The method for determining the acid resistance of erythrocytes

 

The invention relates to medicine, namely to laboratory diagnosis. To evaluate the acid resistance of erythrocytes erythrocyte suspension will standardize the breeding of erythrocyte mass saline solution in a ratio of 1:3 and centrifugation for 15 min, followed by dilution of 0.01 ml of erythrocyte sediment 4.0 ml of physiological solution. Add acid hemolytic, which is used as a 1% solution of acetic acid. Determine the amount of light transmission through the parallel account changes in the average size of red blood cells and the amount of light transmission with graphing, which determine the time of registration the maximum average size of erythrocytes and the average rate of hemolysis. Calculate the index of acid resistance of erythrocytes (X) by the formula: X= t_d max/gVm, where t_d max - time maximum average radius of erythrocytes, sec; gVm - average rate of hemolysis, the river. units, the value of X from 0.50 to 0.68 consider normal. The application of the method allows to estimate the parameters of the rigidity and durability of erythrocyte pool necessary for the diagnosis of anemia of different origin. 1 Il.

This purpose is often used methods to evaluate the resistance of erythrocytes to the influence of various factors that may cause their destruction, that is, to cause hemolysis.

There is a method of determining the osmotic resistance of erythrocytes Limbuko and Ribiero (Manual of clinical laboratory research, Medgiz, 1960, pp. 96-98), in which the use of hypotonic solutions with salt concentrations from 0.56 to 0.28%. They poured in a row of test tubes, then the same pipette from geometry Sali in each tube is poured 0.02 ml of blood taken from the pulp of the finger. Careful shaking of the tubes reach a uniform mist of blood in saline solution. The tubes leave the tripod at room temperature for 1 hour, then within 5 minutes, centrifuged at 2000 rpm, after which mark the maximum and minimum level of osmotic resistance.

The reading of the results.

The maximum and minimum level of resistance is the salt concentration at which hemolysis began and ended.

The maximum level of resistance, that is, the first signs of hemolysis determined by eye light yellow, and the level of minimum durability (complete destruction of erythrocytes) - intensively to the school resistance fluctuations in the range of 0.48-0.46 per cent; for a maximum of 0.34-0.32 per cent.

Lowering the resistance of red blood cells, according to the authors, the method has a place in congenital spherocytosis, and acute hemolytic crisis and vitamin B12 deficiency anemia expanding the boundaries of resistance.

The disadvantage of this method is its inaccuracy related to the fact that

1) results are taken into account through 3-4-24 hours, whereas osmotic hemolysis of erythrocytes is performed rapidly, reaching its maximum after 5 minutes;

2) increased hemolysis long periods of time may occur due to microbial growth and due to the absorption of carbon dioxide in the air;

3) inaccurate visual assessment as the minimum and maximum hemolysis.

The closest achieved a positive result is a method of evaluating the resistance of erythrocytes to acid hemolytica by determining the amount of light transmission of standardized suspension of erythrocytes with graphing hemolysis (Laboratory work. - 1965. No. 9. - P. 530-531).

The course definition.

Taken from a finger puncture 1-2 drops of blood and placed in a tube containing 2-3 ml of saline.

Standardization of conditions is achieved by bringing the optical density of the suspension of erythrocytes to the greates what Amnesty 0,7 D and placed in the cuvette in the right light beam fotoelektrokalorimetry. In the left slot insert a cuvette containing saline solution.

To the suspension of erythrocytes add 2.0 ml of 0.004 N hydrochloric acid and immediately noted the testimony of FEC on the right reel. Then take readings every 30 seconds as long as they do not become permanent. The value of optical density is written in the column opposite corresponding to the timestamp.

To plot the acid resistance of erythrocytes produce the following calculations.

From the first readings of optical density subtract the latter. So recognize the change in optical density, corresponding to 100% hemolysis. The obtained result is divided by 100 and get the change in optical density per 1% of hemolysis.

1. From each of the previous readings of optical density is deducted from a subsequent and receive optical density in each subsequent 30 seconds.

2. Data obtained under paragraph 2, is divided by the optical density corresponding to 1% of hemolysis (item 1), and receive a percentage hemosiderosis erythrocytes for each subsequent 30 seconds.

3. The results obtained are expressed graphically (see drawing).

Possible avtra EFA-1.

The course definition.

In the cuvette is placed in 2.0 ml of the suspension of erythrocytes and 2.0 ml of 0.004 N hydrochloric acid (stylus should be rejected in its rightmost position) and include the recording mechanism. The record is produced on a paper tape with a millimeter grid. With the development of hemolysis recorder trace along the integral curve of optical density of the suspension of erythrocytes.

Processing of the material.

The resulting curve is enclosed in a coordinate system so that the zero mark coincides with the beginning of the recording, the abscissa shows the delay time of hemolysis, and the ordinate axis is the optical density.

The change in optical density corresponding to the hemolysis for each subsequent 30 seconds, easy to find, comparing the segments on the axes of abscissa and ordinate.

The percentage of broken red blood cells per 1 mm of the scale of the ordinate, is found by measuring in millimeters the total change in optical density, which is taken for 100%. Erythrocytes can be divided into the following groups: decreased-persistent - broken for the first third of the entire time of hemolysis, rednisone for the second third and value-resistant, broken for the last third of the time of hemolysis.

In addition, you can calculate the total from the cylinder the appropriate time.

This method, as well as similar, is used for the diagnosis of various anemias.

The disadvantages of this method are:

1) complexity; (2) duration; 3) the inability to assess the dynamics of the elasticity of erythrocytes under the influence of acid hemolytica.

A positive result of the proposed method is the determination of the parameters of the elasticity of red blood cells to fracture and increase the transmittance of the suspension of erythrocytes under the influence of acid hemolytica for a more accurate assessment of resistance of erythrocytes to the effects of acid hemolytica largest index of acid resistance.

A positive result is achieved by the fact that the resulting standardized level of dilution of red blood cells allows to construct curves of hemolysis and the elasticity of red blood cells using analyzer aggregation “Biola”, which is used in many clinical laboratories in Russia only for the evaluation of platelet aggregation, and to calculate an index of acid resistance, decreasing or increasing in the presence of the investigated blood of a large number of red blood cells, prone, or more resistant to destruction by acid hemolytica.

The method is performed as education is ifoga laboratory (ARF-U,2).

Reagents:

1) the anticoagulant of 3.8% solution of sodium citrate (produced by dissolving 3.8 g of sodium citrate (REACHIM OFS-6-4 Mikhailovsky plant chemicals) in 100 ml of distilled water);

2) a 0.85% solution of sodium chloride (produced by dissolving 0.85 grams of sodium chloride (REACHIM OFS-6-4 Mikhailovsky plant chemicals) in 100 ml of distilled water);

3) acid hemolytic - 1% solution of acetic acid (receive, adding to 10.2 ml of 98% acetic acid in 89.8 ml of distilled water).

The course definition.

From the cubital vein of a wide dry needle, blood is taken in a volumetric flask with 3.8% sodium citrate in a ratio of 9:1. A slight rocking or shaking the blood mixed with anticoagulant.

The blood is centrifuged in a centrifuge at 1500 rpm for 15 min, after which the plasma is sucked off by pipette. Received RBC mass bred a 0.85% solution of sodium chloride in the ratio of 1:3 and centrifuged 15 min at 1500 rpm After centrifugation the supernatant is sucked off by pipette, and the remaining RBC mass is thoroughly mixed to a homogeneous state. In a test tube with 4 ml of 0.85% sodium chloride solution, add 0.01 ml of the obtained erythrocyte mass, and further work with this will eritrocitary in a cuvette with a built-in magnetic stirrer analyzer aggregation “Biola”, it's the same with constant stirring at a speed of "2" (temperature 37°C) after the tone, enter 0.3 ml of 0.85% sodium chloride solution. A calibration curve recorded for 2 minutes

Next, write the graph. To do this, in other dry cuvette containing a magnetic stir bar is placed 0.3 ml of erythrocyte suspension with constant stirring at a speed of "2" (temperature 37°C) it after the tone, enter 0.05 ml of 1% solution of acetic acid. Schedule record within 5 minutes

The first curve (1) reflects the dynamics of change in the average size of the particles (red blood cells) when exposed to acid hemolytica. Curve at first, gradually growing up, reflecting, thus, the process of stretching of red blood cells, which under the influence of hydrogen protons penetrating into the cells, there is an increase in oncotic pressure within them and, as a consequence, the increase of the entering current of water molecules. Having reached the peak, the curve begins to descend, which suggests that the process of fragmentation and complete destruction of erythrocytes under the influence of acid hemolytica.

The second curve (2) characterizes the change in the value of transmittance of the laser beam when passing through the erythrocyte in the, the specific point in time, the growth stops and the curve reaches a plateau.

The reading of the results.

To evaluate the acid resistance of erythrocytes using the following parameters.

For curve 1:

1) the initial average size of erythrocytes (srvc.ed.);

2) the maximum average size of erythrocytes (srvc.ed.);

3) the elongation of erythrocytes - magnitude increase in the average size of particles from the initial to the maximum (expressed in percent, with an initial average particle size is taken as 100%);

4) check the maximum average size of red blood cells (sec).

For curve 2:

1) the maximum rate of hemolysis (srvc.ed.);

2) check the maximum speed of hemolysis (s);

3) the average rate of hemolysis is a value expressed by the area bounded by:

the top curve 2;

bottom abscissa;

on the right is the perpendicular from 2 curve at the point x-axis corresponding to time the third minute incubation of erythrocytes (srvc.ed.).

In conclusion, calculate the index of acid resistance (X), representing the parameters of both curves, reflecting the stability of the studied red blood cells to the effects of acid hemolytica. It is determined by the formula:

- gV m - the average rate of hemolysis (srvc.ed.), which is determined by the second curve.

In the study of blood samples of healthy people received the following normal values X: average of 0.59, with the range of normal fluctuations from 0.50 to 0.68.

The claimed method has the following advantages:

the simplicity and ease of execution

- the cost in time,

- parallel recording changes in the average size of red blood cells and the amount of light transmission.

The inventive method will be widely used in the clinic for the diagnosis of various anemias.

Claims

Evaluation method for acid resistance of erythrocytes by determining the amount of light transmission of standardized suspension of erythrocytes after adding acid hemolytica with graphing hemolysis, characterized in that the suspension of erythrocytes will standardize the breeding of erythrocyte mass saline solution in a ratio of 1:3 and centrifugation for 15 min, followed by dilution of 0.01 ml of erythrocyte sediment 4.0 ml of physiological solution, as the acid of hemolytica use 1% solution of acetic acid, conduct parallel change record with the maximum average size of erythrocytes and the average rate of hemolysis and calculate the index of acid resistance of erythrocytes (X) by the formula

X= t_d max / gVm,

where t_d max - time maximum mean radius of the cells;

gVm - average rate of hemolysis, the river. units;

the value of X from 0.50 to 0.68 consider normal.



 

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