JP5785713B2 - Leukocyte fraction collection device - Google Patents

Description

  The present invention relates to an apparatus for concentrating components containing nucleated cells from blood. In the present specification, a component containing nucleated cells is represented by white blood cells.

Undifferentiated stem cells such as hematopoietic stem cells and iPS cells are attracting attention as cell sources applicable to regenerative medicine. For example, hematopoietic stem cells contained in umbilical cord blood are increasingly used for hematopoietic stem cell transplantation, which is a treatment method for leukemia. Against this background, it is also important to improve the results of cell transplantation by establishing a method and facility for storing umbilical cord blood and the like. Regarding the storage of cord blood, there is a mechanism in which it is stored in a public or private cord blood bank. In Japan, there are few providers and the number of preservation is not increasing. Problems include cost and technical aspects.
Umbilical cord blood is collected in a small amount, and from about 30 to 150 cc, on average 70 to 80 cc is collected from one umbilical cord. In the case of bone marrow, about 800 to 1000 cc can be collected per person. Since umbilical cord blood used for transplantation is required to be about 3 to 5 cc per 1 kg body weight of the patient, in Japan, the transplant target is often a child.
Among the components in cord blood, those to be preserved are nucleated cell components excluding plasma and red blood cells, which include stem cell components such as hematopoietic stem cells. Since nucleated cells are mainly white blood cells, they are also called components containing white blood cells. As a technique for concentrating the leukocyte component, a technique that is widely used in cord blood banks is specific gravity sorting. This is a method of collecting a layer containing a large amount of white blood cell components by performing centrifugation twice. This method often does not produce a clear white blood cell layer, and the amount of reagent added in the middle differs depending on the amount of collected umbilical cord blood. Therefore, it is necessary to calculate and determine each time. is there. From the above, it is often dependent on the skill of the person in charge, the processing takes time, and the quality of the white blood cell component is not constant.

The umbilical cord blood treatment process is (1) blood collection → (2) concentration → (3) addition of cryoprotective solution → (4) cryopreservation.
In the concentration step, nucleated cell components are concentrated by removing red blood cells and plasma from the collected cord blood. To remove erythrocytes, 20 vol% of erythrocyte sedimentation agent is added to cord blood in the blood collection bag, and then light centrifugation is performed to precipitate erythrocytes. At this time, the leukocyte layer is slightly formed at the uppermost part of the erythrocyte layer, that is, at the interface with plasma as the supernatant. After centrifugation, the leukocyte-rich layer is transferred to the separation bag by transferring the supernatant plasma to the upper part of the red blood cell layer from the blood collection bag to the separation bag. Next, the separation bag is centrifuged to precipitate blood cells, and the supernatant plasma is discharged to concentrate leukocyte-containing components including hematopoietic stem cells.
In this concentration method, the amount of umbilical cord blood varies from sample to sample, so the amount of erythrocyte sedimentation agent must be calculated each time. In addition, since the white blood cell layer at the interface between the plasma layer and the red blood cell layer may not be clearly formed, the collection and collection rate of unnecessary components will fluctuate. There are problems such as the time and skill required for layer formation by centrifugal sedimentation, which are constraints on cost and quality.

In Patent Document 1 (Japanese Patent No. 4061715), the leukocyte layer fractionated by light centrifugation is further lightly centrifuged, and fractionated into a plasma layer and a leukocyte concentrated liquid layer, and the leukocyte concentrate is collected and frozen. A method is disclosed.
In Patent Document 2 (Japanese Patent No. 39389973), white blood cells or CD34 positive cells are recovered from bone marrow, peripheral blood, umbilical cord blood, or a cell population obtained by roughly separating these blood cells, including red blood cells, platelets and CD34 positive cells. Disclosed is a method for separating cells. In this method, the leukocytes or CD34 positive cells are captured, while the red blood cells and platelets are introduced into a container filled with a non-woven filter having a property of substantially passing, and then the dextran is added to the filter. The leukocytes or the CD34 positive cells captured by the filter are selectively collected by introducing a physiological solution containing. In this prior art document, 25 non-woven fabrics made of polyester are used as cell separation filters. In a filter in which non-woven fabrics are laminated, there is a large possibility that gaps formed due to fiber entanglement are large, leukocytes and the like are entangled between the fibers, and the quality of the collected cells is not stable even if they are made to flow backward. In order to increase the recovery rate, it is considered that many filters and a large amount of recovery liquid are required.
Patent Document 3 (Republished 2007-046501) discloses a method of using a nonwoven fabric for a stem cell separation filter.
Patent Document 4 (Japanese Patent Laid-Open No. 2009-5655) discloses an apparatus and method for separating and recovering nucleated cell components using a filter for blood components after specific gravity sorting of red blood cells by gravity. In this apparatus and method, a non-woven fabric laminated as a cell separation filter is used. The problem of using a non-woven fabric for the filter is the same as that disclosed in the patent document. It is characterized by the recovery of nucleated cells by backwashing using saline containing dextran as a recovery solution, but in order to increase the recovery rate, a large amount of recovery is required to separate the nucleated cells from the non-woven filter. Liquid is required. In addition, it is said that erythrocytes and nucleated cell components are separated by the difference in specific gravity by suspending the blood container for 10 to 30 minutes before passing through the filter, but the interface is not clear and skill is required for identification. In addition, it is difficult to clearly separate the red blood cell fraction by clipping the interface with the container, and there is a risk that red blood cell components may be mixed in the remaining part, and nucleated cells will be included in the red blood cell fraction. It is a device that leaves problems in time, quality, and recovery yield in the pre-process for separating the. In this apparatus and method, it takes time to include a certain level of proficiency in the elimination of red blood cells and the subsequent cell separation and cell recovery operations. In the nucleated cell recovery, the centrifugation operation is performed to promote the specific gravity selection to be allowed to stand, so the method described in this patent document is adjusted as a whole rather than the method using centrifugation. The time will be longer.

Japanese Patent No. 4061715 Japanese Patent No. 39389973 Republished 2007-046501 JP 2009-5655 A

As a concentration technique, concentration by specific gravity selection or centrifugal treatment has disadvantages that the components after preparation are not stable, preparation takes time, and skill is required for operation. Moreover, in the method of collecting leukocytes entangled in the filter using a non-woven fabric as a filter, first, in order to increase the amount of leukocytes attached to the filter and increase the recovery rate of leukocytes, a large number of non-woven fabrics are laminated, It needs to be thick. On the other hand, when collecting the attached leukocytes, it is necessary to efficiently separate and collect leukocytes that have penetrated into the inside of the nonwoven fabric filter, so it is extremely difficult to satisfy these two requirements at the same time. Have difficulty. Furthermore, since the gap formed between the fibers varies, it is difficult to obtain reproducibility.
An object of the present invention is to provide an apparatus for easily concentrating leukocyte components including stem cells in a short time from blood without using specific gravity difference sorting.

The inventor of the present invention pays attention to the deformability of erythrocytes, and leukocytes containing stem cells using a means for passing erythrocytes and plasma through a membrane filter having micropores and recovering components containing leukocytes as non-filtered components. An apparatus for concentrating ingredients is provided. Focusing on the deformability of red blood cells, after passing red blood cells or plasma through a first filter (hereinafter sometimes referred to as a leukocyte capture filter), a second filter (hereinafter referred to as an red blood cell capture filter). A device that removes red blood cells and collects the components containing white blood cells as non-filtered components using the plasma obtained as filtered components with two filters is proposed.
The main configuration of the present invention is as follows.
1. The original blood backed (1) / collecting back (7) and plasma backed (4), two types of blood cells fraction of the original blood back (1) side leukocyte trap in order from (2) and erythrocyte trap (3) The image capture device includes a main circuit (a) disposed between both bags, a bypass circuit (b) that does not pass through the red blood cell capture device (3), and a blood-feeding syringe (5) that is a raw blood. Concentrate and collect leukocyte fraction from raw blood provided between bag (1) / collection bag (7) and leukocyte trap (2) or / and between red blood cell trap (3) and plasma bag (4) A leukocyte fraction collection device (10),
Leukocyte trap (2) is provided with a white blood cell capture filter a large number of micropores of diameter 2~7.8μm is formed,
The red blood cell capture device (3) is a leukocyte fraction collection device comprising a red blood cell capture filter which is a fine hole through which red blood cells cannot pass and which allows plasma components to pass through.
2 . The pore diameter of the leukocyte capturing filter is 5 to 7.8 μm. The leukocyte fraction collection device described.
3. The leukocyte capture filter has an area of 1-1000 cm ² . Or 2. The leukocyte fraction collection device described.
4 . The syringe (5) for blood feeding is arranged on the plasma back (4) side from the bypass circuit,
White blood cell fraction from the original blood back (1) trap (2), red blood cell fraction trap (3), the main circuit for recovering plasma component via the order of the syringe (5) in the plasma back (4) (a ) and blood cell separation circuit using,
Leukocyte fraction collection for collecting the leukocyte fraction containing plasma in the collection bag (7) through the plasma bag (4) through the syringe (5), bypass circuit (b) and leukocyte trap (2) in this order. 1. A circuit is constructed. ~ 3 . The leukocyte fraction collection device according to any one of the above.
5 . Original blood bag (1) / collection bag (7), plasma bag (4), syringe (8) for blood feeding, leukocyte trap (2), red blood cell trap ( 3), a blood-feeding syringe (5) and a plasma bag (4) are arranged in this order,
Syringe for blood feeding (8), leukocyte capturing device (2), red blood cell capturing device (3), syringe for blood feeding (5), plasma bag (4) in order from the original blood bag (1) side A capture and separation circuit for transferring blood components ;
From the plasma back (4) side, blood components are transferred in the order of the bypass circuit (b) that does not go through the red blood cell trap (3), the white blood cell trap (2), and the recovery bag (7) to concentrate the white blood cell fraction. A leukocyte fraction collection device equipped with a circuit to collect,
The leukocyte trap (2) includes a leukocyte trapping filter in which a large number of micropores having a diameter of 2 to 7.8 μm are formed,
The red blood cell capture device (3) is a leukocyte fraction collection device comprising a red blood cell capture filter which is a fine hole through which red blood cells cannot pass and which allows plasma components to pass through.
6 . 1. The leukocyte fraction collection process from raw blood is composed of a closed system. ~ 5 . The leukocyte fraction collection device according to any one of the above.
7 . 3. In the system constituting the leukocyte fraction collection circuit, an auxiliary port is provided between the plasma bag (4) and the leukocyte trap (2) . Or 5. Leukocyte recovery apparatus according to.
8 . 1. The raw blood to be processed is 20 to 150 cc. ~ 7 . The leukocyte fraction collection device according to any one of the above.
9 . 1. The blood volume including the collected leukocyte fraction is 5 to 50 cc. ~ 8 . The leukocyte fraction collection device according to any one of the above.
10 . The suction speed in the filtration step is 0.01 to 4.0 ml / cm ² / min. ~ 9 . The leukocyte fraction collection device according to any one of the above.
11 . 1. The blood to be treated is any one of umbilical cord blood, bone marrow fluid, and peripheral blood. ~ 10 . The leukocyte fraction collection device according to any one of the above.

The present invention mainly has the following effects by having the above features.
(1) A concentrated leukocyte fraction component containing nucleated cells can be collected by attaching the collected raw blood and operating a syringe. This device depends on mechanical operations such as filter diameter and syringe speed, and does not require sensation such as interface discrimination. Basically, it can be closed, so there is little risk of contamination. It can be processed in a short time without the need for specific gravity separation or centrifugation. Since centrifugation and gravity are not used, operations such as upside down are unnecessary, and this is a space-saving device.
(2) Since it can be processed in a closed system without requiring skill, it can be installed in hospitals such as obstetrics and gynecology. Since the concentrated leukocyte fraction can be collected in a short period of time from the collection of the original blood and stored in a frozen state, it can be stored without reducing the activity of the stored leukocyte fraction.
(3) The present invention can provide a technique for easily concentrating nucleated cell components including stem cells in a short time from blood without using specific gravity difference sorting by using a filter having micropores. In particular, utilizing the deformability of erythrocytes, the remaining nucleated cell fraction can be recovered by passing erythrocytes, plasma components, and the like using a filter having a pore size smaller than that of erythrocytes. The recovered liquid can be easily recovered by making it flow backward.
(4) Since filtration is performed using a pore filter, it is not necessary to manually add an erythrocyte sedimentation agent or to select an unclear layer fraction. Basically, nucleated cell components along the diameter of the micropores of the filter can be obtained, and the quality can be made constant.
(5) Since the time required for the centrifugation operation and the stabilization of the layer separation is unnecessary, the preparation for separation and recovery can be completed in a short time. Since separation and preparation can be performed in a short time from collection of blood such as umbilical cord blood, the risk of deterioration of blood components such as contamination can be suppressed.
(6) Concentration accuracy can be easily controlled by suction or changing the pore size.
(7) The device can be operated without being a skilled engineer.
(8) Since a fraction enriched with nucleated cell components of constant quality can be obtained in a short time, it can be put to practical use at low cost.

The figure which shows an example of a leukocyte fraction collection device The figure which shows the outline of the concentration process of a leukocyte fraction collection apparatus. The figure which shows the other example of a leukocyte fraction collection apparatus.

  The present invention is an apparatus for easily concentrating nucleated cell components including stem cells in a short time from blood without using specific gravity difference sorting by using a filter having micropores. In particular, it is an invention that utilizes the deformability of red blood cells to pass red blood cells and plasma components, etc., using a filter having a pore size smaller than that of red blood cells, and collects the remaining nucleated cell fraction. Unlike the method of collecting the leukocyte-rich layer by centrifuging and collecting the leukocyte-rich layer, or the method of collecting the leukocyte component by adsorbing the leukocyte component to the non-woven fabric, The image can be collected. In collecting the nucleated cell component which is a non-filtrate, it is collected by reverse flow using plasma or the like from which red blood cells have been removed. In the present specification, nucleated cell components including stem cells may be referred to as “white blood cells” for convenience.

The leukocyte fraction collection device configuration for concentrating and collecting the leukocyte fraction of the present invention comprises a raw blood bag / collection bag and a plasma bag containing the collected raw blood, and a leukocyte capture device and a red blood cell capture device in order from the original blood bag side. Are equipped with a main circuit (a) interposed between both bags. A bypass circuit (b) that does not go through the red blood cell trap is also provided. The device is provided with a syringe for blood feeding. The leukocyte trap is provided with a filter membrane in which a large number of micropores are formed, and the micropores are provided with a leukocyte capture filter having a pore size smaller than the average diameter of red blood cells, which is a size through which red blood cells can be deformed and passed. ing. The red blood cell trap is a micropore through which red blood cells cannot pass, and includes a red blood cell trapping filter that allows plasma components to pass through. A valve is provided at a portion connecting these components.
A blood cell separation system circuit for separating a white blood cell fraction, a red blood cell fraction, and plasma from the original blood is constituted by the path of the plasma back-white blood cell trap-red blood cell trap-plasma bag.
A white blood cell fraction collection system circuit for collecting white blood cells by backwashing using plasma is constituted by the path of plasma back-leukocyte capture device-recovery bag.
A syringe is provided in the circuit as a means for transferring blood in these two systems.
Since the syringe is used as a drive source, the leukocyte fraction collection device is a stationary type, and the arrangement direction of the constituent devices is not limited. Posture change such as upside down is unnecessary.

  Examples of blood to be treated include bone marrow fluid, peripheral blood, and umbilical cord blood.

The apparatus of the present invention is characterized in that, in the filtration step, red blood cells, plasma components, and the like are passed using a filter having a pore size smaller than that of red blood cells, utilizing the deformability of red blood cells. In order to utilize the deformability of erythrocytes, the suction speed in the filtration step is preferably in the range of 0.01 to 4.0 ml / cm ² / min.

<Leukocyte trap>
The leukocyte trap has a membrane filter having a small container shape and provided with micropores, and functions to temporarily store leukocyte fractions and send leukocytes to the collection bag by backwashing. In the present invention, the filter has a processing capacity of several tens ml or more of blood such as umbilical cord blood.
One or a plurality of the membrane filters provided with the fine holes can be used. Separate the red and white blood cells roughly. As a result, white blood cells are concentrated.
Since erythrocytes can be deformed in a short time, they can pass through pores smaller than erythrocytes. White blood cells also have deformability, but require more time than red blood cell deformation. The present invention focuses on the difference in deformability between red blood cells and white blood cells, and allows only red blood cells to pass through and collects without passing white blood cells.
The membrane used for the separation is a filter having a structure in which a large number of pores having a uniform pore diameter are opened in parallel. The pore diameter is smaller than that of white blood cells and is preferably smaller than that of red blood cells. Since human red blood cells are 7.8 μm, it is preferably less than 8.0 μm. Considering the size and deformability of red blood cells, the minimum pore diameter of the micropores provided in the filter is about 2 μm. Experimentally, it was confirmed that red blood cells can be passed even at 3 μm and leukocytes can be trapped. More preferably, the diameter of the micropores provided in the filter is 5 μm or more and less than 8.0 μm.
When the micropore filter is formed into a multilayer, it is designed to have micropores that can pass the erythrocyte component as a whole. However, in many cases, three or less sheets will function sufficiently.
The size of the filter can be determined in consideration of the blood volume to be processed and the processing speed. The area of the leukocyte capture filter is appropriately designed from the range of 1 to 1000 cm ² .
The suction speed in the filtration step of the leukocyte capture filter is preferably in the range of 0.01 to 4.0 ml / cm ² / min. In order to realize the filtration speed of the leukocyte capture filter, it is determined by the size of the micropore, the area of the filter, and the operation speed of the syringe.

Examples of the material for the filter include those preferable in terms of low moldability, sterility, and cytotoxicity. Synthesis of polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, polyvinylidene fluoride, polyethersulfone, polytetrafluoroethylene, polyvinyl chloride, polyglycolic acid, polylactic acid, polycaprolactone, etc. Examples thereof include polymers such as polymers, cellulose acetate, regenerated cellulose, and cellulose mixed esters, and metals such as stainless steel, silver, and titanium.
In the processing of fine holes in the filter, uniform film holes can be formed by a drilling operation using a laser beam. Multi-layer design of film thickness and filter is easy.

In addition, since the conventional non-woven fabric is thick and entangled in the gap, it adheres to the non-woven fabric fiber, and there are many recovery residues and there is a limit to the recovery rate. In addition, since leukocytes are activated by contacting foreign substances in the body to protect and protect the living body, increasing the chance that leukocytes come into contact with the non-woven fibers increases the risk of activation and is collected. There is also a risk that the white blood cells that have been used will deteriorate to a state where they cannot be used.
The structure of the membrane filter used in the present invention is greatly different from that of a “cotton-like” filter using a nonwoven fabric conventionally used as a leukocyte removal filter. A filter having a structure in which pores are opened in parallel has been used for a limited purpose in the medical field as a “filter for measuring red blood cell deformability”. It has also been used as a “plasma separation filter” for extracting only plasma components from blood. However, there is no report as “a filter for separating red blood cells and white blood cells and collecting white blood cells”. There is an example that has been used for “measurement of erythrocyte deformability”, but it is not known to use it for collection without passing white blood cells. In addition, the blood volume used in the “red blood cell deformability measurement” is about 1 ml. In the present invention, the filter is required to be able to process tens of ml or more of blood such as umbilical cord blood.

<Red blood cell trap>
The erythrocyte trap has a function of capturing a erythrocyte fraction from components that have passed through the leukocyte trap and allowing plasma components to pass through. The red blood cell trap is provided with a filter. The filter can also be used as a membrane filter having fine pores or a filter made of nonwoven fabric. Since the red blood cells captured here are discarded, the filter type is not limited, but it is designed in consideration of the processing time. The erythrocyte separation filter uses micropores having a diameter of 2.0 μm or less that are difficult to pass even if the erythrocytes are deformed. For example, about 0.2 to 0.6 μm is suitable.

<Original blood bag>
The raw blood bag is a bag containing unadjusted whole component blood collected from the umbilical cord or the like. This bag is made of a synthetic resin that is flexible and free to contract / expand. What is used for the existing blood bag can be used. A bag-like bag that is free to shrink / expand does not hinder the movement of the blood being processed. The same applies to the plasma bag and the recovery bag. In a rigid box-shaped container, it is necessary to suck and discharge air as blood components enter and leave, and it is difficult to perform a processing operation in a closed system.
Blood is introduced from the original blood bag to the entrance side of the leukocyte trap. The emptied raw blood bag can be used as a bag for collecting the leukocyte fraction collected by backwashing.

<Plasma bag>
The plasma bag is a bag that stores plasma components that have passed through the red blood cell trap. This bag is made of a flexible synthetic resin. What is used for the existing blood bag can be used. Alternatively, the syringe can have a plasma back function.
This stored plasma component bypasses the red blood cell trap and is introduced from the outlet side of the leukocyte trap, and is used as a backwash liquid component that sends the white blood cell fraction to the collection bag.

<Recovery bag>
The collection bag is a bag that contains a leukocyte fraction collected by backwashing, and is made of a flexible synthetic resin. What is used for the existing blood bag can be used. Similar to the original blood bag, a flexible bag is suitable. The raw blood bag can also be used. Rigid plastic containers and glass containers can also be used. The collected leukocyte component is frozen and stored after necessary post-treatment.
The attachment position of the recovery bag can be exchanged with the original blood bag. Alternatively, it can be provided immediately downstream of the original blood bag.

<Syringe>
In the present invention, the syringe serves as the drive source for the system. The syringe exhibits a suction or pressurization function with respect to the filtering action of the filter depending on the arrangement position. When placed on the plasma back side in the circuit, it performs a suction function. When placed before the leukocyte trap, it performs a pressurizing function.
When the syringe is moved backward, the blood component once flows into the cylinder. When the piston is advanced by controlling the discharge direction by operating the valve, the blood component is sent to the next step. When the amount of blood to be processed is large, the piston is advanced and retracted in several steps.
The processing time of this apparatus can be controlled by the advance / retreat speed of the piston of the syringe. Since the operation can be performed mechanically, it can be automatically set. Alternatively, a suction or pressurization load can be made constant by using a weight.
The filtration speed of the leukocyte capture filter is determined by the size of the micropores and the area of the filter. Since the suction speed of this filtration step is set in the range of 0.01 to 4.0 ml / cm ² / min, it is set to the operation of the syringe that realizes this.
It is also possible to arrange a syringe at the position of the plasma bag to also serve as a temporary plasma storage function.
Although the capacity | capacitance of a syringe is arbitrary, when processing cord blood, it is about 30-200 ml.

<Circuit>
The circuit connects components using tubes and pipes. A valve is interposed in a portion where the direction of blood flow needs to be changed or the flow rate needs to be controlled. A tube used in a medical device can be used.

<Recovered blood>
The blood collected in the collection bag is mainly composed of a leukocyte fraction containing nucleated cells contained in the original blood. Currently, umbilical cord blood is administered using leukocyte-enriched blood containing leukocyte component as an active ingredient and plasma or physiological saline. The red blood cell component is unnecessary, but it is inevitable that it is mixed. In the present invention, the main purpose is to completely capture the leukocyte component with the leukocyte trapping device, and it is not necessary to pass all red blood cells. Since the current cryopreservation capacity is 20 to 30 ml, it is a practical design range in which more leukocyte fractions are collected and converged in this cryopreservation capacity in a state where plasma and red blood cells are mixed.

<Variation>
In the treatment process, when a situation where the recovery process using only the plasma of the filtrate becomes impossible due to some factor occurs, a container or a syringe containing the recovered liquid is connected to the auxiliary port 6 and the recovery process is performed. It is also possible to collect the leukocyte fraction by feeding the collection solution using a syringe. As the recovery solution, a solution with little cell degeneration, for example, a buffer solution such as physiological saline, Dulbecco's phosphate buffer (D-PBS), Hanks' solution or the like can be used. Moreover, in order to collect | recover efficiently, the collection | recovery liquid which has a fixed blood osmotic pressure may be employ | adopted, and dextran etc. may be added.

[Example 1 of leukocyte fraction collection device]
1 and 2 show Example 1 of the leukocyte fraction collection device (10).
According to the direction shown in the drawing, the original blood bag (1) is placed at the left end and the plasma bag (4) is placed at the right end, and the leukocyte capture device (2), red blood cell capture device (3), and syringe (5) are placed in the middle from the left side. It is arranged. Each component device is connected by a tube to constitute the main circuit (a). Further, a bypass circuit (b) that bypasses the red blood cell trap (3) is provided. Directional valves (21) to (24) whose flow paths can be changed at two joints of the bypass circuit (b) and the main circuit, joints of the syringe (5), and joints of the plasma bag (4). Is provided. In the example of this apparatus, the collection bag (7) is also used as the raw blood bag (1).

  FIG. 2 shows a leukocyte fraction concentration process using this apparatus. 2A shows a separation process using the main circuit (a), and FIG. 2B shows a leukocyte fraction collection process using the bypass circuit (b).

In the separation step shown in FIG. 2 (a), the plasma component is transferred from the original blood bag (1) to the plasma bag (4) via two kinds of blood cell fraction traps (2) (3) and a syringe (5). A circuit for separating the white blood cell fraction, the red blood cell fraction and the plasma using the system of the main circuit to be collected is used.
The syringe (5) is pulled at a constant speed, the collected blood contained in the original blood bag (1) is drawn, the leukocyte fraction C is captured by the leukocyte trap (2), and then the erythrocyte content is collected. The fraction D is captured by the red blood cell trap (3), and the plasma component B is sucked into the syringe (5) and filtered. The flow path of the valve (23) is changed to the plasma bag (4) side, the syringe (5) and the plasma bag (4) are communicated, and the filtrate B in the syringe (5) is sent to the plasma bag (4). This process is repeated until all the raw blood has been filtered. At this time, if the amount of blood to be processed is small and only one operation is required, the syringe (5) can also serve as the plasma bag (4). Moreover, even if a throughput increases by making a syringe (5) into a some syringe, a syringe can serve as a plasma bag.

In the leukocyte fraction collection step shown in FIG. 2 (b), the plasma B stored in the plasma bag (4) is once sucked into the syringe (5), and the leukocyte trap (2) is passed through the bypass circuit (b). A collection circuit for backwashing and sending the leukocyte fraction C to the collection bag (7) is used.
The syringe (5) is pulled at a constant speed, the plasma B, which is the filtrate contained in the plasma bag (4), is drawn out, sent to the bypass circuit (b), and enters the leukocyte trap (2) from the outlet side. The captured leukocyte fraction C is backwashed, and the leukocyte fraction C is sent together with plasma B to the collection bag (7) to concentrate and collect leukocyte components. In the illustrated example, the collection bag (7) uses the empty raw blood bag (1).
The syringe (5) can also be recovered by operating it multiple times. Since the frozen storage volume in practical use is about 25 ml, the syringe can be operated once with about 30 ml.
The auxiliary port 6 is used when a physiological saline or the like is used as a cleaning liquid for backwashing.

[Example 2 of leukocyte fraction collection device]
FIG. 3 shows Example 2 of the leukocyte fraction collection device (10).
The original blood bag (1) is placed at the left end and the plasma bag (4) is placed at the right end, and the syringe (8), leukocyte trap (2), erythrocyte trap (3), syringe ( The equipment of 5) is arranged. The difference from Example 1 is that the syringe (8) is provided between the original blood bag (1) and the leukocyte trap (2).
In this apparatus, in the step of separating the leukocyte fraction, the red blood cell fraction and the plasma, first, the blood contained in the original blood bag (1) is once sucked into the syringe (8) and then the leukocyte trap ( It is a point which can employ | adopt the system of the pressure filtration supplied to 2).
Moreover, by using the syringe (8) and the syringe (5) properly, it is possible to select the pressure treatment or the negative pressure treatment for both the separation step and the recovery step, and the degree of freedom of operation can be made depending on the nature of the target blood and the filter. .

The Example using the apparatus structure of FIG. 1 is shown. The apparatus configuration is as described above.
Raw blood was collected from humans. Heparin was used as an anticoagulant to prevent blood from coagulating inside the instrument during blood collection or blood treatment.

[Human blood cell size]
Red blood cell: 7.8 μm
White blood cells ・ Granulocytes: 12-15μm
Monocyte: 12 (15) to 20 μm
・ Lymphocyte small size 6-8μm
Large size 9-12μm
(See “Tissue Cell Biology” by Yasuo Uchiyama, Nanedo, p147-154)

  As the parts of the device, the original blood bag (1) and the plasma bag (4) are paralleled with Nipro bag CBC-20 (manufactured by Nipro Co., Ltd.) and the filter used for the leukocyte trap (2). Two polycarbonate films (isopore filter (registered trademark) manufactured by Millipore Corporation, diameter 47 mm) having a structure opened in parallel were used in parallel. As this filter, four types of filters having pore sizes of 3 μm, 5 μm, 8 μm, and 10 μm were used. As a filter used for the red blood cell trap (3), a glass fiber AP filter (90 mm in diameter) manufactured by Millipore was used. 30 ml of SS-30ESZ (manufactured by Terumo, 30 mL) was used for the syringe (5).

  About 80 ml of human blood was placed in the original blood bag (1), and the blood (about 60 to 75 ml) was filtered using a syringe (5). The blood filtration rate at this time was 10 ml / min.

  After the filtration is completed, the bypass circuit (b) is opened to bypass the red blood cell trap (3), and the syringe (5) is used to remove the filtrate (plasma) B from the plasma bag (4). Plasma was passed through the collection bag (7) via (2), and the leukocyte-rich fraction D captured by the leukocyte trap (2) was collected. The volume of the collected blood was 20.0 ml. The time required for the process from the start of filtration to recovery was 8 minutes 47 seconds to 15 minutes 32 seconds.

  While measuring the volume of the original blood, filtrate, and collected blood, and measuring the concentration of red blood cells and white blood cells in each solution with an automatic blood cell counter (Celltac MEK-5258, Nihon Kohden Industry), The number of contained red blood cells and white blood cells was calculated.

The following indices were used for evaluation of blood cell recovery.
(1) White blood cell recovery rate A
A = (white blood cell count a1 in collected blood) / (original white blood cell count a2)
(2) Red blood cell recovery rate B
B = (white blood cell count b1 in the collected blood) / (original white blood cell count b2)

[Test results]
The results of a concentration test using four types of filters (pore diameters 3, 5, 8, 10 μm) as the filters used in the leukocyte trap (2) will be described. The leukocyte collection rate A was approximately 100% when filters with a pore size of 3 μm and 5 μm were used, whereas the recovery rate with the increase in the amount of filtrate was obtained with the 8 μm and 10 μm filters. Decreased. The recovery rate of leukocytes is preferably less than 8 μm. It can be seen that a pore diameter of 3 to 8 μm is suitable in this test.

  The red blood cell recovery rate B decreased as the filtrate amount increased. The rate of this decrease was almost constant regardless of the pore size of the filter used in the leukocyte trap (2).

  It was observed that red blood cells were captured on the red blood cell separation filter.

  From the above results, it was shown that by using the apparatus of the present invention, it was possible to concentrate without substantially reducing the white blood cell count, and it was possible to significantly reduce the time required for the treatment as compared with the conventional method. That is, the device of the present invention achieves the concentration of leukocytes at a higher recovery rate than the conventional method, and can be processed in a short time with a simple operation.

DESCRIPTION OF SYMBOLS 1 Original blood bag 2 Leukocyte capture device 3 Red blood cell capture device 4 Plasma bag 5 Syringe 6 Auxiliary port 7 Blood collection bag 8 Syringe 10 Leukocyte fraction collection device 21-25 Valve a Tube b Bypass tube A Blood B Filtrate (plasma)
C Leukocyte rich fraction D Rich red blood cell fraction E Concentrated blood

Claims (11)

The original blood backed (1) / collecting back (7) and plasma backed (4), two types of blood cells fraction of the original blood back (1) side leukocyte trap in order from (2) and erythrocyte trap (3) The image capturing device includes a main circuit (a) disposed between both backs,
A bypass circuit (b) that does not go through the red blood cell trap (3), and
A syringe (5) for blood feeding is provided between the original blood bag (1) / collection bag (7) and the leukocyte trap (2) or / and between the red blood cell trap (3) and the plasma bag (4). A leukocyte fraction collection device (10) for concentrating and collecting a leukocyte fraction from the obtained raw blood,
Leukocyte trap (2) is provided with a white blood cell capture filter a large number of micropores of diameter 2~7.8μm is formed,
The red blood cell capture device (3) is a leukocyte fraction collection device comprising a red blood cell capture filter which is a fine hole through which red blood cells cannot pass and which allows plasma components to pass through. Leukocyte trapping filter pore size, leukocyte recovery apparatus according to claim 1, characterized in that the. 5 to 7.8 [mu] m. The leukocyte fraction collection device according to claim 1 or 2 , wherein the leukocyte capture filter has an area of 1-1000 cm ² . The syringe (5) for blood feeding is arranged on the plasma back (4) side from the bypass circuit,
White blood Tamato捉器from the original blood back (1) (2), red blood cell trap (3), the main circuit for recovering plasma component via the order of the syringe (5) in the plasma back (4) (a) The blood cell separation circuit used,
A leukocyte fraction collection circuit for collecting and collecting leukocyte fractions containing plasma in the collection bag (7) from the plasma bag (4) through the syringe (5), bypass circuit (b) and leukocyte trap (2) in this order. The leukocyte fraction collection device according to any one of claims 1 to 3 , wherein: Original blood bag (1) / collection bag (7), plasma bag (4), syringe (8) for blood feeding, leukocyte trap (2), red blood cell trap ( 3), a blood-feeding syringe (5) and a plasma bag (4) are arranged in this order,
Syringe for blood feeding (8), leukocyte capturing device (2), red blood cell capturing device (3), syringe for blood feeding (5), plasma bag (4) in order from the original blood bag (1) side A capture and separation circuit for transferring blood components;
From the plasma back (4) side, blood components are transferred in the order of the bypass circuit (b) that does not go through the red blood cell trap (3), the white blood cell trap (2), and the recovery bag (7) to concentrate the white blood cell fraction. A leukocyte fraction collection device equipped with a circuit to collect,
The leukocyte trap (2) includes a leukocyte trapping filter in which a large number of micropores having a diameter of 2 to 7.8 μm are formed,
The red blood cell capture device (3) is a leukocyte fraction collection device comprising a red blood cell capture filter which is a fine hole through which red blood cells cannot pass and which allows plasma components to pass through. The leukocyte fraction collection apparatus according to any one of claims 1 to 5 , wherein the process of collecting leukocyte fraction from raw blood is constituted by a closed system. In a system which constitutes the leukocyte recovery circuit, leukocyte recovery apparatus according further comprising an auxiliary port to claim 4 or 5, characterized in between the white blood cell trap (2) from the plasma back (4) . The leukocyte fraction collection device according to any one of claims 1 to 7 , wherein the raw blood to be treated is 20 to 150 cc. The leukocyte fraction collection device according to any one of claims 1 to 8 , wherein the blood volume including the collected leukocyte fraction is 5 to 50 cc. The leukocyte fraction collection device according to any one of claims 1 to 9 , wherein a suction speed in the filtration step is 0.01 to 4.0 ml / cm ² / min. The leukocyte fraction collection device according to any one of claims 1 to 10 , wherein the blood to be treated is any one of umbilical cord blood, bone marrow fluid, and peripheral blood.