The Platelet Function Analyzer 100 (PFA100) is an auto mated benchtop instrument that evaluates primary hemostasis in vitro in a shear-stressed blood sample. The PFA-100 uses disposable test cartridges, each containing a nitrocellulose membrane impregnated with collagen plus ADP (Col/ADP) or collagen plus epinephrine (Col/Epi). A sample of citrated blood (0.8 mL) is placed in a cup of the instrument and aspirated through the membrane opening. Shear stress occurs when platelets pass through Col/ADP or Col/Epi, which act as activation agonists, aggregate and progressively reduce until the flow through the membrane is stopped. This represents the endpoint, expressed as the time of closure. The formation of platelet aggregates depends on the following:
• The binding of von Willebrand factor to the collagen- coated nitrocellulose membrane
• Platelet adhesion to von Willebrand factor via activation of the gp Ib receptor
• Platelet aggregation is mediated by the interaction of gp IIb/IIIa with von Willebrand factor and fibrinogen
Normal closure times range from 77 to 133 s for the Col/ADP membrane and from 98 to 185 s for the Col/Epi membrane. The PFA-100 has been tested in patients with bleeding disorders. The closure time using the Col/Epi cartridge is abnormal in patients with congenital defects of platelet function and von Willebrand disease after aspirin intake, whereas the closure time using the Col/ ADP cartridge is abnormal mainly in patients with von Willebrand disease and in congenital diseases. Aspirin prolongs the closing time in 94% of cases with the Col/ Epi cartridge and only in 27% of cases with the Col/ADP cartridge. Glanzmann thrombasthenia, Bernard–Soulier syndrome, and most mild forms of von Willebrand dis ease are associated with prolonged closing time with both cartridges, whereas a storage pool defect and giant platelet thrombocytopathy have prolonged closing time only with the Col/Epi cartridge.
The advantages of this instrument include simplicity and reproducibility. The PFA-100 has been reported to have a coefficient of variation of less than 10%. It may be useful for determining global platelet function and assessing the efficacy of antiplatelet therapy.
Platelet aggregation is measured by turbidimetric methods. When platelets aggregate, the platelet-rich plasma sus pension becomes clearer and allows more light to be transmitted. The degree of aggregation is determined by measuring the progression over time, usually for 5 min, of the increase in light transmission and by reporting the aggregation curve, which evaluates the reversibility/irreversibility of aggregation (primary vs. secondary), the shape change, that is, the change in platelet shape upon stimulation with agonists such as ADP and collagen, and the maximum amplitude of the aggregation wave.
Small doses of ADP (<1 μmol) induce a reversible form of platelet aggregation (primary wave), unaccompanied by thromboxane synthesis and intraplatelet ADP release. However, with increasing doses of ADP, sufficient stimulation of platelets occurs with intraplatelet ADP release and thromboxane A2 synthesis by rapid metabolism of arachidonic acid (released from platelet membrane phospholipids) by the cyclo-oxygenase enzyme; the final consequence is a more pronounced and irreversible aggregation wave (secondary wave). A secondary wave of aggregation is also caused by collagen and directly by arachidonic acid, which is used as an agonist. Ristocetin causes platelet aggregation by inducing von Willebrand factor to bind to the gp Ib protein complex on the platelet.
Platelet aggregation tests are useful in distinguishing various disorders of platelet function. They are also particularly used in the differential diagnosis of von Willebrand disease, in which ristocetin-induced platelet aggregation is defective.
