RU2477953C1 - Combined cryoprotective solution for thrombocyte freezing - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention refers to a combined cryoprotective solution for thrombocyte freezing at low (-80°C) temperature containing hexamethylene bistetraoxyethyl urea, citric acid and water for injections differing by the fact that is additionally contain dimethyl acetamide in the following proportions, wt %: hexamethylene bistetraoxyethyl urea 1.0%; dimethyl acetamide 1.25%; citric acid 0.1%; water for injections the rest.

EFFECT: preparing the solution easy to mix with the thrombocyte concentrate, provides morphological and functional integrity at positive temperature and a high percentage of physiologically adequate thrombocytes after freezing to -80°C and thawing.

1 cl, 2 tbl

Description

The invention relates to medicine, in particular to the preservation of blood cells by freezing. The combined cryoprotective solution for platelet freezing has the following composition (wt.%):

hexamethylene bistetraoxyethylurea 1,0 dimethylacetamide 1.25 lemon acid 0.1 water for injections up to 100 ml pH 5.5-6.4

Experimental studies were performed in the laboratory for the preservation of blood and tissues of the Federal State Budgetary Institution of Science "Kirov Research Institute of Hematology and Blood Transfusion of the Federal Medical and Biological Agency."

As a prototype, we used the work of E.P.Svedentsov and co-authors “An aqueous solution for preserving platelets” (1). A significant disadvantage of the prototype is the presence in the composition of the cooling solution of a toxic antiplatelet agent EDTA Na ₂ . In the proposed cryoprotective solution, the noted drawback is eliminated (EDTA Na _{2 is} excluded). The optimum pH of the medium for platelets is obtained by the production of a tread of 1,1-1,6-hexamethylene-3,3,3'3'-tetrakis (2-hydroxyethyl) bismaurea according to the new method (2), when, after synthesis, they are added to the reaction mixture K2 cation exchange resin and the mixture are evaporated to reduce the pH to 5.6-5.8, a clean protector with a given acidity of the medium is obtained in solution.

Thus, the proposed combined cryoprotective solution for platelet freezing contains the following ingredients, wt.%:

1) hexamethylenebetetraoxyethylurea (GMBTOEM for short), which has the least toxicity of all modern cryoprotectants - its LD ₅₀ is 15.5 ± 0.6 g / kg of mouse weight (Svedentsov E.P. et al. (3)). GMBTOEM is a cryoprotector of predominantly exocellular action;

2) a cryoprotective substance - dimethylacetamide (abbreviated as DMAC), is an endocellular cryoprotectant;

3) citric acid

4) water for injection.

All ingredients included in the cryoprotective solution are produced in the Russian Federation.

Example

Example of use: the proposed solution containing a combination of cryoprotectants GMBTOEM at a concentration of 1% and DMAC - 1.25%, citric acid - 0.1%; water for injection up to 100 ml, having a pH of 5.5-6.4, is mixed with platelet concentrate in a quantitative ratio of 1: 1 in a polymer container "Compoplast 300". Stir the mixture and incubate at room temperature for 20 minutes.

The platelet mixture is frozen with a solution of combined cryoprotectant in an electro-freezer at -40 ° C in a 4-liter bath with 96 ° ethyl alcohol, cooled to -28 ° C, kept in it for 15 minutes. After that, Compoplast with platelets is transferred to an electric refrigerator at -80 ° C for further freezing and storage.

We studied 3 variants of a combined cryoprotective solution for platelet freezing, differing in the concentration of its constituent ingredients. The composition of the solutions is presented in table 1.

Table 1 The composition of the combined cryoprotective solution for freezing
platelet count Combination Cryoprotective Solution Number The composition of the combined cryoprotective solutions GMBTOEM,% DMAC,% Lemon acid, % Water for injection, ml one 2 2,5 0.25 up to 100 2 one 1.25 0.1 up to 100 3 one 0.6 0.1 up to 100

When mixing these solutions with platelets of donated blood in a ratio of 1: 1, the final concentration of substances (Table 1), which are part of 1, 2, 3 solutions, decreased by 2 times compared to the initial one, while the pH of the medium remained within 5.4 -6.5, which is optimal for platelets.

Thawing was carried out after 1-21 days in a 20-liter water bath at + 38 ° C for 40-45 s with active shaking of the container. Heated to + 2 ° C.

The morphological safety of thawed platelets was determined (counting the number of cells in the Goryaev chamber), their functional activity (adhesion to glass, induced aggregation, reaction to hypotonic shock) (table 2). Data are indicated as a percentage in comparison with similar indicators of cells before freezing.

table 2 Preservation of morphological and functional indicators of platelets thawed after cryopreservation at -80 ° C with various combined cryoprotective solutions (in% of the same indices of native platelets) Combination Cryoprotective Solution Number Solution composition Platelet count adhesion ADP adrenalin Rgsh one 2% GMBTOEM + 2.5% DMAC 86.6 ± 9.8 (n = 6) 63.6 ± 13.3 (n = 3) 53.8 ± 19.1 (n = 7) 32.6 ± 14.2 (n = 4) 88.1 ± 7.0 (n = 4) 2 1% GMBTOEM + 1.25% DMAC 93.0 ± 5.4 (n = 6) 63.5 ± 6.1 (n = 3) 67.3 ± 10.9 (n = 5) 77.8 ± 8.9 (n = 6) 84.5 ± 6.4 (n = 3) 3 1% GMBTOEM + 0.6% DMAC 79.2 ± 2.5 (n = 4) 30.1 ± 5.1 (n = 4) 2.5 ± 1.0 (n = 4) 6.8 ± 1.9 (n = 4) 50.2 ± 6.4 (n = 4)

Positive results were obtained after freezing - heating of platelets, especially with solutions No. 2 and No. 1. A large number of morphologically safe cells (93.0% and 86.6%) and the expressed functional usefulness of platelets thawed after cryopreservation with combined cryoprotective solutions No. 2 and No. 1 (adhesion to glass, respectively, 63.5%, 63.6 %, induced aggregation with ADP 67.3% and 53.8%, as well as adrenaline 77.8% and 32.6%).

Solutions No. 1 and No. 2 provided high rates of response to hypotonic shock (84.5 ± 6.4% and 88.1 ± 7.0%, respectively, do not differ statistically) in thawed platelets, which are the leading test for them before administration to the vascular channel.

Given that the chemical load on the body when using the combined cryoprotective solution No. 1 will be 2 times (when using the cryoprotector GMBTOEM) and 2.5 times (when using DMAC) more than when using the combined solution No. 2, then preference should be given to this coolant solution.

A study of it for acute toxicity by the method of GF XII revealed its 100% tolerance and the absence of adverse reactions.

The combined cryoprotective solution developed by us (No. 2) is transparent, odorless, and has a pH of 5.5-6.4. It mixes easily with platelet concentrate, provides morphological and functional safety at a positive temperature and a high percentage of physiologically complete platelets after freezing to -80 ° C and thawing. The positive properties of the developed cryoprotective solution make it possible to use it for preservation by freezing platelets at -80 ° C in medical institutions and blood service institutions that have the specified refrigeration equipment.

Literature

1. Swedentsov E.P., Selezneva O.M., Simkin D.S., Novosadov V.M. An aqueous solution for preserving platelets. Patent No. 1561227 (Rospatent). Register 12.04.93.

2. Baskin Z. L., Murin A. V., Novikova M. D., Toropov A. N., Shabalin D. A., Swedentsov E. P. The method of obtaining 1,1-1,6-hexamethylene-3,3,3 ', 3'-tetrakis (2-hydroxyethyl) -bisma. Patent No. 2376284 of March 31, 2008. Bull. Number 35. Publ. 12/20/2009.

3. Swedentsov EP Obtaining and cryopreservation of bone marrow for clinical use. Abstract of diss. ... Doctors of medical sciences, Leningrad. 1987, 45 (20) (chipboard bar was canceled in 2009).

Claims (1)

Combined cryoprotective solution for platelet freezing at low (-80 ° C) temperature, including hexamethylene bistetraoxyethylurea, citric acid and water for injection, characterized in that it additionally contains dimethylacetamide in the following ratio of ingredients, wt.%:
hexamethylene bistetraoxyethylurea 1,0 dimethylacetamide 1.25 lemon acid 0.1 water for injections rest