RU2027188C1 - Method for recording variation of surface charge of erythrocytes - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: this method prescribes fixing cells in glutaric aldehyde solution, washing and diluting resultant matter in buffered physiological solution, placing it in cuvette, adding lanthanum chloride concentrated to 0.30 to 0.35 mmol, and then calculating extent of variation of surface charge of eryhtrocytes based on aggregatogram representing degree of aggregation of cells. EFFECT: greater simplicity. 3 dwg

Description

 The invention relates to medicine and biology and can be used to record changes in the surface charge of red blood cells in violation of the rheological parameters of the blood.

 A known method for determining the surface charge of red blood cells by measuring their electrophoretic mobility.

 The disadvantages of this method include the relative complexity of determining changes in the surface charge of red blood cells, the inaccessibility of special devices.

 There is also a method of detecting changes in the surface charge of red blood cells, which consists in measuring the degree of aggregation previously fixed with glutaraldehyde red blood cells stimulated by a positively charged substance - polylysine.

 However, the disadvantage of the known method of recording changes in the surface charge of red blood cells is the duration of the determination of aggregation of red blood cells (average 12 minutes).

 The purpose of the invention is to reduce the time of registration of changes in the surface charge of red blood cells.

 This goal is achieved by the fact that red blood cells are fixed with glutaraldehyde, then washed from the fixative, lanthanum chloride (0.30-0.35 mmol) is added and their aggregation is determined, changes in surface charge are calculated by the aggregatogram.

The method is as follows. Fresh blood stored no more than 1-2 hours at 4 ^{° C} in the presence of heparin (100 U / ml). Centrifuge for 10 min at 1000g. Plasma and white blood cells are removed, and red blood cells are washed twice with a 10-fold volume of physiological saline. The washed red blood cells are fixed in a 0.6% solution of glutaraldehyde (0.05-0.1%) for 20 minutes. Fixation of red blood cells with glutaraldehyde is necessary so that the aggregating substance, in this case lanthanum chloride, interacts only with the external, negatively charged surface of the red blood cells. That is, did not penetrate into the cell and did not change its shape. The concentration of glutaraldehyde was chosen in order to carry out “soft” cell fixation, without affecting the charge of red blood cells. After that, the red blood cells were washed twice with saline from the fixative.

 The principle of the method for determining the change in surface charge from the change in red blood cell aggregation induced by lanthanum chloride is based on the fact that the aggregation of red blood cells occurs by the mechanism of bridge formation between negative charges of interacting cells and positively charged lanthanum chloride. The smaller the negative charge of the cells, the less the aggregation of red blood cells, and vice versa.

 The red blood cells washed from glutaraldehyde are diluted in a ratio of 1: 200 in buffered saline (10 mmol Tris-HCl, 146 mmol NaCl, pH 7.4). After this, the red blood cells are placed in a cuvette to study aggregation and lanthanum chloride 0.33 mmol (0.30-0.35 mmol) is added. The concentration of lanthanum chloride was selected experimentally. At concentrations less than 0.30 mmol, red blood cell aggregation does not occur, and starting from 0.30 mmol aggregation rapidly increases and reaches a plateau in the region of 0.33 mmol. With a further increase in the concentration of lanthanum chloride, the degree of aggregation of cells does not change, so the use of concentrations above 0.33 mmol is not advisable. The degree of aggregation is determined quantitatively, as well as other blood cells, such as platelets, by the photometric method on aggregometers. After the addition of the aggregating agent, the optical density of the cell suspension decreases, since enlightenment occurs due to the formation of red blood cell aggregates. The drop in optical density is recorded on the tape of the recorder in the form of a curve, which is called an aggregatogram (densitogram). The stronger the interaction of red blood cells occurs, the greater the amplitude of the aggregatogram (M). The maximum amplitude of the aggregogram is measured in millimeters.

 Erythrocyte aggregation was studied using a device mounted on the basis of a KFK-2 photocolorimeter coupled to a KSP-4 recorder. A test tube with a suspension of red blood cells was inserted into a photocolorimeter, and a stirrer consisting of an electric motor with a shaft was placed in it. The amplitude of the aggregatogram of control intact cells is taken as 100%. Then the amplitude of the aggregate of the experimental cells is measured. The percentage change in surface charge relative to the charge of control intact cells is determined.

 PRI me R 1. The addition of lanthanum chloride to intact red blood cells causes their aggregation M = 49 mm (Fig. 1).

 On the graph below, the ordinate (Y) axis shows the amplitude of the agretogram, the abscissa (X) axis shows time.

 PRI me R 2. The addition of lanthanum chloride to red blood cells pre-treated with trypsin M = 33 mm, ie a decrease in surface charge by 33% (Fig. 2).

 PRI me R 3. The addition of lanthanum chloride to red blood cells pre-treated with neuraminidase M = 25 mm, ie a decrease in surface charge by 49% (Fig. 3).

 The accuracy of the method for measuring changes in surface charge by the proposed method is confirmed in parallel experiments in which charge measurement was determined by the electrophoretic mobility of red blood cells.

 Thus, the proposed method is a sensitive and quick test for changes in the surface charge of red blood cells. More than 12 times faster than the method taken as a prototype (less than 1 min instead of 12 min in the prototype).

 The method can be used to record changes in the surface charge of cells both in experimental experiments and in various hematological diseases of humans and animals.

Claims (1)

 METHOD FOR REGISTRATION OF CHANGES IN THE SURFACE CHARGE OF ERYTHROCYTES, including fixation of the studied cells, their washing and determination of the change in the surface charge by the degree of aggregation of cells induced by the aggregating substance, characterized in that, in order to reduce the study time, lanthanum chloride in concentration 0 is used as an aggregating substance 30 - 0.35 mm, and the change in surface charge is calculated by the aggregatogram.

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