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A Practical Guide to Haemostasis


Inhibitor Assays in Haemostasis

Introduction

Inhibitors are antibodies that in coagulation are usually targeted against either:
 - Specific clotting factors e.g. Factor VIII
 - 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 [commonly the APTT] 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 that target clotting factors may be either time-dependent e.g. Factor VIII inhibitors or immediate acting e.g. Factor IX inhibitors. Inhibitory antibodies may develop in an individual with an inherited deficiency of a clotting factor e.g. Haemophilia A or B - these are termed alloantibodies but may also arise in individuals with no history of an inherited bleeding disorder - termed autoantibodies.

Antibodies that are directed against FVIII or FIX but which are non-inhibitory i.e. non-neutralising antibodies - have been reported.

Factor VIII inhibitors are commonly directed against the A2, C1 and C2 domains of the molecule. Factor IX inhibitors frequently target the GLA domains or protease domains of the molecule.


Principles & Methodology
1. Screening Tests: Factor VIII Inhibitors


Screening for a FVIII 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. Factor VIII inhibitors are time dependent in contrast to FIX inhibitors which are immediate acting and hence the requirement for an incubation step at 37°C for 120 minutes.  This incubation step is not necessary if screening for a FIX inhibitor.
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.

Step Tube 1 Tube 2 Tube 3
  Normal plasma Test plasma Equal volumes of normal + test plasma
1 Incubate at 37°C for 120 minutes Incubate at 37°C for 120 minutes Incubate at 37°C for 120 minutes
2 Place samples on ice Place samples on ice Place samples on ice
3 Prepare equal volumes of normal + test plasma i.e. Tube 1 and Tube 2 [=Tube 4]
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, Tube 3 and Tube 4 - are shown in the table below:

APTT Results
Tube 1
[Normal plasma]
Tube 2
[Test plasma]
Tube 3
[Incubated mix of Normal and Test plasma]
Tube 4
[Immediate mix of Normal and Test plasma]
INTERPRETATION
Normal Normal Normal Normal → Normal
Normal Prolonged Prolonged Prolonged → Clotting factor deficiency
Normal Normal Prolonged Prolonged → Immediate acting inhibitor
Normal Normal Prolonged Normal → 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.

2. 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 neutralise 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.
The source of factor VIII is pooled normal plasma for anti-human titres and in the case of porcine factor VIII, the porcine factor VIII is diluted to 1 U/ml (100%) in factor VIII deficient human plasma.

3. Principles of 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 a 2hr incubation at 37°C.

The diagram below summarises the Bethesda assay and the Nijmegen modification:

A. The Bethesda assay:

Schematic illustrating the principles of the Bethesda assay and the Nijmegen modification


B. The Nijmegen modification of the Bethesda assay:

Schematic outlining the principles of the Njimedgen modification of the Bethesda assay

In more detail, a Bethesda assay is performed as follows:

1. Doubling dilutions of test (patient) plasma [usually 1/2 - 1/1024] are made in Imadazole buffered saline and incubated with an equal volume of a 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. A smaller number of dilutions can be made if the inhibitory titre is already known.
In the Nijmegen modification, the dilutions of patient plasma are made in Factor VIII deficient plasma.

2. A control mixture consisting of an equal volume of normal plasma mixed with Imadazole buffered saline (or in the case of the Nijmegen modification - see below, immunodepleted factor VIII deficient plasma) is prepared. The normal plasma pool will contain ~ 100% [100 IU/dL] Factor VIII. This mixture actually has a starting concentration of 50% [50 IU/dL] 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. The use of the control compensates for the deterioration in Factors VIII and V during the incubation period.

3. At the end of the incubation period the residual factor VIII is assayed using a standard 1-stage APTT based assay using the incubated control as the 100% [100 IU/dL] standard.

4. 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 titre for each dilution and take the average. Any residual factor VIII <25% [25 IU/dL] or >75% [75 IU/dL] should NOT be used for the calculation of inhibitor level.

5. If the residual factor VIII activity is between 80-100% [80-100 IU/dL] the sample does not contain an inhibitor.

6. 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.

7. 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 - see below.

In the table and the graph below, four 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% 1.475 x 100 147.5 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.


Graph showing the calculation of a Factor VIII inhibitor


The equation:
   Bethesda Units [Bu] = (2 Log%RA) ÷ 0.30
- can be used to derive the inhibitory titre in Bu where RA is the Residual FVIII activity. 
Only RA that fall between 25 and 75 IU/dL (%) can be used to convert to BUs. Any residual factor VIII activity <25 IU/dL or  >75 IU/dL should NOT be used for the calculation of inhibitor levels.

Factor Deficient Plasma
For FVIII inhibitor assays, deficient plasma must contain normal levels of VWF.  In the case of immunodepleted Factor VIII deficient plasma, the antibody used to remove the FVIII may still be present in low concentration but sufficient to influence the Bethesda Assay,  Similarly the use of deficient plasma from an individual with severe Haemophilia may contain an inhibitory antibody

4. The Bethesda Assay: Nijmegen Modification - see Figure above

Changes in pH and protein concentration can affect the stability of Factor VIII and its inactivation. By buffering the normal plasma and using Factor VIII deficient plasma in the assay, these variables are minimised/eliminated.

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 to make the serial dilutions of the patient plasma.


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 Nijmegen modified Bethesda assay is only required when the inhibitor value is low.

If a Nijmegen modification of the factor VIII inhibitor assay is used, inhibitory titres should be reported as Nijmegen Bethesda Units [NBu]/mL.

Modifying the Nijmegen Assay to detect very low titre inhibitors

Low titre inhibitors that are undetectable by the Nijmegen-modified assay can reduce the half-life of infused FVIII after/during immune tolerance. It is possible to increase the sensitivity of the Nijmegen assay 20-fold by selective protein filtration which increased the concentration of the inhibitory antibody in the test plasma. Residual FVIII is remove from the test plasma sample by heat treatment at 58°C for 90 minutes.  After centrifugation, the concentrated test plasma in mixed with Imidazole buffered normal plasma in a ration of 3:1 and incubated at 37°C for 120 minutes.  The residual FVIII is then assayed using a Chromogenic assay and from which the FVIII inhibitor level, employing an inhibitory algorithm, can be calculated - see References.

5. The Bethesda Assay: Heat Treatment

In patients receiving treatment with a Factor VIII concentrate, the measurement of inhibitors can be problematic as residual Factor VIII may be present and this can influence the Bethesda assay. Heating the plasma sample 56°C for 30 minutes and then centrifuging the sample leads to a FVIII:C and FVIII:Ag of <0.10 IU/mL. Immunoglobulins are heat resistant so the inhibitor level in the plasma sample is unaffected. This modification allows inhibitor measurement during FVIII replacement therapy and facilitates inhibitor surveillance in patients on FVIII replacement treatment.

6. Factor VIII Inhibitor Detection by ELISA

Factor VIII antibodies can also be detected using an ELISA assay.  Recombinant Factor VIII is immobilised onto a microtitre plate and diluted patient serum or plasma is added to the wells and incubated. If an anti-FVIII antibody is present it will bind to the immobilised Factor VIII. The plate is then washed and any unbound material is removed. An alkaline phosphatase labelled anti-human IgG (Anti-IgG) is added to the wells and incubated. The unbound Anti-IgG removed by washing and the substrate PNPP is added. After a further incubation step, the reaction is stopped [by the addition of sodium hydroxide] and the change in Optical Density [OD] is measured 405 nm and from which the presence or absence of a Factor VIII antibody can be established.

A number of variables have been shown to affect the detection of FVIII inhibitor by ELISA. Albumin-free recombinant FVIII as the target antigen appears to provide the highest sensitivity for the assay, whereas plasma-derived concentrates containing a high level of von Willebrand factor give the lowest sensitivity. Similarly some Factor VIII inhibitors may demonstrate activity against s specific FVIII product but not another due to sequence variations in the FVIII product used as the target antigen. 
Finally the ELISA assay may detect non-neutralising antibodies i.e. the presence of antibodies which are not identified by a functional clotting-based Bethesda Assay and the significance of these remains unclear. It is possible that may influence plasma clearance, survival times and the circulating levels of infused Factor VIII.


7. The Bethesda Assay: Chromogenic Assay

The chromogenic Bethesda assay is very similar to the standard Bethesda assay except the residual factor VIII is measured using a chromogenic FVIII assay. 

1 Chromogenic Bethesda Unit is defined as the level of inhibitor/mL in the patient plasma sample that inactivates 50% FVIII in 1mL of normal plasma.

Interpretation of Results

See above

Factor VIII Inhibitors: Kinetics

Factor VIII Inhibitors are classically divided into Types I or II depending upon the kinetics of the inhibitor:

Type I FVIII Inhibitors: These 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 [but not always] demonstrate Type I kinetics and demonstrate complete inhibition of FVIII activity.
.
Type II FVIII Inhibitors: These show more complex kinetics [type II or second-order kinetics ] and are commonly [but again not exclusively] seen with autoantibodies and 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.


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.

Type Kinetics Inhibition of FVIII Seen with:
Type I Simple - First Order Complete Alloantibodies arising in a person with Haemophilia A 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:

Graphical Illustration of Type I and Type II Inhibitors in Haemophilia A



In cases of haemophilia complicated by an inhibitor, most arise in individuals with severe Haemophilia A i.e. FVIII:C 0.01 IU/mL [<1 IU/dL]. However, in cases of mild-moderate haemophilia A that develop an inhibitor - the inhibitor can be of two types:
i. Basal endogenous 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.

Both Type I and Type II inhibitors have been reported in mild Haemophilia A but there appears to be a predominance of Type II inhibitors.


What Test Next?

1. Autoantibodies: In individuals with an acquired factor inhibitor it is important to investigate possible causes for the development of the inhibitor. The incidence of acquired haemophilia A increases with age, and is very rare in in children.  The age distribution of autoantibodies is biphasic with a small peak between 20 and 30 years due to post-partum inhibitors and a major peak in individuals aged 68 - 80 years.
In ~50% of cases, Factor VIII autoantibodies occur in patients without any obvious cause whilst the remainder may be associated with the post-partum period, autoimmune diseases, malignancy, infections and rarely some drugs.

2. Alloantibodies:  Routine surveillance for inhibitor formation in all individuals with an inherited bleeding disorder on regular replacement therapy is an essential part of their follow-up and there are both national and international guidelines on how such individuals who develop an inhibitor should be managed - see References.