DE202014010872U1 - Composite release agent for blood collection tubes - Google Patents

Abstract

A sample collection tube comprising: a tube having a lumen; and a separation substance disposed within the lumen, comprising: a gel component layer material, the gel component comprising a thixotropic gel; and additionally a photocurable sealant component layer material other than the gel component layer, and (1) an oligomer comprising (2) a photoinitiator and (3) a stabilizer.

Description

Field of the invention

The field of the invention is sample separation technology.

background

The analysis of blood samples often requires the separation of whole blood into a serum fraction and a cell-containing fraction. It is known in the art that a separation of whole blood can be performed by centrifugation, by placing whole blood in a blood collection tube, placing the tube in a centrifuge and centrifuging the blood.

Unfortunately, once this separates the blood, the fractions of whole blood may recombine, causing contamination of the fractions by diffusion, shaking, sample extraction, or other undesirable interaction. Ideally, the two fractions should remain isolated to ensure that no contamination occurs when the desired fraction is accessed.

In an effort to overcome the problems discussed above, efforts have been made to provide separation gels which are placed in a lower portion of a pipe. These release gels are intended to help maintain analyte stability, reduce manual labor (pipetting to a secondary tube), and allow for delayed processing that may result from the need to transport samples from sampling sites to testing facilities. Some performance and performance characteristics of the gels typically include the following: (1) the gel properties prevent flow prior to use to eliminate reflux potential during blood collection; (2) the gel has a density value between cells and serum / plasma (about 1.04 specific gravity); (3) the gel is thixotropic (shear thinned in a centrifuge under normal clinical laboratory conditions); (4) the gel liquefies and passes through blood cells and proteins during centrifugation; and (5) the liquefied gel again gels on a layer between the cells and the serum after centrifugation and adheres to the tube wall. Furthermore, the components of the gel generally should not interfere with blood components or assays used in the clinical laboratory.

Unfortunately, known release gels suffer from one or more of the direct and indirect disadvantages, including, for example: interference (certain assays are known to be problematic); Analyte flow due to permeability, cells are trapped in or on the surface of the gel; physical contamination of analyzer probes or with electrophoresis gels; Inaccuracies caused by another spin; the need for aliquoting (with the associated costs of manual labor and possible errors in relabelling or relabelling); incomplete aspiration, which has a negative effect on standardization and leads to waste; and storage / shipping issues such as gel displacement, freeze-thaw problems and the inability to remix a sample with a soft gel barrier.

In an effort to overcome some of the problems associated with separation gels, efforts have been made to provide compositions and methods for the separation of whole blood which ensure that the separated fractions of whole blood are effective against contamination by unwanted interactions with the sample to be protected. For example, Applicant has obtained several patents for previous efforts on photopolymer serum separators and methods (eg U.S. Patent Nos. 7,674,388 . 7,673,758 . 7,775,962 . 7,971,730 . 7,780,861 . 8,206,638 . 8,282,540 ). Photopolymer serum separators may be advantageous in providing a solid interface (when the photopolymer gel is polymerized) between cells and serum or plasma, which allows for complete aspiration of the sample. Additionally, a tube comprising some photopolymer separator gels (prior to polymerization to form a solid) may be shipped, cooled, or frozen with repeated freeze-thaw cycles. Still further, the tube can be mixed without interruption of the barrier, ensuring consistent sampling of the test fraction (eg, blood does not mix again when tubes are agitated), the tube does not have soft gel material (after polymerization). which may plug the assay probes or pipette tips, or come into contact with the test fraction, resulting in disruption of sensitive laboratory testing procedures (eg, electrophoresis, etc.).

These and all other extrinsic references are incorporated herein by reference in their entirety. If a definition or the use of a term in a recorded reference is inconsistent with or inconsistent with the definition of this term in the present context, the definition of that term given here applies and the definition of that term in the reference does not apply.

Unfortunately, some known release compositions and methods have been problematic for a number of reasons, including the high cost of photohardenable compositions, the need to prepare a thixotropic composition, the heat generated in exothermic polymerization reactions that may affect analytes, the impossibility to sterilize by irradiation while maintaining the fluidity of the release compositions for subsequent UV cure (eg, after shipping the sterilized composition into sample collection tubes) and volume dependent UV exposure requirements.

Thus, there is still a need for improved separation technologies.

Summary of the invention

The present invention provides devices, compositions, systems, and methods that attempt to solve the problems described above by providing a sample collection tube comprising: (i) a tube having a lumen and (ii) a composite separation substance disposed within the lumen, the release substance comprising a gel component layer material and a photohardenable sealant component layer material. More specifically, the gel component layer material may comprise thixotropic separation gel which is prepared to become a liquid when agitated or shaken, and the photocurable sealant component layer material may comprise a photopolymer sealant. In some aspects, both the gel component layer material and the photocurable sealant component layer material may comprise separate layers (for example, prior to sterilization by irradiation, prior to centrifugation, before curing, after sterilization by irradiation, after centrifugation, after curing and after sterilization by irradiation, before and after centrifugation, before and after curing). Additionally or alternatively, the gel component sheet material and the photohardenable sealant component sheet material may comprise a blend.

In some aspects, the photohardenable sealant component may optionally be anti-thixotropic. Additionally or alternatively, the photohardenable sealant component may be formulated to polymerize to a hardness of at least 1 on the Shore-00 hardness scale within 10 minutes when initiated by a suitable source of energy (eg, UV light, etc.). From another perspective, the photohardenable sealant component may include a promoter to allow polymerization within 10 minutes to at least 1 on the Shore 00 hardness scale when triggered by a suitable source of energy. Additionally or alternatively, the photohardenable sealant component may be formulated to polymerize within 10 minutes to at least 10 on at least one of the Shore A hardness scale and the Shore D hardness scale when initiated by a suitable source of energy. From yet another aspect, it may be contemplated that the photohardenable sealant component may be a solid after a polymerization initiated by a suitable energy source with respect to a probe.

The present invention also provides devices, systems and methods in which a collection tube includes a separation substance which can maintain the levels of analytes (e.g., potassium levels and glucose levels, etc.) for longer periods of time within acceptable limits. In one aspect of the subject invention, potassium levels are stable within 10% of an initial pre-centrifuge level and glucose levels are stable within 5%. From another perspective, one or both of the gel component and the photohardenable sealant component (eg, the entire release substance) may be formulated such that a potassium level of a sample placed in the tube is within 25%, within 15%, within 10%, or even within of than 5% of an initial potassium level is stable for at least four days. It is also contemplated that one or both of the gel component and the photohardenable sealant component are formulated such that a glucose level of a sample in the tube is within 25%, within 15%, within 10%, or even within 5% of an initial one Glucose level for at least four days, more preferably for at least five days.

Additionally or alternatively, the separation substance may advantageously be biocompatible with whole blood and formulated to have a density between an average density of a serum / plasma fraction of whole blood and a cell-containing fraction of whole blood (for example about 1.04 g / cm ³ ). The photohardenable sealant component typically has a density that is slightly lower than that of the gel component such that upon curing, the photohardenable sealant component sits on top of the gel component and provides a clear barrier. Additionally or alternatively, the release substance may be flowable in whole blood prior to curing and form a solid sealant layer after curing in immobilized form.

Viewed from a different perspective, the subject invention includes methods of making sample collection tubes. A contemplated step of the methods described herein may include placing a photocurable sealant component layer material in a lumen of the tube. Another step may be to place a gel separation component sheet material in the lumen of the tube. The foregoing steps may be contemplated in any suitable order such that the gel component may be disposed above or below the photohardenable sealant component. In some methods, the photohardenable sealant component sheet material is disposed in front of / below the gel component sheet material and is configured to form a solid sealant layer over the gel component sheet material upon curing. In some contemplated methods, the gel component is first placed in a tube followed by the photohardenable sealant component. Upon centrifugation, the sealant component may rise above the gel component to form a sealant layer which acts as a barrier between the gel component and a fraction of plasma, serum or other sample. It should be noted that the gel component and photocurable sealant component form a composite release substance of the invention as described above.

Some contemplated methods may additionally include placing a sample (e.g., blood, etc.) in the lumen of the tube and centrifuging the sample collection tube with the composite separation substance and the sample disposed therein. Additionally or alternatively, the sample collection tube may be exposed to UV light (eg, during or after centrifugation) to solidify the photohardenable sealant component. Additionally or alternatively, when whole blood is in the tube, a method of the invention may comprise separating a cell-containing fraction of whole blood from the serum fraction (eg, by physically removing a fraction with a pipette, etc.) after the tube has been exposed to UV light or another exposed to appropriate energy source to initiate the polymerization of the photohardenable sealant component.

Other optional steps of some contemplated methods include, but are not limited to, sterilizing a collection tube before or after placing a composite separation substance therein, and creating a vacuum in a lumen of the tube. The step of sterilization may be performed in any suitable manner including, for example, gamma irradiation or by sterilizing the components by heating (eg, to at least 250 degrees Celsius, etc.). The step of creating a vacuum may be performed in any suitable manner, including, for example, by decompressing the volume of the lumen using any suitable pump. The term "vacuum" as used herein means a partial vacuum having a pressure lower than the pressure outside the tube.

It should be understood that a sample collection tube of the invention may be used for the fractionation of any suitable sample, including, for example, whole blood. Suitable separation substances are formulated to have a suitable density between those of the fractions of the separated liquid. For example, when the separated liquid is whole blood, the separation substance may be formulated at a density between an average density of a serum fraction of whole blood and a cell-containing fraction of whole blood, and to be flowable in whole blood. Once the separation substance separates the fractions of the separated liquid, the photohardenable sealant component / layer can be cured by polymerization.

Other components may advantageously be included in a tube of the invention including, for example, an anticoagulant (e.g., when a sample comprises plasma) or a clotting activator (e.g., when a sample contains serum).

The invention also provides apparatus, compositions, systems and methods of providing polymerizable compositions that are sterilizable by irradiation or application of heat while maintaining a predetermined flowability that is effective to cause sedimentation of the composition under a centrifugal force to a position between to allow a cell-poor phase of whole blood and a cell-enriched phase of whole blood. In some aspects For example, the polymerizable composition comprises an oligomer, a photoinitiator, a stabilizer and an antioxidant, and may be disposed in a lumen of a sample collection tube. When the tube and the polymerizable composition are sterilized by irradiation or heat, the predetermined flowability of the composition is preferably maintained in a manner which enables subsequent polymerization of the composition by means of UV curing. Various objects, features, aspects and advantages of the invention will become more apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings, in which like reference characters represent like components.

Short description of the drawing

1 shows a commercial plasma separation tube (BD Vacutainer) as a control and comparison with a photopolymer sealant.

2 Figure 11 is a cross-sectional view of a separator tube comprising a photopolymer separator of the invention.

Detailed description

The following discussion sets forth many exemplary embodiments of the invention. Although each embodiment represents a single combination of elements of the invention, the subject invention is considered to include all possible combinations of the elements disclosed. Thus, if one embodiment comprises elements A, B and C, and a second embodiment comprises elements B and D, then the subject invention will be considered to include the remaining combinations of A, B, C or D, although not expressly disclosed ,

• • Fähigkeit, jegliche Zelleinschlüsse, die innerhalb des Gels auftreten können, von der Testfraktion abzutrennen.
• • Reduzieren der für die vollständige Aushärtung (Verfestigen) der Barriere erforderlichen UV-Lichtintensität und Belichtungszeit, zumindest weil das erforderliche Volumen der photohärtbaren Dichtungsmittelkomponente verringert wird. Diese Verringerung hat die zusätzlichen Vorteile, dass die Wirkung von UV auf lichtempfindliche Pigmente abnimmt, der Arbeitsablauf durch die verkürzte Verarbeitungszeit verbessert wird und die exotherme Wärmeproduktion während der Aushärtung abnimmt, welche bestimmte Blutkomponenten wie etwa Enzyme beeinflussen kann.
• • Ermöglicht ein vollständiges Absaugen einer Probe ohne eine wesentliche, wenn überhaupt, Verunreinigung durch die Gel-Komponente, zumindest weil die verfestigte photohärtbare Dichtungsmittelkomponente als Barriere gegenüber der Gelkomponente wirkt.
• • Ermöglicht das Mischen einer zellfreien Fraktion von Vollblut, um eine gleichförmige Probennahme zu gewährleisten.
• • Verhindert erneutes Vermischen von Blut, das auftreten kann, wenn weiche Gel-Separatoren physikalisch durch Bewegung zerrissen werden, beispielsweise während des Transports oder Mischens.
• • Vermindert Störungen durch die Gel-Komponente oder Zellfraktion von Vollblut.
• • Ermöglicht wiederholte Gefrier/Auftau-Zyklen des Rohres. Einfrieren von Serum oder Plasma ist übliche Praxis, zum Beispiel für zukünftige Tests oder zur Einhaltung bestimmter Vorschriften. Wenn ein Gel-Separator zum Trennen von Fraktionen einer Probe verwendet wird, wird die Fraktion unterhalb des Separators typischerweise vor dem Gel eingefroren, wodurch die Form der Gel-Trennbarriere erweitert und verzerrt wird. Da eine erfindungsgemäße Trennsubstanz eine photohärtbare Dichtungsmittelkomponente einschließt, die so formuliert ist, dass sie sich bei Exposition gegenüber einer geeigneten Energiequelle verfestigt, werden einige oder alle der Probleme, die typischerweise mit Einfrieren und Auftauen von Trennrohren verbunden sind, wesentlich reduziert oder sogar eliminiert.
• • Die photohärtbare Schicht kann im Volumen minimiert werden, wodurch Kosten reduziert werden.
• • Die photohärtbare Schicht muss nicht thixotrop sein, da thixotropes weiches Gel in das Rohr über der photohärtbaren Schicht geladen wird und keine Strömung vor der Verwendung resultiert. Dies ermöglicht einfache Zusammensetzungen mit weniger Zelleinfang oder -einschluss und beseitigt die Notwendigkeit für Additive, die zum Abbau von Blutzellen oder Beeinträchtigung von Labor-Assays beitragen.
• • Verringert die Arbeitskosten im Zusammenhang mit dem Entfernen von Serum oder Plasma hin zu einem Sekundärrohr.
• • Verringert mögliche Fehler bei erneuter Etikettierung im Zusammenhang mit dem Entfernen von Serum oder Plasma hin zu einem Sekundärrohr.

Separating substances according to the invention are advantageous over existing separating substances, at least for the following reasons:

• Ability to separate any cell occlusions that may occur within the gel from the test fraction.
• • Reducing the UV light intensity and exposure time required to completely cure (solidify) the barrier, at least because the required volume of photohardenable sealant component is reduced. This reduction has the added benefits of decreasing the effect of UV on photosensitive pigments, improving the workflow through the shortened processing time, and decreasing the exothermic heat production during cure, which may affect certain blood components, such as enzymes.
• • Allows complete aspiration of a sample without significant, if any, contamination by the gel component, at least because the solidified photocurable sealant component acts as a barrier to the gel component.
• • Allows mixing of a cell-free fraction of whole blood to ensure consistent sampling.
• • Prevents re-mixing of blood that can occur when soft gel separators are physically ruptured by movement, such as during transport or mixing.
• • Reduces interference from the gel component or cell fraction of whole blood.
• • Enables repeated freeze / thaw cycles of the tube. Freezing serum or plasma is common practice, for example, for future testing or compliance. When a gel separator is used to separate fractions from a sample, the fraction below the separator is typically frozen in front of the gel, thereby widening and distorting the shape of the gel separation barrier. Because a release substance of the invention includes a photocurable sealant component that is formulated to solidify upon exposure to a suitable source of energy, some or all of the problems typically associated with freeze-thawing of separation tubes are substantially reduced or even eliminated.
• • The photocurable layer can be minimized in volume, which reduces costs.
• The photohardenable layer need not be thixotropic because thixotropic soft gel is loaded into the tube over the photohardenable layer and no flow results before use. This allows simple compositions with less cell capture or entrapment and eliminates the need for additives that contribute to the breakdown of blood cells or impairment of laboratory assays.
• • Reduces labor costs associated with removing serum or plasma to a secondary tube.
• • Reduces potential re-labeling errors associated with removing serum or plasma to a secondary tube.

Feasibility has been demonstrated when using a photo-curable sealant composition comprising: (1) at least one of a monomer and an oligomer (e.g., a combination thereof (for example Ebecryl, Cytec)), (with (2) a photoinitiator for example, Additol ^® BDK, Additol ^® TPO) and (3) a stabilizer (phenothiazine). It should be understood that any suitable commercially available photocurable sealant component can be used. Suitable photohardenable sealant components are typically at least one of a gel (eg, when a gelling agent is added (eg, DBS or silica, etc.)) and flowable (with whole blood) prior to polymerization, and may solidify if they are a suitable source of energy (For example, UV light, etc.) are exposed. Examples of suitable photohardenable sealants may include, but are not limited to, MLA (eg, M1L1A1), MLAI (eg, M1L1A1 and phenothiazine), MAI (eg, M1A1 and phenothiazine), LAI (eg, L1A1 and phenothiazine), and LMA (eg, L1M1A1). As used herein M = a monomer (eg, M1, which is a monomer trimethylolpropane propoxylate triacrylate from Sigma-Aldrich Cat. No. 407577, etc.); L = an oligomer (eg L1 = Ebecryl 230 from Allnex, previously from Cytec Industries, Inc., etc.); A = a light starter or photoinitiator (eg A1 = Additol BDK); I = a stabilizer (for example phenothiazine, etc.). See Table 1 below. LAI (eg L1, A1 and phenothiazine) may be included in some particularly preferred photocurable sealant components because there cm may have the desired density range of 1.00 to 1.09 g / ³ system.

Other examples of suitable photohardenable sealants include LAIR, LAIE (eg, L1, A1, phenothiazine, and tocopherol) and LAIER, where R = a gelling agent (eg, DBS, silica, etc.), and E = at least one of an antioxidant and a radical scavenger ( for example, vitamin E, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), carotene, bilirubin, ascorbic acid, etc.). While R and E are generally not required, each may provide advantageous features to the photohardenable sealant. More specifically, a gelling agent is generally not a necessary component of the photohardenable sealant because a thixotropic soft gel can be loaded into the tube over the photocurable layer so that no flow results prior to use. Nonetheless, it may be desirable to have a thixotropic sealant, for example, where it is desirable to have the sealant in the tube on top of the soft gel component. In addition, although unnecessary, a radical scavenger such as compounds having the activity of vitamin E (eg, tocopherol) may allow the photocurable sealant to be sterilized by irradiation without curing (for example, by changing the density properties) rather than requiring heat sterilization to maintain flowability which is effective to allow sedimentation between two fractions of a sample. ABBREVIATION SURNAME R Gelling agent (eg DBS, silica, etc.) M monomer M1 trimethylolpropane L oligomer L1 Ebecryl 230 from Allnex A photoinitiator A1 Additol BDK I stabilizer I1 phenothiazine e Antioxidants / Radical scavengers (eg Vitamin E, BHT, BHA, Caroten, Bilirubin, Ascorbic Acid, etc.) D Density adjusting agent (eg Ebecryl 113 from Cytec, etc.) Table 1

It is contemplated that composite release agents of the invention in one or both of the photohardenable component and the gel component may include a polymer such as silicone oil, polyamides, olefinic polymers, polyacrylates, polyesters and copolymers thereof, polysilanes, and polyisoprenes. Additionally or alternatively, composite release agents may include a filler (eg, silica, latex, or other inert material) to adjust a density of the release agent or a component thereof. Additionally or alternatively, composite release agents may include additives or reagents to achieve a desired purpose (eg, EDTA (chelating agent) or heparin, citrate, dextrose (anticoagulants)).

It should also be appreciated that any suitable gel component can be placed in a pipe according to the invention. Suitable gel components include those which become liquid during centrifugation, and then again become gel, and can, for example, the typical laboratory gels (for example, BD Vacutainer ^® SST ^TM, BD Vacutainer ^® PST ^TM, Vacuette ^® blood collection tubes with gel separators, PPMA serum Separating gel tubes, polypropylene serum separating gel tubes, etc.) or any other commercially suitable gel formulated to be present between two fractions of a sample during centrifugation (for example between a serum and blood clots, between serum and a cell-containing fraction of whole blood, etc.).

1 shows a commercial plasma separation tube (BD Vacutainer) as a control or comparison 110A and with a photopolymer sealant 110B , The photo-sealant 120 without gelling agents or thixotropy-modifying components is clear and free of trapped cells or cell entrapment before and after centrifugation and UV-curing. Cell inclusion is undesirable because the analyte seeps into the test fraction. Cells often adhere to the upper layer of soft gels 110 to how it is in 1 is shown. However, they can by the photo sealant 120 be separated from the plasma.

The following data show improved analyte stability using a composite release agent of the invention (Photogel plasma separation tube) as compared to using a soft gel alone. BD-plasma separation tube Photogel plasma separation tube Comprehensive Metabolic Board immediate spin 24 hours 5 days Freezing / thawing immediate spin 24 hours 5 days Freezing / thawing Sodium mVal / L 142 139 140 143 140 140 142 143 Potassium mVal / L 3.7 3.7 4.1 4.5 3.6 3.6 3.7 3.8 Chloride mVal / L 106 104 105 108 106 105 107 108 CO2 mVal / L 29 27 26 22 27 25 25 22 Glucose mg / dL 102 100 93 91 104 105 102 106 Urea nitrogen mg / dL 16 15 16 16 17 16 16 16 Creatinine mg / dL 0.6 0.8 0.8 0.8 0.7 0.8 0.8 0.5 Calcium mg / dL 9.4 9.3 9.5 9.7 9.2 9.4 9.5 9.6 Total protein g / dL 6.8 6.8 6.4 7 6.8 7 6.7 7 Albumin g / dL 4 4 4 4 4 4.1 4.1 4 Alkaline phosphatase IE / L 37 39 36 38 36 38 39 37 AST IE / L 17 15 19 26 17 17 18 16 ALT IE / L 15 16 19 16 16 17 17 13 Bilirubin Mg / dL 0.6 0.7 0.6 0.5 0.5 0.6 0.6 0.4

A sample collection tube according to the invention generally comprises a tube, a stopper and a separating substance having a gel component / layer and a photohardenable sealant component / layer, which are arranged in a lumen of the tube. The header may preferably be made of a suitable rigid material to assist a vacuum within the lumen. Exemplary materials include hard plastics, glass or other similar materials. The lumen is preferably of sufficient volume to receive a desirable sample of whole blood or other fluid. Typical volumes are in the range of a few ml to 10 ml or more. The plug can fit snugly into the tube to hold the vacuum in the lumen. An example of an acceptable pipe that can be used to produce an inventive manifold, the vacutainer ^® specimen collection products includes that (Franklin Lakes, NJ 07417 USA) were developed by Becton, Dickenson and Company.

The term "pipe" is used euphemistically to refer to vessels having a cavity. Although a preferred embodiment includes a tube, it should be appreciated that other voided vessels may also be used, and still fall within the scope of the invention. For example, it is contemplated that the manifold may be replaced with other vessels which may contain a liquid or optionally assist a vacuum. Alternative examples of vessels include flasks, cups, beakers, bottles, blood collection bags or vials. Vessels beyond mere tubes also benefit when the subject invention is applied to alternative markets beyond the blood collection.

In a preferred embodiment, a header is made by placing a composite release within a lumen of the tube and creating a vacuum within the lumen in preparation for sale. It is generally preferred that no more than about 1 ml or about 1 gram of the release agent substance be placed in the lumen for a typical 10 ml collection tube. Additionally or alternatively, it is generally preferred that not more than 50%, more preferably not more than 25%, and even more preferably not more than 10% of the release agent substance comprise the photohardenable sealant layer. It is contemplated that other amounts of the release agent substance (or a layer thereof) may be used in some embodiments to suit a particular application. For example, a smaller version of a pipe may require less release agent substance, while a larger version may require more to form an adequate sealed barrier.

In some embodiments, headers are sterilized before they are sold. For example, tubes (preferably without polymerization of the release agent substance or a portion thereof) may be sterilized using gamma radiation or heat between 100 to 250 degrees Celsius or even more. An optional vacuum can be created by simply decompressing the volume of the lumen of the tube by using a suitable pump.

It is also contemplated that a user may add one or more release agent substances to a collection tube after purchase, rather than a release agent substance predisposed within the tube.

When a sample (e.g., whole blood) is added to a collection tube of the invention, centrifugation may separate the whole blood into a serum fraction and a cell-containing fraction. When each layer (gel / photocurable sealant) of the release agent substance has a density intermediate that of the serum fraction and that of the cell-containing fraction, it migrates between the two fractions during centrifugation, whereby the fractions will be isolated from each other. The release agent substance can then be rapidly cured by polymerization when initiated by a suitable source of energy to create a solid barrier between the two fractions.

In some aspects of the invention, a separation tube may be used with both (1) a polymerizable composition and a thixotropic gel as described in U.S. Pat 1 or (2) a polymerizable composition alone, as in 2 shown to be provided. As illustrated, the sample collection tube comprises 200 a polymerizable composition according to the invention 210 (eg, LAIE) disposed therein and after centrifugation in a position between a cell-poor phase 220 and a cell-enriched phase 230 is cured by whole blood.

The polymerizable composition preferably comprises at least three of the following components: an oligomer (L) (eg, an acrylate-containing oligomer and a methacrylate-containing oligomer, etc.), a photoinitiator (A), a stabilizer (I), and at least one of Radical scavenger and an antioxidant (E).

The polymerizable composition may advantageously be a composition effective to allow sterilization by irradiation without loss of a predetermined flowability (eg, effective to sediment the composition under a centrifugal force into a position between two phases of a sample (e.g. Phase and a cell-enriched phase of whole blood, etc.)). From another point of view, the composition may be effective to allow sterilization by irradiation or heating without curing the composition by more than 40%, more preferably without curing the composition by more than 30%, and then by UV polymerization. Hardening or otherwise allow curing. The subsequent polymerization by UV curing, after sterilization by irradiation after minutes (for example, more than 30 minutes), hours (for example, more than 1 hour, more than 2 hours, more than 5 hours, more than 10 hours), days (more than 1 day, more than 5 days, more than 10 days, more than 15 days, more than 20 days, more than 25 days), months (more than 1 month, more than 2 months, more than 6 months, more than 9 Months) or even years (more than 1 year, more than 2 years, more than 3 years, more than 5 years or even longer). In some embodiments, the polymerizable composition may be injected without loss of the predetermined fluidity of a radiation dose of between 5 and 35 kGy each, more preferably between 10 and 30 kGy each, and even more preferably between 10 and 20 kGy each. From another perspective, the polymerizable composition may be exposed to a radiation dose of less than 30 kGy, more preferably less than 20 kGy, and even more preferably less than 17 kGy to achieve both (1) irradiation sterilization without loss of given flowability and 2) to allow subsequent polymerization by UV curing.

It is contemplated that a photoinitiator (e.g., azobisisobutyronitrile, benzoyl peroxide, camphorquinone, a phosphine oxide photoinitiator, a ketone-based photoinitiator, a benzoin ether photoinitiator, etc.) may be present in the polymerizable composition at any suitable concentration. In some preferred embodiments, the photoinitiator is present in the composition in a concentration of less than 5% by weight, more preferably in a concentration of less than 2% by weight, and even more preferably in a concentration of less than 1.5% by weight. Additionally or alternatively, a polymerizable composition according to the invention may contain a photoinhibitor.

It should be noted that a scavenger or antioxidant is not required in all polymerizable compositions contemplated. However, where included (for example, to facilitate flowability by sterilization by gamma ray or electron beam irradiation), it is contemplated that the at least one of the radical scavenger and antioxidant (eg, tocopherol, etc.) in the polymerizable composition will be in any suitable molar concentration may be present. Applicant has surprisingly found that when a radical scavenger such as tocopherol is included, some compositions of the invention (e.g., LAIE) maintain fluidity during sterilization by irradiation at a radiation dose of greater than 3 kG, while some other compositions (e.g. LAI) under the same radiation dose, fluidity is not maintained. From a different perspective, LAI maintained flowability only during sterilization by irradiation up to a radiation dose of about 3 kG. In some preferred embodiments, the at least one of the radical scavenger and the antioxidant comprises tocopherol and is present in the composition in a molar concentration of at least 75 mM, more preferably at least 100 mM, and even more preferably at least 135 mm.

Additionally or alternatively, the polymerizable composition may be polymerizable to any suitable polymer by UV cure (eg, after sterilization by irradiation (gamma, e-beam, etc.)), including, for example, an acrylate polymer, a methacrylate polymer, an epoxide Polymer, a polyurethane, or a thiol-ene polymer.

From another perspective, a polymerizable composition of the invention may comprise one or more of a polymer containing a terminal epoxide group, a polymer having a pendant epoxy group, an epoxy siloxane resin, an epoxy polyurethane, an epoxy polyester, epichlorohydrin, a polyhydric diol, a polyhydric polyol, a polymer having an end or side isocyanate group, a polymer having at least two hydroxyl groups, a polyhydric diol, a polyhydric polyol, an aliphatic monomeric polythiol, an aliphatic dithiol, an aromatic dithiol, a polymeric polythiol, an acrylate, a Methacrylate, an alkenyl and a cycloalkenyl. When a polymerizable composition of the invention is exposed to a suitable source of energy (for example, a UV energy source, etc.), it is contemplated that the composition will be less than ten minutes, more preferably less than five minutes, more preferably in a period of less than 60 seconds and even more preferably in a period of less than 20 seconds to a hardness of at least 1 (e.g. a hardness of at least 10, etc.) on a Shore A hardness scale. From another perspective, it is contemplated that the composition will last at least 10 minutes, more preferably in a period of less than 5 minutes, more preferably in a period of less than 60 seconds, and even more preferably in a period of less than 20 Seconds to a hardness of at least 1 (for example, a hardness of at least 10, etc.) on the Shore 00 hardness scale can be cured. Viewed from yet another perspective, it is contemplated that the composition will last for a period of less than ten minutes, more preferably less than 5 minutes, more preferably less than 60 seconds, and even more preferably a period of less than 20 seconds to a hardness of at least 1 (for example, a hardness of at least 10, etc.) can be cured on a Shore D hardness scale.

experiments

• a. Enddichte – getestet durch Pyknometrie und Leistung in Vollblut während der Zentrifugation.
• b. Interferenzen mit bzw. Einflüsse auf Labortests – getestet durch Vergleich von Ergebnissen mit jenen, die bei in BD-Rohren gesammeltem Blut gefunden wurden. Es waren keine Einflüsse zu entnehmen, wenn die Mittel der Messungen innerhalb der Assay-Katalogwerte lagen. Der Labortest-Vergleich schloss den gesamten Prozess der Verwendung des Trennmittels in der Zentrifuge und des Härtens mit UV ein. Monomere und Oligomere wurden auch hinsichtlich eines Austretens aus dem Blut bei Kontakt mit dem härtenden Photopolymer getestet, und zwar für bis zu 8 Tage, wobei nach verzögerter Interferenz geschaut wurde. i. Umfassende metabolische Tafel (Natrium, Kalium, Chlorid, CO2, Creatinin, Bilirubin, Gesamtprotein, AST, ALT, alkalische Phosphatase, Glucose, Harnstoff-Stickstoff, Albumin) ii. Immunoassays (PSA, Testosteron, Östradiol, TSH, freies T4, Ferritin iii. Elektrophorese (Serumprotein-Elektrophorese, Lipid-Platten durch Elektrophorese) iv. Lipide (Gesamtcholesterin, LDL-c, HDL-c, VLDL-c, Lp(a), Triglyceride) v. Molekulartests (DNA, RNA Exosom)
• c. Hämolyse – gemessen durch den Index auf automatischen Analysegeräten, die visuelle Beurteilung und erhöhte Kaliumspiegel.
• d. Aushärtungszeiten mit UV-Lampen (Bogenlampen, Mikrowellenleistungslampen, LEDs) i. Zunehmende Antioxidanskonzentration darüber hinaus (zum Beispiel über 500 mM Tocopherol) wirkt sich negativ auf Härtungszeiten aus (verlängert). Erhöhung der Photoinitiatorkonzentration über etwa 3%, um diesen Effekt zu kompensieren, beeinträchtigt die Funktion des Antioxidans bei der Erhaltung der Verbindung während Sterilisation mittels Bestrahlung.
• e. Wärmeerzeugung bei Aushärtung.
• f. Zusammenziehen bei Aushärtung.
• g. Fähigkeit, durch Wärme oder Bestrahlung (e-Strahl und Gamma in verschiedenen Dosen und Dosisverabreichungsschemata) sterilisiert zu werden. i. Für Sterilisation durch Erhitzen waren keine Antioxidantien erforderlich und verschiedene Kombinationen von L1 und M1 erfüllen die anderen Leistungsanforderungen. ii. Für die Sterilisation durch Bestrahlung ist es für eine gegebene Zusammensetzung wahrscheinlicher zu funktionieren, wenn die Dosis schnell verabreicht wird (beispielsweise durch e-Strahl anstatt durch Gamma). 1. LAI kann ohne ein Antioxidationsmittel mit bis zu 3 kGy sterilisiert werden.

Various candidate compositions, variably comprising an oligomer or monomer (or both), a photoinitiator, a stabilizer, an antioxidant, a density adjusting agent, or a gelling agent (or both) were tested to determine, inter alia, whether during sterilization Irradiation (e-beam or gamma) at different radiation doses and over different periods of a predetermined flowability could be maintained. Specifically, the compositions were tested for the following:

• a. Final density - tested by pycnometry and performance in whole blood during centrifugation.
• b. Interference with or interference with laboratory tests - tested by comparing results with those found in blood collected in BD tubes. There were no effects if the mean of the measurements were within the assay catalog values. The laboratory test comparison included the entire process of using the release agent in the centrifuge and curing with UV. Monomers and oligomers were also tested for leakage from the blood on contact with the curing photopolymer for up to 8 days looking for delayed interference. i. Comprehensive Metabolic Table (Sodium, Potassium, Chloride, CO2, Creatinine, Bilirubin, Total Protein, AST, ALT, Alkaline Phosphatase, Glucose, Urea Nitrogen, Albumin) ii. Immunoassays (PSA, testosterone, oestradiol, TSH, free T4, ferritin iii, electrophoresis (serum protein electrophoresis, lipid plates by electrophoresis) iv) lipids (total cholesterol, LDL-c, HDL-c, VLDL-c, Lp (a) , Triglycerides) v. Molecular tests (DNA, RNA exosome)
• c. Hemolysis - measured by the index on automatic analyzers, the visual assessment and increased potassium levels.
• d. Curing times with UV lamps (arc lamps, microwave power lamps, LEDs) i. Increasing antioxidant concentration beyond (for example, over 500 mM tocopherol) has a negative effect on cure times (prolonged). Increasing the photoinitiator concentration above about 3% to compensate for this effect interferes with the function of the antioxidant in maintaining the compound during sterilization by irradiation.
• e. Heat generation during curing.
• f. Contracting on curing.
• G. Ability to be sterilized by heat or radiation (e-beam and gamma in different doses and dose-delivery regimens). i. For sterilization by heating, no antioxidants were required and various combinations of L1 and M1 meet the other performance requirements. ii. For irradiation sterilization, for a given composition, it is more likely to function if the dose is administered rapidly (for example, by e-beam rather than gamma). 1. LAI can be sterilized without an antioxidant up to 3 kGy.

The tested monomers (some in combination for density adjustment) included M1, 1,6-hexanediol ethoxylate diacrytal, poly (ethylene glycol) methyl ether methacrylate, poly (ethylene glycol) diacrylate (cat. No. 437441), poly (ethylene glycol) diacrylate (cat No. 475629), trimethylolpropane propoxylate triacrylate and 1,6-hexanediol ethoxylate diacrylate (Cat. No. 497134). Of the monomers tested, M1 worked best. Unfortunately, compositions containing M1 resulted in a constriction after curing and were therefore considered to be less than optimal for use in sealant compositions.

The oligomers tested were all from the Ebecryl series and included L1-L10 (various oligomers from Cytec). Of the oligomers tested, L1, L2, L6, L8, L9 and L10 were better at maintaining flowability compared to L3-L5 and L7. L1 worked best in terms of, for example, less heat generation during cure, less contraction (if any), density, viscosity, less bubble entrapment, and fewer disturbances that were seen using standard serum assays, and the other oligomers were given up quickly because they did not Did not meet criteria. L8 and L9 resulted in greater disruption of enzymes than L1, while L10 resulted in visible interaction with hardened materials. The visible plasma dissected into the upper layer of the barrier.

The photoinitiators tested at 1 wt% in M1 included (azobisisobutyronitrile): 254 nm; Benzophenone: 254 nm; 4- (dimethylamino) benzophenone: 356 nm; 4,4'-bis (dimethylamine) benzophenone: 370 nm; 4,4'-bis (diethylamino) benzophenone: 379 nm; and A1: 365nm. The wavelength of the photoinitiator was selected by transmission through PET tubes that would be used. A1 was found to have superior performance based on criteria including compatibility with blood, cure times, heat generation, and miscibility with oligomers and monomers. It was found that A1 shows the best performance.

A1 was tested in different concentrations in L1 (between 1-8 wt% each). It was found that 1% ± 0.5% was optimal when antioxidant / antioxidants were present. Additol TPO, which has a longer wavelength absorption to match selected UV sources, was also tested at various concentrations in L1. When Additol TPO was tested at a concentration of 1% with L1, I1 and Vitamin E at a concentration of 200-300mM, the cure time was slightly better than A1 at a concentration of 1% with L1, I1 and Vitamin E in one concentration was tested from 200-300mM. In addition, no interference was detected using a comprehensive metabolic panel.

The tested stabilizer is phenothiazine in a concentration of 0.1% by weight. This stabilizer was present in all samples. However, it is contemplated that any suitable stabilizer may be used, including, for example, suitable stabilizers that would function in an evacuated atmosphere (low O ₂ ).

The antioxidants tested included alpha-tocopherol-2mM-500mM, carotene, bilirubin, BHT, BHA, tempo, and 4-OH tempo. While tocopherol, tempo, and 4-OH tempo maintained fluidity when irradiated with doses above 15 kGy (carotene, bilirubin, BHT, and BHA failed), tempo and 4-OH tempo caused hemolysis and laboratory disorders. It was found that tocopherol had the best performance.

• a. 30% L = EBECRYL 230 und 70% D
• b. wobei: i. 1% (von L + D) A1 (Additol BDK); ii. 8,19% (von L + D) R, pyrogenes Sciliciumdioxid R1 (Aerosil R805 von Degussa); und iii. 0,1% (von L + D) I (Phenothiazin von Sigma).

The density-adjusting agents tested included Ebecryl 113 from CYTEC. A physical gel formulation referred to as LARID (D = Ebecryl 113 from Cytec) was tested and showed good cure time and little or no interference in most laboratory tests. LARID includes:

• a. 30% L = EBECRYL 230 and 70% D
• b. where: i. 1% (from L + D) A1 (Additol BDK); ii. 8.19% (from L + D) R, pyrogenic silica R1 (Aerosil R805 from Degussa); and iii. 0.1% (from L + D) I (phenothiazine from Sigma).

It was found that the LARID formulation was not sterilizable by irradiation (while maintaining flowability) because no antioxidant was present.

The gelling agents tested included fumed silica and DBS (1,3: 2,4-dibenzylidenesorbitol) and co-solvent NMP (N-methylpyrrolidone). DBS showed inferior silica performance with respect to sterilization by irradiation while maintaining flowability. With 0.6% DBS and 1.8% NMP, the L1A1I1 gels. It is shear thinning and has a density lower than a BD gel. The UV curing time was about the same as L1A1I1R1, and the cured gel was able to survive one round in a liquid nitrogen / hot water test, which means that the cured gel can survive many rounds of freeze-thaw cycles.

Examples

The following experimental data is provided to exemplify various aspects of the subject matter of the invention presented herein. More particularly, the data illustrate the surprising effects of tocopherol and additol BDK in certain concentrations in maintaining fluidity of a composition (to permit sedimentation between two phases of whole blood on centrifugation) after sterilization by irradiation with specified radiation doses. As shown, compositions comprising an oligomer (EBECRYL 230), a photoinitiator, Additol BDK, at a concentration of less than 2% by weight, and a radical scavenger, tocopherol, at a concentration of at least 100mM, have fluidity after sterilization Irradiation (gamma or e-beam) at a dose of less than 20 kGy surprisingly upright. In contrast, compositions without a scavenger and compositions with a photoinitiator concentration greater than 5 wt.% Could not maintain flowability after sterilization by irradiation. composition Tocopherol conc. (MM) Photoinitiator conc. (Wt.%) Dose (kGy) Sterilization method Post-sterilization heat Satisfied (retain flowability) or not met L1A1I1-DBS (L1A1I1 = Ebecryl 230, Additol BDK, phenothiazine) (DBS = dibenzylidene sorbitol) 0 1 0 gamma None Not satisfied (worse non-compliance with increased DBS) L1A1I1 0 1 17 Electron n-ray None Not fulfilled L1A1I1 0 1 15 gamma None Not fulfilled L1A1I1 0 1 10 gamma None Not fulfilled LAIE (Ebecryl 230, Additol BDK, phenothiazine, tocopherol) 0.1-2.0 1 25 gamma None Not fulfilled LAYMAN 10, 20 1 25 gamma None Not fulfilled LAYMAN 100, 120, 140 1 16.1 e-beam None Nearly LAYMAN 120 1 16.1 gamma 70 ° C 60 min Not fulfilled LAYMAN 140 1 16.1 e-beam 70 ° C 60 min Not fulfilled LAYMAN 145 1 16 e-beam 50 ° C 2 hours Not fulfilled LAYMAN 145 1 12 e-beam or gamma 70 ° C 60 min Fulfills LAYMAN 145 1 16 e-beam or gamma 70 ° C 60 min Fulfills LAYMAN 200 5 15-20 e-beam or gamma None Not fulfilled LAYMAN 140-200 2-5 15-20 e-beam or gamma None Not fulfilled LAYMAN 140 2-3 17 e-beam None Not fulfilled LAYMAN 140 1 16 e-beam None Close LAYMAN 140, 200 1 16 e-beam 60-70 ° C 60 min Fulfills Larid 140 1 16 e-beam None Not fulfilled LAYMAN 140 1.5 12 e-beam 50 ° C 60 min Fulfills

As illustrated herein, a technical effect of some aspects of the invention is that tocopherol contained in a sealant composition in a suitable concentration range (for example, between 100 to 200 mM) both (1) during irradiation sterilization (e.g., e-beam or gamma) capture or interfere with generated radicals and (2) can not capture or interfere with radicals generated during UV-induced polymerization. From a different perspective, tocopherol must be present in an amount effective to prevent off-line polymerization when radicals are generated during electron beam or gamma sterilization but are not effective to effect polymerization in the presence of UV or another suitable one Prevent energy sources.

In some embodiments of the invention, two types of radical scavengers are included in a release agent composition. A first radical scavenger (for example, E) may be sensitive to radicals that initiate polymerization and that are generated by e-beam or gamma irradiation. A second free radical scavenger (e.g., I) may be sensitive to radicals which initiate polymerization and which are generated by a photoinitiator. Without wishing to be bound by any particular theory or to limit the scope of the invention, a technical effect of some aspects of the invention is that E (eg, tocopherol) can be used to provide an adequate balance between A (photoinitiator) and I (stabilizer). which are required to cure L (oligomer) in the presence of radiation-induced radicals. In other words, if there were an increase in I in an amount that would be suitable for trapping radicals during sterilization by irradiation (over 3 kG), the I in Composition A would predominate and the composition would not cure, if exposed to a source of UV energy. Compositions of the invention may contain I in a lesser amount due to the presence of E, which enables curing / curing of the composition upon exposure to a UV energy source. From another perspective, E can be considered a sacrificial antioxidant that can be dispensed with and does not interfere with an A-induced polymerization reaction.

Without wishing to be bound by any particular theory, it is also contemplated that in some embodiments, a stabilizer I may not be required to facilitate sterilization by irradiation and separate UV cure. Experiments have shown that LAI does not maintain fluidity when sterilized by irradiation. However, it is contemplated that an LAE composition can maintain flowability during sterilization by irradiation to allow subsequent UV cure when E is contained in a concentration that is not effective to increase the radicals generated by A. but effective to consume the radicals generated by irradiation during sterilization.

While the above data are based on L equals L1, it should be understood that other oligomers (eg, an acrylate-containing oligomer and a methacrylate-containing oligomer) are expected to function instead of L1 because of their similar chemical composition and use in free radical polymerization , While the above data are based on A equals A1 (Additol BDK), other photoinitiators are similarly expected to promote their ability to decompose into free radicals when exposed to light and the ability to promote polymerization reactions of A1 (for example, a phosphine oxide photoinitiator, a ketone-based photoinitiator, and a benzoin ether photoinitiator).

Additionally or alternatively, other stabilizers may be used in place of phenothiazine (eg, hydroquinone), as they would be expected to similarly extend the storage and shelf life of the composition. Other radical scavengers are also expected to function in place of tocopherol; however, other tested radical scavengers (e.g. BHA, BHT, carotene, ascorbic acid, bilirubin, bile acid and tempo-nitroxide) that they interfere with some laboratory tests. Nonetheless, these scavengers can be used if the types of tests used are limited to specific clinical laboratory tests. For example, certain antioxidants have been found to interfere with the measurement of some immunoassays but not molecular tests.

Unless the context indicates otherwise, all of the areas outlined here should be interpreted to include their endpoints, and open areas should be interpreted as containing only commercially practical values. Similarly, all lists of values should be considered to include the intermediate values unless the context indicates the opposite. All methods described herein may be performed in any suitable order, unless otherwise stated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language (eg, "such") made in relation to particular embodiments herein is intended merely to better illuminate the invention and does not limit the scope of the invention otherwise claimed. No language in the description should be construed as an indication of any unclaimed element which is essential to the practice of the invention.

As used herein in the specification and in the claims that follow, the meaning of "a" and "the" includes plural reference unless the context clearly dictates otherwise. As used herein, the meaning of "in" may include "in" and "on," unless the context clearly dictates otherwise.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be designated and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included or removed from a group for convenience and / or patentability. When such inclusion or deletion occurs, the description herein is considered to include the group as modified so that the written description of all of the Markush Groups used in the appended claims is met.

It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The subject invention or the invention is therefore not limited except on the spirit of the appended claims. Furthermore, in interpreting both the description and the claims, all terms are to be interpreted in the broadest possible way in accordance with the context. In particular, the terms "comprises" and "comprising" are to be interpreted as referring to elements, components or steps in a non-exclusive manner, indicating that the referenced elements, components or steps exist, or are used, or can be combined with other elements, components, or steps that are not explicitly referenced. When the description and claims refer to at least one of anything selected from the group consisting of A, B, C ... and N, the text should be interpreted as requiring only one element of the group, not A plus N, or B plus N, etc.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 7674388 [0006]
• US 7673758 [0006]
• US 7775962 [0006]
• US 7971730 [0006]
• US 7780861 [0006]
• US 8206638 [0006]
• US 8282540 [0006]

Claims (5)

Sample collection tube, comprising: a tube with a lumen; and a separation substance disposed within the lumen, comprising: a gel component layered material, wherein the gel component comprises a thixotropic gel; and in addition, a photohardenable sealant component layer material other than the gel component layer, and (1) an oligomer comprising (2) a photoinitiator and (3) a stabilizer. The tube of claim 1, wherein the gel component layer material and the photohardenable sealant component layer material each have a density between 1.00 and 1.09 g / cm ³ , respectively. The tube of claim 1 or 2, wherein the separation substance has a density between an average density of a cell-poor fraction of whole blood and a cell-enriched fraction of whole blood and is flowable in whole blood. The tube of claim 3, wherein the photocurable sealant component layer material is sterilizable by irradiation without loss of fluidity for sedimentation of the separation substance under a centrifugal force to a position between the cell-poor phase of whole blood and the cell-enriched phase of whole blood and for the subsequent polymerization by means of UV curing. The tube of any one of claims 1 to 4, wherein the photohardenable sealant component layer material maintains at least one of a potassium level of the sample within 10% of an initial potassium level and a glucose level of the sample within 5% of an initial glucose level for at least four days.

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