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A Practical Guide to Laboratory Haemostasis

 

1-Stage APTT-Based Factor Assays



Introduction

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 [Prothrombin] 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 factors.
2. If the only variable altering the APTT of a sample relative to a normal reference 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 is in (1) and that that the only variable in (2) is the coagulation factor of interest - then 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 & XI.
Factor XII can also be assayed using this technique as can pre-kallikrein and although neither is associated with a bleeding tendency they both result in significant prolongations of the APTT.

Method

To assay a sample using the APTT requires:

Reagents Interpretation
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 are 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 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 are plotted on the logarithmic X-axis and clotting times on the linear 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 then there are several possibilities:

You have plotted the results incorrectly - check again
An inhibitor is present. Often with an inhibitor, the clotting factor being measured increases with increasing dilutions as the inhibitor is diluted out.
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.]

Data used in the movie:

Plasma Source
Dilution
  1/10 1/25 1/50 1/100
APTT [s] Standard Reference Plasma
[FVIII:C Reference Plasma 104 IU/dL

]
47 55 62 67
APTT [s] - Test 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 plotted in red and the various plasma samples in purple. 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 Factor 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 or non-parallel lines 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, in general, change with age above 6 months, however, neonates have physiologically lower normal ranges due to immaturity of the fetal/neonatal liver.

In addition, factor VIII is an acute phase protein and so its levels will increase in response to stress including pregnancy and surgery.

What Test Next

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

Specific Factor Deficiency
FVIII 1. If factor VIII deficiency is a new and unexpected finding then Von Willebrand Factor levels and Factor V levels should be checked. This is unnecessary if a finding of factor VIII deficiency is expected (e.g. known person with a haemophilia A).
2. There are rare cases in which patients with factor VIII deficiency have discrepant factor VIII levels when measured by a 1-stage assay compared to a 2-stage or chromogenic FVIII assay. The levels are generally higher with a 1-stage assay but the bleeding phenotype correlates better with a 2-stage or chromogenic FVIII assay
3. Acquired Factor VIII deficiency. 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.
4. You can also find a low FVIII in acquired Von Willebrand Syndrome [AVS].
5. Factor VIII is an acute phase protein and the levels may be high in individuals who are stressed for any reason and this includes pregnancy.
6. The concentration of FVIII is a major determinant of the APTT and low FVIII levels will prolong the APTT and conversely high FVIII levels will shorten the APTT.
7. In a female with a low FVIII or FIX no relevant family history, the karyotype should be established e.g. Turners syndrome should be considered.
FIX Low factor IX levels are seen in haemophilia B.
Acquired FIX inhibitors [autoantibodies] are rare.
FX 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.
FXI FXI deficiency is rare but significantly more common in individuals of Jewish origin. In individuals of Jewish origin, for mutations within the FII gene account for the majority of mutations.
Any deficiency Consider undertaking:
1. Family screening
2. Mutation analysis

 

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.