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A Practical Guide to Laboratory Haemostasis

 

Inhibitor Assays



Introduction

Inhibitors are antibodies that in coagulation are usually targeted against either:
 - Specific clotting factors e.g. FVIII
 - Phospholipids i.e. a lupus anticoagulant [actually most lupus anticoagulants are directed against a protein-phospholipid combination]
An inhibitor is usually suspected from the clinical history or the finding of a prolonged clotting test that does not correct in a 50:50 mix with normal plasma. The most frequently seen inhibitors are targeted against phospholipids – the lupus anticoagulant – these are discussed elsewhere.
Circulating inhibitors or anticoagulants that target clotting factors may be either time-dependent e.g. FVIII inhibitors or immediately acting e.g. FIX inhibitors.

Principles & Methodology
APTT-Based Screening Tests

Screening for an inhibitor is based upon the APTT and involves measuring the APTT on a plasma sample before and after it has been incubated at 37°C. Three tubes are prepared as shown in the table below. The tubes are incubated at 37°C for 120 minutes and then placed on ice to stop the reaction. A fourth tube is prepared from equal volumes of Tube 1 and Tube 2 and APTTs are performed on all 4 tubes.

  Tube 1 Tube 2 Tube 3
  Normal plasma

Test plasma

Equal volumes of normal + test plasma

Step 1 Incubate at 37°C for 120 minutes

Incubate at 37°C for 120 minutes

Incubate at 37°C for 120 minutes

Step 2 Place samples on ice

Place samples on ice

Place samples on ice

Step 3 Prepare equal volumes of normal + test plasma i.e. Tube 1 and Tube 2 [=Tube 4]
Step 4 Perform APTT on Tube 1, Tube 2, Tube 3 and Tube 4.
 

The interpretation of results of the 4 APTTs i.e. the APTT on Tube, 1, Tube 2 etc - are shown in the table below:

Tube APTT
Tube 1
[Normal plasma]

Normal

Normal

Normal

Normal

Tube 2
[Test plasma]

Normal

Prolonged

Prolonged

Prolonged

Tube 3
[Incubated test + normal plasma]

Normal

Normal

Prolonged

Prolonged

Tube 4
[Non-incubated test + normal plasma]

Normal

Normal

Prolonged

Normal

Interpretation

Normal

Clotting factor deficiency

Immediate acting inhibitor

Time-dependent inhibitor

 
Remember - a screening test should be sensitive but not necessarily specific. So it should be able to identify all factor inhibitors but it may not be very specific but if you do obtain a positive inhibitor screen then this can be investigated in more detail by a Bethesda Assay.

Quantitative Assays: The Bethesda Assay

The Bethesda assay is widely used to quantitate the concentration of a factor VIII inhibitor.
1 Bethesda Unit (Bu) is defined as the amount of an inhibitor that will neutralised 50% of 1 unit of FVIII:C in normal plasma after 120 minutes incubation at 37°C. Factor VIII inhibitors are time dependent (e.g. factor VIII) whilst others are immediate acting (e.g. factor IX) and there is no requirement for an incubation step. However, the basic principles are the same.

As factor VIII inhibitors are time dependent, if exogenous factor VIII is added to plasma and the mixture is incubated, factor VIII will be progressively neutralised. If the amount of factor VIII added and the duration of incubation are standardised then the strength or concentration of the inhibitor may be measured in units according to how much of the added factor VIII is destroyed. The assay can be performed using both human and porcine factor VIII [although the latter is not currently available - a recombinant porcine FVIII concentrate is in development.]
The source of factor VIII is pooled normal plasma for anti-human titres and porcine concentrate diluted in factor VIII deficient plasma for anti-porcine titres. In the case of porcine factor VIII, the porcine factor VIII is diluted to 1 U/ml (100%) in factor VIII deficient human plasma.
The Nijmegen modification of the factor VIII inhibitor assay involves buffering the normal plasma with 0.1M imidazole buffer at pH7.4 and using immunodepleted factor VIII deficient plasma in the control mixture. At low inhibitor titres (<1 Bu) the classical Bethesda assay can result in false positives whereas the Nijmegen modified assays would give zero levels of inhibition.
The assay can also be modified to use factor VIII concentrate and the incubation time can be increased to 4 hours.

The Bethesda Assay:
1 Bethesda Unit (Bu) is defined as the amount of inhibitor in a plasma sample which will neutralise 50% of 1 unit of factor VIII:C in normal plasma after 2hr incubation at 37°C

1. Doubling dilutions of test (patient) plasma [usually 1/2 - 1/1024] in Imadazole buffer are incubated with an equal volume of the normal plasma pool at 37°C. Remember in a plasma sample with an unknown value for the inhibitor a series of dilutions will need to be made.
2. The normal plasma pool will normally contain around 100 IU/dl (1 IU/ml or 100%) factor VIII.
3. A control consisting of an equal volume of normal plasma mixed with buffer (or in the case of the Nijmegen modification, immunodepleted factor VIII deficient plasma) is taken to represent the 100% value. This mixture actually has a starting concentration of 50 IU/dl (50%) factor VIII (because you have performed a 50:50 dilution with buffer) but this does not matter because the same source and volume is added to all incubation mixtures.
4. At the end of the incubation period the residual factor VIII is assayed using a standard 1-stage APTT based assay with the control as the 100% (100 IU/dL or 1 IU/mL) standard.
5. The inhibitor concentration is calculated from a graph of residual factor VIII activity versus inhibitor units. The dilution of test plasma that gives a residual factor VIII nearest to 50% but within the range 30-60% is chosen for calculation of the inhibitor. It is also possible to calculate the inhibitor tire for each dilution and take the average. Any residual factor VIII <25% or >75% should NOT be used for the calculation of inhibitor level.
6. If the residual factor VIII activity is between 80-100% (IU/dL) or 0.8-1.0 IU/mL the sample does not contain an inhibitor.
7. Derive the inhibitor titre from the graph and multiply by the dilution to give the final titre. A positive control plasma of known inhibitor titre should be included.
9. Remember when plotting the residual FVIII against the BU titre – the Y axis is a log scale and the X axis is linear. Residual FVIII is plotted on the Y Log axis and Bu titre on the linear X axis.

In the table and the graph below, 4 plasma samples with varying dilutions have been assayed and the inhibitor titre in Bethesda Units [Bu] calculated.

Plasma Sample Dilution Residual FVIII:C Activity Bu x Dilution Bu Titre
1

1:10

50%

1 x 10

10 Bu

2

1:20

30%

1.75 x 20

35 Bu

3

1:40

60%

0.75 x 40

30 Bu

4

1:100

36%

1.5 x 100

150 BU

Remember you must take into account the dilution when you derive the inhibitor titre from the graph e.g. if the dilution is 1:10 and the residual FVIII is 50% then the value of 1Bu must be multiplied by 10 to give the actual inhibitor titre within the plasma sample of 10Bu.

Interpretation

See above.

Factor VIII Inhibitors

Factor VIII Inhibitors are classically divided into Types I or II depending upon the kinetics of the inhibitor. Type I FVIII inhibitors exhibit linear inhibition kinetics [first-order kinetics], that are both time and concentration dependent i.e. there is a linear relationship between the logarithm of residual FVIII:C and antibody concentration. Alloantibodies arising in individuals with severe haemophilia in response to treatment, classically demonstrate Type I kinetics and demonstrate complete inhibition of FVIII activity.

In contrast, Type II inhibitors show more complex kinetics [type II or second-order kinetics ] and commonly seen with autoantibodies and are therefore in individuals with acquired haemophilia A. Type II inhibitors are unable to completely inactivate FVIII:C even at maximum antibody concentration i.e. undiluted. This may explain why in some patients with an inhibitor [acquired haemophilia] small amounts of FVIII may be detectable.

Type Kinetics Inhibition of FVIII Seen with:
Type I Simple - First Order Complete Alloantibodies arising in a person with haemophilia treated with FVIII concentrates and who makes an antibody to the foreign protein.
Type II Complex - Second order  Incomplete Autoantibodies seen in acquired haemophilia A

The graph below summarises the differences between Type I and Type II FVIII inhibitors:


The Type I FVIII inhibitor shows a linear inactivation of VIII:C by the antibody whilst the Type II FVIII inhibitor shows a non-linear inactivation with a rapid initial inactivation phase followed by a slower phase where residual FVIII can usually be measured. In practice although residual FVIII can be demonstrated it does not prevent haemorrhagic symptoms.

In cases of haemophilia complicated by an inhibitor, most arise in individuals with severe haemophilia A i.e. FVIII:C <1 IU/dL. However, in cases of mild-moderate haemophilia A that develop an inhibitor - the inhibitor can be of two types:
i. Basal FVIII levels decrease i.e. there is a change in phenotype from moderate-mild to severe.
ii. The basal FVIII levels do not decrease but an inhibitor is present. Such patients maintain their moderate-mild phenotype but when treated with FVIII concentrates clear the exogenous FVIII rapidly.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.