RU2311027C1 - Solution for leukocyte preservation at near-zero temperature - Google Patents

Abstract

FIELD: physiology, medicine.

SUBSTANCE: claimed method includes preparation of solution for leukocyte preservation at near-zero temperature and conserving thereof in physiologically active state after warming. Said solution contains in optimal component ratio hexamethylene-bistetraoxyethylurea, glucose, oxymethylethylpyridine succinate, gelatinol, three-substituted sodium citrate and water for injection. Solution has pH 7.0-7.4.

EFFECT: effective solution for preservation of functionally active leukocyte cells.

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Description

The invention relates to physiology and medicine, in particular to the creation of a solution for preserving white blood cells, in particular humans.

Known preservative solution for leukocyte mass of the following composition: sucrose - 9.0 g, glucose - 0.4 g, ascorbic acid - 0.2 g, ethylenediaminetetraacetate Na ₂ - 0.1 g, chloramphenicol - 0.002 g, gelatin 10% solution intravenous administration - 35 ml, double-distilled water up to 100 ml (Materials on the issues of blood service. - M .: Medicine, 1970 - p.306).

A significant drawback of the solution is that it is designed to preserve the leukocyte mass at a positive temperature, which can cause the development of pathogenic microflora, and also contains the toxic anticoagulant ethylenediaminetetraacetate Na ₂ , the antibiotic levomycetin, which can cause allergic reactions, and gelatin, which is due to the large molecular mass is able to sediment red blood cells when administered intravenously. We could not find a solution for preserving white blood cells at near zero temperature from known sources of information.

The technical result of the present invention is the creation of a solution for preserving white blood cells at near zero temperature. This solution ensures the preservation of a high percentage of functionally active white blood cells.

The technical result is achieved by the fact that the following are included in the preservation solution: 1) hexamethylene bistetraoxyethylurea, non-toxic and does not require washing before intravenous administration of the leukocyte concentrate; 2) a restorative additive - hydroxyethylmethylpyridine succinate, which has an antioxidant, membrane stabilizing effect, restores the functions of cell membranes and inhibits lipid peroxidation; 3) gelatinol - a gelatin hydrolyzate that positively affects the microcirculation and disaggregation of blood cells.

The following ingredients are included in the solution for preserving leukocytes at near zero temperature: protector - hexamethylene bistetraoxyethylurea (briefly HMBTOEM); carbohydrate solution for infusion - a glucose solution, which, along with energy and plastic functions (Blood substitutes, preservatives of blood and bone marrow. / Under the editorship of G.N. Khlyabich. - M .: Medicine, 1997. - p.96-99) also has a weak protective effect (Belous A.M., Grishchenko V.I. Cryobiology. - Kiev: Naukova Dumka, 1994. - p. 88-90); the substance is oxymethylethylpyridine succinate (briefly OMEPS), which, in addition to antioxidant, has a membrane-stabilizing effect, restores damaged structures and functions of cell membranes, and also inhibits lipid peroxidation. Succinate is a derivative of succinic acid (Nesmeyanov AN, Nesmeyanov NA The beginning of organic chemistry. Book 1. - M .: Chemistry, 1974. - p. 314-315, p. 436-437) is involved in the energy metabolism of the cell , ensuring its vital activity, and pyridine in nucleic acid exchange (German A.S. Piridin. Big Medical Encyclopedia. - M .; 1981. - T.17. - p. 1054-1055). Also, the gelatinol blood substitute was introduced into the solution - an 8% solution of hydrolyzed edible gelatin in a 0.9% sodium chloride solution (Blood substitutes, blood and bone marrow preservatives. / Ed. By G.N. Khlyabich. - M .: Medicine, 1997. - p. 49-51). Gelatinol has a significantly lower molecular weight (20,000 ± 5,000 D) than gelatin (30 to 100 kD), is non-toxic, does not cause erythrocyte sedimentation upon intravenous administration, has a weak antigenic property, and does not impair liver function. A 0.2% solution of trisubstituted sodium citrate was used as an anticoagulant. The preservation solution has a pH of 7.0-7.4, which is most favorable for blood cells. The ingredients in this solution are tropic to leukocyte cells and are produced in Russia.

An example of using the tool

Preservative solution containing 20% HMBTOEM, hydroxymethyl ethyl pyridine succinate 0.2%, gelatinol 16%, glucose 0.7%, trisubstituted sodium citrate 0.2%, water for injection up to 100 ml and having a pH of 7.0- 7.4, mixed in a 1: 1 ratio with leukocyte concentrate. The mixture, having stood for 20 min at room temperature, was poured into 1.5 ml plastic tubes and transferred for cooling and further storage in an electric freezer with a temperature of 0 + 2 ° С. The samples were warmed up at room temperature (+ 21 ° С) while shaking the container 1-2 times / sec for 5 minutes. We determined the morphological safety of leukocyte cells, their viability by vital dye (with 1% eosin solution) and the phagocytic activity of neutrophils in a sample with latex (according to Potapova S.G. et al., Problems of hematology and blood transfusion. - 1977. - N9. - p. 58-59). The calculation was expressed as a percentage in comparison with similar parameters of cells before cooling.

Experimental studies were performed on the basis of blood cryophysiology laboratories of the Institute of Physiology of the Komi Scientific Center of the Ural Branch of the Russian Academy of Sciences and the preservation of blood and tissues of the Federal State Institution “Kirov Research Institute of Hematology and Blood Transfusion of Roszdrav”.

Three versions of a preservative solution for leukoconcentrate, differing in the concentration of their constituent ingredients, were studied. The composition of the solution options is presented in table 1.

Table 1
Characterization of the composition of the various solutions for preserving white blood cells at near-zero temperature, per 100 g of dry matter (in g). Preservation Solution Option Number The composition of the preservation solution GMBTOEM in% OMEPS in% Glucose in% Gelatinol in% Trisubstituted sodium citrate in% Water for injection, in ml one 14.00 0.15 0.70 16.00 0.2 Up to 100 2 20.00 0.20 0.70 16.00 0.2 Up to 100 3 26.00 0.70 0.70 16.00 0.2 Up to 100

The final concentration of the substances included in the solutions of N1, N2 and N3 after mixing 1: 1 with leukoconcentrate turned out to be 2 times lower than the initial (table 2), the pH of the medium did not change, remaining in the range of 7.0-7.4.

table 2
The final concentration of ingredients in various versions of the preservative solution after mixing with leukocyte concentrate. Preservation Solution Option Number Final concentration of ingredients in various preservative solutions after mixing with the suspension GMBTOEM in% OMEPS in% Glucose in% Gelatinol in% Trisubstituted sodium citrate in% Water for injection in ml one 7.00 0,075 0.35 8.00 0.1 Up to 200 2 10.00 0,100 0.35 8.00 0.1 Up to 200 3 13.00 0.350 0.35 8.00 0.1 Up to 200

Cooling and storage for 6 days was carried out in plastic tubes in an electric freezer at a temperature of 0 + 2 ° C. Samples were warmed at room temperature (+ 21 ° С) while the container was shaken 1-2 times per second for 5 minutes. Leukocyte studies were performed on morphological safety, viability and functional activity (table 3).

Table 3
The results after cooling to 0 + 2 ° C, storage at a given temperature for 6 days and warming of white blood cells with various versions of the proposed preservation solution Preservation Solution Option Indicators, in% of the initial level. n = 5 White blood cell viability The morphological preservation of granulocytes Phagocytic activity of neutrophils The lower limit of the dosage of the ingredients of the solution 84.8 ± 3.6 51.5 ± 6.6 * 32.5 ± 6.2 * The optimal ratio of the ingredients of the solution 86.2 ± 7.8 61.9 ± 2.1 56.7 ± 8.6 The upper limit of the dosage of the ingredients of the solution 66.4 ± 8.2 * 36.4 ± 9.3 * 37.0 ± 1.4 * * - significant difference (p <0.05)

The main criterion for assessing the biological usefulness of leukocytes, primarily neutrophils, is the determination of their phagocytic activity. This test, which most reliably characterizes the physiological properties of cells, was the best after cooling - storing - heating a biological object using a solution with the optimal ratio of ingredients and significantly differed from data obtained using a preservative solution with upper and lower dosage limits of the ingredients.

The ready-made optimal solution is transparent, with a slight yellowish tint, odorless, pH 7.0-7.4, mixes well with leukocyte concentrate in all ratios, does not cause conglomeration of cells and provides functional and morphological preservation at a positive temperature until cooling. The solution has a protective effect when cooling white blood cells to 0 + 2 ° C.

Thus, the proposed preservation solution allows you to maintain a high percentage of physiologically active leukocyte cells at a temperature of 0 + 2 ° C for 6 days. This solution can be used in medical and biological institutions for the preservation of biological objects in a domestic refrigerator.

Claims (1)

A solution for preserving leukocytes at near zero temperature, characterized in that it contains hexamethylene bistetraoxyethylurea, glucose, hydroxymethylethylpyridine succinate, gelatinol, trisubstituted sodium citrate and water for injection in the following ratio of ingredients,%: Hexamethylene bistetraoxyethylurea 26.0-14.0 Glucose 0.7 Oxymethylethylpyridine Succinate 0.7-0.15 Gelatinol 16,0 Trisubstituted Sodium Citrate 0.2 Water for injections up to 100 ml In this case, the pH of the solution 7.0-7.4