1-Stage APTT-Based Factor Assays

A 1-stage APTT-based factor assay is widely used for the measurement of factors VIII, IX, XI and XI. It can also be used for assaying factors X, V and II although a PT-based assay is more commonly used. For the measurement of fibrinogen - see Fibrinogen Assays.

 

Principles

The APTT assesses the interaction of a large number of coagulation factors. For it to be valid all the factor levels must also be normal or nearly so (depending on the methodology the APTT may be insensitive to mildly reduced factor levels). Conversely, when factor levels are low the APTT becomes longer. There are two key principles we can use here:

  1. The APTT has a clear mathematical relationship with the concentration of coagulation factor.
  2. If the only variable altering the APTT of a sample relative to normal plasma is the level of a specific coagulation factor then the value of the APTT can be used to calculate the concentration of that factor.

Therefore, so long as we can determine what the relationship in a) is and that that the only variable in b) is the coagulation factor of interest - we can use the APTT to measure the level of those coagulation factors which influence it, which in practice most commonly means factors VIII, IX, X & XI. Factor XII can also be assayed using this technique as can pre-kallikrein and although neither is associated with a bleeding tendency they can both result in significant prolongations of the APTT.

 

Method

To assay a sample using the APTT requires:

APTT reagents

See APTT

Standard Reference Plasma

This will be used to establish a benchmark from which the factor levels in other plasma can be determined by comparing clotting times. It is assigned a value of 100% of all coagulation factors and may be produced by mixing plasma from a number (e.g. 40) of normal volunteers.

Many labs now purchase commercial reference standards in which the levels the clotting factors are calibrated against international standards and their levels reported in IU/dl or IU/ml and not %.

Factor deficient plasma (substrate plasma)

A plasma completely deficient in a factor relevant to the APTT - most commonly VIII, IX, or XI. Such plasma is usually commercially available but can be prepared from donors (e.g. a known severe haemophilia A patient for FVIII deficient plasma.) If this plasma is prepared from a donor then factor levels must be <1 IU/dL [%] and the absence of any inhibitors should be verified.

Patient plasma

Patient platelet poor plasma [PPP]

Modern analysers perform all of the relevant calculations but for the purposes of illustrating the principles we will use graph paper. There is a link to a worked example you can watch at the end of this section. To take factor VIII as an example:

  1. Determining the mathematical relationship between APTT and concentration of coagulation factors is relatively straightforward. Serial (e.g. doubling 1/10, 1/20, 1/40 etc) dilutions of a standard reference (i.e. normal) plasma are mixed with an equal volume of substrate plasma (i.e. plasma which has normal levels of all the other factors but is deficient in the clotting factor that is being assayed in this case factor VIII) and an APTT performed.

  2. The clotting times for the APTT are then plotted against dilution on Log-Lin graph graph and a best fit line drawn through them. That line allows us to determine the dilution of normal plasma required to produce any given APTT. As the relationship between dilution and APTT is exponential, if the points are plotted on Log-Lin paper a straight line is produced.

    Dilutions
    [of standard]    		 1/10 1/20 1/30 1/40 1/80 1/100 1/1000
    % Activity 100% 50% 33% 25% 12.5% 10% 1%


  3. The test plasma is treated the same way as the reference plasma with serial dilutions mixed with equal volumes of substrate plasma. Since the substrate plasma contains no factor VIII but provides normal amounts of the other factors the only significant difference between our test plasma dilutions and the standard reference plasma dilutions is the factor VIII level of the test sample.

  4. Now, we perform APTTs of the test plasma (patient/factor deficient plasma mix) for each dilution and plot the results on Log-Lin paper [dilutions on the X-axis and clotting times on the Y axis.]

  5. The result should be a straight line running parallel to the line for the standard reference sample unless the factor VIII level in our test sample is exactly the same as that in the standard sample in which case the lines will be superimposed. If the lines are not parallel - there are several possibilities:
    1. You have plotted the results incorrectly - check again
    2. An inhibitor is present - often with an inhibitor the factor being measured increases with increasing dilutions as the inhibitor is diluted out.
    3. Remember you cannot dilute nothing - if the all the clotting times are grossly prolonged and do not change with increasing dilutions - the factor level is probably <1 IU/dl [%].

  6. A vertical line is drawn from the dilution that represents 100 IU/dl - in the case the 1/10 dilution. Where this line intercepts the line of best fit for the reference plasma standard (shown here in blue) - a line at right angle to this is drawn until it intercepts the plasma sample being assayed (shown here in red). A vertical line is then dropped until it intercepts the X-axis. In the case it intercepts the X-axis at 5 IU /dl. This represent the factor level in the unknown plasma sample. If the 1/100 dilution is not 100 IU/dl then a correction can be made at this stage. In the case the plasma sample has a value of 5 IU/dl. if the standard is 104 IU/dl then the corrected factor assay is 5 x 104/100 = 5.2 IU/dl = ~5 IU/dl.

    Remember if the plasma standard is given as IU/dl then the unknown should be reported as IU/dl. If it is given in % or IU/ml use the same units - do not switch!

    Click HERE to see a video which will show you how to plot a factor assay based on the PT [The assays in the website are based on Quicktime movies and you may need to download the Quicktime Player from the Apple Website -click HERE to go to the Apple Website.]

Data used in the movie:

 
Dilutions
1/10 1/25 1/50 1/100

APTT (s)
Standard Reference Plasma [FVIII:C 104 IU/dL]

 47 55 62 67s

APTT (s)
Patient Plasma

 72 81 85 94

This graph below shows the results of a factor FVIII assay with varying factor FVIII levels. The reference plasma standard is plotting in red.
Note that the lines are parallel.

 

Interpretation

In general the level of factors VIII and IX correlate with bleeding risk.

Severity Factor Levels
Severe haemophilia A or B Factor VIII or IX <1 IU/dL [%]
Moderate haemophilia A or B actor VIII or IX is >1 IU/dL but less than <5 IU/dL [%]
Mild haemophilia A or B factor VIII or IX is between 5-30 IU/dL [%]

In factor XI deficiency the bleeding risk varies between individuals with the same factor level but is consistent for a given individual.

Failure to obtain a straight line when plotting the APTT/dilution of the test sample may indicate the presence of an inhibitor.

 

Reference Ranges

Reference ranges may be expressed as either a percentage or in IU/dl. Many factors, including factors VIII, IX, X, have reference ranges of 50-150% alternatively expressed as 0.50-1.50 IU/ml or 50-150 IU/dl. The range for factor XI is narrower at 0.65 – 1.25 IU/ml.

Reference ranges for a given factor do not change with age above 6 months, however, neonates have physiologically lower normal ranges due to immaturity of the fetal/neonatal liver. These ranges vary with age and a full list is available at: http://www.bcshguidelines.com/pdf/Neonatal020703.pdf.

 

Comments

This method is subject to all the pre-analytical variables which can affect the APTT (see pre-analytical variables) including variations in method (e.g. different sources of phospholipid or factor deficient plasma) and instrument performance. The preparation of the reference curve and the evaluation of patient values also increase the potential for test result variation. As a result there may be considerable inter-laboratory variation. This situation is improving with better automation and international standardisation as well as commercially available plasma preparations but should still be born in mind when comparing results from different laboratories.

The APTT based assays have an inherent degree of imprecision especially due to interference in optical density measurements from lipids or traces of heparin in samples and its sensitivity to preactivation of factor VIII. Alternative methods for assaying factor levels exist and are used under certain circumstances, these include a chromogenic assay and a two-stage coagulation assay which are used by the pharmaceutical industry to determine coagulation factor concentrate potencies.

 

What Test Next?

The following table provides a summary of what tests might be undertaken finding an abnormal factor assay.

Specific factor deficiencies

 

1. Factor VIII

If factor VIII deficiency is a new and unexpected finding then factor V levels should be checked. This is unnecessary if a finding of factor VIII deficiency is expected (e.g. child within a haemophilia A pedigree).

There are rare cases in which patients with factor VIII deficiency have factor VIII levels inconsistent with their bleeding history but in whom a 2-stage factor assay provides a different factor VIII level (usually lower) more consistent with their clinical state.

Acquired Factor VIII deficiencies. An otherwise healthy individual may develop on autoantibody against factor VIII leading to acquired haemophilia A. Such antibodies are also seen in patients with immunological disorders e.g. rheumatoid arthritis.

If you find a low FVIII level - you need t check the Von Willebrand Factor [VWF] to exclude Von Willebrands Disease [VWD] as the cause of the low FVIII.

You can also find a low FVIII in acquired Von Willebrands Syndrome [AVS].

The finding of a low FVIII or FIX in a female with no family history may require establishing whether there is a normal karyotype e.g. Turners syndrome.
2. Factor IX Low factor IX levels are seen in haemophilia B. Acquired FIX inhibitors are rare.

2. Factor X

If factor X deficiency is suspected but an APTT- based assay does not give the expected results consider using an alternative technique (e.g. PT based assay, a chromogenic assay, immunoassay) as mutations causing deficiencies exist which are detectable only with certain techniques. Acquired FX inhibitors are are rare but a low factor X level may be seen in some patients with amyloidosis.

3. Factors VIII, IX, XI

Mutation analysis may facilitate pedigree analysis and antenatal screening.

Inhibitors

 

1. Specific factor inhibitors

Can be assayed. The Bethesda Assays and its variants are commonly employed for this

2. Phospholipid dependent

A lupus anticoagulant.
Reagents with a high phospholipid content can be used to overcome this.

 

Useful Links & References

Normal factor ranges in neonates www.bcshguidelines.com/pdf/Neonatal020703.pdf
F9 mutation database www.factorxi.org/
F8 mutation database http://europium.csc.mrc.ac.uk/Web Pages/Main/main.htm
F11 mutation database
http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html

 

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.