Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5021—Test tubes specially adapted for centrifugation purposes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/16—Reagents, handling or storing thereof
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0609—Holders integrated in container to position an object
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0633—Valves, specific forms thereof with moving parts
  • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves

Definitions

• the invention relates to a device, a container with the device and a method for fluid separation by means of density gradient centrifugation.
• the invention relates to a kit for carrying out the method.
• the invention is used to separate body fluids e.g. animal blood, human blood, for further analyses like clinical diagnostics or research.
• This invention relates to the fields of containers for laboratory use, in particular to a specialized centrifugal tubes/containers.
• Another purpose of the invention relates to the area associated with testing or analyzing of materials by determining their chemical, physical or biological properties, in particular the analysis of liquid biological material, for example blood.
• Collection, purification, separation into fractions and/or preservation of fluid samples, including blood play an important role in medical diagnostics as well as in clinical trials.
• a blood sample obtained from a patient can be separated into different fractions by centrifugation, filtration, or elutriation and stored for later use or further testing.
• the separated blood components typically include fractions of red cells, white cells, platelets and plasma.
• Blood separation into its fractions can be performed continuously during collection of blood or in steps after it has been collected. It is critical for a number of therapeutic applications and for purposes of clinical trials that blood separation into its various fractions takes places in a highly sterile conditions.
• a technique is known, from the international patent application WO8805331, to separate white blood cells (leukocytes) from red blood cells (erythrocytes). It involves mixing a blood sample with a working solution which then aggregates the red blood cells, as a result the sedimentation rate of agglutinated red blood cells increases. The density of the separation fluids is adjusted such that the sedimentation process of white blood cells is only slightly altered. This prevents the sedimentation of the white blood cells on bottom of container, after separation white blood cells can be collected from the upper portion of the separated blood sample, while at the same time red blood cells sediment to the bottom of the container.
• PBMC mononuclear white blood cells
• density gradient centrifugation In the first step of this method, a mixture of Isopaque-Ficoll (Nyegaard & Co., Norway) with metrizoat as a main component, is being used.
• the second step of this method enables isolation of PMN fraction from blood employing dextran or gelatin, which causes increased sedimentation of red blood cells.
• Another method uses a discontinuous density gradients where two or more working fluids are carefully layered on top of each other. Densities are chosen such that the
• noncontinuous gradient is in the optimal required range - it is being chosen according to the density of separated substance.
• U.S. Patent Application No. US4824560 A discloses methods and means of rotation of the tubular container having at least two adjacent chambers which are connected to each other by a narrow, capillary-like opening. Operation principles are as following: the working fluid is placed in the lower chamber, and the fluid to be separated into fractions is applied in the upper chamber. There is no need for any special precautions to avoid mixing of the fluids before centrifugation. This method has several advantages over the manual methods described above. It also possess a disadvantage because the narrow opening between the two chambers prevents efficient passage of blood cells between the two chambers, even during centrifugation, as a result the efficiency of the blood separation is reduced.
• the aim of the embodiments of the present invention is to provide a tool for the rapid and partly automated separation of fluids into fractions of various densities like in case of biological fluids, including blood, which also may allow for purification, isolation and preservation of biological samples.
• Container includes any receptacle for collecting liquid, which is adapted to use in centrifuges, for example centrifugal tubes.
• Guide is a part of the device which controls fluid flow speed and direction while flowing from the upper chamber to the lower chamber through the opening in the partition disk, wherein the guide should have an adequate size, to allow flow and layering of one liquid from the upper chamber on top of the other liquid in the lower chamber in particular the fluid sample on the separation fluid/medium already located at the bottom of the container.
• Guide in accordance to this invention can be a container wall or other structure within the container e.g. a spiral elongated sleeve, etc.
• the invention relates to a device for a centrifugation container, particularly to a tube, for separation of liquid fractions having a desired density range, in particular invention applies to biological and / or liquids forming suspensions, characterized by the device having a partition that separates the interior of the container into at least two chambers in a vertical arrangement - an upper chamber and a lower chamber, and the device having the partition has an aperture which can be lined up with the guide, on which liquids, in particular fluid sample, can flow down from upper chamber to lower chamber, of the container for centrifugation.
• the guide is the inner wall of centrifuge container, a spiral, funnel or vertical elements in the shape of an elongated cylinder.
• the partition disk consists of two adjacent surfaces with apertures, in particular in the shape of flattened disks fitted to a container having a cross section similar to the wheel, where the surfaces are movable with respect to each other and their positioning relative to each other can be adjusted allowing for closing communication via partition apertures.
• the upper chamber additionally have a vertical partition or partitions dividing it into sub-chambers, each of the sub-chambers having an aperture.
• the invention in another aspect, relates to a container for centrifugation comprising device for centrifugation container, particularly for a tube, for separation of liquid samples having a desired density range, particularly liquid forming a suspension or biological fluids, the device has a partition that separates the interior of the container into an upper chamber and a lower chamber, and the partition has an aperture, and near the aperture there is a guide along which the down-flow of liquids takes place, especially separation liquids flow to the lower chamber of the centrifugation container.
• the partition has a aperture where a guide is placed close by along which the down-flow of liquids takes place, especially separation liquids flow to the lower chamber of the centrifugation container.
• the invention also includes the method for separating out a fraction having the desired density range from the sample containing fractions of different density, especially from a biological sample, comprising:
• step (b) is followed by an additional step or steps of (b) which entails addition of an additional medium for density gradient separation, additional media are added in the order from highest to lowest density.
• step (d) selected fractions of different density from separated liquid sample can be studied, tested and analyzed, these fractions can also be preserved by freezing.
• each separated fraction contains different blood elements including: leukocytes
• lymphocytes and granulocytes lymphocytes and granulocytes
• platelets erythrocytes
• bone marrow cells megakaryocytes, erythroblasts
• cells suspended in homogenate including endothelial cells, neurons, fungus, viruses, microparticles including exosomes, cellular fragments, cell organelles including nuclei, mitochondria, chloroplasts.
• the invention also relates to a kit comprising:
• the device for the container for centrifugation in particular for tubes for separation of liquid sample to fractions of different density by density centrifugation, particularly liquids forming a suspension or biological fluids
• device has a partition dividing the interior of the container to the upper chamber and a lower compartment, wherein the partition has an aperture, the aperture 's guide, along which fluids flow- down, in particular liquid sample, to the lower chamber of the container for centrifugation
• Fig. 1 illustrates a container in the shape of a centrifuge tube, intended for collecting fluids, especially biological material, it also illustrates the device which together with the container is used for density gradient liquids separation, according to the invention - the device enables layering of liquids in the container, prior centrifugation, one on top of another with maintaining clear interphase between them.
• Fig. 2 and Fig. 3 illustrate respectively a longitudinal sectional view and a side view of a container in the shape of a centrifuge tube, wherein, for a better understanding of the invention- the discs that the partition is built of are spaced apart;
• Fig. 4 and Fig. 5 illustrate respectively a side view and a longitudinal section of the tube- shaped container with visible narrowing of the inner diameter of the tube and with increasing wall thickness.
• Fig. 6 illustrates a cross-section through the container-shaped tubes in the embodiment without vertical partition, and illustrates the air duct in the device partition
• Fig. 7a and Fig. 7b illustrate respectively a side view and cross-section of the upper part of the device in the form of a disk with incomplete vertical partition
• Fig. 8a and Fig. 8b illustrate respectively a side view and cross-section of the upper part of the device in the form of a disk with vertical partition of rectangular shape
• Fig. 9a and Fig. 9b illustrate respectively a side view and cross-section of the upper part of the device with vertical partition build of three rectangles
• Fig. 10a and Fig. 10b illustrate respectively a side view and cross-section of the upper part of the device with vertical partition build of two intersecting rectangles forming a cross shape.
• Fig. 11a and Fig. l ib illustrate a sectional and a side view of the partition disc with a cutout.
• Fig. 12 illustrates one embodiment of the invention, wherein the device is fitted onto the container for centrifugation.
• Fig. 13 and Fig. 13a shows the device in a sectional side and from top view, which allows fitting separate upper chamber on top the device with guide in a form of elongated cylinder
• Fig. 14 and Fig. 14a and illustrates the device in a sectional side and top view with a guide in the form of eight elongated cylinders.
• Fig. 15 and Fig. 15a shows the insert in a sectional side and top view equipped with a guide in the form of spiral
• Fig. 16 and Fig. 16a shows the insert in a sectional side and top view, equipped with a guide in a form of a funnel.
• device 6 for the centrifuge container in a form of a centrifuge tube is built of a flat circular disc 7, tightly fitted to the inner walls of the tube partition, and another circular disc 8 which both 7 and 8 constitute the device partition and of a full vertical partition 11 which is attached to disc 8.
• the device in this embodiment of the invention is placed inside the centrifugation container 1 which is a centrifugation tube with 0.23" diameter.
• the device 6 in this embodiment is made of plastic, but could also be made of other materials.
• a shown in fig. 12 the device 6 can be placed in another container that can be fitted on to the centrifuge container 1, in this case device is outside of the centrifuge container 1.
• inner walls of the centrifuge container 1 are at the same time the guide 12 and that centrifuge container walls thickens, inner diameter of the centrifuge container decreases gradually toward its' bottom.
• inner wall of the container 1 is the guide 12, which directs the down-flow of liquids from upper chamber 2 to the lower chamber 3 via the aperture 4.
• Flow -down of liquids along or on the guide 12 prevents mixing of liquids, which otherwise would impair separation of these liquids.
• partition 7 has shape of circular disc which in transverse section has shape of a circle (fi, 1 la, fig. 1 lb) and its' shape is tightly fitted to the transverse section on the container 1, therefore the diameter of the partition is longer on the top side compared to the bottom side, and its' longitudinal section closely resembles the shape of flattened inverted trapezium.
• Partition 7 divides container 1 to upper chamber 2 and lower chamber 3.
• Partition in this embodiment has an aperture 4 which is a notch in the shape resembling semicircle.
• vertical partition 11 may have a shape of rectangle, which adheres tightly to the inner walls of the container 1, whereupon vertical partition 11 attached to the disc 8 separates upper chamber 2 of the container 1 in the shape of a tube to two sub-chambers 10a, 10b.
• apertures 5 in a shape of a notch in disc 8 are in a shape of semicircle.
• the shape of apertures 4, 5 and their positioning against each other determines the speed of liquids down-flow from upper chamber 2 to lower chamber 3.
• apertures 4, 5 are in shape of a semicircular notch with 0.115" radius and have identical shape. In different embodiments of the invention apertures 4, 5 can have various shapes, and shapes can be different from one another, however their diameter should not be bigger than 0.1". In such arrangement of the partition 7 and disc 8 that apertures 4, 5 are not overlapping, down-flow of liquids between upper chamber 2 and lower chamber 3 is blocked and flow of liquids cannot take place.
• container 1 is equipped with lid 9.
• lid 9 has a gap, through which protrudes upper part of the vertical partition 11 of the device 6.
• Container 1 and lid 9 has a thread and is a nut.
• lid without a gap 91 can be used, wherein vertical partition 11 of the device is adjusted to the length of the container 1 in such a way that after screwing down the lid 9 vertical partition 11 tightly adheres to the inner side of the lid 9.
• Lid 9 may be made of polymers and can have calibrated scale for turning/screwing the lid 9. On the container 1 for centrifugation and on the lid 9 labels may be present to facilitate correct adjusting/arranging of the apertures 4, 5 positions against each other.
• vertical partition 11 does not have to adhere to the inner walls of the container 1, in which case vertical partition 11 placed on disc 8 separates the tube only to two chambers - upper chamber 2 and lower chamber 3 and upper chamber 2 is not further divided to additional sub -chambers.
• disc 8 is equipped in one aperture 5 in a shape of a notch, in the other embodiment of the invention shape of the disc 8 could be limited to the size that would enable closure of the apertures 4 in the disc 7.
• vertical partition 11 can be built of three elements in the shape of a rectangle connected with each other with longer edges, which other edges adhere tightly to the inner wall of the container 1, in this embodiment vertical partition 11 placed on the disc 8 divides upper chamber 2 of the container 1 in the shape of the tube to three sub-chambers.
• disc 8 has three notches 5, one in each of the sub-chambers.
• vertical partition 11 may be built of four rectangles connected with each other, which edges adhere tightly to the inner wall of the container 1, in this embodiment vertical partition 11 placed on the disc 8 divides upper chamber 2 of the container 1 in the shape of the tube to four sub-chambers.
• disc 8 has four notches 5, one in each of the sub-chambers.
• Device 6 may also be used in containers 1 shaped differently than centrifuge tube presented in this example of invention embodiment, however there has to be a method that allows to centrifuge this container.
• Embodiment 2 Fig. 13 and 13a show another embodiment of the invention, wherein the device 6 has a baffle 7, which does not have an upper chamber but allows the connection through a tube (see part 16) down to upper partition in a form of a container (for example, a test tube, pouch, bag) with separation medium or separation liquid.
• the partition is equipped with a guide 12 in a form of an elongated cylinder which is attached to the partition 7 and is situated at a distance from the aperture 4. This allows fluid flow from the upper chamber 16 through the tube followed by the aperture in the partition along the guide the lower chamber 3.
• the elongated cylinder forms a guide 12 and its length is such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.
• Fig. 14 and 14a show another embodiment of the invention, wherein the insert 6 has a partition 7, equipped with a guide 12 in a form of eight elongated rollers which are anchored to partition 7 and are located at such distance from the aperture 4, which allows the liquid to flow from the upper chamber through the aperture, in the partition along the guide, to the lower chamber 3.
• the length of the guide for the elongated rollers 12 is such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.
• Fig. 15 and 15a show yet another embodiment of the invention, wherein the device 6 has a partition 7 equipped a guide 12 in the shape of a spiral.
• the length of the coil should be such that the test material spreads gently on a surface of the centrifugal medium used in the gradient separation method and it does not cause significant disturbances to the separation medium.
• Fig. 16, 16a and 16b show yet another embodiment of the invention, wherein the insert 6 has a partition 7 provided with a guide 12 in the shape of a funnel. Wherein the four holes in the partition 7 directs the fluids from the upper chamber so as to roll down the outer surface of the funnel to the bottom of the lower chamber 3.
• the length of the coil should be such that the test material spread over a surface of the medium to the gradient centrifugation thereby causing no significant adverse to the separation medium.
• Method for separation of fractions of given density from fluid sample with fractions of different density can be achieved by, filling two sub-chambers 10a, 10b of the upper chamber 2 with two media for separation in on density gradient, first medium has density of 1.119 g/mL second medium has density of 1.077g/mL (respectively Histopaque 1.119 and Histopaque 1.007 Sigma Aldrich), at the same time apertures 4, 5 being notches- respectively in disc 7 and disc 8 - are not overlapping and remain in closed position. Next by changing the position of disc 8 by its' turning, apertures 4, 5 overlap each other in such a way that enables down-flow of mediums from the upper chamber 2 to the lower chamber 3.
• Down-flow occurs on and along the guide 12 which in this embodiment is the internal wall of the container 1.
• Media are added one by one starting from the highest density to the lowest density, and interface is established between media of different densities.
• the size of the clearance created by apertures 4, 5 being the notches of respectively disc 7 and disc 8 can be controlled by regulation of positions of disc 7 and disc 8 against each other. Slow turning of the upper part of the vertical partition 11, and subsequently disc 8, causes gradual increase of the down-flow velocity up to the moment when expected velocity, of liquid down- flow from the upper chamber 2 to the lower chamber 3, is achieved. By regulation of positions of disc 7 and disc 8 against each other, liquid down-flow can be controlled in order to achieve stable laminar flow of liquid on and along the internal wall 12 of the centrifuge container 1.
• discs 7 and disc 8 ensures very gentle down-flow of the liquid from the upper chamber 2 to the lower chamber 3 of the centrifuge container 1 in such a way that the surface of the liquid is intact and subsequently added liquids which down-flows from the upper chamber 2 does not mix with the liquid already present in the lower chamber 3.
• analyzed sample was added - blood in this case- although it is possible to use different types of separation liquids, including native or diluted biological samples.
• Blood was first placed in sub-chamber 10a, and next after turning the disc 8 of the device 6 in such a way that aperture 4 of the disc 7 was overlapping at least partially with respective aperture 5 in the disc 8 of the device 6 and enables down-flow of the blood on and along the inner wall 12 of the container 1 from the sub-chamber 10a to the lower chamber 3 layering it on the surface of previously placed separation media. Because of the device 6 construction it is not necessary to place the biological material in the container 1 with extraordinary precision and care.
• Insert and method of the invention is used, for example, for separating the desired subset of blood cells.
• EDTA versene acid
• the volume of the centrifuge tube 1 of which the essence of the invention was 50 ml was also used for the separation of two media of different densities (Histopaque 1119 and Histopaque 1077 Sigma Aldrich).
• the separation of fluids used have a neutral pH, be isotonic to body fluids, the first separation medium to have a density of 1.119 g/ml, while the second had a density of 1.077 g/ml.
• PBMC mononuclear white blood cells
• PMN white blood cells with a segmented nucleus
• Czerwonki cells Purity fraction of PBMC and PMN was confirmed by flow cytometry. Purity PBMC and PMN in the fractions was 95% and 92%. PBMC and PMN were undetectable in plasma fractions. Isolated plasma, PBMC and PMN were suitable for further analysis, including, but not limited to aPatryk, nalysis: RNA, micro-RNA, mitochondrial DNA, nuclear DNA, proteins and phenotyping of the cells.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Hematology (AREA)
• Clinical Laboratory Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Centrifugal Separators (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• External Artificial Organs (AREA)

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• 2015
  • 2015-09-15 PL PL413910A patent/PL237582B1/pl unknown
• 2016
  • 2016-09-15 CN CN201680065865.3A patent/CN108367288A/zh active Pending
  • 2016-09-15 ES ES16791676T patent/ES2910923T3/es active Active
  • 2016-09-15 DK DK16791676.6T patent/DK3349897T3/da active
  • 2016-09-15 EP EP16791676.6A patent/EP3349897B8/de active Active

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