[Evaluation of platelet intracellular calcium ion concentrations with flow cytometry]

Platelet intracellular Ca++ ([Ca++] i) was measured by flow cytometry, using a new Ca(++)-sensitive fluorescent dye, fluo 3. The acetoxymethyl derivative of fluo 3, fluo 3 AM, was incorporated into platelets most efficiently in the presence of 1.5 micrograms/ml pluronic F-127, a surfactant often used to facilitate intracellular incorporation of lipophilic agents. [Ca++] i measurement with flow cytometry proved to be more sensitive than that with ordinary fluorescence spectrophotometers, detecting cells with elevated [Ca++] i at much lower agonist concentrations. Two-dimensional analysis using forward scatter intensities and [Ca++] i more clearly defined a subset of platelets responsive to low concentrations of agonists. In normal subjects [Ca++] i hardly changed in response to low-dose thrombin (0.002 U/ml), while it invariably showed a marked increase in response to high-dose thrombin (0.02 U/ml). Thus, these dose of thrombin were used to evaluate clinically whether platelets was hyper-reactive or hypo-reactive. Of 44 patients with diabetes mellitus, 6 patients had hypo-responsive platelets, while platelets were sensitive to low concentrations of thrombin in 3 patients. Since such differences cannot be detected by conventional spectrophotometric methods, [Ca++] i measurement by flow cytometry may prove useful tool for clinical evaluation of platelet function.