RU2280689C2 - Method for retention of viable blood leukocytes - Google Patents

Abstract

FIELD: biology, immunology.

SUBSTANCE: method involves preparing a stabilizing solution for providing the maximal retention of isolated viable blood leukocytes and prevention of their sticking. Method involves taking off a blood sample and isolation of leukocytes by using the concentrate Haenx without phenol red that is used in the ratio with water = 1:9 and containing additionally glucose, lyophilized bovine serum albumin in the dose 10-15 mg per 100 ml of each. Method provides retaining the constant parameters of cells and to enhance retention period of viable leukocytes (90-95%) in carrying out the chemiluminescent analysis.

EFFECT: improved retaining method.

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Description

The invention relates to biology, namely to the section of immunology, and can be used for chemiluminescent analysis in medicine and veterinary medicine.

The well-known "Stabilizing composition for producing lyophilized preparations based on the culture virus of Venezuelan encephalomyelitis horses", copyright certificate No. 1761800 A1, C 12 N 7/00, A 61 K 39/125 from 09/15/92. Bull. Number 34. To obtain dry viral preparations, a stabilizing composition is used that contains (g / l of the culture virus suspension): maltose 28-60; peptone 44-100; gelatin 10-20; bovine serum albumin 11-25, sodium thiosulfate 1.25-3.0.

However, the proposed stabilizing composition can be used only after lyophilization of the viral material, which requires expensive equipment.

Of those described in the literature, the closest to the invention is the "Method of preservation of immunocompetent cells by culturing them in a nutrient medium", copyright certificate No. 1073281, C 12 N 5/00, G 01 N 54/57 from 02.15.84. Bull. No. 6.

The method is carried out by isolating immunocompetent cells or organ tissue homogenate by layering ficoll-verographin on a standard density gradient, the isolated cells are washed three times from ficoll and verographin with 199 medium. Then, the obtained lymphocytes are placed in glass containers with nutrient medium 199 previously cooled to 5-15 ° C. additionally containing jelly-forming components, fixing immunocompetent cells in suspension, as well as inactivated serum I group cro and man a 1% solution of 1-glutamine, and 2% solution of L-asparagine. The containers with the culture medium and cells are placed in a household refrigerator and periodically shaken from time to time until the culture medium transitions from a liquid to a jelly-like state with subsequent visual control of the uniform distribution of cells.

However, the proposed method allows you to select only one type of "white" cells - lymphocytes, to carry out visual (subjective) control of the uniform distribution of cells, in addition, the method is expensive.

An important role in bacterial infections in the interaction of immunocompetent cells is played by phagocytes (neutrophils, etc.) that process the antigen (microbe, foreign cell), translating it into a more immunogenic form and thereby participating in the specific sensitization of the corresponding lymphoid elements of the humoral and cellular systems (I .E. Kolyakov, 1986).

The objective of the invention is to increase the shelf life of viable blood leukocytes and prevent their adhesion during chemiluminescent analysis.

The method is as follows. Preliminary blood is taken with Trilon B in test tubes treated with a 3% solution of polymethylsiloxane liquid in chloroform. The destruction of red blood cells is carried out by hypotonic lysis. Centrifuged at 1200 rpm for 15 minutes Additionally, leukocytes are washed with a 0.02% solution of sodium salt of ethylenediaminetetraacetic acid (Trilon B). It is centrifuged under the above conditions, the supernatant is drained. The cell suspension pellet is resuspended in a stabilizing solution of the following composition: Hanks concentrate without phenol red in a ratio of 1: 9 with water.

Hanks concentrate without phenol red, used for research, is industrially produced by the Sverdlovsk Research Institute of Virus Infections bacterial production enterprise, consists of concentrated solution A containing sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, chloroform for preservation, bidistilled water; concentrated solution B containing disubstituted sodium phosphate, monosubstituted potassium phosphate, glucose, bidistilled water; solution B containing sodium carbonate.

While stirring on a magnetic stirrer, to 100 ml of Hanks concentrate without phenol red in a ratio of 1: 9 water add 10-15 mg of glucose until completely dissolved, then the solution is heated to 50 ° C and 10-15 mg of bovine serum albumin lyophilized is added until homogeneous colloidal solution. The resulting solution is filtered, packaged, stored in a freezer at -20 ° C for up to 1 month.

A standard Hanks concentrate without phenol red in a 1: 9 ratio with water as a buffer provides a favorable environment for white blood cells, has the ability to maintain pH (pH) and isotonicity at the same level. We suggest using Hanks concentrate without phenol red, as phenolic compounds quench chemiluminescence (Nelson et al., 1977).

Additional leukocyte washing with a standard solution of Trilon B, which is a 0.02% solution of sodium salt of ethylenediaminetetraacetic acid in bidistilled water, provides a homogeneous suspension of cells, because the basis of the action of this standard solution is the ability to bind complexes containing calcium. As a result of the binding of calcium and magnesium ions, the intercellular bonds weaken and then, using a weak mechanical effect, a homogeneous suspension of cells can be obtained (Z. Lyarski, 1980).

Silicone, or organosilicon compounds, is characterized by a low pour point, hydrophobicity, chemical inertness, high thermal and thermo-oxidative stability, a relatively small change in viscosity with temperature, good dielectric properties, which prevents the adhesion of blood cells to the walls of the tube, thereby increasing the output of leukocytes.

Albumin - supports colloid osmotic (oncotic) pressure, as well as blood pH. Complexes of albumin easily dissociate with a change in pH, ionic strength, or in the presence of any component in the tissues, form stronger bonds with the ingredients of the complex (BME, v. 1. S.906).

Albumin is added to the Hanks concentrate without phenol red in a 1: 9 ratio with water, mixed; to dissolve albumin, the mixture is heated to 50 ° C, obtaining a homogeneous colloidal solution. An increase in temperature violates the tertiary structure of albumin, which determines its specificity (P. Zengbush, 1982).

Glucose - creates osmotic pressure, protects the allocated microstructures from destruction (L.M. Model, 1962).

Example 1. The working solution is prepared from a standard 10-fold Hanks concentrate without phenol red as a buffer solution, which has a pH of 7.4-7.6. The composition of Hanks is composed in such a way that it is balanced by air, and not by CO ² (V. Lyarski, 1980). Below is a table of selection of dilutions of Hanks concentrate and distilled water.

Table 1
Preservation of viable white blood cells at various ratios of Hanks concentrate and water Option Preservation of viable white blood cells (%) 10 cr Hanks concentrate 30-40 1 part Hanks concentrate + 1 part distilled water 60-70 1 part Hanks concentrate + 9 parts distilled water 90-95

From table 1 it can be seen that a dilution of 1 part of Hanks concentrate + 9 parts of distilled water provides the greatest preservation of viable white blood cells.

Example 2. We give a comparative assessment of the preservation of viable leukocytes in liquid nutrient media used for stabilizing solutions.

table 2 Options Preservation of viable white blood cells (%) Wednesday 199 90-95 1 part Hanks concentrate without phenol red + 9 parts distilled water 90-95

Table 2 shows that both media have a large percentage of viable leukocyte retention, however, medium 199 contains phenol, which dampens the chemiluminescent glow, therefore, for chemiluminescent analysis, we offer Hanks concentrate without phenol red in a ratio of 1: 9 with water.

Example 3. We give a comparative assessment of the safety of viable leukocytes when adding various doses of glucose and albumin in a stabilizing solution

Table 3
Preservation of viable leukocytes depending on the dose of glucose and albumin Option B 100 ml (1 part Hanks concentrate + 9 parts water) Preservation of viable white blood cells (%) 1-5 mg glucose
1-5 mg albumin 70-75 10-15 mg glucose
10-15 mg albumin 90-95

From table 3 it is seen that the addition of glucose and serum albumin at a dose of 10-15 mg provides the highest yield of viable leukocytes.

In the Goryaev’s chamber, leukocyte viability is determined by 1% trypan blue, while living cells should be at least 90-95% at a concentration of 2 × 10 ⁶ cells / ml.

The proposed method allows to maintain the constancy of cell parameters: chemical composition of the medium, temperature, acidity, osmotic pressure. Due to the permeability of membranes for water, the concentration of dissolved substances inside and outside the cell does not lead to a gradient of hydrostatic pressure on the membrane: due to changes in the volume of the cell, it remains equal to zero. At the same time, biomembranes have high stereospecificity: only D-isomers of Sugars and L-amino acids penetrate through them (Y. Kagawa, 1985).

Thus, the entire set of essential features increases the shelf life of viable leukocytes (90-95%), which is 20-25% higher compared to known methods for chemiluminescent analysis.

Claims (1)

A method of preserving viable white blood cells in a stabilizing solution, including blood sampling, isolation of white blood cells using Hanks concentrate without phenol red, characterized in that Hanks concentrate without phenol red is used in a ratio of 1: 9 with water, which additionally contains glucose and bovine serum albumin in dose of 10-15 mg per 100 ml.