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A Practical Guide to Laboratory Haemostasis

 

1-Stage PT-Based Factor Assays



Introduction

To establish the levels of factors V, VII, X and II [prothrombin] a method based upon the Prothrombin Time (PT) is used.

Principles

The assay of a clotting factor relies upon measuring the degree of correction of the prothrombin time (PT) when plasma is added to a plasma sample specifically deficient in the factor to be measured.

Method

You will need a sharp pencil, a clear ruler, possibly an eraser (!) and some Log-Log graph paper.
Prepare dilutions of a plasma standard and control plasma samples in buffer e.g. 1/5, 1/10, 1/20, 1/40, 1/80 etc.

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


Check the concentration of the standard reference plasma and the units that are used:
1. Is the standard reference plasma 100 IU/dl? If not you will need to make a correction to take this into account with any factor assays you derive using this standard.
2. Are the units for the reference plasma IU/dl or IU/ml or %? Make sure you use the same units and do not switch % or IU. The use of 'IU' implies that the plasma has been calibrated against an international standard. The use of '%' suggests that a calibration against an international standard has not been performed.
3. Plot the results – usually on Log-Log paper i.e. a logarithmic scale on the X-axis and a logarithmic scale on the Y axis. Dilutions are always plotted on the X axis and clotting time (s) on the Y axis. Dilutions start at the right with the smallest dilution. Clotting times start at the bottom of the Y axis and as the clotting times increase you move up the Y axis.
4. REMEMBER – the axes are LOG scales (for plotting PT-based factor assays) and you need to make sure you orientate the scales correctly - see the movie below.
5. The smallest dilution of the standard is usually assigned a value of 100% (100 IU/dl). However, you can assign any dilution of the standard a value of 100% so long as all the dilutions take this into account e.g. if a 1/10 dilution is assigned a value of 100 IU/dl (or 100%) then 1/20 dilution will have a value of 50 IU/dl (50%), a 1/40 dilution of 25 IU/dl (25%) and so on. If a 1/20 dilution is assigned a value of 100 IU/dl (100%) then 1/40 dilution will have a value of 50 IU/dl (50%) and so on.
6. Click HERE to see a video which will show you how to plot a 1-stage FVII assay based on the PT [The assays in the website are in a QuickTime format and you may need to download the QuickTime Player from the Apple Website.]

In the table below is the raw data that we have used to plot the assay shown in the movie.

Raw data used in the movie

  Dilutions
  1/10 [100%] 1/25 [40%] 1/50 [20%] 1/100 [10%]
PT [s] Reference Plasma [FVII:C = 95 IU/dL] 28 32 37 42
PT [s] Patient Plasma 55 59 73 80

 

Interpretation

The graph below shows the results of a 1-stage PT-based factor X assay with varying factor X levels - note the parallel lines. The reference plasma is shown in red. The axes are both logarithmic and the dilutions are plotted on the X-axis and the clotting time in seconds on the Y axis.

A vertical line is drawn from the dilution that represents 100% - in the case the 1/10 dilution - any dilution can be taken as 100% so long as you treat the patient samples in exactly the same way as the reference plasma standard. Where this line intercepts the line of best fit for the reference plasma standard (shown here in red) - a line at right angles to this is drawn until it intercepts the plasma sample being assayed (shown here in green). A vertical line is then dropped until it intercepts the X-axis. In the case it intercepts the X-axis at 2.75 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 2 IU/dl. if the standard is 110 IU/dl then the corrected factor assay is 2.75 x 110/100 = 2.5 IU/dl.

Remember for a log scale if the 1/10 dilution then the concentration is 100% [100 IU/dL] then a 1/100 dilution is 10% [100 IU/dL] and a 1/1000 dilution is 1% [1 IU/dL].

Reference Ranges

Laboratories should establish their own reference ranges although it is probable that many do not and choose to use either published ranges or those provided with a commercial standard. For most 1-stage PT derived factor assays, the reference range will lie between 50-150% [50-150 IU/dL or 0.5-1.50 IU/mL.]

What Test Next

1. If you find a low FV result (and similarly if you find a low FVIII result) you should request a FVIII (or FV) assay to exclude these rare autosomally inherited disorders.
2. If you find a low FVII result make sure that the test was performed using human TF in the PT. Some F7 gene mutations e.g. FVII Padua can give rise to varying factor levels depending upon the source of TF used in the PT. Wherever possible, human recombinant TF should be employed as this gives a result that more closely relates to the situation found in vivo.
3. If you find a low level of a vitamin K dependent clotting factor - consider the possibility that that the patient is on warfarin, has true vitamin K deficiency or may have a mutation within the genes involved in encoding the proteins involved in the vitamin K cycle.
4. Remember - PT-based factor assays will be reduced in patients with vitamin K deficiency and in patients on oral vitamin K antagonists such as warfarin.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.