RU2580300C1 - Method of determining osmotic resistance of erythrocytes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and provides a method of determining osmotic erythrocyte resistance by blood examination, characterised by that blood in equal volumes is placed in measuring test tube N1 and N2 with sodium citrate is centrifuged, then plasma is sucked off with pipette, erythrocyte mass produced from measuring test tube N1 is diluted with 0.85 % sodium chloride solution at a ratio of 1:1, erythrocyte mass from measuring test tube N2 is diluted with 0.425 % solution of sodium chloride in ratio 1:1, both measuring test tubes are simultaneously centrifuged; further, by swinging or light shaking, blood is mixed with supernatant liquid to a homogeneous state, then from each test tube using a device for investigation of blood samples taken Microvette 200 mcl of erythrocyte mass and erythrocyte count in automatic blood analyser and finally osmotic resistance index (OIC), which is determined by formula OIC = RBC₁/RBC₂, where RBC₁ -erythrocyte count (× 10¹²/l) from measuring test tube N1, RBC₂ -erythrocyte count (× 10¹²/l) from measuring test tube N2, and if OIC 1.50-1.60 erythrocytes is assessed as average-resistant to osmotic hemolysis, 1.30-1.49 - as low-resistant, 1.61-1.8 - as highly resistant.

EFFECT: invention simplifies method for determining resistance of erythrocytes.

1 cl

Description

The invention relates to medicine, namely to the study of blood, and can be used for laboratory diagnosis of various anemia.

Currently, many methods are used to evaluate the resistance of red blood cells to the effects of various factors that can provoke their destruction, that is, cause hemolysis. However, the known methods are often inaccurate, time-consuming and extended over time. Therefore, there is the problem of creating a simplified method to reduce the complexity of determining the resistance of red blood cells to hemolysis.

A known method for assessing the resistance of red blood cells to acid hemolytic by determining the transmittance of a standardized suspension of red blood cells with the construction of a hemolysis schedule (Laboratory work. - 1965. - No. 9. - S. 530-531).

The course of determination.

Take 1-2 drops of blood from a finger puncture and place in tubes with 2-3 ml of physiological saline.

The standardization of the conditions is achieved by bringing the optical density of the suspension of red blood cells to a value equal to 0.7 units of extinction (D) in a cell with a working length of 30 mm Take 2.0 ml of a suspension of erythrocytes with an optical density of 0.7 D and place in a cuvette located in the right light beam of a photoelectric colorimeter. In the left nest insert a cuvette with physiological saline.

To a suspension of red blood cells add 2.0 ml of a 0.004 hydrochloric acid solution and immediately read the FEC on the right drum. Then the readings are taken every 30 seconds until they become constant. The optical density value is recorded in a column opposite the corresponding time stamps.

To plot the acid resistance of red blood cells, the following calculations are made.

The latter is subtracted from the first absorbance reading. In this way, a change in optical density corresponding to 100% hemolysis is recognized. The result is divided by 100 and get a change in optical density per 1% hemolysis.

1. Subtract the next from each previous reading of the optical density and obtain changes in the optical density in each subsequent 30 seconds.

2. The data obtained in paragraph 2 is divided by the optical density corresponding to 1% hemolysis (paragraph 1), and the percentage of hemolyzed red blood cells for each subsequent 30 seconds is obtained.

3. The results are expressed graphically. The disadvantages of this method are:

1) labor input;

2) time duration.

The closest technical result achieved is the method for determining the osmotic resistance of red blood cells according to Limbek and Ribier (Guide to clinical laboratory research. - M .: Medgiz, 1960, S. 96-98), in which hypotonic solutions with salt concentrations from 0.56 are used up to 0.28%. They are poured into a series of test tubes, then 0.02 ml of blood taken from the pulp of a finger is poured into each tube with the same pipette from the Sali hemometer. By gently shaking the tubes, the suspension of blood in the saline is uniform. The tubes are left in a rack at room temperature for 1 hour, then centrifuged for 5 minutes at 2000 rpm, after which the maximum and minimum levels of osmotic resistance are noted.

Reading the results.

The maximum and minimum levels of resistance are those salt concentrations at which hemolysis began and ended.

The level of maximum resistance, that is, the first signs of hemolysis, is determined by eye with a slight yellowness, and the level of minimum resistance (complete destruction of red blood cells) is determined by the intensely red color and complete transparency of the solution.

Normal limits of osmotic resistance:

il minimum resistance to fluctuation in the range of 0.48-0.46%; for the maximum - 0.34-0.32%.

Reducing the resistance of red blood cells, according to the authors of the method, occurs with congenital spherocytosis, and in acute hemolytic crisis and B ₁₂ deficiency anemia expansion of the boundaries of resistance.

The disadvantages of the known method are its complexity and duration:

1) an increase in hemolysis in remote periods of time can occur due to microbial growth and due to the absorption of carbon dioxide in the air;

2) the results are taken into account after 3-4-24 hours, while osmotic erythrocyte hemolysis takes place quickly, reaching its maximum after 5 minutes.

The authors propose a method for determining the osmotic resistance of erythrocytes, which allows one to evaluate the tendency of erythrocytes to osmotic hemolysis using an automatic hematological analyzer and calculate the osmotic resistance index, which decreases or increases when there are a large number of red blood cells in the blood sample that are prone or more resistant to destruction by osmotic hemolytic.

The technical result of the proposed method is to simplify the method.

The technical result is achieved by the fact that blood in equal volumes is placed in volumetric tubes No. 1 and No. 2 with 3.8% sodium citrate in a ratio of 9: 1, centrifuged at 1500 rpm for 15 minutes, after which the plasma is aspirated with a pipette; the obtained erythrocyte mass from the volumetric tube No. 1 is diluted with a 0.85% sodium chloride solution in a ratio of 1: 1, the erythrocyte mass from a volumetric tube No. 2 is diluted with a 0.425% sodium chloride solution in a 1: 1 ratio; both measuring tubes are simultaneously centrifuged for another 15 min at 1,500 rpm, after which the blood is mixed with a shaking or gentle shaking with a supernatant until homogeneous, then 200 μl of red blood cell are taken from each tube using a Microvette blood sampling device and counted erythrocytes in an automatic hematology analyzer: first, red blood cells are counted in a sample taken from a volumetric tube No. 1, then from a volumetric tube No. 2, and finally, the inspection index is calculated cal resistance (IER) which is defined by the formula:

IOS = RBC ₁ / RBC ₂ , where

RBC ₁ - the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 1,

RBC ₂ - the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 2;

and with an IOS value of 1.50-1.60, erythrocytes are evaluated as moderate to osmotic hemolysis; 1.30-1.49 - as low resistance; 1.61-1.8 - as highly resistant.

The method is as follows.

Equipment: automatic hematology analyzer, for example, "Hemolux 19" (Mindray Bio-Medical Electronics Co., Ltd., China); laboratory centrifuge (OPN-3U4,2); Microvette blood sampling devices with anticoagulant 200 µl K3E (K3-EDTA) Sarstedt AG & Co., Germany.

Reagents:

1) anticoagulant - 3.8% sodium citrate solution (obtained by dissolving 3.8 g of sodium citrate (REACHIM OSCH-6-4 Mikhailovsky Chemical Reagents Plant) in 100 ml of distilled water);

2) a 0.85% solution of sodium chloride (obtained by dissolving 0.85 grams of sodium chloride (REACHIM OSCH-6-4 Mikhailovsky Chemical Reagents Plant) in 100 ml of distilled water);

3) osmotic hemolytic - 0.425% sodium chloride solution (obtained by diluting a 0.85% sodium chloride solution with an equal volume of distilled water).

The course of determination.

Blood is taken from the ulnar vein with a wide dry needle and placed in equal volumes into measuring tubes No. 1 and No. 2 with 3.8% sodium citrate in a ratio of 9: 1. By shaking or lightly shaking, the blood is mixed with an anticoagulant.

Blood is centrifuged in a centrifuge at 1,500 rpm for 15 minutes, after which the plasma is aspirated with a pipette. The resulting red blood cell mass from the measuring tube No. 1 was diluted with a 0.85% sodium chloride solution in a ratio of 1: 1; the erythrocyte mass from the measuring tube No. 2 is diluted with a 0.425% sodium chloride solution in a ratio of 1: 1; both measuring tubes are simultaneously centrifuged for another 15 min at 1500 rpm. After centrifugation by shaking or gentle shaking, the blood is mixed with the supernatant until homogeneous. 200 μl of red blood cells are taken from each measuring tube using a Microvette blood sampler and the red blood cells are counted in a Hemolux 19 automated hematology analyzer.

First, the red blood cells are counted in the sample taken from the measuring tube No. 1, then the red blood cells are counted in the sample taken from the measuring tube No. 2.

In conclusion, calculate the osmotic resistance index (IOS), which is determined by the formula:

IOS = RBC ₁ / RBC ₂ , where

RBC ₁ - the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 1 obtained in the device for the study of blood samples Microvette when counting in an automatic hematological analyzer "Hemolux 19",

RBC ₂ - the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 2, obtained in the device for the study of blood samples Microvette when counting in an automatic hematological analyzer "Hemolux 19"; and with an IOS value of 1.50-1.60, erythrocytes are evaluated as moderate to osmotic hemolysis; 1.30-1.49 - as low resistance; 1.61-1.8 - as highly resistant.

The inventive method is simple and convenient to use, economical in time and will be widely used in the clinic for the diagnosis of various anemia.

Claims (1)

A method for determining the osmotic resistance of red blood cells by blood testing, characterized in that the blood is placed in equal volumes in measuring tubes No. 1 and No. 2 with 3.8% sodium citrate in a ratio of 9: 1, centrifuged at 1500 rpm for 15 minutes , then the plasma is aspirated with a pipette; the obtained erythrocyte mass from the measuring tube No. 1 is diluted with a 0.85% sodium chloride solution in a ratio of 1: 1, the erythrocyte mass from a measuring tube No. 2 is diluted with a 0.425% sodium chloride solution in a ratio of 1: 1, both measuring tubes are centrifuged for another 15 minutes at the same time 1500 rpm, after which, by shaking or lightly shaking, the blood is mixed with the supernatant until homogeneous, then 200 μl of erythrocyte mass is taken from each tube using a Microvette blood sampling device and counted erythrocytes in an automatic hematological analyzer: first, red blood cells are counted in a sample taken from a measuring tube No. 1, then from a measuring tube No. 2, and finally, the osmotic resistance index (IOS) is calculated, which is determined by the formula
IOC = RBC ₁ / RBC ₂ ,
where RBC ₁ is the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 1,
RBC ₂ - the number of red blood cells (× 10 ¹² / l) from the measuring tube No. 2,
and with an IOS value of 1.50-1.60, erythrocytes are assessed as medium resistant to osmotic hemolysis, 1.30-1.49 as low resistant, 1.61-1.8 as high resistant.