Testing for FVIII Inhibitors

1
Testing for FVIII Inhibitors
Several options exist in the laboratory for
identifying FVIII inhibitors. A solid-phase
ELISA assay (microtiter plates coated with
recombinant FVIII) may be useful as a screen for
FVIII inhibitors. Once identified, it is
necessary to quantify the strength of the
inhibitor. The three assays in use today
include 1) Bethesda Assay, 2) Nijmegen
modification of the Bethesda Assay, and 3) the
New Oxford Method. All assays measure the amount
of FVIII inactivated by inhibitors (antibodies,
predominantly IgG) present in patient plasma.
The Bethesda Assay is the most widely used in the
US and is outlined in detail in the Slide 3. It
is readily apparent that all three quantitative
assays have the potential to suffer from a large
number of analytical variables due to testing
complexity as well as inherent variables that
affect an APTT test system.

• Several options exist in the laboratory for
  identifying FVIII inhibitors. A solid-phase
  ELISA assay (microtiter plates coated with
  recombinant FVIII) may be used as a screen for
  FVIII inhibitors. Once identified, it is
  necessary to quantify the strength of the
  inhibitor. Three assays currently used include
  1) Bethesda Assay, 2) Nijmegen modification of
  the Bethesda Assay, and 3) the New Oxford Method.
  All assays measure the amount of FVIII
  inactivated by inhibitors (antibodies,
  predominantly IgG) present in patient plasma.
  The Bethesda Assay is the most widely used in the
  US and is outlined in detail in the Slide 3. It
  is readily apparent that all three quantitative
  assays have the potential to suffer from a large
  number of analytical variables due to testing
  complexity and variables inherent in APTT test
  systems.

Alloantibodies Develop in 15-35 of Hemophilia
A patients who have received exogenous FVIII
through replacement therapy. These antibodies
demonstrate type 1 kinetics (FVIII inactivation
is directly proportional to antibody
concentration).
Autoantibodies Found in acquired hemophilia
and exhibit type 2 kinetics (rapid inactivation
of FVIII followed by a plateau in which some
FVIII remains unneutralized). Most autoantibodies
are idiopathic though others are seen post partum
or in older patients with an underlying
autoimmune disorder/hematologic malignancy.
2
Bethesda vs Nijmegen Assays
Nijmegen-Bethesda Assay
Classical Bethesda Assay
Slide concept courtesy of Drs HW Verbruggen and
Piet Meijer, ECAT Foundation
3
Classical Bethesda Assay
Step 5 Patient FVIII / Control FVIII Corrected
Residual FVIII Activity (RA) Read RA from Graph
(RA x Dilution BU)
Triplett DA, Harms CS. Procedures for the
Coagulation Laboratory. ASCP Press 1981, pp
61-63.