From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Related Information
Antiphospholipid Antibody (Lupus Anticoagulant and/or Anticardiolipin Antibody)
Coagulation Factor Assays
Mixing Studies
Synonyms Bethesda Assay; Circulating Anticoagulant; Modified Bethesda Assay
Abstract Specific factor inhibitors are antibodies that inhibit the activity of a specific coagulation factor. A severe acquired bleeding disorder may develop.
Specimen Plasma
Container Three blue top (sodium citrate) tubes
Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled. Deliver tubes immediately to the laboratory. Avoid heparin contamination of specimens during specimen collection.
Storage Instructions Separate plasma from cells as soon as possible. Plasma may be stored on ice for up to 4 hours; otherwise, store frozen.
Causes for Rejection Specimen received more than 4 hours after collection, tubes not filled, clotted specimens
Turnaround Time 1 full day or longer (usually performed in a specialized laboratory)
Special Instructions Specific factor inhibitors, such as factor VIII inhibitors, can cause a severe bleeding disorder and treatment can be difficult. Therefore, the specimen should be sent to a laboratory that can perform the assay promptly.
Reference Interval The inhibitor is quantitated in Bethesda units (BU). Each Bethesda unit of inhibitor decreases the factor concentration in the assay by 50%. For example, one unit of factor VIII inhibitor decreases factor VIII from 100% (normal) to 50%, two units decrease it to 25%, three units decrease it to 12.5%, and so on.
Use Performed when mixing studies and factor assays suggest the presence of a specific factor inhibitor, and the findings are not due to a lupus anticoagulant, heparin, or other anticoagulants (see Mixing Studies). For example, if a mixing study shows the characteristic pattern for a factor VIII inhibitor, and factor VIII is the only markedly decreased factor (usually <1% to 10%; mean normal value is 100%), a Bethesda assay should be performed to identify and titer the factor VIII inhibitor.
Methodology Bethesda assay for factor VIII inhibitor:1 Serial patient plasma dilutions in citrated saline are prepared, from 1:1 up to 1:160 (or higher if necessary for high-titer factor inhibitors). The purpose of these dilutions is to dilute out the inhibitor. The patient plasma dilutions are then mixed with an equal volume of normal plasma containing a normal amount of coagulation factors. The mixed dilutions are usually incubated for up to 2 hours, because certain inhibitors show an inhibitory effect only after prolonged incubation (particularly factor V and factor VIII inhibitors). Factor VIII assays are then performed on each mixed dilution. The dilution that inhibits 50% of factor VIII in the assay defines the titer of the inhibitor. For example, if the 1:40 dilution inhibits 50% of the factor VIII in the assay, the patient is reported to have a titer of 40 BU of factor VIII inhibitor.
Porcine factor VIII can be substituted for normal plasma (which contains human factor VIII) in the Bethesda assay to determine if the factor VIII inhibitor cross-reacts with porcine factor VIII. If there is little or no cross-reactivity, porcine factor VIII is often used to treat bleeding due to a factor VIII inhibitor.
The Bethesda assay can be modified to identify and titer other specific factor inhibitors. For example, if a factor V inhibitor is suspected, factor V assays are performed on the mixed dilutions instead of factor VIII assays.
Additional Information Antibodies that inhibit the activity of a specific coagulation factor can develop spontaneously or in association with certain medications, autoimmune diseases, or other conditions. These antibodies may also arise when a patient with a hereditary factor deficiency is transfused with a product containing the factor, such as a factor concentrate or fresh frozen plasma. The immune system in the patient with the deficiency views the transfused factor as foreign, and forms an antibody against the transfused factor. This complication makes treatment of bleeding episodes difficult in such patients. The most common clinically significant factor inhibitor is a factor VIII inhibitor. Factor VIII inhibitors develop in approximately 10% to 20% of patients with severe hemophilia A and less commonly with mild or moderate hemophilia A, following the infusion of factor VIII-containing products. Rarely, factor VIII inhibitors can also arise spontaneously in persons without hereditary hemophilia. Factor VIII inhibitors cause decreased factor VIII activity and consequently a prolonged PTT. Factor VIII inhibitors exhibit a characteristic pattern in the PTT mixing study where the mixed plasma PTT is initially normal (or significantly more normal than the patient plasma’s PTT) but becomes prolonged (typically by increasing at least 8-10 seconds) over the course of a 1- to 2-hour incubation.
Factor IX inhibitors develop in approximately 2% to 12% of patients with severe hemophilia B, and less commonly with mild or moderate hemophilia B, following transfusion of factor IX-containing products.2 Very rarely, factor IX inhibitors can also arise spontaneously in persons without hereditary hemophilia B. Factor IX inhibitors cause decreased factor IX activity and consequently a prolonged PTT. The prolonged PTT caused by a factor IX inhibitor is immediately prolonged in the PTT mixing study.
Other factor inhibitors arise occasionally following exposure to “fibrin glue” preparations, which are administered topically and intraoperatively to help achieve hemostasis. Fibrin glue is prepared by adding bovine thrombin to human fibrinogen, in the form of cryoprecipitate. The affected patient’s immune system views the bovine thrombin as foreign, and forms an antibody against it. Frequently, traces of bovine factors V, VII, or X are also present and antibodies can be generated against these factors as well. The antibodies to bovine coagulation factors sometimes cross-react against the corresponding human coagulation factor, which can lead to bleeding. In one series, 1.7% of patients exposed to bovine thrombin preparations developed a clinically significant inhibitor with bleeding.3
Other specific factor inhibitors have also been observed, but most are exceedingly rare. These include inhibitors to factors I (fibrinogen), II, V, VII, X, XI, XII, XIII, and prekallikrein.
Factor V inhibitors may behave like factor VIII inhibitors in the mixing study, with increasing PTT (or PT) prolongation over a 1- to 2-hour incubation.4 Other factor inhibitors most likely behave like factor IX inhibitors in mixing studies, with immediate prolongation of the PTT (or PT) in the mixed plasma.
Note: Factor inhibitors can cause artifactual decreases in the in vitro factor level of other coagulation factors. Therefore, laboratories should perform factor assays at multiple dilutions. At higher dilutions, the inhibitor interference will decrease due to dilution of the inhibitor. For example, a factor VIII inhibitor sometimes causes false decreases in factor IX, XI, or XII assays. Typically, the false decreases, if any, are mild to moderate, whereas the decrease in the truly inhibited factor is typically severe.
Rarely, nonspecific factor inhibition is found with monoclonal gammopathy (paraproteinemia) which can appear to nonspecifically inhibit clotting reactions in the laboratory without targeting any particular coagulation factor. The PT and PTT may be prolonged, and multiple factor assays are nonspecifically inhibited.
Footnotes
1. Brown BA, Hematology: Principles and Procedures, 6th ed, Philadelphia, PA: Lea & Febiger, 1993, 256-8.
2. Shapiro SS and Hultin M, “Acquired Inhibitors to the Blood Coagulation Factors,”Semin Thromb Hemost, 1975, 336-85.
3. Dorion RP, Hamati HF, Landis B, et al, “Risk and Clinical Significance of Developing Antibodies Induced by Topical Thrombin Preparations,”Arch Pathol Lab Med, 1998, 122(10):887-94.
4. Crowell EB, “Observations on a Factor-V Inhibitor,”Br J Haematol, 1975, 29(3):397-404.
References
Sahud MA, “Laboratory Diagnosis of Inhibitors,”Semin Thromb Hemost, 2000, 26(2):195-203.
