Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes.

Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3(-)/Cl(-) transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg(2+) (taking into account Mg(2+) membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg(2+) with Band 3 as KB~0.07mM, which is in agreement with known values of the apparent Mg(2+) dissociation constant (from 0.01 to 0.1mM) that reflects experiments on enrichment of Mg(2+) at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia.

Accurate measurement of volume and shape of resting and activated blood platelets from light scattering.

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.

Light-scattering flow cytometry for identification and characterization of blood microparticles.

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.

Methylation-based analysis of circulating DNA for breast tumor screening.

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.