WO2010102632A2 - Improved rna purification method - Google Patents

Abstract

The present invention provides a device comprising a detachable filter section and at least one tube section. Also provided is a method for collecting a sample in a non-toxic easy-to-use collection media and efficiently extracting said sample from said collection media thereby obtaining sufficient quantity and quality of RNA, DNA or protein from said samples, such as single in vivo fine-needle aspirates.

Description

Improved RNA purification method

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to a device comprising a filter and the use of a device comprising a filter for efficiently extracting a sample from a collection media, thereby increasing the yield obtained from a sample in a collection media.

Background of invention

Messenger RNA (mRNA) is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes, where the nucleic acid polymer (mRNA) s translated into a polymer of amino acids: a protein. microRNAs (miRNAs) are small, non-coding, single-stranded RNA gene products that regulates translation and stabilization of specific messenger RNAs (1 ).

Both RNA and miRNA profiling of tumors has rapidly become a method to gain new information about tissue or tumor biology and a way to improve tissue or tumor classification and prognostics (2-4). As an auxiliary diagnostic tool, identification of differentially regulated RNAs and miRNAs also offers the potential of improving the distinction between benign and malignant tumors (5).

Biopsy material for RNA and miRNA profiling of patient samples can be obtained during surgery or by needle biopsies. Fine needle aspiration (FNA) is a diagnostic procedure used to investigate masses accessible by a needle. A thin, hollow needle is inserted into the mass to extract cells that are subsequently examined microscopically. Cytopathological examination of fine needle aspirates is widely used as a diagnostic tool in e.g. mammary, hepatic and pancreatic tumors, thyroid nodules and processes of unknown origin (1 1 -15). An increasing number of publications have confirmed the feasibility of extracting RNA from fine needle aspirates (FNA) (6-10). However, the majority of these studies are based on multiple ex vivo tumor aspirates collected in toxic preservative media unsuited for routine clinical use. Recently, Szafranska et al. showed the diagnostic potential of a PCR-based quantification of miRNA levels extracted from multiple samples obtained by in vivo endoscopic ultrasound-guided fine- needle aspiration (1 1 ).

If mRNA and miRNA profiling on samples, such as samples obtained by FNA, is to be established as a routine analysis, it is crucial that collection can be done in a non-toxic medium and that the resulting sample is stable at room temperature for a sufficient period. One way to accomplish this is to use an RNA preservation solution, such as RNAIater®, as a preservative. However, isolating RNA from a single in vivo fine needle aspirate gives insufficient amounts of RNA for subsequent miRNA and mRNA array expression analyses.

Dunmire et al. has modified the procedure of single in vivo FNAs kept in RNAIater, by extracting RNA from both the cell pellet obtained from centrifugation of RNAIater with tne FNA sample, and from the resulting supernatant. This method increases the RNA yield 2-fold to an average of 3 ug per sample, of which 53% is extracted from the pellet.

The present invention discloses a simple and efficient method to overcome the above- cited problems by employing a simple and non-toxic filtration technique for efficiently extracting a sample from a collection media in which said sample is collected, said method drastically increases the yield of RNA, DNA or protein obtained from the sample collected in a collection media while maintaining the integrity of the RNA, DNA or protein.

As disclosed herein, capturing FNA tissue samples on the filter device according to the present invention increased the RNA yield 10 fold while maintaining RNA pureness.

Summary of invention

The present invention provides a device comprising a detachable filter section and at least one tube section, and a method for collecting a sample in a non-toxic easy-to-use collection media and extracting said sample from said collection media thereby obtaining sufficient quantity and quality of RNA, DNA or protein from said sample, such as single in vivo fine-needle aspirates.

One embodiment of the invention is directed at a simple method for capturing a sample stored in a collection media such as an RNA preservation solution on a filter, such as a 0.45 μm filter. The captured sample is subsequently collected from the filter by changing the direction of movement of the sample, for example by inverting the filter or the device comprising the filter section.

The collected sample may be analysed further, either directly or by extracting RNA, DNA or protein from the sample and analysing said RNA, DNA or protein.

The sample collected by the disclosed method gives a markedly higher yield than simple centrifugation and direct pelleting or precipitation of a sample collected in a collection media, which is today the predmominant method for extracting a sample from a collection media such as an RNA stabilisation solution.

Description of Drawings

Figure 1 . RNA Extraction using RNAIater with a Modified Protocol according to the present invention.

Figure 2: Median values of total RNA yield (A) and median 260/280 ratios (B) from single in vivo fine-needle aspirates.

Figure 3: A device comprising a detachable filter section and at least one tube section.

Figure 4: A method for efficiently extracting a sample from a collection media by using a device comprising a detachable filter section and at least one tube section.

Figure 5: Correlations between the Iog2 normalized miRNA expression values from fine needle aspirates and corresponding surgical biopsies from the target nodule tissue.

Definitions

Collection media: Is used herein to denote any solution suitable for collecting and storing of a sample for later retrieval of e.g RNA, DNA or protein from said sample. When attempting to extract RNA from the sample, an RNA preservation solution is preferred, such as commercially available solutions comprising RNAIater® (Ambion and Qiagen), PreservCyt medium (Cytyc Corp), PrepProtect™ Stabilisation Buffer (Miltenyi Biotec), Allprotect Tissue Reagent (Qiagen) and RNAprotect Cell Reagent (Qiagen). or homemade solutions according to available protocols. Individual: Any species or subspecies of bird, mammal, fish, amphibian, or reptile, including human beings. As used herein, 'subject' and 'individual' may be used interchangeably.

Pellet: small particles typically created by compressing an original material; also a precipitate formed by centrifugation of a sample. As used herein a pellet is the part of a sample that is formed by centrifugal forces.

Sample: A portion, piece, or segment that is representative of a whole, an actual part of something larger. A sample may for example be a sample from an individual or from a cell culture.

Detailed description of the invention

The present invention provides a device comprising a detachable filter section and at least one tube section. Also provided is a method for collecting a sample in a non-toxic easy-to-use collection media and extracting said sample from said collection media thereby obtaining sufficient quantity and quality of RNA, DNA or protein from said samples, such as single in vivo fine-needle aspirates. The details of the device and method according to the present invention are further specified herein below.

Collection media

A collection media according to the present invention is any solution suitable for collecting and storing of a sample for later retrieval of e.g. RNA, DNA or protein from said sample.

Preferably, the collection media will preserve the sample and maintain its components, such as cells and the interior components of the cells (i.e. RNA, DNA and/or protein) in a largely unaltered state from the point of collection of the sample in the collection media to the point of extraction of the sample from the collection media.

RNA preservation solution

When the object is to retrieve RNA from the sample, the collection media is most preferably an RNA preservation solution or reagent suitable for containing samples without the immediate need for cooling or freezing the sample, while maintaining RNA integrity prior to extraction of RNA from the sample. An RNA preservation solution or reagent may also be known as RNA stabilization solution or reagent or RNA recovery media, and may be used interchangeably herein.

The RNA preservation solution may penetrate the harvested cells of the collected sample and retards RNA degradation to a rate dependent on the storage temperature.

In one embodiment, the RNA preservation solution may be any commercially available solutions or it may be a solution prepared according to available protocols.

In one embodiment, the commercially available RNA preservation solutions is selected from RNAIater® (Ambion and Qiagen), PreservCyt medium (Cytyc Corp), PrepProtect™ Stabilisation Buffer (Miltenyi Biotec), Allprotect Tissue Reagent (Qiagen) and RNAprotect Cell Reagent (Qiagen).

Protocols for preparing a RNA stabilizing solution may be retrieved from the internet (e.g. L.A. Clarke and M. D. Amaral: 'Protocol for RNase-retarding solution for cell samples', provided through The European Workin Group on CFTR Expression), or may be produced and/or optimized according to techniques known to the skilled person.

Other collection media

When the object is to retrieve RNA, DNA and/or protein from the sample, the collection media may be any media such as water, sterile water, denatured water, saline solutions, buffers, PBS, TBS, Allprotect Tissue Reagent (Qiagen), cell culture media such as RPMI-1640, DMEM (Dulbecco's Modified Eagle Medium), MEM (Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), BGjB (Fitton-

Jackson modification), BME (Basal Medium Eagle), Brinster's BMOC-3 Medium, CMRL Medium, CO₂-lndependent Medium, F-10 and F-12 Nutrient Mixture, GMEM (Glasgow Minimum Essential Medium), IMEM (Improved Minimum Essential Medium), Leibovitz's L-15 Medium, McCoy's 5A Medium, MCDB 131 Medium, Medium 199, Opti-MEM, Waymouth's MB 752/1 , Williams' Media E, Tyrode's solution, Belyakov's solution, Hanks' solution and other cell culture media known to the skilled person, tissue preservation media such as HypoThermosol®, CryoStor™ and Steinhardt's medium and other tissue preservation media known to the skilled person. Dynamics of the collection media

The sample of the present invention may be kept in the collection media for a variable period of time and at various temperature ranges. In one embodiment, the sample is kept in collection media for between 15 minutes and 100 years prior to collecting the sample from said collection media by the method disclosed herein, such as between 15 minutes and 1 hour, for example 1 to 2 hours, such as 2 to 5 hours, for example 5 to 10 hours, such as 10 to 24 hours, for example 24 hours to 48 hours, such as 48 to 72 hours, for example 72 to 96 hours, such as 4 to 7 days, such as 1 week to 2 weeks, such as 2 to 4 weeks, such as 4 weeks to 1 month, such as 1 month to 2 months, for example 2 to 3 moths, such as 3 to 4 months, for example 4 to 5 moths, such as 5 to 6 months, for example 6 to 7 moths, such as 7 to 8 months, for example 8 to 9 moths, such as 9 to 10 months, for example 10 to 1 1 moths, such as 1 1 to 12 months, for example 1 year to 2 years, such as 2 to 3 years, for example 3 to 4 years, such as 4 to 5 years, for example 5 to 6 years, such as 6 to 7 years, for example 7 to 8 years, such as 8 to 9 years, for example 9 to 10 years, such as 10 to 20 years, for example 20 to 30 years, such as 30 to 40 years, for example 40 to 50 years, such as 50 to 75 years, for example 75 to 100 years prior to collecting the sample from said collection media by the method disclosed herein.

In one embodiment, the sample is kept in collection media at a temperature of between -8O⁰C to 37⁰C, such as between -80 to -4O⁰C, for example -40 to O⁰C, such as 0 to 5⁰C, for example 5 to 10⁰C, such as 10 to 15⁰C, for example 15 to 2O⁰C, such as 20 to 25⁰C, for example 25 to 3O⁰C, such as 30 to 37⁰C prior to collecting the sample from said collection media by the method disclosed herein.

As used herein, 'kept' is meant to cover both the collection stage, an optional storage stage and an optional transportation stage.

The sample may be kept at different temperatures during collection, transportation and/or storage in the collection media. Thus, the sample may for example be collected at ambient temperature or on ice, kept in a refrigerator (i.e. 4⁰C) for a while and subsequently shipped to a research facility at ambient temperatures, refrigerated (i.e. 4⁰C), kept on ice or frozen. Device

It is an aspect of the present invention to provide a device comprising a detachable filter section and at least one tube section, wherein the filter section is detachably attached to a first tube section having an elongated shape and a proximal opening and a distal opening, and the filter section is detachably attached to a second tube section having an elongated shape and a proximal opening and an optionally detachably attached distal closure unit.

As used herein, 'distal' means the part furthest away from the filter section, and 'proximal' means the part closest to the filter section of the device.

The device according to the present invention is suitable for collecting a sample dispersed in a collection media. The device has been developed for use in a method for efficiently extracting a sample from a collection media in which said sample was collected, thus increasing the yield of RNA, DNA or protein obtained from a sample.

The device of the present invention and a method for using the device is depicted in figures 3 and 4.

The tube sections of the device

In one embodiment, the distal closure unit of the second tube section is an integrated part of the second tube section. In other words, the second tube section is in one embodiment closed in one end; the distal end facing away from the filter. This makes the second tube section suitable for collection of liquid flow-through without the need for a second collection tube.

In another embodiment, the distal closure unit of the second tube section is in the form of a detachable unit; such as a click-on unit, screw-on unit, add-on unit or press-on unit. This makes the second tube section suitable for connection with a collection device, a pump or other means for creating a vacuum, suction or pressure.

In one embodiment, the distal opening of the first tube section is closable by a closure unit. Said closure unit is in one embodiment in the form of a detachable unit; such as a click-on unit, screw-on unit, add-on unit or press-on unit. This makes the first tube section suitable for collection of the sample from the filter section without the need for a second collection tube.

The first and second tube sections may be made of for example a medical grade polymer such as plastic or glass or any other suitable material. In one embodiment, the first and second tube sections are made of or comprise transparent plastic.

The first and second tube sections of the device of the present invention may be made of or comprise one or more of the materials selected from the group consisting of Biodegradable plastic, Bioplastics obtained from biomass e.g. from pea starch or from biopetroleum, Polypropylene (PP), Polystyrene (PS), High impact polystyrene (HIPS), Acrylonitrile butadiene styrene (ABS), Polyethylene terephthalate (PET), Polyester (PES), Fibers, textiles, Polyamides (PA), (Nylons), Polyvinyl chloride) (PVC), Polyurethanes (PU), Polycarbonate (PC), Polyvinylidene chloride (PVDC) (Saran), Polyvinylidene Fluoride (PVDF), Polyethylene (PE), Polymethyl methacrylate (PMMA), Polytetrafluoroethylene (PTFE) (trade name Teflon), Fluorinated ethylene propylene (FEP), Polyetheretherketone (PEEK) (Polyetherketone), Polyetherimide (PEI) (Ultem), Phenolics (PF), (phenol formaldehydes), Perfluoroalkoxy (PFA), Poly(methyl methacrylate) (PMMA), Urea-formaldehyde (UF), Melamine formaldehyde (MF), Polylactic acid and Plastarch material or any mixture thereof.

In one particular embodiment, the first and second tube sections are made from polypropylene.

The first and second tube sections of the device of the present invention may be made of or comprise one or more materials selected from the group consisting of TECAFORM™ AH MT, CELCON® (Acetal Copolymer), RADEL®, TECASON™ P XRO (Polyphenylsulfone, also Radio Opacifer), UDEL® Polysulfone, ULTEM® (Polyetherimide), UHMW Lot Controlled, LENNITE® UHME-PE, TECANAT™ PC (USP Class Vl Polycarbonate Rod), ZELUX® GS (Gamma Stabilized Polycarbonate),

ACRYLIC (Medical grade Cast Acrylic), TECAMAX™ SRP (Ultra High Performance Thermoplastic), TECAPRO™ MT (Polypropylene Heat Stabilized), TECAPEEK™ MT (USP Class Vl compliant), TECAFORM™ AH SAN, ANTIMICROBIAL filled plastics, TECASON™ P XRO (Biocompatible Radio Opacifer PPSU), TECAPEEK™ CLASSIX, POLYSULFONE® (Medical grade), TECANYL™ (Medical grade Noryl®), TYGON® (Medical grade Tubing), TEXOLON™ Medical Grade PTFE (USP CLASS Vl), PROPYLUX HS and HS2, ABS (FDA Approved Medical Grades), TOPAS® (Medical grade), and other Medical Grade/FDA approved plastic products.

The first and second tube sections may be of any suitable size.

In one embodiment, the first and second tube sections may each hold a volume of between 0.1 ml and 100 ml; such as 0.1 to 1 ml, for example 1 to 2 ml, such as 2 to 3 ml, for example 3 to 4 ml, such as 4 to 5 ml, for example 5 to 6 ml, such as 6 to 7 ml, for example 7 to 8 ml, such as 8 to 9 ml, for example 9 to 10 ml, such as 10 to 1 1 ml, for example 1 1 to 12 ml, such as 12 to 13 ml, for example 13 to 14 ml, such as 14 to 15 ml, for example 15 to 20 ml, such as 20 to 25 ml, for example 25 to 30 ml, such as 30 to 35 ml, for example 35 to 40 ml, such as 40 to 50 ml, for example 50 to 60 ml, such as 60 to 70 ml, for example 70 to 80 ml, such as 80 to 90 ml, for example 90 to 100 ml.

The diameter of the first and second tube sections may in one embodiment be between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

The length of the first and second tube sections from the proximal end to the distal end may in one embodiment be between 0.5 cm mm to 20 cm, such as 0.5 to 1 cm, for example 1 to 2 cm, such as 2 to 3 cm, for example 3 to 4 cm, such as 4 to 5 cm, for example 5 to 6 cm, such as 6 to 7 cm, for example 7 to 8 cm, such as 8 to 9 cm, for example 9 to 10 cm, such as 10 to 1 1 cm, for example 1 1 to 12 cm, such as 12 to 13 cm, for example 13 to 14 cm, such as 14 to 15 cm, for example 15 to 16 cm, such as 16 to 17 cm, for example 17 to 18 cm, such as 18 to 19 cm, for example 19 to 20 cm.

The first and second tube sections are detachably attached to a filter section. Attachment between the first tube section and the filter section, and between the second tube section and the filter section may in one embodiment be a add-on, click- on, screw-on or press-on system.

The filter section of the device The filter section in one embodiment comprises a material such as PVDF

(Polyvinylidene Fluoride), Nitrocellulose, Cellulose, Cellulose Acetate (CA), PTFE (Polytetrafluoroethylene), Nylon, PES (Polyethersulfone), MCE (Mixed Cellulose Ester) and Glass fiber (GF). In a preferred embodiment, the filter section comprises PVDF.

The filter section may be any suitable shape. In a preferred embodiment, the filter section comprises an annular filter. In another preferred embodiment, the filter section comprises a flat filter. In a particular embodiment, the filter section comprises a flat, annular filter.

In one embodiment, the filter section has a pore size of between 0.01 to 5.0 urn, such as 0.01 to 0.02 urn, for example 0.02 to 0.03 urn, such as 0.03 to 0.04 urn, for example 0.04 to 0.05 urn, such as 0.05 to 0.06 urn, for example 0.06 to 0.07 urn, such as 0.07 to 0.08 urn, for example 0.08 to 0.09 urn, such as 0.09 to 0.1 urn, for example 0.1 to 0.2 urn, such as 0.2 to 0.3 urn, for example 0.3 to 0.4 urn, such as 0.4 to 0.5 urn, for example 0.5 to 0.6 urn, such as 0.6 to 0.7 urn, for example 0.7 to 0.8 urn, such as 0.8 to 0.9 urn, for example 0.9 to 1.0 urn, such as 1.0 to 1.5 urn, for example 1.5 to 2.0 urn, such as 2.0 to 2.5 urn, for example 2.5 to 3.0 urn, such as 3.0 to 3.5 urn, for example 3.5 to 4.0 urn, such as 4.0 to 4.5 urn, for example 4.5 to 5.0 urn. The pore size of the filter is in one particular embodiment 0.22 μm, and in another particular embodiment 0.45 μm.

The diameter of the filter section is in one embodiment between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm. The length of the filter section is in one embodiment between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

Sample

The sample according to the present invention is collected in a collection media. Subsequently, the sample is extracted from the collection media by the method disclosed herein.

The sample preferably comprises whole, intact cells that may be collected from the collection media using a device according to the present invention and/or the method according to the present invention. It follows that the samples may further comprise some ruptured cells and genomic material and/or protein; however the device according to the present invention is directed mainly at collecting intact cells and tissues from the sample.

Retention of the sample on the filter according to the present invention is achieved due to the size of the sample or due to chemical properties of the sample.

Sample type

In one embodiment, the sample comprises cells and/or tissue.

The sample may be collected from an individual or a cell culture, preferably an individual. The individual may be any animal, such as a mammal, including human beings. In a preferred embodiment, the individual is a human being.

The sample may comprise cells, either eukaryotic or prokaryotic, such as mammalian cells, bacteria cells, fungus cells, yeast cells; and/or virus particles. In one embodiment, the sample is taken from a cancer selected from the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Anal Cancer, Astrocytoma (e.g. Childhood Cerebellar or Childhood Cerebral), Basal Cell Carcinoma, Extrahepatic Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumor, Breast Cancer, Male Breast Cancer, Bronchial Adenomas/ Carcinoids, Burkitt's Lymphoma, Carcinoid Tumor, Carcinoma of Unknown Primary, Primary Central Nervous System Lymphoma, Cerebral Astrocytoma/ Malignant Glioma, Cervical Cancer, Childhood Cancers, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic

Myeloproliferative Disorders, Colon Cancer, Cutaneous T-CeII Lymphoma, Endometrial Cancer, Ependymoma (such as Childhood Ependymoma), Esophageal Cancer, Ewing's Family of Tumors, Extracranial Germ Cell Tumor (such as Childhood Extracranial Germ Cell Tumor), Extragonadal Germ Cell Tumor, Eye Cancer (Intraocular Melanoma or Retinoblastoma), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma (such as Childhood Hypothalamic and Visual Pathway Glioma), Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Lung Cancer (Non-Small Cell or Small Cell), Lymphoma (such as AIDS-Related Lymphoma, Burkitt's Lymphoma, Cutaneous T-CeII Lymphoma, Non-Hodgkin's Lymphoma), Macroglobulinemia (such as Waldenstrom's Macroglobulinemia), Malignant Fibrous Histiocytoma of Bone/ Osteosarcoma, Medulloblastoma (such as Childhood Medulloblastoma), Melanoma, Merkel Cell Carcinoma, Mesothelioma (such as Adult Malignant Mesothelioma or childhood Mesothelioma), Metastatic Squamous Neck Cancer with Occult Primary, Multiple Endocrine Neoplasia Syndrome (such as occurring in childhood), Multiple Myeloma/ Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/ Myeloproliferative Diseases, Myeloma (such as Multiple Myeloma), Chronic myeloproliferative disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer (such as Childhood Nasopharyngeal Cancer), Neuroblastoma, Oropharyngeal Cancer, Osteosarcoma/ Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (such as Childhood Ovarian Cancer), Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, Pleuropulmonary Blastoma, Prostate Cancer, Renal Pelvis and Ureter Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma (such as Childhood Rhabdomyosarcoma), Salivary Gland Cancer, Adult-onset soft tissue Sarcoma, Soft Tissue Sarcoma (such as Childhood Soft Tissue Sarcoma), Uterine Sarcoma, Sezary Syndrome, Skin Cancer (such as non-Melanoma skin cancer), Merkel Cell Skin Carcinoma, Small Intestine Cancer, Supratentorial Primitive Neuroectodermal Tumors (such as occurring in Childhood), Cutaneous T-CeII Lymphoma, Testicular Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer,

Transitional Cell Cancer of the Renal Pelvis and Ureter, Trophoblastic Tumor (such as Gestational Trophoblastic Tumor), Urethral Cancer, Endometrial Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma (such as Childhood Visual Pathway and Hypothalamic Glioma), Waldenstrom's Macroglobulinemia and Wilms' Tumor.

In a preferred embodiment, the sample is taken from thyroid cancer, thyroid nodules, breast cancer, breast nodules, pancreatic cancer, pancreatic nodules, liver cancer, liver nodules and processes of unknown origin.

In a particular embodiment, the sample is taken from thyroid cancer and/or thyroid nodules.

In another embodiment, the sample is taken from a tissue selected from the group consisting of the nervous system, the musculoskeletal system, the circulatory system, the respiratory system, the gastrointestinal system, the integumentary system, the urinary system, the reproductive system, the immune system and the endocrine system.

Specific examples of tissues from which a sample may be obtained comprises muscle, bone, bone marrow, ligaments, tendons, skin, hair, nails, sweat glands, sebaceous glands, liver, pancreas, spleen, kidney, bladder, urethra, ureters, heart, lungs, nasopharynx, trachea, stomach, esophagus, intestine, mouth, rectum, gall bladder, salivary glands, breast, testis, ovary, uterus, cerebrospinal fluid, blood, thyroid gland, parathyroid gland, adrenal gland, thymus, lymph nodes, lymph channels, pituitary, cerebellum, cerebrum, spinal cord, eyes, ears, tongue and nose.

The tissue from which the sample derives may be healthy or diseased.

Sample collection

In one embodiment, the sample is collected in a collection media from an individual by any available means, such as fine-needle aspiration (FNA) using a needle with a maximum diameter of 1 mm; core needle aspiration using a needle with a maximum diameter of above 1 mm (also called coarse needle aspiration, large needle aspiration or large core aspiration); cutting biopsy; open biopsy; or any other means known to the person skilled in the art.

In another embodiment, the sample is collected from an in vitro cell culture.

In a preferred embodiment, the sample is a fine-needle aspirate from an individual. The fine-needle aspiration is performed using a needle with a diameter of between 0.2 to 1.0 mm, such as 0.2 to 0.3 mm, for example 0.3 to 0.4 mm, such as 0.4 to 0.5 mm, for example 0.5 to 0.6 mm, such as 0.6 to 0.7 mm, for example 0.7 to 0.8 mm, such as 0.8 to 0.9 mm, for example 0.9 to 1.0 mm in diameter.

The diameter of the needle is indicated by the needle gauge. Various needle lengths are available for any given gauge. Needles in common medical use range from 7 gauge (the largest) to 33 (the smallest) on the Stubs scale. Although reusable needles remain useful for some scientific applications, disposable needles are far more common in medicine. Disposable needles are embedded in a plastic or aluminium hub that attaches to the syringe barrel by means of a press-fit (Luer) or twist-on (Luer-lock) fitting.

The fine-needle aspiration of the present invention is in a preferred embodiment performed using a needle gauge of between 20 to 33, such as needle gauge 20, for example needle gauge 21 , such as needle gauge 22, for example needle gauge 23, such as needle gauge 24, for example needle gauge 25, such as needle gauge 26, for example needle gauge 27, such as needle gauge 28, for example needle gauge 29, such as needle gauge 30, for example needle gauge 31 , such as needle gauge 32, for example needle gauge 33. In a particular embodiment, the gauge of the needle is 23.

The fine-needle aspiration may in one embodiment be assisted, such as ultra-sound (US) guided fine-needle aspiration, endoscopic ultra-sound (EUS) guided fine-needle aspiration, Endobronchial ultrasound-guided fine-needle aspiration (EBUS), ultrasonographically guided fine-needle aspiration, stereotactically guided fine-needle aspiration computed tomography (CT)-guided percutaneous fine-needle aspiration and palpation guided fine-needle aspiration.

The sample may in one embodiment be collected in a volume of collection media of between 0.1 ml to 100 ml, such as 0.1 to 0.5 ml, for example 0.5 to 1.0 ml, such as 1.0 to 1.5 ml, for example 1.5 to 2.0 ml, such as 2.0 ml to 3.0 ml, for example 3.0 to 4.0 ml, such as 4.0 ml to 5.0 ml, for example 5.0 to 6.0 ml, such as 6.0 ml to 7.0 ml, for example 7.0 to 8.0 ml, such as 8.0 ml to 9.0 ml, for example 9.0 to 10.0 ml, such as 10 to 15 ml, for example 15 to 20 ml, such as 20 to 30 ml, such as 30 to 40 ml, for example 40 to 50 ml, such as 50 to 60 ml, for example 60 to 70 ml, such as 70 to 80 ml, for example 80 to 90 ml, such as 90 to 100 ml of collection media.

Method for extracting a sample from a collection media

The present invention discloses a method for increasing the yield of RNA, DNA or protein obtained from samples collected in a collection media.

The present invention also discloses a method for efficiently extracting a sample from a collection media in which said sample was collected.

It is thus an aspect of the invention to provide a method for extracting a sample from a collection media, comprising the steps of: a. obtaining a sample from an individual and diverting said sample to a collection media, b. contacting all or part of said collection media comprising the sample with a device comprising a filter section, by diverting the sample in the collection media in a first direction onto and/or into the filter section so that the sample is collected on and/or in the filter section of the device, c. optionally discarding the collection media flow-through while the sample is in contact with the filter section, d. separating the sample and the filter section by diverting the sample from the filter section to a container in a second direction different from the first direction, thereby extracting said sample from said filter section.

In one embodiment, the second direction different from the first direction is the opposite direction of the first direction.

If the whole volume of the collection media comprising a sample is transferred to the device, then steps b) and c) are performed once. If only part of the collection media comprising a sample is transferred to the device, then steps b) and c) may be performed more than once (i.e. they are repeated using the same device before moving on to step d)). For example, if half the volume of the collection media comprising a sample is transferred to the device, then steps b) and c) may be repeated once i.e. performed twice for both half volumes, using the same device twice.

In one embodiment, steps b) and c) are performed more than once. In another embodiment, steps b) and c) are performed twice.

The sample in collection media is diverted in a first direction onto and/or into the filter section so that the sample is collected on the filter section of the device.

In one embodiment, the collection media flow-through is collected in the second tube section. In another embodiment, the collection media flow-through is collected in a microtube.

After the sample is collected from the collection media into and/or onto the filter section, the sample is diverted in a second and opposite direction from the filter section so that the sample is removed from the filter and collected.

In one embodiment, the sample is collected in the first tube section. In another embodiment, the sample is collected in a second microtube. The opposing directions of the sample may be achieved by inverting the filter section and/or at least one tube section. In one embodiment, the filter section is inverted, and the direction of the sample is unaltered. In another embodiment, at least one tube section comprising the filter section is inverted, and the direction of the sample is unaltered.

The opposing directions of the sample may be achieved by reversing the direction of the sample into and/or onto the filter section. Thus, the filter section and/or the tube section are not inverted. In one embodiment, the direction of the sample is changed by reversing the direction of the sample from a first direction to a second opposite direction. In one embodiment, the direction of the sample is reversed between step b) and d) above.

The movement of the sample comprised in the collection media (step b above) or in the filter section (step d above) may be achieved by any suitable means.

In one embodiment, the movement of the sample comprised in the collection media or in the filter section is achieved by centrifugation.

In another embodiment, the movement of the sample comprised in the collection media or in the filter section is achieved by suction.

In another embodiment, the movement of the sample comprised in the collection media or in the filter section is achieved by a partial vacuum or low pressure.

The term movement as used herein may be defined as a flow of a sample and/or a collection media.

The filter containing the sample in step c) above may optionally be washed by applying a suitable volume of liquid through the filter before extracting the sample from the filter. The liquid may be any suitable liquid such as water, saline, buffers, PBS, TBS, TBS-T, PBS-T, or any other known to the skilled person.

In a particular embodiment, the invention is related to a method for extracting a sample from a collection media, comprising the steps of: a. collecting a sample from an individual in a collection media, b. transferring all or part of the collection media of step a) to a device comprising a filter section and at least one tube section, c. centrifuging the device of step b) so that the sample is transferred into and/or onto the filter section of the device, d. discarding the collection media flow-through while the sample is in contact with the filter section, e. inverting the filter of step c) f. centrifuging the tube with the inverted filter comprising the sample to pellet the sample.

In one particular embodiment, an optional step of placing the inverted filter into a clean microtube is added between steps e) and f) in the particular method disclosed above.

In one particular embodiment, an optional step of removing an extended base of the filter is added between steps d) and f).

In one particular embodiment, an optional step of washing the filter is added between steps d) and e).

In another particular embodiment, the invention is related to a method for extracting a sample from a collection media, comprising the steps of: a) collecting a fine-needle aspirate from an individual in an RNA stabilisation solution, b) transferring half the volume of the RNA stabilisation solution comprising a fine- needle aspirate to a device comprising a microtube comprising a 0.45 μm PVDF-filter, c) centrifuging the device of step b) so that the fine-needle aspirate is collected on the filter of the device, d) discarding the collection media flow-through while the fine-needle aspirate is in contact with the filter section, e) transferring the second half volume of the RNA stabilisation solution comprising a sample to the device comprising a microtube comprising a 0.45 μm PVDF- filter as used in step b) f) centrifuging the device of step e) so that the fine-needle aspirate is collected on the filter of the device g) discarding the collection media flow-through while the fine-needle aspirate is in contact with the filter section, h) inverting the filter of step f) and placing it into a clean microtube, i) centrifuging the microtube with the inverted filter comprising the fine-needle aspirate, j) obtaining a pellet comprising the fine-needle aspirate k) extract RNA from the fine-needle aspirate pellet

After the sample is collected by any method disclosed herein, it may be subjected to analysis of any kind, as detailed herein below.

It is also an aspect to provide a method for extracting one or more biological molecules from a biological sample comprising a plurality of biological cells, said method comprising the steps of: i) obtaining a biological sample comprising a plurality of biological cells comprising one or more biological molecules ii) suspending said biological sample in a fluid or liquid composition, iii) diverting said sample suspension to a filter section in a predetermined orientation and contacting said filter section with said plurality of biological cells, iv) separating said biological cells from said filter section by diverting said biological cells to a collection chamber, wherein the biological cells are diverted from the filter section to the collection chamber in an orientation different from the first orientation, and optionally v) extracting one or more biological molecules from the biological sample diverted to the collection chamber.

Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the separation of mixtures. Increasing the effective gravitational force will more rapidly and completely cause a precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is called the "supernate" or "supernatant". The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or g (relative centrifuge force or RCF). The particles' settling velocity in centrifugation is a function of their size and shape, centrifugal acceleration, the volume fraction of solids present, the density difference between the particle and the liquid, and the viscosity.

A centrifuge is a piece of equipment, generally driven by a motor that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration is used to separate substances of greater and lesser density.

Microcentrifuges are small and have rotors than can quickly change speeds. Microcentrifuge tubes generally hold 0.5-2 ml_ of liquid, and are spun at maximum angular speeds of 12000-13000 rpm. Superspeed centrifuges work similarly to microcentrifuges, but are conducted via larger scale processes. These centrifuges are used to purify 25-30 ml_ of solution within a tube, and reach higher angular velocities (around 30000 rpm), and also use a larger rotor. Ultracentrifuges can reach maximum angular velocites in excess of 70000 rpm.

Protocols for centrifugation typically specify the amount of acceleration to be applied to the sample, rather than specifying a rotational speed such as revolutions per minute. The acceleration is often quoted in multiples of g, the standard acceleration due to gravity at the Earth's surface. This distinction is important because two rotors with different diameters running at the same rotational speed will subject samples to different accelerations.

The acceleration can be calculated as the product of the radius and the square of the angular velocity.

Relative centrifugal force is the measurement of the force applied to a sample within a centrifuge. This can be calculated from the speed (RPM) and the rotational radius (cm) using the following calculation. g = RCF = 0.00001 1 18 x r * hf where: g = Relative centrifuge force r= rotational radius (centimetre, cm)

N= rotating speed (revolutions per minute, r/min)

To avoid having to perform a mathematical calculation every time, nomograms for converting RCF to rpm for a rotor of a given radius are readily available. A ruler or other straight edge lined up with the radius on one scale, and the desired RCF on another scale, will point at the correct rpm on the third scale.

In one embodiment, the device according to the present invention is centrifuged at a relative centrifuge force (RCF) of between 1 to 100 RCF; such as 1 to 2 RCF, for example 2 to 3 RCF, such as 3 to 4 RCF, for example 4 to 5 RCF, such as 5 to 6 RCF, for example 6 to 7 RCF, such as 7 to 8 RCF, for example 8 to 9 RCF, such as 9 to 10 RCF, for example 10 to 1 1 RCF, such as 1 1 to 12 RCF, for example 12 to 13 RCF, such as 13 to 14 RCF, for example 14 to 15 RCF, such as 15 to 20 RCF, for example 20 to 25 RCF, such as 25 to 30 RCF, for example 30 to 35 RCF, such as 35 to 40 RCF, for example 40 to 45 RCF, such as 45 to 50 RCF, for example 50 to 60 RCF, such as 60 to 70 RCF, for example 70 to 80 RCF, such as 80 to 90 RCF, for example 90 to 100 RCF.

In another embodiment, the device according to the present invention is centrifuged at a relative centrifuge force (RCF) of 1 RCF, such as 2 RCF, for example 3 RCF, such as 4 RCF, for example 5 RCF, such as 6 RCF, for example 7 RCF, such as 8 RCF, for example 9 RCF, such as 10 RCF, for example 1 1 RCF, such as 12 RCF, for example 13 RCF, such as 14 RCF, for example 15 RCF.

In one embodiment, the device according to the present invention is centrifuged for between 5 seconds to 10 minutes; such as 5 seconds to 15 seconds, for example 15 to 30 seconds, such as 30 to 45 seconds, for example 45 to 60 seconds, such as 1 minute to 1.5 minutes, for example 1.5 to 2 minutes, such as 2 to 2.5 minutes, for example 2.5 to 3 minutes, such as 3 to 3.5 minutes, for example 3.5 to 4 minutes, such as 4 to 4.5 minutes, for example 4.5 to 5 minutes, such as 5 to 5.5 minutes, for example 5.5 to 6 minutes, such as 6 to 6.5 minutes, for example 6.5 to 7 minutes, such as 7 to 7.5 minutes, for example 7.5 to 8 minutes, such as 8 to 8.5 minutes, for example 8.5 to 9 minutes, such as 9 to 9.5 minutes, for example 9.5 to 10 minutes. In another embodiment, the device according to the present invention is centrifuged for 15 seconds, such as 30 seconds, for example 45 seconds, such as 60 seconds, for example 1 minute, such as 1.5 minutes, for example 2 minutes, such as 2.5 minutes, for example 3 minutes, such as 3.5 minutes, for example 4 minutes, such as 4.5 minutes, for example 5 minutes, such as 5.5 minutes, for example 6 minutes, such as 6.5 minutes, for example 7 minutes, such as 7.5 minutes, for example 8 minutes, such as 8.5 minutes, for example 9 minutes, such as 9.5 minutes, for example 10 minutes.

Suction (or vacuum) Suction is the flow of a fluid into a partial vacuum, or region of low pressure. The pressure gradient between this region and the ambient pressure will propel matter toward the low pressure area.

A vacuum is a volume of space that is essentially empty of matter, such that its gaseous pressure is much less than atmospheric pressure. The word comes from the Latin term for "empty," but in reality, no volume of space can ever be perfectly empty. A perfect vacuum with a gaseous pressure of absolute zero is a philosophical concept that is never observed in practice. The quality of a vacuum refers to how closely it approaches a perfect vacuum. The residual gas pressure is the primary indicator of quality, and is most commonly measured in units called torr, even in metric contexts. Lower pressures indicate higher quality, although other variables must also be taken into account. Quantum theory sets limits for the best possible quality of vacuum, predicting that no volume of space can be perfectly empty. Outer space is a natural high quality vacuum, mostly of much higher quality than can be created artificially with current technology. Low quality artificial vacuums have been used for suction for many years. Vacuums are thus commonly used to produce suction. A vacuum pump is a device that removes gas molecules from a sealed volume in order to leave behind a partial vacuum.

Vacuum is measured in units of pressure. The SI unit of pressure is the pascal (symbol Pa), but vacuum is usually measured in torrs, named for Torricelli, an early Italian physicist (1608 - 1647). A torr is equal to the displacement of a millimeter of mercury (mmHg) in a manometer with 1 torr equaling 133.3223684 pascals above absolute zero pressure. Vacuum is often also measured using inches of mercury on the barometric scale or as a percentage of atmospheric pressure in bars or atmospheres. Low vacuum is often measured in inches of mercury (inHg), millimeters of mercury (mmHg) or kilopascals (kPa) below atmospheric pressure. "Below atmospheric" means that the absolute pressure is equal to the current atmospheric pressure (e.g. 29.92 inHg) minus the vacuum pressure in the same units. Thus a vacuum of 26 inHg is equivalent to an absolute pressure of 4 inHg (29.92 inHg - 26 inHg).

Vacuum quality is subdivided into ranges according to the technology required to achieve it or measure it. Atmospheric pressure is variable but standardized at 101 .325 kPa (760 torr). Low vacuum ranges from 760 to 25 torr (100 to 3 kPa), medium vacuum ranges from 25 to 1 χ10^'3 torr (3 kPa to 100 mPa) and high vacuum ranges from 1 χ10^'3 to 1 χ10⁹ torr (10O mPa to 100 nPa).

The device according to the present invention is subjected to a vacuum. In one embodiment the vacuum may be in the range of 760 to 1 χ10^"9 torr, such as 760 to 25 torr, for example 25 to 1 x 10³ torr, such as 1 x 10³ to 1 x 10⁹ torr.

Pressure

Pressure (symbol: p or P) is the force per unit area applied to an object in a direction perpendicular to the surface. Pressure is an effect which occurs when a force is applied on a surface. The symbol of pressure is p (lower case). Pressure is a scalar quantity, and has SI units of pascals; 1 Pa = 1 N/m².

The device according to the present invention is subjected to a pressure. In one embodiment, the pressure may be in the range of 101 .325 Pa 1000 Pa, such as 101 .325 to 200 Pa, for example 200 to 300 Pa, such as 300 to 400 Pa, for example

400 to 500 Pa, such as 500 to 600 Pa, for example 600 to 700 Pa, such as 700 to 800 Pa, for example 800 to 900 Pa, such as 900 to 1000 Pa.

Method for diagnosing The present invention discloses a method for efficiently extracting a sample from a collection media in which said sample was collected. Said method is suited for use in the ex vivo diagnosing of a clinical indication in an individual from which the sample was taken. In one aspect, the present invention relates to a method for performing a diagnosis of a clinical indication in an individual, said method comprising the steps of performing the method for extracting a sample from a collection media according to the present invention, and performing a diagnostic assay on the cells or the biological molecules collected in the collection chamber.

In another aspect, the present invention relates to a method for performing a diagnosis of a clinical indication in an individual, said method comprising the steps of performing the method for extracting one or more biological molecules from a biological sample comprising a plurality of biological cells according to the present invention, and performing a diagnostic assay on the cells or the biological molecules collected in the collection chamber.

Use of sample After the sample is collected according to the present invention, it may be subjected to analysis of any kind.

Extracting RNA, DNA or protein

In one embodiment, the sample may be used for RNA isolation, DNA isolation and/or protein isolation. It follows that one, two or all three components of the sample may be isolated simultaneously.

In one embodiment, the sample is used for isolating DNA according to any conventional methods known in the art.

In one embodiment, the sample is used for isolating protein according to any conventional methods known in the art.

In one preferred embodiment, the sample is used for isolating RNA according to any conventional methods known in the art.

The RNA extracted or isolated from the sample may be total RNA, mRNA, microRNA, tRNA, rRNA or any type of RNA. Conventional methods and reagents for isolating RNA from a sample comprise Trizol (invitrogen), Guanidinium thiocyanate-phenol-chloroform extraction, RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol- chloroform extraction, TCA/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, Pure Yield™ RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell^® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™ Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods, commercially available or not, known to the skilled person.

The RNA may be further cleaned-up, concentrated, DNase treated or subjected to any other post-extraction method known to the skilled person.

In one preferred embodiment, the sample is used for isolating RNA and DNA.

In one preferred embodiment, the sample is used for isolating RNA and protein.

In one preferred embodiment, the sample is used for isolating RNA, DNA and protein.

Analysis of the extracted RNA, DNA or protein

The isolated RNA, DNA and/or protein may in one embodiment be further analysed by any method known in the art, such as by DNA microarray analysis (spotted array or oligonucleotide array), miRNA microarray analysis, quantitative 'real-time' PCR (QPCR), northern blotting, polymerase chain reaction (PCR), agarose gel electrophoresis, reverse transcriptase PCR (RT-PCR), western blotting, southern blotting, dot blotting, ELISA assays, Serial analysis of gene expression (SAGE), ligase chain reaction (LCR), proximity ligation assay, oligonucleotide lligation assay (OLA), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), or a combination of any of the above. Methods for analysing a sample are disclosed in Molecular Cloning, A Laboratory Manual (Sambrook and Russell (ed.), 3^rd edition (2001 ), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.

In one embodiment the DNA microarray analysis is used to detect mRNA (as cDNA after reverse transcription) known as gene expression profiling. The DNA microarray for detection of mRNA may be a commercially available array platform, such as GeneChip Array (Affymetrix), BeadChip Array (lllumina), Geniom® Biochips (Febit Inc.), mRNA Array (Oxford Gene Technology) or any other commercially available array.

In another embodiment, the DNA microarray for detection of mRNA is custom made.

In one embodiment, the microarray analysis is used to detect microRNA, known as microRNA expression profiling.

The microarray for detection of microRNA may be a commercially available array platform, such as miRCURY LNA™ microRNA Arrays (Exiqon), microRNA Array (Agilent), μParaflo^® Microfluidic Biochip Technology (LC Sciences), MicroRNA Profiling Panels (lllumina), Geniom® Biochips (Febit Inc.), microRNA Array (Oxford Gene

Technology), Custom AdmiRNA™ profiling service (Applied Biological Materials Inc.), microRNA Array (Dharmacon - Thermo Scientific), LDA TaqMan analyses (Applied Biosystems), Taqman Low Density Array (Applied Biosystems) or any other commercially available array.

In another embodiment, the microarray for detection of microRNA is custom made.

In another embodiment, the DNA microarray analysis is used to detect DNA (such as Comparative genomic hybridization). In yet another embodiment, the microarray analysis is Chromatin lmmunoprecipitation (ChIP) on Chip (ChIP on Chip), SNP detection, Alternative splicing detection or Genome Tiling array.

A microarray is a multiplex technology that consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides or antisense miRNA, called features, each containing picomoles of a specific sequence. This can be a short section of a gene or other DNA or miRNA element that are used as probes to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of nucleic acid sequences in the target. In standard microarrays, the probes are attached to a solid surface by a covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others). The solid surface can be glass or a silicon chip, in which case they are commonly known as gene chip. DNA arrays are so named because they either measure DNA or use DNA as part of its detection system. The DNA probe may however be a modified DNA structure such as LNA (locked nucleic acid).

Direct analysis

In another embodiment, the collected sample is analysed directly (without extracting RNA, DNA or protein from the sample), i.e. by techniques sush as flow cytometry analysis, FACS, immune cytochemistry, immune histochemistry, in situ hybridisation or any other applicable methods in the art.

Methods for analysing a sample are disclosed in Molecular Cloning, A Laboratory Manual (Sambrook and Russell (ed.), 3^rd edition (2001 ), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.

Device for use in the method of the present invention

In one embodiment, the device of the present invention is a device comprising a detachable filter section and at least one tube section, as detailed herein above.

In one embodiment, the method disclosed herein employs a device comprising a detachable filter section and at least one tube section, as detailed herein above

In another embodiment, the device is a commercially available tube, such as a microtube, comprising a commercially available filter, such as a PVDF filter. Commercially available tubes and filters are specified herein below.

In another embodiment, the method disclosed herein employs a commercially available tube, such as a microtube, comprising a commercially available filter, such as a PVDF filter.

Commercially available tubes

The tube according to the present invention is shaped to have an opening and a closed bottom. A lid is optionally associated with the opening, which may be attached to for example the opening (i.e. a 'safe-lock' tube) or may be separate from the tube (i.e. a 'screw-top' tube). The bottom of the tube may be conical or pointed, round (convex) or flat. In a preferred embodiment, the bottom is conical.

The tube is preferably a microtube, a microfuge tube, a microcentrifuge tube or a micro test tube, optionally with a lid, suitable for centrifugation.

The tube may be made of for example a medical grade polymer such as plastic or glass or any other suitable material. In one embodiment, the tube is made of or comprises transparent plastic. In one particular embodiment, the tube is made from polypropylene.

Tubes that may be used according to the present invention are commercially available. These include Eppendorf® tubes such as safe-lock tubes or screw-cap tubes, Trefflab microcentrifuge tubes (Anachem) such as ClickFit or LockFit cap tubes, or any other commercially available microtube known to the skilled person.

The tube may be of any size, made for containing between 0.1 ml and 100 ml; such as 0.2 ml tubes, 0.5 ml tubes, 1.0 ml tubes, 1 .5 ml tubes, 2.0 ml tubes, 5.0 ml tubes, 10 ml tubes, 15 ml tubes and 50 ml tubes.

The diameter of the tube may be between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

Commercially available filters

The commercially available tube has in its inner cavity inserted a filter for use in the collection of sample from the collection media. The filter, or membrane, may be made of a material such as PVDF (Polyvinylidene Fluoride), Nitrocellulose, Cellulose, Cellulose Acetate (CA), PTFE (Polytetrafluoroethylene), Nylon, PES (Polyethersulfone), MCE (Mixed Cellulose Ester) and Glass fiber (GF). In a preferred embodiment, the filter is a PVDF filter.

The filter, or membrane, may have a pore size of between 0.01 to 5.0 urn, such as 0.01 to 0.02 urn, for example 0.02 to 0.03 urn, such as 0.03 to 0.04 urn, for example 0.04 to 0.05 urn, such as 0.05 to 0.06 urn, for example 0.06 to 0.07 urn, such as 0.07 to 0.08 urn, for example 0.08 to 0.09 urn, such as 0.09 to 0.1 urn, for example 0.1 to 0.2 urn, such as 0.2 to 0.3 urn, for example 0.3 to 0.4 urn, such as 0.4 to 0.5 urn, for example 0.5 to 0.6 urn, such as 0.6 to 0.7 urn, for example 0.7 to 0.8 urn, such as 0.8 to 0.9 urn, for example 0.9 to 1 .0 urn, such as 1.0 to 1.5 urn, for example 1.5 to 2.0 urn, such as 2.0 to 2.5 urn, for example 2.5 to 3.0 urn, such as 3.0 to 3.5 urn, for example 3.5 to 4.0 urn, such as 4.0 to 4.5 urn, for example 4.5 to 5.0 urn. The pore size of the filter is in one particular embodiment 0.22 μm, and in another particular embodiment 0.45 μm.

The diameter of the filter is adjusted according to the inner cavity of the tube and may be between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm. The size of the filter is adjusted so as to enable the insertion into the cavity of the tube; therefore the main part of the filter should have a slightly smaller diameter that the cavity of the tube.

The length of the filter is adjusted according to the inner cavity of the tube and may be between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

The filter may be any shape to be adapted to the tube in which it is inserted. The filter may be cylindrical, that is; circular in its circumference and flat or curved in both ends.

In one embodiment, the end of the filter facing the opening of the tube may have an extended base, which is wider that the inner cavity of the tube, to ensure that the filter is not completely immersed into the tube but retains a portion outside of the tube, resting on the opening of said tube.

In the embodiment where the method disclosed herein employs a commercially available tube, such as a microtube, comprising a commercially available filter, such as a PVDF filter, an optional step of removing an extended base of the filter may be included. The extended base is removed after capturing the sample on the filter of the device, and before inverting the filter in the tube to extract the sample from the filter. The extended base of the filter may be removed by any means, such as by a hot scalpel, a scalpel, scissors, or the filter may be adapted so that it may be removed manually.

Detailed description of the drawings

Figure 1 . RNA Extraction using RNAIater with a Modified Protocol according to the present invention. A Durapore PVDF 0.45 μm filter was inserted into a 1.5 ml

Eppendorf tube and half of the RNAIater-sample (500μl) was added on the filter (1A).

The tube was spun at 8 rcf for 1 minute (1 B). The flow-through was discarded. The procedure was repeated on the same filter with the remaining 500μl of the sample. The top of the filter was cut away with a hot scalpel (1 C) and the remaining filter was inverted, placed in a clean 2 ml Eppendorf tube (1 D) and spun at 8 rcf for 2 minutes

(1 E). Figure 1 provides an example of an illustrated embodiment, and is meant as a non-limiting illustration of the present invention.

Figure 2: Median values of total RNA yield (A) and median 260/280 ratios (B) from single in vivo fine-needle aspirates as obtained according to the present invention. Figure 3: A device comprising a detachable filter section and at least one tube section, A) with an integrated closure unit of the second tube section, B) with a detachably attached closure unit of the second tube section.

Figure 4: A method for efficiently extracting a sample from a collection media by using a device comprising a detachable filter section and at least one tube section. The collection media comprising a sample is added to the first tube section, and contacted with the filter section, thus collecting the sample into and/or onto the filter section and allowing the collection media to pass the filter. To retrieve the sample from the filter, one of three possible steps may be used: A) The filter is inverted and the direction of the sample is unaltered; B) The filter is inverted, the second tube section is removed, a closure unit is added for collecting the sample from the filter and the direction of the sample is unaltered, or C) the direction of the sample is reversed. Figure 4 provides an example of an illustrated embodiment, and is meant as a non-limiting illustration of the present invention. As such, the second tube section of the device used for illustration could also have a detachably attached closure unit. The diversion of the collection media comprising sample and separating the sample from the filter section may be achieved by e.g. centrifugation, suction, vacuum and/or pressure.

Figure 5: Correlations between the Iog2 normalized expression values from the four patients from whom miRNA expression profiles were generated from both fine needle aspirates and corresponding surgical biopsies from the target nodule tissue. Correlation coefficients ranged from 0.84 to 0.91.

Examples

Example 1 : RNA purification from fine-needle aspirates from thyroid nodules

microRNA (miRNA) expression profiling and classification of tissue obtained from fine- needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules. Before this can be implemented in the clinical setting, a robust nontoxic method for obtaining sufficient quantity and quality of RNA from single in vivo FNA has to be established. RNAIater is a non-toxic stabilization agent that preserves RNA. However, due to the high density of RNAIater, pelleting of the tissue samples is difficult, and causes a low recovery of RNA insufficient for subsequent miRNA array expression analyses. We therefore developed a simple centrifugation method for capturing tissue stored in RNAIater on a 0.45 μm filter. FNA from 24 patients with a solitary cold thyroid nodule was stored in Trizol, liquid nitrogen, or RNAIater. The tissue stored in RNAIater was either pelleted by centrifugation or captured on the 0.45 μm filters. RNA was extracted using the Trizol method. Capturing FNA tissue samples on the filters increased the RNA yield 10 fold, maintained RNA pureness, thus permitting microRNA array expression profiling. We recommend this modified RNAIater protocol for isolating RNA from single in vivo FNA in the clinical setting. For validation, we have examined the correlation in miRNA expression profiles between individual in vivo FNA's and their corresponding tumors, removed by subsequent surgery for histopathological diagnosis.

MATERIALS AND METHODS

Patients and in vivo Fine Needle Aspirates. In vivo fine-needle aspirates were collected from 24 consecutive patients with a solitary solid cold thyroid nodule referred to ultrasound-guided FNA at the outpatient clinic at the Department of Endocrinology and Metabolism, Herlev Hospital (Denmark). The inclusion took place in April and May 2008 and patients provided signed, informed consent. For fine-needle aspirates a 23- gauge needle attached to a 2 ml syringe was used. Each procedure comprised three aspirates for routine cytology and a fourth aspirate for our RNA-collecting and - extraction study. The 24 aspirates were randomized to 4 different RNA isolation protocols: RNAIater, RNAIater modified, Snap-frozen and Trizol. After collection, the RNAIater samples were stored and transported at room temperature and protected from sunlight, whereas the samples in Trizol, as well as liquid nitrogen snap frozen samples, were put on dry ice and stored and transported at -8O⁰C. Extractions and miRNA microarray analyses were done at the Department of Clinical Biochemistry, Rigshospitalet (Denmark).

Finally, we collected thyroid nodule tissue from 4 patients from whom FNA's had been obtained prior to surgical treatment at Gentofte Hospital to examine how well the expression profiles based on miRNA from the FNA correlated with those generated from miRNA extracted surgical biopsies from target nodule tissue.

Sample Collecting and RNA Extraction.

1 ) RNA Extraction using RNALater standard protocol: Twelve in vivo fine needle aspirates were immediately washed out in a 2 ml Eppendorf® tube containing 1 ml

RNAIater® RNA Stabilization Reagent (Ambion, Austin, TX), kept at room temperature for up to 10 hours and afterwards stored at + 5⁰C for a maximum of 4 weeks before RNA extraction procedures. The first six of the RNAlater® -samples were handled according to the manufacturers RNAlater extraction protocol (RNAIater®Tissue Collection: RNA Stabilization Solution) followed by isolation of RNA according to the standard TRIzol®-protocol (Invitrogen, Carlsbad, CA). In the remaining six RNAlater® - samples the RNA extraction procedure was performed using our modified protocol as described below.

2) RNA Extraction using RNAlater with Modified Protocol: A Durapore PVDF 0.45 μm filter (Millipore, Billerica, MA) was inserted into a 2.0 ml Eppendorf tube and half of the RNAIater-sample (500μl) was added to the filter (figure 1A). The tube was spun at 8 rcf (relative centrifugal force) for 1 minute (figure 1 B). If remaining liquid was observed on the filter the sample was stirred gently and the spin repeated. The flow-through was discarded. The procedure was repeated on the same filter with the remaining 500μl of the sample. The top of the filter was cut away with a hot scalpel (figure 1 C) and the remaining filter was inverted, placed into a clean 2 ml Eppendorf tube (figure 1 D) and spun down at 8 rcf for 2 minutes (figure 1 E). The inverted filter was removed and the sample subjected to standard RNA isolation according to the TRIzol®-protocol.

3) RNA Extraction using Liquid Nitrogen: After obtaining the in vivo fine needle aspirate it was instantly transferred to an empty Eppendorf tube, frozen in liquid nitrogen and stored at -8O⁰C until RNA isolation following the standard TRIzol®-protocol.

4) RNA Extraction using Trizol®: The in vivo fine-needle aspirate was transferred directly to a closed Eppendorf tube containing 1 ml of Trizol by piercing the cap by the needle and washing the tissue out. Subsequently the tube was placed on ice and transported to a fume hood were the needle was extracted and the tube sealed with an intact cap. Samples were subsequently stored at -8O⁰C until RNA isolation following the standard TRIzol®-protocol.

RNA Extraction from Tumor samples. Surgical biopsies were obtained from tumors during surgery and snap frozen in liquid nitrogen. RNA was subsequently extracted using the standard TrizolΘ-protocol as described above. Total RNA miRNA microarray analysis. Following RNA isolation the quantity was measured on a NanoDrop®ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the samples with a sufficient amount and pureness of total RNA (>500ng and 260/280 ratio > 1.5) were processed further. Around 600 -1 OOOng of total RNA was labeled with the NCode™Rapid miRNA Labeling System according to the manufacturer's protocol (cat. no. MIRLSRPD-20, Invitrogen) and added on the NCode™Human miRNA Microarray V3 (cat.no.MIRAH3-05, Invitrogen). Upon overnight hybridization in a MAUI 4-Bay System (BioMicro Systems, Inc.), the arrays were washed and finally scanned using Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, CA). The resulting images were analyzed with Genepixe Pro 6.0 software (Molecular Devices, Sunnyvale, CA). Artifactual spots were eliminated manually. Image intensities were measured as median of foreground intensities and processed further as described below.

Normalization of individual miRNA expression results

Foreground intensities from the raw data gpr-files were normalized in "R" using the "smida" software package, spatial normalization and dye normalization were performed. Logarithmic transformation of ratios between sample and reference was done. Each sample was determined in triplicate and represented by the median value in the further calculations.

Statistical analyses: Unpaired t-test with Welch's correction for unequal variance was used. For correlations between FNA and corresponding frozen tumors Pearson correlation analysis was used.

RESULTS The total RNA yield from twelve fine needle aspirates from solitary solid cold thyroid nodules either snap frozen in liquid nitrogen or collected directly in Trizol were qualitatively and quantitatively (median = 1950 ng (552-3168) and 380 ng (280-800) respectively) sufficient for further miRNA array analysis (Figure 2A and B). However, while the yield for further array analysis is often sufficient, these methods are either toxic (Trixol) or impractical in the clinical routine (Liquid nitrogen). Preservation of FNA samples in RNAIater therefore seemed as a good alternative, but the total RNA yield from six fine needle aspirates obtained from solitary solid cold thyroid nodules, preserved in RNAIater and using the recommended protocol from the manufacturer, were qualitatively and quantitatively (median = 165 ng (40-340)) insufficient for miRNA expression analyses (Figure 2). However, after using the present filter method on tissue recovered in RNAIater, the total RNA yield (median = 1998 ng (728-5088)) was significantly higher compared to the original RNALater protocol (Figure 2A) with no difference in 260/280 ratios (Figure 2B). The quality of the miRNA microarray analysis based on the median signal-to-noise ratios ((F635median-B635median)/B635 SD) gave acceptable results, which did not differ significantly between protocols. The acceptability threshold for the signal-to-noise ratio according to the GenePixPro6 Array Quality Report is 10 and our median values in the three different RNA-collecting groups were 12.1 in the RNAIater modified protocol, 12.6 in the Snap Frozen group, and 8.6 in the group where RNA was recovered in Trizol.

Finally we correlated the expression profiles obtained on the fine needle aspirates with the profiles generated from the corresponding biopsy target nodule tissue obtained after surgery. This produced correlation coefficients from 0.84 to 0.91 (see Figure 5) which demonstrates that the miRNA profiles generated from single pass in vivo FNA are comparable to those obtained after surgery on the biopsy target tissue. Prior investigations have shown that FNA's have an overall accuracy rate around 75 to 80% in detecting thyroid malignancy when one compared the FNA assessment with the definitive histological diagnosis. This level of accuracy is well compatible with the present level of agreement, which was good, but not complete.

CONCLUSION

The results presented herein show that miRNA array analyses can be successfully generated from single in vivo fine-needle aspirates from thyroid nodules using the present modified RNAIater protocol. miRNA array analysis adds a new diagnostic possibility to the current panel of diagnostic tools, and miRNA array analysis on larger biopsies has already proven valuable in molecular tumor diagnosis in several types of malignancies (2, 3). In order to apply a molecular diagnostic tool in a daily clinical setting the tissue stabilization media has to be non-toxic (patient, nurse and operator). This makes Trizol and liquid nitrogen less attractive when dealing with a large number of biopsies as in a thyroid outpatient clinic. The modified RNAIater protocol as used herein permits miRNA microarray profiling from a single fine-needle aspirate obtained in an expedient way and with fair agreement with the profile of the biopsy target tissue, which is essential for improved molecular diagnosis of thyroid nodules prior to a possible operation. Reference List

1. Bartel,D.P.2OO4. microRNAs: genomics, biogenesis, mechanism, and function. Cell 1 16:281 -297.

2. Rosenfeld,N., R.Aharonov, E.Meiri, S.Rosenwald, Y.Spector, M.Zepeniuk, H.Benjamin, N.Shabes, et al.2008. microRNAs accurately identify cancer tissue origin. Nat. Biotechnol. 26:462-469.

3. Lu,J., G.Getz, E.A.Miska, E.varez-Saavedra, J. Lamb, D. Peck, A.Sweet-Cordero, B.L.Ebert, et al.2005. microRNA expression profiles classify human cancers. Nature 435:834-838. 4. Yu,S.L, H.Y.Chen, G.C.Chang, C.Y.Chen, H.W.Chen, S.Singh, C.L.Cheng, C.J.Yu, et al.2008. microRNA signature predicts survival and relapse in lung cancer. Cancer Cell 18213:48-57.

5. Bloomston,M., W.L.Frankel, F.Petrocca, S.Volinia, H.AIder, J.P.Hagan, C.G.Liu, D.Bhatt, et al.2007. microRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297:1901 - 1908.

6. Lubitz,C.C, S.K.Ugras, J.J.Kazam, B.Zhu, T.Scognamiglio, Y.T.Chen, and T.J.Fahey, III. 2006. Microarray analysis of thyroid nodule fine-needle aspirates accurately classifies benign and malignant lesions. J. MoI. Diagn. 8:490-498. 7. Catrina,R.F., E.J.Schlette, J.M.Stewart, M.J.Keating, R.LKatz, and N.P.Caraway. 2007. Fine-needle aspiration biopsy findings in patients with small lymphocytic lymphoma transformed to hodgkin lymphoma. Am. J. Clin. Pathol. 128:571 -578.

8. Dunmire,V., CWu, W.F.Symmans, and W.Zhang. 2002. Increased yield of total RNA from fine-needle aspirates for use in expression microarray analysis. Biotechniques 33:890-2,894, 896.

9. Sotiriou,C, T.J.Powles, M.Dowsett, A.A.Jazaeri, A.L.Feldman, LAssersohn, C.Gadisetti, S.K.Libutti, and E.T.Liu. 2002. Gene expression profiles derived from fine needle aspiration correlate with response to systemic chemotherapy in breast cancer. Breast Cancer Res. 4:R3. 10. Chen,Y.T., N.Kitabayashi, X.K.Zhou, T.J.Fahey, III, and T.Scognamiglio. 2008. microRNA analysis as a potential diagnostic tool for papillary thyroid carcinoma. Mod.

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11. Szafranska,A.E., M.Doleshal, H. S. Edmunds, S. Gordon, J.Luttges, J.B.Munding,

R.J.Barth, Jr., E.J.Gutmann, et al.2008. Analysis of microRNAs in Pancreatic Fine- Needle Aspirates Can Classify Benign and Malignant Tissues. Clin. Chem. 12. Lui.P.C., P.P.Lau, G.M.Tse, P.H.Tan, R.C.Lo, V.W.Tang, W.K.Ng, A.Somali, et al.2007. Fine needle aspiration cytology of invasive micropapillary carcinoma of the breast. 207Pathology 39:401 -405.

13. Kandil,D., G.Leiman, M.AIIegretta, W.Trotman, L.Pantanowitz, R.Goulart, and M.Evans. 2007. Glypican-3 immunocytochemistry in liver fine-needle aspirates : a novel stain to assist in the differentiation of benign and malignant liver lesions. Cancer 1 1 1 :316-322.

14. Yassa,L, E.S.Cibas, C.B.Benson, M.C.Frates, P.M.Doubilet, A.A.Gawande, F.D.Moore, Jr., B.W.Kim, et al.2007. Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation. Cancer 1 1 1 :508-516.

15. Ustun,M., B.Risberg, B.Davidson, and A.Berner. 2002. Cystic change in metastatic lymph nodes: a common diagnostic pitfall in fine-needle aspiration cytology. Diagn. Cytopathol. 27:387-392.

Claims

Claims

1. A device comprising a detachable filter section and at least one tube section, wherein the filter section is detachably attached to a first tube section having an elongated shape and a proximal opening and a distal opening, and the filter section is detachably attached to a second tube section having an elongated shape and a proximal opening and an optionally detachably attached distal closure unit.

2. The device of claim 1 , wherein the filter section comprises an annular filter.

3. The device of claim 1 , wherein the filter section comprises a flat, annular filter.

4. The device of claim 1 , wherein the distal closure unit of the second tube section is an integrated part of the second tube section.

5. The device of claim 1 , wherein the distal opening of the first tube section is closable by a closure unit.

6. The device of claim 5, wherein the closure unit is a detachable closure unit.

7. The device according to claim 1 , wherein the first and second tube sections are made of or comprise one or more of the materials selected from the group consisting of Biodegradable plastic, Bioplastics obtained from biomass e.g. from pea starch or from biopetroleum, Polypropylene (PP), Polystyrene (PS), High impact polystyrene (HIPS), Acrylonitrile butadiene styrene (ABS), Polyethylene terephthalate (PET), Polyester (PES), Fibers, textiles, Polyamides (PA), (Nylons), Polyvinyl chloride) (PVC), Polyurethanes (PU), Polycarbonate (PC), Polyvinylidene chloride (PVDC) (Saran), Polyvinylidene Fluoride (PVDF),

Polyethylene (PE), Polymethyl methacrylate (PMMA), Polytetrafluoroethylene (PTFE) (trade name Teflon), Fluorinated ethylene propylene (FEP), Polyetheretherketone (PEEK) (Polyetherketone), Polyetherimide (PEI) (Ultem), Phenolics (PF), (phenol formaldehydes), Perfluoroalkoxy (PFA), Poly(methyl methacrylate) (PMMA), Urea-formaldehyde (UF), Melamine formaldehyde (MF), Polylactic acid and Plastarch material or any mixture thereof.

8. The device according to claim 7, wherein the first and second tube sections are made of or comprise polypropylene.

9. The device according to claim 1 , wherein the first and second tube sections each holds a volume of between 0.1 ml and 100 ml; such as 0.1 to 1 ml, for example 1 to 2 ml, such as 2 to 3 ml, for example 3 to 4 ml, such as 4 to 5 ml, for example 5 to 6 ml, such as 6 to 7 ml, for example 7 to 8 ml, such as 8 to 9 ml, for example 9 to 10 ml, such as 10 to 1 1 ml, for example 1 1 to 12 ml, such as 12 to 13 ml, for example 13 to 14 ml, such as 14 to 15 ml, for example 15 to 20 ml, such as 20 to 25 ml, for example 25 to 30 ml, such as 30 to 35 ml, for example 35 to 40 ml, such as 40 to 50 ml, for example 50 to 60 ml, such as 60 to 70 ml, for example 70 to 80 ml, such as 80 to 90 ml, for example 90 to 100 ml.

10. The device according to claim 1 , wherein the diameter of the first and second tube sections are between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example

3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

1 1. The device according to claim 1 , wherein the length of the first and second tube sections from the proximal end to the distal end are between 0.5 cm mm to 20 cm, such as 0.5 to 1 cm, for example 1 to 2 cm, such as 2 to 3 cm, for example 3 to 4 cm, such as 4 to 5 cm, for example 5 to 6 cm, such as 6 to 7 cm, for example 7 to 8 cm, such as 8 to 9 cm, for example 9 to 10 cm, such as 10 to 1 1 cm, for example 1 1 to 12 cm, such as 12 to 13 cm, for example 13 to 14 cm, such as 14 to 15 cm, for example 15 to 16 cm, such as 16 to 17 cm, for example 17 to 18 cm, such as 18 to 19 cm, for example 19 to 20 cm.

12. The device according to claim 1 , wherein the filter section comprises a material such as PVDF (Polyvinylidene Fluoride), Nitrocellulose, Cellulose, Cellulose

Acetate (CA), PTFE (Polytetrafluoroethylene), Nylon, PES (Polyethersulfone), MCE (Mixed Cellulose Ester) and Glass fiber (GF).

13. The device according to claim 12, wherein the filter section comprises PVDF.

14. The device according to claim 1 , wherein the filter section comprises an annular filter.

15. The device according to claim 1 , wherein the filter section comprises a flat filter.

16. The device according to claim 1 , wherein the filter section comprises a flat, annular filter.

17. The device according to claim 1 , wherein the filter section has a pore size of between 0.01 to 5.0 urn, such as 0.01 to 0.02 urn, for example 0.02 to 0.03 urn, such as 0.03 to 0.04 urn, for example 0.04 to 0.05 urn, such as 0.05 to 0.06 urn, for example 0.06 to 0.07 urn, such as 0.07 to 0.08 urn, for example 0.08 to 0.09 urn, such as 0.09 to 0.1 urn, for example 0.1 to 0.2 urn, such as 0.2 to 0.3 urn, for example 0.3 to 0.4 urn, such as 0.4 to 0.5 urn, for example 0.5 to 0.6 urn, such as 0.6 to 0.7 urn, for example 0.7 to 0.8 urn, such as 0.8 to 0.9 urn, for example 0.9 to 1.0 urn, such as 1.0 to 1.5 urn, for example 1 .5 to 2.0 urn, such as 2.0 to 2.5 urn, for example 2.5 to 3.0 urn, such as 3.0 to 3.5 urn, for example 3.5 to 4.0 urn, such as 4.0 to 4.5 urn, for example 4.5 to 5.0 urn.

18. The device according to claim 1 , wherein the filter section has a pore size of

0.22 μm.

19. The device according to claim 1 , wherein the filter section has a pore size of

0.45 μm

20. The device according to claim 1 , wherein the diameter of the filter section is between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as

13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

21 . The device according to claim 1 , wherein the length of the filter section is between 1 mm to 10 cm, such as 1 mm to 2 mm, for example 2 to 3 mm, such as 3 to 4 mm, for example 4 to 5 mm, such as 5 to 6 mm, for example 6 to 7 mm, such as 7 to 8 mm, for example 8 to 9 mm, such as 9 to 10 mm, for example 10 to 1 1 mm, such as 1 1 to 12 mm, for example 12 to 13 mm, such as 13 to 14 mm, for example 14 to 15 mm, such as 15 to 16 mm, for example 16 to 17 mm, such as 17 to 18 mm, for example 18 to 19 mm, such as 19 to 20 mm, for example 2 cm to 2.5 cm, such as 2.5 cm to 3 cm, for example 3.5 to 4 cm, such as 4 to 4.5 cm, for example 4.5 to 5 cm, such as 5 to 6 cm, for example 6 to 7 cm, such as 7 to 8 cm, for example 8 to 9 cm, such as 9 to 10 cm.

22. A kit-of-parts comprising a device according to claim 1 and a collection media.

23. The kit of claim 22 wherein the collection media is an RNA stabilisation solution.

24. The kit of claim 23 wherein the RNA stabilisation solution is a commercially available RNA stabilisation solution.

25. The kit of claim 24 wherein the RNA stabilisation solution is selected from the group consisting of RNAIater® (Ambion and Qiagen), PreservCyt medium (Cytyc Corp), PrepProtect™ Stabilisation Buffer (Miltenyi Biotec), Allprotect Tissue Reagent (Qiagen) and RNAprotect Cell Reagent (Qiagen).

26. The kit-of-parts of claim 22 further comprising a needle for fine-needle aspiration of a sample from an individual.

27. A method for extracting a sample from a collection media, comprising the steps of: a. obtaining a sample from an individual and diverting said sample to a collection media, b. contacting all or part of said collection media comprising the sample with a device comprising a filter section, by diverting the sample in the collection media in a first direction onto and/or into the filter section so that the sample is collected on and/or in the filter section of the device, c. optionally discarding the collection media flow-through while the sample is in contact with the filter section, d. separating the sample and the filter section by diverting the sample from the filter section to a container in a second direction different from the first direction, thereby extracting said sample from said filter section.

28. The method of claim 27, wherein the collection media is an RNA stabilisation solution

29. The method of claim 28, wherein the RNA stabilisation solution is a commercially available RNA stabilisation solution.

30. The method of claim 29, wherein the RNA stabilisation solution is selected from the group consisting of RNAIater® (Ambion and Qiagen), PreservCyt medium

(Cytyc Corp), PrepProtect™ Stabilisation Buffer (Miltenyi Biotec), Allprotect Tissue Reagent (Qiagen) and RNAprotect Cell Reagent (Qiagen).

31. The method of claim 27, wherein steps b) and c) are performed more than once.

32. The method of claim 27, wherein steps b) and c) are performed twice.

33. The method of claim 27, wherein the second direction is the opposite direction of the first direction.

34. The method of claim 27, wherein the sample is kept in collection media for between 15 minutes and 100 years prior to collecting the sample from said collection media, such as between 15 minutes and 1 hour, for example 1 to 2 hours, such as 2 to 5 hours, for example 5 to 10 hours, such as 10 to 24 hours, for example 24 hours to 48 hours, such as 48 to 72 hours, for example 72 to 96 hours, such as 4 to 7 days, such as 1 week to 2 weeks, such as 2 to 4 weeks, such as 4 weeks to 1 month, such as 1 month to 2 months, for example 2 to 3 moths, such as 3 to 4 months, for example 4 to 5 moths, such as 5 to 6 months, for example 6 to 7 moths, such as 7 to 8 months, for example 8 to 9 moths, such as 9 to 10 months, for example 10 to 1 1 moths, such as 1 1 to 12 months, for example 1 year to 2 years, such as 2 to 3 years, for example 3 to 4 years, such as 4 to 5 years, for example 5 to 6 years, such as 6 to 7 years, for example 7 to 8 years, such as 8 to 9 years, for example 9 to 10 years, such as 10 to 20 years, for example 20 to 30 years, such as 30 to 40 years, for example

40 to 50 years, such as 50 to 75 years, for example 75 to 100 years prior to collecting the sample from said collection media.

35. The method of claim 27, wherein the sample is kept in collection media at a temperature of between -8O⁰C to 37⁰C, such as between -80 to -4O⁰C, for example -40 to O⁰C, such as 0 to 5⁰C, for example 5 to 1 O⁰C, such as 10 to 15⁰C, for example 15 to 2O⁰C, such as 20 to 25⁰C, for example 25 to 3O⁰C, such as 30 to 37⁰C prior to collecting the sample from said collection media.

36. The method of claim 35, wherein the sample is kept in collection media during collection, storage and/or transportation of said sample in collection media.

37. The method of claim 27 wherein the movement of the sample comprised in the collection media or in the filter section is achieved by centrifugation.

38. The method of claim 37 wherein the device is centrifuged at a relative centrifuge force (RCF) of between 1 to 100 RCF; such as 1 to 2 RCF, for example 2 to 3 RCF, such as 3 to 4 RCF, for example 4 to 5 RCF, such as 5 to 6 RCF, for example 6 to 7 RCF, such as 7 to 8 RCF, for example 8 to 9 RCF, such as 9 to 10 RCF, for example 10 to 1 1 RCF, such as 1 1 to 12 RCF, for example 12 to 13 RCF, such as 13 to 14 RCF, for example 14 to 15 RCF, such as 15 to 20 RCF, for example 20 to 25 RCF, such as 25 to 30 RCF, for example 30 to 35 RCF, such as 35 to 40 RCF, for example 40 to 45 RCF, such as 45 to 50 RCF, for example 50 to 60 RCF, such as 60 to 70 RCF, for example 70 to 80 RCF, such as 80 to 90 RCF, for example 90 to 100 RCF.

39. The method of claim 37 wherein the device is centrifuged at a relative centrifuge force (RCF) of 1 RCF, such as 2 RCF, for example 3 RCF, such as 4 RCF, for example 5 RCF, such as 6 RCF, for example 7 RCF, such as 8 RCF, for example 9 RCF, such as 10 RCF, for example 1 1 RCF, such as 12 RCF, for example 13 RCF, such as 14 RCF, for example 15 RCF.

40. The method of claim 37 wherein the device is centrifuged for between 5 seconds to 10 minutes; such as 5 seconds to 15 seconds, for example 15 to 30 seconds, such as 30 to 45 seconds, for example 45 to 60 seconds, such as 1 minute to 1.5 minutes, for example 1.5 to 2 minutes, such as 2 to 2.5 minutes, for example 2.5 to 3 minutes, such as 3 to 3.5 minutes, for example 3.5 to 4 minutes, such as 4 to 4.5 minutes, for example 4.5 to 5 minutes, such as 5 to 5.5 minutes, for example 5.5 to 6 minutes, such as 6 to 6.5 minutes, for example 6.5 to 7 minutes, such as 7 to 7.5 minutes, for example 7.5 to 8 minutes, such as 8 to 8.5 minutes, for example 8.5 to 9 minutes, such as 9 to 9.5 minutes, for example 9.5 to 10 minutes.

41 . The method of claim 37 wherein the device is centrifuged for 15 seconds, such as 30 seconds, for example 45 seconds, such as 60 seconds, for example 1 minute, such as 1 .5 minutes, for example 2 minutes, such as 2.5 minutes, for example 3 minutes, such as 3.5 minutes, for example 4 minutes, such as 4.5 minutes, for example 5 minutes, such as 5.5 minutes, for example 6 minutes, such as 6.5 minutes, for example 7 minutes, such as 7.5 minutes, for example 8 minutes, such as 8.5 minutes, for example 9 minutes, such as 9.5 minutes, for example 10 minutes.

42. The method of claim 27, wherein the movement of the sample comprised in the collection media or in the filter section is achieved by suction.

43. The method of claim 27, wherein the movement of the sample comprised in the collection media or in the filter section is achieved by a partial vacuum or low pressure.

44. The method of claim 43, wherein the vacuum is in the range of 760 to 1 χ10^'9 torr, such as 760 to 25 torr, for example 25 to 1 χ10^~3 torr, such as 1 χ10^~3 to 1 x10⁹ torr.

45. The method of claim 27, wherein the movement of the sample comprised in the collection media or in the filter section is achieved by pressure.

46. The method of claim 43, wherein the pressure is in the range of 101.325 Pa 1000 Pa, such as 101.325 to 200 Pa, for example 200 to 300 Pa, such as 300 to 400 Pa, for example 400 to 500 Pa, such as 500 to 600 Pa, for example 600 to 700 Pa, such as 700 to 800 Pa, for example 800 to 900 Pa, such as 900 to

1000 Pa.

47. The method of claim 27, wherein the sample comprises cells and/or tissue.

48. The method of claim 27, wherein the sample is from an animal.

49. The method of claim 27, wherein the sample is from a human being.

50. The sample of claim 47 wherein the sample comprises eukaryotic or prokaryotic cells.

51 . The sample of claim 50 wherein the sample comprises mammalian cells, bacteria cells, fungus cells or yeast cells.

52. The method of claim 27, wherein the sample comprises virus particles.

53. The method of claim 27, wherein the sample is taken from a cancer selected from the group consisting of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, AIDS-Related Cancers, Anal Cancer, Astrocytoma (e.g. Childhood Cerebellar or Childhood Cerebral), Basal Cell Carcinoma, Extrahepatic Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumor, Breast Cancer, Male Breast Cancer, Bronchial Adenomas/ Carcinoids, Burkitt's Lymphoma, Carcinoid Tumor, Carcinoma of Unknown Primary, Primary Central Nervous System Lymphoma, Cerebral Astrocytoma/ Malignant Glioma, Cervical Cancer, Childhood Cancers,

Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Chronic Myeloproliferative Disorders, Colon Cancer, Cutaneous T-CeII Lymphoma, Endometrial Cancer, Ependymoma (such as Childhood Ependymoma), Esophageal Cancer, Ewing's Family of Tumors, Extracranial Germ Cell Tumor (such as Childhood Extracranial Germ Cell Tumor), Extragonadal Germ Cell

Tumor, Eye Cancer (Intraocular Melanoma or Retinoblastoma), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gestational Trophoblastic Tumor, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver) Cancer, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma (such as Childhood

Hypothalamic and Visual Pathway Glioma), Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Lung Cancer (Non- Small Cell or Small Cell), Lymphoma (such as AIDS-Related Lymphoma, Burkitt's Lymphoma, Cutaneous T-CeII Lymphoma, Non-Hodgkin's Lymphoma),

Macroglobulinemia (such as Waldenstrom's Macroglobulinemia), Malignant Fibrous Histiocytoma of Bone/ Osteosarcoma, Medulloblastoma (such as Childhood Medulloblastoma), Melanoma, Merkel Cell Carcinoma, Mesothelioma (such as Adult Malignant Mesothelioma or childhood Mesothelioma), Metastatic Squamous Neck Cancer with Occult Primary, Multiple Endocrine Neoplasia

Syndrome (such as occurring in childhood), Multiple Myeloma/ Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/ Myeloproliferative Diseases, Myeloma (such as Multiple Myeloma), Chronic myeloproliferative disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer (such as Childhood Nasopharyngeal Cancer),

Neuroblastoma, Oropharyngeal Cancer, Osteosarcoma/ Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (such as Childhood Ovarian Cancer), Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Pituitary Tumor, Pleuropulmonary Blastoma, Prostate Cancer, Renal Pelvis and Ureter Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma (such as Childhood Rhabdomyosarcoma), Salivary Gland Cancer, Adult-onset soft tissue Sarcoma, Soft Tissue Sarcoma (such as Childhood Soft Tissue Sarcoma),

Uterine Sarcoma, Sezary Syndrome, Skin Cancer (such as non-Melanoma skin cancer), Merkel Cell Skin Carcinoma, Small Intestine Cancer, Supratentorial Primitive Neuroectodermal Tumors (such as occurring in Childhood), Cutaneous T-CeII Lymphoma, Testicular Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and

Ureter, Trophoblastic Tumor (such as Gestational Trophoblastic Tumor), Urethral Cancer, Endometrial Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma (such as Childhood Visual Pathway and Hypothalamic Glioma), Waldenstrom's Macroglobulinemia and Wilms' Tumor.

54. The method of claim 27, wherein the sample is taken from thyroid cancer, thyroid nodules, breast cancer, breast nodules, pancreatic cancer, pancreatic nodules, liver cancer, liver nodules and processes of unknown origin.

55. The method of claim 27, wherein the sample is taken from a tissue selected from the group consisting of the nervous system, the musculoskeletal system, the circulatory system, the respiratory system, the gastrointestinal system, the integumentary system, the urinary system, the reproductive system, the immune system and the endocrine system.

56. The method of claim 27, wherein the sample is taken from muscle, bone, bone marrow, ligaments, tendons, skin, hair, nails, sweat glands, sebaceous glands, liver, pancreas, spleen, kidney, bladder, urethra, ureters, heart, lungs, nasopharynx, trachea, stomach, esophagus, intestine, mouth, rectum, gall bladder, salivary glands, breast, testis, ovary, uterus, cerebrospinal fluid, blood, thyroid gland, parathyroid gland, adrenal gland, thymus, lymph nodes, lymph channels, pituitary, cerebellum, cerebrum, spinal cord, eyes, ears, tongue and nose.

57. The method of claim 27, wherein the sample is taken by using fine-needle aspiration (FNA); core needle aspiration; cutting biopsy; open biopsy; or any other means known to the person skilled in the art.

58. The method of claim 27, wherein the sample is taken by using fine-needle aspiration (FNA).

59. The method of claim 58 wherein the fine-needle aspiration (FNA) sample is a single fine-needle aspiration sample.

60. The method of claim 58 wherein the fine-needle aspiration is performed using a needle with a diameter of between 0.2 to 1.0 mm, such as 0.2 to 0.3 mm, for example 0.3 to 0.4 mm, such as 0.4 to 0.5 mm, for example 0.5 to 0.6 mm, such as 0.6 to 0.7 mm, for example 0.7 to 0.8 mm, such as 0.8 to 0.9 mm, for example 0.9 to 1.0 mm in diameter.

61 . The method of claim 58 wherein the fine-needle aspiration is performed using a needle gauge of between 20 to 33, such as needle gauge 20, for example needle gauge 21 , such as needle gauge 22, for example needle gauge 23, such as needle gauge 24, for example needle gauge 25, such as needle gauge 26, for example needle gauge 27, such as needle gauge 28, for example needle gauge 29, such as needle gauge 30, for example needle gauge 31 , such as needle gauge 32, for example needle gauge 33. In a particular embodiment, the gauge of the needle is 23.

62. The method of claim 58 wherein the fine-needle aspiration is assisted, such as ultra-sound (US) guided fine-needle aspiration, endoscopic ultra-sound (EUS) guided fine-needle aspiration, Endobronchial ultrasound-guided fine-needle aspiration (EBUS), ultrasonographically guided fine-needle aspiration, stereotactically guided fine-needle aspiration computed tomography (CT)- guided percutaneous fine-needle aspiration and palpation guided fine-needle aspiration.

63. The method of claim 27, wherein the sample is collected from an in vitro cell culture.

64. The method of claim 27, wherein the sample is collected in a volume of collection media of between 0.1 ml to 100 ml, such as 0.1 to 0.5 ml, for example 0.5 to 1.0 ml, such as 1.0 to 1 .5 ml, for example 1 .5 to 2.0 ml, such as 2.0 ml to 3.0 ml, for example 3.0 to 4.0 ml, such as 4.0 ml to 5.0 ml, for example 5.0 to

6.0 ml, such as 6.0 ml to 7.0 ml, for example 7.0 to 8.0 ml, such as 8.0 ml to 9.0 ml, for example 9.0 to 10.0 ml, such as 10 to 15 ml, for example 15 to 20 ml, such as 20 to 30 ml, such as 30 to 40 ml, for example 40 to 50 ml, such as 50 to 60 ml, for example 60 to 70 ml, such as 70 to 80 ml, for example 80 to 90 ml, such as 90 to 100 ml of collection media.

65. The method of claim 27, wherein the extracted sample is used for isolation of RNA, DNA and/or protein.

66. The method of claim 27, wherein the extracted sample is used for isolation of

RNA.

67. The method of claim 27, wherein the extracted sample is used for isolation of DNA.

68. The method of claim 27, wherein the extracted sample is used for isolation of protein.

69. The method of claim 27, wherein the extracted sample is used for isolation of RNA and DNA.

70. The method of claim 27, wherein the extracted sample is used for isolation of RNA and protein.

71. The method of claim 27, wherein the extracted sample is used for isolation of

RNA, DNA and protein.

72. The method of claim x wherein the isolated RNA, DNA or protein is used for further analysis.

73. The method of claim 72 wherein the further analysis comprises DNA microarray analysis (spotted array or oligonucleotide array), miRNA microarray analysis, quantitative 'real-time' PCR (QPCR), northern blotting, polymerase chain reaction (PCR), agarose gel electrophoresis, reverse transcriptase PCR (RT- PCR), western blotting, southern blotting, dot blotting, ELISA assays, Serial analysis of gene expression (SAGE), ligase chain reaction (LCR), proximity ligation assay, oligonucleotide lligation assay (OLA), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), or a combination of any of the above.

74. The method of claim 73 wherein the DNA microarray analysis is used to detect mRNA known as gene expression profiling.

75. The method of claim 74 wherein the DNA microarray for detection of mRNA is a commercially available array platform, such as GeneChip Array (Affymetrix),

BeadChip Array (lllumina), Geniom® Biochips (Febit Inc.), mRNA Array (Oxford Gene Technology) or any other commercially available array.

76. The method of claim 73 wherein the microarray analysis is used to detect microRNA, known as microRNA expression profiling.

77. The method of claim 76 wherein the microarray for detection of microRNA is a commercially available array platform, such as miRCURY LNA™ microRNA Arrays (Exiqon), microRNA Array (Agilent), μParaflo^®Microfluidic Biochip Technology (LC Sciences), MicroRNA Profiling Panels (lllumina), Geniom®

Biochips (Febit Inc.), microRNA Array (Oxford Gene Technology), Custom AdmiRNA™ profiling service (Applied Biological Materials Inc.), microRNA Array (Dharmacon - Thermo Scientific), LDA TaqMan analyses (Applied Biosystems), Taqman Low Density Array (Applied Biosystems) or any other commercially available array.

78. The method of claim 27 wherein the extracted sample is analysed directly.

79. The method of claim 78 wherein the collected sample is analysed by flow cytometry analysis, FACS, immune cytochemistry, immune histochemistry or in situ hybridisation.

80. A method for extracting a sample from a collection media, comprising the steps of: a. collecting a sample from an individual in a collection media, b. transferring all or part of the collection media of step a) to a device comprising a filter section and at least one tube section, c. centrif uging the device of step b) so that the sample is transferred into and/or onto the filter section of the device, d. discarding the collection media flow-through while the sample is in contact with the filter section, e. inverting the filter of step c) f . centrif uging the tube with the inverted filter comprising the sample to pellet the sample.

81. The method of claim 80 wherein an optional step of placing the inverted filter into a clean microtube is added between steps e) and f).

82. The method of claim 80 wherein an optional step of removing an extended base of the filter is added between steps d) and f).

83. The method of claim 80 wherein an optional step of washing the filter is added between steps d) and e).

84. A method for extracting a sample from a collection media, comprising the steps of: a. collecting a fine-needle aspirate from an individual in an RNA stabilisation solution, b. transferring half the volume of the RNA stabilisation solution comprising a fine-needle aspirate to a device comprising a microtube comprising a 0.45 μm PVDF-filter, c. centrifuging the device of step b) so that the fine-needle aspirate is collected on the filter of the device, d. discarding the collection media flow-through while the fine-needle aspirate is in contact with the filter section, e. transferring the second half volume of the RNA stabilisation solution comprising a sample to the device comprising a microtube comprising a 0.45 μm PVDF-filter as used in step b) f. centrifuging the device of step e) so that the fine-needle aspirate is collected on the filter of the device g. discarding the collection media flow-through while the fine-needle aspirate is in contact with the filter section, h. inverting the filter of step f) and placing it into a clean microtube, i. centrifuging the microtube with the inverted filter comprising the fine- needle aspirate, j. obtaining a pellet comprising the fine-needle aspirate k. extract RNA from the fine-needle aspirate pellet

85. A method for performing a diagnosis of a clinical indication in an individual, said method comprising the steps of performing the method for extracting a sample from a collection media according to claim 27, and performing a diagnostic assay on the cells or the biological molecules collected in the collection chamber.