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Specific Factor Inhibitor Testing
Coagulation factor inhibitor assays are used to identify inhibitors occurring in individuals with inherited factor deficiencies (alloantibodies) and those not congenitally deficient (autoantibodies). Alloantibodies are sought when congenitally deficient patients fail to respond to appropriate therapy. Autoantibodies are suspected when low factor levels occur in previously unaffected patients, particularly when multiple dilutions of patient plasma give different factor activity levels, suggesting that the patient’s dilution curve does not parallel the normal.
Coagulation factor inhibitors are detected in the laboratory primarily through their ability to neutralize specific coagulation factors and must be distinguished from nonspecific inhibitors, such as the lupus anticoagulant. Recognition of specific inhibitors may be complicated by their presentation as multifactor deficiencies ( Table 132.1 ). At dilutions used in most clinical factor assays, these antibodies may interfere with tests of other factors in their pathway. When patient plasma is tested in higher dilution, accurate measurement of the noninhibited factors is obtained.
Table 132.1
Spurious Effects of Factor Inhibitors on Factor Activity Assays
| Dilution | Factor VIII | Factor IX | Factor XI | Factor XII | |
|---|---|---|---|---|---|
| Factor VIII inhibitor | 1:5 | <1 | 23 | 19 | 40 |
| 1:20 | <1 | 45 | 38 | 55 | |
| 1:100 | <1 | 78 | 65 | 82 | |
| Factor IX inhibitor | 1:5 | <1 | <1 | <1 | – |
| 1:20 | 110 | <1 | 70 | – |
Factor VIII (FVIII) inhibitor quantitation was standardized in 1974, when investigators meeting in Bethesda, Maryland, agreed on a standard method and a unit of measure: the Bethesda unit (BU), defined as the amount of inhibitor, which destroys one half of the FVIII activity (VIII:C) in 1 mL of normal plasma within 2 hours when incubated at 37°C. Modifications to increase the sensitivity and specificity of the Bethesda method have been adopted, particularly the Nijmegen-Bethesda (NB) method and use of chromogenic measurement of FVIII. Preanalytical heat inactivation of patient plasma to allow testing with FVIII present has also been introduced. The Bethesda method has been extended to the quantitation of inhibitors to other coagulation factors.
Antibody detection methods, including enzyme-linked immunosorbent assays (ELISA) and fluorescence immunoassays, detect most inhibitors that neutralize factor activity and those that bind to the factor but fail to neutralize its activity in vitro.
Methods and Test Interpretation
A factor inhibitor is detected by performing the relevant coagulation factor assay on a mixture of patient plasma and normal pool plasma (NPP) and comparing the result to a control mixture. Inhibitors to FVIII and some inhibitors to factors V and XI are time-dependent and require incubation. Inhibitors to other factors usually react immediately.
Antibodies with type 1 or “simple” kinetics show high affinity and can be saturated. In antibody excess, all activity is neutralized. Antibodies with type 2 or “complex” kinetics show lower affinity and can dissociate from antigen. Both free antigen and free antibody may be present. Most hemophilic inhibitors have type 1 kinetics; type 2 is more common among autoantibodies.
FVIII Inhibitors
The Bethesda assay is performed by incubating a 1:1 mixture of patient plasma and NPP and a control 1:1 mixture of NPP with imidazole buffer at 37°C for 2 hours and measuring the VIII:C remaining. In the NB method, FVIII-deficient plasma or 4% bovine serum albumin is substituted for buffer in patient dilutions and the control mixture, and NPP is buffered with imidazole to pH 7.4 to maintain protein concentration and pH of the mixtures during incubation. In either method, the activity in the patient mixture is divided by the activity remaining in the control mixture and multiplied by 100 to give the % residual activity (RA). The RA is converted to BU using a graph plotting the logarithm of RA against BU or the equation:
If RA = 100%, BU = 0. For RA 25%–100%, BU of the lowest dilution in that range is reported. If no dilution falls above 25% RA, patient plasma is tested serially diluted with imidazole buffer, and the first dilution falling between 25% and 75% RA is multiplied by the dilution factor and reported. Examples of tests on inhibitors with type 1 and type 2 kinetics are shown in Table 132.2 . For Type 2 inhibitors, which do not respond linearly to dilution, the dilution closest to an RA of 50% is often reported, but quantitation is inexact. Some laboratories report the first dilution falling within the 25%–75% RA range. It is useful to compare the same dilution in subsequent assays to document rise or fall. Results are reported as BU or Nijmegen-Bethesda units. To test patients with measurable VIII:C, such as recently treated hemophilia patients or nonhemophilic patients, plasma may be heated to 56°C for 30 minutes and centrifuged before testing to destroy VIII:C. A chromogenic FVIII assay may also be used in the Bethesda or NB assay.
Table 132.2
Calculation of Results for Type 1 and Type 2 Inhibitors Measured in Nijmegen-Bethesda Units (NBU)
| Patient Dilution | Patient Mix VIII:C a | Control Mix VIII:C a | % Residual Activity b | Calculated NBU c | Total NBU d | |
|---|---|---|---|---|---|---|
| Type 1 inhibitor | Undiluted | 19 | 45 | 42.2 | 1.24 | 1.2 |
| 1:2 | 30 | 45 | 66.7 | 0.58 | 1.2 | |
| 1:4 | 35 | 45 | 77.8 | 0.36 | 1.4 | |
| Type 2 inhibitor | Undiluted | 27 | 45 | 60.0 | 0.74 | 0.7 |
| 1:2 | 27 | 45 | 60.0 | 0.74 | 1.5 | |
| 1:4 | 27 | 45 | 60.0 | 0.74 | 3.0 |
a Units per deciliter of FVIII activity.
b Patient mix VIII:C/control mix VIII:C X 100.
c NBU read from graph or calculated as NBU = (2 − log % residual activity) (0.301) −1 .
An ELISA for FVIII inhibitors is commercially available, and other immunologic methods have been described. Because they also detect nonneutralizing antibodies, quantitation using these methods is not directly comparable to that of clot-based tests. They may be used to confirm the presence of specific antibodies.
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