WO2019056060A1 - Appareil, kit et méthode de prélèvement de cellules - Google Patents

Appareil, kit et méthode de prélèvement de cellules Download PDF

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Publication number
WO2019056060A1
WO2019056060A1 PCT/AU2018/051019 AU2018051019W WO2019056060A1 WO 2019056060 A1 WO2019056060 A1 WO 2019056060A1 AU 2018051019 W AU2018051019 W AU 2018051019W WO 2019056060 A1 WO2019056060 A1 WO 2019056060A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell sampling
support structure
sampling component
cell
sampling apparatus
Prior art date
Application number
PCT/AU2018/051019
Other languages
English (en)
Inventor
Robyn Alice LINDLEY
Original Assignee
Gmdx Co Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2017903814A external-priority patent/AU2017903814A0/en
Application filed by Gmdx Co Pty Ltd filed Critical Gmdx Co Pty Ltd
Publication of WO2019056060A1 publication Critical patent/WO2019056060A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0096—Casings for storing test samples
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0045—Devices for taking samples of body liquids
    • A61B2010/0061—Alimentary tract secretions, e.g. biliary, gastric, intestinal, pancreatic secretions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • A61B2010/0216—Sampling brushes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B42/00—Surgical gloves; Finger-stalls specially adapted for surgery; Devices for handling or treatment thereof
    • A61B42/10—Surgical gloves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention generally relates to an apparatus, kit and method for safely collecting samples of exfoliated cells, for example from the colorectal mucosal surface of a subject, for DNA or RNA extraction and sequencing.
  • Diagnostic testing procedures typically involve the collection of a sample from a patient, the extraction of one or more cell types of interest from the sample, and analysis of the extracted cells to infer a clinical status of the patient. These steps may vary widely according to the test under consideration.
  • faecal tests such as faecal occult blood test (FOBT), faecal immunochemical test (FIT) and stool DNA or RNA analysis.
  • FOBT faecal occult blood test
  • FIT faecal immunochemical test
  • stool DNA or RNA analysis a sample of cells
  • faecal tests are presently the screening method of choice.
  • Other methods of screening for colorectal cancer include full or partial structural exams such as colonoscopy, flexible sigmoidoscopy (FS), and imaging techniques including computed tomographic colonography (CTC) and double contrast barium enema (DCBE).
  • CTC computed tomographic colonography
  • DCBE double contrast barium enema
  • Colorectal screening using a colonoscopy procedure involves clinical risks and significant costs, and a large number of patients are found to have no abnormal pathology. There is also a cumulative risk of complications, especially if applied in elderly patients or repeatedly performed for screening purposes. Concerns about cost, required specialist skill and patient radiation exposure are also present for imaging techniques such as CTC and DCBE.
  • Exfoliation of colonocytes i.e. the spontaneous detachment of cells from the epithelial layer of the colonic mucosa
  • Their collection and use for cytological testing is well known in the art and has shown that morphologically distinct exfoliated neoplastic cells could be detected in colorectal cancer patients.
  • the mucocellular layer covering the human rectal mucosa is rich in exfoliated colonocytes.
  • the number appears to be much higher than in healthy individuals, primarily due to greatly increased presence of malignant colonocytes.
  • Clinical observations describing distal (e.g. anal) implantation of persisting exfoliated cells from removed colorectal tumours strongly corroborate this hypothesis.
  • DNA or RNA analyses of the colonocytes will be able to provide a direct indication of the likely presence (or not) of pre-cancerous and cancerous conditions.
  • Current methods for obtaining samples of rectal tissue or colonocytes are physically invasive and involve the use of a range of surgical instrument attachments which carry a risk of perforation, unless the user has considerable specialist expertise.
  • the most commonly used method of obtaining samples is to attach a brush to the end of a colonoscopic instrument, with a torch also being attached to the instrument in order to illuminate the area to be sampled from.
  • This method requires the involvement of a specialist surgeon, the use of general anaesthetic, and bowel preparation by the patient.
  • there is a risk of material loss and serious contamination with other tissue elements which would have an adverse effect on DNA sequencing.
  • the present invention seeks to alleviate one or more of the above difficulties, or at least to provide a useful alternative to existing cell sampling kits and methods.
  • a first aspect of the present disclosure relates to a cell sampling apparatus, comprising: a support structure; and
  • a cell sampling component removably attached to the support structure, the cell sampling component having an exfoliation portion
  • the support structure is manipulable by a user to harvest cells by the exfoliation portion from a mucous membrane of a subject.
  • the support structure may be a glove, and the cell sampling component may be removably attached to a forefinger of the glove, for example near the tip of the forefinger.
  • the apparatus may be used to sample cells from a subject, and the cell sampling component then removed such that it can be immediately placed in a stabilisation buffer in a sealed tube for downstream analyses on the harvested cells, such as DNA or RNA analysis. Cells can therefore be sampled in a simple, highly efficient and non-intrusive way.
  • the cell sampling component may be removably attached to the support structure by a flexible polymer layer.
  • the cell sampling apparatus comprises a liner removably attached to the exfoliation surface.
  • the liner may be attached during manufacture of the apparatus, for example, and left in place to ensure that the exfoliation surface remains free of contamination until a user is ready to sample cells from a subject.
  • the exfoliation portion comprises a plurality of bristles, hooks or loops. These may be made of nylon, for example. Bristles may act as a capillary system in order to draw cells from the surface being sampled.
  • the exfoliation portion is at least partly hydrophobic.
  • the exfoliation portion may be at least partially formed from, or be coated with, a hydrophobic material.
  • an at least partially hydrophobic exfoliation portion may facilitate the release of the harvested cells into the stabilising buffer once the cell sampling component is immersed.
  • the present disclosure relates to a cell sampling kit, comprising:
  • a sample collection tube containing a stabilising buffer for receiving the cell sampling component when removed from the support structure.
  • the present disclosure relates to a method of collecting cells from a subject using the cell sampling kit, comprising:
  • the mucous membrane may be rectal mucosa, for example.
  • Figure 1 shows an embodiment of a cell sampling apparatus
  • Figure 2 shows a sample collection tube suitable for use with the cell sampling apparatus as part of a cell sampling kit
  • Figure 3(a) shows a side-on view of part of the cell sampling apparatus of Figure 1, prior to use in a cell sampling method
  • Figure 3(b) is similar to the view of Figure 3(a), but shows the cell sampling apparatus with a protective liner removed;
  • Figure 4 is a partial exploded view of an alternative embodiment of a cell sampling apparatus.
  • FIGS. 5(a) through 5(d) schematically depict steps of an exemplary cell sampling method. Detailed description
  • kits used for the collection of sampling exfoliated colonocytes from the rectal mucosa of a human subject, and have considerable advantages over other kits used for this purpose.
  • the invention may have application in many other contexts, including sampling cells from other locations such as the oral mucosa or cervical mucosa, or from various mucosal surfaces of non-human subjects.
  • the kit, apparatus and method of the present invention may be employed during a surgical procedure in order to collect cells for DNA sequencing.
  • the cell sampling apparatus 10 is in the form of a glove, which may be made from a plastics or rubber material, for example.
  • the glove 10 has a forefinger 11 which carries a cell sampling component generally indicated at 14.
  • the cell sampling component 14 is removably attached to the forefinger 11 of glove 10. This can be by means of one or more patches or strips of a flexible polymer, for example.
  • the cell sampling component 14 has disposed thereon a liner 12, which may be formed from a paper, polymer or polymer composite material, which may be transparent or translucent, and which is releasably attached to the cell sampling component 14.
  • the liner 12 may have a portion that is attached to the cell sampling component 14, and an unattached portion, generally indicated at 16, which is not attached to the cell sampling component 14 or to the glove 10, such that the unattached portion 16 can be easily lifted free of the glove 10 to release the liner 12, whereby the surface of the cell sampling component 14 is exposed.
  • the unattached portion 16 may carry indicia 17 to indicate to a user that the user should pull the unattached portion 16 to release the liner 12.
  • the purpose of liner 12 is to ensure that the surface of cell sampling component 14, which is typically sterilised prior to packaging, remains free of contamination.
  • the glove 10 may be packaged together with a sample collection tube 20 to form a single-use, sterile cell sampling kit.
  • the sample collection tube 20 has a tube body 22 sealed by a cap 24, and contains a buffer solution 28.
  • the tube body 22 may carry a label 26.
  • the buffer solution 28 is a stabilising buffer solution suitable for stabilising colonocytes prior to DNA or RNA extraction, by inhibiting microbial activity that occurs following any rectal cell sampling.
  • Any suitable DNA/RNA stabilising buffer may be used, such as DNA/RNA ShieldTM of Zymo Research, or BuccalFixTM of Isohelix, for example.
  • the stabilising buffer may also contain a lysing agent, such that it also acts as a lysis buffer.
  • the cell sampling component 14 comprises a backing layer 32 from which extends a plurality of bristles 34, which may be formed from nylon, for example. Bristles 34 may be integral with the backing layer 32 or may be permanently attached to backing layer 32 in any suitable fashion, for example by stapling, sewing, or adhesives.
  • the bristles 34 extend substantially across the plane of the cell sampling component 14, and can be brushed across a mucous membrane to harvest cells, such as exfoliated colonocytes, therefrom. The bristles 34 therefore constitute an exfoliation portion of the cell sampling component 14.
  • the soft nylon bristles 34 constitute a capillary system that facilitates the collection of colonocytes without damage.
  • the cell sampling component 14, or at the very least the bristles 34 may be made from a hydrophobic material, or have a hydrophobic coating applied to it or them. This facilitates the release of colonocytes collected by the bristles 34 when the cell sampling component 14 is immersed in a surrounding stabilising buffer fluid 28.
  • the design also reduces the chance of perforation or tissue damage while collecting colonocytes.
  • the fibres of bristles 34 may have thicknesses in the range 20 ⁇ to 200 ⁇ , and may have lengths in the range 100 ⁇ to 5 mm, for example.
  • the cell sampling component 14 is protected by a liner 12 having an unattached portion (tab) 16 that can be pulled away from the glove 10 to expose the exfoliation portion (bristles) 34, as shown in Figure 3(b).
  • the liner 12 is attached to the exfoliation portion by a thin layer of adhesive 42.
  • the liner 12 is bonded electrostatically or thermally to the exfoliation portion.
  • the liner 12 may envelop the exfoliation portion, such as by wrapping around and being adhered to the forefinger 11 of glove 10.
  • the underside of the backing layer 32 is bonded to the glove surface by a flexible polymer layer 46, such that the backing layer 32 (and thus the cell sampling component 14) does not come free of the glove 10 while cells are being collected.
  • the flexible polymer may be applied in one or more patches or strips in liquid or semi-liquid formed, and then cured in any suitable fashion to bond the backing layer 32 to the forefinger 11 of glove 10.
  • the flexible polymer of layer 46 may be any adhesive which adheres to both the glove 10 and the material of backing layer 32, yet is peelable such that the backing layer 32 can be removed to detach the cell sampling component 14.
  • Suitable polymers may be polyethylene, polyester, Teflon, epoxy, or polyurethane-based, for example.
  • the backing layer 32 may have at least a portion 50 that is not bonded to the glove 10, such that portion 50 forms a small tab that can be gripped to peel off the cell sampling component 14 to place it into the collection tube 20.
  • the flexible polymer layer 46 may bond more strongly to the material of glove 10 than to the material of backing layer 32.
  • the exfoliating portion includes a plurality of loops 60 of a hook-and-loop fastener.
  • the liner 12' carries a plurality of hooks 62 that can mate with loops 60 to attach the liner 12' to the backing layer 32.
  • the loops 60 may be formed from a similar material to bristles 34, and may have substantially the same thickness such that they harvest cells in substantially the same way when brushed over a mucous membrane.
  • the exfoliating portion also forms part of the mechanism for attaching the liner 12' to the backing layer 32, and the liner 12' is able to remain firmly in place whilst also being readily removed (via pulling tab 16') when the cell sampling device is ready for use.
  • the placement of hooks and loops can be reversed, such that the hooks 60 are carried on backing layer 32, and loops 62 are carried on liner 12'.
  • each of the backing layer 32 and liner 12' may carry a mixture of hooks 62 and loops 60.
  • the hooks 62 and loops 60 may have fibre thicknesses and lengths in the same respective ranges as for bristles 34.
  • the cell sampling component 14 is attached to a support structure in the form of a glove.
  • the support structure may be a (flexible or inflexible) handle, rod or the like, in which case the cell sampling component 14 need not be substantially planar as shown in the Figures, but may be substantially cylindrical, or may include a central linear part from which the bristles 34 (for example) radiate.
  • the support structure may be a sleeve which is adapted to slide over a finger of a standard glove worn by a user.
  • the support structure may be sized and shaped such that both it and the cell sampling component can be inserted directly into a sample collection tube.
  • a method of sampling cells is illustrated schematically.
  • a user dons the glove 10 and grasps tab 16 of liner 12 to pull it away from the forefinger 1 1 to expose the cell sampling component 14.
  • the user introduces forefinger 11 into the rectum of a subject 70 such that the exfoliation portion 34 of cell sampling component 14 contacts the anterior rectal wall.
  • the user gently scrapes the cell sampling component 14 against the anterior rectal surface, about 4-6 cm past the sphincter, to collect rectal mucosa containing exfoliated colonocytes, and removes the forefinger 11 from the rectum.
  • the user grasps tab 50 of backing layer 32, and pulls it away from the forefinger 11 to remove the cell sampling component 14 from the glove.
  • the entire cell sampling component 14 (backing layer 32 and exfoliation portion formed by bristles 34, for example) is placed into a rigid container containing a DNA or RNA fixative buffer solution 28, such that at least the exfoliation portion is immersed in the buffer 28.
  • the rigid container is sealed by cap 24 and labelled (26), and can be stored until DNA or RNA is required to be extracted from the lysate (if the buffer is a lysis/stabilisation buffer) for sequencing.
  • the collected cells isolated can also be used for both further molecular or biochemical analysis and cytological investigation (to look for tumour cells with specific morphological features) in colorectal cancer patients.
  • each of the components of the cell sampling kit including all components of the glove 10, will be disposable. Accordingly, after the cell sampling component 14 is stored in the sample collection tube 20, the remaining components may be discarded.
  • the sample can readily be screened using standard techniques, without the need for any additional isolation steps, such that bacterial or stool contaminants are disregarded during the library preparation before sequencing.
  • standard procedures for screening saliva prior to DNA sequencing may be used.
  • Embodiments of the invention may be useful for sampling cells for a variety of purposes, including:
  • At least some embodiments of the cell sampling kit, apparatus and method described herein have one or more of the following advantages:
  • gloves of varying sizes may be provided in order to accommodate different users, and the size and shape of the cell sampling component 14 may also vary.
  • Many different materials may be used to fabricate the bristles 34 or hooks 62 and loops 60.
  • a number of different buffer solutions may be used as a fixative.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medical Informatics (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Un appareil de prélèvement de cellules (10) comprend une structure de support (11), et un composant de prélèvement de cellules (14) fixé de façon amovible à la structure de support, le composant de prélèvement de cellules ayant une partie d'exfoliation (34) ; la structure de support pouvant être manipulée par un utilisateur pour récolter des cellules par la partie d'exfoliation à partir d'une muqueuse d'un sujet. Le composant de prélèvement de cellule (14) est fixé de manière amovible à un index du gant (11) et comprend un revêtement (12) fixé de manière amovible à la surface d'exfoliation.
PCT/AU2018/051019 2017-09-19 2018-09-18 Appareil, kit et méthode de prélèvement de cellules WO2019056060A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2017903814 2017-09-19
AU2017903814A AU2017903814A0 (en) 2017-09-19 RECTAL CELL SAMPLER A method and kit for collecting a sample of exfoliated colonocytes collected directly from the surface of the rectal mucosa of a subject, and wherein the collected colonocytes are introduced to a buffer solution in a sealable rigid container to form a lysate for obtaining a high DNA or RNA yield after extraction.

Publications (1)

Publication Number Publication Date
WO2019056060A1 true WO2019056060A1 (fr) 2019-03-28

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ID=65810121

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2018/051019 WO2019056060A1 (fr) 2017-09-19 2018-09-18 Appareil, kit et méthode de prélèvement de cellules

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WO (1) WO2019056060A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643232A (en) * 1996-01-19 1997-07-01 James P Villotti Jr PAP smear glove
US7938776B2 (en) * 2008-03-18 2011-05-10 Mederistic Solutions, Inc. Examination device for blood detection
US20110179887A1 (en) * 2008-02-15 2011-07-28 Cobian Paul J Sample acquisition device
US8690767B2 (en) * 2005-06-22 2014-04-08 Speculus (Uk) Limited Speculum
WO2016178189A1 (fr) * 2015-05-06 2016-11-10 Caldbeck Overseas Limited Dispositif d'échantillonnage clinique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643232A (en) * 1996-01-19 1997-07-01 James P Villotti Jr PAP smear glove
US8690767B2 (en) * 2005-06-22 2014-04-08 Speculus (Uk) Limited Speculum
US20110179887A1 (en) * 2008-02-15 2011-07-28 Cobian Paul J Sample acquisition device
US7938776B2 (en) * 2008-03-18 2011-05-10 Mederistic Solutions, Inc. Examination device for blood detection
WO2016178189A1 (fr) * 2015-05-06 2016-11-10 Caldbeck Overseas Limited Dispositif d'échantillonnage clinique

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