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.
Index of Tests
Factor Inhibitors [CO000900]
Synonyms Bethesda Assay; Circulating Anticoagulant; Modified
Abstract Specific factor inhibitors are antibodies that inhibit
the activity of a specific coagulation factor. A severe acquired
bleeding disorder may develop.
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,
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
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.
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,
Sahud MA, "Laboratory Diagnosis of Inhibitors,"Semin Thromb
Hemost, 2000, 26(2):195-203.