Indirect hemagglutination test at the ultrastructural level]

A study on ultra-thin sections was made of the preparations of agglutinate produced during the reaction of the immunoglobulin erythrocytic diagnostic agent with dry corpuscular Rickettsia prowazeki antigen, fluoresceine isothiocyanate labeled, and also SRBC used for the preparation of the diagnostic agent after formalinization, tannin treatment, sensitization with hyperimmune horse serum immunoglobulins and lyophilization, respectively. Formalin and tannin treatment of erythrocytes failed to be reflected on the ultrastructure of their cellular membranes; the treatment with hemosensitin was accompanied by the appearance of spheroid protrusions of the erythrocyte cytoplasmic membrane with the preservation of its three-layer structure. Specific interaction of sensitized erythrocytes with the antigen corpuscles was expressed morphologically in their apposition or connection through a gap of 20--30 nm.     

Study of hemosensitization characteristics for the purpose of standardizing erythrocyte flagellin diagnostica]

The work is devoted to the study of conditions required for the purpose of creation of standard and the most active preparations of erythrocytic H-diagnostic agents. The dependence of binding of the H-antigen by erythrocytes on the pH and the ionic power of the medium was investigated. It was demonstrated that in the process of erythrocyte sensitization with flagellin of great importance were electrostatic, hydrogen, and hydrophobic powers.     

Molecular aspects of the interaction between albumin and tannin-treated erythrocytes in the process of hemosensitization. Report 1. Stable and unstable effects. Quantitative parameters]

A study was made of the process of interaction between the tannin-treated sheep erythrocytes and the human serum albumin. Protein binding increased, whereas the loose/stable binding ratio decreased with the rise of the initial protein concentration. The process of stable albumin binding is described by the Langmour equation, this permitting to assess the limiting binding values -- about 6-10(5) molecules per erythrocyte. Albumin molecules were bound by erythrocyte with their greatest surface. At any concentration albumin was incapable of occupying more than 85% of the erythrocyte surface; at 90% binding level it blocked only 51--52% of the tannin-treated erythrocyte. The data obtained were regarded as a methodical basis for the preparation of erythrocytic diagnostic agents with the proteins of albumin type.     

Determination of meningococcal antibodies by a radial hemolysis method

The possibility of using, on principle, the reaction of radial hemolysis for the determination of antibodies to meningococci has been shown. The sensitivity and resolution of this method has been found to depend on the dose of the antigen used for the sensitization of erythrocytes, on the concentration of the erythrocyte suspension introduced into the gel and on the amount of complement. The optimum conditions for the reaction of optimum hemolysis, used for the determination of antibodies to serogroup A Neisseria meningitidis polysaccharide, have been established: the sensitizing dose of the antigen must be 50-100 micrograms/ml, the concentration of sensitized erythrocytes 25%, and the amount of complement 20-40 HU.     

Nonspecific means of increasing the sensitivity of the passive hemagglutination reaction]

The effect of some methods of preliminary treatment of erythrocytes on the PHAT depended on the sensitin náture and the method of erythrocyte load. In case of erythrocyte load with nonprotein and immunoglobulin sensitins without any conjugating agents the simulating effect of heating and periodate treatment was caused not by increase of stable sensitin binding, but by the reduction of physico-chemical resistance of erythrocytes. This effect of erythrocyte treatment permitted to increase the sensitivity of the antibodies and antigens determination. In loading the erythrocytes with the aid of conjugating agents and in sensitization with protein antigens after Boyden no stimuating effect of the treatment was noted.     

Membrane-bound enzymes. III. Protease activity in leucocytes in relation to erythrocyte membranes.

Protease activity was detected in membranes of human bovine erythrocytes prepared by the conventional procedures which include washing and removal of the "buffy layer". The enzyme was extracted by 0.75 M KCNS or (NH4)2SO4 and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease. Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.     

Interaction of erythrocytes with human serum proteins. I. Analysis of the effect of pH and ionic strength of the medium.

The effect of ionic parameters of the medium (pH and ionic strength) on the processes of interaction of tannin-treated erythrocytes and the protein fractions of human serum (macroglobulins, microbulins and albumin) was studied in factorial experiments. Complex effect of these parametres on the processes under investigation and optimum conditions of erythrocyte sensitization were established. Subsequent fixation of antibodies by the erythrocyte diagnostic and their agglutinating activity are manifested in different mannera depending on the conditions of preceding sensitization. Important peculairities were discovered in the mechanism of interaction between the erythrocytes and various serum proteins. The obtained results should be taken into account in the production of erythrocyte antigen and antibody diagnosticums.     

Comparative evaluation of different globulin preparations and of the means for blood sensitization in obtaining antibody erythrocytic diagnostica]

In comparing the hemosensitizing activity of various immunoglobulin preparations with different methods of erythrocyte loading the greatest activity and specificity was revealed in IgG isolated by means of DEAE-Sephadex A-50. The method of erythrocyte sensitization with the use of alizarin blue indicator was more advantageous in respect to sensitivity and specificity of Sh. sonnei and Newcastle indication over Boyden's, Jandl and Simmons', Bing's methods.     

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.     

Erythrocytic diagnostic agents for determining antibodies to Proteus O antigens

Highly sensitive and specific erythrocyte diagnostic agents (ED) for the determination of antibodies to Proteus O-antigens have been obtained by the sensitization of formolated sheep red blood cells (SPBC) with activated lipopolysaccharides (LPS) without the use of mediators. The tannin treatment of formolated SRBC and/or the increase of temperature from 45 degrees C to 100 degrees C in the process of the preparation of ED have been found to produce no increase in effectiveness. Antibody ED permitting the detection of Proteus O- and H-antigens has been obtained by the sensitization of formolated chick red blood cells with immunoglobulin preparations to Proteus hydroxylamine antigens, carried out with the use of amidol. The experiments have shown the possibility of using this antibody ED for the determination of O-antibodies in the antigen neutralization test with nonactivated LPS used as an agglutinating agent. The passive hemagglutination test with antibody ED has proved to be a more sensitive method for the detection of O-antibodies than the antigen neutralization test with antigenic ED. The determination of Proteus etiology in the passive hemagglutination test with the use of antigenic ED has been shown to be highly effective in the examination of patients with chronic osteomyelitis at the stage of exacerbation.     

Membrane-bound enzymes: III. Protease activity in leucocytes in relation to erythrocyte membranes

Protease activity was detected in membranes of human and bovine erythrocytes prepared by the conventional procedures which include washing and removal of the “buffy layer”. The enzyme was extracted by 0.75 M KCNS or (NH₄)₂SO₄ and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease.Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with the content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.     

Principles of constructing efficient erythrocyte immunoglobulin diagnostics

Selection of the method of loading erythrocytes with preparations of immunoglobulins (IG) should be determined by the properties of the sensitins to be used. For the production of efficient antibody diagnostics it is advisable to use amidol while in the case of highly active and concentrated sera, amidol, CrCl3 and glutaraldehyde should be employed. The application of IgG preparations considerably enhances the sensitivity of PHAR in the indication of antigens. Immunoglobulin diagnostics on the basis of IG preparations of normal sera should be produced by means of rivanol, tannin or previously heated erythrocytes. In the multiform group of erythrocyte immunoglobulin diagnostics it is necessary to distinguish antibody and non-antibody preparations. Such a classification is justified with respect to differences in the purpose and the optimum methods of production of immunoglobulin diagnostics.     

Changes in the Architectonics and Morphometric Characteristics of Erythocytes under the Influence of Magnetite Nanoparticles

A new high-precision technique for calculating the ratio of the erythrocyte area/volume using atomic-force microscopy has been developed. The method was tested on erythrocytes of healthy donors. Scanning electron microscopy, atomic-force microscopy, and X-ray microanalysis revealed that magnetite nanoparticles can interact with erythrocyte membranes in vitro. This interaction resulted in the development of a pathology of erythrocytes typical for poikilocytosis and anisocytosis. When the magnetite was incubated with erythrocytes in a serum-free medium, nanoparticles aggregated.     

Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

Requirements of drug-induced endocytosis by intact human erythrocytes.

Study of drug-induced endocytosis in intact human erythrocytes continues to provide an opportunity for correlating membrane functions such as invagination and fusion with erythrocytic energetics and other determinants of plasma membrane function like Ca++. The studies reported indicate that high concentrations of vinblastine and chlorpromazine can produce endocytic vacuoles, albeit in reduced amounts, even in severely ATP depleted erythrocytes. In contrast, primaquine-induced endocytosis seems definitely dependent upon persistence of erythrocytic ATP stores. The ionophore mediated entry of Ca++ into erythrocytes potentiates primaquine endocytosis, inhibits vinblastine endocytosis, and has no regular effect on chlorpromazine endocytosis. Sodium lactate enhances primaquine endocytosis, probably by causing an increase in the entry of primaquine into erythrocytes. Cytochalasin B neither enhances nor inhibits erythrocytic endocytosis, thereby suggesting that microfibrils or analogues of microfibrils in erythrocytes are not involved in endocytosis. Cyclic nucleotide inhibition of endocytosis is confined to a very high concentration range of nucleotides in the medium. Primaquine and chlorpromazine endocytosis are inhibited by cyclic nucleotides as is vinblastine endocytosis.     

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells.     

A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2–3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages.     

Progressive muscular dystrophy type Duchenne. II. Viscosity and resistence to erythrolytic compounds of erythrocyte membranes

The transition temperature of erythrocyte ghosts of normal subjects is about 18-20 degrees C. We have studied the viscosity of erythrocyte ghosts of dystrophic children, showing that the transition shifts to lower temperatures (17-18 degrees C). After treatment with erythrocytic compounds like L-Lyso phosphatidyl-Choline dystrophic erythrocytes hemolize at lower Lysophosphatidyl-Choline concentration and at a greater extents than these of normal and carriers subjects.     

The development of a complex Leptospira antigenic erythrocyte diagnostic agent

Blood-sensitive activity of antigenic extracts obtained from leptospiras using the supersound and sodium dodecyl sulphate has been studied. Erythrocytic diagnostica, obtained on the basis of the mentioned sensitines, possess the genus and serogroup specificity, which expands their diagnostic spectrum. New diagnostica are shown to possess higher sensitivity as compared with the previously developed leptospirosis genus-specific erythrocytic diagnostica. The data are presented confirming the prospects of development of erythrocytic diagnostica based on antigens of leptospiras obtained by means of a supersound and sodium dodecyl sulphate. These preparations may be used in the diagnosis of human and animal leptospirosis.     

Characteristics of endogenous proteolysis in preparations of human erythrocyte membranes

Endoproteolytic activity in human erythrocyte membrane preparations has been examined at 37 degrees C by one- and two-dimensional electrophoresis. Two-dimensional mapping has shown that the presence of leukocyte enzymes in erythrocytes prepared in a regular manner (centrifugation) cannot be excluded. Sedimentation in the 1.5% dextran 500,000 with the following erythrocyte purification on HBS-cellulose has made it possible to prepare erythrocyte membranes characterized by low level endoproteolytic activity without leukocyte enzymes. The marker peptide has been found. It is likely to be a specific product of the enzyme activity of membrane localization.