Fluorescence-free flow cytometry for measurement of shape index distribution of resting, partially activated, and fully activated platelets.

Whereas commercially available hematological analyzers measure volume of individual platelets, angle-resolved light-scattering provides unique ability to additionally measure their shape index. We utilized the scanning flow cytometer to measure light-scattering profiles (LSPs) of individual platelets taken from 16 healthy donors and the solution of the inverse light-scattering problem to retrieve the volume and shape index of each platelet. In normal conditions, the platelet shape index distribution (PSID) demonstrates three peaks, which relate to resting, partially activated, and fully activated platelets. We developed an algorithm, based on fitting PSID by a sum of three peak functions, to determine the percentage, mean platelet shape index, and distribution width of each platelet fraction. In total, this method gives eight additional parameters of platelet morphology and function to be used in clinical hematological analysis. We also stimulated the platelets with adenosine diphosphate (ADP) and measured the dependence of equilibrium PSID, including the total percentage of activated platelets, on ADP concentration. © 2016 International Society for Advancement of Cytometry.

National Outcomes of Liver Transplantation for Model for End-Stage Liver Disease Score ≥40: The Impact of Share 35.

In certain regions of the United States in which organ donor shortages are persistent and competition is high, recipients wait longer and are critically ill with Model for End-Stage Liver Disease (MELD) scores ≥40 when they undergo liver transplantation. Recent implementation of Share 35 has increased the percentage of recipients transplanted at these higher MELD scores. The purpose of our study was to examine national data of liver transplant recipients with MELD scores ≥40 and to identify risk factors that affect graft and recipient survival. During the 12-year study period, 5002 adult recipients underwent deceased donor whole-liver transplantation. The 1-, 3-, 5- and 10-year graft survival rates were 77%, 69%, 64% and 50%, respectively. The 1-, 3-, 5- and 10-year patient survival rates were 80%, 72%, 67% and 53%, respectively. Multivariable analysis identified previous transplant, ventilator dependence, diabetes, hepatitis C virus, age >60 years and prolonged hospitalization prior to transplant as recipient factors increasing the risk of graft failure and death. Donor age >30 years was associated with an incrementally increased risk of graft failure and death. Recipients after implementation of Share 35 had shorter waiting times and higher graft and patient survival compared with pre-Share 35 recipients, demonstrating that some risk factors can be mitigated by policy changes that increase organ accessibility.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

[Monoclonal antibodies to recombinant human laminin-binding protein. Production and immunochemical description]. / Monoklonal'nye antitela k rekombinantnomu lamininsviazyvaiushchemu belku cheloveka. Poluchenie i iimmunokhimicheskaia kharakteristika.

Thirteen murine hybridoma lines producing monoclonal antibodies (Mabs) to recombinant human laminin-binding protein (rLBP) were developed. All 13 Mabs reacted with affinity purified 43 kDA rLBP in ELISA and Western blotting. Mab class determination showed 9 Mabs as belonging to IgM class, 2 Mabs--to IgG2 subclass, 1 Mab--to IgG1 and 1 Mab--to IgG2b. Ten Mabs of different classes were capable to react with LBP on the surface of Vero cells. Mabs displayed a high and simultaneously varying affinity to rLBP (10(8) 10(9) M(-1)). The Mab affinity was found to be comparable with the mean affinity of mouse and rabbit antibodies isolated from hyperimmune sera. The possibility of using the produced Mabs in mapping the LBP domains involved in virus attachment, cell differentiation and cancer metastases progression as well as in the systemic response to bacterial protozoan and parasitic infection is under discussion.

Mathematical modeling the kinetics of cell distribution in the process of ligand-receptor binding.

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand-receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand-receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.

[Effects of mental adaptation on vegetative, hormonal and sensory reactions in preoperative period]. / Vliianie psikhicheskoi adaptatsii na vegetativnoe, gormonal'noe i sensornoe reagirovanie v predoperatsionnom periode.

Fifty-seven urological patients whose emotional behavioral reactions were characterized by somatic anxiety caused by mental adaptation were examined under conditions of preoperative emotional stress. On day 5 after admission to hospital the hormonal, autonomic, and sensory responses decreased in patients with previously manifest anxiety, which was due to decrease of anxiety because of appearance of some psychopathological features. Satisfactory hormonal, autonomic, and mental adaptation was observed on day 5 after admission to hospital in patients with moderately expressed anxiety. In patients with previously predominating parasympatotonia and a low level of anxiety, the hormonal and autonomic areactivity and mental adaptation observed in the same terms were due to augmenting psychopathological features.

[Correlation between the effectiveness of premedication and neuroanatomic protection during surgery with NLA and the initial state of the autonomic nervous system]. / Zavisimost' éffektivnosti premedikatsii i neirovegetativnoi zashchity pri operatsiiakh v usloviiakh NLA ot iskhodnogo sostoianiia vegetativnoi nervnoi sistemy.

The study was performed on 94 urological patients, aged 15 to 78 years, subjected to planned operations. The initial autonomic tone, autonomic reactivity, autonomic maintenance of the activity, ACTH and cortisol content have been investigated in the ward and in the operation room with concomitant premedication with pipolphen in combination with promedol and diazepam in combination with promedol. Tactile and pain thresholds were studied in patients on premedication in the ward and the operation room. It has been noted that positive effect of premedication, an increase in the pain threshold and adequate neuroautonomic protection do not only depend on the presence or absence of diazepam in premedication, but also on the initial autonomic tone, autonomic reactivity, and autonomic maintenance of the activity. The above parameters in their turn depend on the functional state of the autonomic nervous system, the patient's age, concomitant diseases, and other factors determining general physical status of the patient.

[Video display terminals: their electromagnetic safety]. / Videodispleinye terminaly: élektromagnitnaia bezopasnost'.

Widespread application of video terminals (VT) has led to accumulating some unambiguous data on harmful effect of electromagnetic and electrical fields on the health. An analysis indicates that the level of electromagnetic and electrical fields is below permissible limits. IRPA/INIC concludes that the electromagnetic effects on VT users health are unharmful. However, the problem of VT use is an integrated one and involves not only radiation but ergonomic aspects and in this context requires further studies.

[The dependence of sensory perceptions on the predominance of parasympathicotonia or sympathicotonia during preoperative psychoemotional stress]. / Zavisimost' sensornykh vospriiatii ot preobladaniia para- ili simpatotonii v usloviiakh predoperatsionnogo psikhoémotsional'nogo napriazheniia.

Low tactile and pain thresholds and high Kerdo index were observed in patients with sympathicotonia predominance during preoperative emotional stress. Low Kerdo index and high tactile and pain thresholds were observed in patients with parasympathicotonia predominance during preoperative psychoemotional stress.

[Effectiveness of hyperbaric oxygenation as a factor of increasing the resistance of the human body to space flight conditions]. / Effektivnost' giperbaricheskoi oksigenatsii kak sredstva povysheniia ustoichivosti organizma cheloveka k vozdeistviiu faktorov poleta.

In four experimental runs (30 test subjects in 407 tests), the beneficial effect of hyperbaric oxygenation (HBO) on flight tolerance was demonstrated. HBO sessions (7-8 sessions of an hour in duration during 10-12 days at pO2 = 2 atm) helped subjects with poor altitude tolerance to increase the altitude ceiling by 1 km and reserve time at altitudes of 6-7 km by 3-4 min, the effect being persistent for 2-3 months. Statokinetic tolerance of subjects with medium motion sickness susceptibility grew by 2-2.5 times after the above HBO sessions. As compared to regular rest, the HBO treatment shortened two-fold the period of recovery of the health state and work capacity of operators, which went down during flight, and increased 15-20% the time of repeated work prior to failure in a stressful environment.

[Use of the micronucleus test in the screening and monitoring of mutagens]. / Ispol'zovanie mikroiadernogo testa v skrininge i monitoringe mutagenov.

Data from literature on the use of micronuclear test to determine mutagenicity in agents of physical, chemical and biological nature are presented. The objects on which this method is used most frequently are enumerated. Great attention is paid to the analysis of micronuclei in blood erythrocytes and bone marrow of animals. It is shown that the animal sex, age and the way of mutagen injection are of great importance in micronuclear testing of mutagens. Methodical papers concerning the peculiarities of fixation colour, analysis and mathematical testing are given. Mutagen factors tested by the method of micronuclear analysis are enumerated. A high resolution and small labour input of the micronuclear test are shown.