DE102018219379A1 - Device and method for obtaining a cell-free sample fraction - Google Patents

Abstract

The invention describes a blood collection tube with a cylinder which, with its first end, which is closed by a wall which is impermeable to cells and permeable to serum, can be inserted into the tube and is arranged between the outer surface of the cylinder and the inner surface of the tube To form a seal. The cylinder is accessible at its second end opposite the first end for the removal of liquid, optionally provided with a pouring edge, or covered by a pierceable diaphragm.

Description

The present invention relates to a device and a method for obtaining a cell-free fraction of a cell-containing sample, in particular for obtaining a cell-free plasma fraction from whole blood, and the use of the device as a separation device for the production of a cell-free fraction from a cell-containing sample. The device enables a method which has at most only one centrifugation step, preferably exactly one centrifugation step, in order to separate a cell-free plasma fraction from whole blood. In a preferred embodiment, the use of the device allows the method to be carried out, and further preferably subsequent storage, within a tube which is set up for taking blood, that is to say without transferring a fraction from the one tube which contains a whole blood sample.

For obtaining a cell-free plasma sample it is known to take a whole blood sample in a centrifuge tube, which can be formed by a blood collection tube, in a first centrifugation step, e.g. 10 min at 2500 x g to separate into a cell-containing fraction and a lighter fraction. The lighter fraction still contains cells. Therefore, the lighter fraction generated with the first centrifugation step is removed, e.g. removed from the cell-containing fraction by means of a pipette, filled into a further centrifuge tube and in a second centrifugation step, e.g. 15 min at 15000 x g, separated into a cell fraction that forms a pellet and a cell-free lighter plasma fraction. For the storage of the cell-free fraction, this is separated from the heavier cell fraction, e.g. removed from the heavier fraction by means of a pipette and filled into a third vessel.

The object of the invention is to provide an alternative device for isolation, preferably also for storage, of a cell-free sample fraction, e.g. from whole blood, and to provide a process for producing a cell-free sample fraction, which is in particular a cell-free plasma fraction. The device is preferably intended to enable a method which contains at most or exactly one centrifugation step and in which the cell-free plasma fraction preferably remains connected to the blood collection tube in which the original whole blood sample was contained.

The invention achieves the object with the features of the claims and, in particular, provides a device which has a tube set up for blood sampling, also referred to as a blood sampling tube. In one embodiment, the tube has at its first end a guide for a piston which is displaceable in the tube and at a second end opposite the first end has a detachably attached cover with an inlet opening arranged thereon. In a further embodiment, the tube has a preset vacuum and can have a closed bottom firmly connected to the wall at the first end and accordingly no piston and a detachably attached cover at the opposite second end. A cover which is detachably attached to the second end of the tube is also referred to as the first cover and generally preferably has a connector for a cannula as an inlet opening, the connector being preferably covered by a diaphragm which is pierced by the cannula when the connector is connected. The first cover is preferably attached to the second end of the tube by means of a thread.

According to the invention, the device has a cylinder which, with its first end, which is closed by a wall which is impermeable to cells and permeable to serum, can be inserted into the tube and is arranged, has a seal between the outer surface of the cylinder and the inner surface of the tube to build.

The cylinder is accessible at its second end opposite the first end for the removal of liquid, e.g. open, optionally provided with a pouring edge, or covered by a pierceable diaphragm and can optionally have a second cover covering its second end.

The inlet opening is preferably formed by a connector for a cannula. The second lid can be arranged to connect to the tube, e.g. a thread on the outer surface of the tube, and / or to engage the second end of the cylinder. The second cover can optionally be set up to close the entire cross-sectional opening at the second end of the tube, optionally including the opening at the second end of a cylinder inserted therein, only to close a gap which is formed between the tube and the cylinder inserted therein. or just to close the opening of the cylinder at its second end.

The cylinder may have a pouring edge at its second end. Optionally, the cylinder can be driven along the tube by means of a screw nut. Such a nut engages, for example, the cylinder and the tube, once by means of a thread and once on a stationary guide, so that the rotation of the nut drives the movement of the cylinder along the tube, for example the movement of the cylinder into the tube. Optionally, a nut can be attached to the cylinder and engaged with a thread on the tube. Alternatively or additionally, the Cylinder by means of a thread directly with the tube, which preferably has a preset vacuum and has no piston, so that the cylinder can be driven by rotating relative to the tube along the tube, in particular into the tube.

In general, the tube preferably has an individual identification, e.g. a label.

The device can have, as an assembly of the blood collection tube, which optionally has a piston guided at its first end or a preset negative pressure in its internal volume, a first cover arranged at its second end, with a separate cylinder, optionally a screw nut, which is connected to the cylinder and the tube is to be brought into engagement, wherein further optionally the cylinder can be directly engaged with the tube by means of a thread, in particular for a tube which has a preset vacuum and has no piston, and / or optionally with a separate second cover be trained.

The device preferably forms a blood collection tube which has at a first end a guide for a piston which is displaceably and liquid-tightly guided in the tube, a first cover being arranged at the opposite second end of the tube, e.g. by means of a screw thread which has an inlet opening, e.g. in the form of a connector for a cannula, e.g. has a bayonet connection. The inlet opening is preferably covered by a diaphragm which is pierced by a cannula connected to the connector. Such a blood collection tube is available under the Monovette brand from Sarstedt, Nümbrecht.

The blood collection tube can optionally contain a stabilizing composition for cell-free nucleic acids and / or for cells. Such a stabilizing composition placed in the blood collection tube, e.g. when a sample is drawn in, mixed with the latter, has the advantage of stabilizing the content of cell-free nucleic acids contained in the cell-free fraction produced, or of reducing the degradation or the re-synthesis of cell-free nucleic acids. Another advantage of such a stabilizing composition can be the reduction in the proportion of cells which are destroyed when the cylinder is inserted into the tube and which can release the nucleic acids.

Generally preferred is the sample whole blood, e.g. is drawn into the tube immediately after removal from a human or animal body.

1. a) Mercaptoethanol, Dithiotreitol (DTT), optional zusätzlich Glycin, ein Alkylamin und/oder Polyamin als Fänger für freies Formaldehyd, oder
2. b) Urotropin und zumindest eine Puffersubstanz, die auf eine pH-Wert von 7 oder darunter puffert, bevorzugt Urotropin und als Puffersubstanz Citrat,

jeweils optional zusätzlich eine Puffersubstanz und/oder einen Gerinnungshemmer, der ein Chelatbildner für zweiwertige Kationen sein kann, insbesondere EDTA und/oder Citrat, optional PEG,
aufweisen oder daraus bestehen.The stabilizing composition, or a solution of the stabilizing composition in water or in the sample, can, for example

1. a) mercaptoethanol, dithiotreitol (DTT), optionally additionally glycine, an alkylamine and / or polyamine as scavenger for free formaldehyde, or
2. b) urotropin and at least one buffer substance which buffers to a pH value of 7 or below, preferably urotropin and citrate as buffer substance,

each additionally optionally a buffer substance and / or an anticoagulant, which can be a chelating agent for divalent cations, in particular EDTA and / or citrate, optionally PEG,
exhibit or consist of.

The stabilizing composition for the cell-free nucleic acids contained in the biological sample and / or for the nucleated cells can be a composition which contains at least one buffer substance which buffers to a pH of 7 or below, at least one anticoagulant, which is preferably a chelating agent. and urotropin, optional PEG, e.g. dissolvable in dry mixture or in aqueous solution or in the sample, or consists of them. The stabilizing composition can consist of the at least one buffer substance, the at least one anticoagulant, which is a chelating agent, and urotropin, optionally PEG, in aqueous solution. This stabilizing composition preferably contains no scavenger compound for free or dissolved formaldehyde. The buffer substance can buffer the aqueous solution to a pH in the range from 3.5 to 7 and have a buffer capacity which corresponds to a concentration of 10 to 100 mM citrate, with 1 to 30% by weight / volume in the solution. Urotropin is dissolved. The at least one buffer substance and the at least one anticoagulant, which is a chelating agent, can be formed by citrate buffers. The at least one buffer substance can e.g. citrate buffer, acetate buffer, MES (2-N-morpholinoethanolsulfonic acid), PIPES (piperazine-N, N'-bis-2-ethanosulfonic acid), MOPS (3-N-morpholinopropanesulfonic acid), phosphate buffer and mixtures of at least two of these and the at least one anticoagulant that is a chelating agent can be selected from citrate and / or EDTA and mixtures thereof.

An aqueous solution of the stabilizing composition, also called a stabilizing solution, can be in the tube in a volumetric proportion of e.g. up to 20% of the maximum sample volume for which a tube is set up can be contained in the tube.

The device according to the invention is suitable for use as a separation device for isolation, preferably also for storage, of a cell-free sample fraction, in particular a cell-free plasma fraction from whole blood. The invention provides a method which can be carried out with the device for producing a cell-free sample fraction, which is in particular a cell-free plasma fraction.

The method for producing a cell-free fraction, which is in particular a plasma fraction, or for separating cell-free plasma from a cell-containing fraction, provides for the tube with a sample contained therein, which is in particular a whole blood sample, and optionally with a stabilizing composition for free nucleic acids and / or for cells is centrifuged in exactly one centrifugation step in order to separate the sample into a heavy fraction which contains cells and a light fraction which has a substantially lower content of cells. The cylinder is then pushed into this tube, ie without removing the light fraction from the tube or without decanting the light fraction into another container. Since the cylinder has a seal between its outer surface and the inner surface of the tube and is fluid-tightly slidable along the tube, the light fraction is inserted into the tube by pushing the cylinder into the tube through the wall at the first end of the cylinder Internal volume of the cylinder is pressed and cells are held back by the wall. The wall can e.g. Have pores that have a maximum diameter of 1 µm in order to allow only cell-free liquid, in particular only cell-free serum, to pass through.

The wall that covers the first end of the cylinder is preferably supported against a pressure load that is directed against the outside of the wall. This is because when the cylinder is inserted into the tube containing the cell-containing sample, a pressure load is created in the direction of the inner volume of the cylinder on the outside of this wall, which covers the first end of the cylinder. The sample is divided into a heavy, cell-containing fraction and a light, cell-depleted fraction, in particular by exactly one centrifugation step. The wall can e.g. supported by a grid which is arranged between the inner volume of the cylinder and the wall and covers the clear cross section of the cylinder. Such a grid can e.g. connected to the inside of the cylinder, e.g. be formed by a clamp connection, or in one piece with the cylinder.

The wall preferably consists of material which does not bind nucleic acids, in particular nucleic acids of the sample, or binds only to a small extent, e.g. made of hydrophobic material.

In the process, since the light fraction obtained by centrifugation has passed through the wall of the cylinder into the cylinder when the cylinder is inserted into the tube, the light fraction through the wall located at the first end of the cylinder is from the cell-containing heavy fraction Cut. The arrangement of the cylinder within the tube has the advantage that the light fraction is still connected to the same tube in which the original sample was contained. This prevents mix-ups of samples that are possible by transferring fractions from one container to another, since a label attached to the tube also remains connected to the cylinder.

Optionally, the method may include the step of removing the cell-free fraction from the cylinder, e.g. by suction or pouring, and to store this fraction in another container and / or to analyze it directly.

Another advantage of the device and the method is that the centrifugation is limited to one centrifugation step and the method can therefore be carried out more quickly than a method which contains a second centrifugation step.

The insertion of the cylinder into the tube can be driven by loading the cylinder on its second end towards its first end or against the first end of the tube. Optionally, the insertion of the cylinder into the tube is driven by rotating a second cover against the tube, the second cover engaging with a screw thread of the tube and the second cover being arranged at least in sections, preferably completely, against the second end of the cylinder the second cover can load the second end of the cylinder towards its first end or against the first end of the tube be arranged against an end edge at the second end of the cylinder. Alternatively, the insertion of the cylinder into the tube can be driven by rotating a screw nut which is engageable with the tube and the cylinder, or by rotating the cylinder relative to the tube in a thread with which the cylinder directly engages the tube is feasible.

A latching device can be arranged between the second cover and the tube and / or the cylinder, which allows the second cover to be fixed on the tube and / or on the cylinder in the direction of screwing the second cover onto the tube and which locks in the opposite direction, in particular, unscrewing the second cover from the tube and / or the cylinder is prevented.

The second cover preferably has a pierceable section, for example a pierceable diaphragm, in an area which is to be arranged above the clear cross section of the cylinder. This area can be pierced, for example, by a hollow needle. The pierceable area is preferably formed by a diaphragm which closes automatically after a hollow needle is pulled out.

The sealing of the outer surface of the cylinder to the inner surface of the tube can be done by a sealing edge formed on the cylinder and / or a sealing ring, e.g. an O-ring. A sealing edge and / or a sealing ring can e.g. be arranged circumferentially on the outer surface of the cylinder at the first end of the cylinder, preferably at a distance from the first end of the cylinder. The arrangement of a sealing edge and / or a sealing ring at a distance from the first end of the cylinder has the advantage that the section of the cylinder between its first end, e.g. the wall arranged thereon, and the sealing edge or the sealing ring can form a guide when the cylinder is inserted into the tube. This distance can e.g. 1 to 50%, e.g. 2 to 20% of the length of the cylinder by which the cylinder can be inserted into the tube at most.

In general, the cylinder can preferably be attached coaxially to the tube, and the cylinder has a rotationally symmetrical outer surface and the tube has a rotationally symmetrical inner surface.

• - 1 eine Ausführungsform der erfindungsgemäßen Vorrichtung,
• - 2 eine weitere Ausführungsform einer erfindungsgemäßen Vorrichtung,
• - 3 eine bevorzugte weitere Ausführungsform einer erfindungsgemäßen Vorrichtung

zeigen.The invention will now be described in more detail by means of examples and with reference to the figures which are shown schematically in

• - 1 one embodiment of the device according to the invention,
• - 2nd another embodiment of a device according to the invention,
• - 3rd a preferred further embodiment of a device according to the invention

demonstrate.

The 1 shows a tube 1 , which can have a preset negative pressure or at its first end 3rd a piston (not shown) is slidably guided. At the second end 4th the tube 1 a first cover is provided (not shown), which has an attachment piece for a cannula as an inlet opening. A first cover is, for example, by means of a thread 7 , which is attached to the outside of the tube, releasably attached to the tube. The tube 1 is centrifuged, if necessary after removing a protruding plunger which engages a piston, with the filled-in sample, so that the sample is divided into a heavy fraction and a light fraction.

Following this single centrifugation step of the method, the first cover is removed from the tube, for example unscrewed, and a cylinder 9 will end with its first 10th ahead, that of a wall impermeable to cells 11 is covered in the tube 1 inserted, for example by exerting a force on the second end 8th of the cylinder 9 ..

The cylinder 9 points at its second end in this embodiment 8th a pouring edge 14 on, for example, as a spout over the circumference of the cylinder 9 extends.

The 2nd shows the device in the form of an assembly of a tube 1 for blood sampling, a screw nut 5 and a cylinder 9 . The nut 5 engages in the external thread in the embodiment shown here 7 that at the second end 4th the tube 1 is arranged. The nut 5 is with the cylinder 9 connected and not along the cylinder 9 slidable so that turning the nut 5 inserting the cylinder 9 in the tube 1 drives. A seal is optional 16 on the screw nut 5 arranged against the second end of the tube 1 and / or against the cylinder 9 is present when the screw nut 5 on the tube 1 is set

After inserting the cylinder 9 in the tube 1 forms, for example, a sealing edge that extends around the cylinder 9 at its first end 10th is attached, a seal to the inner surface of the tube 1 . In the embodiment shown here, the seal is a sealing ring 13 formed the one at the first end 10th of the cylinder is fixed. Optionally, the cylinder 9 generally have multiple circumferential seals that span the length of the cylinder 9 are distributed.

The second cover is optional 12th have a seal (not shown) that bears against the second end of the tube 1 is present when the second cover 12th on the tube 1 is set.

Generally the seal 13 be formed by at least one circumferential sealing ring.

The 3rd shows an embodiment in which the insertion of the cylinder 9 is driven by a second cover 12th who the cylinder 9 covered and that in the thread 7 at the second end 4th the tube 1 engages against the tube 1 is rotated so that the second cover 12th towards the first end 3rd the tube 1 moves while doing the cylinder 9 in the same direction in the tube 1 pushes. The second lid 12th points in the area where he has the open second end 8th of the cylinder 9 covered, a diaphragm 15 which can be pierced with a hollow needle for taking cell-free samples. Such a second cover 12th closes the second end 4th the tube 1 and the open second end 8th of the cylinder 9 .

Example: Preparation of a cell-free serum fraction from a whole blood sample

Whole blood was drawn into a monovette as a blood collection tube as a sample. In one embodiment, the tube optionally contained an aqueous composition for stabilizing cell-free nucleic acids and for stabilizing cells. The stabilization composition consisted of 15% hexamethylenetetramine, 8% trisodium citrate dihydrate and 3.8% citric acid. 10% of the composition, based on the blood volume drawn in, could have been placed in the tube.

The tube had a guide for a piston at a first end and a first cover with a connector for a cannula at the opposite second end.

The whole blood sample or the mixture of whole blood and the stabilizing composition was centrifuged until a heavy, cell-containing and red fraction and above that a yellow light fraction formed. The centrifugation was e.g. run at 2500 to 3000 x g for 10 min. Immediately after this centrifugation step, the first lid was removed from the tube and a cylinder, the first end of which was covered by a grid-supported membrane as a cell-impermeable wall, was pushed into the tube with its first end first. The cylinder had a circumferential sealing edge which was attached to its outer surface 1 to 2 mm from its first end and which formed a liquid-tight seal to the inner surface of the tube. The cylinder wall was arranged coaxially and at a distance of approximately 0.2 to 0.5 mm from the inner surface of the tube. During the insertion of the cylinder into the tube, at least a portion of the light fraction was pressed through the membrane and was found inside the cylinder.

For the insertion of the cylinder, a second cover was placed on the second end of the tube, which had an internal thread that engaged the external thread on the second end of the tube. The second cover covered the second end of the tube and also the second end of the cylinder, so that when the second cover was screwed onto the tube, the cylinder was pushed into the tube. The second lid had the advantage of preventing sample from leaking out of the second end of the tube or cylinder.

An analysis of the plasma, e.g. after unscrewing the second cover or after piercing the second cover using a hollow needle, cfDNA was isolated using kits from Macherey-Nagel or Qiagen. Analysis of the isolated cfDNA e.g. with the Agilent Bioanalyzer 2100 (High Sensitivity DNA Chips) or via the PCR detection of ALU fragments (such as from Umetani N., Kim J., Hiramatsu S., Reber HA, Hines OJ, Bilchik AJ, Hoon DSB ( 2006 a) Increased Integrity of Free Circulating DNA in Sera of Patients with Colorectal or Periampullary Cancer: Direct Quantitative PCR for ALU Repeats.Clinical Chemistry, 52 (6), 1062-1069) showed that the filtration also removes cells and cell fragments Samples removed, like a second centrifugation step (15 min, 15,000 xg) carried out for comparison.

Reference symbol list

1

tube

3rd

first end of the tube

4th

second end of the tube

5

Nut

6

Inlet opening

7

thread

8th

second end of the cylinder

9

cylinder

10th

first end of the cylinder

11

wall

12

second lid

13

poetry

14

Pouring edge

15

Diaphragm

16

poetry

Claims (20)

Device with a tube (1) for drawing blood for use as a separating device for producing a cell-free fraction from a cell-containing liquid sample, the tube (1) having a first end (3) and at the opposite second end (4) one detachably attached to one has an inlet opening arranged with a cylinder (9) which can be inserted between its outer surface and the inner surface of the tube (1) after the sample has been drawn into the tube (1) and after the first lid has been removed into the tube (1) ) to form a seal (13) and which can be moved with its first end (10) forward into the tube (1), the first end (10) of the cylinder (9) having a wall impermeable to cells and permeable to serum ( 11) is covered. Device after Claim 1 , characterized in that a piston is displaceably guided at the first end (3) of the tube (1) or the tube (1) is provided with a preset negative pressure. Device according to one of the preceding claims, characterized in that the tube (1) contains a stabilizing composition which a) mercaptoethanol, dithiotreitol (DTT), optionally additionally glycine, an alkylamine and / or polyamine as scavenger for free formaldehyde or b) urotropin and at least one buffer substance which buffers to a pH value of 7 or below, preferably urotropin and citrate as buffer substance, each optionally additionally having or consisting of a buffer substance and / or an anticoagulant and / or PEG. Device according to one of the preceding claims, characterized by a second cover (12) which is set up to be fixed at the second end (4) of the tube (1) and at least the edge of the cylinder (9) at its second end (8) to cover. Device after Claim 4 , characterized in that the second cover (12) has a diaphragm (15) in the area that covers the second end (8) of the cylinder (9). Device after Claim 4 or 5 , characterized in that the second cover (12) is arranged to engage the tube (1) and to close a gap formed between the tube (1) and the cylinder (9). Device according to one of the Claims 4 to 6 , characterized in that the second cover (12) is arranged to engage with the tube (1) and to cover the second end (8) of the cylinder (9) in a liquid-tight manner when engaged with the tube (1). Device according to one of the Claims 4 to 7 , characterized in that the second cover (12) is arranged to engage the tube (1) and in engagement with the tube (1) upon rotation against the tube (1) the cylinder (9) towards the to push the first end (3) of the tube (1). Device according to one of the Claims 4 to 8th , characterized in that the second cover is guided liquid-tight on the tube. Device according to one of the preceding claims, characterized in that the seal (13) is formed by a sealing ring fixed to the cylinder (9). Device according to one of the preceding claims, characterized in that the cell-impermeable and serum-permeable wall (11) has only pores smaller than 1 µm in diameter. Device according to one of the preceding claims, characterized in that the wall (11) which covers the first end (10) of the cylinder (9) is supported against a pressure load which is directed against the outside of the wall. Device according to one of the preceding claims, characterized in that the cylinder (9) is driven along the tube by means of a screw nut which engages the cylinder (9) and the tube (1). Device according to one of the preceding claims, characterized in that the cylinder (9) is to be brought directly into engagement with the tube (1) by means of a thread, so that the cylinder (9) is rotated relative to the tube (1) along the tube (1) can be driven Device according to one of the preceding claims, characterized in that as a combination of the tube (1), which has a piston guided at its first end (3) or a preset negative pressure in its internal volume, and one at the second end (4) of the Tube (1) arranged first cover, is formed with a separate cylinder (9) and optionally a separate second cover (12) and / or optionally a screw nut (5). Method for producing a cell-free fraction from a blood sample, in particular using a device according to one of the preceding claims, with the steps of a) centrifuging a tube (1) for taking blood, into which a blood sample has been drawn, the tube (1) having a displaceably guided piston at a first end (3) or a preset negative pressure in its internal volume, and at its opposite second end (4) has an inlet opening arranged on a detachably attached first cover, b) removing the first cover from the tube (1), c) inserting a cylinder (9) with its first end (8) ahead into the tube (1), which forms a seal (13) between its outer surface and the inner surface of the tube (1), wherein d) the first end (8) of the cylinder (9) is covered with a wall (11) which is impermeable to cells and permeable to serum and when the cylinder (9) is inserted cell-free plasma through the wall (11) into the cylinder (9) flows. Procedure according to Claim 16 , characterized in that in step c) the cylinder (9) is inserted by rotating a second cover (12) which closes the second end (8) of the cylinder (9) in a liquid-tight manner. Procedure according to one of the Claims 16 to 17th , characterized in that the second cover (12) engages in a thread (7) arranged on the tube (1). Procedure according to Claim 16 , characterized in that in step c) the insertion of the cylinder (9) is driven by turning a screw nut (5) which can be brought into engagement with the cylinder (9) and the tube (1). Procedure according to one of the Claims 16 to 19th , characterized in that the second cover closes the cylinder and the second cover is pierced to remove serum from the cylinder.

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