Bethesda assay


Consult pathologist prior to collection.


Test plasma is mixed with a source of Factor VIII and is incubated for 2 hours at 37oC. A control mixture is prepared by mixing Factor VIII deficient plasma and buffered-normal plasma pool and is also incubated. After 2 hours the Factor VIII activity of each mixture is measured. The Factor VIII of the test mixture is compared to that of the control and the percentage of residual Factor VIII is calculated. One Bethesda unit (BU) is defined as that amount of inhibitor in the test plasma (patient) that results in 50 % residual Factor VIII activity. Dilutions of patient plasma are also tested.

Reference Interval:

Results are expressed as Bethesda units/mL.

Consult laboratory/pathologist


To quantitate Factor VIII inhibitory antibodies (inhibitors) in patients with classical, inherited haemophilia or acquired haemophilia.

May be used to estimate the level of inhibitors to human Factor VIII or adapted to measure inhibitors for Factors IX, XI, X, V etc.


A patient plasma producing a residual Factor VIII activity of 50 % in an incubation mixture is considered to contain one Bethesda unit per mL.


Dacie JV and Lewis SM. Practical Haematology. 11th ed. Churchill Livingstone 2012