The Importance of Using an Acceptable Linear Calibration Curve for Coagulation Factor Assays
Haemostasis is the balance of maintaining blood in a fluid state within blood vessels, limiting blood loss after trauma and the removal of blood clots in the process of wound healing. In an healthy status, haemostasis tends towards an anticoagulant state to maintain blood flow and prevent inappropriate thrombus formation. However after tissue injury, an intricate series of events are activated, involving platelets and specific blood proteins (known as coagulation factors) to promote clot formation and prevent excessive blood loss. Knowledge of haemostasis is important in understanding major disease states associated with thrombosis, such as venous thromboembolism (VTE or blood clots), atherothrombosis (thrombosis triggered by plaque rupture) or cardioembolic stroke. Haemostatic disorders can develop for many different reasons and can be congenital or acquired in nature.
The complex interplay of cellular and molecular components of haemostasis achieves a crucial yet fine balance of procoagulant and anticoagulant mechanisms. Deficiency or defect of any of the coagulation proteins disrupts clot formation and can give rise to a bleeding or thrombotic disorder, depending on where the defect occurs.
Coagulation factor assays are performed to determine the level of a particular factor which will indicate if a patient has sufficient coagulation activity to maintain haemostasis. A deficiency in any of the coagulation factors is associated with reduced clot formation and excessive bleeding, leading to the dysregulation of haemostasis. One or more coagulation factor activity tests may be ordered to evaluate the function of specific factors.
Accurate determination of clotting factor activity is essential in understanding why a patient may present with a prolonged activated partial thromboplastin time (APTT) or prothrombin time (PT). The screening tests employed to determine the haemostatic potential of each patient will include PT, APTT and Fibrinogen in the first instance, followed by mixing studies as a guide to whether the cause of prolongation is likely to be a factor deficiency, clotting factor inhibitor or lupus anticoagulant. Second line testing will then include intrinsic or extrinsic one stage clotting factor assay or inhibitor analysis, as appropriate.
One-stage clotting assays for determining coagulation factor function are based on the ability of dilutions of patient's plasma to correct the clotting time of a plasma that has normal levels of all clotting factors except the one being measured, in which it is totally deficient. Clotting times are derived via prothrombin time reagents for factors II, V, VII & X, and APTT-derived reagents for factors VIII, IX, XI, XII, prekallikrein and HMWK. Results are assessed from a parallel line bioassay graphical plot against that of standard plasma.
It is crucial to scrutinise dose response curves in order to establish a central linear component with acceptable differences between plasma dilutions. Also at least three dilutions of test plasma are needed to determine linearity and parallelism. The coagulation data must be checked to confirm the slope ratio or R squared on the print out but also by looking at the shape of the curve. By using the parallel line method to determine relative factor potency this may show evidence of inhibition (most commonly because of the presence of antibodies to coagulation factors or lupus anticoagulants). A point to point curve or a single point result can give a misleading result and inhibitors can be missed.
The importance of using a linear calibration curve was demonstrated in a recent factor IX NEQAS survey (sample 15.24). When using the current linear parallel line curve the results achieved were 117.3, 122.2 and 125.3 u/d L for the 1/1, 1/2, and 1/4 MDA dilutions respectively (mean 121.6 u/d L). Investigation revealed loss of linearity below approximately 15% factor IX activity, leading to subsequent protocol amendments which included altering the initial sample dilution from 1/5 to 1/20 and additional dilutions added to the calibration curve. Re-analysis of NEQAS sample 15.24 gave a mean of 104.8u/dL which would have been a Grade A EQA result.
For further assay results and information on the importance of using an acceptable linear calibration curve for factor assays, click here
One-Stage Factor Assay Service at Viapath
The one-stage factor assay is offered at Viapath’s Diagnostic Haemostasis & Thrombosis laboratory, based at St Thomas’ Hospital (http://www.viapath.co.uk/our-tests/factor-assays-one-stage-clotting-fii-fv-fvii-fviii-fix-fx-fxi-fxii-pk-hmwk). A Sodium citrate (light blue lid) sample is required and approximately four factor assays can be carried out from each 3.5mL sample. The turnaround time is 7 - 10 days.
For further information, please contact:
Clare Dunsmore, Senior Biomedical Scientist
Email: clare [dot] dunsmore [at] viapath [dot] co [dot] uk (subject: pathology%40viapath)
Dr Gary W. Moore, Consultant Biomedical Scientist, Head of Diagnostic Haemostasis
Email: gary [dot] moore [at] viapath [dot] co [dot] uk (subject: pathology%40viapath)
Elaine Bromidge, Senior Biomedical Scientist
Email: elaine [dot] bromidge [at] viapath [dot] co [dot] uk (subject: pathology%40viapath)
Haematology (Fundamentals of Biomedical Science) 1st Edition by Dr Gary Moore, Gavin Night & Andrew Blann (2010).