Haemostasis and coagulation: plasmas, purified factors and assay reagents

Haemostasis is the set of mechanisms that stop blood loss from an injured vessel while keeping it patent. It has three phases: primary haemostasis, in which platelets adhere and aggregate through von Willebrand factor; coagulation, a cascade of serine proteases that ends in thrombin generation and the conversion of fibrinogen into fibrin; and fibrinolysis, in which plasmin dissolves the clot. Opposing the system are the natural anticoagulants — antithrombin, protein C, protein S, TFPI — whose deficiency shifts the balance towards thrombosis. In the laboratory, haemostasis is studied with global clotting-time tests, single-factor assays, antigen assays and thrombin generation: each approach calls for different reference materials.

Why does a factor assay need a deficient plasma?

The one-stage assay measures how far the test plasma corrects the prolonged clotting time of a plasma lacking the factor of interest, against a calibrator. The deficient plasma therefore acts as the substrate: it must contain every other factor at normal concentration and the measured factor at a negligible level, conventionally below 1 IU/dL. If the residual level is higher, the curve flattens and severely deficient samples can no longer be told apart.

Deficient plasmas are obtained either from subjects with a severe congenital deficiency or by immunodepletion, selectively adsorbing the factor from a normal pool. The two origins behave comparably as substrate; what differs is availability, the residual content of associated proteins — in factor VIII deficient plasma, for instance, the presence or absence of von Willebrand factor — and the format, frozen or lyophilised.

Which materials are needed, and for which test

The choice of material follows the information sought: functional activity, antigen quantity, presence of an inhibitor, or quality control of the analytical system.

How is a factor VIII inhibitor measured?

When a patient develops an antibody that neutralises factor VIII, activity can no longer be measured by the one-stage assay alone: the Bethesda assay is required. Dilutions of the test plasma are incubated for two hours at 37 °C with a known amount of factor VIII in normal plasma, and residual activity is measured. One Bethesda unit is defined as the amount of inhibitor that leaves 50% residual activity in the test mixture. The Nijmegen modification, introduced in 1995, buffers the normal plasma with imidazole at pH 7.4 and uses immunodepleted factor VIII deficient plasma for the dilutions: by standardising pH and protein concentration it improves discrimination between positive and negative samples. Inhibitor plasmas at mild, moderate and severe titre serve as control material for the assay.

Purified proteins, substrates and activators

Alongside plasmas, defined systems are built from purified proteins: prothrombin, thrombin, factors VII, IX, X, XI, XII and XIII, protein C, antithrombin, plasminogen and plasminogen-depleted fibrinogen, also available free of von Willebrand factor and fibronectin. To these are added the activators and inhibitors in routine use: Russell's viper venom, which activates factor X and underlies the dRVVT test for lupus anticoagulant, and corn trypsin inhibitor (CTI), which blocks factor XIIa and suppresses contact activation in thrombin generation assays. Animal versions — bovine, murine, rabbit, porcine, canine — serve in experimental models where the human reagent does not apply.

Plasmas and factors in the catalogue

The references below come from Affinity Biologicals and Enzyme Research Laboratories. The list is partial: the full range of deficient plasmas, inhibitor plasmas, controls and paired antibody sets is in the product catalogue and in the deficient and control plasmas category. The intended use of each reference is the one stated by the manufacturer in its own documentation.

To identify the reference suited to a given factor or test, it is enough to state the code or the analyte and write to CABRU.