JPH0698178B2 - Platelet storage method and platelet storage device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a platelet storage method and a platelet storage device capable of storing platelets for a long period of time.

[0002]

2. Description of the Related Art For patients who are required to supply platelets due to infections, thrombocytopenia, trauma, radiation damage, etc., it is necessary to carry out a component blood transfusion of Platelet Concentrate (PC). The platelet concentrate is plasma containing platelets richly, and can be obtained by centrifuging blood collected from the donor or by donating blood components. The function of platelets obtained by either method is extremely unstable, and it is necessary to quickly infuse the platelet concentrate to the patient.

As a method for storing concentrated platelets, there is a method of storing in a liquid state by shaking at room temperature (22 ° C.) in a plastic blood bag such as polyvinyl chloride (PVC) or polyolefin (PO). This liquid storage has a shelf life of 3 days in Japan from the viewpoint of maintaining platelet function.
It is said to be 5 days in other countries. Moreover, the storage period of the platelet concentrate (pheresis PC) obtained by the component blood donation is further limited. In addition, HLA (lymphocyte antigen) -matched platelets are
It is desired to store the liquid in a better condition for a long period of time.

Therefore, the inventor of the present invention continues to study the long-term liquid preservation of platelets by using the double bag system proposed in JP-A-63-56275 and JP-A-63-248382. Came. Initially, a cell culture medium (MCDP-151, SIG
It was clarified that the platelet aggregating ability can be maintained at a high value when human serum albumin is added and preserved by using MA Inc. ( JP-A-2-255601 ). However, the results showed that the platelet count retention, platelet mean volume (MPV), and osmotic shock recovery rate (% HSR) were similar to those of the PVC bag-preserved platelets.

In order to investigate these causes, autologous platelet plasma (PPP: Platelet PoorPlasma blood preservation solution CPD added) was injected into the outer bag to preserve the platelets. The platelet count, MPV and% HSR were remarkably improved. Was recognized.
From these results, it is necessary to equilibrate the osmotic pressure between the platelets and the preservative solution in order to reduce the volume fluctuation of the platelets in preserving the platelets in the double bag. That is, the present invention has been achieved.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a concentrated platelet obtained by preparing whole blood collected from a donor, or a concentrated platelet obtained by component blood donation for a long period of time without degrading its function. Its purpose is to preserve it in liquid form.

[0007]

[Means for Solving the Problems] A means of the present invention for solving the above-mentioned problems is a method of storing platelets in a liquid state, which comprises platelets, sodium bicarbonate and glucose.
Look, and a platelet storage solution obtained by adding an osmotic pressure adjusting agent is contacted through the semipermeable membrane, while applying shaken platelets and platelet storage solution in a state in which balance the osmotic pressure difference of platelets and platelet storage solution platelets It is characterized by storing.

An instrument for carrying out the method for preserving platelets is an instrument for preserving platelets in a liquid state, and includes a semipermeable membrane inner bag for preserving platelets and an outer bag for inserting the inner bag therein. It has a platelet preservative solution stored in the space of the inner bag and the outer bag, and the platelet preservative solution is sodium bicarbonate and
Those containing lucose and added with an osmotic pressure adjusting agent to balance the osmotic pressure difference with the platelets in the inner bag
And characterized in that.

Examples of the material of the semipermeable membrane include cellulose membranes such as regenerated cellulose and cellulose acetate, or polyacrylonitrile, polymethylmethacrylate, polysulfone, polycarbonate, It is a synthetic film of polyolefin such as polyamide, polyethylene and polypropylene. The outer bag may be made of a soft plastic material such as polyvinyl chloride, vinyl acetate copolymer and polyolefin.

Examples of the osmotic pressure adjusting agent include dextran, polyethylene glycol, polyvinyl ridone and the like, but any substance that balances the osmotic pressure of the concentrated platelets with the preservation solution may be used. When the molecular weight of dextran is 40,000, the amount added to the platelet preservation solution is 2.5-3.0 W / V
%, Preferably 2.5 W / V%. When the dextran addition amount is 2.5 W / V% or less, the platelet count decreases, MP
An increase in V and a decrease in% HSR were observed, and at 3.0 W / V% or higher, the concentration of the preservative solution in the concentrated platelets increased, and the platelet volume increased. At 2.5 W / V%, platelet volume fluctuation due to colloid osmotic pressure is the smallest.

As a platelet preservation solution, Holme, Murphy,
Some have been formulated by Rock, and among them, the Holme formulation containing sodium bicarbonate and glucose is preferable.
The Holme formulation stabilizes the pH of the preservation solution for a long period of time and maintains good platelet function. Also, sodium bicarbonate dissociates into HC
It becomes O ₃ and complements the plasma buffer system.

[0012]

EXAMPLES Next, examples of the present invention will be specifically described. FIG. 1 is a configuration diagram of a double bag for storing platelets used in this embodiment. Reference numeral 1 is an outer bag, 2 is an inner bag inserted in the outer bag, and a space portion for storing a storage solution is formed between the outer bag 1 and the inner bag 2.

The outer bag 1 is formed by sealing the periphery of two soft synthetic resins in a bag shape, and a plurality of operation ports 3 are provided at the lower end portion. In addition , mounting holes 4 for a rotary agitator, which will be described later, are opened in the seal portions at the corners of the outer bag 1.

The inner bag 2 is made by sealing the openings at both ends of a tube-shaped semipermeable membrane, and the upper bag has an operation port 3 for injecting and removing platelets at the upper end. It is provided so as to project to the outside of the. The inner bag 2 is entirely a protective net 5.
The outer space of the inner bag 2 and the inner surface of the outer bag 1 are not covered in close contact with each other, and a predetermined space can be maintained.

The outer bag 1 of this embodiment is made of soft polyvinyl chloride and has a sheet thickness of 0.4 mm and a capacity of 2.000.
is l. The inner bag 2 is composed of a semipermeable cellophane film, and has a film thickness of 20 μm, a molecular weight cutoff of 10.000, and a volume of 1
It is 00 ml.

FIG. 2 is a perspective view of the rotary agitator used in this embodiment. It is a box 10 in which a drive mechanism, a control mechanism and the like are built, and the box 10 is mounted at a predetermined angle. The rotary plate 11 is provided with a rotary plate 11, and the rotary plate 11 is rotated by a drive mechanism built in the box 10. Stoppers 12 for fixing the storage bag are provided at the four corners of the rotary plate 11, and the double bag for storing platelets is mounted on the rotary plate 11 by inserting the mounting holes 4 of the outer bag 1 into the stopper 12. Fixed. The stopper 12 is movable along the mounting position adjusting groove 13.

The experimental method was to inject 100 ml of concentrated platelets prepared from whole blood according to a conventional method into the inner bag 2 of the above-mentioned double-plate storage bag through the operation port 6. Also, the outer bag 1 has Ho
Dextran (MW: 40,00) for lme prescription (see Table 1)
0) was added to adjust the pH to 7.0. Platelet preservation solution 50
0 ml and sterile air-500 ml were injected from the operation port 3 and were stored in the space portions of the outer bag 1 and the inner bag 2.

The double bag for storing platelets is fixed to the rotary agitator and the rotary plate 11 is rotated (5 rpm).
Let the double bag for platelet storage be shaken while applying 2
It was stored in a thermostatic chamber at 2 ° C for 2 weeks.

As a comparative example, using a commercially available PO (polyolefin) bag (600 ml, pre-bag) for storing concentrated platelets, 100 ml of concentrated platelets prepared in the same manner as described above was injected, and horizontal reciprocal shaking was performed by a platelet shaker ( 60rp
m 2) for 2 weeks in a constant temperature room at 22 ° C.

The concentrated platelets stored according to the above conditions were sampled, and the pH and gas partial pressure, platelet count and MP were sampled.
V,% HSR and aggregation capacity were measured. The pH and gas partial pressure were measured by a pH / blood gas analyzer. Platelet count and MPV were measured using a counter.

For the% HSR and agglutination ability, the sampled concentrated platelets were diluted with PPP which had been cryopreserved in advance to obtain platelet rich plasma (PRP: Platelet).
PoorPlasma, average platelet concentration 3.5 × 10 ⁵ / μ
1, pH 7.4) was prepared and used as a test solution. % HSR was determined from the change in transmittance for 5 minutes at 610 nm when 0.7 ml of distilled water was added to PRP1.4 using a spectrophotometer. Also, the aggregating ability is A in the presence of CaCl ₂ (final concentration 3 mM).
DP (final concentration 10 μM), collagen (final concentration 5 μM
g / ml) alone or ADP (5 μM) and collagen (1 μm)
The maximum aggregation rate for 10 minutes by co-stimulation (g / ml) was measured by a platelet aggregometer. The results of these experiments will be described below.

[PH and Gas Partial Pressure] As shown in FIG. 3, when stored in the bag of the present invention, the fluctuation of pH of the concentrated platelets was small, was stable until the 14th day, and was maintained between 7 and 7.3. Was done. On the other hand, the conventional bag was relatively stable until the 3rd day, but then dropped, and the pH dropped sharply after the 7th day and dropped to 6.00 ± 0.07 on the 14th day. did.

As shown in FIGS. 4 and 5, the carbon dioxide gas partial pressure PCO ₂ and the oxygen gas partial pressure PO ₂ of the concentrated platelets were always higher in the bag of the present invention than in the conventional bag. It was a similar change. After the 10th day, the bag of the present invention
PCO ₂ gradually decreased, but remained until the 14th day, and PO ₂ increased. On the other hand, in the conventional bag, PCO ₂ increased from the 7th to 10th days, and both the PCO ₂ and the PO ₂ showed a value close to the saturation value with air on the 14th day.

[Platelet count and MPV] As shown in FIG. 6, the variation in platelet count was always higher in the bag of the present invention than in the conventional bag. The total number of platelets is 10.1 on the first day
0 ± 3.23 × 10 ¹⁸ pieces / bag on the 14th day with the present invention bag 8.40 ± 2.84 × 10 ¹⁰ pieces / bag (83.04 ± 4.54% on the first day), with the conventional bag 7.72 ±
1.75 × 10 ¹⁰ pieces / bag (77.94 ± 7.
47%).

As shown in FIG. 7, the MPV increased in the bag of the present invention for 1 to 3 days, but after that, it was almost stable, and it was 7.12 ± 0.29 μm ³ on the 14th day. On the other hand, in the conventional bag storage, the value on the first day was almost maintained until the 5th day, but it increased after that, and significantly increased from the 7th day. Then, on the 14th day, the value was 8.66 ± 0.36 μm ³ .

As shown in FIG. 8, [% HSR]% HSR was almost the same in the bag of the present invention and the conventional bag until the 7th day. However, after that, the decrease in% HSR in the present invention was gradual, and was maintained at about 76% on the first day on the 14th day, whereas it decreased sharply after the 7th day on the conventional bag and on the 14th day. Had no response to changes in osmotic pressure.

[Aggregating Ability] As shown in FIGS. 9, 10 and 11, the aggregating ability includes ADP, collagen alone and AD.
In both cases of P and co-stimulation of collagen, the same decreasing tendency was exhibited until the 10th day. From the 10th day onward, in the bag of the present invention, the aggregation of ADP alone and the co-stimulation of ADP and collagen showed a substantially constant value from the 10th day to the 14th day. On the other hand, in the conventional bag storage, the aggregation due to any stimulus was drastically reduced from the 7th to 10th day, and the response to the stimulus was lost on the 14th day.

[0028]

[Table 1]

When platelets are preserved by the above-mentioned bag of the present invention, platelet function is maintained while maintaining metabolic function. In addition, concentrated platelets are stored in a large capacity including the semipermeable membrane of the outer bag 1 via the semipermeable membrane of the inner bag 2, and the allowable range of plasma buffering ability, accumulation of metabolites, etc. is widened, As a result, it was possible to store the liquid for a long period of at least 2 weeks.

[0030]

According to the present invention described above, bicarbonate
It contains sodium and glucose, and contains an osmotic pressure regulator.
By bringing the added platelet preservation solution into contact with the platelets through the semipermeable membrane, the concentrated platelets can be stored in a liquid state for a long time while maintaining the platelet function.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a platelet storage bag of the present invention.

FIG. 2 is a perspective view of a rotary agitator used in the present invention.

FIG. 3 is a graph comparing the results of pH measurement of platelets by the platelet preservation method of the present invention with those of the conventional example.

FIG. 4 is a graph comparing the results of measuring the carbon dioxide partial pressure of platelets by the platelet storage method of the present invention with those of the conventional example.

FIG. 5 is a graph comparing the results of measuring the oxygen gas partial pressure of platelets by the platelet preservation method of the present invention with those of the conventional example.

FIG. 6 is a graph comparing the results of platelet count measurement of platelets by the platelet storage method of the present invention with those of a conventional example.

FIG. 7 is a graph comparing the platelet average volume measurement results of platelets according to the platelet preservation method of the present invention with that of a conventional example.

FIG. 8 is a graph comparing the results of measuring the osmotic shock recovery rate of platelets by the platelet storage method of the present invention with those of the conventional example.

FIG. 9 is a graph comparing the results of measuring the aggregation rate of platelets by ADP stimulation according to the platelet preservation method of the present invention with those of the conventional example.

FIG. 10: Platelet color according to the platelet preservation method of the present invention
The graph which compared the aggregation rate measurement result by a gen stimulus with the conventional example.

FIG. 11: ADP of platelets according to the platelet preservation method of the present invention
And a graph comparing the results of measurement of the aggregation rate by co-stimulation of collagen with conventional examples.

[Explanation of symbols]

 1 Outer bag 2 Inner bag 3 Outer bag operation port 5 Inner bag protection mesh 6 Inner bag operation port

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kazunori Ichinohe 1-26-7 Yokoyamadai, Sagamihara-shi, Kanagawa Kawasumi Chemical Co., Ltd. Sagamihara Works (72) Inventor Tetsuo Ota 1-26 Yokoyamadai, Sagamihara-shi, Kanagawa Address 7 Kawasumi Chemical Co., Ltd. Sagamihara Works (72) Inventor Maruyama Kamo 1-26 Yokoyamadai, Sagamihara City, Kanagawa Prefecture Kawasumi Chemical Co., Ltd. Sagamihara Works

Claims (2)

[Claims] The method according to claim 1 Platelets A method of liquid storage, and platelets, comprises sodium bicarbonate and glucose, and contact with a platelet storage solution added with <br/> osmotic agent through the semipermeable membrane The platelet preservation method is characterized in that the platelets and the platelet preservation solution are shaken while the platelets are preserved while the osmotic pressure difference between the platelets and the platelet preservation solution is balanced. 2. A device for storing platelets in a liquid state, which comprises a semipermeable membrane inner bag for storing platelets, an outer bag for inserting the inner bag, and a space portion between the inner bag and the outer bag. And a platelet preservative stored therein, the platelet preservative being sodium bicarbonate
It includes arm and glucose, and adding osmotic agent for balancing the osmotic pressure difference between the platelets in the inner bag
Platelet storage device, characterized in that to those were.