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.
Highlights
IntroductionPlatelets are quasi-cellular particulate blood components circulating in blood vessels
We aimed to evaluate platelet division by measuring the platelet count before and after culture in a liquid medium using an automated hematology analyzer (Coulter counter)
* No significant difference in division between platelets strongly and weakly labeled with carboxyfluorescein diacetate N‐succinimidyl ester (CFSE)
Summary
Platelets are quasi-cellular particulate blood components circulating in blood vessels. The most important and well-known function of platelets is hemostasis, or prevention of blood loss at sites of tissue injury. Platelets function as the first-line defense mechanism against blood loss by promptly aggregating around vascular breakages. These platelet aggregates are often called “the primary hemostatic plug” because this structure clogs the hole in a blood vessel in a pure physical sense. For the hemostatic plug to be effective, it must form a mass of a certain size, and this is one of the reasons why the platelet count is important
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