The method of determination of reticulocytes incubated in the blood of birds


G01N1/30 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)
G01N1/28 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

 

The invention relates to the field of veterinary medicine. The method includes stabilizing the blood of 3.8% sodium citrate in a ratio of 1 part anticoagulant and 4 parts of blood, incubation at rest with 39With the mixing of blood with the dye in the ratio of 3:1 laminar flow in a confined space for 30 s, staining with 1% solution of the dye diamond crazily blue, cooked physiological solution, at pH 2,95-3.0 V for 40 min at room conditions. Then prepare strokes that are fixed with methanol. The counting of reticulocytes produced per 1000 erythrocytes by maturity using an image analyzer. The proposed method allows to detect reticulocytes 1, 2, 3 and 4 classes and erythrocytes. table 2. 4 Il.

The invention relates to the field of veterinary medicine and can be used to determine the functional activity of bone marrow (red blood cell balance) in birds under physiological conditions and under the action of extreme factors.

Red blood cell is a heterogeneous cytological system consisting of morphologically and functionally kinetically distinct cells [1, 2]. Definition of colistoole erythrocytes [3].

Maintaining erythrocyte equilibrium in normal or altered level by mechanisms that increase the intensity of erythropoiesis with fewer red blood cells and reduce it by increasing their number. Well-known practice of counting of reticulocytes and determine the time of maturation to quantify the intensity of erythropoiesis and thus indirectly determine the lifespan of erythrocytes [4].

For the prototype accepted way of determining the number of daily maturing reticulocytes, developed by E. N. Mosalini [1, 4, 5] on mammalian animals, including the selection of peripheral blood in an amount of 0.4 ml and kept sterile conditions in a well-waxed tube in which add a few grains of powdered heparin; incubation of heparinized blood in the incubator for 4 h, and to get closer to the physiological conditions of the test tube is mounted on the Board, receiving every 3 to move up and down from a small electric motor to create a simulation of the motion of red blood cells in blood stream, which, according to the authors, affects the process of maturation of reticulocytes; color incubated blood to the thief (pH 7,3) with preliminary mixing on waxed hour glass waxed glass rod. Watch glass placed on a 35 min in a humid chamber. Then the mixture of paint and blood again stirred and make broad strokes on the net low-fat glasses; the expense of reticulocytes in smears conducted in all fields.

Erythrocytes are in the first ten fields of view. After the account is brought up to 10 000, counting reticulocytes advanced in nine fields of view, without separating them into separate groups according to the degree of maturity [4].

The method is not widely used due to low precision and physiologically unexplained differences in the data on normal values of the number of reticulocytes in humans and animals, obtained by different techniques for staining reticulocytes. Moreover, in the literature there are no data regarding the study of the intensity of erythropoiesis in birds based on the counting of reticulocytes incubated, and the norms of the number of reticulocytes in physiological conditions also vary considerably according to different authors. Because reticulocytes are in the peripheral blood of all stages of maturation, they reticulum gradually disappears, and the perfect color and microscopic techniques, the later ripening stages can be distinguished reticulocyte from tretazicar balance in birds, because in the process of electron microscope coloring heparinised incubated blood 1% dye diamond crazylove blue accelerates the clotting of blood, smears detected shadow cells, around which there are characteristic halos. Most of the cells die, the reticulocytes are not identified. The counting of reticulocytes in all of the fields of view is limited by the specificity of smear preparation, which involves the presence of thin and thick areas; in this regard, when using the microscope the thicker parts of the stroke, where cells overlap each other (in birds besides erythrocytes nuclear, which creates unnecessary interference), the separation of reticulocytes becomes quite difficult.

Literary analysis has allowed to reveal the reasons for the unsuitability of the existing method, due to the peculiarities of chemical reactions in the environment between the dye, anticoagulant and biological tissue of birds: 1) the blood of birds compared to mammals differs enhanced coagulation due to the high content of CA2+[6], performing at the initial stage of blood coagulation function of prothrombin activators. Adding on Wednesday anticoagulants leads to the precipitation of the ion is, s calcium metabolism and the degradation of cellular components, contributing to the increase in extracellular CA2+in the environment, increased blood clotting, and cell death [8]; 3) the molecule of heparin, with a strong acidic properties, carries a negative charge and reacts with basic dyes (including diamond crazily blue), while it is inactivated and loses the ability to slow the clotting process [7].

All this leads to cell death, the appearance of smears halos and shadows cells contaminated with a complex of heparin with dye. Thus, staining of blood birds dye and heparin lose their properties, which leads to the impossibility of identifying reticulocytes.

Object of the present invention is to provide a noninvasive method for determining the number of reticulocytes incubated in the blood of birds to assess the functional activity of bone marrow.

To achieve these objectives in the way, including the stabilization of blood heparin, incubation of blood for 4 h at body temperature with periodic shaking, staining with 1% solution of the dye diamond crazily blue prepared in saline solution (pH of 7.3), in the ratio of 1:1 on waxed dscat the number of reticulocytes per 10,000 erythrocytes, proposed stabilization of blood to produce a 3.8% sodium citrate in a ratio of 1 part anticoagulant and 4 parts of blood; incubation of blood to produce biological unit 39C for 4 h in a quiescent state; mixing the blood with 1% dye diamond crazylove blue (3:1) to produce laminar flow in a confined space, such as in a test tube for 30 s; staining at pH 2,95-3,0 be made within 40 min in air at 18-20With; the fixation of smears prepared on a clean, fat-free glasses, make 2-3 drops of methanol to complete the spreading of the alcohol on the surface of the glass; the expense of reticulocytes to pay on maturity groups per 1000 erythrocytes using an image analyzer.

When used as an anticoagulant sodium citrate is not detected morphologically modified erythrocytes (swollen, star shaped, etc.,), reticular substance clearly identified. Stabilization incubated blood citrate also allows you to avoid excessive dirt on smears and artifacts, so how do we prevent the increased coagulability of the blood of birds, to avoid inactivation of the dye molecules; in the end rc="https://img.russianpatents.com/chr/176.gif">With allows you to maintain in vitro close to natural physiological parameters of blood. Prior to staining laminar mixing of blood with the dye in a confined space, such as in vitro, the effect of reducing the traumatized cells and helps to avoid artifacts on the smear, because the red blood cells of birds less resistant to mechanical damage than mammals.

Breeding incubated blood with the dye in the ratio of 3:1 reduces the error of the experiment (when using a dilution of 1:1 are obtained strokes with a limited number of red blood cells, which implies the need to prepare two smears for the same object). Fixation of the smear allows long-term storage and, if necessary repeatedly to explore.

Use in the process of counting of reticulocytes analyzer images can count reticulocytes at maturity per 1000 erythrocytes instead of 10000 through a special program, developed by the company “Videotest”, and high-precision device that allows to obtain reliable results and to save time of the study. Determining the number of reticulocytes different degrees of maturity providing the basis [9].

Distinctive features of the claimed method are used to stabilize the blood as an anticoagulant of 3.8% sodium citrate in a ratio of 1 part anticoagulant and 4 parts of blood, incubating the biological thermostat at rest at a temperature of 39With, laminar mixing in a confined space, such as in vitro blood dye (3:1) for 30 s with painting in vitro at pH 2,95-3.0 V for 40 min in air at room conditions (18-20C) fixing cooked on a clean, non-fatty glass smear two or three drops of methanol to full flow, the counting of reticulocytes at maturity per 1000 erythrocytes using an image analyzer.

Example determine the number of reticulocytes incubated in the blood of birds. In a test tube pour 0.1 ml of 3.8% sodium citrate and 0.4 ml of blood, gently but thoroughly. Incubate at rest in the biological unit 39C for 4 h In a test tube pour 0.1 ml of 1% aqueous solution of brilliant Krasilovka blue, cooked in physiological solution (0.9% sodium chloride) and 0.3 ml of the incubated blood, �176.gif">C). Make strokes on the net low-fat glasses. Fix two or three drops of methanol to complete the spreading of the alcohol on the surface of the glass. Air dried. To microscopical under immersion. The counting of reticulocytes on the maturity of lead per 1000 erythrocytes using an image analyzer.

The proposed method allows to detect reticulocytes and erythrocytes. The reticulocytes in the form of elongated ellipsoids, painted in pale green color with kernel from light blue to purple and blue mesh having a different configuration in the reticulocytes of different degrees of maturity: reticulocytes 1st class - mesh fills the cytoplasm and is superimposed on the core (Fig.1); reticulocytes 2nd class - fishnet densely fills the cytoplasm, it is not imposed on the core (Fig.2); reticulocytes 3rd class - mesh is mainly around the nucleus, sometimes in the form of an eccentric lying tangles (Fig.3); reticulocytes 4th class - mesh in the form of filiform formations and individual inclusions scattered throughout the cytoplasm (Fig.4). Erythrocytes - pale-green intensely blue core, without intracellular inclusions. The research results are summarized in tables 1 and 2.

As you can see, in slavescom type of blood involves the receipt of immature forms of erythrocytes in the peripheral blood, where the process of their formation, there are reticulocytes third and fourth classes (see tab. 1). Moreover, the rhythm of the regenerative processes of the system Eritrea (intensity of erythropoiesis) individuals will depend on the lability and adaptive capabilities of the nervous structures, and the functional reserve of bone marrow birds.

When perturbing effects indicators of bone marrow hematopoiesis indicate the powerful emission of young forms cells in the bloodstream. The increased catabolic processes during stress leads to changes in the rate of maturation of reticulocytes to reduce the time of their maturation and accelerated half-life of red blood cells from the bloodstream due to the high damage of cells erythrocyte populations under the influence of stress hormones. In accordance with this change of intensity of erythropoiesis, manifested in the increase in bone marrow production and appearance of reticulocytes first and second classes (see tab. 2).

Literature

1. Mosyagina E. N. The kinetics of blood cells/Mosyagina E. N., Vladimir E. B., Torubarov N. A., Mysina N. In. - M.: Medicine, 1976, 272 S.

2. Ilyukhin A. C. Cytokinetic and morphology of blood in chronic about the SKOV I. A. The study Eritrea as controlled by the body's cellular system/Issues Biophysics, biochemistry and pathology of erythrocytes/Ed. by G. M. Frank. - M.: Nauka, 1967, S. 48-62 per.

4. Mosyagina E. N. Erythrocyte balance in health and disease. - M.: Medgiz, 1962, 271 S.

5. Ilyukhin A. C., Burkovskaya I.e., Shafirkan A. C., Klyuchanskaya N. In. Some methodological issues of the study of erythrocyte balance according to the counting reticulocytes incubated//Space biology and aerospace medicine. - 1982, so 16, No. 3, S. 86-88.

6. Agricultural bird/Ed. by E. E. of Personnalize. - M.: publishing house of the agricultural literature, 1962, T. 1, 383 S.

7. Zubairov D. M. Biochemistry of blood coagulation. - M.: Medicine, 1978, 174 S.

8. Ashkenazi I. J. erythrocyte Destruction/Manual physiology of the blood system. Fisiologia erythropoiesis. - L.: Nauka, 1979, S. 274-334.

9. Yanovsky, A. N. Picture of blood and its clinical significance. - Kiev, 1957, 543 S.

Claims

The method of determination of reticulocytes, including stabilization, incubation of blood within four hours, the color incubated in 1% of blood dye brilliantissima prepared with physiological solution, the stabilizing of 3.8% sodium citrate in the ratio of 4:1, incubation of blood performed at rest with 39With, the blood is mixed with a dye laminar in the ratio of 3:1 in closed volume, for example, in a test tube for 30 s, dyeing is carried out at a pH of 2.95 and-3.0 V for 40 min in air at a temperature of 18-20With dried smears fixed with methanol, the counting of reticulocytes are on their degree of maturity per 1000 erythrocytes using an image analyzer, and the reticulocytes appear as elongated ellipsoids pale green with a kernel from light blue to purple and blue mesh having a different configuration in the reticulocytes of different degrees of maturity: in reticulocytes class 1 - net fills the cytoplasm and is superimposed on the core, reticulocytes class 2 mesh densely fills the cytoplasm, it is not imposed on the kernel, the reticulocytes 3 class - mesh is mainly around the nucleus, sometimes in the form of an eccentric lying tangles, reticulocytes class 4 - mesh in the form of filiform formations and individual inclusions scattered throughout the cytoplasm, and erythrocytes pale-green intensely blue kernel without intracellular inclusions.

 

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