Biometric Image Analysis for Quantitation of Dividing Platelets

Abstract

(1) Background: Quantification of platelet division is challenging because automated Coulter cell counters produce equivocal platelet counts. (2) Methods: We applied the flow cytometric cell tracking dye dilution assay as a popular immunological method to evaluate lymphocyte proliferation to prove and quantitate platelet division. We also devised a method relying on platelet culture in a semisolid medium which enabled dividing platelets to be identified by limiting the diffusive movement of platelets. Mixing platelets of different labeling colors in semisolid medium and counting the platelet doublets of each color combination enabled us to prove and quantitate platelet division. (3) Results: The tracking dye dilution assay revealed that 75.5 to 85.6% of platelets were dividing after 20 hours in culture. Platelets labeled with two different tracking dyes were mixed and cultured in semisolid medium for differential doublet counting. We counted platelet singlets and doublets of each color and color combination using confocal microscopy after six hours of culture and compared the relative number of two-colored doublets with binomial prediction to prove platelet division (P < 0.01). Division was suppressed by taxol, nocodazole, or cytochalasin D treatment. We derived a formula for determining the fraction of dividing platelets using the numbers of singlets and doublets of each color and color combination. The platelet division fraction ranged from 8.8 to 17.5%. (4) Conclusion: We successfully measured platelet division using a simple biometric image analysis method with possible future application to microfluidic devices.