JPH0859489A - Freezing damage inhibitor for frozen blood - Google Patents

Abstract

PURPOSE: To obtain the inhibitor with no need of long time for the glycerin removing treatment after thawing frozen blood. CONSTITUTION: This freezing damage lnhibitor to be added for the long-term preservation of thick erythrocytes or bone marrow fluid is such that a substitution has been made so that at least one kind selected from saccharides and biopolymers account for 20-80wt.% of the whole inhibitor. The saccharide is, e.g. a common monosaccharide, disaccharide; while, the biopolymer is, e.g. a dextran, phosphoric ester salt such as phosphorylcholine chloride, polyvinylpyrrolidone.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition of an antifreezing agent for frozen blood, which reduces the ratio of conventional antifreezing agents such as glycerin and ethylene glycol, and therefore, the antifreezing agent after thawing. For facilitating removal.

[0002]

2. Description of the Related Art Typical long-term storage of blood and blood components includes a freezing method at -80 ° C., a freezing method using liquid nitrogen (about -196 ° C. storage), and a freezing method using liquid helium (- 270 ° C storage). In all of these blood storage methods, it is necessary to mix a cryopreservative with a frost damage inhibitor such as glycerin or ethylene glycol, and to remove the frost damage inhibitor such as glycerin or ethylene glycol after thawing before use. The blood storage method is performed as follows. The following are concentrated red blood cells (hematocrit value * 70%
˜90%) is frozen at −80 ° C. as an example (* hematocrit value = ratio of blood components to total blood components, normal value of blood of healthy subjects is about 45%).

1) Blood collected with sodium citrate or PBS (phosphate buffer) or heparin as an anticoagulant is centrifuged to obtain a hematocrit value of 70% to 90%.
To obtain concentrated red blood cells. 2) Add an equal amount of a preservative solution containing glycerin or ethylene glycol as a main component. The composition of the preservation solution is, for example, in the case of a solution containing glycerin as a main component, as follows: a) Glycerin 60 g b) 70% sodium lactate 2.57 g c) KCl 0.02 g d) NaCl 0. 26 g is dissolved in pure water, and finally 100 ml is used [for example, Frozen Blood Freezing Damage Protection Liquid SF-60 manufactured by Fuso Yakuhin Kogyo Co., Ltd.]. 3) The concentrated red blood cells to which the protective solution is added are placed in a storage container and cooled. The cooling rate is the same as a normal blood transfusion container (volume 2
It is about several ° C / min for blood in (00 ml). This is because it is usually simply placed in a freezer at -80 ° C without special temperature control. Here, the cooling rate of blood and the concentration of glycerin have a correlation with the hemolysis rate of blood after thawing (corresponding to the mortality rate of blood), and when trying to keep it below a certain hemolysis rate, a high cooling rate In the case of, a relatively thin concentration of glycerin is sufficient,
Lower cooling rates require a relatively high concentration of glycerin. 4) Store in a freezer at -80 ° C. 5) When using frozen blood, remove it from the freezer and thaw it rapidly in a water bath at about 40 ° C. 6) After thawing, wash with physiological saline or the like to remove glycerin. It takes several hours for this work. The same applies to ethylene glycol.

[0004]

In the above-mentioned conventional method for preserving blood, a protective solution containing glycerin, ethylene glycol or the like as a main component is used, and therefore it is necessary to remove this when using blood. Therefore, it takes time for deglycerylation before being thawed and used. This treatment time is longer as the concentration of glycerin or ethylene glycol is higher. In addition, as described above, if an attempt is made to increase the cooling rate, an expensive refrigerant substance such as liquid nitrogen is required, or a special container therefor is required, which is complicated and expensive. As illustrated above, the conventional method for preserving blood has a problem that it cannot be applied to, for example, an emergency because it requires complicated and time-consuming deglycerination. An object of the present invention is to provide an antifreezing agent for frozen blood which requires less time for deglycerin treatment after thawing even in a simple method of cooling by leaving it in a freezer at -80 ° C. .

[0005]

The present invention will be described in brief. The present invention relates to a cryoprotective agent for frozen blood, which is added for the long-term storage of concentrated red blood cells, bone marrow fluid, etc. 20 to 8 of the antifreezing agents
It is characterized in that 0% by weight is substituted so as to be at least one kind of sugar and biopolymer.

The present invention will be described in detail below. Blood is
For example, concentrated red blood cells having a hematocrit value of 70 to 90% are used. Examples of sugars are mannose, xylose,
Examples thereof include those containing at least one kind of sugar selected from glucose, trehalose, sucrose and maltose. Examples of the biopolymer include dextran, phosphate ester salt, polyvinylpyrrolidone (PV
Examples thereof include those containing at least one biopolymer selected from P). Here, examples of the phosphoric acid ester salt include phosphorylcholine chloride [Sigma reagent # P0378], 5-phosphorylribose-1-pyrophosphate (Sigma reagent # P8296),
In addition, C ₃ H ₃ NO ₆ P (Sigma reagent # P5506),
C ₃ H ₅ NO ₆ P (Sigma reagent # P0753), C ₃ H
₈ NO ₆ P (Sigma Reagent # P0878), C ₃ H ₁₀ NO
₆ P (Sigma reagent # P1003), and examples of their salts include sodium salt and calcium salt.

[0007]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. In each of the following examples, PVP, phosphoric acid ester salt, and dextran each had a molecular weight of about 40,000. As a result, similar results were obtained with the sodium salt and the calcium salt in the phosphate ester salt.

Example 1 The antifreezing liquid (50 ml) shown in Table 1 was prepared and mixed with an equal amount of concentrated red blood cells (50 ml) and frozen. The frozen blood obtained was stored in a freezer (temperature: about -80 ° C) for 1 month, then taken out and thawed in hot water at 37 ° C for a short time. The obtained blood was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected, and the blood counter [sysmecs]
The amount of hemoglobin was measured with NE-8000]. A value obtained by dividing (dividing) this by the hemoglobin value of the mixed solution before freezing (mixed solution of concentrated red blood cells and antifreezing solution) which was measured in advance was defined as the hemolysis rate. Based on the hemolysis rate at this time, the survival rate after freezing was approximated to (100-hemolysis rate) (%). The results are shown in Table 1.

[0009]

[Table 1] Table 1 (mixing ratio is relative value) ─────────────────────────────────── Liquid (percentage) ───────────────────────── Cryoprotection liquid after freeze-thawing ^{* 1} Sugars Biopolymers Survival rate of red blood cells ───── ────────────────────────────── # 0 50 (conventional method) 0 0 95-98% # 1 40 10 ^{* 2} 0 95-98% # 2 30 20 ^{* 2} 0 95-98% # 3 20 30 ^{* 2} 0 85-90% # 4 10 40 ^{* 2} 0 80-85% # 5 30 0 20 ^{* 3} 80-95% # 6 30 20 ^{* 4} 0 95-98% ────────────────────────────────────

^{* 1} Frozen blood frost damage protection liquid [SF-60: Fuso Yakuhin Kogyo Co., Ltd.] The composition is glycerin (60 g), 70% sodium lactate (2.57 g), KCl (0.02 g), NaCl
(0.26 g) was dissolved in pure water to finally make 100 ml. ^{* 2} 2M-glucose solution ^{* 3 At} least one (% by weight) of dextran, PVP and phosphate ester salt. ^{* 4} A solution containing at least one of 1M to 2M glucose, mannose, xylose, trehalose, sucrose, and maltose (concentrations are all 1M to 2M).

Example 2 The washing time required for deglycerination after thawing was tested for the above-mentioned preventive solution. The result of # 0 in Table 1 is 1 (reference value)
As a result, the time for which the glycerin concentration was similar to # 1 to # 6 was measured. Table 2 shows the results.

[0012]

[Table 2] Table 2 ─────────────────────────
── Antifreeze liquid Cleaning time (relative value) ──────────────────────────
── # 01 (standard value) # 1 0.8 # 2 0.5 # 3 0.5 # 4 0.3 # 5 0.5 # 6 0.5 0.5 ──────────── ──────────────
──

By replacing 20-80% by weight of the glycerin solution with sugars or biopolymers, the time for the deglycerin treatment was significantly shortened.

[0014]

According to the present invention, since the content ratio of glycerin or ethylene glycol can be reduced, the time for deglycerin (ethylene glycol) conversion can be greatly shortened. Further, since the survival rate of erythrocytes does not change, there are very few restrictions on the use of the antifreezing liquid, and it is simple and applicable to emergency blood transfusion.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area A61K 47/36 J

Claims (3)

[Claims] 1. An antifreezing agent for frozen blood, which is added for long-term storage of concentrated red blood cells, bone marrow fluid, etc., wherein 20-80% by weight of the antifreezing agent comprises at least one of sugars and biopolymers. An antifreeze agent for frozen blood, characterized in that it is replaced so that 2. The antifreezing agent for frozen blood according to claim 1, wherein the saccharide is a monosaccharide or a polysaccharide. 3. The saccharide is mannose, xylose,
Glucose, trehalose, sucrose, at least one kind of sugar selected from maltose, the biopolymer, dextran, phosphate ester salt, at least one kind of biopolymer selected from polyvinylpyrrolidone The antifreezing agent for frozen blood according to claim 1, which is characterized in that.