JPS5854125B2 - Leukocyte separation filter and leukocyte separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a leukocyte separation filter and a method for separating leukocytes from blood, body fluids, or a blood cell suspension obtained by processing these in a short time using simple operations.

More specifically, the column contains at least one type of synthetic fiber, semi-synthetic fiber, regenerated artificial fiber, inorganic fiber, or natural fiber that does not denature blood and has an average diameter of 10 microns or less. This invention relates to a leukocyte separation filter packed to a density of less than /, ffl, and a method using the same to separate leukocytes with high purity and high yield in a short time and with simple operations.

In recent years, with the development of hematology and immunology, instead of conventional whole blood transfusions, it has become possible to extract red blood cells, white spot fluid, and only granulocytes and lymphocytes from whole blood and transfuse them to various patients. It has become popular.

This component transfusion has the great advantage that components unnecessary or harmful to the patient can be removed from whole blood, and only the necessary components can be concentrated in large quantities and transfused.

Currently, common component transfusions include leukocyte transfusions and granulocyte transfusions for patients with abnormally low white blood cell counts and fever due to bacterial infection, lymphocyte transfusions for various immunotherapies, and those that only require red blood cells. This includes red blood cell transfusions for patients with severe symptoms.

Conventional leukocyte separation methods that can be used for this purpose include methods that use aggregating agents that act on various types of blood cells, centrifugation operations, and methods that use substances that stick, adhere, or adsorb blood cells. ing.

An example of a method using an agglutinating agent is to add a red blood cell aggregating agent such as dextran or hydroxyethyl starch to blood and sediment the red blood cells to obtain white blood cells, but the white blood cells obtained are not very pure and are time consuming.

Examples of methods using centrifugation include layering liquids with different densities, placing blood on top of the layers, and centrifuging the layers to separate blood cells based on the differences in specific gravity; however, it is difficult to process large amounts of blood. However, it is difficult.

In addition, granulocytes and monocytes are commonly used to attach blood cells to nylon fibers, polyester fibers, cotton, silicon-treated glass wool, etc. Because of their weak adhesion to these substances, it was not possible to separate all white blood cells at once using these substances alone.

Therefore, the present inventors conducted extensive research on a method for separating white blood cells from a large amount of blood with simple operations and high yields. As a result, the inventors found that a filter in which specific fibers were packed in a column at a specific bulk density was used to separate white blood cells. Found that it captures well,
This led to the completion of the present invention.

In other words, the present invention provides that at least one of synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, and natural fibers that do not denature blood and have an average diameter of 10 microns or less is added to the column.
．． A leukocyte separation filter packed at a density of 15 f/i or less, and a method for separating white blood cells using the same.

In the present invention, blood, body fluid, or a blood cell suspension obtained by processing these refers to blood or body fluid, such as ascites fluid or bone marrow fluid itself, or to these fluids through some treatment, such as adding red blood cells such as dextran or hydroxyethyl starch. A blood cell suspension obtained by adding a hemagglutinating agent such as a coagulant,
Refers to blood cell suspensions obtained by centrifugation operations such as density gradient centrifugation, blood cell suspensions obtained by cell electrophoresis, etc.

Furthermore, the term fibers used in the present invention are fibers that are much longer in length than their average diameters.

The average diameter (D) is the weight of the object x2 and the length y
α, density ρ? /art is defined as D=277÷wa (α).

According to the present invention, fibers with an average diameter of 10 microns or less
．． A filter packed in a column with a density of 15 f/cy or less can selectively capture white blood cells from blood in a short time.
Can be separated.

That is, when blood is passed through this filter, a significant portion of the white blood cells and some red blood cells remain within the filter.

Next, when a physiological solution such as physiological saline or phosphate buffered saline that hardly collects the white blood cells in the filter is passed through the filter, most of the red blood cells remaining in the filter are removed from the filter. They come out, but very few white blood cells come out.

Next, by collecting the leukocytes captured by the filter through some kind of treatment, a leukocyte suspension containing few components other than leukocytes can be obtained.

At this time, some remaining red blood cells are mixed in, but compared to the amount of red blood cells in the blood, the amount is so small that it does not pose a problem.

In this way, fibers with an average diameter of 10 microns or less have 0.
15? A filter packed in a column with a density of /cril or less can trap a considerable amount of white blood cells, but if the fibers are easy to trap white blood cells due to their surface charge state, morphology, or fiber constituents, then one filter can trap white blood cells easily. The leukocyte trapping power of is increased.

The state of charge may be related to the unevenness of the fiber surface, and even fibers made of the same components may have areas with a lot of positive charge and places with a lot of negative charge depending on the location.

Granulocytic cells, in systems that do not contain much plasma protein, generally have a high affinity for negatively charged parts, and lymphoid cells have a high affinity for positively charged parts, so they have a high affinity for the charged parts of fiber components. Together, it is possible that a significant amount of both these granulocytes, monocytes, and lymphocytes can be attached to a type of fiber.

Of course, it may also be effective to mix separate fibers of different components in one column.

In terms of surface morphology, when granulocytic cells adhere to a foreign object with a smaller diameter than themselves, they deform to wrap around the foreign object and try to eat it, so they have minute protrusions on the fiber surface. It is thought that the granulocytes tend to stick to the granulocytes, and once they stick, they are difficult to separate.

On the other hand, filters filled with small fibers with an average diameter of 10 microns or less have a larger surface area than filters filled with the same amount of large-diameter fibers, so they are less likely to adhere to granulocytes, monocytes, and lymphocytes. In addition to being advantageous, the reduced interfiber distance reduces the space between the fibers, making it easier for white blood cells to get caught.

In addition, phagocytic mechanisms may also function in granulocytic cells, making granulocytes more likely to adhere.

Further, in this case, even if the filter is a mixture of small diameter fibers and a small amount of large diameter fibers, leukocytes can be sufficiently captured.

On the other hand, fibers that capture such leukocytes well are placed in the column at a concentration of 0.15? If the filter is filled with more than /cril, the captured white blood cells may become clogged and cannot be collected when blood is passed through the filter, or the filter may become clogged due to denatured substances such as fibrin, making it impossible to perform efficient separation for a long time.

The fibers used in the present invention may be any fiber having an average diameter of 10 microns or less and capable of capturing white blood cells without denaturing blood (organic synthetic fibers such as polyamide, polyester, polyacrylonitrile, polytetrafluoroethylene, etc.).
Semi-synthetic fibers such as acetate, copper ammonia rayon,
Recycled man-made fibers such as, inorganic fibers such as glass fiber, cotton,
Natural fibers such as silk and wool can be used.

Among these, synthetic fibers such as polyamide, polyester, and polyacrylonitrile, and semi-synthetic fibers such as acetate are preferred materials, and these can be used to capture white blood cells by changing their surface morphology or introducing copolymerized components. You can further improve your strength.

When performing transfusion of blood components using a filter using such fibers, it is desirable to remove finishing agents on the fiber surface with a surfactant or organic solvent, as well as remove dust adsorbed on the fiber surface.

When filling the column with fibers, cut the fibers into appropriate lengths and spread them uniformly, or use secondary products such as non-woven fabrics, woven fabrics, knitted fabrics, etc. to have a specific bulk density. This is used as a filter for leukocyte separation.

As described above, by using the leukocyte separation filter of the present invention, granulocytes, monocytes, and lymphocytes in blood can be removed in a short time with easy operations, and a red blood cell suspension containing few leukocytes can be obtained. I can do it.

It is thought that by using this red blood cell suspension, side effects such as GVH reactions caused by differences in histocompatibility antigens and fever due to destroyed leukocyte components due to whole blood transfusion can be prevented.

On the other hand, it is also possible to take out the leukocytes captured by the filter to obtain a leukocyte suspension, or to remove granulocytes and monocytes from this to obtain a lymphocyte suspension.

If the white blood cells are attached to the fibers in the filter by some mechanism, the methods for recovering the white blood cells in the filter include adding a substance that inhibits the adhesion, or applying physical external force to remove the adhesion. Alternatively, leukocytes can be collected by placing the filter under conditions that do not cause adhesion.

Furthermore, if white blood cells are caught in the space created by the fibers in the filter, the white blood cells can be recovered by expanding this space or by flowing the liquid while vibrating the fibers.

In addition, before passing the blood cell suspension through this filter, a hemagglutinating agent such as dextran is added in advance to obtain a blood cell suspension containing few red blood cells, which is then passed through the leukocyte separation filter of the present invention, and the inside of the filter is thoroughly washed. By leaving it for a long time, the number of red blood cells in the collected white blood cell suspension can be further reduced.

Granulocytes and monocytes among the leukocytes obtained in this way have well-preserved phagocytosis and chemotaxis, and lymphocytes are treated with various mitogens such as phytohemagglutinin and Bork-weed mitogen. , can be immature to the same extent as lymphocytes obtained by other methods.

Also, the ratio of T cells and B cells is not much different from the original blood.

As described above, according to the present invention, leukocytes can be removed from a hemocyte suspension such as blood with a simple operation, and by recovering the leukocytes in the filter, the leukocyte suspension can be removed with high purity and yield with a simple operation. These components can be easily transfused at various medical facilities.

Example 1 Acrylonitrile synthetic fibers with a diameter of 6 microns and a length of 4 cm to 7 cIrL were taken.
A leukocyte separation filter was obtained by uniformly filling an IrLO column with a density of 0.08 f/-.

5ml/# of 100ml of healthy blood into this filter
Then, 29 ml of physiological saline was flowed at a flow rate of 5 ml/win to obtain a total volume of 100 ml.

This fluid contains 7% white blood cells and 94% red blood cells.
% was included.

Example 2 Diameter: 4.5 microns, length: 4CrrL to 7Crn
Take 2.8279 polyester synthetic fibers and have a diameter of 2.
A leukocyte separation filter was obtained by uniformly packing the mixture into an α column at a density of 0.09 f/i.

100 ml of blood from a healthy person was poured through this filter, and then 29 ml of physiological saline was poured into the filter to obtain a total volume of 100 TLl of liquid.

This fluid contained 7% of the original white blood cells and 92% of the red blood cells.

Example 3 Diameter: 9.8 microns, length: 4crrL to 7crr
Take L acrylonitrile synthetic fiber 6.0329,
A leukocyte separation filter was obtained by uniformly filling a column with a diameter of 2 CTL to a density of +28 P/ffl.

Put 150ml of blood from a healthy person into this filter at 5ml/v.
tm flow rate, and then physiological saline at 1°r/Ll/
The red blood cells remaining in the filter were mostly washed out by flowing 250 ml at a flow rate of #.

Thereafter, 150 ml of a physiological solution containing plasma was flowed at a flow rate of 5 ml/yniyt, and the white blood cells in the filter were collected while applying physical external force to the filter.

This fluid contains 64% of its original white blood cells and 0 of its original red blood cells.
．． 1%, and platelets contained 4% of the original.

Claims (1)

[Claims] 1. At least one type of synthetic fiber, semi-synthetic fiber, regenerated artificial fiber, inorganic fiber, or natural fiber that does not denature blood and has an average diameter of 10 microns or less is added to the column. A leukocyte separation filter packed with a density of /- or less. 2. A leukocyte separation filter according to claim 1, in which the fibers are made of at least one of polyamide, polyester, polyacrylonitrile, and acetate. 3. A leukocyte separation filter according to claim 1, in which the fibers are made of at least one of polyamide, polyester, polyacrylonitrile, and acetate. 3. Blood, body fluid, or suspended blood cells obtained by processing these Synthetic fibers, semi-synthetic fibers, recycled man-made fibers, inorganic fibers that do not denature blood and have an average diameter of 10 microns or less.
At least one type of natural fiber is added to the column at a concentration of 0.15S'/
CI! A method for separating leukocytes, which comprises passing the leukocytes through a leukocyte separation filter packed at the following density, and trapping the leukocytes in the filter. 4. Synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, which do not denature blood, have an average diameter of 10 microns or less, and which do not denature blood, body fluids, or the blood cell suspension obtained by processing these.
At least one type of natural fiber is added to the column at 0.15 P/f.
A method for separating leukocytes, which comprises passing through a leukocyte separation filter packed at a density of less than fl to separate leukocytes from other components, and then recovering the leukocytes captured in the filter.