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A Practical Guide to Haemostasis


Factor Assays: Introduction

Introduction

Factor assays are commonly undertaken when there is prolongation of the APTT or PT suggesting a deficiency of one or more clotting proteins. However, assays of other clotting factors e.g. FXIII or of the natural anticoagulants e.g. Protein C, Protein S and Antithrombin also constitute factor assays. Thrombophilia testing is covered in a separate section of the website.

Factor assays can be broadly divided into:

  • - 1-Stage PT-Based assays
  • - 1-Stage APTT-Based assays
  • - 2-Stage APTT/Chromogenic assays
  • - 1-Stage RVV Factor X assay
  • - Factor XIII Assays

Assays of Von Willebrand Factor are also included in this section although a functional assay for Von Willebrand factor that uses platelets is covered in the section on Platelets. Inhibitor assays are covered in a separate section.


Principles

The general principles of all functional clotting factor assays are the same and involve plotting the clotting times (generally derived from either a PT or an APTT test depending upon which factor is being assayed) against sample dilution. The degree of correction of the clotting time when the plasma is added to a clotting system specifically deficient in the clotting factor to be measured, allows the level of that clotting factor to be determined e.g. Factor IX deficient plasma is used to assay the level of Factor IX. The Factor deficient plasma should have a level of the factor being assayed of <0.01 IU/mL and normal levels of all the other clotting factors that affect that particular test.

In each case a reference plasma with a known level of a specific clotting factor is required. Historically this was a pool of normal plasma and it was assumed that the levels of each of the clotting factors was 100%. This has now been replaced by commercial plasma standards and the concentrations of the various factors in this reference material are provided by the manufacturer. In most cases, where these exist, the standard is calibrated against an international clotting factor standard and the factor assay result plotted in International Units per ml [IU/mL] or International Units per dl [IU/dL].

Methodology

The methods for the various assays are covered throughout Practical-Haemostasis.com.  It is important to remember that in many laboratories, factor assays are now fully automated. Historically coagulation tests were based on a visual assessment of the formation of a fibrin clot within a test tube.  The time between initiation of clotting and the formation of the fibrin clot assessed visually, represented the clotting time for a particular tests e.g. PT,  APTT etc.   However most labs now employ auto-analysers and there are a number of different methods employed to detect the formation of the fibrin clot including both mechanical and photo-optical approaches.  The photo-optical method involves the detection of a change in optical density of the plasma sample as the fibrin clot forms whereas in the mechanical method the movement of a magnetic ball or the movement of two probes in relation to each other is retarded as the fibrin clot forms and this change is recorded as the clotting time.

A series of dilutions of both the Reference plasma and the Test plasma are performed.  Dilutions are chosen to reflect the clotting factor activity in the sample to be assayed. At least three dilutions of both the Reference Plasma and the Test Plasma are required for a factor assay.  Additional dilutions may be required if the clotting factor levels fall outwith the range of the assay i.e. the test clotting factor level is very high or very low.
The use of three dilutions improves accuracy and allows for the detection of inhibitors - these can include specific clotting factor inhibitors [e.g. a Factor VIII inhibitor] non-specific inhibitors  or an anti-phospholipid antibody i.e. a Lupus Anticoagulant.  In cases in which an inhibitor is present, dilution of the test plasma sample may dilute the inhibitory antibody sufficiently such that its effect is minimised and the underlying clotting factor can be assayed, Similarly in the presence of a Lupus Anticoagulant the use of Lupus Anticoagulant insensitive reagents may allow the accurate measurement of the clotting factor.

A single point assay of a test plasma sample will always generate a parallel line even if it is not parallel. The use of a single point assay also significantly increases the CV of the assay - see Quality Assurance.

Interpretation

An assay must be both specific [i.e. it measures only the factor of interest and under investigation] and it must be sensitive i.e. it can detect low levels of the clotting factor under investigation.