WO2018060366A1

WO2018060366A1 - Vessel

Info

Publication number: WO2018060366A1
Application number: PCT/EP2017/074689
Authority: WO
Inventors: Bruno Ehrler
Original assignee: 1 Cryobio Ag
Priority date: 2016-09-30
Filing date: 2017-09-28
Publication date: 2018-04-05
Also published as: GB201616642D0; EP3519095A1

Abstract

A multi-specimen storage vessel comprising a container comprising a first volume and a second volume separated by a filter; and a plurality of storage specimens. A method of filtering blood and a method of analysing a sample of blood are disclosed.

Description

VESSEL

The present invention relates to a vessel for containing, filtering and storing a sample and subsequent easy access to individual specimens of the sample, in particular specimens of biological samples. The multi-specimen storage vessel according to the present invention may be applied within a cryogenic storage application.

BACKGROUND OF INVENTION The listing or discussion of a prior-published document or any background in the specification should not necessarily be taken as an acknowledgement that a document or background is part of the state of the art or is common general knowledge.

Biological samples, such as substances in solution, e.g. blood samples, water tests and tissue samples such a fertilised embryos, can often be effectively stabilised by freezing. The frozen fluid and/or sample will remain stable for extended periods of time as long as it is kept in the frozen state. Frequently these samples are collected in relatively large quantities, or collective sample, but could be utilised in smaller quantities, or specimens e.g. for test purposes.

When a specimen is needed, it often requires thawing of the entire collective sample to obtain the specimen currently needed, and then refreezing the remainder of the collective sample. However, frequent freezing and thawing cycles can be detrimental to the often unstable ingredients in the collective sample.

One solution is to store the collective sample in multiple small individual vessels. Then, when a specimen is needed, the necessary number of individual vessels may be thawed to provide the specimen needed without thawing and refreezing other individual samples of the collective sample. However, manual separation and freezing in individual vessels is cumbersome and time consuming, while the provision of an automatic aliquoting machine is expensive and requires ongoing maintenance. This approach inherently requires a larger amount of individual vessels and thus larger storage facilities. The chance of confusion and mix-up of the individual vessels in also present. Another solution is to store the collective sample in a single breakable storage vessel. US 6,383,453 discloses a multi-specimen storage vessel provided with a number of equally distanced ring-shaped "notches" that extend around the perimeter of the vessel to constitute breakpoints. External screw threads for closing each specimen with screw caps may be provided on the ends of each specimen, i.e. surrounding the breakpoints. WO 2009/086829 discloses a breakable multi-specimen storage vessel comprising a container provided with break portions at predetermined positions alongside whereby the container is adapted to be broken into a plurality of specimens. The vessel and a broken off specimen may be closed by closure caps.

WO 2012/107046 discloses a breakable multi-specimen storage vessel comprising a container having break portions at predetermined positions to allow the container to be broken into a plurality of specimens. Each specimen has a first fastening mechanism at a first end and a second fastening mechanism at a second end, the configuration of the first fastening mechanism being different from the configuration of the second fastening mechanism. The approach of storing a collective sample in multiple small individual vessels, either by manual or automated aliquoting, is known in the art. Furthermore, breakable storage vessels are known in the art that allow a specimen containing a biological sample to be broken off and subsequently closed at each end with e.g. caps. The present invention seeks to provide an improved multi-specimen storage vessel.

STATEMENT OF INVENTION

According to the present invention there is provided a multi-specimen storage vessel comprising a container comprising a first volume and a second volume separated by a filter; and a plurality of storage specimens.

Conveniently, the container has predetermined break points and is adapted to be broken into the plurality of storage specimens. Advantageously, the first volume and/or the second volume of the container is at sub- atmospheric pressure.

Preferably, the predetermined break points are provided with a fastening mechanism for the attachment of a cap.

Conveniently, the vessel comprises a manifold having a single inlet and one or more outlets. Advantageously, the inlet of the manifold is reversibly coupled to the container. Preferably, the outlet of the manifold is reversibly coupled to a specimen.

Conveniently, the inlet of the manifold and the container are reversibly coupled by a push- fit connection, a snap fit-connection, a bayonet connection, or a screw threaded connection. Advantageously, the outlet of the manifold and a specimen are reversibly coupled by a push-fit connection, a snap-fit connection, a bayonet connection, or a screw threaded connection.

Preferably, the outlet of the manifold comprises a needle, and wherein a specimen comprises a pierceable cover.

Conveniently, each of the plurality of storage specimens is at sub-atmospheric pressure.

Advantageously, the volume of the manifold is greater than the volume of the first volume of the container.

Preferably, the volume of the manifold is greater than the volume of the second volume of the container. Conveniently, the volume of the manifold is greater than the volume of a specimen. Advantageously, the outlets of the manifold are in a linear arrangement. Preferably, the outlets of the manifold are in a circular arrangement.

Conveniently, the filter mesh size is from 0.1 to 50 μηη, preferably from 0.5 to 1.5 μηη.

Advantageously, the filter is suitable for separating blood components. Preferably, an anticoagulant is present in the first volume and/or the second volume of the container. Conveniently, the anticoagulant is selected from heparin, oxalic acid, citric acid, ethylenediaminetetraacetic acid, or salts thereof.

Conveniently, a first portion of the container comprising the filter and a second portion of the container may be reversibly coupled together.

Advantageously, the first portion and second portion are coupled together by a snap-fit connection, a bayonet connection or a screw threaded connection. Preferably, the first portion of the container and second portion of the container are of different volumes.

Conveniently, the vessel comprises a plunger. Conveniently, part or all of the inner surface of the container comprises one or more coatings.

Advantageously, the one or more coatings comprise a component of one or more assays. Preferably, the component is suitable for the analysis of blood.

Conveniently, the vessel further comprises a reagent for an assay.

According to an aspect of the invention, there is provided a method of filtering blood, comprising the steps of:

providing a sample of blood in a vessel; and

centrifuging the vessel such that at least a portion of the blood passes through the filter. According to an aspect of the invention, there is provided a method of filtering blood, comprising the steps of:

providing a sample of blood in a vessel; and

moving the plunger in the vessel such that at least a portion of the blood passes through the filter.

Preferably, centrifuging the vessel or moving the plunger leads to substantially all of the blood plasma passing through the filter. Advantageously, the further comprises the step of freezing the vessel, or a portion thereof.

According to an aspect of the invention, there is provided a method of analysing a sample of blood, comprising the steps of:

providing a sample of blood in a vessel;

allowing the blood sample to interact with the one or more coatings; and performing analysis of the sample and/or the one or more coatings.

DESCRIPTION OF DRAWINGS

In the following, the invention is described with reference to some embodiments shown in the accompanying schematic drawings, in which:

Figure 1 A shows a side-view schematic illustration of a breakable multi-specimen storage vessel;

Figure 1 B shows a side-view schematic illustration of another breakable multi-specimen storage vessel;

Figure 1 C shows a side-view schematic illustration of a further breakable multi-specimen storage vessel; Figure 2 is a perspective illustration of a broken off specimen of the storage vessel of Figure 1A, illustrated with matching closure caps;

Figure 3 shows a side-view schematic illustration of the breakable multi-specimen storage vessel with coatings;

Figure 4 shows a side-view schematic illustration of the breakable multi-specimen storage vessel with a blood sample in the first volume;

Figure 5 shows a side-view schematic illustration of the breakable multi-specimen storage vessel with blood cells in the first volume and plasma in the second volume;

Figure 6 shows a side-view schematic illustration of the first portion and second portion of the breakable multi-specimen storage vessel; Figure 7 shows a side-view schematic illustration of the breakable multi-specimen storage vessel with an integral identifying device;

Figure 8 shows a side-view schematic illustration of a multi-specimen storage vessel comprising a reversibly coupled manifold;

Figure 9 shows a side-view schematic illustration of another multi-specimen storage vessel comprising a reversibly coupled manifold; Figure 10 shows a side-view cross-sectional schematic illustration of the manifold of the multi-specimen storage vessel.

Figure 1 1 shows a perspective illustration of the manifold of the multi-specimen storage vessel.

Figure 12 shows a perspective illustration of the manifold of the multi-specimen storage vessel. Figure 13 shows a side-view schematic illustration of a multi-specimen storage vessel with a blood sample in the first portion.

Figure 14 shows a side-view schematic illustration of a multi-specimen storage vessel with blood cells in the first portion and plasma in the second portion.

Figure 15 shows a side-view schematic illustration of a multi-specimen storage vessel with blood plasma in the manifold.

Figure 16 shows a side-view schematic illustration of a multi-specimen storage vessel with blood plasma in the manifold and in the plurality of storage specimens.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1A shows a breakable multi-specimen storage vessel 2 of the invention which is suitable for filtering and containing a collective sample of a biological fluid sample storage at cryogenic temperatures, as will be explained in more detail below. It is noted that the vessel may be suitable for use in other applications, for example filtering and storing household or industrial cooking stock, as the vessel is used for storing a fluid, which is fluid at room temperature, but solid at temperatures around 5 °C, or for storing water bound samples, where the fluid is crystalline at -3 °C, but fluid above. Further, the samples kept within the vessel may be non-fluid and/or non-biological, depending on the application.

The storage vessel 2 comprises a tubular container 4 having a container bottom 22 at a closed lower end thereof and an open upper end 24 provided with a cap 12, with a filter 26 separating the container into a first volume 28 and a second volume 30. The first portion 27 of the container comprises the first volume 27 and the filter 26; the second portion 29 comprises substantially all of the second volume 30. The second portion 29 of container 4 in Figure 1 is provided with four externally located ring-shaped break portions 6 on a perimeter thereof which provides the possibility of breaking off four specimens 10 in total from the vessel 2 comprising the collective sample. The break portions 6 are formed as predetermined areas of weakness extending approximately partly through the thickness of the wall material of the tubular container 4. The plurality of break portions 6 is formed along the length of the container 4 comprising the second portion 29. The break portions 6 separate the multi-specimen container 4 into individual specimens 10, which by breaking can be controllably separated from the remainder of the collective sample as needed. The container wall interior opposite the break portions 6 has a longitudinally plane, smooth surface in order to provide as large an interior volume as possible and for facilitating low- cost production thereof. By the term smooth it is meant that the surface provided is substantially without recesses and/or projecting parts outside production tolerances, such as less than a few hundredth to less than a few thousandth of the wall thickness. The break portions 6 are designed to facilitate breakage of the container 4 at the break portions 6 since they constitute reduced wall thickness areas of the container 4. It is contemplated that the predetermined areas of weakness may be constructed in other ways to achieve the same result. Further, said container 4 is provided with parts 8, 8' of a fastening mechanism which allows a cap 12 to be removably secured on the open upper end 24 of the container 4. In the illustrated embodiment, the parts 8, 8' are the male parts of bayonet mounts which may interact with the female parts of bayonet mounts on the inside of the cap 12 to removably secure it on the container 4. Parts 8, 8' of a fastening mechanism are also provided on the outer surface of the container 4 adjacent to each break portion 6 to allow the securing of a cap 12 on the open ends of specimens 10 after breaking of a break portion 6. Further detail is found in the discussion relating to Figure 2.

The plurality of break portions 6 may be formed along the length of the container 4 comprising the second portion 29, as illustrated in Figure 1 , and alternatively or additionally along the first portion 27. The distance between specimens may be selected appropriately having regard to the intended use, specimen volume, and user needs. They may be of equal length ranging from between about 1 to about 100%, preferably from between about 10 to about 50%, more preferably from about 33 to about 40% of the length of first volume 28 or second volume 30 of the container 4. The length of a specimen may be selected in many uses to correspond to about 0.1 to about 0.5 mL volume of sample within the container, depending of course on the container diameter at hand, which in principle may be any diameter at hand, but in practice, in particular when applied to cryo tubes, often ranges from 1 mm to 50 mm in diameter. Any number of break portions needed for any type of application is conceivable. Also, different numbers of break portions between the first portion 27 and the second portion 29 are conceivable, as are differing lengths of a specimen between the first portion 27 and the second portion 29. Non-equally distanced break portions are also conceivable for the first portion 27 and/or the second portion 29, e.g. for special applications using increasing or decreasing amounts of volume for each test sample. The bottom 22 of the container 4 shown in Figure 1 gently curves inwards to form a rounded bottom such as semicircular, convex, cone shaped or pyramidal, in order to provide space e.g. for housing a needle end. The container bottom wall in the container bottom 22 extends beyond the outer surface of the bottom 22 in order to provide stability, if vessel 2 is placed on a plane surface.

In an alternative arrangement, the storage container may be provided with both ends open, rather than with a closed bottom 22 at the lower end as in Figure 1. Preferably, part of a fastening mechanism is provided at each end of the container to allow the removable securing of closure caps or reversible coupling to a manifold, which is described in further detail in relation to Figures 8 to 10. In this arrangement, it is particularly preferable for at least one of the closure caps to be adapted to allow the vessel to stand stably on one end. The storage vessel 2 may be broken into two or more specimens during use. The container 4 is designed so that a radially directed manual or machine-operated force will break the container 4 along one of the pre-defined break portions 6. Further, the container 4 is designed such that when the container 4 is divided in a manner that leaves both the lower part and the upper part with a new open end, see Figure 2, two caps 12 can be attached to these open ends of the container 4.

The first volume 28 and/or second volume 30 of container 4 may contain an anticoagulant to prevent clotting when a sample of blood is introduced into the container 4. The anticoagulant may be selected from any known in the art, and is preferably selected from heparin, oxalic acid, citric acid, ethylenediaminetetraacetic acid, or salts thereof.

Figure 1 B is another embodiment of a container 7 of the invention, which is similar to the container 4 shown in Figure 1 A. Whilst the container 4 is constructed in an integral manner (having a substantially unitary structure), the container 7 is manufactured in 2 parts, namely an upper portion 27 and a lower portion 29. The upper portion 27 defines a first volume 28 of the container and comprises a filter 26. The lower portion 29 defines a second volume 30 with a number of specimen segments 10 separated by areas of weakness 6 as described above. In another embodiment, the lower portion defines a second volume 30 with only a single specimen (i.e. with no pre-defined break points 6), and may additionally provide at its lower end means for the fastening of a cap, manifold or needle, as is described in more detail in relation to Figures 8 to 10. The lower portion 29 is provided at its upper end with an upwardly projecting circumferential collar 5 which allows the lower portion 29 to be coupled to the upper portion 27. Other means of coupling the lower portion and upper portion together may be used.

So, the lower portion 29 and the upper portion 27 are manufactured separately, and then coupled together, provided with a cap, and also, if desired, provided with a sub- atmospheric pressure. This method of manufacturing the container can provide advantages over the manufacture of a unitary container.

Figure 1 C shows a container 9 of the invention which is similar to the container 4 described above. However, the container 9 is provided with specimen portions 10 in the upper portion 27 in addition to the lower portion 29. In the embodiment shown, a coating 32 is provided in each of the specimen portion 10 in the upper portion 27 of the container 9. In use, this allows for a sample in the upper portion 27 of the container 9 to be frozen and subsequently broken into individual specimens as desired. In addition, each of the individual specimen sections 10 is provided with a coating 32 to facilitate analysis or an assay to be performed. The coatings 32 in each specimen 10 may be the same or different.

Figure 2 shows an individual tubular specimen 10 isolated from the container 4. A pin 8 is provided on the outer surface of the specimen 10 adjacent to one open end, and a pin 8' is provided on the outer surface adjacent to the other open end. Pin 8' is provided with a protrusion 16 whereas the surface of pin 8 is substantially flat. Even though the pins 8, 8' are different, they match the same female part of a bayonet mount, which is provided in closure caps 12. The closure caps 12 are adapted to engage with the open end of the container 4 and with broken off specimens from the container 4, e.g. the specimen 10 illustrated in Figure 2 by means of a bayonet mount. The female part of the bayonet mount in the closure cap 12 comprises a groove 14 adapted to both pins 8, 8'. A closure cap is mounted by fitting the pins into the groove 14 and subsequently rotating the closure cap 12 and the specimen 10 (or container 4) in relation to each other. The pins 8, 8' then follow inside the groove 14 to couple the specimen 10 and cap 12 in a closed configuration. However, in this closed configuration the protrusion 16 on the pin 8' engages with protrusion 18 in groove 14 and provides a locking effect of the cap.

The locked configuration occurs with the two engaged protrusions 16, 18 aligned against each other. A closure cap 12 mounted on the opposite end of the specimen 10 with the pin 8 (without protrusion) also provides a closed and tight configuration between the cap 12 and specimen 10. However, with the substantially planar surface of the pin 8 there is no locking effect and the cap 12 can be detached. Thus, if a specimen 10 is provided with closure caps 12 at both ends and one cap is detached by holding the other cap, the cap which is mounted on the end of the specimen 10 with the pin 8 will open because the other cap is locked. When a specimen 10 is closed by two caps it may be difficult to distinguish the different fastening mechanism. However, by somehow indicating this difference on the surface of the specimen 10 (e.g. by means of an arrow or a simple marking) a user accessing a sample inside the specimen is sure to be able to keep the specimen in the correct vertical orientation and concurrently open the correct cap.

The interchanging pins 8, 8' are illustrated along the length of the container 10 in Figure 1 and it can be also seen that two pins 8, 8' are located opposite each other across a break portion 6 and pins are provided for every 180 degrees round the perimeter of the container 4. Thus, four pins (two of type 8 and two of type 8') are provided adjacent to each break portion 6 in the embodiment shown. As seen in these Figures, the cap 12 may be provided with indentations or knurling on the outer surface to provide a better grip when mounting and detaching the cap.

Figure 2 shows a single specimen 10 that has been broken from the storage vessel of Figure 1. A biological sample is not shown within the storage vessel 2 and specimen 10. However, it may advantageously be used for storing a fluid biological collective sample (not shown) filling substantially the entire inside volume of the container 4. Further, the vessel comprising the collective sample may preferably be frozen, e.g. for cryogenic use, in order to provide a clean surface when the specimen is broken off. When broken in a frozen condition, each broken off part will ideally contain frozen specimens, where the exposed surface thereof lies in a substantially flat planar perpendicular relationship to the outer wall surface of the container 4.

The depth of a recess of a break portion 6 is preferably selected relative to the hardness of the material of the container 4 in such a way that both safe storage and handling, and an easy break operation is achieved. The depth of a recess may range between from about 5 to about 95%, preferably from about 50 to about 95%, more preferably from about 75 to about 95% of the total wall thickness of the tubular container 4, depending on the container material selected. A remaining wall thickness of about 5 to about 25% is sufficient for maintaining container stability and securing handling. The shape of the recess may be v- shape, u-shape, ]-shape or any other appropriate shape, and/or may differ or be of uniform shape along the container 4.

The storage vessel, e.g. the tubular container 4 and one or more caps 12, should all be made of materials which can withstand deep freezing temperatures and which have got reduced resistance against radial breakage at least deep frozen. In general, a chemical resistant material is preferred, where some preferred materials include plastic materials such as polypropylene (PP), polyethylene (PEHD), polystyrene, or polycarbonate, but some glass materials resistive to temperature variances may also come into use. Preferably, the material is polypropylene, more preferably Bormed RF830MO polypropylene available from Borealis AG. The caps and/or the container may further comprise rubber or plastic gaskets suitable for sealing during cryogenic temperatures.

The material used for the tubular container 4 may preferably be chosen as to be easily mouldable and/or workable for providing break portions, such as cuts and/or threads therein, which are both durable during storage and handling, and easily breakable during dividing. The material may then preferably be chosen as polypropylene, because this material has increased brittleness during freezing temperatures.

Further, in order to provide excellent security against spillages, the cap 12, at least in room temperatures, may be of a more or less resilient and/or more or less hard material than the container 4, or vice versa. The material of the cap 12 may be softer than the material of the container 4 at room temperature and/or during breakage temperature, such as cryogenic temperatures, i.e. around -70°C, or even higher temperatures, e.g. around 0°C, or higher yet. Further, the material of the cap 12 may be harder than the material of the container 4 at room temperature and/or during freezing temperature. That is to say that the hardness of the material of the cap 12 and/or container 4 may be chosen as to ease the application and detachment of the cap 12 from the container 4, while at the same time provide a secure fit there between. In order to increase readability of the volume or ID markings upon the container 4, the cap 12 or caps may be provided in a transparent material.

The container may be produced by moulding, e.g. blow or injection moulding or the like, as is known to the skilled person, and different elements of the container, such as the threads, the markings, the recesses and/or the side extensions may be provided at the same time or machined after moulding. If more than one material is needed, e.g. two materials of different hardness, multi component injection moulding is a good choice.

The pre-defined break portions 6 of the container 4 may be specifically indicated, e.g. using peripheral colour line markings, metal or magnetic band marking, e.g. for use in further processing, or the like, for a further visual indication of the position of the break portion. A storage vessel according to the invention is designed to be broken off into specimens using a manual break operation, but may also broken using a break tool, in which case, the risk of breakage in a wrong position or damage to the container is decreased. Examples of such possible break tools are described in WO 2009/086829.

During use, the cap 12 is applied, for example during a frozen state by mounting the cap 12. Then the cap 12 and container 4 is held by the user in each hand and broken into two parts by using the necessary break force. Other alternatives are conceivable, for example a break tool is held fixed against a surface, such as a table or a wall, and the user breaks the specimen off using manually applied force, or the breaking off is performed automatically or manually using a force providing means, such as a motor operated winch, pawl or pin (not shown).

Figure 3 shows a variation of the vessel 2 of Figure 1 , vessel 2', wherein the first portion 27 is provided with four internally located ring-shaped coating zones 32 on the internal surface thereof which may interact with a sample in the first volume 28. Advantageously, the coating zones 32 may be internally located on the first portion 27 and/or the second portion 29 of the container 4 which may interact with a sample in the first volume 28 and/or the second volume 30. The number of coating zones 32 located internally on the first portion 27 and/or the second portion 29 may be selected independently from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 (i.e. the number of coating zones internally provided on first volume 28 and the second volume 30 of the container 4 may be different).

The coating zones 32 may comprise one or more components which form part of one or more assays for in-situ analysis of the sample.

Alternatively, or in addition to a coating, the vessel of the invention may contain a reagent. The reagent may be a component of an assay, or may be a material which prepares the sample for an assay or analysis. Also, the vessel may contain other material to help the stabilisation and/or storage of the sample. This may include, for example, inhibitors of enzymes that would otherwise degrade components of the sample. The reagents may help with the stabilisation and/or storage of components such as cells, proteins, peptides, hormones and/or nucleic acids. Figure 4 shows the storage vessel 2 of Figure 1 containing a sample 34 (illustrated as a sample of blood), in the first volume 28 of the container 4 above the filter 26. The sample 34 may be introduced into the first volume 28 of the container 4 through the open end 24 of the container 4 in the absence of a cap 12. Alternatively, a portion of the cap 12 may comprise a reversibly sealing material suitable for low temperature application, for example rubber or Suba-Seal ®, allowing the direct introduction of the sample, e.g. with a needle, into the first volume 28 of the container 4 without the need to remove the cap 12. More advantageously, the first volume 28 and/or second volume 30 of the container is at sub- atmospheric pressure, allowing direct introduction of the sample 34, e.g. from a syringe via a needle, into the first volume 28 without the need to equalise the pressure inside the container 4 with the external atmosphere. These embodiments advantageously minimise the risk of ingress of contaminants, for example microorganisms or air, into the container 4, which might otherwise lead to the degradation of the sample or interfere with any subsequent analysis.

After the introduction of the sample 34 into the first volume 28 of the container 4 (shown as a sample of blood in Figure 4), a cap 12 may be affixed to the open end 24 of the container 4 (if not already in place), followed by centrifugation of the vessel 2. Centrifugation should be carried out to provide a longitudinal force on the sample 34 in the direction of the filter 26, causing smaller components to pass through the filter 26 into the second volume, separating them from the larger components which are unable to pass through the filter. Smaller components are those which are able to pass through the filter mesh, whereas larger components are those which are unable to pass through the filter mesh.

Figure 5 shows the storage vessel 2 of Figure 4 after centrifugation, wherein the sample 34 has been separated into the filtrate 36 (illustrated as blood plasma) which has passed through the filter 26 into the second volume 30 of the container 4, and the retentate 38 (illustrated as the remaining blood components, e.g. red blood cells, white blood cells and platelets) which have remained in the first volume 28 of the container 4 due to being too large to pass through the filter 26.

The size of the mesh of the filter 26 may be chosen according to the nature of the specific sample and the separation needs of that sample. For example, in the separation of blood samples, the filter may be chosen to separate white blood cells from the remainder of the blood, or to separate white blood cells and red blood cells from the platelets and plasma, or to separate the plasma from the remainder of the blood. Consequently, the filter mesh size may preferably be between from about 0.1 to about 50 μηη, or preferably between from about 0.5 to about 1 .5 μηη, where the lower limit is selected from about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1.5, 2.0. 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, 45 and 50 μηη and the upper limit is selected from about 1 .0, 1.1 , 1 .2, 1.3, 1.4, 1 .5, 2.0, 2.5, 3.0, 4.0, 5.0, 7.0, 8.0, 9.0, 10.0, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200 and 250 μηι.

The filter 26 may be made from any suitable material known in the art, giving regard to the need for compatibility with the intended sample and the temperature conditions that are likely to be experienced during, for example, storage. Materials used in filters for the separation of blood may, for example, be polyvinylidenefluoride, polyamide 6.6, polyethylene and polyethersulfone. After centrifugation, the container 4 may be frozen, with the retentate 38 contained in the first volume 28 of the container 4 and the filtrate 36 contained in the second volume 30 of the container 4. Either before or after freezing, the container 4 may be broken along a break portion 6 to divide the storage vessel 2 into the first portion 27 substantially comprising the first volume 28, filter 26 and retentate 38, and into the second portion 29 substantially comprising the second volume 30 and the filtrate 36. Caps 12 may be used to seal the revealed open ends of the two specimens, allowing the first portion 27 and the second portion 29 to be dealt with independently. For example, one portion may be frozen and stored whereas the other portion may be disposed of.

According to another embodiment of the invention, the storage vessel may have a modular design, allowing the interchange of different components. Figure 6 shows the storage vessel 40 of the invention comprising a first portion 44 of the container 42 provided with a cap 46, and a number of different second portions 48, 50 and 52 of the container 42 which may be reversibly coupled to the first portion 44 by means of a fastening mechanism 54. The first portion 44 comprises the first volume 56 of the container 42 and the filter 58. The second portion 60 comprises substantially all of the second volume 62 of the container 42. An advantage of this modular design is that it allows the independent selection of first portion 44 and the second portion 60. Depending on the application, the first portion 44 may be selected to optimise the filter material and mesh size, as well as the number and spacing of break portions 64. The second portion 60 may be selected to optimise the number and spacing of break portions 64, the volume of each specimen 66, and even the diameter of the second volume 62. For example, in situations where it is expected that there will be a substantial volume of retentate relative to filtrate, a tapered second portion (as illustrated in second portion 52) may be employed to allow subsequent subdivision of the filtrate into a suitable number of specimens 66. On the other hand, for example, in situations where it is expected that there will be a substantial volume of filtrate relative to retentate, a second portion with additional length (as illustrated in second portion 50 compared to second portion 48) may be used.

The fastening mechanism 54 allowing reversible coupling of the first portion 44 to the second portion 60 may be any suitable fastening mechanism known in the art, preferably a snap-fit connection, a bayonet connection or a screw threaded connection. According to another embodiment of the invention, as shown in Figure 7, the storage vessel 68 may additionally comprising a radio-frequency identification (RFID) tag 70 embedded within the bottom 72 of the container 74. The RFID tag 70 may be introduced during the moulding process and serves to provide a means of uniquely identifying each vessel 68. The RFID tag 70 may be read-only, having a factory-assigned serial number that is used as a key into a database, or may be read/write, where object-specific data can be written into the tag by the system user. The RFID tag 70 is preferably located in the bottom 72 of the container 74, but may be located anywhere within the vessel 68, including within the cap 76. Further, RFID tags 70 may be located within each specimen 76 of the container 74.

Other forms of identification may be used, such as bar codes, QR codes and serial numbers. An outer surface of at least a part of said container 74 or specimen 78 may further include information or indicia such as markings identifying at least the specimen 78 taken, and/or identifying which vessel said specimen has been taken from, e.g. a three to give digit (number, letter, symbol) code or codes in sequence extending peripherally and/or longitudinally along the longitudinal side thereof or the like. When being delivered in a collection of 100 to 1000 pieces of such storage vessel, the sequence of digits is preferably selected in such batch as not to result in any duplicate digit combination thereon. Thus, the risk of mix-up between specimens broken off and the remainder of the vessel before labelling thereof has been performed may be reduced, as it could happen in the case of dropping or misplacing one or more of said specimens.

Further, at least part of an outer surface of container 74 and/or the outer surface of cap 76 may be provided with volume indication markings of the volume within, in sequence or using simple perimeter line markings, as is known to the skilled person. They may correspond to relatively small volumes, such as 0.1 mL each or larger volumes, such as from 0.1 mL up to 100 mL, depending on the length and diameter of the container being used.

Further, the vessel may be provided with further information, such as trademarks, producer name, and the like. The markings may include a planar longitudinally extending section for provided an adhesive ID label or barcode, e.g. for individual specimen identification, date and/or user initials. The different types of markings may for example comprise written information, a number, barcode, and/or sign indication sequence, or any combination thereof, also stating production info, producer ID, and may be provided by labelling, moulding, etching, cutting or milling.

Alternatively or additionally to the specimens provided as part of the container, the vessel may also comprise a manifold to allow the distribution of filtrate that has been filtered through the container into individual specimens. In general, the manifold comprises: an inlet suitable for reversibly coupling to the container; a chamber suitable for containing filtrate that has been filtered through the container; and one or more outlets suitable for reversibly coupling to a plurality of storage specimens to permit transfer of filtrate from the chamber into a specimen. The manifold is described in further detail in the discussion relating to Figures 8 to 14.

Figure 8 shows the storage vessel 80 comprising a tubular container 82 having a container bottom 84 at a lower end thereof and an open upper end 86 provided with a cap 88, with a filter 90 separating the container into a first volume 92 and a second volume 94. The container bottom 84 is at least partially open and provided with means to allow the container to be reversibly coupled to a manifold 96, cap and/or needle.

In the illustrated embodiment, the container bottom 84 is provided with a narrowed protruding portion 98, such as a Luer lock or a Luer slip fitting, to allow a push-fit connection with the manifold 96 or a needle. Alternatively, the container bottom 84 may be provided with parts of a fastening mechanism, as described in relation to Figures 1 A and 2, to allow the reversible coupling of either a cap or the manifold 96. In the illustrated embodiment, the inlet of the manifold comprises a narrowed depression, such as a female Luer lock or Luer slip fitting, which allows the reversible coupling of the narrowed protruding portion 98 of the container bottom 84. However, as will be described in relation to the other Figures, the inlet of the manifold may comprise alternative means suitable for reversibly coupling to the container, such as other push-fit connections, a snap- fit connection, a bayonet connection, or a screw threaded connection.

When the container 82 and manifold 96 are reversibly coupled together, filtrate may be transferred from the second volume 94 of the container 82 into the chamber of the manifold 96 by gravity or by centrifugation. In another embodiment (not shown), the cap 88 may be replaced with a plunger, which, when moved into the container 82, forces filtrate from the container 82 into the chamber of the manifold 96. The movement of the plunger into the container 82 may also expel filtrate from the chamber of the manifold 96 through an outlet, and, if reversibly coupled to the manifold, into a storage specimen.

Figure 9 shows the container 4 of Figure 1A in which the terminal break portion has been broken and the terminal specimen (comprising the container bottom 22) removed. The container is provided with parts of a fastening mechanism 8' which allows the open lower end 100 of the container 4 to be reversibly coupled to a manifold 102.

In the illustrated embodiment, the parts 8' are the male parts of bayonet mounts which may interact with the female parts of bayonet mounts on the inside of the manifold 102 to removably secure the manifold 102 to the container 4. Further detail of the bayonet mounts is found in the discussion relating to Figure 2.

Figure 10 shows the manifold 96 of Figure 8 in more detail, comprising a chamber 104, an inlet 106 and six outlets 108. The inlet 106 of the manifold 96 is configured so that it may be reversibly coupled to the partially open container bottom (e.g. 84 of Figure 8) or the open lower end of the container (e.g. 100 of Figure 9), and to permit the transfer of filtrate from the container 4, 82 to the chamber 104 of the manifold 106. The body of the manifold is preferably formed from a chemical resistant material, where some preferred materials include plastic materials such as polypropylene (PP), polyethylene (PEHD), polystyrene, or polycarbonate, but some glass materials resistive to temperature variances may also come into use. Preferably, the material is polypropylene, more preferably Bormed RF830MO polypropylene available from Borealis AG.

In the illustrated embodiment, the inlet 106 of the manifold comprises a female Luer lock or Luer slip connector which is suitable for reversibly coupling to the male Luer lock or Luer slip connector of the partially open container bottom 84. However, the inlet of the manifold may alternatively comprise the female part of a bayonet mount, comprising a groove suitable for engagement with the pins located on the outer circumference of the open lower end of the container (e.g. 100 of Figure 9), as is described in detail in relation to the closure cap 12 of Figure 2, or means for an interference fit. Alternatively, the inlet of the manifold may comprise a reversibly sealing material, for example rubber or Suba-Seal ®, allowing the direct transfer of filtrate from the container, e.g. with a needle.

The chamber 104 of the manifold 96 is of sufficient volume to contain substantially all the filtrate from the container, or even from more than one container. For example, the chamber 104 of the manifold 96 may be of sufficient volume to permit the transfer and mixing of filtrate from a number of containers.

When the manifold 96 is in the desired orientation for use, the outlet 108 is configured to be in fluid communication with the chamber 104, even when the chamber 104 is substantially empty. For example, in the illustrated embodiment, the outlets 108 are located on the upper surface 1 10 of the manifold 96, with each outlet 96 further comprising a tube 1 12 extending from the outlet 108 inside the manifold 96 towards the bottom of the chamber 104. The application of negative pressure to an outlet 108 will therefore allow filtrate to be drawn from the chamber 104 through the tube 1 12, before being expelled via the outlet 108, even when the filtrate level in the chamber 104 is low.

In some embodiments (not shown), there are no tubes 1 12 extending from the outlet 108 into the chamber 104. For example, the outlets may be located on the lower surface of the manifold 96 when the manifold is in the desired orientation for use. In this configuration, fluid communication between the outlet 108 and the chamber 104 is maintained by gravity.

The exterior of each outlet 108 may be provided with means to allow it to be reversibly coupled to a specimen. For example, an outlet 108 may be provided with a male Luer lock or Luer slip connector to permit the reversible attachment of a needle. Alternatively, as shown in the illustrated embodiment, an outlet 108 comprises a fixed needle 1 16.

Each outlet 108 may be integrally formed from the same material as the manifold, such as from chemical resistant plastic materials such as polypropylene (PP), polyethylene (PEHD), polystyrene, or polycarbonate. However, the outlet 108 may also be formed from other materials, such as metal, for example when the outlet 108 comprises a fixed needle 1 16.

As shown in Figure 1 1 , the manifold 96 may be cylindrical in shape. However, it may also be formed in any other suitable shape, for example cuboid, frustrum or prism. The inlet (not shown) may be located on one planar surface of the manifold and an outlet located on the other planar surface of the manifold. Alternatively, the inlet may be located on a curved surface of the manifold, or on the same planar surface as the outlet. In the illustrated embodiment, the six outlets 108 are arranged in a circular manner. Alternatively, where there is more than one outlet, the outlets may be arranged in any other suitable configuration, for example in a single line, around the perimeter of a shape (such as a triangle, square, or irregular shape), or spaced equally within the boundary of a shape (such as a triangle, square, or irregular shape).

Figure 12 shows another embodiment of the manifold 1 18, in which the manifold 1 18 is substantially cuboid in shape and in which the six outlets 120 are arranged in a linear manner on its upper face 122.

In embodiments of the invention where the container is not provided with predetermined break points and is not adapted to be broken into a plurality of storage specimens, such as in the embodiment illustrated in Figure 8, the vessel further comprises a plurality of storage specimens extraneous to the container. In embodiments of the invention where the container is provided with predetermined break points and is adapted to be broken into a plurality of storage specimens, such as in the embodiment illustrated in Figure 9, the vessel may also further comprise a plurality of storage specimens extraneous to the container. These storage specimens are discussed in further detail in relation to Figures 13 to 15.

Figure 13 shows the storage vessel 80 of Figure 8 containing a sample 124 (illustrated as a sample of blood), in the first volume 92 of the container 82 above the filter 90. The sample 124 may be introduced into the first volume 92 of the container 82 through the open upper end 86 of the container 82 in the absence of a cap 88. Alternatively, a portion of the cap 88 may comprise a reversibly sealing material suitable for low temperature application, for example rubber or Suba-Seal ®, allowing the direct introduction of the sample (e.g. with a needle, into the first volume 92 of the container 82 without the need to remove the cap 88.

After the introduction of the sample 124 into the first volume 92 of the container 82 (shown as a sample of blood in Figure 13), a cap 88 may be affixed to the open end 86 of the container 82 (if not already in place). A stopper 126 may also be applied to the narrowed protruding portion 98 of the container bottom 84 (shown as a Luer lock stopper in Figure 13), followed by centrifugation of the vessel 80. Alternatively, in place of a stopper 126, the manifold 96 may be reversibly coupled to the container bottom 84 (not shown), followed by centrifugation of the vessel. Centrifugation should be carried out to provide a longitudinal force on the sample 124 in the direction of the filter 90, causing smaller components to pass through the filter 90 into the second volume 94 (or into the manifold 96, if reversibly coupled to the container 82), separating them from the larger components which are unable to pass through the filter. Smaller components are those which are able to pass through the filter mesh, whereas larger components are those which are unable to pass through the filter mesh.

Figure 14 shows the storage vessel 80 of Figure 13 after centrifugation, wherein the sample 124 has been separated into the filtrate 128 (illustrated as blood plasma) which has passed through the filter 90 into the second volume 94 of the container 82, and the retentate 130 (illustrated as the remaining blood components, e.g. red blood cells, white blood cells and platelets) which have remained in the first volume 92 of the container 82 due to being too large to pass through the filter 90.

The stopper 126 has been removed from the narrowed protruding portion 98 of the container bottom 84, and the manifold 96 has been reversibly coupled (illustrated as push- fit Luer slip connections). Additionally, the cap 88 has been removed from the upper end 86 of the container 82 and replaced with a plunger 132.

Figure 15 shows the storage vessel 80 of Figure 14 after insertion of the plunger 132 into the first portion 92 of the container 82, wherein the filtrate 128 (illustrated as blood plasma) has passed from the second volume 94 of the container 82 through the narrowed protruding portion 98 of the container bottom 84 and into the chamber 104 of the manifold 96. In some configurations, further insertion of the plunger into the first portion 92 of the container 82 may be used to prime the outlets 108 by expelling any air from the chamber 104, outlets 108 and fixed needles 1 16. Also illustrated is a plurality of empty storage specimens 134 which are at sub-atmospheric pressure and comprise a pierceable covering comprising a reversibly sealing material.

The storage specimens may have a volume appropriate to the intended use and user needs. For example, each of the storage specimens may have a volume of about 0.1 to about 0.5 ml_, particularly when applied to cryo tubes. However, volumes less than about 0.1 mL and greater than about 0.5 mL are also envisaged, depending on the intended use. Each of the storage specimens may have the same volume. Alternatively, storage specimens of different volumes are also conceived e.g. for special applications using increasing or decreasing amounts of volume for each test sample.

The storage specimen is provided with means to allow each to be reversibly coupled to an outlet of the manifold. In the illustrated embodiment, the storage specimen comprises a pierceable covering comprising a reversibly sealing material, for example rubber or Suba-Seal ®, allowing the direct transfer of filtrate from the manifold, e.g. with a needle. In other embodiments, the storage specimen may comprise a hinged push-fit cap (such as in an Eppendorf ® tube), a screw-fit cap or a crimp seal (such as in an HPLC vial).

In the illustrated embodiment, the storage specimen is at sub-atmospheric pressure, allowing direct introduction of the filtrate into the storage specimen without the need to equalise the pressure inside the storage specimen with the external atmosphere, and without the need to exert a positive pressure on the chamber of the manifold to expel filtrate through an outlet. These embodiments advantageously minimise the risk of ingress of contaminants, for example microorganisms or air, into the manifold or storage specimen, which might otherwise lead to the degradation of the sample or interfere with any subsequent analysis.

Figure 16 shows the storage vessel 80 of Figure 15 after insertion of each of the fixed needles 1 16 of the manifold 96 into a storage specimen 134. Due to the sub-atmospheric pressure in each storage specimen 134, filtrate 128 (illustrated as blood plasma) has been drawn from the chamber 104 of the manifold 96 through each outlet 108 comprising a fixed needle 1 16 and into the storage specimen 134. In some embodiments, there is a sufficient volume of filtrate 128 remaining in the chamber 104 of the manifold 96 to allow the filling of one or more further sets of storage specimens 134.

Embodiments of the invention are set out in the following numbered paragraphs:

1 . A breakable multi-specimen storage vessel comprising a container having predetermined break points whereby the container is adapted to be broken into a plurality of specimens and wherein the container comprises a first volume and a second volume separated by a filter.

2. A vessel according to numbered paragraph 1 wherein the filter mesh size is from 0.1 to 50 μηι, preferably from 0.5 to 1 .5 μηι.

3. A vessel according to numbered paragraphs 1 or 2 wherein the filter is suitable for separating blood components.

4. A vessel according to any preceding numbered paragraph wherein an anticoagulant is present in the first volume and/or the second volume of the container.

5. A vessel according to numbered paragraph 4 wherein the anticoagulant is selected from heparin, oxalic acid, citric acid, ethylenediaminetetraacetic acid, or salts thereof. 6. A vessel according to any preceding numbered paragraph wherein the first volume and/or the second volume of the container is at sub-atmospheric pressure.

7. A vessel according to any preceding numbered paragraph wherein the predetermined break points are provided with a fastening mechanism for the attachment of a cap.

8. A vessel according to any preceding numbered paragraph comprising a first portion of the container comprising the filter and a second portion of the container reversibly coupled together.

9. A vessel according to numbered paragraph 8 wherein the first portion and second portion are coupled together by a snap-fit connection, a bayonet connection or a screw threaded connection. 10. A vessel according to numbered paragraph 8 or 9 wherein the first portion of the container and second portion of the container are of different volumes. 1 1. A vessel according to any preceding numbered paragraph wherein part or all of the inner surface of the container comprises one or more coatings.

12. A vessel according to numbered paragraph 1 1 wherein the one or more coatings comprise a component of one or more assays.

13. A vessel according to numbered paragraph 12 wherein the component is suitable for the analysis of blood.

14. A vessel according to any preceding numbered paragraph further comprising a reagent for an assay.

15. A method of filtering blood, comprising the steps of:

providing a sample of blood in a vessel as defined in any of the previous claims; and centrifuging the vessel such that at least a portion of the blood passes through the filter.

16. A method of according to numbered paragraph 15 wherein centrifuging the vessel leads to substantially all of the blood plasma passing through the filter.

17. A method according to numbered paragraph 15 or 16 further comprising the step of freezing the vessel, or a portion thereof.

18. A method of analysing a sample of blood, comprising the steps of:

providing a sample of blood in a vessel as defined in any of numbered paragraph 1 1 to 14; allowing the blood sample to interact with the one or more coatings; and

performing analysis of the sample and/or the one or more coatings.

Claims

1 . A multi-specimen storage vessel comprising:

a. a container comprising a first volume and a second volume separated by a filter; and

b. a plurality of storage specimens.

2. The vessel according to claim 1 wherein the container has predetermined break points and is adapted to be broken into the plurality of storage specimens.

3. A vessel according to any preceding claim wherein the first volume and/or the second volume of the container is at sub-atmospheric pressure.

4. A vessel according to any preceding claim wherein the predetermined break points are provided with a fastening mechanism for the attachment of a cap.

5. The vessel according to claim 1 comprising a manifold having a single inlet and one or more outlets.

6. The vessel according to claim 5 wherein the inlet of the manifold is reversibly coupled to the container.

7. The vessel according to claims 5 or 6 wherein an outlet of the manifold is reversibly coupled to a specimen.

8. The vessel according to claims 6 or 7 wherein the inlet of the manifold and the container are reversibly coupled by a push-fit connection, a snap-fit connection, a bayonet connection, or a screw threaded connection.

9. The vessel according to claims 6 to 8 wherein an outlet of the manifold and a specimen are reversibly coupled by a push-fit connection, a snap-fit connection, a bayonet connection, or a screw threaded connection.

10. The vessel according to claims 6 to 8 wherein an outlet of the manifold comprises a needle, and wherein a specimen comprises a pierceable cover.

1 1. The vessel according to claims 5 to 10 wherein each of the plurality of storage specimens is at sub-atmospheric pressure.

12. The vessel according to claims 5 to 1 1 wherein the volume of the manifold is greater than the volume of the first volume of the container.

13. The vessel according to claims 5 to 12 wherein the volume of the manifold is greater than the volume of the second volume of the container.

14. The vessel according to claims 5 to 13 wherein the volume of the manifold is greater than the volume of a specimen.

15. The vessel according to claims 5 to 14 wherein the outlets of the manifold are in a linear arrangement

16. The vessel according to claims 5 to 15 wherein the outlets of the manifold are in a circular arrangement.

17. A vessel according to any preceding claim wherein the filter mesh size is from 0.1 to 50 μηι, preferably from 0.5 to 1.5 μηι.

18. A vessel according to any preceding claim wherein the filter is suitable for separating blood components.

19. A vessel according to any preceding claim wherein an anticoagulant is present in the first volume and/or the second volume of the container.

20. A vessel according to claim 19 wherein the anticoagulant is selected from heparin, oxalic acid, citric acid, ethylenediaminetetraacetic acid, or salts thereof.

21. A vessel according to any preceding claim comprising a first portion of the container comprising the filter and a second portion of the container reversibly coupled together.

22. A vessel according to claim 21 wherein the first portion and second portion are coupled together by a snap-fit connection, a bayonet connection or a screw threaded connection.

23. A vessel according to claim 20 or 21 wherein the first portion of the container and second portion of the container are of different volumes.

24. A vessel according to any preceding claim comprising a plunger.

25. A vessel according to any preceding claim wherein part or all of the inner surface of the container comprises one or more coatings.

26. A vessel according to claim 25 wherein the one or more coatings comprise a component of one or more assays.

27. A vessel according to claim 26 wherein the component is suitable for the analysis of blood.

28. A vessel according to any preceding claim further comprising a reagent for an assay.

29. A method of filtering blood, comprising the steps of:

providing a sample of blood in a vessel as defined in any of the previous claims; and

centrifuging the vessel such that at least a portion of the blood passes through the filter.

30. A method of filtering blood, comprising the steps:

providing a sample of blood in a vessel as defined in claim 24; and moving the plunger in the vessel such that at least a portion of the blood passes through the filter.

31 . A method of according to claims 29 or 30 wherein centrifuging the vessel or moving the plunger leads to substantially all of the blood plasma passing through the filter.

32. A method according to claim 29 to 31 further comprising the step of freezing the vessel, or a portion thereof.

33. A method of analysing a sample of blood, comprising the steps of:

providing a sample of blood in a vessel as defined in any of claims 25 to 28; allowing the blood sample to interact with the one or more coatings; and performing analysis of the sample and/or the one or more coatings.