Comparative investigations on the quantitative determination of ADP- and adrenaline-induced aggregation by means of a microscopic test and an electronic platelet counting method

Abstract

During platelet aggregation by photometric methods or during retention tests, platelets are subjected to varying mechanical forces. Already at a strength of about 50 dynes/cm 2, shear forces can induce the release of substances from platelet organelles. Forces of this magnitude may develop in photometric tests by stirring process or during passage of platelets through glass bead filters (retention tests). In contrast, such forces are not likely to arise in the microscopic aggregation method (“chamber test”). In the “chamber test” ADP or adrenaline is added to platelet rich plasma and the aggregation sample transferred to a counting chamber. In given time intervals the number of free platelets is counted by phase contrast microscopy and aggregation is calculated in terms of percentage. This method yields reproducible results and aggregation kinetics can be quantified. When 5 mM TRIS-buffer, pH 7.4, is added instead of aggregation inducers, and upon incubation of this plasma in the counting chamber for about one hour, spontaneous aggregation was noted, the kinetics of which resemble that of ADP-or adrenaline-induced aggregation. With higher concentrations of citrate (0.76% in blood) spontaneous aggregation can be inhibited to a large extent while ADP and adrenaline still induce aggregation (lower concentrations of calcium ions). By modification of the “chamber test” the contact activation time of the platelets can be reduced. After the addition of ADP or adrenaline, the platelet rich plasma is stored in a plastic tube and samples are transferred to the counting chamber 15 or 30 minutes before the actual platelet count. Counting is conducted in the usual way or by means of an electronic device (Thrombocounter, Coulter Electronics). With the thrombocounter method it is possible to determine the aggregation kinetics without subjecting the platelets to shear stresses.