Presentations

Background: The cellular origin of microparticles is usually established by fluorescent antibody labeling, which is laborious, expensive, and involves practical problems (JTH 2010, 8: 2596–607). Using an ultra-modern Raman microspectroscopy setup, we have distinguished platelet from erythrocyte microparticles without labeling.

Methods: Platelet and erythrocyte microparticles were isolated from blood bank concentrates by differential centrifugation and analyzed by flow cytometry, transmission electron microscopy, and Raman microspectroscopy. For Raman microspectroscopy, a 35 mW krypton ion laser operating at a wavelength of 647 nm was focused to a probe volume smaller than 1 μm³ by an Olympus microscope objective with an NA of 0.95. The Stokes shift from light scattered by microparticles diffusing through the probe volume was measured by a sensitive spectrograph dispersing in the range 646–849 nm.

Results: The figure shows that platelet microparticles have a Raman spectrum containing spectral transitions characteristic of phospholipids. The Raman spectrum of erythrocyte microparticles, however, is entirely different.

Conclusions: For the first time, platelet microparticles are distinguished from erythrocyte microparticles without fluorescent antibody labeling.

We have studied the ability of two new, advanced methods (nanoparticle tracking analysis and resistive pulse sensing with a pore diameter of 1 μm), to measure the size and concentration of a standard population of human urine vesicles by comparison with two established techniques (transmission electron microscopy and flow cytometry). Nanoparticle tracking analysis and resistive pulse sensing detected a concentration of 1.4 × 10¹⁰ ml^-1 and 3 × 10¹⁰ ml^-1, respectively, which is 1,000-fold higher than the observed concentration with flow cytometry. By describing the data with a model based on Mie theory, we have obtained the relation between the measured light scattering signal, the refractive index, and the diameter of beads and vesicles. In addition, we provided evidence that the detection of vesicles by flow cytometry is partly attributed to the simultaneous presence of multiple vesicles in the laser beam.