The method for estimating change of status of erythrocyte membranes

 

The invention relates to medicine, namely to laboratory methods of research. The essence of the method: the red blood cells separated from the blood, is placed in the cuvette of aggregometry, as an aggregation inductor using picric acid, which is added to a final concentration of 3-5 mm, about the status of erythrocyte membranes is judged by the degree of their aggregation in comparison with the control. The method reduces the time to research and record changes in the state of erythrocyte membranes when exposed to both external and internal factors. 1 Il.

The invention relates to medicine and biology and can be used to assess changes in the condition of the membranes of erythrocytes in stored blood and various anemias.

Currently there is no single universal method, allowing to adequately assess changes in the state of erythrocyte membranes. Usually use several methods together.

Known methods of assessment of erythrocyte membranes, including the determination of osmotic fragility, acid resistance, concentration of intracellular ATP, K+, Na+, CA2+(Stus L. K., and others Hematology and Transfusiology - 1988. T. 33. N4. S. 86-91), including the allocation of erythrocytes from the blood, determination of the degree of their aggregation, caused by the simultaneous decrease in ionic strength and pH of the incubation medium. About the state of erythrocyte membranes judge agregarme, which is constructed graphically by points of index aggregation determined visually under the microscope by counting the cells included in the units.

However, the method is tedious and takes a long time, because the process of aggregation of erythrocytes lasts 30-40 minutes

The task of creating a simple and fast enough for the performance evaluation of changes in the state of erythrocyte membranes is important.

The task is achieved by the method, which is that the red blood cells separated from the blood, is placed in the cuvette of aggregometry, where as the agent that causes the aggregation of erythrocytes, add picric acid to a final concentration of 3-5 mm. The aggregation process is logged photocolorimetric recording on the recorder in the form of agregatogramme. About the status of erythrocyte membranes are judged by their degree of aggregation compared to control.

The method reduces the time of the research through the use of picric acid, in which the aggregation process is faster (1.5 to 2.0 min) than in SAC and internal factors.

The method is as follows.

The erythrocytes washed three times from plasma and white blood cells by centrifugation in a 10-fold volume of saline. The washed erythrocytes resuspension in the ratio of 1:200 in buffered physiological solution (10 mm Tris-Hcl,146 mm NaCl, pH 7,4), after which the cell suspension is placed in a cell for the study of aggregation and add picric acid to a final concentration of 3-5 mm. Registration aggregation carried out by the photometric method (Sheremetiev Y. A. and others Biophysics - 2000. So 45. No. 1. S. 79-82). Picric acid is prepared in physiological solution. After adding picric acid pH of the medium decreases from 7.4 to values of 1.5-2.0.

At a concentration of 3-5 mm picric acid is a strong echinocandin agent (Meiselman, H. J. - Biorheology. 1978. 1978. V. 5. N. 3-4. P. 225-238). At concentrations below 3 mm, the change in the shape of red blood cells is less pronounced and therefore less aggregation of cells. At concentrations of picric acid above 5 mm there is a strong change in the shape of red blood cells, with the advent of spherocytes. It also reduces the degree of aggregation of erythrocytes.

To change the state of the membranes washed erythrocytes were subjected to various pre-treatment. For this purpose, the erythrocytes are processed the level of ATF.

Example 1. Assessment of status changes of erythrocyte membranes treated with glutaraldehyde.

Fixation of erythrocytes was performed in 0.1% buffered solution of glutaraldehyde for 1 h After fixing the erythrocytes washed three times with saline and examined for aggregation. The drawing shows agregatogramme normal (1) fixed and (2) red blood cells, by analyzing which you can conclude that a strong state changes of the membranes after treatment with glutaraldehyde.

Example 2. Assessment of status changes of the membranes after incubation of erythrocytes with 50C.

Suspension of erythrocytes incubated with 50C for 15 minutes and Then the suspension was cooled to room temperature. Analysis of agregatogramme (drawing) shows significant changes in the state of erythrocyte membranes after incubation at 50(3) relative to normal (1).

Example 3. Assessment of status changes of membrane ATP-depleted erythrocytes.

The decrease of ATP levels in red blood cells were obtained by incubation of cells in buffered saline solution for 20 hours

The level of intracellular ATP was monitored by enzymatic method and it was 30% from USENIX erythrocytes (4) relative to normal (1).

These examples show the possibility of application of the method of assessment of status changes of erythrocyte membrane and its advantages over the prototype (time is reduced to 40 min in the prototype to 2 min on the implementation and evaluation of aggregation in the proposed method).

Claims

The method for estimating change of status of erythrocyte membranes, including the allocation of erythrocytes from the blood and check their aggregation, characterized in that as an aggregation inductor using picric acid, and the red blood cells are placed in a cuvette of aggregometry add picric acid to a final concentration of 3-5 mm, about the status of erythrocyte membranes is judged by the degree of their aggregation in comparison with the control.

 

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