JPS5854126B2 - Leukocyte separation material - Google Patents

Description

[Detailed description of the invention] The present invention relates to a leukocyte separation material.

More specifically, the present invention relates to a leukocyte separation material made of synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, or natural fibers that do not denature blood and have an average diameter of 10 microns or less.

In recent years, with the development of hematology and immunology, in place of conventional whole blood transfusion, transfusions of red blood cells, white blood cells, and granulocytes among white blood cells are being used.
Component transfusion, in which only specific components of blood, such as lymphocytes, are transfused to various patients has come to be performed.

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

Currently, the most common treatments are 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 transfusions that require only red blood cells. such as red blood cell transfusions for patients who

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.
The purity of the white blood cells obtained is not good and it takes a long time.

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. is difficult.

In addition, there are methods that utilize the property of granulocytes and monocytes to adhere well to nylon fibers, polyester fibers, cotton, silicone-treated glass wool, etc., but lymphocytes generally do not adhere to these materials. Due to their weak adhesion to substances, it was not possible to separate all white blood cells at once using these substances alone.

Therefore, the present inventors conducted extensive research into a method for separating white blood cells from a large amount of blood with simple operations and high yield.As a result, they were able to capture large amounts of not only granulocytes and monocytes, but also lymphocytes, and remove red blood cells. They found a separation material that does not trap much, and completed the present invention.

In other words, the present invention provides a material having an average diameter of 10 microns or less.
This leukocyte separation material is characterized by being made of synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, or natural fibers that do not denature blood.

In the present invention, blood, body fluids, or a blood cell suspension obtained by processing these are blood or body fluids, such as ascites or bone marrow fluid themselves, and these fluids may be treated in some way, such as by 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 fiber as used in the present invention refers to a fiber whose length is much longer than its average diameter.

The average diameter (D) is the weight of the object x? , the length is y
If the density is ρt/crrt, then D=21'';(
m) πy.

The synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, natural fibers, etc., which do not denature blood and have an average diameter of 10 microns or less according to the present invention, can selectively capture and separate leukocytes from blood in a short time.

In other words, when blood is passed through a filter containing these fibers in columns, most of the white blood cells and some red blood cells remain in 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 will come out of the filter. However, almost no white blood cells come out.

Next, the captured leukocytes are recovered by some treatment to obtain a leukocyte suspension.

At this time, some remaining red blood cells are mixed in, but the amount is incomparably small compared to the initial number of red blood cells.

In this way, fibers with an average diameter of 10 microns or less capture white blood cells well, but one possible mechanism is that when thin fibers are packed into a column, the spacing between the fibers is much smaller than that of conventional thick fibers. For this reason, not only granulocytes and monocytes, but also lymphocytes are trapped in the gaps between the fibers, and because the fibers are thin, the fiber surface area can be made extremely large, and lymphocytes, granulocytes, and monocytes are size is 8
~15 microns, which means that they tend to stick to the fibers of the present invention, which have a diameter smaller than white blood cells and have a diameter of 10 microns or less.

The fibers used in the present invention have an average diameter of 10 microns or less,
Any material that does not denature blood may be used, such as synthetic fibers such as polyamide, polyester, polyacrylonitrile, and polytetrafluoroethylene, semi-synthetic fibers such as acetate, recycled artificial fibers such as copper ammonia rayon, inorganic fibers such as glass fiber, Natural fibers such as cotton, silk, and wool are used.

Among them, 1. Synthetic fibers such as polyamide, polyester, and polyacrylonitrile, and semi-synthetic fibers such as acetate, have an average diameter of 10 microns or less.
Shows significantly high leukocyte capture ability.

Even fibers with an average diameter larger than 10 microns exhibit considerable leukocyte-capturing power when packed in large quantities into a column.

However, even under conditions of the same surface area, ie, when the amount of thread used is larger than that of fibers with an average diameter of 10 microns or less, fibers with an average diameter of more than 10 microns have inferior leukocyte capture power.

When such a separation material made of fibers with an average diameter of 10 microns or less is used for blood transfusion, it is desirable to remove finishing agents and the like on the fiber surface as well as remove dust adsorbed to the surface.

Further, when making a filter, a separation material in which the above-mentioned fibers are made into a secondary product may be used.

Figure 1 shows the leukocyte-capturing power of various fibers.

Figure 1 shows that 20 drops of blood containing heharin were first poured into a filter filled with various fibers in a column with a diameter of 1 cIfL at a density of 0.085 S'/-, and then 50 ml of physiological saline was poured. This is the result obtained.

The leukocyte capture rate is expressed as a percentage of the number of leukocytes remaining in the filter when the physiological saline has finished flowing, relative to the number of leukocytes in the original blood.

Figure 2 shows the results obtained by first flowing 20 ml of heparinized blood and then 5 oml of physiological saline through a filter filled with polyacrylic fibers packed in a column with a diameter of 1 crfL so that the fiber surface area was constant at 2677 ctA. This is the result.

The lymphocyte capture rate is the ratio of the number of lymphocytes remaining in the filter to the original number of lymphocytes in the blood when the physiological saline has finished flowing, expressed as a percentage.

Note that in both FIGS. 1 and 2, the linear velocity at which the liquid flows through the filter is assumed to be constant.

As is clear from these figures, if fibers with an average diameter of 10 microns or less are used as a filter and have a certain surface area, most of the leukocytes in the blood can be captured, and even under the conditions of the same surface area and the same average distance between fibers, , filters filled with fibers with an average diameter of 10 microns or less are better.
It has a stronger ability to capture Linha spheres than a filter filled with fibers larger than 10 microns in diameter.

This filter only needs to contain a certain amount of such fibers with an average diameter of 10 microns or less, and may be mixed with other substances to form a filter.

For example, it goes without saying that the fibers having an average diameter of 10 microns or less may be of different types, have different average diameters, or a mixture of both.

As described above, by using the leukocyte separation material 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 no leukocytes can be obtained. can.

By using this, side effects such as GVH reactions based on differences in histocompatibility antigens and fever due to destroyed white blood cell components, which occur when whole blood transfusions are given to patients who require only normal red blood cells, can be prevented. It is considered possible.

On the other hand, it is also possible to elute the leukocytes captured in the leukocyte separation material to obtain a leukocyte suspension, or to remove granulocytes and monocytes from this to obtain a lymphocyte suspension.

If the leukocytes are stuck to the fibers of the separation material, methods for recovering the leukocytes captured in the separation material include adding a substance that inhibits adhesion, applying physical external force, or using conditions that make it difficult to cause adhesion. If the white blood cells are stuck in the space created by the fibers of the separation material, it is possible to collect the white blood cells by expanding this space or by flowing the liquid while vibrating the fibers. .

Furthermore, before bringing the blood cell suspension into contact with this separation material, a red blood cell agglutinating agent such as dextran is added in advance to obtain a blood cell suspension containing few red blood cells. By washing, the number of red blood cells in the collected white blood cell suspension can be further reduced.

The granulocytes and monocytes among the white blood cells obtained in this way have well-preserved poor phagocytic ability and chemotaxis, and the lymphocytes contain various mitogens such as phytohemagglutinin, Po-7, weed, and mitogen. By this method, lymphocytes obtained by other methods can be transformed to the same extent as lymphocytes.

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

As described above, the leukocyte separation material according to the present invention can efficiently remove leukocytes from blood, and by recovering leukocytes captured by the separation material, leukocyte suspension can be achieved with high purity and yield with simple operation. These components can be easily transfused at various medical facilities.

Example 1 Diameter: 5.2 microns, length: 4crfL to 7crr
A leukocyte separation filter was made by uniformly packing 2.7z L of acrylonitrile synthetic fibers into a column with a diameter of 2 cm and a length of 10 crIL.

Put 5rrLl/rfuI of healthy human blood into this filter.
The red blood cells in the filter were collected by flowing 100 TI'Ll at a flow rate of 10 L, and then flowing 30 ml of physiological saline at a flow rate of 5 ml/yniyi.

This fluid contains 3% of the original white blood cells and 93% of the original red blood cells.
% was included.

Example 2 A polyester synthetic fiber with a diameter of 9.6 microns and a length of 4 crfL to 7 cm was fabricated with a diameter of 2 CIrL and a length of 1 cm.
A leukocyte separation filter was prepared in which 41 cells were uniformly packed in a 0α column.

100ml of healthy blood was passed through this filter at a flow rate of 5'/nl/-, and then physiological saline was poured into the filter at a flow rate of 100ml/nl/-.
Most of the red blood cells remaining in the filter were washed out by flowing 200 ml at a flow rate of mlyt.

Thereafter, 100 TILl of physiological solution containing plasma was flowed at a flow rate of 5 ml/mltt to apply physical external force to the filter, and leukocytes in the filter were collected.

This fluid contains 55% of the original white blood cells and 0 of the original red blood cells.
．． 2%, and platelets were 8% of the original.

Example 3 Acrylonitrile synthetic fibers with a diameter of 9.8 microns and a length of 4 cffL to 7 cm were made into 2 CrrL in diameter and 1 cm in length.
A leukocyte separation filter was made in which 4z cells were uniformly packed in a 0 column.

A solution obtained by adding a dextran solution with a molecular weight of 200,000 to 100 ml of blood from a healthy person, with a concentration of 3% red blood cells and 65% white blood cells, was passed through this, and then physiological saline was added at 200 mA'. Most of the remaining red blood cells in the filter were washed out by flushing.

Thereafter, 100 ml of a physiological solution containing plasma was flowed to collect the white blood cells inside the filter while applying physical external force to the filter.

This fluid contains 40% white blood cells and 0 red blood cells.
．． It contained 0.02% and platelets 0.6%.

Example 4 Acrylonitrile synthetic fibers with lengths from 4crIL to 7α having various average diameters were made from 0.085? A column with a diameter of 1 crrL and a length of 10 crIL is packed at a packing density of /c11t, and 20 - of healthy human blood is poured into this column at a packing density of 2 trtl/c11t.
Rinse with mlyr, then add 30WLl of raw food to 5rrtl/
Figure 3 shows the leukocyte capture rate in the filter when flowing with vtin.

Curve a in FIG. 3 is the capture rate of lymphocytes, and curve b is the capture rate of granulocytes.

As is clear from this figure, when the average diameter is IOμ or less, the capturing rate of lymphocytes and granulocytes is better than when the fiber diameter is larger than 10μ.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the leukocyte capture rate of various fibers. FIG. 2 is a graph showing the relationship between the average interfiber distance and the lymphocyte capture rate. FIG. 3 is a graph showing the relationship between fiber diameter and leukocyte capture rate.

Claims (1)

[Scope of Claims] 1. A leukocyte separation material made of synthetic fibers, semi-synthetic fibers, regenerated artificial fibers, inorganic fibers, or natural fibers that do not denature blood and have an average diameter of 10 microns or less. 2. The leukocyte separation material according to claim 1, wherein the fibers are selected from polyamide, polyester, po'J7crylonitrile, and acetate.