JPS6326089B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a filter for capturing and collecting leukocytes.

More specifically, the present invention relates to a filter that can capture and collect white blood cells from blood, body fluids, or a blood cell suspension obtained by processing these in a short time with simple operations.

In recent years, with advances in hematology and immunology, transfusion of blood components instead of conventional whole blood transfusion, functional tests of leukocytes, and tests of surface antigens of leukocytes have been carried out, and applied to the treatment and diagnosis of various diseases. are hospitals in various places,
It is carried out at a research institution.

Conventional leukocyte capture and collection techniques that can be used for such purposes include methods using red blood cell agglutinants, centrifugation, and methods that utilize the adhesive force of blood cells to fibers.

More specifically, the method using a hemagglutinating agent involves adding a hemagglutinating agent such as dextran or hydroxyethyl starch to the blood, and then leaving it for a certain period of time to obtain a supernatant rich in white blood cells.The centrifuging method involves centrifuging the blood. There are two methods: a method in which blood is separated and a buffer coat rich in white blood cells is collected, and a density gradient centrifugation method in which blood is layered in a liquid with a specific gravity of 1.077 and then centrifuged to collect a lymphocyte layer. Known methods that utilize adhesion to fibers include attaching monocytes and granulocytes to fibers and collecting the attached blood cells using physiological saline, phosphate buffered saline, etc., and using a flocculant or centrifugation. A leukocyte-rich fraction was obtained by
Then, this white blood cell fraction was placed in a column packed with fibers such as nylon or glass wool, and kept at 37℃ for 30 minutes.
This is a method in which lymphocytes are collected after being left in a portion.

However, these methods had the following drawbacks. In other words, the method using a hemagglutinating agent allows a suspension of white blood cells to be obtained through a relatively simple operation, but this suspension contains several times as many red blood cells as white blood cells, several tens of times as many platelets, and a large amount of plasma as well as the hemagglutinating agent. It could not be used directly as a white blood cell suspension, let alone for blood transfusions or for testing purposes. In order to obtain a leukocyte suspension in a condition that can actually be used, several washing operations and various operations for removing mixed impurities are required, which ultimately takes a lot of time and effort. The centrifugation method requires a large centrifuge to process the amount of blood required for transfusion, and a fairly expensive centrifuge is required even to process a small amount of blood for testing. Become. In addition, several more centrifugation operations are required to remove impurities from the obtained leukocytes, and when these operations are performed, some leukocytes are destroyed, resulting in a poor recovery rate of leukocytes. . Furthermore, since it is a very complicated operation, it is difficult to operate aseptically, and even an experienced person requires a great deal of labor and time. Conventional methods that utilize the adhesion of blood cells to fibers have generally been unable to separate all leukocytes at once because the adhesion of lymphocytes to fibers is weak.

Therefore, we can improve the purity and purity of blood with simple operations.
As a result of intensive research aimed at capturing and collecting leukocytes with a high yield, we found that a porous filter with an average pore diameter of 5 to 20 μm captures leukocytes well, and that the captured leukocytes can be recovered with simple operations. Heading This has led to the present invention.

That is, the present invention is a filter for trapping and collecting white blood cells, the main part of which is a porous filter having continuous pores with an average pore diameter of 5 to 20 μm.

Any material can be used for the porous filter as long as it does not cause damage to blood cells.
Synthetic rubber, synthetic resin foam, etc. are easy to use as the pore diameter can be easily adjusted.

The present invention will be explained below using the drawings. FIG. 1 is a schematic cross-sectional view of a porous filter used in the present invention. The porous filter used in the present invention refers to a filter in which the pores 1 are randomly opened as shown in FIG. 1, and the pores are continuous from the surface of the porous structure 2 to other surfaces. Even though it is porous, it does not necessarily have to be flexible. The average pore diameter is the number of pores with the largest number when cutting a porous filter arbitrarily and measuring the diameter of each pore dispersed throughout the cross section to examine the relationship between the diameter and the number of pores. It represents the diameter of the hole converted into a circle. In other words, the pores dispersed on any cut surface of a porous filter have various shapes and diameters, but each pore is converted into a circle with the same area as the cross-sectional area of the pore, and the If you draw a graph with the diameter on the horizontal axis and the number of pores on the vertical axis, you will generally get a curve close to a normal distribution. At this time, it is preferable to randomly count 1000 or more pores. The diameter corresponding to the peak of the curve is the average pore diameter in the present invention. Therefore, when various particles are passed through a porous filter, particles with a diameter larger than the average diameter of the porous filter are difficult to pass through, and particles with a larger diameter absolutely do not pass through. It's nothing.

Further, it is desirable that the porous filter has a higher pore porosity than a porous structure, and it is also desirable that the pore size distribution of the pores be narrower.

Hereinafter, the filter for capturing and collecting clear blood cells of the present invention will be explained using an example. FIG. 2 is a schematic diagram showing an example of a filter for capturing and collecting clear blood cells of the present invention, and FIG. 3 is a schematic diagram showing an example of an apparatus when using the filter for capturing and collecting clear blood cells of the present invention.

The filter for trapping and collecting blood cells of the present invention is constructed by housing a porous filter 6 in a container 5 having an inlet 3 and an outlet 4, as shown in FIG. 2, for example. When actually using this filter, for example,
The experimental equipment shown in the figure is used. Taking as an example an experiment in which leukocytes are collected from blood, blood 7 is first sent by a pump 8 to a filter 9 for capturing and collecting leukocytes, where the leukocytes are captured.
Red blood cells, plasma, etc. are not captured by the filter 9 and are sent to the container 10.

There are two reasons why white blood cells are selectively captured by the filter 9: one is due to the adhesiveness of the white blood cells, and the other is due to the pores. is hardly captured. In addition, because white blood cells are captured using two types of adhesion and pore filtration, there is very little chance of clogging, which is common in general membrane filters. There is almost no chance of large amounts of lymphocytes leaking out, unlike with filters that use filters.

Next, when the blood 7 in the container 11 is emptied, the blood introduction tube 12 is placed in the washing liquid 13 to wash away the red blood cells, plasma, etc. remaining in the filter 9, and then the white blood cells trapped in the filter 9 are washed away. By collecting the white blood cells using an appropriate method, white blood cells can be collected with high purity and high yield. Here, cleaning liquid 13
may be physiological saline, phosphate buffered saline, etc. Furthermore, if blood from which monocytes and granulocytes have been removed by some method is used instead of blood 7, the only blood cells captured by the filter 9 will be lymphocytes, and it goes without saying that lymphocytes can be collected with high purity and high yield. None.

As described above, by using the filter for capturing and collecting leukocytes of the present invention, it has become possible to capture and collect leukocytes with high purity and high yield in a short time with simple operations.

Here, the average pore diameter of the porous filter is from 5 to
The diameter is in the range of 20 μm, and from the viewpoint of both purity and yield of white blood cells, 10 to 15 μm is preferable. Average pore size is 5μ
m or less, the number of red blood cells remaining in the filter increases rapidly and the filter becomes easily clogged, making it difficult to use as a filter for capturing and collecting white blood cells, and the average pore size increases. If the diameter exceeds 20 μm, lymphocytes with low adhesion will easily leak out of the filter, making it difficult to capture and collect all white blood cells.

In addition, in order to prevent excessive leakage of blood cells to be captured due to non-uniformity of the pore diameter of the porous filter, the thickness of the porous filter, that is, the linear distance from the blood inlet to the outlet of the porous filter, is set to 10 mm.
The above shall apply.

Example 1 Polyester foam with an average pore size of 15 μm was
It was placed in a 20 mm x 100 mm long container and used as a filter for capturing and collecting leukocytes. The experimental apparatus shown in FIG. 3 was used. Add 50ml of heparinized blood from a healthy person to this filter.
The blood was washed away by flowing physiological saline at the same flow rate to obtain a total of 150 ml of liquid.
This fluid contained only 7% of the original blood white blood cells, but 99.7% red blood cells.
Thereafter, 50 ml of a physiological solution containing plasma was flowed through the filter at a flow rate of 5 ml/min, and the white blood cells in the filter were collected while vibrating the filter. When we examined the collected fluid, we found that 55% of the white blood cells were recovered, 0.2% of the red blood cells, and 0.2% of the red blood cells.
Platelet count was 7%.

Example 2 A polyether urethane foam material with an average pore diameter of 10 μm was placed in a container with an inner diameter of 10 mm and a length of 25 mm and used as a filter for capturing and collecting leukocytes. This filter was used to remove most of the monocytes and granulocytes from the heparinized blood of healthy people using polyamide fibers (the ratio of lymphocytes in white blood cells was 95%).
2 ml was flowed at a flow rate of 4 ml, and then 15 ml of physiological saline was flowed at the same flow rate to wash the red blood cells in the filter. Thereafter, 2 ml of physiological saline was rapidly poured into the filter, and the white blood cells in the filter were collected.
When the collected fluid was tested, the ratio of lymphocytes in white blood cells was 95%, that is, 95% pure lymphocytes compared to blood excluding most of monocytes and granulocytes.
A recovery rate of 60% was obtained. Furthermore, the amount of contaminated red blood cells at this time was 0.5% of the original blood, and the amount of mixed platelets was 0.8%.

As described above, by using the filter for capturing and collecting leukocytes of the present invention, it has become possible to capture and collect leukocytes with good purity and yield in a short time with simple operations. In addition, the filter for capturing and collecting clear blood cells of the present invention can easily form a sealed circuit, making aseptic operation extremely simple.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view of the porous filter of the present invention, and FIG. 2 is a cross-sectional view of the porous filter of the present invention.
FIG. 3 is a schematic diagram showing an example of a collection filter, and FIG. 3 is a schematic diagram showing an example of an apparatus when using the clear blood cell capture/collection filter of the present invention. 1... Pore, 2... Porous structure, 3... Inlet, 4... Outlet, 5, 10, 11... Container, 6...
... Porous filter, 7 ... Blood, 8 ... Pump, 9 ... Filter for capturing and collecting white blood cells, 1
2...Blood introduction tube, 13...Washing liquid.

Claims (1)

[Claims] 1. The average pore diameter is from 5 to 20 micrometers (μ
m) A filter for capturing and collecting white blood cells, the main part of which is a porous filter having continuous pores. 2. The filter for capturing and collecting white blood cells according to claim 1, wherein the porous filter has a thickness of 10 mm or more.