Kinetic titration method in flow cytometry for quantification of cell receptors.

For the quantitative determination of cell receptors by fluorescence flow cytometry, we proposed a new method, which takes into account the reaction kinetics. The binding reaction of the ligand with receptors begins after placing the cells in the ligand solution. In the proposed method, there are several samples with the same concentration of cells and different initial concentrations of fluorescently labeled ligand, and each sample is measured by a flow cytometer once at the time when the following condition is met: the product of the incubation time (cells with ligand) and the initial concentration of ligand is the same for all samples. The proposed approach eliminates disadvantages and combines advantages of both kinetic and titration methods for quantification of receptors on single cells without the use of traditional calibration fluorescent beads. Practical application of the method was demonstrated in quantification of CD8 and CD14 on peripheral blood human leukocytes. Particularly, we found decreased (by a factor of two) mean number of CD14 on monocytes and granulocytes in patients with atherosclerosis (treated in the hospital) compared to conditionally healthy donors, whereas no difference was found in the mean CD8 expression on leukocytes between the same patient and donor groups.

Proposed Dynamics of CDB3 Activation in Human Erythrocytes by Nifedipine Studied with Scanning Flow Cytometry.

Nifedipine is calcium channels and pumps blocker widely used in medicine. However, mechanisms of nifedipine action in blood are not clear. In particular, the influence of nifedipine on erythrocytes is far from completely understood. In this work, applying scanning flow cytometry, we observed experimentally for the first time the dynamics behind a significant increase of HCO3- /Cl- transmembrane exchange rate of CDB3 (main anion exchanger, AE1, Band 3, SLC4A1) of human erythrocytes in the presence of nifedipine in blood. It was found that the rate of CDB3 activation is not limited by the rate of nifedipine binding and/or Ca2+ transport. In order to explain the experimental data, we suggested a kinetic model assuming that the rate of CDB3 activation is limited by the dynamics of the balance between two intracellular processes (1) the activation of CDB3 limited by its interaction with intracellular Ca2+ , and (2) the spontaneous deactivation of CDB3. Thus the use of scanning flow cytometry allowed to clarify quantitatively the molecular kinetic mechanism of nifedipine action on human erythrocytes. In particular, the efficiency (~30) and rates of activation (~0.3 min-1 ) and deactivation (~10-3 min-1 ) of CDB3 in human erythrocytes was evaluated for two donors. © 2019 International Society for Advancement of Cytometry.

Cell therapy of critical limb ischemia-problems and prospects.

Cell therapy is proposed for indirect revascularization for the patient's incurable by endovascular or surgical revascularization. The therapy with stem cells (SCs) or progenitor cells is assumed to be more efficient as compared with protein or gene therapy not only because of their direct vasculogenic properties, but also thanks to their paracrine effect via secretion of manifold biologically active substances. This review gives an overview of the potential of SC-based therapy for critical limb ischemia (CLI), putative mechanism underlying cell therapy, and comparison of cell therapy to angiogenesis gene therapy in CLI treatment. Human trial data and meta-analysis, as well as some problems of clinical trials and considerations for future SC-based therapy in CLI are also discussed.

Calibration-free quantitative immunoassay by flow cytometry: Theoretical consideration and practical implementation for IgG antibody binding to CD14 receptors on human leukocytes.

We propose a calibration-free method to determine the number of receptors per cell, as well as the direct and the reverse reaction rate constants for a single receptor. The method is based on the analysis of the temporal evolution of the cells mean fluorescent intensity measured by a flow cytometer during the ligand-receptor (antigen-antibody) binding under the conditions of their comparable concentrations. We developed the kinetic approach accounting both for the delay between the dilution and the measurement and for the practical duration of the measurement itself. The method was applied to determine thenumber of CD14 receptors on human blood mononuclear (granulocytes, monocytes, lymphocytes) cells of several donors. We also obtained the direct ( k+= (5.6 ± 0.2) × 107 M-1 min-1 ) and reverse ( k-= (1.3 ± 0.2) × 10-2 min-1 ) rate constants of ligand-receptor interaction, and estimated the size of the binding site as b = 0.5 nm. The latter allows one to recalculate the rate constants for a different ligand, fluorescent label, medium viscosity, and/or temperature. The knowledge of the rate constants is essential for the calibration-free determination of the number of receptors per cell from a single kinetic curve of the cells mean fluorescence intensity.

Chylomicrons against light scattering: The battle for characterization.

Chylomicrons (CMs) are lipoprotein particles circulating in blood and transporting dietary lipids. Optically speaking, CMs are small compared to the wavelength of visible light and widely distributed by the size and refractive index (RI). Consequently, intensity of light scattered by the CMs scales with up to the sixth power of their size, hampering simultaneous analysis of 60 and 600 nm CMs. We present an accurate method for quantitative characterization of large-size CM subpopulation by the distributions over size and RI. For the first time the CM characteristics have been determined at a single particle level based on angle-resolved light-scattering measurements. We applied the developed method to 2 key processes relating to CM metabolism, namely in vivo dynamics of CMs in blood plasma after a meal and in vitro lipolysis of CMs by the lipoprotein lipase in postheparin plasma. We have observed the substantial variations in CM concentration, size and RI distributions. This opens the way for a multitude of medical applications involving screening of CM metabolism, which we exemplified by revealing large differences in CM characteristics after a 12-hour fast between a healthy volunteer and a patient with atherosclerosis.

Remote Endarterectomy and Lamina Vastoadductoria Dissection Improves Superficial Femoral Artery Biomechanical Behavior during Limb Flexion.

BACKGROUND: Superficial femoral artery (SFA) remote endarterectomy offers the advantage of preserving anatomy and geometry of the native artery, but the risk of restenosis still exists. The particular role of the adductor canal (AC) in mechanical constraints has been highlighted. The aim of this study was to assess if a surgical protocol associating remote SFA endarterectomy and AC freeing would modify the SFA geometrical changes during physiological limb flexion. METHODS: From January 2015 to March 2015, 10 patients (Rutherford 3-5) with unilateral SFA occlusion were included. Functional postoperative assessments were performed through duplex ultrasound (DUS) examinations with flow velocity measurements in both straight and flexed positions and anatomical measurements through 3-dimensional computed tomography angiography (CTA) reconstructions with arterial angulations examination. Functional results were compared with similar findings in healthy volunteers, and anatomical results were compared with contralateral limb findings. RESULTS: Mean occlusion length was 243.0 ± 17.7 mm. Technical success was achieved in all cases. No difference of peak flow velocities was noticed between operated patients and volunteers. CTA results showed that limb flexion induced SFA shortening in all segments, with a maximal value for the popliteal artery (PA) (10.4 ± 4.4%). Comparisons between the operated and contralateral limbs showed that angles were less sharp during bending in the operated limb. CONCLUSIONS: This preliminary study demonstrates that freeing the AC modifies the biomechanical properties of the SFA. These results could potentially help in proposing future hybrid techniques that could improve technical performances for SFA occlusive disease treatment.

Light-scattering gating and characterization of plasma microparticles.

Flow cytometry method (FCM) is widely used for analysis of cell-derived microparticles (MPs). Numerous efforts are currently aimed to standardize these measurements among different instruments. We push the FCM characterization of MPs to the limit based on rigorous simulation of measured signals. We measured forward- and side-scatter (FSC/SSC) signals and angle-resolved light-scattering profiles (LSPs) of polystyrene microspheres and MPs, including their aggregates, using a scanning flow cytometer (SFC). We used the Mie theory to (1) accurately evaluate instrument detection limits; (2) construct FSC/SSC gates for MPs in absolute scales of size and refractive index (RI); and (3) determine size and RI of individual spherical MPs. LSPs were used for advanced characterization, including differentiation of spherical and nonspherical particles. The proposed absolute FSC/SSC gating is naturally standardized for any FCM instrument, given the knowledge of its optical system and leads to instrument-independent analysis of MPs. The inverse Mie problem has a unique solution only for some regions of size and RI and uncertainties rapidly increase with decreasing size and RI. The developed methods are applicable to any flow cytometer, but are limited by assumption of particle sphericity. The latter can be relaxed only if additional signals, such as LSP, are measured.