JPS5929563B2 - Red blood cell fractionation and purification method - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for removing white blood cells and platelets from blood to separate and purify red blood cells. The purpose of this invention is to provide a preparation method and an apparatus for the same.

Traditionally, whole blood was often used in blood transfusions, but
Recently, along with medical advances, a treatment method called component transfusion, in which only the necessary components are transfused, is being promoted both domestically and internationally.

However, red blood cell transfusions are not always carried out frequently because there is no excellent method for completely removing leukocytes containing HL-A antigens and platelets containing platelet antigens. Also, in a biochemical function test of red blood cells, the presence of other blood cell components does not yield good results. Known techniques for fractionating red blood cells for the purpose of blood transfusion include the following methods.

Namely, (1) centrifugation method (2) saline washing method (3) erythrocyte sedimentation method using dextran or HES (4) nylon column passage method (5) Preparation of thawed red blood cell suspension [Platt Components Therapy, American Published by Association of Pratt Punks, Centennial Press U, 5
,A(BloodComponentTherapy7
, American Association of Blo.
odBanks,,TwentythCenturyP
ressU, S, A), pp. 9-13 1969; Japanese Red Cross Blood Center Business Standards 1975; Blood Component Transfusion Era Handbook Published by Osaka Red Cross Center 1975], in which (3) is white blood cell-depleted red blood cells. Although it is actually carried out under the name "Japanese Red Cross Red Blood Cell", neither method is satisfactory in terms of collecting red blood cells and removing white blood cells and platelets. In addition, as a method of packing an adsorbent into a column as described in (4) above, a method using cotton and cotton thread [Flemimg, British Journal]
of Experimental Pathology (Britis)
hJournalofExperimentalPat
Diepenhorst et al., Vox Sang, Vol. 7, p. 281, 1926; Vox Sang, Vol. 23, 308-3
20, 1972], a method using Danelon fibers [Langfelder et al., Vox Samg, Vol. 19, p. 57;
1970], but the separation of lymphocytes from platelets and white blood cells is not good, and the amount of adsorbent is too large compared to the amount of blood, making it difficult to recover red blood cells sufficiently.

Nakao et al.
reNewBiology), vol. 246, p. 94, 197
3 years], red blood cells were purified from heparinized whole blood using cation-exchanged cellulose or cation-exchanged Cephadex, but ion exchangers were expensive, magnesium ions were impermeable, and blood It has drawbacks such as the amount of adsorbent being large relative to the amount of adsorbent, making it impractical.

On the other hand, Wright (TheL)
Ancet), Vol. 1, p. 4, 1926] found that white blood cells were adsorbed to filter paper, but Garv.
in) [Journal of Experimental Medicine
Medicine), Vol. 114, p. 51, 1961) states that the adsorption to filter paper is not so large. As a result of intensive research in view of the above circumstances, the present inventor has found that human whole blood,
Cellulose powder from erythrocyte sediment layer and mammalian blood,
The present invention was completed by successfully recovering red blood cells with high purity and high yield by almost completely adhering white blood cells and platelets to crystalline cellulose and anion exchange cellulose.

The blood used in the practice of the present invention includes fresh human blood, heparin, ACD. These include those containing a suitable anticoagulant such as CPD, and an erythrocyte sedimentation layer (Nisseki), but are not necessarily limited thereto, and any preparation in which red blood cells are suspended may be used.

When using an erythrocyte sedimentation layer, add 1/3 to 1/2 volume of physiological saline or isotonic buffer. In addition, the adsorbents used in the practice of the present invention include cellulose powder, crystalline cellulose, anion exchange cellulose,
Examples include KC floc manufactured by Sanyo Kokusaku Pulp, filter paper powder manufactured by Toyo Roshi, Avicel manufactured by Asahi Kasei Industries, and diethylaminoethyl cellulose. An anion exchanger such as diethylaminoethylcellulose may be used in the OH type or may be used in advance as the C1 type, but in consideration of stability, the salt type is preferred. These adsorbents are washed in advance with physiological saline before use. In the practice of this invention, the adsorbent is supported by a nylon mesh having a 25 micron opening in a column made of hard plastic such as polypropylene. To fill the adsorbent column, the adsorbent suspended in physiological saline is poured into the column, and if necessary, the column is pressurized and packed at a rate of 2 to 6111/1V. The amount of adsorbent is 10,077 blood! 10-50'l for l! 11
is within the range of Washing after passing blood through the adsorbent should be done to increase the recovery rate of red blood cells, and the washing solution should be physiological saline. The appropriate amount of physiological saline for washing is 1 to 3 ml per ml of adsorbent. The time for passing through the column is in the range of 5 to 120 minutes, and if the flow rate is slow, elution may be accelerated by applying pressure with a force of 1 kg/Cd or less.
As shown in Examples 1 to 5 below, as a result of fractionating red blood cells from human whole blood, red blood cell sediment layer, and mammalian blood using the method of the present invention, the recovery rate of red blood cells was more than 95% white blood cells. The results showed that the platelet removal rate was over 99%.

When the recovered red blood cells were observed under a scanning electron microscope, they showed exactly the same disk shape as before contact with the adsorbent, and when viewed by Perpart's method, there was no change in osmotic fragility before and after contact with the adsorbent. In addition, after storing the samples that were in contact with the adsorbent and those that were not at 4°C for one week, the amounts of adenosine triphosphate and 2,3-diphosphoglycenic acid were determined.
No difference was observed in the quantitative values. As an apparatus for carrying out the method of the present invention, there is an apparatus as shown in FIG. 1, in which a blood bag 1 is connected to a column 3 filled with an adsorbent 5.

Blood products such as human whole blood or an erythrocyte sedimentation layer are guided from a hollow needle 7 through a conduit 2 to a column 3 filled with an adsorbent 5, and white blood cells and platelets adhere to the adsorbent 5. Red blood cells that do not adhere to the adsorbent 5 are collected into the blood bag 1 through the conduit 2. Additionally, this device can be autoclaved for 1
Sterilization can be achieved by processing at 15°C for 15 minutes. This heat treatment changes diethylaminoethylcellulose, an anion exchanger, and other celluloses are not affected by this treatment. As explained above, the present invention uses an inexpensive adsorbent to easily and economically prepare red blood cells for component transfusion from which white blood cells and platelets containing tissue antigens have been completely removed from human whole blood or red blood cell sediment layers. be able to do so,
This produces effects such as facilitating the preparation of pure red blood cells for clinical tests.

The present invention will be specifically described below with reference to Examples. Example 1 KC-Flock W-50 (S) (manufactured by Sanyo Kokusaku Pulp Co., Ltd.) 0.5y was suspended in approximately 5ml of physiological saline, and this was added to a nylon mesh 2 having an internal volume of 10ml and an opening of 25 microns. Pack into a lined polypropylene column.

Pressure is applied to bring the volume of the adsorbent to 2.51 n1. 6.0 ml of fresh human whole blood containing 101 U/ml of heparin is run.
Furthermore, saline red blood cell IL? ? ? ? ? −? -1X? - Blood cell counts were compared at the same time when 2ml of physiological saline was added to 6me red blood cells.

White So ball So small board Add 2.0 ml of saline to 4.0 ml of wood and drain.

The cells were treated in the same manner as in Example 1, and blood cells were counted. −
---One]-One? -H8IU/ml added 6
．． Flow 0ml.

Thereafter, the same treatment as in Example 1 was carried out, and blood cells were counted. Instep −? − ] ★ru.

Add to this 4.0 ml of erythrocyte sedimentation layer (Japan Red Cross) and 20 ml of raw wild saline.
Add m1 and let it flow. Thereafter, the cells were treated in the same manner as in Example 1, and blood cells were counted. 0.57 of Hakujyukyujinol\ plate (made by Tokyo Chemical Industry Co., Ltd. purified and dried as Cl type 1) was suspended in about 5 ml of physiological saline and packed in a column in the same manner as in Example 1, and the red blood cell sediment layer was added. (Japan Red Cross) 4.0m
Add 2.0 TfL1 of physiological saline to 1 and drain.

Thereafter, the cells were treated in the same manner as in Example 1, and blood cells were counted.

[Brief explanation of the drawing]

FIG. 1 shows an embodiment of an apparatus for carrying out the method of the invention. 1... Blood bag, 2... Conduit, 3.
...Column, 4...Nylon mesh,
5...adsorbent, 6...branch pipe, 7...
...Hollow needle.

Claims (1)

[Claims] 1. It is characterized by removing mixed white blood cells and platelets from anticoagulated whole blood or red blood cell sediment layer by attaching white blood cells and platelets to an adsorbent made of at least one of cellulose powder, crystalline cellulose, and anion exchange cellulose. A method for fractionating and purifying red blood cells.