Assay of Plasma Clotting Factors Using Parallel-Line Bioassay Principles

• Assay of Plasma Clotting Factors Using Parallel-Line Bioassay Principles

AUTHOR(S)

AS LAWRIE, G PURDY, IJ MACKIE, and SJ MACHIN

Haemostasis Research Unit, Department of Haematology, University College London

SUMMARY

The specific quantification of clotting factors is central to the laboratory investigation of a prospective haemorrhagic diathesis or prothrombotic state. Plasma clotting factors are assayed by assessing the degree to which dilutions of a test and reference plasma correct the clotting time of a substrate plasma ( specifically deficient in the factor ) measured in a suitable test system. The test system used is dependant on the clotting factor investigated. The prothrombin time ( PT ) system is commonly used for the assay of factors II, V, VII, X and the activated partial thromboplastin time ( APTT ) for factors VIII, IX, XI, XII. The range of sample dilutions to be used in an assay system are determined by testing a wide range of normal plasma dilutions ( e.g. doubling dilutions 1/2 _ 1/1024 ). This process produces a classical sigmoid dose response curve with a central linear component. The sample dilutions to be used are selected to produce a series of clotting times which fall towards the middle of that central linear component. Data produced in an assay of this type can be analysed by graphical means ), comparing the dose response of serially diluted test plasma with corresponding dilutions of a standard ( or reference preparation ). Plotting the clotting times against log concentration in this procedure allows not only quantification, but also reveals evidence of inhibition or sample activation, processes which can either be of pathological or artefactual origin.

In the absence of an inhibitor or sample activation, the relationship between the logarithm of the dose ( i.e. sample dilution ) and the response ( i.e. clotting time ) is represented by a straight line over the range of dilutions tested. For any unknown preparation the straight line is parallel to that for the standard. Using these procedures the experienced scientist can make a visual assessment of the relationship ( linearity and parallelism ) between the reference preparation and the sample under test. However, data analysed solely by graphical means introduces subjectivity associated with fitting lines to data points visually and ignoring the information which optimal experimental design together with subsequent statistical analysis would yield ). To obtain a more complete analysis of coagulation assay data a number of computer programs have been described ), but these descriptions supply only general principles of bioassay programs ) or are for use on large computer systems ), and are not readily introduced into clinical haemostasis laboratories.

KEY WORDS

Plasma Clotting Factors, Assay Validation