Inhibitor assays

The Bethesda assay is used to quantify the concentration of inhibitory antibodies in a plasma sample. Lupus anticoagulant (LA) tests can be used to investigate the presence of antiphospholipid antibodies.

The Bethesda¹ assay is used to quantify the concentration of inhibitory antibodies in a plasma sample. By mixing serial dilutions of test plasma with normal plasma, any inhibitory alloantibodies or autoantibodies in the test plasma will neutralise the FVIII or FIX (or other coagulation factor) activity in the normal plasma. FVIII or FIX activity in the normal plasma will be detectable at sufficiently high dilutions of the test plasma containing the inhibitor, permitting a quantification of the inhibitor titre based on the residual activity and the sample dilution factor. The Nijmegen modification of the Bethesda assay² reduces the incidence of false positive results and is now widely used for inhibitor quantification.

Nijmegen-modified Bethesda assay

Serial dilutions made in FVIII-deficient plasma are mixed with an equal volume of pooled normal plasma buffered with 0.1 M imidazole pH 7.4. Normal plasma mixed with immunodepleted, factor-deficient plasma is used as the negative (no inhibitor; 100% activity) control. After incubation, the residual factor activity is measured using a standard one-stage activated partial thromboplastin time (aPTT)-based clotting assay.

The principle behind Nijmegen-modified Bethesda assays to quantify inhibitor titre.

Inhibitor titre estimation

One Bethesda unit (BU) is defined as the amount of inhibitor in a plasma sample that can neutralise 50% of one unit (1 IU/mL) of FVIII activity in normal plasma after 2 hours at 37°C. Measurement of FIX activity in the presence of inhibitors requires no incubation time, but the principle is the same. The dilution(s) that generate factor activity measurements closest to 50% (but >25% and <75%) of the control are used to calculate the inhibitor titre. Generally, if the residual factor activity is >80%, no inhibitor is considered to be present.

As an example, a test sample diluted 1:16 that has 50% activity relative to the control reaction has an inhibitor titre of 16 BU (50% activity = 1 BU multiplied by the 16-fold dilution). The inhibitor titre can also be calculated using a graph that plots BU vs. residual factor activity multiplied by the sample dilution factor.

Inhibitor kinetics

Inhibitory alloantibodies, typically observed in patients with haemophilia A (HA) or B (HB) treated with factor concentrates, exhibit linear first-order (type 1) time versus activity kinetics.³ These antibodies are able to completely neutralise FVIII or FIX activity.

In contrast, autoantibodies such as those observed in individuals with acquired haemophilia A (AHA) typically exhibit non-linear, second-order (type 2) kinetics and are unable to completely inhibit factor activity, even at maximum concentrations.³ In the absence of personal expertise, the assessment of autoantibody assay kinetics is best undertaken in consultation with an expert with appropriate experience.

Care should be taken in cases of patients with measurable residual FVIII activity, as the Nijmegen-modified Bethesda assay may not detect the presence of inhibitors accurately. An additional heat inactivation step can help improve the accuracy of the assay in such cases⁴.

Lupus anticoagulant (LA; 2017 ICD-10-CM: D68.312) is an autoantibody that binds to phospholipids and cell membrane proteins that can lead to prothrombotic effects. Conversely, in vitro LA is associated with increased clotting times using the aPTT assay as a result of binding to phospholipids in the reaction.⁵

More sensitive tests to confirm the presence of LA antiphospholipid antibodies include the dilute Russel viper venom time (dRVVT), platelet neutralisation procedure (PNP) and kaolin clotting time (KCT).

Dilute Russel viper venom time ⁶

Russel viper venom (RVV) directly activates coagulation FX, which in turn converts prothrombin to thrombin in the presence of FVa and phospholipids. 

Pooled normal plasma or test plasma is incubated with phospholipids at 37°C followed by the addition of diluted RVV and calcium to initiate coagulation and the clotting time measured. The ratio of normal:test plasma clotting time determines whether coagulation is affected. A ratio of >1.05 suggests the presence of LA, once a deficiency in common coagulation pathway factors (I, II, V, X) is excluded.

The presence of a lupus anticoagulant interferes with this reaction by binding to the phospholipids, resulting in prolonged clotting times. A corrected clotting time during mixing studies using a 1:1 mixture of test and normal plasma suggests a factor deficiency, however because RVV activates FX directly, the test is not affected by the intrinsic (or contact-activation) pathway. A failure to correct the clotting time using mixing studies and correction with the addition of excess phospholipid indicate the presence of LA.

Silica clotting time

Silica clotting time (SCT) is essentially an aPTT assay that uses silica as the contact activator in the presence of plateler-poor plasma and a low concentration of phospholipids followed by the addition of calcium as a sensitive test to screen for LA.⁷

Platelet neutralisation procedure⁸

The platelet neutralisation procedure uses washed, disrupted platelet membranes as a source of excess phospholipid to test whether the aPTT can be normalised by neutralising the effect of a LA antibody.

Kaolin clotting time⁹

The KCT is sensitive for the detection of LA and follows the same principle as the aPTT, but lacks phospholipids, relying instead on plasma lipids and cell membrane fragments. The test can be used to detect all classes of coagulation inhibitors, including antibodies directed against phospholipids and FVIII.

Normal and patient plasma are mixed in ratios ranging from 1:10 to 10:1 in the presence of kaolin and calcium and clotting time measured. A test sample:control ratio >1.2 suggests the presence of an inhibitor. A factor deficiency can be excluded in the presence of a prolonged KCT by adding excess normal plasma. A KCT that does not correct in the presence of normal clotting factors suggests a coagulation inhibitor, but may not exclude a coagulation factor autoantibody. The Rosner index or Chang ratio can be applied to a KCT analysis to calculate limits that distinguish between correction and no correction.

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