2. Coagulation Inhibitor Potential [CIP] Assay
Introduction
The Overall Haemostatic Potential [OHP] assay is a global assay of Fibrin generation and Fibrinolysis and which has been evaluated in a number of clinical situations.
Principles & Methodology
The Test involves the generation of a Fibrin Time Curve in which the generation of Fibrin and its subsequent lysis is measured by changes in absorbance at 405nm.
1a. Normal or Test platelet poor plasma is diluted with a Buffer containing CaCl2, Thrombin [IIa] and t-PA [tissue Plasminogen Activator. The absorbance at 405nm is measured every minute from Time = 0 to Time = 30 minutes. The Fibrinogen in the sample is converted to Fibrin and the added t-PA converts the Plasminogen in the sample to Plasmin and which digests the Fibrin into soluble fibrin fragments.
1b. A 'Blank' is created by diluting Normal or Test plasma with a Buffer that contains no CaCl2, Thrombin [IIa] or t-PA.
2. The Area under the curve is determined by adding together the Absorbance values recorded during the 30 minutes of the test - termed the ABS-sum and then deducting the background absorbance, which corresponds to the initial optical density of the plasma sample before initiation of clotting. The ABS-sum is calculated from the formula:
..where ABS-sum [X] is a mean summation of ABS values obtained from duplicate samples of an unknown plasma sample and ABS-sum [N] is a mean summation of ABS values obtained from duplicate samples of a known pooled plasma sample run in parallel.
When the Fibrin curve is analysed, each ABS value on the graph equates to a specific amount of Fibrin generated at a specific time point.
The Overall Fibrinolysis Potential [OFP] can be calculated from:
.. and represents the data derived from two fibrin aggregation curves - the OHP assay containing t-PA and the OCP that does not contain t-PA. An additional parameter - the Clot Lysis Time [CLT] can be derived and represents the time from 50% of the maximum clotting to 50% of clot lysis.
The Coagulation Inhibitor Potential (CIP) assay is a further development of the Overall Haemostatic Potential [OHP] assay in which a Pentasaccharide is added to the plasma sample to activate Antithrombin and so increases the sensitivity of the assay to functional abnormalities/deficiencies of Antithrombin and in addition Protac is included which activates Protein C to activated Protein C [APC]. In addition omitting t-PA allows more rapid testing recognising that abnormalities of Fibrinolysis are a rare cause of an inherited thrombophilia.