JP3182145B2 - Platelet preservation solution - Google Patents

Description

The present invention relates to a platelet preservation solution.

[Conventional technology and its problems]

In recent years, component transfusion has become mainstream. For example, platelet transfusion generally uses concentrated platelet plasma (hereinafter, referred to as PC), and its expiration date is three days from the blood collection date. Further, blood components such as other red blood cells also have respective expiration dates, and must be preserved during that period.

Recently, the demand for plasma products has increased significantly, and domestic plasma products have relied on imported products. However, some imported plasma preparations are contaminated with acquired immunodeficiency syndrome (AIDS), non-A, non-B hepatitis virus, etc., and this is a major social problem at present. At present, studies are being made to utilize plasma as much as possible in order to self-supply plasma products in Japan. As one of the methods for this, it has been studied to reduce the plasma in PC as much as possible and to add a platelet preservative instead.

Platelets continuously produce adenosine triphosphate (ATP) in response to their energy needs for their survival. There are two pathways for this production, glycolysis and oxidative phosphorylation. In the glycolytic action, one molecule of glucose is converted into two molecules of lactic acid, and two molecules of AT
Produces P. Oxidative phosphorylation is far more than glycolysis because glucose, amino acids, or fatty acids are converted to carbon dioxide (CO ₂ ) and water while consuming oxygen (O ₂ ), producing 36 molecules of ATP. It is an efficient system.

During storage, platelets can only produce ATP by oxidative phosphorylation, and continue to produce lactic acid by glycolysis. Sodium bicarbonate in plasma alone does not have a buffering action commensurate with the production of lactic acid, leading to a drop in pH. If the pH drops below 6.0, platelet viability is irreversibly lost. Thus, glucose is
Because of the idea that lactic acid is produced, resulting in a decrease in pH and loss of platelet viability, many platelet preservatives so far have not added glucose (for example, Transfusion 1988). , vol28, No.3, p.217-21
9).

However, platelet preservation solution without glucose cannot generate enough energy for platelets, and during storage, platelet swelling and low osmotic shock recovery (% HSR), which is highly correlated with the survival of platelets after transfusion, The decline was observed.

[Means for solving the problem]

The present inventors, in order to solve the above problems of the prior art,
As a result of intensive research, the present invention has been completed,
The present invention is a platelet preservation solution having the following configuration.

1) An aqueous solution having the following composition, wherein the molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1: 3 to 10: 1, and substantially isotonic and the pH is 6.2 to 7.8. A platelet storage solution consisting of an aqueous solution.

Disodium hydrogen phosphate 1-50 mmol / sodium dihydrogen phosphate 1-50 mmol / glucose 5-30 mmol / sodium acetate 10-30 mmol / membrane impermeable saccharide 5-50 mmol / physiologically compatible Electrolyte 50-150 mmol / 2) The membrane-impermeable saccharide is a disaccharide or a sugar alcohol 1
The platelet preservation solution according to the above item.

3) The platelet preservation solution according to claim 1, wherein the physiologically compatible electrolyte comprises one or more of alkali metal or alkaline earth metal mineral acids.

4) The platelet preservation solution according to claim 1, wherein the electrolyte comprises one or more of sodium chloride, potassium chloride, magnesium chloride, and magnesium sulfate.

5) the electrolyte is sodium chloride 50-150 mmol /;
The platelet preservation solution according to claim 1, comprising 1 to 10 mmol / of potassium chloride and 1 to 5 mmol / of magnesium chloride or magnesium sulfate.

6) The platelet preservation solution of claim 1, further comprising 10 to 20 mmol / trisodium citrate / and / or dextran.
Platelet stock solution with 0.05-1 percent (w / v) added.

In the above platelet preservation solution, disodium hydrogen phosphate and sodium dihydrogen phosphate are pH buffers, and the preservation solution is maintained at pH 6.2 to 7.8, preferably 6.8 to 7.4, and lactic acid generated by glycolysis of glucose is used. This is for preventing the pH of the storage solution from being lowered. Further, phosphoric acid has an action of promoting metabolism, and can maintain the energy of platelets high. The molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is from 1: 3 to 10: 1, preferably from 1: 1 to 3: 1. Sodium acetate is for suppressing the production of lactic acid by platelets. Membrane-impermeable saccharides have the effect of preventing platelets from expanding during storage and protecting the platelet membrane. The sugars that do not penetrate the platelet membrane include sugars or sugar alcohols that are disaccharides or more.
Typical examples are maltose, mannitol,
Sucrose, sorbitol, lactose and the like. Physiologically compatible electrolytes are for adjusting the preservation solution to be isotonic, and alkali metal or alkaline earth metal mineral salts such as sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate are appropriately used. Used in combination. Trisodium citrate and dextran have the effect of preventing aggregation of platelets during storage.

In the storage solution of the present invention, the composition ratio of each of the above components is not critical, but in order to effectively preserve platelets, disodium hydrogen phosphate and sodium dihydrogen phosphate are:
1 to 50 mmol /, preferably 2 to 30 mmol /, respectively, glucose is 5 to 30 mmol /, preferably
15-25 mmol /, sodium acetate 10-30 mmol /, preferably 20-25 mmol /, membrane-impermeable saccharides 5-50 mmol /, preferably 15-35 mmol /,
The total amount of the electrolyte is 50 to 150 mmol / mol, preferably 50 to 150 (preferably 80 to 130) mmol / sodium chloride, 1 to 10 (preferably 3 to 7) mmol / potassium chloride, magnesium chloride or magnesium sulfate 1
-5 (preferably 2-4) mmol /. Trisodium citrate 10-20 mmol /, dextran
The concentration is between 0.05 and 1 w / v percent.

In the present invention, the most preferred platelet preservation solution has the following composition.

Disodium hydrogen phosphate 16-22 mmol / sodium dihydrogen phosphate 4-9 mmol / glucose 23.5 mmol / sodium acetate 23 mmol / maltose (or mannitol) 29 mmol / sodium chloride 90 mmol / potassium chloride 5 mmol / magnesium chloride ( Alternatively, a solution obtained by adding 17 mmol / trisodium citrate / 0.1% w / v dextran to the platelet preservation solution having the above composition is also preferable.

The platelet preservation solution of the present invention is prepared according to a conventional method. That is, it is easily prepared by adding predetermined amounts of the above components to distilled water for injection with stirring and dissolving.

To preserve platelets using the preservation solution of the present invention, platelet-rich plasma or concentrated platelet solution is further centrifuged to precipitate platelets, and this platelet precipitate is added to the preservation solution of the present invention for about 10 days.
The cells are suspended at a concentration of 0 to 3,000,000 cells / μ, and stored at 20 to 24 ° C under horizontal shaking according to a conventional method. The supernatant plasma obtained by centrifugation can be used for other purposes. The platelets stored in the preservation solution of the present invention are used as they are at the time of use, or are appropriately diluted and used as component infusions.

Next, the present invention will be described more specifically with reference to examples and comparative test examples.

[Examples and Comparative Test Examples]

Platelet-rich plasma (PRP) or platelet-rich plasma (PC)
Was prepared from human blood anticoagulated with citrate-phosphate-dextrose (CPD) or citrate-dextrose (ACD). PRP or P obtained
C was centrifuged at 3000 g for 6 minutes to remove the plasma, leaving about 5 ml of the supernatant plasma. 5 ml of platelet button and 35 ml of the platelet preservation solution or autologous plasma shown in Table 1 are aseptically added to a 300 ml polyvinyl chloride bag (PVC bag), stored at room temperature for 1 hour, and then stirred with a stirrer. After stirring for 1 hour, the platelets were resuspended in the platelet stock solution. After storage at 22 ° C. with horizontal shaking, sampling was performed on days 3 and 5 for measurement.

The platelet count and average platelet volume (MPV) were measured using a multi-item automatic blood analyzer (Sysmex: NE-6000).

The pH was measured using a fully automatic blood gas analyzer (Radometer: A
BL30).

Lactate dehydrogenase (LDH) activity was measured by centrifuging PC at 3500 g for 10 minutes, and measuring the LDH activity of the supernatant using an LDH UV test Wako (Wako Pure Chemical Industries).

The recovery rate of low osmotic shock (% HSR) was measured using a hematotracer II (Nikko Bioscience) with twice the volume of autologous plasma added to the volume of PC1.

The platelet morphology was fixed with a 1% glutaraldehyde-0.2 M sodium cacodylate solution (pH 7.4), dehydrated with alcohol, observed with a scanning electron microscope, and the proportion of platelets in the form of discs (Disc) was measured.

Table 2 shows the results of Example 1, Comparative Example 1, and the control.

During the storage period, in Comparative Example 1, although the pH and platelet morphology were maintained, cell swelling and a decrease in% HSR were observed. In Comparative Example 2, the amount of lactic acid produced was large and the pH was lowered. Further, in Comparative Example 3, cell swelling was observed. On the other hand, in Example 1 of the present invention, platelets were favorably maintained in all test items. In particular, the LDH activity was very low. The LDH in the supernatant was obtained by releasing the LDH inside the cell together with the collapse of the cell.
Shows that almost no platelet disruption occurred during storage. In addition, it was confirmed that all of Examples 2 to 4 exhibited good platelet preservability as in Example 1.

From the above, it can be seen that the platelet preservation solution of the present invention can maintain platelets more favorably than the conventional platelet preservation medium by storing platelets at 22 ° C. in an oxygen-permeable container. . In addition, the use of a platelet preservation solution is expected to substantially reduce virus transmission by blood transfusion and side effects after blood transfusion.

(Effects)

Since the platelet preservation solution of the present invention contains glucose, both anaerobic and aerobic metabolism of platelets is performed as in a living body, so that platelets obtain a sufficient amount of metabolic energy. Can be. Lactic acid is produced by anaerobic metabolism, but the pH of the preservation solution does not decrease because a phosphate buffer is added. Furthermore, since the preservation solution of the present invention contains a membrane-impermeable saccharide, the platelet membrane is protected, and platelet swelling during storage is prevented.

Further, since the preservation solution of the present invention contains dextran and trisodium citrate, aggregation of platelets during storage is prevented.

Thus, the platelet preservation solution of the present invention has an extremely excellent platelet preservation effect.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Koji Shibata, Inventor 1500, Inokuchi, Nakai-machi, Ashigara-gun, Kanagawa Prefecture Inside Terumo Corporation (56) References JP-A-63-146824 (JP, A) , A) WO 89/2274 (WO, A1) (58) Fields investigated (Int. Cl. ⁷ , DB name) A01N 1/00 A61K 35/14 A61K 31/19 A61K 31/70 A61K 33/42 CAPLUS (STN) BIOSIS (STN) CA (STN) MEDLINE (STN) EMBASE (STN)

Claims (6)

(57) [Claims] An aqueous solution having the following composition, wherein the molar ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 1: 3 to 10: 1, substantially isotonic and the pH is 6.2 to A platelet stock solution consisting of an aqueous solution of 7.8. Disodium hydrogen phosphate 1-50 mmol / sodium dihydrogen phosphate 1-50 mmol / glucose 5-30 mmol / sodium acetate 10-30 mmol / membrane impermeable saccharide 5-50 mmol / physiologically compatible Electrolyte 50-150 mmol / 2. The platelet preservation solution according to claim 1, wherein the membrane-impermeable saccharide is a disaccharide or a sugar alcohol. 3. The platelet preservation solution according to claim 1, wherein the physiologically compatible electrolyte comprises one or more alkali metal or alkaline earth metal mineral acids. 4. The platelet preservation solution according to claim 1, wherein said electrolyte comprises one or more members selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, and magnesium sulfate. 5. The electrolyte according to claim 1, wherein the electrolyte is 50 to 150 mmol / sodium chloride, 1 to 10 mmol / potassium chloride, and 1 to 5 mmol / magnesium chloride or magnesium sulfate.
The platelet preservation solution according to claim 1, comprising: 6. A platelet preservation solution obtained by further adding 10 to 20 mmol / trisodium citrate / and / or 0.05 to 1 percent (w / v) of dextran to the platelet preservation solution according to claim 1.