We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 μm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 μm due to used laser wavelength of 0.66 μm.

We introduce a novel approach for determination of volume and shape of individual blood platelets modeled as an oblate spheroid from angle-resolved light scattering with flow-cytometric technique. The light-scattering profiles (LSPs) of individual platelets were measured with the scanning flow cytometer and the platelet characteristics were determined from the solution of the inverse light-scattering problem using the precomputed database of theoretical LSPs. We revealed a phenomenon of parameter compensation, which is partly explained in the framework of anomalous diffraction approximation. To overcome this problem, additional a priori information on the platelet refractive index was used. It allowed us to determine the size of each platelet with subdiffraction precision and independent of the particular value of the platelet aspect ratio. The shape (spheroidal aspect ratio) distributions of platelets showed substantial differences between native and activated by 10 μM adenosine diphosphate samples. We expect that the new approach may find use in hematological analyzers for accurate measurement of platelet volume distribution and for determination of the platelet activation efficiency.

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The applicability of cyclin D2 and RARbeta2 methylated markers for the development of a breast tumor screening assay was evaluated. Overall, 76 volunteers (mean age, 50.4 years), including clinically healthy women and women with breast lesions, were enrolled in a blind study of methylation of the cyclin D2 and RARbeta2 genes. Methylation-specific PCR (MSP) was conducted using total circulating DNA (cirDNA) from the blood, including the cell-free and cell-surface-bound DNA fractions. Only 1 of the 25 women of the clinically healthy group displayed methylated cyclin D2 gene (4%). However, 42% of the patients with primary diagnosis of breast fibroadenoma showed an aberrant methylation of at least one of the tested genes in the cirDNA. The number of patients with breast lesions (mastopathy) not diagnosed as fibroadenoma that displayed methylation was lower (33%). A long-term follow-up study based on a quantitative evaluation of cyclin D2 and RARbeta2 methylation in cirDNA will provide the data on applicability of these markers for breast tumor predictive diagnostics.

Resistance to hemolysis of erythrocytes in lysing isotonic water solution of ammonium chloride becomes apparent in the dynamics of the change of their optical properties. Optical properties of erythrocytes were registered on light scattering from single cells with a scanning flow cytometer. It was shown experimentally during hemolysis the dynamics of the change of the integral intensity of light scattering of erythrocytes is different for irradiated and non-irradiated human blood. Qualitatively, the effect is similar for the both sources of pulse radiation: at the beginning of hemolysis the mean integral intensity of irradiated erythrocytes is increasing more than that is of non-irradiated ones. Quantitatively, at the same mean power density the interrupted irradiation of CO2 laser caused greater effect than continuous one, but less than pulsed terahertz irradiation, i.e. the effect is greater for shorter pulse. On the base of the obtained results one can suggest that the observed effect on pulse infrared radiation of low mean power caused by unsteady heating of erythrocytes.