EP2346606A1 - Dispositif, kit et méthode d activation par impulsion d échantillons biologiques avec un agent, et stabilisation de l échantillon ainsi activé - Google Patents

Dispositif, kit et méthode d activation par impulsion d échantillons biologiques avec un agent, et stabilisation de l échantillon ainsi activé

Info

Publication number
EP2346606A1
EP2346606A1 EP08875202A EP08875202A EP2346606A1 EP 2346606 A1 EP2346606 A1 EP 2346606A1 EP 08875202 A EP08875202 A EP 08875202A EP 08875202 A EP08875202 A EP 08875202A EP 2346606 A1 EP2346606 A1 EP 2346606A1
Authority
EP
European Patent Office
Prior art keywords
vessel
control
agent
mrna
blood
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08875202A
Other languages
German (de)
English (en)
Inventor
Patrick Stordeur
Michel Goldman
Marius Tuijnder
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Libre de Bruxelles ULB
Original Assignee
Universite Libre de Bruxelles ULB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite Libre de Bruxelles ULB filed Critical Universite Libre de Bruxelles ULB
Publication of EP2346606A1 publication Critical patent/EP2346606A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/56—Labware specially adapted for transferring fluids
    • B01L3/563—Joints or fittings ; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/048—Function or devices integrated in the closure enabling gas exchange, e.g. vents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0481—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure squeezing of channels or chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the present invention relates to devices, method and kits for use in diagnostic assays, and has applications in the field of immunology.
  • nucleic acid levels for example, those of mRNA is valuable in ascertaining directly the effect of an agent on a biological system. For example, if an agent is introduced into a biological system for a defined length of time, the reaction of the system to the agent can be determined by measuring the levels of mRNA. This may be useful in monitoring immunity wherein, for example, the agent is an antigen and the mRNA monitored is cytokine mRNA e.g. interleukins.
  • RNA degradation and in vitro gene induction can lead to an under- or over-estimation of the in vivo gene transcript number.
  • a challenge in the art is to manage the process of mRNA degradation which begins immediately after the blood is withdrawn and resumes after the introduction of antigen. Because the 'before' degradation can have an impact on the 'after' degradation, the error is coupled to two processes; hence the potential for error is greater and the error is more difficult to delineate.
  • Another problem in the art is the necessity for several pieces of equipment in performing an exposure of a biological sample to an agent followed by a subsequent nucleic acid analysis thereof.
  • reagent bottles, accurate pipettors, refrigeration means are at least required to perform quantitative measurements. If samples are being taken in the absence of suitable laboratory facilities, for example in the home of an individual or in a basically-equipped surgery, it may not be suitable or convenient to perform accurate substrate additions, and furthermore refrigeration facilities might not be available.
  • RNA nucleic acids therein
  • Some methods allow even the determination of low-level transcripts out of a pool of transcripts.
  • none of them provide the possibility to determine the level(s) of transcript(s) present in the biological sample at the time of the sampling.
  • Even under refrigerated conditions the storage of biological samples leads to incorrect mRNA levels. Indeed, in practice, the analysis of fresh sample is not feasible as the place of sampling and the place of RNA analysis are located differently.
  • PreAnalytiX (a joint venture between Becton Dickinson and Qiagen) produced the PAXgeneTM Blood RNA System.
  • the PAXgeneTM Blood RNA System (also referred to as the Qiagen method) is an integrated and standardized system for the collection and stabilization of whole blood specimens and isolation of cellular RNA.
  • PreAnalytiX in the PAXgeneTM Blood RNA System, blood is collected directly into PAXgeneTM Blood RNA Tubes and RNA is subsequently isolated using the PAXgeneTM Blood RNA Kit. Using this system, intact cellular RNA can be retrieved from whole blood.
  • the PAXgeneTM Blood RNA Tube is a plastic, evacuated tube, for the collection of whole blood and stabilization of the cellular RNA profile.
  • the tubes contain an additive (a proprietary blend of reagents) that stabilizes cellular RNA and may eliminate ex vivo induction of gene transcription and prevents the drastic changes in the cellular RNA expression profiles that normally take place in vitro.
  • RNA is then isolated using silica-gel- membrane technology supplied in the PAXgeneTM Blood RNA Kit. According to PreAnalytiX, the resulting RNA accurately represents the expression profile in vivo and is suitable for use in a range of downstream applications. According to the supplier, accurate quantification of gene transcripts is possible using this system.
  • PAXgeneTM Blood RNA System A major disadvantage of this PAXgeneTM Blood RNA System is that respective PAXgeneTM Blood RNA Tube needs to be combined with the PAXgeneTM Blood RNA Kit (see instruction manual of the PPAAXXggeenneeTMTM BBlloooodd RRNNAA T Tubes). This obliged combination, however, limits further improvement of the system.
  • TempusTM Blood RNA Tube (Applied Biosystems) which is similarly is a plastic, evacuated tube, for the collection of whole blood and stabilization of the cellular RNA profile.
  • Each tube contains an additive (a proprietary blend of reagents) that stabilizes cellular RNA and may eliminate ex vivo induction of gene transcription and prevents the drastic changes in the cellular RNA expression profiles that normally take place in vitro.
  • One aim of the present invention is to provide a device, kit and method for exposing a biological sample to an agent and stabilising the nucleic acid in the sample so exposed.
  • One aim of the present invention is to provide a device, kit and method for exposing a biological sample to an agent, stabilising the nucleic acid in the sample so exposed, which device, kit and method also provide a control reaction.
  • Another aim of the present invention is to provide a device, kit and method which reduces, makes constant, or makes nearly constant the time between obtaining the biological sample and exposing said sample to an agent.
  • Another aim of the present invention is to provide a device, kit and method which reduces, makes constant or makes nearly constant the time from which the sample is exposed to an agent and the nucleic acid in said sample is stabilised.
  • Another aim of the present invention is to provide a device, kit and method which exposes an agent to a sample without the need to measure an amount of sample and/or agent.
  • Another aim of the present invention is to provide a single device, which exposes an agent and a control to a sample, and makes constant or makes nearly constant the time from which the sample and control are exposed to an agent and the nucleic acid in said sample is stabilised.
  • Another aim of the present invention is to provide a device, kit and method which exposes an agent to a sample without the need to measure an amount of stabilising agent.
  • Another aim of the present invention is to provide a device, kit and method for exposing a biological sample to an agent, stabilising the nucleic acid in the sample so exposed and extracting the nucleic acid therefrom for further analysis.
  • Another aim of the present invention is to provide a device for exposing a biological sample to an agent, stabilising the nucleic acid in the sample so exposed, which device also provide a control reaction and facilitates robotic automation.
  • Another aim of the present invention is provide a device, kit and method which addresses a combination of one or more of the aforementioned aims.
  • One embodiment of the invention is a kit for assaying a liquid biological sample comprising: a vessel suitable for accepting liquid biological sample, exposing said sample to a first substance and subsequently a nucleic acid stabilising agent, said vessel comprising: a) a first substance present inside said vessel, b) a container in which said stabilising agent is present, c) a connection between the inside of said vessel and the inside of said container, d) a physical barrier that temporarily blocks said connection; and a control vessel suitable for accepting liquid biological sample, exposing said sample to a control substance and subsequently a nucleic acid stabilising agent, said control vessel comprising: a) a control substance present inside said control vessel, b) a control container in which said stabilising agent is present, c) a connection between the inside of said control vessel and the inside of said control container, d) a physical barrier that temporarily blocks said connection.
  • Another embodiment of the invention is a kit as described above, wherein said first substance is immobilised on part or all of the inside surface of said
  • Another embodiment of the invention is a kit as described above, wherein said first substance is immobilised on a solid support.
  • Another embodiment of the invention is a kit as described above, wherein said first substance is a liquid.
  • Another embodiment of the invention is a kit as described above, wherein said first substance is a solid.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise one or more areas suitable for puncture by a syringe needle.
  • Another embodiment of the invention is a kit as described above, wherein said area is a re-sealable septum.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise a fitting suitable for receiving a syringe and transmitting the contents therein to the interior of said vessel or control vessel.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise a fitting suitable for receiving a syringe needle.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise a valve which is capable of minimising the flow of gas/liquid from vessel, and allowing the flow of liquid biological sample into the vessel.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise a means through which displaced gas may be expelled.
  • Another embodiment of the invention is a kit as described above, wherein the vessel and/or control vessel comprise is held under negative pressure.
  • Another embodiment of the invention is a kit as described above, wherein the physical barrier of item d) is opened by the application of physical force to said vessel or control vessel.
  • Another embodiment of the invention is a kit as described above, wherein said force transmits an opening means to said physical barrier.
  • Another embodiment of the invention is a kit as described above, wherein said force irreversibly opens said physical barrier.
  • Another embodiment of the invention is a kit as described above, wherein said vessel and/or control comprises an indication for dispensing a known volume of stabilising agent therein.
  • Another embodiment of the invention is a kit as described above, wherein said first substance comprises one or more immune system antigens.
  • Another embodiment of the invention is a kit as described above, wherein said immune system antigens are vaccine components.
  • Another embodiment of the invention is a kit as described above, wherein said immune system antigens are antigens which provoke a hyperallergenic response.
  • Another embodiment of the invention is a kit as described above, wherein said immune system antigens are one or more selected from histocompatibility antigens, bacterial LPS, tetanous toxoid, a cancer immunotherapy antigen, MAGE-3, a cat allergen, FeIdI , antigen presenting cells from an organ donor, an autoantigen, GAD65.
  • said immune system antigens are one or more selected from histocompatibility antigens, bacterial LPS, tetanous toxoid, a cancer immunotherapy antigen, MAGE-3, a cat allergen, FeIdI , antigen presenting cells from an organ donor, an autoantigen, GAD65.
  • Another embodiment of the invention is a kit as described above, wherein said stabilising agent is an inhibitor of cellular RNA degradation and/or gene induction.
  • Another embodiment of the invention is a kit as described above, wherein said inhibitor of cellular RNA degradation and/or gene induction is that as found in a PAXgeneTM or Tempus TM Blood RNA Tube.
  • Another embodiment of the invention is a kit as described above, wherein the exterior of the vessel and control vessel are joined to form a single entity.
  • Another embodiment of the invention is an assay device for a liquid biological sample, which facilitates exposure of said sample separately to a first substance and a control substance, and subsequently to a nucleic acid stabilizing agent, the device comprising:
  • Another embodiment of the invention is an assay device as described above, wherein one or more of the compartments is sealed, and comprises one or more areas suitable for puncture by a hollow needle.
  • Another embodiment of the invention is an assay device as described above, wherein said area is a re-sealable septum.
  • Another embodiment of the invention is an assay device as described above, further comprising a support for demountable attachment to a transfer tubing, which transfer tubing comprises a hollow flexible or rigid tubing disposed with a hollow needle at either end, adapted for transfer of liquid between any two compartments, or between a compartment and the biological sample tube.
  • Another embodiment of the invention is an assay device as described above, further comprising the transfer tubing as defined above.
  • Another embodiment of the invention is an assay device as described above, wherein one needle of the transfer tubing is attached in parallel alignment to the hollow needle of a pressurising tubing, which pressurising tubing comprises a flexible hollow tubing disposed at one end with said needle, and is adapted to apply vacuum or pressure to a compartment or biological sample tube to propel liquid through the transfer tubing.
  • Another embodiment of the invention is an assay device as described above, wherein said first substance is immobilised on part or all of the inside surface of said vessel.
  • Another embodiment of the invention is an assay device as described above, wherein one or more of the compartments comprises an air vent.
  • Another embodiment of the invention is an assay device as described above, wherein the first and/or second compartments are held under negative pressure.
  • Another embodiment of the invention is an assay device as described above, wherein the first and/or second compartments comprise an indication for dispensing a known volume of stabilising agent therein.
  • Another embodiment of the invention is an assay device as described above, wherein said first substance is as defined above.
  • Another embodiment of the invention is an assay device as described above, wherein said stabilizing agent is as defined above.
  • One aspect of the present invention is related to a vessel suitable for holding a biological sample, said vessel holding a predetermined amount of a first substance which may be a pulsing agent.
  • pulsesing agent comprises any substance to which a biological sample may be exposed.
  • substances include peptides, nucleic acids, antigens.
  • the pulsing agent may comprise other components besides the substance, such as stabilising agents, indicators, linkers, matrices, etc.
  • biological sample means a sample containing nucleic acids/biological agents such as clinical (e.g. cell fractions, whole blood, plasma, serum, urine, tissue, cells, etc.), agricultural, environmental (e.g. soil, mud, minerals, water, air), food (any food material), forensic or other possible samples.
  • clinical e.g. cell fractions, whole blood, plasma, serum, urine, tissue, cells, etc.
  • agricultural e.g. soil, mud, minerals, water, air
  • food any food material
  • forensic or other possible samples e.g. cell fractions, whole blood, plasma, serum, urine, tissue, cells, etc.
  • 'whole blood' is meant blood such as it is collected by venous sampling, i.e. containing white and red cells, platelets, plasma and eventually infectious agents; the infectious agents may be viral, bacterial or parasitical.
  • the clinical samples may be from human or animal origin.
  • the sample analysed can be both solid or liquid in nature.
  • RNAIater reagent sold by Qiagen.
  • this solution is not always a real "buffer" with at least two well balanced components. It may be a strong hypotonic solution such as NaCI alone or an extraction solution such as with alcohol.
  • the vessel also known herein as a reaction vessel, may hold the pulsing agent in several ways.
  • the pulsing agent may be immobilised on the inside wall of the vessel.
  • the inside wall of the vessel may be lined with a suitable coating enabling the pulsing agent to be attached.
  • the pulsing agent may be attached directly to part or all of the inside wall of the vessel. Suitable coatings, methods and vessel materials for suitable for such attachments are known in the art.
  • the pulsing agent is present as a solid.
  • the solid may be a powder, a freeze-dried pellet, a gel, a cream. Suitable solid compositions and method of their preparation are known in the art.
  • the pulsing agent is immobilised on a solid support.
  • the solid support may be attached to the inside of the vessel.
  • the solid support may be free of the inside of the vessel.
  • solid supports include, but are not limited to, chromatography matrix, magnetic beads.
  • pulsing agent is present as a liquid. Suitable liquid compositions and method of their preparation are known in the art.
  • Performing an analytical pulsing experiment outside of laboratory conditions requires calibrated measuring equipment such as pipettors. Errors due to uncalibrated measuring devices can lead to inherent errors and also human error in dispensing can lead to error between different samples, making comparative analysis invalid.
  • Providing a vessel supplied with a pre-determined amount of pulsing agent obviates the need for additional equipment and eliminates human measuring errors.
  • the provision of a separate control vessel allows normalisation of results, and facilitates the detection of errors.
  • the use of a control is important since RNA is labile and rapidly degrades once it is withdrawn from the circulation. Inconsistent delays in pulsing whole blood would lead to un- comparable results.
  • the present invention allows measurement of such a biological process by quantifying the messenger RNA of biomarkers such as cytokines, chemokines, transcription factors and other gene products or surrogate markers representative for a specific process. Without a reference value, the quantification of these biomarkers in the assay has little meaning; the use of an integrated control permits rapid execution of a control reaction. By comparing assay and control, the meaning of the quantity of the biomarker and the influence of the pulsing agent on the biological system. As an example, it is general accepted that the Mx1 gene is under the control of IFN type I.
  • the Mx1 gene product is quantified after incubating whole blood with IFN type I.
  • the quantity of Mx1 gene product can be expressed as a number of mRNA copies compared to a number of mRNA copies of a normalization gene product, i.e. a gene whose transcription activity is not influenced by the action of IFN. But this quantification would be meaningless without a control reaction being a quantification of the surrogate marker under non-stimulated control conditions i.e. same treatment without IFN type I.
  • the same reasoning can be made for any immune reaction.
  • control sample is treated the same way, i.e. is from the same withdrawal, same incubation time and temperature and parallel treatment; the combination of assay and control compartments in a single device makes this feasible.
  • Types of vessel according to the invention can be any suitable for storage of biological samples.
  • the vessel containing the pulsing agent is sealed.
  • the vessel containing the pulsing agent has a resealing means such as a screw-cap, push-on cap, a flip-cap. See, for example, FIG. 5.
  • the biological sample or other fluids may be introduced into the vessel by puncture, using a syringe needle into the wall of the vessel.
  • the wall of the vessel may be resealable after puncture, or the wall of the vessel may not be resealable after puncture, or the wall of the vessel may be provided with a resealable area such as a septum. See, for example, FIG. 4.
  • the biological sample, or other liquids are introduced by means of a transfer tubing comprising a hollow tube disposed with a needle either end (e.g. a cannular needle).
  • a needle is configured to puncture a septum of a sample tube holding the sample, or other container, and the other needle is configured to puncture the septum of the vessel; biological sample is able to be transferred from the sample tube to the vessel using the needle.
  • the tubing may be flexible or rigid.
  • the shape of the transfer tubing may be U-shaped. See, for example, FIG. 22.
  • the biological sample may be introduced into the vessel by means of one or more fittings attached to vessel for receiving a syringe or other container fitted with a coupling means.
  • the vessel might be fitted with a Luer fitting that can receive a needleless syringe. See for example, FIG. 3.
  • the vessel might be fitted with a non-Luer fitting which can mate with a container having a reciprocating non-Luer design of coupling.
  • the biological sample may be introduced into the vessel by means of a cannular or hypodermic needle fitted to said vessel, suitable for directly withdrawing biological samples from an individual. See, for example, FIG. 6.
  • the biological sample may be introduced into the vessel by opening the resealing means. See, for example, FIG. 5.
  • the vessel may be provided with a suitable means to allow displaced gas to exit from said vessel, or to accommodate the build up of pressure.
  • Said means are known the art and include valves, non-drip holes, vents, clothed-vents, expandable vessel walls, use of negative pressure within said vessel. See, for example, arrow 31 on FIG. 11.
  • the pressure inside the sealed vessel is negative.
  • the negative pressure may be utilised to relieve the pressure build-up upon introduction of biological sample into said sealed vessel.
  • the negative pressure may be at a predetermined level and may be utilised so as to allow the introduction of a fixed volume of biological sample.
  • the vessel is provided with a sealable septum as described above, adapted to receive a first needle (cannular) through which liquid biological sample can enter the vessel and a second needle through which positive or negative pressure can be applied to the inside of the vessel.
  • a first needle cannular
  • a second needle through which positive or negative pressure can be applied to the inside of the vessel.
  • negative (vacuum) pressure is applied through the second needle, biological sample can be drawn into the vessel through the first needle.
  • the first needle is part of the transfer tubing described earlier.
  • the second needle may be part of a pressurizing tubing described below, which is connected to an air (evacuating or pressuring) pump.
  • the first and second needles may be joined in parallel alignment. See, for example, FIG. 24.
  • the vessel in which a pre-determined quantity of pulsing agent is already supplied allows diagnostic tests to be performed on individuals without the necessity for apparatus for measuring out said antigen. Furthermore, when diagnostic test are performed outside laboratory conditions, problems with contamination and dispensing accuracy can lead to false results in a quantitative assay.
  • a vessel as described herein overcomes these problems.
  • control vessel that may be the vessel as described above, except instead of containing a biological pulsing agent, it contains a control pulsing agent (control substance).
  • control substance control substance
  • the vessel as described herein is used to hold pulsing agent and is sometimes also called the "reaction vessel", while the control vessel as described herein is used to hold control pulsing agent.
  • the vessel and control vessel operate independently; they independently receive sample and stabilizing agent, and interiors are not connected.
  • the vessel and the control vessel may be present together as part of a kit.
  • the exterior surfaces of the vessel and control vessel may be mechanically connected to form a single entity comprising both a vessel and control vessel.
  • the control vessel containing the control pulsing agent is sealed.
  • the control vessel may have a resealing means such as a screw-cap, push-on cap, a flip-cap for example.
  • the control vessel may have a breakable seal such as a peel- back adhesive seal, a snap-off seal.
  • the wall of the control vessel may be provided with a resealable area such as a septum, through which substances may be introduced into the control vessel by puncture, using a syringe needle.
  • the septum of the control vessel may be resealable after puncture.
  • the biological sample or other liquids are introduced by means of a transfer tubing described above having disposed with a needle either end (e.g. a cannular needle).
  • a needle e.g. a cannular needle
  • One needle is configured to puncture a septum of a tube holding the sample tube or other container, and the other needle is configured to puncture the septum of the control vessel; liquid biological sample is able to be transferred from the sample tube to the control vessel using the needle.
  • the tubing may be flexible or rigid.
  • the shape of the transfer tubing may be U-shaped.
  • control vessel may have a means to draw liquid biological sample from a biological sample tube when in fluid connection therewith; examples include but are not limited to a syringe-type plunger, or any means to apply negative pressure.
  • control vessel receives a portion of the same biological sample introduced into the reaction vessel.
  • the biological sample may be divided into two portions, one for the control and the other for the reactions vessel.
  • one aliquot of the biological sample may be introduced into the reaction vessel and a second equal aliquot may be introduced into the control vessel.
  • the control vessel is provided with a sealable septum as described above, adapted to receive a first needle (cannular) through which liquid biological sample can enter the control vessel and a second needle through which positive or negative pressure can be applied to the inside of the control vessel.
  • a first needle cannular
  • a second needle through which positive or negative pressure can be applied to the inside of the control vessel.
  • biological sample can be drawn into the control vessel through the first needle.
  • the first needle is part of the transfer tubing described earlier.
  • the second needle may be part of a pressurizing tubing described below, which is connected to an air (evacuating or pressuring) pump.
  • the first and second needles may be joined in parallel alignment. See, for example, FIG. 24.
  • control pulsing agent will depend on the pulsing agent, and the parameter under investigation, as is well understood by the skilled artisan.
  • the control pulsing agent may comprises a peptide or peptides of the same length, with the exception that the sequence of the peptide(s) is randomized and/or is not identical to a segment of an existing protein or peptide.
  • This control pulsing agent would be incubated with the same whole blood as the pulsing agent, with the same quantity of agent, same volume of blood, for the same period of time and at the same temperature.
  • the pulsing agent may be a protein therapeutic agent, such as IFN-beta dissolved in an excipient such as water, phosphate-buffered saline, or saline to the desired concentration.
  • the control pulsing agent may then be the excipient.
  • the pulsing agent may be an antigen, such as FeIdI dissolved in an excipient such as water, phosphate-buffered saline, or saline to the desired concentration; the control pulsing agent may then be excipient of the antigen.
  • the control vessel may devoid of control agent or is empty in which case an induced immune response, or induced gene expression, for example, is compared to an untreated sample.
  • Another aspect of the present invention is related to a vessel as described herein, further comprising a container in which a stabilising agent is present; the stabilising agent is temporarily prevented from coming into contact with the pulsing agent or the biological sample exposed to said agent.
  • the stabilising agent comprises a nucleic acid stabilising agent and/or cellular RNA degradation inhibiting agent and/or a gene induction inhibiting agent, and/or the stabilising agent is as that found in a PAXgeneTM Blood RNA Tube and/or stabilising agent is as that found in a TempusTM Blood RNA Tube. Agents and combinations thereof are known in the art, or can be deduced by the skilled artisan.
  • the PAXgeneTM and TempusTM Blood RNA Tubes are supplied with a solution containing an additive that stabilises cellular RNA and may eliminate ex vivo induction of the gene transcription. No detailed information is provided describing the nature of this additive.
  • the brochure provided with PAXgeneTM tubes refers to patent US 5,906,744 for this purpose. Nevertheless, the tube described in this patent allows a person skilled in the art to prepare nucleic acids from plasma and not from whole blood as performed in the present invention.
  • the device of US 5,906,744 preferably comprises a plastic or glass tube, a means for inhibiting blood coagulation and a means for separating plasma from whole blood (US 5,906,744 column 2, 1.42-43). Therefore, according to the present invention, the content as described in US 5,906,744 does not relate to the real content of the PAXgeneTM Blood RNA Tube as it relates to a different use.
  • the solution held in the PAXgeneTM Blood RNA Tubes may contain a quaternary amine surfactant. Therefore, according to the present invention, a quaternary amine surfactant may be used as a stabilising agent.
  • a quaternary amine surfactant in order to stabilise nucleic acids in a biological sample has been previously described in US 5,010,183. This patent provides a method for purifying DNA or RNA from a mixture of biological materials.
  • Said method comprises the step of adding a cationic detergent to a mixture containing the RNA or DNA in an amount sufficient to dissolve cells, solubilize any contaminating proteins and lipids in the mixture, and form insoluble hydrophobic complex between the nucleic acid and the detergent.
  • the complex which comprises the RNA or DNA with the detergent thus becomes separated from the solubilised contaminants.
  • quaternary amine which can be used in the method of the present invention is tetradecyltrimethyl-ammonium oxalate. (US 5,985,572).
  • said cationic detergent may be Catrimox-14TM (US5,010,183) as shown in the example 1 of the present invention.
  • said applications describe the isolation of the nucleic acids using conventional separation techniques such as column chromatography. Due to the obliged combination of the PAXgeneTM Blood RNA Tube with the PAXgeneTM Blood RNA kit (which also applies column chromatography) the supplier gives the impression that the compounds present in the PAXgeneTM Blood RNA Tube may only be compatible with said chromatographic method.
  • the stabilising agent is contained in said container until such as time as the biological sample has mixed with the pulsing agent and/or a user requires introduction of the stabilising agent.
  • the container may be integrated into the vessel, meaning that it the container may be mechanically joined to the vessel so forming a one-piece unit.
  • the inside of said container and the inside of said vessel are connected, and a physical barrier that blocks the connection is present.
  • an application of force opens the physical barrier, allowing the stabilising agent to mix with the biological sample so-pulsed.
  • a physical barrier reversibly opens and closes in accordance with the physical force applied.
  • the force applied may transmit to the physical barrier itself, or via the stabilising agent to the physical barrier.
  • a physical barrier may be any mechanical barrier. Examples of such physical barriers include rotary valve, aperture valve, slit valve, diaphragm valve, ball valve, flap valve.
  • the physical barrier may be irreversibly opened by the application of force.
  • a physical force may be any mechanical force.
  • the force applied may transmit to the physical barrier itself (see for example, FIG. 7), or via the stabilising agent to the physical barrier (see for example, FIG. 8).
  • Another example of such physical barriers include a plug which is forced out of position (See, for example, FIG. 1 ), a barrier which shatters upon the application of force (see, for example, FIG. 7).
  • the inside of said container and the inside of said vessel are connected, and the flow of stabilising agent from the container to the vessel is prevented by the surface tension of the stabilising agent in combination with the aperture size of the connection.
  • an application of force which transmits to the stabilising agent forces the stabilising agent from the container into the vessel.
  • the force may be applied, for example, by squeezing, continually inverting, and agitating.
  • Another aspect of the present invention is related to a control vessel as described herein, further comprising a control container in which a stabilising agent is present; the stabilising agent is temporarily prevented from coming into contact with the control pulsing agent or the biological sample exposed to said control pulsing agent.
  • the vessel with integrated container described herein, together with the control vessel with integrated control container may be present together as part of a kit.
  • the exterior of the vessel with integrated container described herein, together with the exterior of the control vessel with integrated control container may be joined or connected using a bridging member to form a single device (FIGs. 19 and 20).
  • the control container may be integrated into the control vessel, meaning that it the control container may be mechanically joined to the control vessel so forming a one-piece unit.
  • the inside of said control container and the inside of said control vessel are connected, and a physical barrier that blocks the connection is present.
  • an application of force opens the physical barrier, allowing the stabilising agent to mix with the biological sample so-pulsed.
  • a physical barrier reversibly opens and closes in accordance with the physical force applied.
  • the force applied may transmit to the physical barrier itself, or via the stabilising agent to the physical barrier. Examples of such physical barriers include rotary valve, aperture valve, slit valve, diaphragm valve, ball valve, flap valve.
  • the physical barrier may be irreversibly opened by the application of force.
  • the force applied may transmit to the physical barrier itself (see for example, FIG. 17), or via the stabilising agent to the physical barrier (see for example, FIG. 18).
  • Another example of such physical barriers include a plug which is forced out of position (See, for example, FIG. 12), a barrier which shatters upon the application of force (see, for example, FIG. 17).
  • the inside of said control container and the inside of said control vessel are connected, and the flow of stabilising agent from the control container to the control vessel is prevented by the surface tension of the stabilising agent in combination with the aperture size of the connection.
  • an application of force which transmits to the stabilising agent forces the stabilising agent from the control container into the control vessel.
  • the force may be applied, for example, by squeezing, continually inverting, and agitating.
  • the vessel or control vessel as described herein which comprises a container or control container respectively for dispensing a stabilising agent, allows an untrained technician to pulse blood using a pulsing agent or control, and stabilise the blood so pulsed for analysis by a skilled artisan at a later stage.
  • a vessel or control vessel as disclosed herein allows a cost saving since unskilled operators can be employed to pulse and stabilise the blood.
  • the vessel or control vessel allow reproducibility because known amounts of pulsing agent and stabilising agent may be pre-supplied in said vessel, so minimising errors associated with pipetting.
  • the time between withdrawing a biological sample and exposing said sample to a pulsing antigen or control is greatly reduced since the sample can be drawn directly into said tube, or via a syringe for example. Furthermore, the time between pulsing the biological sample and stabilising the biological sample can be accurately set, since the introduction of stabilising agent to the sample is achieved simply by the application of force; hence there are no delays due to pipetting stabilising agent.
  • kits suitable for pulsing a biological sample with a pulsing agent, and subsequently introducing a stabilising agent thereto and testing the RNA components in the biological sample so pulsed comprising one or more vessels as disclosed above and one or more containers in which said stabilising agent is present.
  • the kit may further comprise a control vessel in which a biological control pulsing agent is present.
  • the container in which said stabilising agent is present is not integrated into the vessel in which the pulsing agent in present, and/ or the control container in which said stabilising agent is present is not integrated into the control vessel in which the control substance is present.
  • the container or control container can thus be separate and may be any container of the art, suitable for holding a stabilising agent in a kit.
  • the container or control container containing the stabilising agent is sealed.
  • the container or control container may have a resealing means such as a screw-cap, push-on cap, a flip-cap for example.
  • the container or control container may have a breakable seal such as a peel-back adhesive seal, a snap-off seal.
  • the wall of the container or control container may be provided with a resealable area such as a septum; stabilizing agent may drawn from the container or control container by puncture, using a syringe needle.
  • the septum of the container or control container may be resealable after puncture.
  • the container or control container is used in conjunction with a transfer tubing disposed with a needle either end (e.g. a cannular needle).
  • a needle is configured to puncture a septum of the container or control container and the other needle is configured to puncture the septum of the vessel or control vessel (described below) respectively; stabilising agent is able to be transferred from the container or control container to the vessel or control vessel respectively using the transfer tubing.
  • the tubing may be flexible or rigid.
  • the shape of the transfer tubing may be U-shaped.
  • the container or control container might comprise one or more fittings suitable for attachment of said vessel fitted reciprocal coupling means and transfer of stabilising agent to said vessel or control vessel.
  • the container or control container might be fitted with a Luer fitting that can receive a reciprocal Luer fitting attached to said vessel as described above (see, for example, FIGs. 9, 10 and 11 ), or attached to the control vessel.
  • the container or control container might be fitted with a non-Luer fitting which can mate with a vessel or control vessel having a reciprocating non-Luer design of coupling.
  • the stabilising agent might be transferred to the vessel or control vessel by opening the resealing means of the vessel; the stabilising agent may exit the container or control container via any of the aforementioned fittings or openings.
  • the container or control container may optionally have a means to allow air to enter while stabilising agent exits.
  • the container or control container may have a means to force stabilising agent from said container or control container; examples include but are not limited to a syringe-type plunger, squeezable walls of the container or control container or any means to apply positive pressure.
  • the container or control container optionally has a measuring means to determine the volume of stabilising agent being dispensed, for example, a scale.
  • the container or control container holds a volume of stabilising agent sufficient for single use.
  • the container or control container holds a volume of stabilising agent sufficient for multiple pulsing experiments.
  • the container or control container is provided with a sealable septum as described above, adapted to receive a first needle (cannular) through which stabilizing agent can exit the vessel and a second needle through which positive or negative pressure can be applied to the inside of the vessel.
  • first needle cannular
  • second needle through which positive or negative pressure can be applied to the inside of the vessel.
  • stabilising agent can be propelled from the container or control container through the first needle.
  • the first needle is part of the transfer tubing described earlier.
  • the second needle may be part of a pressurizing tubing described below, which is connected to air (evacuating or pressuring) pump.
  • the first and second needles may be joined in parallel alignment.
  • the container in which said stabilising agent is present is connected to the vessel in which the pulsing agent is present; embodiments of the vessel are described above.
  • the control container in which said stabilising agent is present is connected to the control vessel in which the control substance is present; embodiments of the control vessel are described above.
  • kits of the present invention may comprise an instruction manual comprising a description of a method for pulsing a biological sample.
  • Another aspect of the present invention is related to a vessel, and a kit comprising said vessel as disclosed herein wherein said pulsing agent comprises an antigen.
  • said antigen is bacterial LPS.
  • said antigen is an immune response recall antigen.
  • said antigen is tetanous toxoid.
  • said antigen is a cancer immunotherapy antigen.
  • said antigen is MAGE-3.
  • said antigen is a cat allergen.
  • said antigen is FeIdL
  • said antigen is antigen presenting cells from an organ donor.
  • said antigen is an autoantigen.
  • said antigen is GAD65.
  • control pulsing agent will depend on the pulsing agent, and the parameter under investigation, as is well understood by the skilled artisan.
  • the control pulsing agent may comprises a peptide or peptides of the same length, with the exception that the sequence of the peptide(s) is randomized and/or is not identical to a segment of an existing protein or peptide.
  • This control pulsing agent would be incubated with the same whole blood as the pulsing agent, with the same quantity of agent, same volume of blood, for the same period of time and at the same temperature.
  • the pulsing agent may be a protein therapeutic agent, such as IFN-beta dissolved in an excipient such as water, phosphate-buffered saline, or saline to the desired concentration.
  • the control pulsing agent may then be the excipient.
  • the pulsing agent may be an antigen, such as FeIdI dissolved in an excipient such as water, phosphate-buffered saline, or saline to the desired concentration; the control pulsing agent may then be excipient of the antigen.
  • the control vessel may devoid of control agent or is empty in which case an induced immune response, or induced gene expression, for example, is compared to an untreated sample.
  • Another aspect of the present invention is related to a method of pulsing a biological sample with an antigen, and subsequently stabilising the nucleic acid therein and testing the RNA components in the stabilising biological sample so pulsed.
  • the method comprises the use of a vessel, control vessel and/or kit as disclosed herein to optionally collect, pulse and stabilise the sample.
  • One embodiment of the invention is a device for accepting a liquid biological sample, which facilitates exposure of said sample separately to a first substance and a control substance, and subsequently to a nucleic acid stabilizing agent, the device comprising:
  • FIGs. 21a and 21 b An example of a device is shown in FIGs. 21a and 21 b.
  • at least one, (e.g. 1 , 2, 3, all), preferably all the compartments are sealed.
  • One or more (e.g. 1 , 2, 3, all) compartments may have a resealing means such as a screw- cap, push-on cap, a flip-cap for example.
  • One or more (e.g. 1 , 2, 3, all) compartments may have a breakable seal such as a peel-back adhesive seal, a snap-off seal.
  • compartments is provided with a resealable area such as a septum, through which substances may be introduced into the compartment by puncture, using a syringe needle.
  • a resealable area such as a septum
  • the septum of the compartment may be resealable after puncture.
  • one or more (e.g. 1 , 2, 3, all) compartments and the biological sample tube is used in conjunction with a transfer tubing - a hollow tubing disposed with a hollow needle either end (e.g. a cannular of hypodermic needle) as shown, for instance, in FIG.22.
  • a transfer tubing - a hollow tubing disposed with a hollow needle either end e.g. a cannular of hypodermic needle
  • One needle is configured to puncture a septum of one compartment (e.g. the first compartment) and the other needle is configured to puncture the septum of the other compartment (e.g. the third compartment).
  • one needle is configured to puncture a septum of one compartment (e.g.
  • the fluid connection allows stabilising agent, for example, to be transferred from the third compartment to the first and/or second compartment using the transfer tubing.
  • the tubing may be flexible or rigid.
  • the transfer tubing may be U-shaped.
  • the biological sample tube according to the invention can be any suitable for storage of a biological sample.
  • the biological sample tube is sealed.
  • the biological sample tube has a resealing means such as a screw-cap, push-on cap, a flip-cap.
  • a biological sample may be introduced into the tube by puncture, using a syringe needle into the wall of the tube.
  • the wall of the tube may be resealable after puncture, or the wall of the tube may not be resealable after puncture, or the wall of the sample tube may be provided with a resealable area such as a septum.
  • the biological sample may be drawn from the tube introduced by means of a transfer tubing comprising a hollow tube disposed with a needle either end (e.g. a cannular needle) as described elsewhere herein.
  • a transfer tubing comprising a hollow tube disposed with a needle either end (e.g. a cannular needle) as described elsewhere herein.
  • One needle is configured to puncture a septum of a sample tube holding the sample or other liquids, and the other needle is configured to puncture the septum of a compartment; liquid biological sample is able to be transferred from the sample tube to one of the compartments using the tubing.
  • the tubing may be flexible or rigid.
  • the shape of the transfer tubing may be U-shaped.
  • the biological sample may be introduced into the sample tube by means of one or more fittings attached to sample tube adapted to a syringe or other container fitted with a coupling means.
  • the tube might be fitted with a Luer fitting that can receive a needleless syringe.
  • the tube might be fitted with a non-Luer fitting which can mate with a container having a reciprocating non-Luer design of coupling.
  • the biological sample may be introduced into the tube by means of a cannular or hypodermic needle fitted to said tube, suitable for directly withdrawing biological samples from an individual.
  • the biological sample may be introduced into the sample tube by opening the resealing means.
  • the first compartment may have a means to draw liquid biological sample from a biological sample tube into the first compartment when in fluid connection therewith.
  • the second compartment may similarly have a means to draw liquid biological sample from a biological sample tube into the second compartment when in fluid connection therewith using the transfer tubing for instance.
  • Such means include but are not limited to the use of a syringe-type plunger, or any means to apply negative pressure to the receiving compartment, or positive pressure to the supplying compartment.
  • pressure inside the compartment is controlled using an insertable pressurising tubing described below.
  • the first and/or second compartments may partially held under vacuum.
  • the first compartment may have a means to draw stabilising agent from the third compartment into the first compartment when in fluid connection therewith.
  • the second compartment may similarly have a means to draw stabilising agent from the third compartment into the second compartment when in fluid connection therewith using the transfer tubing for instance.
  • Examples of such means include but are not limited to the use of a syringe-type plunger, or any means to apply negative pressure to the receiving compartment, or positive pressure to the supplying compartment.
  • pressure inside the compartment is controlled using an insertable pressurising tubing described below.
  • the first and/or second compartments may partially held under vacuum.
  • the third compartment may have a means to propel stabilising agent from the third compartment into the first and/or into the second compartment when in fluid connection therewith using the transfer tubing for instance.
  • a means to propel stabilising agent from the third compartment into the first and/or into the second compartment when in fluid connection therewith using the transfer tubing for instance.
  • examples of such means include but are not limited to a syringe-type plunger, or any means to apply positive pressure to the third compartment.
  • pressure inside the compartment is controlled using an insertable pressurizing tubing described below.
  • the third compartment may be held under positive pressure.
  • the first compartment is sterile.
  • the second compartment is sterile.
  • the third compartment is sterile.
  • all the compartments are sterile.
  • the each compartment is provided with a sealable septum as described above, adapted to receive a first needle through which liquid can pass and a second needle through which positive or negative pressure can be applied to the inside of the compartment.
  • a sealable septum as described above, adapted to receive a first needle through which liquid can pass and a second needle through which positive or negative pressure can be applied to the inside of the compartment.
  • positive pressure is applied through the second needle
  • liquid can be propelled from the compartment through the first needle.
  • negative pressure is applied through the second needle
  • liquid can be draw into the compartment through the first needle.
  • the first needle is part of the transfer tubing described earlier.
  • the second needle is part of the pressurising tubing. The first and second needles may be joined in parallel alignment. See, for example, FIG. 24.
  • one or more compartments may optionally be disposed with a vent that allows pressure in the compartment to be equalised with ambient pressure.
  • the vent may comprise a one way valve (e.g. rotary valve, aperture valve, slit valve, diaphragm valve, ball valve, flap valve) configured to provide a passage of gas in only one direction, for example, it may allow the release of expelled gas.
  • the device is further provided with a support for the biological sample tube, also known herein as a sample tube support.
  • the sample tube support may be any suitable structure, mechanical or otherwise, that holds the sample tube in an upright position.
  • the sample tube support is a walled aperture having a shape reciprocal to at least the base region of the sample tube.
  • the device is further provided with a support for a transfer tubing, also known herein as a tubing support.
  • the tubing support may be any suitable structure, mechanical or otherwise, adapted for demountable attachment of the transfer tubing to the device.
  • the tubing support allows storage of the transfer tubing during transport and non-use. It also provides a clean environment for the needle ends of the transfer tube.
  • the transfer tubing support comprises two slots each having a shape reciprocal to each end of the transfer tubing.
  • the device may take any form which provides the requirements for the compartments and supporting elements. It will be understood that it should preferably be optimized for size, weight, supporting ability, durability and recyclability.
  • the compartments are mechanically connected to each other.
  • the sample tube support is also mechanically connected to the compartments.
  • the tubing support is also mechanically connected to the compartments. Being so connected, the device is a single entity that has the convenience of being an autonomous unit.
  • Mechanical connection may be achieved using any suitable arrangement as known in the art, for example, employing compartments formed from a single block of biologically inert material such as polycarbonate, polypropylene, cyclic olefin copolymer (COC) or glass mechanically connected to a support of the same or different material.
  • biologically inert material such as polycarbonate, polypropylene, cyclic olefin copolymer (COC) or glass mechanically connected to a support of the same or different material.
  • FIG. 21a A preferred embodiment of the invention is shown in three dimensional view in FIG. 21a and in cross-section in FIG. 21 b, which depict a device 30 formed from a solid block 44 of a material such as cyclic olefin copolymer (COC), provided with a first compartment 32 in which a first substance 2 is present, a second compartment 34 in which a control substance 35 is present, and a third compartment 36 in which a stabilizing agent 5 is present.
  • the compartments 32, 34, 36 are cylindrical openings provided in the solid block, 44 each sealed with a lid 46, 48, 50 which may also be made at least partly from COC.
  • the compartments 32, 34, 36 may each be formed from a cylindrical vial made from a suitable material (e.g.
  • a sample tube 52 support is provided on the device.
  • Said sample tube support 52 is a cylindrical opening configured to support the base region of a sample tube.
  • the device is further disposed with a tubing support 54, 56 comprising two slots each having a shape reciprocal to each end of the transfer tubing.
  • the contents of the sample tube can be transferred to each of first (pulsing agent) compartment 32 and second (control) compartment 34, said compartments being under negative pressure.
  • the same needle may be used to transfer the stabilizing agent from the third compartment 36 into each of the first 32 and second 34 compartments.
  • a transfer tubing comprising a hollow tube disposed with a needle at either end (e.g. a cannular needle) is used to transfer liquid from one compartment or vessel to another, the liquid either being propelled by the application of positive pressure to the providing compartment (or container or control container), or being drawn by application of negative pressure in the receiving compartment (or vessel or control vessel,).
  • the transfer tubing needles are suited to piercing a resealable (e.g. rubber or silicone) septum so that fluid or air may only pass into the compartment (vessel or control vessel) through the needle.
  • the pointed end of the needle is preferably beveled with both sides sharp, and may be non-coring.
  • the needle may be 22 G, 23 G, 24, G or 25 G, or a value in the range between any two of the aforementioned values, preferably 23 G.
  • Each needle may be protected with a detachable water-impermeable cover to prevent the ingress of micro-organisms prior to use.
  • Each needle cover may reside in and be attached to the tubing support such that when the U-shaped needle is lifted from the device, the cover detaches from each needle, leaving the cover still within the tubing support.
  • the length (L) of the transfer tubing needles in the transfer tubing - which refers to the length of the straight part of the needle - may be the same or different. Their lengths will depend on the depth of the compartments and on the volume of liquid that is to be transferred. As a general guidance, the length (L1 ) of one transfer tubing needle may be 25, 26, 28, 30, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 55 mm, or a value in the range between any two of the aforementioned values, preferably between 25 and 35 mm, more preferably between 30 and 35 mm.
  • the length (L2) of the other transfer tubing needle may be 25, 26, 28, 30, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 55 mm, or a value in the range between any two of the aforementioned values, preferably between 40 and 50 mm, more preferably between 42 and 46 mm.
  • the height of the tubing (L3) will depend on the clearance space available. As a general guidance, the length (L3) of one transfer tubing may be 5, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 or 17 mm, or a value in the range between any two of the aforementioned values, preferably between 10 and 15 mm, more preferably between 12 and 14 mm.
  • the hollow tubing may be rigid or flexible. Preferably it is rigid.
  • the transfer tubing is formed from a long needle, bent in a U form, whereby a second needle is attached to the non-pointed end so as to form the transfer tubing of the invention, having a rigid hollow tube.
  • the distance (D) between both needles where rigid tubing is employed will depend on the spacing between the compartments.
  • the distance (D) between both needles is essentially the same as the distance between two adjacent compartments; this is generally achieved when the compartments and sample support are arranged in a square 2 x 2 array.
  • the distance between the needles may be 20, 22, 24, 26, 28, 30, or 32 mm, or a value in the range between any two of the aforementioned values, preferably between 24 and 28 mm, more preferably between 26 mm.
  • the transfer tubing may be adapted for attachment to a robotic system (e.g. robotic arm or platform) to facilitate automated sample processing.
  • a robotic system e.g. robotic arm or platform
  • the robotic system is attached to the transfer tubing via the rigid hollow tubing.
  • the pressure to a compartment, vessel, or container may be provided by a hollow pressurizing tubing (FIG. 23) comprising a flexible tubing having a needle disposed at one end for insertion into the septum of a compartment, vessel, or container, the other end adapted for attachment to an air pump that can provide the requisite controlled pressure to the compartment, vessel or container.
  • This pressurizing tubing needle may be mechanically attached to one of the needles of the transfer tubing by a joint such as a welded joint, adhesive joint, or clamp (see FIG. 24).
  • the pressurizing tubing needle is suited to piercing a resealable (e.g. rubber or silicone) septum so that air or gas may only enter or leave the compartment, vessel or container through the needle.
  • the pointed end of the needle is preferably beveled with both sides sharp, and may be non-coring.
  • the needle may be 22 G, 23 G, 24, G or 25 G, or a value in the range between any two of the aforementioned values
  • the device is disposed with additional sealed compartments.
  • the compartments may be arranged along a straight line or in an array (e.g. 2 x 2, 2 x 4 etc).
  • the compartments and sample support are arranged in a 2 x 2 square array, the distance between adjacent compartments and sample support being equal.
  • a method of pulsing a biological sample comprises the steps of: i) introducing a biological sample into said vessel, ii) optionally agitating said vessel, iii) introducing stabilising agent into said vessel after a pre-determined period of time, and iv) testing the levels of nucleic acid therein.
  • Another embodiment of the present invention is method of testing the immune response of an individual towards an antigen against which the individual has been pre-immunised comprising the use of a vessel as disclosed herein wherein the pulsing agent is the antigen under investigation and the steps of: a) introducing a sample of blood taken from said individual into the vessel, b) optionally agitating said vessel, c) after a pre-determined period of time, introducing said nucleic acid stabilising agent into said vessel d) testing the levels of cytokine mRNA.
  • the cytokine is one or more of IL-2, IL-4, IL-13, IFN-gamma.
  • Another embodiment of the present invention is method of testing an individual for hyperallergenicity towards an antigen comprising the use of a vessel as disclosed herein wherein the pulsing agent is the antigen under investigation and the steps of: e) introducing a sample of blood taken from said individual into the vessel, f) optionally agitating said vessel, g) after a pre-determined period of time, introducing a nucleic acid stabilising agent into said vessel h) testing the levels of IL-4 mRNA.
  • Another embodiment of the present invention is method of testing an individual for rejection of an organ transplant comprising the use of a vessel as disclosed herein wherein the pulsing agent is a histocompatibility antigen of the donor and the steps of: i) introducing a sample of blood taken from said individual into the vessel, j) optionally agitating said vessel, k) after a pre-determined period of time, introducing a nucleic acid stabilising agent into said vessel I) testing the levels of IL-2 mRNA.
  • one embodiment of the present invention is a method of pulsing a liquid biological sample present in a biological sample tube using a device of the invention comprising the steps of: i) transferring liquid biological sample from the biological sample tube to the second compartment in which control agent is present, using the U shaped needle, ii) transferring liquid biological sample from the biological sample tube to the first compartment in which pulsing agent is present, using the U shaped needle, iii) transferring stabilizing agent from the third compartment to the biological sample tube, using the U shaped needle, iv) transferring stabilizing agent from the third compartment to the second compartment in which control agent and liquid biological sample are present, using the U shaped needle, v) transferring stabilizing agent from the third compartment to the first compartment in which pulsing agent and liquid biological sample are present, using the U shaped needle, thereby pulsing the liquid biological sample with a pulsing and control agent, and stabilizing the sample so-pul
  • Another aspect of the invention relates to a method of pulsing a biological sample with an antigen, and subsequently stabilising the nucleic acid therein and testing the RNA components in the stabilising biological sample so pulsed, comprising the steps of:
  • step (B) forming a precipitate comprising nucleic acids, C) separating said precipitate of step (B) from the supernatant,
  • step (C) dissolving said precipitate of step (C) using a buffer, forming a suspension
  • step (E) isolating nucleic acids from said suspension of step (D) using an automated device
  • step (F) dispensing/distributing a reagent mix for RT-PCR using an automated device
  • G) dispensing/distributing the nucleic acids isolated in step (E) within the dispensed reagent mix of step (F) using an automated device
  • step (G) determining the in vivo levels of transcripts using the nucleic acid/RT-PCR reagent mix of step (G) in an automated setup.
  • the above methods may optionally employ a control described herein to which the biological sample is exposed.
  • the control contains containing no reagent or contains a control pulsing agent.
  • the method is performed essentially at the same time as exposure to the pulsing agent, and in an identical way to the method employing the vessel.
  • the results of the control vessel may be used to normalize the results obtained from the vessel.
  • RNA degradation and/or gene induction at the moment of the biological sampling is crucial in order to retrieve a pool of RNAs which can be used to determine the in vivo transcript levels.
  • Cellular RNA can be purified using the PAXgeneTM Blood RNA System in its complete form, however, the present invention proves that real in vivo levels can not be measured using this system 'as such' (see example 2).
  • the present invention shows that the in vivo levels of nucleic acid transcripts can only be measured/determined/quantified when starting from a pool of RNA prepared from a stabilised biological sample, using a compound inhibiting extra- and/or intracellular RNA degradation and/or gene induction; whereby the isolation of the nucleic acids is performed using an automated device, whereby the reagent mix and the isolated nucleic acids, used for the RT-PCR reaction, are dispensed using an automated device, and whereby the determination of the transcript levels is performed in an automated setup.
  • this approach allows the quantification of in vivo RNA in a reproducible manner. The number of steps performed in said method is reduced to a minimum in order to avoid errors.
  • An 'error' may be a pipetting-, a handling-, a procedural- and/or a calculation error or any error which can be made by a person skilled in the art.
  • the present invention suggests to perform the RT and the PCR reaction in one step.
  • the method of the present invention will even be more accurate when combining more intermediate steps. For example, in the method of the present invention steps (A) and (B) can be combined.
  • the dispension of the nucleic acids may be performed after, before or simultaneously with the dispension of the reagent mix needed for RT-PCR (step (F)).
  • the obtained suspension when dissolving the formed precipitate in step (D) of the method according to the present invention, can be used in combination with an RNA extraction method and an analyzing method which are fully automated. It is only this combination which allows the accurate optimisation and reproducibility of the performed method and which allows the accurate and reproducible determination of RNA levels after pulsing.
  • the brochure of the PAXgeneTM Blood RNA System describes that the corresponding tubes can not be used in combination with other isolation methods, and no detailed information is available describing the different compositions of the kit, it is not obvious for a person skilled in the art to use parts of this PAXgeneTM Blood RNA System and develop a new method therefrom.
  • RNA extractors There exist only few commercial systems which allow the isolation of RNA fully automatically. Examples of such automated nucleic acid extractors are: the MagNA Pure LC Instrument (Roche Diagnostics), The AutoGenprep 960 (Autogen), the ABI PrismTM 6700 Automated Nucleic Acid Workstation (Applied Biosystems), WAVE® Nucleic Acid Analysis System with the optional WAVE® Fragment Collector FCW 200 (Transgenomic) and the BioRobot 8000 (Qiagen).
  • MagNA Pure LC Instrument Roche Diagnostics
  • AutoGenprep 960 Autogen
  • ABI PrismTM 6700 Automated Nucleic Acid Workstation Applied Biosystems
  • WAVE® Nucleic Acid Analysis System with the optional WAVE® Fragment Collector FCW 200 Transgenomic
  • BioRobot 8000 BioRobot 8000
  • the present invention points towards the fact that for all these systems it is essential to start with material which is as fresh as possible or which is stabilised in order to allow the determination transcript level after pulsing, wherein RNA degradation is minimised.
  • the problem for all these systems is that the biological sample is collected and brought to the laboratory in tubes that contain no or only a conventional additive, so that mRNA can still be rapidly degraded. Consequently, mRNA quantification using these methods will undoubtedly lead to the quantification of the transcripts present in the tube, but this quantification does not represent the transcript levels present in the cells/biological agent at the moment of sampling. Experimental evidence of this is provided in FIG. 32.2 of example 1 of the present invention.
  • the term 'quantification' is meant accurate and reproducible determination of RNA copy numbers; but it is trivial for a person skilled in the art that also qualitative or semiquantitative studies can be performed using RNA isolated via a method as described by the present invention.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA as well as total RNA can be extracted. This allows to get a correct estimation of the in vivo nuclear RNA, providing a powerful tool to evaluate gene transcription.
  • nucleic acid' refers to a single stranded or double stranded nucleic acid sequence, said nucleic acid may consist of deoxyribonucleotides (DNA) or ribonucleotides (RNA), RNA/DNA hybrids or may be amplified cDNA or amplified genomic DNA, or a combination thereof.
  • a nucleic acid sequence according to the invention may also comprise any modified nucleotide known in the art.
  • the nucleic acid may be present extra- or intracellular ⁇ in the biological sample.
  • the 'separation' of the precipitate from the supernatant in step (C) of present method can be performed via centrifugation, filtration, absorption or other means known by a person skilled in the art.
  • Said precipitate may include cells, cell/debris, nucleic acids or a combination thereof.
  • the basis of the concept is to stop the nucleic-acid-containing-agent (or biological agent) from having contact with external sources/pulses/signals. This can be performed by fixing, lysing and/or disintegrating the nucleic-acid-containing-agent, or by any other means known by a person skilled in the art.
  • the buffer used in step (D) of the method of present invention may be a buffer to dissolve the precipitate obtained in step (C) of said method.
  • This buffer may have additional effects such as lysis or further lysis of the nucleic-acid-containing-agent.
  • the 'automated device' used may be an automated pipetting device or another automated device known by a person skilled in the art suitable for carrying out the indicated actions.
  • a 'reagent mix for RT-PCR' is meant all reagents needed for a simultaneous RT and PCR reaction (with the exception of the oligonucleotides when explicitly mentioned).
  • Oligonucleotides' may comprise short stretches of nucleic acids as found in for example primers or probes. According to the present invention, this method can be used in combination with micro-arrays or RNase protection assays.
  • the method according to the present invention allows the transport biological samples from a remote site to a suitable laboratory without any effect on their in vivo transcript content.
  • Transport of the biological sample can be performed after step (A) or step (B) in the method of the present invention.
  • red blood cells are preferentially eliminated before the nucleic acids are isolated.
  • Red blood cells are rich in haemoglobin and their presence results in the production of highly viscous lysates. Therefore, removal of these allows to isolate nucleic acids in a more improved fashion.
  • this step is eliminated as an insoluble precipitate is immediately formed comprising the nucleic acids, separating these from all other components of the biological sample. This illustrates that, in addition to other advantages, the method of the present invention is a superior method in comparison with most prior art methods.
  • said buffer used in step (D) of a method of the present invention may be a guanidine-thiocyanate-containing buffer.
  • the precipitate formed in the PAXgeneTM Blood RNA Tubes is dissolved in the lysis buffer as provided by the MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals). Therefore, it is suggested in the present invention that one of the possible buffers which may be used in the method of the present invention is a guanidine-thiocyanate-containing lysis buffer as provided by MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals).
  • the MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, Molecular Biochemicals) is especially designed for use on the MagNA Pure LC Instrument, to guarantee the isolation of high quality and undegraded RNA from whole blood, white blood cells, and peripheral blood lymphocytes. According to its product description, obtained RNA is suitable for highly sensitive and quantitative LightCycler RT-PCR reactions, as well as for standard block cycler RT-PCR reactions, Northern blotting and other standard RNA applications. Nevertheless, the present invention proves that the use of this method 'as such' could not result in the determination of correct transcript levels. The present invention shows that there is a need to stabilize the RNA prior to the RNA isolation (see example 1 ). The present invention describes the unique combination of the use of RNA stabilizing compounds and an automated isolation/analysis procedure.
  • the method of the present invention may follow the procedure as described for the MagNA Pure LC mRNA Isolation Kit I.
  • a lysis buffer such as the one provided by MagNA Pure LC mRNA Isolation Kit I.
  • streptavidin-coated magnetic particles are added together with biotin-labeled oligo-dT, and the mRNA binds to the surface of the particles. This is followed by a DNase digestion step.
  • mRNA is then separated from unbound substances using a magnet and several washing steps. Finally, the purified mRNAs are eluted.
  • This isolation kit allows the automated isolation of pure mRNA as a "walk away" system. It allows to isolate mRNA of high quality and integrity suitable for all major downstream applications regarding gene expression analysis. Different protocols are offered depending on the sample material used.
  • the samples may be set directly on the MagNA pure LC Instrument stage. When using whole blood, cells present in the samples are preferentially lysed manually. mRNA isolation may then be postponed or directly further processed on the instrument.
  • RNA-capturing beads such as magnetic beads, coated with oligo-dT via a streptavidin-biotin system or an equivalent system, may be applied in the method of the present invention in order to separate mRNA from the cellular debris.
  • automated devices such as the ABI PrismTM 6700 Automated Nucleic Acid Workstation (Applied Biosystems) or any other automated device that can be used for this purpose.
  • red blood cells are preferentially lysed after step (A) in the method of the present invention.
  • MagNA Pure LC mRNA Isolation Kit I Roche Diagnostics, Molecular Biochemicals
  • the present inventors decided to use the stabilising agent contained in the PAXgeneTM Blood RNA Tube in conjunction with the MagNA Pure mRNA Isolation Kit on the MagNA Pure Instrument.
  • PAXgeneTM Blood RNA Tubes provides a precipitate of nucleic acids that is not supposed to be soluble in the lysis buffer of the MagNA Pure mRNA Isolation Kit. Despite of this observation, the inventors found that it is actually possible. Following this observation, the inventors combined the use of the stabilising agent in the PAXgeneTM Blood RNA Tubes with the use of an automated RNA isolation system. The inventors found surprisingly that this combination is possible and that this combination provides a powerful technique for the accurate mRNA quantification from biological samples.
  • RNA isolated using the method according to the present invention is ready for use in a wide range of downstream applications, including for instance nucleic acid amplification technologies, such as RT-PCR and NASBA®, Expression-array and expression-chip analysis, Quantitative RT-PCR, including TaqMan® technology, cDNA synthesis, RNase and S1 nuclease protection, Northern, dot, and slot blot analysis and primer extension.
  • nucleic acid amplification technologies such as RT-PCR and NASBA®
  • Expression-array and expression-chip analysis Quantitative RT-PCR, including TaqMan® technology, cDNA synthesis, RNase and S1 nuclease protection, Northern, dot, and slot blot analysis and primer extension.
  • the present inventors showed in the example 1 and example 2 of the present invention that the use of a compound inhibiting RNA degradation and/or gene induction in conjunction with an automated RNA isolation and an automated analysis method such as real time PCR allows the determination of in vivo levels of transcripts. Nevertheless, according to present invention analysis methods other than real-time PCR may be applied as long as they are provided in an automated setup.
  • a main advantage of the method according to the present invention is the fact that by using this method small sample volumes can be analyzed. This is of prime importance when only small volumes are available, for example when analyzing neonatal blood samples or in cases of high blood loss.
  • RNA quantification may be performed using a biological sample as small as 100 ⁇ l. The analysis of RNA from a sample as small as 100 ⁇ l is not possible with the Qiagen kit (PAXgenTM Blood RNA System) which requires a larger volume of blood (2.5 ml following the kit handbook).
  • one aspect of the present invention is a kit suitable for pulsing a biological sample with a pulsing agent, and subsequently stabilising the nucleic acid from the biological sample so pulsed.
  • the said kit comprises additional components for isolating quantifiable RNA from the stabilised, pulsed biological samples.
  • the kit may comprise additional components such as: reagents for automated RNA isolation, a reagent mix for a simultaneous RT and real-time PCR reaction or separate compounds thereof , allowing the automated dispension of said mix, optionally, specific oligonucleotides to perform said RT-PCT reactions, and, - optionally, an instruction manual describing a method for an automated RNA isolation, a method for the automated dispension of a reagent mix and the dispension of the isolated nucleic acids for RT- real time PCR, and a method for automated RNA analysis.
  • additional components such as: reagents for automated RNA isolation, a reagent mix for a simultaneous RT and real-time PCR reaction or separate compounds thereof , allowing the automated dispension of said mix, optionally, specific oligonucleotides to perform said RT-PCT reactions, and, - optionally, an instruction manual describing a method for an automated RNA isolation, a method for the automated
  • the present inventors apply the "Lightcycler mRNA hybridisation probes kit" from Roche Diagnostics, Molecular Biochemicals (cat # 3 018 954) to perform the RT-PCR reactions in one step. All reagents needed are included in this kit, except the oligonucleotides (synthesized by Biosource). Nevertheless, real time PCR as described in the present invention can also be performed on other instruments such as the Applied Biosystems instruments.
  • the kit may additionally comprise a buffer such as a guanidine- thiocyanate-containing buffer which can be used in step (b) of the method according to the present invention.
  • the method according to the present invention can also be used for the quantification/ detection of DNA (double or single stranded) in biological samples. Therefore, the present invention also relates to a method for the quantification of DNA from a biological sample wherein a method is used as performed for the quantification of RNA according to the present invention, wherein the RT reaction is skipped and wherein the compound of step (a) also protects the DNA from being degraded. As these nucleic acids are more stable than RNA, its stabilization is less important than for RNA.
  • the present invention relates to a kit for isolating quantifiable DNA from a biological sample according to the present invention, wherein a reagent mix/ compounds for performing said RT reaction is absent.
  • Situations where exact DNA levels need to be determined in biological samples may be to determine the 'presence' of infection(s)/ contamination(s) in biological samples by unexpected genes, pathogens or parasites; and/or to determine the 'level' of said infection/contamination.
  • the method may be used to determine the percentage of transgenic material in a cereal batch.
  • the present invention also relates to the use a device, kit and method, according to the present invention, for the monitoring/detection of changes of in vivo nucleic acids of a biological marker in a biological sample after pulsing with an agent, in order to diagnose a certain disease.
  • the present invention also relates to the use a device, kit and method according present invention, for the monitoring/detection of changes of in vivo nucleic acids of a biological marker in a biological sample after pulsing with an agent, in order to screen for a compound, said compound used for the production of a medicament for curing a disease. Therefore, the invention also relates to a compound identifiable by a method according to present invention.
  • the devices, kits, and methods disclosed herein may be used to treat and/or diagnose diseases.
  • An example of the disease to be cured or diagnosed is an immuno-related disease.
  • examples of immuno-related diseases may be autoimmunity, rheumatoid arthritis, multiple sclerosis, Type 1 diabetes mellitus, cancer (e.g. in cancer immunotherapy), immunodeficiencies (e.g. in AIDS), allergy, graft rejection or Graft versus Host Disease (GVHD) (e.g. in transplantation).
  • GVHD Graft versus Host Disease
  • a immunomodulatory compound or agent may influence one of said diseases; the change of the immuno-related transcripts or the epitope specific CTLs-related or T Helper lymphocyte-related transcripts may indicate the presence and/or the status of one of said diseases; as well as the immunological status which may illustrate the status of one of said diseases.
  • Nucleic acids which may be quantified using the devices, kits, and methods of the present invention in order to study said immuno-related disease may be nucleic acids coding for, for example, chemokines, cytokines, growth factors, cytotoxic markers, transcription factors, members of the TNF-related cytokine-receptor superfamily and their ligands, apoptosis markers, immunoglobulins, T-cell receptor, and any marker related to the activation or the inhibition of the immune system known or to be discovered.
  • said nucleic acids may code for a marker such as IL-1 ra, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ , and any interleukin or cytokine involved or not in the immune response.
  • a marker such as IL-1 ra, IL-1 ⁇ , IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ , TGF- ⁇ , and any interleukin or cytokine involved or not in the immune response.
  • ⁇ -actin or GAPDH glycose
  • said epitope specific CTLs-related or T Helper lymphocyte- related transcripts be a nucleic acid coding for cytokines, cytokine receptors, cytotoxines, inflammatory or anti-inflammatory mediators, members of the TNF-related cytokine- receptor superfamily and their ligands, G-protein coupled receptors and their ligands, tyrosine kinase receptors and their ligands, transcription factors, and proteins involved in intra-cellular signaling pathways.
  • said nucleic acid may code for a marker for any of granzyme, perforines, prostaglandins, leukotrienes, immunoglobulin and immunoglobulin superfamily receptors, Fas and Fas-ligand, T cell receptor, chemokine and chemokine receptors, protein-tyrosine kinase C, protein-tyrosine kinase A, Signal Transducer and Activator of Transcription (STAT), NF-kB, T-bet, GATA-3, Oct-2.
  • the present invention also describes a use of a device, method or a kit according to the present invention, for the detection/monitoring/screening of a compound, wherein said compound is an immunomodulatory compound which may be chosen from the group consisting of eukaryotic cells, prokaryotic cells, viruses, phages, parasites, drugs (natural extracts, organic molecule, peptide, proteins, nucleic acids), medical treatment, vaccine and transplants.
  • an immunomodulatory compound which may be chosen from the group consisting of eukaryotic cells, prokaryotic cells, viruses, phages, parasites, drugs (natural extracts, organic molecule, peptide, proteins, nucleic acids), medical treatment, vaccine and transplants.
  • the use of such a method is not limited to detect/monitor/screen a single compound. Synergetic effects of group of substances can also be studied.
  • the present invention also relates to the use of any of the devices, kits, and methods as described above, for the detection/monitoring of epitope specific CTLs or immuno-related transcripts.
  • the devices, kits, and methods according to the present invention can also be applied for the monitoring of in vivo immunological responses after the treatment of patients with a drug/treatment/vaccine susceptible to modify their immune status.
  • the detection of cytokine mRNA can be extended to chemokine, growth factors, cytotoxic markers, apoptosis markers, or any marker relate to the activation of the immune system known or to be discovered
  • cytokine mRNA can be extended to chemokine, growth factors, cytotoxic markers, apoptosis markers, or any marker relate to the activation of the immune system known or to be discovered
  • an immunomodulator drug or treatment or with a vaccine therapeutic or prophylactic
  • the present invention also relates to a devices, kits, and methods for the detection of in vivo immunological status for the diagnostic/prognostic of diseases affecting the immune system (cancer, auto-immune diseases, allergy, transplant rejection, GVHD, etc.)
  • the detection of cytokine mRNA (can be extended to chemokine, growth factors, cytotoxic markers, apoptosis markers, or any marker relate to the activation of the immune system known or to be discovered) with the described method in whole blood of patients suffering a disease that affects directly of indirectly their immune system with the aim to dress a diagnosis or prognosis.
  • the present invention also describes a method to identify an agent capable of modifying the immunological status of a subject via the analysis of epitope specific CTLs comprising the steps of:
  • step (b) sampling whole blood from said subject, (c) pulsing blood cells present in the whole blood sample of step (b) with an identical/ similar and/or different immunomodulatory agent as applied in step (a), using a device as described above,
  • step (d) collecting pulsed blood cells of step (c) or non-pulsed blood cells of step (b) in a tube comprising a compound inhibiting RNA degradation and/or gene induction, or adding said compound to the pulsed/non-pulsed cells,
  • step (f) separating said precipitate of step (e) from the supernatant
  • step (g) dissolving said precipitate of step (f) using a buffer, forming a suspension
  • step (h) isolating nucleic acids from said suspension of step (g) using an automated device
  • step (j) dispensing/distributing the nucleic acids isolated in step (h) within the dispensed reagent mix of step (i) using an automated device
  • step (k) detecting/ monitoring/ analyzing the in vivo levels of epitope specific CTLs-related transcripts in the dispensed solution of step (j) in an automated setup, and,
  • step (I) identify agents able to modify the immunological status of said subject, whereby, in case the agent of step (a) is already present in the subject, step (a) is omitted.
  • the present invention also relates to a kit comprising components enabling execution of at least step (c) above.
  • the kit may contain additional reagents and instructions to enable one or more of the other steps to be executed.
  • the disclosures made herein instruct the skilled artisan of the components required to build the desired kit.
  • the immunomodulatory agent(s) may be present in case of a disease or in the presence of a transplant in said subject.
  • the 'epitope specific CTLs-related transcripts' may be transcripts coding for cytokines, cytokine receptors, cytotoxines (like granzyme, perforines, etc.), members of the TNF-related cytokine-receptor superfamily and their ligands (ex: Fas and Fas-ligand) or other cellular receptors.
  • the present invention also describes a method to identify an agent capable of modifying the immunological status of a subject:
  • step (c) pulsing blood cells present in the whole blood sample of step (b) with an identical/ similar and/or different immunomodulatory agent as applied in step (a), using a device or kit as described above,
  • step (d) collecting pulsed blood cells of step (c) or non-pulsed blood cells of step (b) in a tube comprising a compound inhibiting RNA degradation and/or gene induction, or adding said compound to the pulsed/non-pulsed cells, (e) forming a precipitate comprising nucleic acids,
  • step (f) separating said precipitate of step (e) from the supernatant
  • step (g) dissolving said precipitate of step (f) using a buffer, forming a suspension
  • step (h) isolating nucleic acids from said suspension of step (g) using an automated device, (i) dispensing/distributing a reagent mix for RT-PCR using an automated device,
  • step (j) dispensing/distributing the nucleic acids isolated in step (h) within the dispensed reagent mix of step (i) using an automated device
  • step (k) detecting/ monitoring/ analyzing the in vivo levels of immuno-related transcripts in the dispensed solution of step (j) in an automated setup, and, (I) identify agents able to modify the immunological status of said subject, whereby, in case the agent of step (a) is already present in the subject, step (a) is omitted.
  • the present invention also relates to a kit comprising components enabling execution of at least step (c) above.
  • the kit may contain additional reagents and instructions to enable one or more of the other steps to be executed.
  • the disclosures made herein instruct the skilled artisan of the components required to build the desired kit.
  • the 'immuno-related transcripts' may be transcripts coding for e.g. cytokine(s), chemokines(s), growth factors, cytotoxic markers, transcription factors, members of the TNF-related cytokine-receptor superfamily and their ligands, or any markers related to activation of the immune system known or to be discovered.
  • the immunomodulatory agent(s) may be present in case of a disease or in the presence of a transplant in said subject.
  • the subject according to the present invention may be of both human or animal origin.
  • the present invention also provides a method for the diagnosis/ prognosis/ monitoring of a clinical status affecting the immune system in a subject comprising the steps of :
  • step (b) pulsing blood cells present in the whole blood sample of step (a) with an identical/ similar and/or different immunomodulatory agent as present in the subject, using a device or kit as described above,
  • step (c) collecting pulsed blood cells of step (b) in a tube comprising a compound inhibiting RNA degradation and/or gene induction, or adding said compound to the pulsed cells,
  • step (e) separating said precipitate of step (d) from the supernatant, (f) dissolving said precipitate of step (e) using a buffer, forming a suspension,
  • step (g) isolating nucleic acids from said suspension of step (f) using an automated device, (h) dispensing/distributing a reagent mix for RT-PCR using an automated device, (i) dispensing/distributing the nucleic acids isolated in step (g) within the dispensed reagent mix of step (h) using an automated device, (j) detecting/ monitoring/ analyzing the in vivo levels of immuno-related transcripts in the dispensed solution of step (i) in an automated setup, and,
  • the present invention also relates to a kit comprising components enabling execution of at least step (c) above.
  • the kit may contain additional reagents and instructions to enable one or more of the other steps to be executed.
  • the disclosures made herein instruct the skilled artisan of the components required to build the desired kit.
  • FIGs. 1a to 1d depict an example a vessel and method according to the invention.
  • FIG. 1a to 1d depict an example a vessel and method according to the invention.
  • FIG. 1a shows a vessel 1 wherein antigen particles 2 are present.
  • the vessel is fitted with a resealable means of entry 3 for a syringe needle.
  • a container 4 is also part of the vessel 1 ; stabilising agent 5 being present in the container, and the connection between the inside of the vessel 1 and the inside of the container 4 being temporarily blocked by a physical barrier, in this case, a plug 25.
  • a means of transmitting physical force to dislodge the plug is provided in the form of a plunger 28. In this example, the plunger is covered with a cap 23.
  • biological sample 24 is introduced into the vessel by way of a syringe needle 6, through the resealable means of entry 3, and the biological sample 24, is allowed to be exposed to the antigen 2.
  • the plunger cap 23 is removed 26.
  • a shaft 7 is introduced and pressure applied thereto 27, so forcing the plunger 28 to push the plug 25 away from the container 4.
  • stabilizing agent 5 is released into the vessel 1 and allowed to mix with the biological sample 24 and antigen 2.
  • FIG. 2 depicts an example of a vessel 1 according to the invention in which antigen 2 is present.
  • the vessel may be open-topped 7, as shown here, or may be fitted with closures or means to introduce sample or stabilizing agent, examples of which are shown in FIGs. 3 to 6.
  • the body of the vessel 1 may also comprise a container in which stabilising agent is present as shown in FIGs. 7 and 8.
  • FIG. 3 depicts an example of a fitting suited for a type of vessel shown in FIG. 2.
  • the top of the vessel 7, is fitted with a Luer-type fitting 8 that can receive a reciprocal Luer type fitting on a syringe 11.
  • the syringe may contain biological sample or stabilising agent according to embodiments of the invention.
  • FIG. 4 depicts an example of a fitting suited for a type of vessel shown in FIG. 2.
  • the top of the vessel 7, is fitted with a resealable septum 9 that can receive a syringe needle.
  • the syringe may contain biological sample or stabilising agent according to embodiments of the invention.
  • FIG. 5 depicts an example of a fitting suited for a type of vessel shown in FIG. 2.
  • the top of the vessel 7, is fitted a means to receive with a screw cap 10.
  • FIG. 6 depicts an example of a fitting suited for a type of vessel shown in FIG. 2.
  • the top of the vessel 7, is fitted a hypodermic syringe needle 19.
  • the vessel might be used directly to withdraw a sample from an individual.
  • FIG. 7 depicts an example of a body of a vessel 1 which could, for example, be used in combination with the vessels and fittings shown in FIGs. 2 to 6.
  • the vessel 1 in which antigen 2 is present, comprises a container 12 in which stabilizing agent 5 is present.
  • the wall of the container is made entirely or in part 15 from a material which shatters upon the application of a certain force.
  • the container is fitted with a means to transmit force from the user to shatter part or all of the container, comprising a depressable area 13 attached to a sharp point 14. Upon depression of the area 13, the sharp point 14 contacts the shatterable material 15, causing it to shatter, so removing the physical barrier between the container and the vessel, allowing the stabilising agent to flow into the vessel 1 at a time determined by the user.
  • FIG. 8 depicts an example of a body of a vessel 1 which could, for example, be used in combination with the vessels and fittings shown in FIGs. 2 to 6.
  • the vessel 1 in which antigen 2 is present, comprises a container 16 in which stabilizing agent 5 is present.
  • the connection between the inside of the container and the vessel 17 is physically blocked by a septum 18 which is breachable by the application of force.
  • pressure is transmitted to the septum, causing the septum to breech so allowing the entry of stabilising agent 5 into the vessel 1.
  • FIG. 9 depicts an example of a container 20 of the invention which is not connected to a vessel.
  • the stabilising agent 5 is present in the container 20 and the container 20, is fitted with a Luer-type fitting 22 suitable for coupling with a vessel having a reciprocal fitting (for example as shown in FIG. 3 and FIG. 11 ).
  • the walls of the container 20 may be squeezable, allowing the stabilizing agent to exit upon the application of force thereto.
  • FIG. 10 depicts an example of a container 29 of the invention which is not connected to a vessel.
  • the stabilising agent 5 is present in the container 29 and the container 29, is fitted with a Luer-type fitting 22 for coupling with a vessel having a reciprocal fitting (for example as shown in FIG. 3 and FIG. 11 ).
  • the vessel is further fitted with a plunger 30, the application of force on which allows the stabilizing agent 5 to exit.
  • FIG. 11 depicts an example of a kit according to the invention, comprising a vessel 1 fitted with a Luer type fitting 8 and, in this instance, a valve 31 allowing the exit of displaced air from vessel.
  • the kit also comprises a container 29, similar to that depicted in FIG. 10, in which stabilising agent 5 is present.
  • the fitting on the vessel 8 is capable of coupling to the fitting on the container 22.
  • FIGs. 12a to 12d depict an example a control vessel and method according to the invention.
  • FIG. 12a shows a control vessel 37 wherein control substance 35 present.
  • the control vessel is fitted with a resealable means of entry 3 1 for a syringe needle.
  • a control container 4 1 is also part of the control vessel 37; stabilising agent 5 1 being present in the control container, and the connection between the inside of the control vessel 37 and the inside of the control container 4 1 being temporarily blocked by a physical barrier, in this case, a plug 25'.
  • a means of transmitting physical force to dislodge the plug is provided in the form of a plunger 28'. In this example, the plunger is covered with a cap 23'.
  • FIG. 12a shows a control vessel 37 wherein control substance 35 present.
  • the control vessel is fitted with a resealable means of entry 3 1 for a syringe needle.
  • a control container 4 1 is also part of the control vessel 37; stabilising agent
  • biological sample 24 is introduced into the control vessel by way of a syringe needle 6 1 , through the resealable means of entry 3 1 , and the biological sample 24, is allowed to be exposed to the control substance 35.
  • the plunger cap 23' is removed 26'.
  • a shaft T is introduced and pressure applied thereto 27', so forcing the plunger 28' to push the plug 25' away from the control container 4 1 .
  • stabilizing agent 5 1 is released into the control vessel 37 and allowed to mix with the biological sample 24 and control substance 35.
  • FIG. 13 depicts an example of a control vessel 37 according to the invention in which a control substance 35 is present.
  • the control vessel 37 may be open-topped T, as shown here, or may be fitted with closures or means to introduce sample or stabilizing agent, examples of which are shown in FIGs. 14 to 16.
  • the body of the control vessel 37 may also comprise a control container 12', 16' in which stabilising agent is present as shown in FIGs. 17 and 18.
  • FIG. 14 depicts an example of a fitting suited for a type of control vessel shown in FIG. 13.
  • the top T of the control vessel 35 is fitted with a Luer-type fitting 8 1 that can receive a reciprocal Luer type fitting on a syringe 11'.
  • the syringe may contain biological sample or stabilising agent according to embodiments of the invention.
  • FIG. 15 depicts an example of a fitting suited for a type of control vessel shown in FIG. 13.
  • the top T of the control vessel 37 is fitted with a resealable septum 9 1 that can receive a syringe needle.
  • the syringe may contain biological sample or stabilising agent according to embodiments of the invention.
  • FIG. 16 depicts an example of a fitting suited for a type of control vessel 37 shown in FIG. 37.
  • the top T of the control vessel 37 is fitted a means to receive with a screw cap 10'.
  • FIG. 17 depicts an example of a body of a control vessel 37 which could, for example, be used in combination with the control vessels and fittings shown in FIGs. 14 to 16.
  • the control vessel 37 in which control substance 35 is present, comprises a control container 12' in which stabilizing agent 5 1 is present.
  • the wall of the control container is made entirely or in part 15' from a material which shatters upon the application of a certain force.
  • the control container is fitted with a means to transmit force from the user to shatter part or all of the control container, comprising a depressable area 13' attached to a sharp point 14'.
  • the sharp point 14' contacts the shatterable material 15', causing it to shatter, so removing the physical barrier between the control container and the control vessel, allowing the stabilising agent to flow into the control vessel 37 at a time determined by the user.
  • FIG. 18 depicts an example of a body of a control vessel 37 which could, for example, be used in combination with the control vessels and fittings shown in FIGs. 14 to 16.
  • the control vessel 37 in which control substance 35 is present, comprises a control container 16' in which stabilizing agent 5 is present.
  • the connection between the inside of the control container and the control vessel 37 is physically blocked by a septum 18' which is breachable by the application of force.
  • pressure is transmitted to the septum, causing the septum to breech so allowing the entry of stabilising agent 5 into the control vessel 37.
  • FIG. 19 depicts an example of a device of the invention comprising a control vessel 37 as described in FIG.
  • the device permits biological sample to be delivered separately to a first substance 2 and control substance 35, prior to a stabilizing agent 5. Because the reaction and control samples are held side-by-side, errors that can arise with mixing control and reaction samples are avoided.
  • FIG. 20 depicts an example of a device of the invention comprising a control vessel 37 as described in FIG. 18, exteriorly mechanically connected to a vessel 1 as described in FIG. 8 by way of a bridging member 70.
  • the device permits delivery of biological sample separately to a first sample 2 and control substance 35, prior to a stabilizing agent 5. Because the reaction and control samples are held side-by-side, errors that can arise with mixing control and reaction samples are avoided.
  • FIG. 21a shows a schematic illustration of an example of a device 30 of the invention for accepting a liquid biological sample, which facilitates exposure of said sample separately to a first substance and a control substance, and subsequently to a nucleic acid stabilizing agent, the device comprising. It comprise a first compartment 32 where a first substance 2 is present, a second compartment 34 in which a control substance 35 is present, a third compartment 36 where a stabilizing agent 5 is present, a support 52 for a biological sample tube that is a cylindrical aperture. Each compartment is formed as an opening in a block of solid material 44.
  • Each compartment is sealed with a lid 46, 48, 50 disposed with a resealable septum 38, 40, 42 that may be breeched with a hollow needle such as a hypodermic cannular or needle.
  • the device is further disposed with a support for a transfer tubing, which support takes the form of two slots 54, 56 adapted to accept the needle ends of the transfer tubing.
  • FIG. 21 b depicts a cross-section through a device of the invention along line A-A', which shows in detail the contents of the second (control) compartment 34 and third (stabilizing agent) compartment 36.
  • FIG. 22 shows a cross-sectional view of a transfer tubing 60 of the invention, comprising a hollow elongate tubing 64, whereby each end of the tubing is disposed with a hollow (e.g. cannular or hypodermic) needle 62, 66.
  • the transfer tubing 60 is configured such that liquid can flow through one needle 62 along the tubing 64 to the other needle 66.
  • the needles 62, 66 are of unequal length.
  • FIG. 23 shows a cross-sectional view of a pressurising tubing 88 of the invention, comprising a hollow flexible elongate tubing 82, whereby one end of the tubing is in connection with a hollow (e.g. cannular or hypodermic) needle 80 and the other end 85 is adapted for connection the outlet coupling 87 of a (pressurizing or evacuating) gaseous media (e.g. air) pump 86.
  • the pressurising tubing 88 is configured to pierce the septum of a vessel, or compartment, and create a differential pressure therein by the evacuation or introduction of gaseous medium (e.g. air).
  • FIG. 24 shows a cross-sectional view of a combined set comprising the pressurising tubing 88 of the invention and the transfer tubing 60.
  • One needle 62 of the transfer tubing 60 is connected using one or more joints 84 to the needle 80 of the pressurising tubing 88 such that both needles 62, 80 are in parallel alignment.
  • a septum is simultaneously pierced by both needles 62, 80.
  • FIG. 25 shows a cross-sectional view of a transfer tubing 60 of the invention, with dimensions indicated.
  • D represents the minimum distance between two needles 62, 66.
  • L1 is the length of one needle, particularly the straight part
  • L2 is the length of the other needle particularly the straight part
  • L3 is the distance on a first imaginary straight line 65, that extends from the straight part of a needle 62, 66 towards the tubing 64, which distance L3 is defined by end 67 of the straight part of the needle, and the point 69 where a second imaginary straight line 63 crosses the first imaginary straight 65, which second imaginary straight line 63 intersects the top of the tubing 64.
  • FIGs. 26 to 29 shows an example of the device 30 as shown in FIG. 21a in use.
  • a biological sample tube 58 comprising liquid biological sample (e.g. blood, urine) is inserted into the tubing support 52.
  • liquid biological sample e.g. blood, urine
  • One end of the transferring tubing 60 is inserted into the biological sample tube 58 through the septum 61 , and the other end is inserted into the second (control) compartment 34, through the septum 40.
  • Liquid biological sample flows from the biological sample tube 58, along the transfer tubing 60 in the direction of the arrow, and into the second compartment 34, where it contacts the control substance 35.
  • Liquid biological sample may be propelled by the use of pressurizing tubing 88 (not shown), the needle of which may pierce either the septum of the 38 of the second compartment 34 and a vacuum created, or the septum 61 of the biological sample tube 58 and pressure therein increased.
  • the transfer tubing has been removed and reinserted, such that the end of transfer tubing 60 in the second compartment 34 is positioned in the first compartment 32, through the septum 38, while the end of transfer tubing 60 in the biological sample tube 58 remains there.
  • Liquid biological sample flows from the biological sample tube 58, along the transfer tubing 60 in the direction of the arrow, and into the first compartment 32, where it contacts the first substance 2 (pulsing agent).
  • Liquid biological sample may be propelled by the use of pressurizing tubing 88 (not shown), the needle of which may pierce either the septum of the 40 of the first compartment 32 and a vacuum created, or the septum 61 of the biological sample tube 58 and pressure therein increased.
  • FIG. 28 shows rinsing of the transfer tubing 60 after transfer of liquid biological sample, by removal and reinsertion of the transfer tubing 60 such that the end of the transfer tubing 60 previously in contact with the biological sample tube 58, is inserted into the third (stabilizing agent) compartment 36 through the septum 42, and the other end of the transfer tubing 60 is inserted into the biological sample tube 58 through the septum 61.
  • Liquid stabilizing agent 5 flows from the third compartment 36, along the transfer tubing 60 in the direction of the arrow, and into the biological sample tube 58, where it is stored as waste or for further processing.
  • Liquid stabilizing agent 5 may be propelled by the use of pressurizing tubing 88 (not shown), the needle of which may pierce either the septum of the 42 of the third compartment 36 and pressure therein increased, or the septum 61 of the biological sample tube 58 and a vacuum created. It is noted that after rinsing, the samples are allowed to incubate. The period of incubation and the temperature will depend on the several factors including the pulsing agent, the sample and the volume and concentration of reagents. For general guidance, incubation may be performed at 37 deg C for between 30 minutes and 16 hours, most typically between 2 and 4 hours.
  • the transfer tubing 60 is removed and reinserted, such that one end is inserted into the third (stabilizing agent) compartment 36, through the septum 42, and the other end is inserted into the second (control) compartment 34through the septum 40.
  • Liquid stabilizing agent 5 flows from the third compartment 36, along the transfer tubing 60 in the direction of the arrow, and into the second compartment 34, where it contacts the mixture of biological sample and control substance 2.
  • Liquid stabilizing agent 5 may be propelled by the use of pressurizing tubing 88 (not shown), the needle of which may pierce either the septum of the 42 of the third compartment 36 and pressure therein increased, or the septum 40 of the second compartment 34 and a vacuum created.
  • the transfer tubing 60 is removed and reinserted such that one end is inserted into the third (stabilizing agent) compartment 36, through the septum 42, and the other end is inserted into the first (pulsing reaction) compartment 32, through the septum 38.
  • Liquid stabilizing agent flows from the third compartment 36, along the transfer tubing 60 in the direction of the arrow, and into the first compartment 32, where it contacts the mixture of biological sample and first substance 35.
  • Liquid stabilizing agent 5 may be propelled by the use of pressurizing tubing 88 (not shown), the needle of which may pierce either the septum of the 42 of the third compartment 36 and pressure therein increased, or the septum 38 of the first compartment 32 and a vacuum created.
  • both the first and second compartments have received biological sample and stabilizing agent.
  • the movements by the transfer tubing 60 and pressurizing tubing 88 where used may be actuated robotically, e.g. by use of a robotic arm having several degree of freedom of movement or by an X-Y robotic table.
  • the transfer tubing will typically comprise a rigid tubing 64 that permits the transfer tubing 60 to be gripped and positioned with accuracy.
  • FIG. 31.1 to 31.4 Strategies followed in the given examples FIG. 31.1 Ex vivo monitoring of immune response against tetanus toxoid.
  • FIG. 32.1 RT-PCR for spontaneous production of IFN- ⁇ and IL-10 mRNAs in peripheral blood.
  • Total RNA was extracted from whole blood and from PBMC, as stated, from six different healthy volunteers (columns 1 to 6).
  • Whole blood 0.6 ml of whole blood were mixed with 6 ml of Catrimox-14TM, within the minute that follows sample collection. The samples were then centrifuged at 12000 g for 5 min. The resulting nucleic acids pellet was carefully washed with water, and dissolved in 1 ml of TripureTM. RNA extraction was then carried out according to TripureTM manufacturer's instructions.
  • PBMC peripheral blood mononuclear cells
  • FIG. 32.2 Real time PCR for IFN- ⁇ and IL-10 mRNA stability in whole blood.
  • a sample of citrated venous blood was collected from healthy donors. From this sample, a 100 ⁇ l aliquot was mixed with 900 ⁇ l of Catrimox-14TM, within the minute that follows blood collection, and every hour after during five hours, the blood sample being simply kept at room temperature between each aliquot taking.
  • the resulting nucleic acids pellet (see legend to FIG.13.1 ) was dissolved in 300 ⁇ l lysis buffer from the "MagNA Pure LC mRNA Isolation Kit I" (Roche Diagnostics, Molecular Biochemicals).
  • FIG. 33 Schematic comparison of the RNA extraction method from whole blood as suggested by PreAnalytiX compared to method as proposed by the present invention.
  • FIG. 34 Cytokine blood mRNA ex vivo induction by tetanus toxoid.
  • Tetanus toxoid (10 ⁇ g/ml, Aventis) was added to 500 ⁇ l whole blood collected from healthy volunteer vaccinated against tetanus seven years ago. After different time periods at 37°C in a 5% CO 2 atmosphere, 1.4 ml of the reagent contained in the PAXgene tube was added. 300 ⁇ l of the obtained lysate were used to isolate total mRNA on the MagNA Pure instrument, and RT-PCR was performed as described in the present invention.
  • FIG. 35 IL-1 ⁇ and IL- 1 RA mRNA kinetics after whole blood stimulation with LPS.
  • 200 ⁇ l of heparinized blood were incubated with 10 ng/ml LPS for 0 (beginning of the culture), 0.5, 1 , 2 and 6 hours.
  • 500 ⁇ l of the PAXgeneTM tube's reagent were added for total cell lysis and nucleic acid precipitation.
  • RT and real time PCR for IL-1 ⁇ , IL-1 RA and ⁇ -actin mRNAs were performed in one step as described in the present invention. Results are expressed in mRNA copy numbers per million of ⁇ -actin mRNA copies. The mean and standard error on the mean of five independent experiments are shown.
  • FIG. 36 Linear regression: mRNA copy numbers on starting blood volume.
  • Various whole blood volumes ranging from 20 to 200 ⁇ l, X-axis) were cultured in the presence of 10 ng/ml LPS for six hours.
  • RT and real time PCR for IL-1 ⁇ and ⁇ -actin mRNAs were performed as described in the present invention.
  • the Y-axis represents the raw copy numbers.
  • the line is for linear regression. One experiment representative of six is shown.
  • FIG. 37 mRNA cytokine kinetics after whole blood stimulation with tetanus toxoid.
  • Heparinized blood has been taken from five healthy volunteers who were vaccinated against tetanus at least five years ago.
  • 200 ⁇ l whole blood aliquots were incubated with 10 ⁇ g/ml tetanus toxoid for 0 (beginning of the culture), 4, 8, 16, 24 and 48 hours.
  • 500 ⁇ l of the reagent contained in the PAXgeneTM tube were added, and the different transcripts quantified using the methodology of the present invention.
  • Results are expressed in mRNA copy numbers per million of ⁇ -actin mRNA copies. The mean and standard error on the mean of five independent experiments are shown.
  • FIG. 38 In vivo modulation of blood cytokine mRNAs after intravenous injection of LPS.
  • Five healthy volunteers were injected with a single dose of 4 ng/kg LPS.
  • Ten minutes before, and 0.5, 1 , 1.5, 2, 3 and 6 hours after the LPS injection a 2.5 ml sample of blood was taken in a PAXgeneTM tube.
  • Quantification of cytokine mRNAs was performed according to the method of the present invention. Results are expressed in mRNA copy numbers per million of ⁇ -actin mRNA copies. The mean and standard error on the mean for each time point are represented.
  • FIG. 39 follow-up of anti-tetanus vaccine response.
  • Six healthy volunteers were selected to receive an anti-tetanus recall.
  • IL-2 mRNA levels were quantified from whole blood cultured for 20 hours with (full circles) or without (open circles) 10 ⁇ g/ml tetanus toxoid, and performed at the moment of the recall (day 0), 14 days before, and 3, 7, 14, 21 and 90 days after (X-axis). Results are expressed in mRNA copy numbers per million of ⁇ -actin mRNA copies (Y-axis).
  • Each of the six panels represents individual data from 6 different donors (one donor per panel).
  • FIG. 40 Summary of the procedure followed in examples 7, 8, 9, 10 and 1 1 for analysing blood cytokine mRNA expression.
  • FIG. 41 Automated mRNA extraction and reagent mix preparation on the MagNA Pure: direct correlation between amount of starting biological material and found copy number.
  • the Y-axis represents the raw copy numbers.
  • the line is for linear regression.
  • FIG. 42 Automated mRNA extraction and reagent mix preparation on the MagNA Pure, direct correlation between amount of starting biological material and found copy number
  • the Y-axis represents the raw copy numbers.
  • the line is for linear regression.
  • FIG. 43 Summarised case report of the patient enrolled for cancer immunotherapy.
  • the melanoma was diagnosed in July 1999. In Augusts 2001 , multiple metastasis were evidenced, and directly after an orchydectomy in April 2002, the patient was enrolled for receiving a cancer vaccine.
  • the vaccine consisted in several injections of the MAGE-3 purified protein (an antigen specifically expressed by melanoma cells) in combination with an adjuvant.
  • FIG. 44 Schematic representation of the vaccination protocol and the monitoring of immune response by real-time PCR.
  • Whole blood was stimulated in vitro to assess immune response to MAGE-3
  • the patient received 3 injections of the vaccine, while a blood sample was taken once a week during 9 weeks.
  • a 200 ⁇ l aliquot of each patient's whole blood sample was incubated in the presence of 10 ⁇ g/ml MAGE-3 protein or 10 ⁇ g/ml TRAP (Plasmodium falciparum antigen) as a negative control.
  • the reagent contained in the PAXgene tube was added to allow IL-2 mRNA quantification as described in example 6.
  • the results are presented in FIG. 45.
  • FIG. 45 IL-2 mRNA in whole blood following MAGE-3 vaccination in Patient #3. Higher IL- 2 mRNA levels are observed in MAGE-3-stimulated whole blood after MAGE-3 vaccine boost.
  • the Y-axis represents the IL-2 mRNA copy numbers per million of ⁇ -actin mRNA copies, and the X-axis the weeks at which blood samples were taken. The vaccine injections were administrated at the weeks 0, 2 and 6.
  • the solid columns are for whole blood incubated in the presence of MAGE-3, and the hatched columns for whole blood incubated in the presence of TRAP.
  • FIG. 46 In vitro stimulation of whole blood: evaluation of immune response to allergen. The experiment was performed for IL-4 mRNA quantification after whole blood incubation with an allergen. Blood samples were taken from a subject allergic to cat, and from two healthy subjects. Whole blood was then incubated in absence or in the presence of the cat allergen (namely FeIdI ), for different time periods of culture, at the end of which the reagent contained in the PAXgene tube was added to allow IL-4 mRNA quantification as described in example 6. The results are presented on FIG. 47.
  • the cat allergen namely FeIdI
  • FIG. 47 In vitro stimulation of whole blood: evaluation of immune response to FeIdL FeIdI allergen significantly induces higher IL-4 mRNA levels in whole blood coming from the subject allergic to the cat compared to non allergic subjects.
  • the Y-axis represents the IL-4 mRNA copy numbers per million of ⁇ -actin mRNA copies, and the X-axis the different incubation times.
  • Green columns represent IL-4 mRNA levels found in normal whole blood incubated with the allergen, IL-4 mRNA levels found in whole blood of the allergic subject being represented by the red columns (blood incubated in the presence of FeIdI ) and the yellow columns (blood incubated without FeIdI ).
  • FIG. 48 The response to FeIdI in this whole blood system is specific and dose-related.
  • Whole blood from the allergic subject was incubated for two hours 1 ) in the presence of increasing concentrations of FeIdI (diagonal hatched columns); 2) in the presence of another allergen, ⁇ -lactoglobulin (BLG) at 10 ⁇ g/ml (horizonal broken hatched column); 3) crossed-linked IgE (dotted column).
  • the Y-axis represents the IL-4 mRNA copy numbers per million of ⁇ -actin mRNA copies.
  • FIG. 49 IL-4 mRNA levels after whole blood stimulation with FeIdI are higher in patients allergic to the cat compared to healthy controls.
  • the experiment described on slides 9 to 1 1 was repeated on blood samples from 10 healthy subjects (CTR columns) and 10 patients allergic to the cat (ALL columns).
  • Whole blood samples were incubated for two hours in the presence of 10 ⁇ g FeIdI , or in the presence of crossed-linked IgE as positive controls. The mean and standard error on the mean are represented.
  • FIG. 50 In vitro stimulation of whole blood: assessment of T cell response to GAD65. Schematic representation of the experiment performed for IL-2 mRNA quantification after whole blood incubation with purified GAD65 protein. Blood samples were taken from six type 1 diabetes patients, and from five healthy subjects.
  • FIG. 51 In vitro stimulation of whole blood: assessment of T cell response to GAD65.
  • Whole blood from type 1 diabetes patients shows higher IL-2 mRNA levels after GAD65 stimulation compared to healthy subjects. Results are expressed in IL-2 mRNA copy numbers calculated relatively to the copy numbers found in whole blood cultured without GAD65, after correction against ⁇ -actin. A logarithmic scale is used. The mean and standard error on the mean are represented. Healthy donors: CTR column; autoimmune diabetes patients: PAT column.
  • FIG. 52 Monitoring of alloreactive immune response : quantification of IL-2 mRNA in a whole blood + dendritic cells system.
  • IL-2 mRNA quantification after whole blood incubation with unrelated dendritic cells (DC) to assess alloreactive T cell response.
  • Dendritic cells from two unrelated healthy volunteers (MT and MA) were generated in vitro in the presence of IL-4 and GM-CSF.
  • a whole blood sample from each donor was cultured in the presence of the dendritic cell population of the other donor (1 ) or in the presence of their own dendritic cells (2).
  • Whole blood samples from both donors were mixed (3), as well as both dendritic cell preparations (4). After 12 hours incubation, the cultures were stopped by adding the reagent contained in the PAXgene tube.
  • IL-2 mRNA levels were then quantified as described in example 6. The results are shown in FIG. 53.
  • FIG. 53 Monitoring of alloreactive immune response quantification of IL-2 mRNA in a whole blood + dendritic cells system. Assessment of alloreactive T cell response by IL-2 mRNA quantification in whole blood. IL-2 mRNA copy numbers per million of ⁇ -actin mRNA copies are shown. The conditions are, from left to right: whole blood from donor MA alone, whole blood from donor MA + DC from donor MA, whole blood from donor MA + DC from donor MT, whole blood from donor MT alone, whole blood from donor MT + DC from donor MT, whole blood from donor MT + DC from donor MA, whole blood from donor MT + whole blood from donor MA, DC from donor MT + DC from donor MA.
  • cytokine mRNAs synthesized by peripheral blood cells should make it possible to estimate a "peripheral immune statute".
  • an accurate quantification can only be performed from a fresh whole blood sample in which mRNA is protected against nuclease digestion, and where gene transcription is inhibited.
  • surfactant reagents such as tetradecyltrimethylammonium oxalate.
  • RT-PCR for the quantification of IL-10 and IFN- ⁇ mRNAs spontaneously produced in peripheral blood was performed.
  • this reagent induces nucleic acids precipitation and nuclease inhibition, in the minute that follows sample collection.
  • This provides a total RNA preparation that is probably the nearest of in vivo mRNA status. This is especially important for cytokine mRNA, which are made sensitive to endogenous nucleases by their AU-rich sequences located in their 3' untranslated region.
  • cytokine mRNA which are made sensitive to endogenous nucleases by their AU-rich sequences located in their 3' untranslated region.
  • peripheral blood IFN- ⁇ mRNA is spontaneously and rapidly degraded, the levels being decreased by roughly 50 % already one hour after blood collection.
  • this phenomenon is not necessary true for all the cytokines, as it was found that IL-10 mRNA level is stable for at least the five hours that follow blood sampling (FIG. 32.2).
  • no significant differences in whole blood IL-10 mRNA levels were found, compared to those of PBMC (FIG. 32.1 ).
  • the nucleic acids pellet obtained after Catrimox-14TM lysis can be dissolved in the guanidium/thiocyanate solution described by Chomczynski and Sacchi (1987), as well as in its commercially available version, such as TripureTM Roche Diagnostics, Molecular Biochemicals, Brussels, Belgium), making the use of this surfactant particularly easy.
  • PAXgeneTM Blood RNA Tubes (Qiagen, Westburg, Leusden, The Netherlands) could be used in the place of Catrimox-14TM.
  • the resulting pellet can be dissolved in the lysis buffer of the "MagNA Pure LC mRNA Isolation Kit I", as described for Catrimox- 14TM in legend to FIG. 32.2.
  • a strong IL-2 mRNA induction was also observed after addition of ionophore A23187 + phorbol myristate acetate to whole blood (not shown), suggesting its use for in vitro studies on whole blood.
  • 'PAXgeneTM Blood RNA System is meant the combination of the PAXgeneTM Blood RNA Tube' with the 'PAXgeneTM Blood RNA Kit'.
  • 'Qiagen Method it is meant 'PAXgeneTM Blood RNA Kit'.
  • the present invention proposes a new procedure to isolate mRNA from whole blood which allows to determine in vivo transcript levels using an easy and reproducible method.
  • the PAXgeneTM blood RNA System and the method according to present invention are schematically compared in FIG. 33.
  • the nucleic acid pellet was then: a) PAXgeneTM Blood RNA Tube + PAXgeneTM Blood RNA Kit- ... washed in water before being dissolved in BR1 buffer for total RNA extraction, as recommended in the corresponding instruction manual.
  • the procedure of the PAXgeneTM Blood RNA System is as follows: Blood samples (2.5 ml) are collected in PAXgene Blood RNA Tubes, and may be stored or transported at room temperature if desired. RNA isolation begins with a centrifugation step to pellet nucleic acids in the PAXgene Blood RNA Tube. The pellet is washed, and Proteinase K is added to bring about protein digestion.
  • RNA is selectively bound to the silica-gel membrane as provided by the PAXgeneTM Blood RNA Kit as contaminants pass through. Following washing steps, RNA is eluted in an optimized buffer. Reverse transcription and real time PCR for IFN- ⁇ and ⁇ -actin mRNAs were conducted as described by Stordeur et al. ("Cytokine mRNA Quantification by Real Time PCR” J Immunol Methods, 259 (1-2): 55-64, 2002).
  • PAXgeneTM Blood RNA Tubes PAXgeneTM Blood RNA System
  • MagNA Pure LC Instrument extraction method also in combination with the PAXgeneTM Blood RNA Tubes was performed. In both methods the use of the PAXgeneTM blood RNA Tubes allows to stabilize RNA from blood cells.
  • Table 1.1 and 1.2 The results of this experiment show a better reproducibility for the MagNA Pure LC Technique (coefficients of variation for IFN- ⁇ mRNA copy numbers corrected against ⁇ -actin are 26 versus 16 % for Qiagen versus MagNA Pure LC, respectively).
  • Example 2 illustrates the possibility to use the PAXgeneTM Blood RNA Tubes in combination with the MagNA Pure LC mRNA Isolation Kit I, or more precisely, the possibility to dissolve the precipitate from the PAXgeneTM Blood RNA Tube in the lysis buffer contained in that kit, this lysis buffer necessarily having to be used with the other components of the kit.
  • this example it is proven that in contrast to other combinations, only the combination as described in the present invention, leads to correct/real in vivo transcript quantification.
  • Example 3 Ex vivo monitoring of immune response against tetanus toxoid.
  • example 3 blood is stimulated ex vivo with an antigen (i.e. tetanus toxoid) against which the blood donor is supposed to be immunised (because vaccinated seven years ago).
  • RT-PCR is performed according to the method (FIG. 31.1 ).
  • Cytokine mRNA is measured as a read out of the ability of the volunteer's immune system to react against the antigen.
  • the IL-2, IL-4, IL-13 and IFN- ⁇ mRNAs are preferentially analysed, but all potentially reactive proteins can be analysed via the quantification of their corresponding mRNA. Results of example 3 is shown in FIG. 34.
  • the strategy followed in this example can be schematically represented as shown in FIG. 31.2.
  • the difficulty to measure the immunological efficiency of these vaccines resides essentially in the absence of assays sufficiently sensitive to detect a cellular immune response in vivo.
  • the used techniques implied the intensive in vitro culture of the PBMC of patients on of long periods times in the presence of antigen and of co-stimulating susceptible to induce a modification of the original functional characteristics of lymphocytes.
  • the analyses of the anergic states or tolerant states of the lymphocyte precursors directed against the tumor antigens is extremely difficult being given the reversible nature of their functional state after their extended in-vitro incubation in the presence of antigen.
  • Example 4 Detection of the activation of the immune system of the recipient by the histocompatibility antigens of the donor.
  • an organ (ex. liver, kidney, bone marrow, etc.) from a donor is transplanted to a recipient.
  • Whole blood the recipient is collected in a tube comprising a compound inhibiting RNA degradation and/or gene induction according to present invention.
  • RT-PCR is performed according to the method.
  • Cytokine mRNA is measured as a read out of the activation of the immune system of the recipient by the histocompatibility antigens of the donor (FIG. 31.3).
  • Example 5 Detection of the reactivity of the immune system of the recipient to the histocompatibility antigens of the donor.
  • an organ ex. liver, kidney, bone marrow, etc from a donor is transplanted to a recipient.
  • Whole blood of the recipient is collected on a tube and incubated ex-vivo with the histocompatibility antigens of the donor.
  • a compound inhibiting RNA degradation and/or gene induction according to present invention is added to the blood.
  • RT-PCR is performed according to the method.
  • Cytokine mRNA is measured as a read out of the response of the immune system of the recipient by the histocompatibility antigens of the donor (FIG. 31.4).
  • transplant rejection is essentially based on the detection of markers measured in the urine or the blood of patients (blood urea nitrogen-BIN- or creatinine in the case of kidney transplants) or at the time of the analyses of biopsies of the grafted organ. These indicators are however only detected when the rejection mechanism is already well advanced. In fact, transplant rejection is the result of an immunological mechanism that precedes the deterioration of the grafted organ. The detection of these immunological mechanisms before the grafted organ is damaged would allow to reduce in a considerable manner the loss of the grafted organ by adapting more earlier the immunosuppressive treatments. On the other side, it is also recognized that of sub-clinical episodes of rejections (with no induction of clinical signs) occur themselves frequently after transplantation.
  • cytotoxic activated T-lymphocytes T activated (granzyme B, perforine, different cytokines) by sensitive methods of quantitative PCR were showed to be excellent tools to measure the triggering of a rejection.
  • messengers coding for different kinds of cytokines may be studied, preferential targets may be IL-2, IFN-gamma, IL-4, IL-5, Granzyme, perforine and FasFas-ligand.
  • Example 6 Immune monitoring in whole blood using real time PCR.
  • example 6 a whole blood method is described allowing the measure of the induction of cytokine synthesis at the mRNA level.
  • the originality of this method consists in the combination of PAXgeneTM tubes containing a mRNA stabilizer for blood collection, the MagNA PureTM instrument as an automated system for mRNA extraction and RT-PCR reagent mix preparation, and the real time PCR methodology on the LightcyclerTM for accurate and reproducible quantification of transcript levels.
  • This example first demonstrate that this method is adequate to measure the induction of IL (interleukin)-i ⁇ and IL-1 receptor antagonist (IL-1 RA) mRNA upon addition of bacterial lipopolysaccharide (LPS) to whole blood.
  • IL interleukin
  • IL-1 RA IL-1 receptor antagonist
  • This example further demonstrates that this approach is also suitable to detect the production of mRNA encoding T cell-derived cytokines in whole blood incubated with tetanus toxoid as a model of in vitro immune response to a recall antigen.
  • this methodology can be used successfully to assess inflammatory as well as T cell responses in vivo, as it allowed to detect the induction of IL-1 ⁇ and IL-1 RA after injection of LPS in healthy volunteers, and also the induction of IL-2 upon recall immunisation with tetanus vaccine.
  • the blood lysate obtained in the PAXgeneTM tube or at the end of whole blood culture was briefly mixed before transferring a 300- ⁇ l aliquot in a 1.5-ml eppendorf tube for centrifugation at maximal speed for 5 minutes (12,000 to 16,000 g, depending on the device). The supernatant was discarded, and the nucleic acid pellet thoroughly dissolved by vortexing in 300 ⁇ l of the lysis buffer contained in the MagNA PureTM mRNA extraction kit (Roche Applied Science). mRNA was then extracted from 300 ⁇ l of this solution, using this kit on the MagNA PureTM instrument (Roche Applied Science) following manufacturer's instructions ("mRNA I cells" Roche's protocol, final elution volume 100 ⁇ l). The quality of the extracted mRNA was previously documented by Northern blot analysis (Roche Applied Science, unpublished data).
  • the mRNA was quantified for ⁇ -actin and IL-1 ⁇ from different volumes of LPS-stimulated whole blood, ranging from 20 to 200 ⁇ l. As shown in FIG. 36, the mRNA copy numbers of both ⁇ -actin and I L-1 ⁇ were indeed directly correlated with the starting volume of blood. In vitro response to tetanus toxoid. To determine whether this method might be suitable for the analysis of T cell responses, cytokine mRNA levels in whole blood culture after addition of tetanus toxoid, a well established recall antigen as all individuals were vaccinated in childhood, was quantified.
  • IL-2 mRNA was the most sensitive parameter to monitor antitetanus toxoid responses
  • this parameter was chosen to analyse the changes in the T cell responses to tetanus toxoid in whole blood upon recall vaccination in vivo.
  • whole blood incubation in absence or presence of tetanus toxoid was performed before and at several time points after administration of the vaccine.
  • FIG. 39 the production of IL-2 mRNA in whole blood exposed to the antigen significantly increased in all vaccinated individuals.
  • IL-2 mRNA induction was already apparent 7 days post vaccination, maximal levels being reached at day 14 or 21.
  • Real time PCR is so called because the amplicon accumulation can be directly monitored during the PCR process, using fluorogenic molecules that bind the PCR product. This leads to the generation of a fluorescence curve for each sample, from which it is possible to determine the (c)DNA copy number of the sample, by comparison to fluorescence curves obtained with calibrated standards.
  • the fluorogenic molecule can be an oligonucleotide complementary to a sequence of the PCR product, localised between the two primers.
  • RNA extraction One of the difficulties encountered using whole blood for RT-PCR analysis is the cell lysis that precedes RNA extraction. Because of the high amount of proteins present in plasma and erythrocytes, the majority of the methods that isolate RNA from whole blood involve the purification of the potential cellular sources of the analysed mRNA or the elimination of the red blood cells, before performing the RNA extraction. These intermediate steps can be associated with mRNA degradation and/or gene induction and thus with changes in mRNA levels. Furthermore, the simple fact of taking blood can lead to degradation of some mRNAs. This is especially true for cytokine mRNAs, which are sensitive to endogenous nucleases via the AU-rich sequences located in their 3' untranslated region.
  • peripheral blood IFN- ⁇ mRNA levels indeed decreased by roughly 50 % already one hour after blood collection (Stordeur et al., (2002) J. Immunol Meth. 261 :195).
  • This can be avoided using quaternary amine surfactants such as tetradecyltrimethylammonium oxalate, a cationic surfactant called Catrimox-14TM (Qiagen, Westburg, Leusden, The Netherlands) that induces whole cell lysis and, in the same time, nucleic acid precipitation.
  • the present example observes that the nucleic acid precipitate obtained with the PAXgeneTM tubes can surprisingly be dissolved in a guanidium/ thiocyanate solution.
  • Another possible application of this whole blood method is the monitoring of T cell responses upon vaccination, as suggested by the clear induction of IL-2 mRNA observed after in vitro rechallenge in individuals vaccinated with tetanus toxoid. This might be of special interest for large-scale vaccination studies in which cell isolation might be difficult to organise in good conditions, especially in developing countries where several new vaccines are under evaluation.
  • FIG. 40 The procedure followed in this example is summarized in FIG. 40.
  • a linear regression of mRNA copy number on starting cell number was calculated (FIG. 41 ).
  • mRNA was extracted from various peripheral blood mononuclear cell (PBMC) numbers (ranging from 100,000 to 600,000 cells, X-axis) and one step RT-real time PCR for ⁇ -actin mRNA was performed as described in the "Material and Methods" section of the present example 6.
  • PBMC peripheral blood mononuclear cell
  • FIG. 42, panels B and D This experiment has been repeated from PBMC for ⁇ -actin and TNF- ⁇ mRNAs
  • FIG. 42, panel A whole blood
  • CD4 + purified T cells FIG. 42, panel C
  • Example 8 Cancer immunotherapy The procedure followed in this example is summarized in FIG. 40. The methodology was applied to the monitoring of immune response induced by cancer vaccine. FIGs. 43, 40 and 41 illustrate the results obtained in this field with a melanoma patient.
  • Example 9 Allergy The procedure followed in this example is summarized in FIG. 40. The methodology was then applied in Allergy. The response induced by in vitro incubation of whole blood of an allergic subject with the relevant allergen was analysed by IL-4 mRNA quantification using real-time PCR. FIGs. 46, 47, 48 and 49 illustrate the results obtained in this field.
  • FIG. 40 The procedure followed in this example is summarized in FIG. 40.
  • the methodology was then applied in Autoimmunity.
  • IL-2 mRNA quantification using this whole blood system was applied to assess T cell response to glutamic acid decarboxylase 65 (GAD65), an autoantigen being the target of auto-reactive T cells in type 1 autoimmune diabetes.
  • FIGs. 40 and 41 illustrate the results obtained in this field.
  • FIG. 40 The procedure followed in this example is summarized in FIG. 40.
  • the methodology was then applied in Transplantation.
  • IL-2 mRNA quantification by real time PCR after whole blood incubation with alloreactive non-T cells provides an alternative to the classical mixed lymphocytes reaction (MLR) to monitor alloreactive T cell response.
  • FIG.s 52 and 53 illustrate the results obtained in this field.
  • IFN-gamma mRNA copy numbers per million of beta-actin mRNA copies result 1 192 result 2 170 result 3 153 result 4 139 result 5 138 result 6 160 result 7 105 result 8 142 result 9 142 mean 149
  • F, R and P indicate forward and reverse primers and probes, respectively; numbers indicate the sequence position from Genebank accession numbers X01586 for IL-2 and NM_000589 for IL-4.
  • Standard curves were generated from serial dilutions of PCR products prepared by "classical" PCR, for which specific conditions were as follows: denaturation at 95°C for 20 s, annealing at 58°C for 20 s and elongation at 72°C for 45 s, for a total of 35 cycles. MgCI2 final concentration was 1.5 mM. Table 3.

Abstract

Cette invention concerne un dispositif et un kit permettant d’activer par impulsion un échantillon biologique avec un agent d’impulsion, puis à stabiliser l’échantillon biologique ainsi activé, ledit dispositif ou kit assurant une réaction témoin. L’invention a des applications dans le domaine des diagnostics médicaux, notamment ceux concernant l’immunité.
EP08875202A 2008-10-17 2008-10-17 Dispositif, kit et méthode d activation par impulsion d échantillons biologiques avec un agent, et stabilisation de l échantillon ainsi activé Withdrawn EP2346606A1 (fr)

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PL2547259T3 (pl) 2010-03-19 2016-04-29 Atomo Diagnostics Pty Ltd Układ diagnostyczny
PT2627255T (pt) 2010-10-15 2016-07-13 Atomo Diagnostics Pty Ltd Montagem para amostragem
WO2015075677A1 (fr) 2013-11-21 2015-05-28 Atomo Diagnostics Pty Limited Contrôle de fluide dans des dispositifs de test intégrés
EP2959971A1 (fr) * 2014-06-27 2015-12-30 Euroimmun Medizinische Labordiagnostika AG Procédé et dispositif de transfert de liquides
EP3206724B1 (fr) * 2014-10-15 2018-12-19 Merck Patent GmbH Récipient de préparation d'échantillon
CN105039548A (zh) * 2015-07-28 2015-11-11 深圳爱生再生医学科技有限公司 Il-4检测引物、试剂盒和半定量检测方法
WO2019094835A1 (fr) * 2017-11-10 2019-05-16 Juno Therapeutics, Inc. Réservoirs cryogéniques à système fermé
CN110012899A (zh) * 2019-04-12 2019-07-16 南京科佰生物科技有限公司 细胞用rna稳定剂及其使用方法
CN110176165B (zh) * 2019-06-20 2022-02-15 中国石油大学(华东) 一种“烃-水-岩”相互作用的热模拟综合实验方法
CN116519968B (zh) * 2023-06-25 2023-09-08 成都云芯医联科技有限公司 一种一体化多试剂样本混合加样装置

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IL212082A0 (en) 2011-06-30
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CN102186590A (zh) 2011-09-14
CA2740150C (fr) 2012-09-25
JP2012505639A (ja) 2012-03-08

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