Practical-Haemostasis.com

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

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

So: 

The dilutions you may be given in an exam-type setting for the standard and test samples may not be the same – be aware of this when you come to plot the data. For example if the dilutions for the reference plasma are 1/64, 1/128 and 1/256 but 1/4, 1/8 and 1/16 for the test sample the you will have to make an adjustment to the final answer to take into account these differing dilutions. If the 1/64 dilution is taken as 100% and used to derive the factor assay in the test sample - the value in the latter will have to be divided by 4 to take into account the differences in dilution - see the video and data below for clarification.

1. Check the concentration of the standard reference plasma and the units that are used:
• 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.
• Are the units for the reference plasma IU/dl or IU/ml or %? Make sure you use the same units and do now 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.
2. 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.
3. 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 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.]
   
   In the table below is the raw data that we have used to plot the assay shown in the movie.
   
   

   	Dilutions
   	1/10	1/25	1/50	1/100
   PT (s) Standard Reference Plasma [FX:C 95 IU/dL]	28	32	37	42
   PT (s) Patient Plasma	55	59	73	80

Interpretation

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 blue) - a line at right angles 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 2%. This represent the factor level in the unknown plasma sample. If the 1/100 dilution is not 100% 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 x 110/100 = 2.2 IU/dl = ~2 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!

The graph below shows the results of a factor X assay with varying factor X levels. Note the parallel lines.

Reference Ranges

Establishing reference ranges is a 'hot topic' in haemostasis. The reference range for most PT-based factor assays is 50-150 IU/dL.

Comments

1. Remember – you cannot dilute nothing! So if all dilutions in a plasma sample give a prolonged but similar clotting time the factor level is likely to be less than 1 IU/dl (or <1%).
2. If you plot the standard reference material using a series of plasma dilutions but only a single point for your unknown plasma sample - you can always generate a parallel line! Some automated machines do this assuming that the lines for the control and plasma sample are parallel. However, the lines may not be parallel e.g. in patients with an inhibitor.
3. Some labs favour calculating the factor level for each sample dilution separately and then deriving the mean of these but there is little advantage to this if the lines are parallel.
4. To have accuracy the lines must have an adequate slope. The minimum slope should not be less that that produced by a 50% prolongation of clotting times as a result of a 10-fold dilution of the test sample. So - if a 1/10 dilution gives a clotting time of 40s then a 1/100 dilution should give a clotting time of at least 60s (40 [+ 50% of 40] = 60s).
5. Make sure you report the values for your test sample using the same units as the standard e.g. U/ml, IU/ml, IU/dl, % etc.
6. If you decide to undertake a common pathway factor assay e.g. II, V or X - because both the PT and APTT were prolonged - don't forget that this may be a case of multiple clotting factor deficiency e.g. in a patient on oral vitamin K antagonists or a case of combined factor V and VIII deficiency.

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 and Yamomoto, 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 related 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 invovled in encoding the proteins involved in the vitamin K cycle.

Useful Links & References

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.