.1 The TT is also very sensitive to heparin, and will also be prolonged by elevation of fibrin degradation products. The prolongation of TT in dysfibrinogenemia is due to the inhibition of fibrinopeptide-A and/or -B release or fibrin monomer polymerization by dysfibrinogens.12 The prolongation of the TT by these fibrinogens strongly correlates with the increased sialic acid content.7

Reptilase time: An additional screening test is the RT. Reptilase is a snake venom enzyme, obtained from the Bothrops atrox viper, which catalyzes only the release of fibrinopeptide-A from fibrinogen. RT is sensitive to both quantitative and qualitative fibrinogen abnormalities, specifically those that are due to defects in fibrinopeptide-A release and fibrin polymerization. RT measures the rate of fibrin clot formation after the addition of reptilase to citrated plasma. The prolongation of RT in dysfibrinogenemia is due to the inhibition of fibrinopeptide-A release or fibrin monomer polymerization by dysfibrinogens. It is recommended to use both the TT and RT when screening for dysfibrinogenemia. The TT and RT can also be used as part of a mixing study protocol to check for heparin in the patient sample. The finding of a prolonged TT with a normal RT suggests heparin in the sample, as the RT is not affected by heparin.1 A procedure to neutralize heparin can then be used to confirm the presence of heparin in the sample.

Fibrinogen activity-antigen ratio: A fibrinogen activity-antigen ratio that is below the reference range is considered positive evidence for the diagnosis of dysfibrinogenemia. Usually this ratio is near 1:1.12 The fibrinogen activity is determined by two commonly known methods: a) Clauss method (measures the rate of clot formation after adding a high concentration of thrombin to diluted citrated plasma), and b) PT-based method (activity is determined from optical density change and compared to a curve derived from protimes of standards having known fibrinogen activity).1,13,14 The Clauss method is preferred for the detection of dysfibrinogenemia.1

Thrombin time 1:1 mixing study: This test is used in diagnosing acquired dysfibrinogenemia due to antibodies to fibrinogen in patients with immunologic disorders. The test may be done as a simple mixing study; correction into the reference range suggests fibrinogen deficiency, failure to correct a possible fibrinogen inhibitor. A flow chart for the diagnosis of acquired dysfibrinogenemia is shown in Figure 1. It is based in part on criteria established by the International Society of Thrombosis and Hemostasis (Subcommittee on Fibrinogen; Scientific and Standardization Committee).15

Immunologic aspects
Acquired dysfibrinogenemia due to inhibiting antibodies is most common in the setting of multiple myeloma and systemic lupus erythematosus. In these cases, acquired dysfibrinogenemia is attributed to non-specific interactions by inhibitor antibodies. Several case reports have identified monoclonal autoantibodies that specifically target fibrin monomer polymerization, providing a clear cut mechanism for the patient's dysfibrinogenemia.6,16,17

Conclusions
Acquired dysfibrinogenemia may be a contributing factor to bleeding seen in liver diseases, obstruction of the biliary tract, or in immunologic disorders. The mechanism of the bleeding tendency is due to fibrinogen alterations that inhibit fibrinopeptide release or fibrin monomer polymerization, leading to poor clot formation. Laboratory testing is crucial for the accurate diagnosis of dysfibrinogenemia. The TT and RT are used for screening in suspected cases of dysfibrinogenemia; but caution should be exercised, since these tests may be abnormal in other disorders of coagulation, such as hypofibrinogenemia or elevation of fibrin split products. Thus, additional laboratory studies should be done to confirm the diagnosis. Liver-function tests may be useful to confirm the diagnosis of acquired dysfibrinogenemia secondary to liver disease. If the liver disorder can be reversed, documentation of resolution of the dysfibrinogenemia is also evidence of an acquired clotting abnormality. The bleeding in the patient described in this report may be attributable to a number of possibilities, including esophageal varices, coagulation-factor deficiencies, thrombocytopenia, and acquired dysfibrinogenemia, all of which may be present in chronic liver disease. The laboratory investigation of the patient's prolonged PT and aPTT allowed recognition of dysfibrinogenemia so that optimal therapeutic decisions could be made to best control future episodes of abnormal bleeding.

Santosh K.S. Math, MD, PhD; Mary Ann Sanders, MD, PhD; and Sandra C. Hollensead, MD, are colleagues at the Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY.

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