Inhibitors are antibodies that in coagulation are usually targeted against against either:

1. Specific clotting factors e.g. FVIII
2. Phospholipids

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 may be either time-dependent or immediately acting.

Principles

Screening tests based upon the APTT

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 37C.

Three tubes are prepared as shown in the table below. The tubes are incubated at 37C for 120 minutes and then placed on ice to stop the reaction. A 4th tube is prepared from equal volumes of Tube 1 and Tube 2 and APTTs are performed on all 4 tubes.

The interpretation of results of the 4 APTTs is shown in the table below:

Quantitative Measurement of Factor Inhibitors: The Bethesda Assay

Method

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 37C.

Some factor 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.

Factor VIII Inhibitors

1. Factor VIII inhibitors are usually time dependent so 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 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.
   
   
2. 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 1U/ml (100%) in factor deficient human plasma.
   
   
3. 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) the classical Bethesda assay can result in false positives whereas the Nijmegen modified assays would give zero levels of inhibition.
   
   
4. The assay can also be modified to use factor VIII concentrate and the incubation time can be increased to 4 hours.
   
   

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.
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 the control as the 100% (100 IU/dl or 1 IU/ml) standard.
5. The inhibitor strength calculated from a graph of residual factor VIII activity versus inhibitor units.
6. 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. One can also calculate the result for each dilution and take the average. Any residual factor VIII <25% or >75% should NOT be used for the calculation of inhibitor level.
7. 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.
8. Derive the inhibitor titre from the graph and multiply by the dilution to give the final titre.
9. A positive control plasma of known inhibitor titre should be included.
10. 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 inhibitory titre calculated.

Remember you must take into account the dilution when you have derived 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.

Comments

1. The Bethesda assay can give rise to false positives at low inhibitor levels [<1 Bu] whereas the Nijmegen modification would give zero levels. Change in pH and protein concentration can affect the stability of the FVIII and its inactivation increases as the pH increases and at low protein concentration. By buffering the normal plasma and using FVIII deficient plasma 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 in the control mixture.
   
   
2. To assay for a porcine FVIII inhibitor involves an identical protocol but the use of porcine FVIII rather than normal human plasma - see Bethesda Assay above.
   
   
3. The Bethesda assay was designed to measure FVIII inhibitors in patients with haemophilia A. However, it is used to test and report inhibitors in association with other clotting factors e.g. FIX, FXI etc. The principles are similar although in the case of some proteins in which the inhibitors are immediately acting e.g. FIX – there is no need for the incubation step.
   
   
4. In patients with complex inhibitor kinetics as commonly seen in patients with acquired inhibitors e.g. acquired FVIII inhibitors – the calculated inhibitor titre may increase as the dilutions increase.

Useful Links & References

1. Hay, C.R., Brown, S., Collins, P.W., Keeling, D.M. & Liesner, R. (2006) The diagnosis and management of factor VIII and IX inhibitors: a guideline from the United Kingdom Haemophilia Centre Doctors Organisation. Br J Haematol, 133, 591-605.
2. Goodeve, A. (2003) The incidence of inhibitor development according to specific mutations--and treatment? Blood Coagul Fibrinolysis, 14 Suppl 1, S17-21.
3. Goodeve, A.C. & Peake, I.R. (2003) The molecular basis of hemophilia A: genotype-phenotype relationships and inhibitor development. Semin Thromb Hemost, 29, 23-30.
4. Goodeve, A.C., Williams, I., Bray, G.L. & Peake, I.R. (2000) Relationship between factor VIII mutation type and inhibitor development in a cohort of previously untreated patients treated with recombinant factor VIII (Recombinate). Recombinate PUP Study Group. Thromb Haemost, 83, 844-848.
   
   

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.