EP2798349A1

EP2798349A1 - Method for separating suspension components or colloid components, and device for separating suspension components or colloid components

Info

Publication number: EP2798349A1
Application number: EP12813006.9A
Authority: EP
Inventors: Manuel KEMMLER; Oliver POPPE
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 2011-12-29
Filing date: 2012-12-21
Publication date: 2014-11-05
Also published as: WO2013098256A1; US20140374360A1; DE102011122579A1

Abstract

The invention relates to a method and a device for separating suspension components or colloid components. A sample (11) is moved in at least one flow direction at at least one flow speed over a period of time in a line arrangement (8) with an axial extension until an axial separation of the components has occurred, said components subsequently being separated from one another.

Description

Process for separating suspension or colloid components and

Device for separating suspension or colloid components

The invention relates to a method for separating suspension or colloid components, in particular blood components. The invention further relates to a device for separating suspension or colloid components, in particular blood components.

WO 96/31 270 A1 discloses a method and a device for separating whole blood, wherein agglutinins are used to improve the separation.

DE 1 03 05 050 A1 discloses a test element and a method for blood tests, wherein a microfluidic channel structure is used for the flow transport of the blood sample, and liquid constituents are obtained from the blood sample. Valve elements serve to control the flow transport. Agglutination is provided in a reaction chamber. From EP 2 41 3 1 38 A2 a device and a method for separating components of blood are known, in particular blood cells are to be separated. For this purpose, a microfluidic device is used which has a separating device, preferably in the form of a membrane or another filter element. For pretreating the sample liquid prior to feeding to the separator, the sample liquid is treated with an agglutinating agent, thereby causing blood cell aggregation. DE 1 03 52 535 A1 discloses a method and an apparatus for separating blood plasma and white blood cells from the remaining blood or from cellular components of the blood. For this purpose, a microstructured separation device is provided with separation areas. The transport path of the liquid consists of a channel system with capillary dimensions, which flows through the liquid at a flow rate in a transport direction from an inlet to a collecting section. To form complexes, agglutinating substances are added to remove certain particles from the suspension. The liquid or the separated parts of the liquid are transported by capillary force and / or another comparable force. The particle transport of larger constituents is slowed down by geometric structures present in the line arrangement.

From the scientific article "High flow rate microfluidic device for blood plasma separation using a rangeoftemperatures" by Al Rodriguez-Villarreal, M. Arundell, M. Carmona et al., Lab Chip, 201 0, 10, 21 1 -21 9 discloses a method and apparatus for separating blood components based on separating blood components due to lateral separation of the blood components towards a constriction in a conduit arrangement. The yield of plasma of this process is about 4 percent in this prior art process.

From the scientific article "Passive Microfluidic Devices for Plasma Extraction of Human Blood" by E. Sollier, H. Rostaing, P. Pouteau et al., Sensors and Actuators B 1 41 (2009) 61 7-624, there are three methods and three devices for Separation of blood components known. The methods are based on microfiltration and centrifugation in microfluidic devices. The best of the methods disclosed here is the so-called "corner edge" Design in which whole blood is diluted by a factor of 20. The yield is less than 1 1 percent.

The requirements for the separation consist in the duration of the process, the amount of yield of blood components, the amount of dead volume, automatic performance with little manual actions, low production costs, variable sample size, size and robustness. The invention has for its object to provide a method and an apparatus for separating components in suspensions or colloids in residual particle constituents and a weiterzuverarbeitenden particle component, resulting in a relatively simple construction of the line arrangement by efficiently separating the components of a relatively small and variable amounts of a sample characterized relatively short time with low dead volume.

This object is achieved in a method for separating suspension or colloid components with the features of claim 1.

This object is achieved in a device for separating suspension or colloid components having the features of patent claim 5.

Due to the fact that, according to the invention, the separation of suspension or colloid constituents, in particular blood constituents, from a specimen containing agglutinated constituents is effected by a purely axial flow in a line arrangement until an axial separation of the agglutinated constituents and the remaining colloid or the remaining suspension is achieved, a relatively simple result Structure of the line arrangement, the one allows efficient separation with a relatively small amount of the sample.

The presented invention uses, as known from WO 96/31 270, so-called agglutinines. In the medical language is meant agglutination (Latin: agglutinare: tack) the sticking or clumping of, for example, cells, or of antibodies with antigens. By adding substances such as cationic polymers hexadimethrine bromide agglutination can be further enhanced, resulting in a faster process of separation in the present invention.

For example, whole blood, urine, saliva or culture medium serve as a sample. Agglutinated components are, for example, blood cells, microorganisms or viruses.

Examples of application are the separation of whole blood as suspension with anticoagulant in plasma and blood cells, separation of whole blood or suspension without anticoagulant in serum and blood cells. The piping has diameters in the millimeter range for sample volumes in the upper microliter range to ensure a certain axial length of the sample in the pipe. Too small amounts of sample can not be separated efficiently.

For this reason, for the separation of sample volumes from, for example, subjects with low sample volumes such as infants, diameters of the micron-order line arrangement are preferred. To minimize the cable length and the consequent space requirement, diameters in the centimeter range are preferred for samples in the milliliter range. For the separation of whole blood, urine, saliva, culture medium components, the channel arrangement is separated for reasons of hygiene in a fluidic chip. The arrangement is largely closed and ensures a low risk of contamination for the user of the device. For a particularly cost-effective reaction, the separation in a tube solution is preferred. For putting in the hose solution is preferred for the separation of less risky material.

Further expedient embodiments of the invention are the subject of the dependent claims. Further expedient refinements and advantages of the invention will become apparent from the following description of exemplary embodiments with reference to the figures of the drawing.

Show it:

1 is a schematic view of a first embodiment of the invention with a line arrangement formed by a branch-free line,

Fig. 2 is a schematic representation of the line in the

Embodiment of FIG. 1 during various steps in carrying out the method according to the invention,

3 shows a schematic representation of a further embodiment of a device according to the invention with a line arrangement designed as a fluidic chip,

4 shows a top view of the fluidic chip in the embodiment according to FIG. 3 after filling with inert fluid solution and with a sample containing agglutinated blood cells, FIG.

5 is a plan view of the fluidic chip according to the Ausfüh- 4 after moving the sample in a first flow direction at a first flow rate over a first period of time and

6 shows a plan view of the fluidic chip according to FIG. 4 after moving the sample in a second flow direction opposite the first flow direction at a second flow velocity different from the first flow velocity over a second time duration.

Fig. 1 shows a schematic representation of a first embodiment of a device according to the invention for separating in particular blood components. The exemplary embodiment according to FIG. 1 has a pump unit 1, which can be controlled via a control unit 2 for pumping in two pumping directions, each with adjustable pump power. Furthermore, the exemplary embodiment according to FIG. 1 is equipped with a two-way valve unit 3 as a valve unit, which is fluid-dynamically connected to an inert fluid container 5 via an inert fluid connection 4. In the inert fluid container 5, an inert fluid solution 6 is stored in the illustration according to FIG. 1. The stock solution 6 serves the purpose of providing the pumping unit 1 with an inert fluid in order to act hydraulically and not pneumatically.

Furthermore, the two-way valve unit 3 is provided with a line connection 7, to which in the embodiment according to FIG. 1 a line arrangement 8 in the form of a non-branching and perforation-free line 9 having a length in an axial direction is provided with at least sections , is preferably connected with a total consistent cross-section of, for example, about 1, 5 millimeters in diameter with a round cross-section. The line 9 is for a the largest possible extent in conjunction with a small footprint in the axial direction expediently coiled or spirally formed. The line connection 7 opposite end of the line 9 is connected to a supply / collecting container 1 1, here consisting of a collecting container and from a storage container, which, as shown in Fig. 1, on the one hand, the function of a storage container for an already with a Reagent for agglutinating blood cells staggered sample 1 1 and, on the other hand, fulfills the function of a collecting container, as explained in more detail below, for receiving blood constituents. For the agglutination of the blood cells, the reagent used is, for example, phytohemagglutinin (PHA-E), which belongs to the so-called lectins.

FIG. 2 shows, in various partial images (a), (b), (c) and (d), the conduit 9 in the exemplary embodiment according to FIG. 1 after carrying out various steps of the method according to the invention. In the uppermost part of FIG. 2 (a), the line 9 is filled with inert fluid solution 6 at its end facing the inert fluid connection 4. At its the storage / Auffangbehältnisanordnung 1 0 facing the end by cooperation of the pump unit 1, the control unit 2 and the two-way valve unit 3 receiving an air cushion 1 2 from the storage container now used storage / collecting container arrangement 1 0, the sample 1 1 with agglutinated blood cells with a Volume of, for example, 200 microliters has been introduced. Behind and in front of the sample 1 1 is the air cushion 1 second

After pumping Inertfluidlösung 6, pumping back the recorded inert fluid 6 with entrainment of the air cushion 1 2 and then picking up the sample 1 1 to adjust a first separation of blood components according to the upper middle partial image (b) of Fig. 2 with a very low flow rate is adjacent to the air cushion 1 2 adjacent a first approach of plasma 1 3 at the supply / Auffangbehältnisanordnung 1 0 opposite end of the line 9 before while a mixing volume 1 4 enriched with agglutinated blood cells is present at the end facing the supply / collecting container arrangement 10. Through this process, the agglutination process is enhanced at this point.

After a relatively short first pumping break, which enhances the agglutination of the components by sedimentation, starting from the arrangement according to the partial image (b) in a first pumping interval first in the direction of the inert fluid port 4 with a relatively high first flow rate in this embodiment over a first period of time in the direction of the inert fluid port 4 as the first flow direction, until the air cushion 1 2 is arranged near the inert fluid port 4. After completion of this first pumping operation, as shown in the lower middle part of the image (c), the plasma 1 3 now on the supply / collecting container arrangement 1 0 side facing the line 9 before.

After a further second pumping pause, the arrangement of plasma 1 3, which has already been enriched in volume relative to sub-image (b), and the amount of mixing volume 1 4 enriched with agglutinated blood cells is then carried out in a second pumping process over a second period of time with a second flow velocity which is smaller than the first flow velocity, is pumped back in the direction of the supply / collecting container arrangement 10 as the second flow direction until, as shown in the lower partial image (d), the plasma 1 3 has a now relatively large volume at the rats / collecting container arrangement 1 0 facing end of the line 9 adjacent. Separation of the blood components is now complete after a typical processing time of about 15 minutes and a plasma yield of typically between about 30 percent and about 40 percent, and by further pumping at a withdrawal rate, the plasma 1 3 is allowed to enter the reservoir / containment assembly 10 drive the collection container now used for subsequent processing from the line 9.

3 shows a schematic representation of a further embodiment of a device according to the invention for separating blood constituents, wherein in the exemplary embodiment according to FIG. 1 and in the exemplary embodiment according to FIG. 2 corresponding elements are provided with the same reference numerals and below some are not explained again in detail. The exemplary embodiment according to FIG. 3 has as a valve unit a multi-way valve unit 15, to which, in addition to the inert fluid container 5 filled with inert fluid solution 6, a storage container 1 filled with pure whole blood 16 as suspension or colloid, which is optionally mixed with an anticoagulant, is filled 7 of a storage container arrangement and a Agglutinierlösung 1 8 for agglutinating blood cells receiving further storage container 1 9 of the storage container arrangement each have a supply connection 20, 21 are connected.

Furthermore, the multiway valve unit 1 5 is equipped with a first outlet connection 22, with a second outlet connection 23 and with a third outlet connection 24, via the first outlet connection 22, via a two way valve unit 25 also controllable by the control unit 2 and via an outlet connection 26 Two-way valve unit 25 Inertfluidlösung 6 from the Inertfluidbehältnis 5 a multi-functional connection 27 of a fluidic chip 28, in which the line assembly 8 is formed, can be fed. The two-way valve unit 25 is also connected via a further outlet port 29 with a collecting container 30, with which, as explained in more detail below, separated blood components can be accommodated.

The second output port 23 is connected to the corresponding position of the multi-way valve unit 1 5 with the whole blood 1 receiving receiving container 1 1 7 and placed on a first feed connection 31 of the fluidic 28. Finally, the third output connection 24 of the multiway valve unit 15 is connected to the additional storage container 1 9 receiving the agglutinating solution 18 with a corresponding position of the multiway valve unit 15 and connected to a second supply connection 32 of the fluidic chip 28.

The rectangularly formed fluidic chip 28, which is expediently designed as a plastic injection-molded part and is intended for single use, has a Y-shaped, closed with a cover line assembly 8, which has an inlet portion 33, a blind portion 34 and an outlet portion 35, which are connected together in a connecting region 36. At an end facing away from the connection region 36, the inlet section 33 is provided with the feed connections 31, 32, while the outlet section 35 has the multifunction connection 27 at its end facing away from the connection region 36. In order to reduce the risk of contamination at its end facing away from the connecting region 36, the blind section 34 is provided with a blank connection 37 which is permeable solely to gaseous, but not to liquid fluid. The diameter of the sections 33, 34, 35 is for example about 1, 3 millimeters. The long side of the fluidic chip 28 is, for example, 75 millimeters and the short side, for example, 25 millimeters long, so that for the inlet section 33 and for the blind section 34 results in a length of about 1 40 millimeters.

FIG. 4 shows the fluidic chip 28 of the exemplary embodiment according to FIG. 3 after pumping in inert fluid solution 6 into the outlet section 35 close to the connection region 26 and due to corresponding actuation of the two-way valve unit 25 under fluid-dynamic separation of the multifunctional connection 27 after pumping in previously the feed connections 31 , 32 submitted whole blood 1 6 and presented Agglutinierlösung 1 8 in the inlet section 33 with mixing to an agglutinated blood cells having sample 1 1. After introduction of the agglutinated blood cells containing sample 1 1, the inlet section 33 is connected via appropriate actuation of the multi-way valve unit 1 5 including an air cushion 1 2 to the inert fluid container 5. The areas of the inlet section 33 and the outlet section 35 around the connecting region 36 and the blind section 34 are filled with residual air 38. FIG. 5 shows the fluidic chip 28 of the exemplary embodiment according to FIG. 3 after moving the sample 1 1 from the arrangement according to FIG. 4 over a first period of time with a first flow velocity in a first flow direction from the inlet section 33 into the blind section 34 1 is pumped under separation into a volume with predominantly plasma 1 3 and a mixing volume 1 4 as far as the air cushion 1 2 and inert fluid solution 6 into the blind section 34, while expelling residual air 38 through the blind port 37, the mixing volume 1 4 has come close to the blind terminal 37. In this case, residual air forming through a further air cushion 39 and the inert fluid solution 6 already present in the outlet section 35 ensure that the sample 11 enters exclusively into the blind section 34. Fig. 6 shows the fluidic chip 28, starting from the arrangement of FIG. 5 with corresponding wiring of the multi-way valve unit 15 and the pump unit 1 over a second period of time in a first flow direction opposite second flow direction at a different from the first flow velocity second flow velocity, the In this embodiment, smaller than the first flow rate is, with closing the connected to the feed ports 31, 32 output ports 23, 24 and suction of existing in the outlet section 35 inert fluid 6 after a process time of typically about 9 minutes in the outlet section 35 now with the volume Plasma 13 is present, wherein the present in the inlet portion 33 inert fluid solution 6 together with the present in the connection region 36 air cushion 12 is a fluid dynamic barrier. Now, with appropriate control of the two-way valve unit 25 via the control unit 2, the present with a yield of about 40 percent plasma 13 from the outlet section 35 via the multi-functional connection 27 into the collecting container 30 transfer.

Claims

A method for separating suspension or colloid components, in particular blood components, with the steps

Adding the suspension or the colloid (1 6) with a reagent (1 8) for agglutinating constituents to give a sample (1 1),

Moving the sample (1 1) with the agglutinated components at a flow rate in an axially extending conduit assembly (8) in a flow direction for a period of time until axial separation of the agglutinated components from the remaining sample (1 3, 1 4).

A method according to claim 1, characterized in that after moving the sample (1 1) in the flow direction moving the sample (1 1) takes place in a direction opposite to the flow direction further flow direction at a different flow rate from the flow rate over a further period of time.

A method according to claim 1 or claim 2, characterized in that the suspension and the colloid is selected from a group containing whole blood, urine, saliva or culture media.

Method according to one of claims 1 to 3, characterized in that the agglutinated components in a Blood cells, microorganisms or viruses containing group are included.

Device for separating suspension or colloid components, in particular for carrying out a method according to one of claims 1 to 4, with a pumping unit (1), with a line arrangement (8) connected to the pumping unit (1), with a storage container arrangement ( 1 0, 1 7, 1 9) for storing a suspension or a colloid (1 6) and a reagent (1 8) for agglutination of suspension or colloid components or a sample (1 1) with agglutinated components, with an inert fluid container (5 ) for storing an inert fluid solution (6), with a collecting container (1 0, 30) for receiving suspension o- the colloid components, with a valve unit (3, 1 5, 25) with which optionally the storage container assembly (1 0, 1 7, 1 9) or the inert fluid container (5) can be connected to the line arrangement (8), and to a control unit (2) for actuating the valve unit (3, 15, 25) and the pump unit (1) such that nac h introducing first of inert fluid solution (6) and then the sample (1 1) in the conduit assembly (8), the sample (1 1) with a flow velocity in a flow direction through the conduit assembly (8) flows.

Apparatus according to claim 5, characterized in that the line arrangement (8) is formed by a continuous branch-free line (9) with a constant cross-section.

Apparatus according to claim 5, characterized in that the line arrangement (8) Y-like manner with an inlet portion (33) and with one in each case to the inlet portion (33) and an outlet portion (35) Blindab- Section (34) is formed, wherein at the outlet portion (35) at its end remote from the inlet portion (33) the collecting container (30) can be coupled, wherein at the inlet portion (33) facing away from the end of the blind portion (34) an air-permeable closure ( 37) and in which the storage container (5, 17, 19), the inert fluid container (5), the valve unit (15) and the pump unit are located at the end of the inlet section facing away from the blind section (34) or the outlet section (35). 1) are connected.

Device according to one of claims 5 to 7, characterized in that the pump unit (1) is arranged to generate a flow direction opposite another flow direction, wherein the prevailing in the further flow direction further flow velocity is different from the flow rate adjustable.