RU2328741C1 - Method of erythrocytes osmoresistivity evaluation - Google Patents

Abstract

FIELD: medicine; laboratory diagnostics.

SUBSTANCE: method includes blood testing for quantification of haemolysis severity in sodium chloride solution of various concentrations. This implies analysis of three capillary blood samples for supernatant optical density: with distilled water, 0.9% sodium chloride solution and 0.45% sodium chloride solution using spectrophotometer at wave length 414 nm in dish of absorption length 1 cm against distilled water. Haemolysis severity is evaluated on basis of original physiological physical-chemical composition of erythrocyte membranes in 0.9% sodium chloride solution and osmotic stress testing in 0.45% sodium chloride solution compared to haemolysis severity in distilled water testing which is considered to be 100%. Haemolysis severity values, 1.61±0.23% tested in 0.9% sodium chloride solution and 25.16±1.83% tested in 0.45% sodium chloride solution erythrocytes osmoresistivity is interpreted as normal.

EFFECT: haemolytic state screening.

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Description

The invention relates to medicine, namely to laboratory diagnostics.

Determination of osmotic resistance of red blood cells (REM) is one of the most accessible methods in laboratory diagnostics for assessing the physicochemical properties of erythrocyte membranes. Changes in the REM are observed in a number of diseases, so with hemolytic anemia (hereditary microspherocytic, autoimmune), the values of this indicator decrease, with thalassemia, hemoglobinopathies, and obstructive jaundice - they increase.

As a prototype, the authors propose a method for determining the WEM in the modification of L.I. Idelson (Medical laboratory technology. Handbook in 2 volumes / Edited by Professor A.I. Karpishchenko. - St. Petersburg: Intermedika, 2002. - T. 1. - 408 p., Ill.). The essence of the method is that red blood cells under the influence of solutions with different concentrations of sodium chloride are capable of destruction, depending on their osmotic resistance. The method is based on a quantitative determination of the degree of hemolysis in buffered hypotonic sodium chloride solutions, in which, as is known, red blood cells swell and hemolysis. The method consists in preparing a basic solution, which according to the osmotic concentration corresponds to a 10% solution of sodium chloride with a pH of 7.4, for which 27.31 g of disubstituted anhydrous sodium phosphate (Na ₂ HPO ₄ ) or 34.23 g are dissolved in 2 liters of distilled water two-water (Na ₂ HPO ₄ · 2H ₂ O), 4.86 g of sodium phosphate monosubstituted two-water (NaH ₂ PO ₄ · 2H ₂ O) and 180 g of sodium chloride (NaCl). The solution can be stored in a sealed container in the refrigerator for several months. The basic solution is diluted 10 times with distilled water, and a solution is obtained that, in its osmotic concentration, corresponds to a 1% solution of sodium chloride. From this solution, 14 working solutions are prepared corresponding to sodium chloride solutions of the following concentrations: 1%, 0.85%, 0.75%, 0.70%, 0.65%, 0.60%, 0.55%, 0, 50%, 0.45%, 0.40%, 0.35%, 0.30%, 0.20%, 0.10%. It is recommended to prepare 100 ml of each working solution, which, when stored in the refrigerator, is suitable for two weeks. In two sterile tubes with 2 drops of heparin (500 Units) add 1.5 ml of venous blood, mix and one of them is used for research, and the second is left for a day in an thermostat at 37 ° C. In 14 centrifuge tubes placed 5.0 ml of all working solutions of sodium chloride from 1% to 0.10%. Then, 0.02 ml of heparinized venous blood is added to each tube and mixed. The tubes are incubated for 30 minutes at room temperature, and then centrifuged for 5 minutes at 2000 rpm. The optical density of the supernatant from each tube is measured on a photoelectrocolorimeter at a wavelength of 500-560 nm (green filter) in a cuvette with an absorbing layer thickness of 1 cm. As a blank sample, a supernatant from a tube containing 1% sodium chloride solution is used. ORE, expressed as a percentage, is determined by calculating the degree of hemolysis in each tube, taking the optical density of the supernatant in a tube with 0.1% sodium chloride solution as the 100% degree of hemolysis. The percentage of hemolysis in each tube is calculated by comparing the extinction value of the supernatant with the extinction, taken as 100%, according to the formula

100 · E _x / E ₁ ,

where: E ₁ is the extinction of the supernatant in a test tube with 0.1% sodium chloride solution,

E _x is the extinction of the test sample,

100 - complete hemolysis in vitro with a 0.1% sodium chloride solution.

A similar study with a second portion of blood incubated for 24 hours in an thermostat is performed the next day.

In healthy people, in fresh blood, hemolysis (minimum erythrocyte resistance) begins at a concentration of sodium chloride 0.50-0.45%, and complete hemolysis (maximum erythrocyte resistance) is observed in a 0.40-0.35% sodium chloride solution.

The disadvantages of the prototype include:

- the need for the use of venous blood in a volume of 3 ml;

- the complexity of the preparation of 14 solutions with different concentrations of sodium chloride, which requires a significant investment of time, a large consumption of reagents and is associated with possible inaccuracies in salt samples, measurement of solvent volumes, which may affect the accuracy of obtaining the necessary concentrations of solutions;

- the use of twice 14 test tubes with blood requires an appropriate amount of laboratory glassware, the number of measurements on a photoelectrocolorimeter, which increases the test time, the time of disinfection, washing of laboratory glassware, and the consumption of disinfectants;

- the need for re-setting the test the next day with a second portion of blood, incubated for a day in the thermostat, and the use of the results of this re-study is not known;

- to determine small concentrations of hemoglobin, the wavelength used for photometry of 500-560 nm does not provide sufficient measurement sensitivity, since the millimolar hemoglobin extinction coefficient at this wavelength is lower than at another wavelength;

- when interpreting the results, only the concentrations of sodium chloride are taken into account, which cause the onset of hemolysis (minimum erythrocyte resistance) and complete hemolysis (maximum erythrocyte resistance), while the degrees of hemolysis at all working concentrations of sodium chloride are calculated, but not taken into account when evaluating the results.

The authors propose a method for determining the osmotic resistance of capillary red blood cells using pharmacy solutions - isotonic sodium chloride solution (0.9% NaCl solution), water for injection (distilled water), which are used independently, as well as for the preparation of 0.45% sodium chloride solution .

The technical result of the proposed method for determining the osmotic resistance of capillary blood erythrocytes is the identification of hemolytic states by assessing the physicochemical properties of erythrocyte membranes in screening studies.

The authors propose to determine the WEM of capillary blood in 0.45% and 0.9% sodium chloride solutions. A study using an isotonic 0.9% sodium chloride solution, which is physiological for the human body, characterizes the initial physico-chemical state of the erythrocyte membranes. However, the determination of the WEM with only 0.9% sodium chloride solution is not enough to fully characterize it. The concentration of 0.45% NaCl was selected based on the results of numerous determinations of the WEM using prototype technology. It was found that the configuration of the hemolysis dynamics curves of various blood samples is of the same type, and that the main differences are observed at concentrations of 0.4% -0.6% sodium chloride. A study using a 0.45% sodium chloride solution is an osmotic stress test, which characterizes the degree of resistance, that is, the reserve capacity, of red blood cell membranes with a change in osmotic pressure.

According to the literature (Kushakovsky MS. Clinical forms of hemoglobin damage: etiology, pathogenesis, spectrophotometric and biochemical methods of research, diagnostics, treatment. - L .: Medicine, 1968, - 325 p.) In the visible hemoglobin absorption spectrum among three well noticeable bands there is the most intense band (Soret) in the region of 400-430 nm with a maximum absorption at a wavelength of 414 nm, at which the millimolar extinction coefficient is maximum, which ensures the necessary sensitivity for determining the extinction and measuring low concentrations of hemoglobin, therefore the authors suggest the use to study the degree of hemolysis when determining the erythrocyte osmotic resistance of wavelength 414 nm.

To determine the osmotic resistance of red blood cells, capillary blood is used, taken for a clinical blood test in a test tube with potassium EDTA (ethylenediaminetetraacetic acid). Three centrifuge tubes are used for the study. 2.5 ml of distilled water are placed in the first tube, 2.5 ml of isotonic sodium chloride solution in the second, 1.25 ml of distilled water and isotonic sodium chloride solution in the third (to obtain 0.45% NaCl).

Ingredients 1 test tube 2 test tube 3 test tube Distilled water 2.5 ml - 1.25 ml Isotonic (0.9%) NaCl solution - 2.5 ml 1.25 ml Capillary blood 0.01 ml 0.01 ml 0.01 ml

Then, 0.01 ml of capillary blood is added to all tubes. Stirred and centrifuged for 10 minutes at 2000 rpm. The optical density of the supernatant of the three samples was measured on a spectrophotometer at a wavelength of 414 nm in a cuvette with an absorbing layer length of 1 cm against distilled water.

ORE is estimated by the degree of erythrocyte hemolysis in solutions with different concentrations of sodium chloride compared to the degree of hemolysis in a sample with distilled water, which was taken as 100%, taking into account that the optical density of the supernatant liquids of the samples is directly dependent on the degree of hemolysis.

The degree of hemolysis was calculated twice in samples with 0.9% and 0.45% sodium chloride solutions according to the formula

H = 100 · E _op. / E _to. ,

where H is the degree of hemolysis (%),

100 - the degree of hemolysis in the sample with distilled water,

E _op. the optical density of the supernatant in the sample,

E _to. - the optical density of the supernatant in a test tube with distilled water.

Applying the proposed method for the determination of ORE in a group of 70 healthy people, the osmotic resistance of capillary blood erythrocytes was determined, which amounted to 1.61 ± 0.23% in the initial physiological state for red blood cells in a 0.9% solution of sodium chloride, and in the osmotic loading test in 0.45% sodium chloride solution - 25.16 ± 1.83%.

The authors examined, using a clinical analysis, the capillary blood of 122 people with clinical signs of anemia to confirm the diagnosis of the disease. To clarify the genesis of the disease for all samples was carried out the determination of the WEM. 5 people were found to have deviations of the WEM, which amounted to 10.13 ± 1.57% in 0.9% sodium chloride solution and 93.58 ± 3.08% in 0.45% sodium chloride solution, which served as the basis for establishing the presence of hemolytic state, the appointment of additional confirmatory studies and adequate therapy.

The obvious advantages of the proposed method for determining the osmotic resistance of red blood cells are:

- the use of 30 μl of capillary blood, which allows the use of a blood sample obtained for clinical analysis without additional blood collection;

- the method is technically simple and allows you to maintain the accuracy of the concentration of sodium chloride, since only one solution of 0.45% sodium chloride is prepared using pharmacy solutions of an isotonic sodium chloride solution and distilled water;

- application of the method for screening studies to detect hemolytic conditions;

- the method is economically advantageous, as it allows to reduce the working time of personnel for preparing solutions, measuring optical densities, washing laboratory glassware, and the number of disinfectants.

Claims (1)

A method for determining the osmotic resistance of red blood cells, including blood testing and quantitative determination of the degree of hemolysis in sodium chloride solutions of various concentrations, characterized in that the absorbance of the supernatant liquids of three samples of capillary blood is measured: with distilled water, with a 0.9% sodium chloride solution and with a 0.45% sodium chloride solution on a spectrophotometer at a wavelength of 414 nm in a cuvette with an absorbing layer length of 1 cm against distilled water, the degree of hemolysis at initial the physiological physical and chemical state of erythrocyte membranes in a 0.9% sodium chloride solution and with an osmotic load sample in a 0.45% sodium chloride solution compared with the degree of hemolysis in a sample with distilled water, which is taken as 100%, and with a degree of hemolysis of 1.61 ± 0.23% in a 0.9% solution of sodium chloride and 25.16 ± 1.83% in a 0.45% solution of sodium chloride, the osmotic resistance of red blood cells is interpreted as normal.