EP0017127B1

EP0017127B1 - A method for separating blood and a barrier device therefor

Info

Publication number: EP0017127B1
Application number: EP19800101547
Authority: EP
Inventors: Tatsuhiko Ikeda; Sohichiro Terada
Original assignee: Terumo Corp
Current assignee: Terumo Corp
Priority date: 1979-03-23
Filing date: 1980-03-24
Publication date: 1985-01-23
Also published as: EP0017127A2; AU5678680A; EP0017127A3; AU542204B2; DE3069996D1

Description

This invention relates to a method for separating blood collected in a blood-collecting tube into a serum part and a solid component part by centrifugation, as defined in the preamble of claim 1. Further, this invention relates to a barrier used for such method.
In a blood test, blood is generally separated by centrifugation into serum and cellular solid matters such as blood corpuscles, and only the serum is collected for analysis and examination. According to a well-known method for separating the serum, blood collected in a test tube is centrifuged, material such as gel material composed of silicone-silica which has an intermediate specific gravity between those of the serum and cellular solid matters is put in the test tube, the gel material is interposed between the serum and cellular matters by centrifugation, and the serum is separated by decantation. In this case, however, it is difficult to perfectly prevent fibrin and other solid matters from being mixed in the serum.
Such mixing of blood corpuscles, fibrin, etc. in the serum is undesirable because it may cause clogging of instrument nozzles as well as errors in measurement.
Accordingly, as a blood separator capable of preventing such mixing in the serum, there is proposed a piston member in which a solid weight for specific gravity adjustment is coupled with a flexible filter member which is large enough to be in slidable contact with the inside wall of a blood-collecting tube, and having a specific gravity of from 1.03 to 1.09 as a whole is inserted in the blood-collecting tube (United States Patent No. 3,931,018). Formed of two submembers with different specific gravities, porous and solid submembers that are bonded together, the piston member is not an entirely satisfactory structure, requiring much labor in manufacture.
From US-A-3 972 812 a porous barrier is known having a pore size of about 70 to 300 ,um. Further, in US-A-3 960 727 a filter member is described having pore sizes of ca. 50 p or less. However, both documents disclose only columnar disc-like barriers having the same diameters at both lower and upper portions. In US-A-3,972,812 the disc-like barrier is thin, so the resistance of blood clot passing through the barrier is low. Further, the barrier has a smaller diameter than the blood tube, so the separated serum is apt to mix with blood corpuscles through a gap along the side surface thereof. Therefore, it is necessary to control the specific gravity precisely, and to coat the barrier with a hydrophobic film.
Further, US-A-3,931,018 discloses a columnar disc-like barrier having the same diameter at both lower and upper portions and having a mass attached to the disc by a bolt. This mass serves to stabilize the barrier and to prevent tumbling. However, this barrier is difficult to manufacture.
The invention as claimed is intended to provide a remedy by a method for separating blood, and a device therefore capable of simplifying manufacture and reducing production costs without any possibility of causing blood cells, fibrin, and other solid matters to be mixed with serum.
According to the present invention, this aim is achieved with the characterizing features of claim 1.
Further, according to the invention, there is provided a barrier for centrifugation of blood to be introduced into a blood-collecting tube and having an overal true specific gravity greater than that of serum, comprising an elastic porous member which is characterized in that said elastic porous member has a porosity of 40% or more, a continuous-pore size of 50 to 400%, the pores being thermally treated for dissolving a filmy material formed around the pores in the foaming step of fabrication thereof, and a cross-section at an upper portion of the barrier coming into contact with the inside wall of the tube which is substantially larger than the cross-section at a lower portion thereof.
The barrier according to the present invention contacts the inner wall of the tube at the upper portion and does not contact that of the tube at the lower portion, and has larger contact surface with blood. Further, in a preferred embodiment, said elastic porous member is formed in the shape of a truncated cone and is bottomed with a hard layer, and the overall specific gravity of said elastic porous member including said hard layer is greater than that of said serum part. In the preferred embodiment, the center of gravity moves to the bottom, whereby ensuring the descending movement of the barrier, and causing it to move stably during centrifugation.
Preferred ways of carrying out the invention are described in detail below with reference to drawings, in which:

Figure 1 is a sectional view of a blood separator according to one embodiment of the invention wherein a barrier is disposed in a vacuum blood-collecting tube in advance;
Figure 2 is a perspective view illustrating the shape of a barrier for use in a blood separation;
Figures 3 to 5 are sectional views showing several modifications of the barrier;
Figure 6(A) is a perspective exploded view of the barrier in combination with a tube member;
Figure 6(B) is a sectional view showing the members of Figure 6(A) in their assembled state;
Figure 7 is a sectional view showing another modification of the barrier of the invention;
Figure 8 is a perspective view showing still another modification of the barrier; and
Figure 9 is a sectional view as taken along line A-A of Figure 8.

One point of this invention resides in that an elastic member with continuous pores of a specified size is used with a blood collecting tube. Another point of the invention is that, although the true specific gravity of the barrier formed of such elastic member need be greater than that of serum it need not always be smaller than that of the solid-phase part of the blood in separating the serum, unless hemolysis is caused. This may be attributed to the fact the whole or principal part of the barrier of the invention, being a porous member, may have extremely small mass (e.g. 100 to 300 mg).
The elastic porous member constituting at least the principal part of the barrier of the invention may be formed of elastic plastics foam, such as polyurethane foam, rubber foam (e.g. silicone rubber latex), polyvinyl chloride foam, polyformal resin, having porosity of 40% or more, preferably 97 to 98%, and a continuous-pore size of 50 to 400 µm, preferably 250 to 400 µm. If porosity and pore size are smaller than those as specified, the isolation of the serum would not be obtained in the ordinary centrifugal operation of 1000 to 1200 G for 10 minutes. And centrifugation under the condition exceeding the above condition of the centrifugal operation would give rise to hemolysis. A pore size of more than 400 µm is not desirable, since blood corpuscles would pass through a foam of such a large pore size, thereby contaminating the serum phase obtained.
In this case, the 25% compressive hardness (JIS K-6401 Tst Method established in 1974) of the barrier should preferably be 5 to 150 kg/cm². Moreover, it is expressly desirable that the barrier of the invention should be hydrophilic by nature or be made hydrophilic by some treatment for hydrophilicity. Such hydrophilic property is preferred because it will enable the serum to quickly penetrate the pores when the barrier is brought in contact with the blood, thereby facilitating the movement of the barrier.
Elastic porous no-woven cloth may also be useful as far as the pores thereof substantially meet the above conditions.
The overall specific gravity of the barrier should preferably be adjusted to 1.2 or more, more preferably to from 1.2 to 1.4.
The barrier may be of any shape as long as at least an upper part of the barrier has a cross-section a little larger than that of a blood-collecting tube for centrifugation used with the barrier and the lower part of the barrier has a cross-section a little smaller than that of the blood-collecting tube, so that the outer periphery of the large-diameter portion of the barrier may rub against the inside wall of the tube during centrifugation. According to this invention, as described above, a single elastic porous member can be directly used for the barrier. Alternatively, however, the outer peripheral portion of the barrier may be coated with silicone, or two or more elastic porous members may be combined with one another or with other materials. For example, a tube member with the outside diameter somewhat smaller than the inside diameter of the blood-collecting tube used, e.g. a plastic tube, may be fitted on the lower peripheral surface of a columnar or cylindrical barrier so as to reduce the area of contact and hence the frictional resistance between the barrier and the inside wall of the blood-collecting tube, thereby facilitating the sliding movement of the barrier during centrifugation. The tube member may be formed of any thermally contractive material, such as polyolefin, polyvinyl chloride, nylon, polyester, polycarbonate, polyurethane or ethylenevinyl acetate copolymer.
As another modified example, there may be used a columnar elastic porous member in the form of e.g. a truncated cone which has cross- sections substantially larger and smaller than that of the interior of the blood-collecting tube used, at its upper and lower portion, respectively, and is bottomed with a solid or porous hard layer. The hard layer may be formed by impregnating relatively hard plastic into the bottom portion of the porous member and solidifying the plastic, or by glueing a solid or porous, relatively hard plastic sheet to the bottom portion. Having the hard bottom portion, the barrier of such construction exhibits extremely large deformation resistance during centrifugation, so that it may be prevented from turning sideways or being distorted while sliding down the tube thereby ensuring the descending movement of the barrier in a properly erected state during centrifugation. Furthermore, the shape of the final product may be obtained directly by stamping out a truncated- cone-shaped member after glueing a hard plastic sheet to one side of an elastic porous sheet or after impregnating a solution of hard plastic into the porous sheet to a predetermined thickness, so that the manufacture of the barrier may be simplified substantially, so as to permit for reduction in production cost.
In view of the yield of serum the volume of the barrier should be minimized. The porous member may be joined with the tube member, or hard plastic sheet by using adhesives, heat sealing or any other suitable means.
In combining the elastic porous member with the additional member, the materials and designs for these members should be selected so that a relationship
may be obtained where the volume and specific gravity of the elastic porous member are X and d respectively, the volume and specific gravity of the additional member are Y and d' respectively, and the overall specific gravity required is A.
Operations required for centrifugation the blood by means of the above-mentioned barrier are not essentially different from the conventional case. That is, the barrier is introduced into the blood-collecting tube before or after collecting the blood, the blood is centrifuged, and then the serum part is easily separated by decantation.
For the process of centrifuging blood serum by using the blood separator according to the invention, as shown in Figure 1, whole blood (not shown) is collected in a blood-collecting tube 21, a barrier 23 formed of an elastic porous member is fitted in the opening of the tube 21, and the tube 21 is set in a centrifugal separator for centrifugation. When the centrifugation is started, the barrier 23 is caused gradually to slide down the inside wall of the blood-collecting tube 21 toward the bottom of the tube 21 by centrifugal force. When the bottom end of the barrier 23 touches the surface of the blood, the serum is caused to penetrate into pores of the barrier 23 by capillarity. When centrifugation is continued, the pores of the barrier 23 are substantially filled with the serum and the barrier 23 is further moved down until it is finally held substantially midway between a serum layer and a solid component layer. In this case, solid constituents such as blood corpuscles and fibrin are trapped in the pores of the barrier 23 and will never be mixed with the serum. This is ensured because the solid constituents are retained in the continuous pores of the barrier 23 the framework of which has a complicated three-dimensional structure.
Thus, the barrier 23 slides relatively slowly down the inside wall of the blood-collecting tube 21 by its elasticity, so that blood corpuscles, fibrin, etc. stuck to the inside wall can be cleared or swept away substantially thoroughly. As a result, there may be obtained serum which does not contain blood corpuscles, fibrin or any other solid matters. The barrier 23 stopped at the interfacial position sticks fast to the inside wall of the blood-collecting tube 21 by its own elasticity, pressing against the inside wall, so that only the serum part can be separated by decantation.
The barrier of this invention may be inserted into the blood-collecting tube prior to centrifugation after blood collection, or otherwise be held in the tube beforehand. Figure 1 shows an example of the latter case. In Figure 1, a barrier 23 having an annular hard layer 27 on its bottom is held by a rubber stopper 24 within a vacuum blood-collecting tube 21 the inside of which is kept at a vacuum. That is, the rubber stopper 24 has a cavity 25 in the lower end, while the barrier 23 has on its top a truncated cone-shaped projection 26 with the outside diameter larger than the diameter of the cavity 25. The projection 26 is fitted and held in the cavity 25 so that the barrier 23 will not be removed from the rubber stopper 24 if the stopper 24 is pierced with a needle for blood collection.
Alternatively, there may be adopted any other suitable methods for previously fixing the barrier in the blood-collecting tube in connection with the shapes of the tube and the barrier itself. For example, a barrier may be fixed to one end of a blood-collecting tube sealed with a rubber stopper at each end, the one end being opposite to the blood intake side of the tube.
Figures 2 to 9 illustrate the respective shapes of several modifications of the barrier. A barrier (42) (Figure 2) with a pair of parallel annular flanges 41; a barrier 62 (Figure 3) similar to the barrier of Figure 2 but with a cavity 51 on one side thereof; a barrier 82 (Figure 4) tapered at the lower portion; a barrier (Figure 5) of the same structure of Figure 4 but with the cavity 51; a barrier formed by fitting a small-diameter tube member 100 on the lower peripheral surface of the columnar porous member 31 as shown in Figure 6(A) to restrict the lower portion of the porous member 31 as shown in Figure 6(B) so as to reduce the area of contact with the blood-collecting tube; a barrier (Figure 7) of the same structure of Figures 6(A) and 6(B) but with the cavity 51; and a barrier 112 formed by bonding a hard layer 11 to one small-diameter end of an elastic porous member 110 substantially in the form of a truncated cone as shown in Figures 8 and 9. The upper portion of the barrier 112, which is brought in close contact with the inside wall of the blood-collecting tube at centrifugation, preferably has a thickness of from 3 mm to 5 mm. Available materials for the hard layer 111 include plastics such as polyolefin, polyvinyl chloride, nylon, polyester, polycarbonate, and polyurethane, fluorine-contained polymers and other organic and inorganic substances. These materials should be hard and have a small contact resistance relative to the blood-collecting tube. Alternatively, hard layer may be porous such as mesh-like. The thickness of the hard layer preferably ranges from 0.1 mm to 5.0 mm, and more preferably from 0.1 mm to 1.0 mm.
Thus, the barrier shape may lend itself to various modifications. The point is that the barrier should have porosity, pore size, and apparent or real specific gravity within prescribed ranges, and be of such suitable size that an upper portion thereof may rub against the inside wall of the blood-collecting tube when it slides thereon during centrifugation.
According to this invention, as described above, the barrier, being a simple elastic porous member with or without a plastic tube member or a hard layer attached thereto, is so simple in construction that it can be manufactured very easily at reasonable cost. Since the elastic porous member transmits only the serum to be separated, there may be obtained pure serum containing no solid matters such as blood corpuscles and fibrin.
Below the invention is described in Examples.

Example 1

A test for separating serum from blood was conducted by using the barrier 31 shown in Figure 6. Polyurethane foam with a porosity of 98%, a pore size of 300 µm, a true specific gravity of 1.2, a 25% compressive hardness (based on KIS-K-6401 Test Method) of 20 kg/cm², and a number of barrier cells, i.e. the number of the pores on an optimal line at a cut surface when the barrier is cut, of approximately 75/25 mm was used for the barrier. Since the framework of the polyurethane foam has continuous pores of complicated three-dimensional structure and reduces the passage resistance of serum it had previously been removed by thermally dissolving filmy material formed around the pores at foaming, as described in Japanese Patent Publication No. 752/66 (January 25, 1966), and US-A-3 329 759, corresponding to Canadian Patent Nos. 737,836 and 870,716. According to these documents in a reticulated material the cell membranes of the cellular material are destroyed by rapidly raising the temperature of the membranes in a gaseous atmosphere under pressure to the destruction point (i.e. to at least the thermoplastic point) without destroying the strands of the skeletal structure and the skeletal structure remaining after destruction of the cell membranes is heat treated.
In this case, the tube 100 of 3 mm height, 12.2 mm inside diameter and 13.0 mm outside diameter was fitted on the lower portion of the columnar porous member 31 (polyurethane foam) of 13.7 mm diameter and 12 mm height. The tube 100 was made of polyethylene, and was provided at the bottom end with an abutment portion (not shown) to engage the bottom end of the porous member 31.
This barrier was inserted through the opening of the blood-collecting tube containing blood, which had been kept at normal temperature for 60 minutes, to a depth where the barrier touched the blood surface. After leaving the barrier to stand for a while, centrifugation was carried out under normal conditions so that the centrifugal force at the central portion of the blood-collecting tube might become approximately 1200 G. In this case, there was noticed no eduction of fibrin. The yield of serum proved to be approximately 4.0 ml.
Also, decantation caused neither shifting of the barrier nor mixing of blood corpuscles or fibrin.
The outside diameter of the tube 100 was smaller than the inside diameter of the blood-collecting tube, and the upper side wall of the porous member 31 was so designed as to form a slope. Therefore, the barrier touched the inside wall of the blood-collecting tube only at the opening portion thereof when it was fitted in the tube. Consequently, the barrier was never prevented from descending by the viscosity of blood sticking to the upper portion of the inside wall of the blood-collecting tube after being left to stand for a while.

Example 2

The barrier shown in Figure 7 was manufactured to obtain the same effect as the barrier of Example 1 and to maximize the yield of serum. The porous member 31 used was just the same as the porous member used in Example 1 in material, dimensions and shape, except that it was provided with the cavity 51 defined therein at the lower portion. Also, the tube 100 made of thermally contractive polyvinyl chloride was fitted on the lower portion of the porous member 31. The tube 100 measured about 13 _jMm in thickness, 12.0 mm in outside diameter, and 6 mm in height when it was fitted on the porous member 31. The bottom end of the tube 100 and the bottom joint part of the porous member 31 were bonded together at several portions by thermal fusion.
When the same test as in Example 1 was conducted by using this barrier, satisfactory yield (approx. 4.5 ml) of serum was obtained with quite the same effect.

Example 3

Serum separation was conducted in the same manner as Example 1 by using the barrier 112 consisting of the elastic porous member 110 which is formed of the same polyurethane foam of Example 1 and has the form of a truncated cone as shown in Figures 8 and 9, measuring 15.5 mm in diameter across the upper large-diameter section, 12.8 mm in diameter across the lower small-diameter section, and 9 mm in height, and the hard layer 111 which is formed of a hard polyvinyl chloride film of 200 µm thickness bonded to the bottom face of the porous member 110. As a result, serum with no fibrin or blood corpuscles mixed therein could be obtained by decantation.
In connection with this example, substantially the same results were obtained when serum separation was conducted in the same manner as aforesaid, except that the hard layer 111 was formed instead of the hard polyvinyl chloride film, by impregnating two-liquid polyurethane resin into the bottom portion of the porous member 110 to a thickness of approximately 1 mm and hardening the resin, or by bonding a polyester mesh (mesh size being 14, diameter of each strand 450 ,um and specific gravity 1.38, sold under trademark TB-15 by NBC Industries Ltd. in Japan) to the bottom face of the porous member 110.

Claims

1. A method for separating blood collected in a blood-collecting tube into a serum part and a solid component part by centrifugation, comprising the steps of introducing a barrier having an overall true specific gravity greater than that of said serum part and comprising an elastic porous member into an upper part of said blood-collecting tube; moving said elastic porous member to the interface between a serum part layer and a solid component layer in the blood by centrifugal force produced in centrifuging the blood; and separating the serum in the blood; characterized in that said elastic porous member has a porosity of 40% or more, a continuous-pore size of 50 to 400 micrometres, the pores being thermally treated for dissolving a filmy material formed around the pores in the foaming step of fabrication thereof, and a cross-section at an upper portion of the barrier coming into contact with the inside wall of the tube, and perpendicular to the axial direction thereof which is larger than that of said blood-collecting tube, and a cross-section at a lower portion thereof and perpendicular to the axial direction thereof which is smaller than that of the blood-collecting tube.

2. A method according to claim 1, wherein said elastic porous member is previously fixedly disposed in said blood-collecting tube kept at a vacuum, before the blood is collected in said blood-collecting tube.

3. A method according to claim 2, wherein the fixed position of said elastic porous member in said blood-collecting tube lies at one end of said tube on the blood intake side thereof.

4. A method according to claim 2, wherein the fixed position of said elastic porous member in said blood-collecting tube lies at the other end of said tube opposite to said blood intake side.

5. A method according to claim 1, wherein said elastic porous member is fitted in said blood-collecting tube after the blood is collected in said tube.

6. A method according to any one of claims 1 to 5, wherein a tube member having smaller outside diameter than the inside diameter of said blood-collecting tube is fitted on part of the peripheral side of said elastic porous member, the combination of said tube member and said elastic porous member having greater true specific gravity than that of said serum part.

7. A method according to any one of claims 1 to 5, wherein the true specific gravity of said elastic porous member is greater than that of said serum part and is also greater than that of the solid component layer in the blood to such a degree that hemolysis substantially does not occur to said solid component layer during centrifugation.

8. A method according to claim 6, wherein the true specific gravity of the combination of said tube member and said elastic porous member is greater than that of said serum part and is also greater than that of the solid component layer in the blood to such a degree that hemolysis substantially does not occur to said solid component layer during centrifugation.

9. A method according to claim 1, wherein said elastic porous member is formed in the shape of a truncated cone which is bottomed with a hard layer, and wherein the overall specific gravity of said elastic porous member including said hard layer is greater than that of said serum part.

10. A method according to claim 1, wherein the overall true specific gravity of said barrier is greater than that of blood corpuscles.

11. A barrier for centrifugation of blood to be introduced into a blood-collecting tube and having an overall true specific gravity greater than that of serum, comprising an elastic porous member (23, 31, 42, 62, 82, 110), characterized in that said elastic porous member has a porosity of 40% or more, a continuous-pore size of 50 to 400 micrometres, the pores being thermally treated for dissolving a filmy material formed around the pores in the foaming step of fabrication thereof, and a cross-section at an upper portion of the barrier coming into contact with the inside wall of the tube which is substantially larger than the cross-section at a lower portion thereof.

12. A barrier according to claim 11, wherein a tube member (100) having smaller outside diameter than the inside diameter of said blood-collecting tube (1) is fitted on part of the peripheral side of said elastic porous member (31), the combination of said tube member (100) and said elastic porous member (31) having a greater true specific gravity than that of said serum part.

13. A barrier according to claim 11, wherein said elastic porous member (42, 72) has one or more annular projections (41,71) formed on the peripheral surface thereof.

14. A barrier according to any one of claims 11 to 13, wherein said elastic porous member (3, 24, 31, 42, 52, 62, 72, 82, 92, 110) is made of elastic plastic foam.

15. A barrier according to claim 11, wherein said elastic porous member (82 or 110) is formed in the shape of a truncated cone and is bottomed with a hard layer (111), and wherein the overall specific gravity of said elastic porous member (82 or 110) including said hard layer (111) is greater than that of said serum part.

16. A barrier according to claim 15, wherein said hard layer (111) is formed of hard plastic which is impregnated into the bottom portion of said elastic porous member (e.g. 110) and solidified.

17. A barrier according to claim 15, wherein said hard layer (111) is formed of a hard plastic sheet which is put on the bottom surface of said elastic porous member (e.g. 110).

18. A barrier according to claim 15, wherein said hard layer (111) is formed of a hard plastic mesh which is put on the bottom surface of said elastic porous member (e.g. 110).