WO2013111025A1 - Flow through device for staining and/or analyzing a biological sample - Google Patents

Flow through device for staining and/or analyzing a biological sample Download PDF

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Publication number
WO2013111025A1
WO2013111025A1 PCT/IB2013/050142 IB2013050142W WO2013111025A1 WO 2013111025 A1 WO2013111025 A1 WO 2013111025A1 IB 2013050142 W IB2013050142 W IB 2013050142W WO 2013111025 A1 WO2013111025 A1 WO 2013111025A1
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WO
WIPO (PCT)
Prior art keywords
sample
biological sample
liquid reagent
conduit
staining
Prior art date
Application number
PCT/IB2013/050142
Other languages
French (fr)
Inventor
David Halter
Roland Cornelis Martinus VULDERS
Gwenola Sabatte
Robertus Leonardus Maria IN'T GROEN
Jacob Marinus Jan Den Toonder
Markus Laubscher
Original Assignee
Koninklijke Philips N.V.
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.)
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Publication date
Application filed by Koninklijke Philips N.V. filed Critical Koninklijke Philips N.V.
Publication of WO2013111025A1 publication Critical patent/WO2013111025A1/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1095—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices for supplying the samples to flow-through analysers

Definitions

  • the invention relates to a flow through device for staining and/or analyzing a biological sample.
  • Cancer arising from cervix is the number one cancer in women in many developing countries. About 30% of cancers in women are due to cervical cancer with more than 100,000 new cases diagnosed every year, e.g., in India. The estimated compounded annual growth rate (CAGR) for cervical cancer cases is 2.56% and at this growth rate 174,001 new cases of cervical cancer will be detected in the year 2012.
  • CAGR compounded annual growth rate
  • One of the recommended tools for screening for cervical cancer is to detect cyto logical precursors of cancer in Papanicolaou tests (also called PAP smear, PAP test, cervical smear, or smear test), which is a screening test used in gynecology to detect premalignant and malignant processes in the endo cervical canal.
  • Papanicolaou tests also called PAP smear, PAP test, cervical smear, or smear test
  • a speculum is used to gather cells from the outer opening of the cervix of the uterus and the endocervix.
  • the cells are examined under a microscope to look for abnormalities.
  • the test aims to detect potentially pre-cancerous changes, which are usually caused by sexually transmitted human papillomaviruses.
  • the test remains an effective, widely used method for early detection of pre-cancer and cervical cancer.
  • the test may also detect infections and abnormalities in the endocervix and endometrium.
  • a flow through device for staining and/or analyzing a biological sample comprising a support for the biological sample, and a conduit in fluid communication with the support to which at least one liquid reagent can be delivered in such way that a biological sample provided on the support engages and/or interacts with the at least one liquid reagent.
  • liquid reagent relates to a reagent which is either liquid as such, or solved in a liquid solvent to obtain the liquid form.
  • the latter can apply both for gaseous and solid reagents, which are thus brought into the liquid form.
  • the reagents can also be provided in dried form which is then solubilized on the fly prior to use, thus avoiding the oxidation and light degradation.
  • biological sample is defined as a biological or body fluid sample or a biological tissue sample.
  • biological or body fluid samples include lymph, blood, saliva, cervical fluid, cervicovaginal fluid, vaginal fluid, synovial fluid, semen, stool, sputum, mucus, lung sputum and lavage or samples derived therefrom (e.g., reagent-modified and/or fractionated samples).
  • samples derived therefrom e.g., reagent-modified and/or fractionated samples.
  • samples which consist of cells taken up in a liquid e.g., a buffer and/or preservation liquid, in order to make a cell suspension.
  • Fluid samples can be collected with a swab.
  • exemplary swab samples include cervicovaginal swab samples, including, but not limited to swab of the point of a possible cervicovaginal lesion, the cervical canal, the cervical os, the ectocervix, the transition zone on the cervix between squamous and columnar cells (i.e., the squamo columnar junction), the vagina, the posterior fornix, the portion of the vagina below the posterior fornix such as the lower third of the vagina, the labia, or combinations thereof.
  • Biological tissue samples are samples containing an aggregate of cells, usually of a particular kind, together with intercellular substances that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues.
  • biological tissue samples also include organs (e.g., breasts), tumors, lymph nodes, arteries and individual cells.
  • the sample can be a tissue sample suspected of being cancerous.
  • the device according to the invention has particular advantages over the prior art. First, it avoids the artifacts caused by subsequent dipping of the sample into different reagents, as it is currently performed both in manual and automated processes of, e.g., PAP staining. Further, it avoids the manual handling of liquid reagents, which can be toxic, and thus reduces the risk of intoxication and/or environmental pollution.
  • the device can be built up as a closed system, it can work independently of environmental conditions (humidity, pollution), and thus provides reproducible results under different environmental conditions, e.g. in points of care where there is no air conditioning, or no clean laboratory space.
  • environmental conditions humidity, pollution
  • the system can be built up as a self sustained system, and thus work independent of laboratory periphery and/or infrastructure. This makes the device suitable for use in regions with poor infrastructure, where access to laboratories and/or access to qualified personnel is absent.
  • the staining process can by highly automated, in order to avoid artifacts caused by faulty operation through poorly trained and/or overworked personnel. Further, the staining process can be highly standardized, in order to make analytic results more reproducible and comparable.
  • the device can be integrated, e.g., combined with a magnifying unit (e.g., a microscope), in order to increase its independence from local periphery and/or infrastructure. Further, the device can be combined with an automatic image processing and analysis device, in order to increase reproducibility and comparability of the analytic results.
  • a magnifying unit e.g., a microscope
  • staining process can be observed in real time, in order to either modify or adapt the process, if needed (e.g., extend exposure to a given stain in case it turns out, under real time control, that a particular sample has unexpectedly poor staining behavior), or to carry a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dye (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counter stain like a cytoplasmic dye).
  • a first dye e.g., a nuclear dye
  • a second dye e.g., a counter stain like a cytoplasmic dye
  • the device further comprises at least one pumping means for the delivery of at least one liquid reagent to the conduit, at least one valve and/or one manifold, and/or at least one storage means for providing at least one liquid reagent.
  • Pumping means can be, e.g., Syringe pumps (also called syringe drivers), i.e. small infusion pumps comprising a syringe as storage and delivery means.
  • Syringe pumps also called syringe drivers
  • a peristaltic pump which is a type of positive displacement pump used for pumping a variety of fluids.
  • Such pump is, e.g., a syringe pump or a peristaltic pump that either pulls or pushes liquid reagents through the conduit.
  • Storage means can be, e.g., canisters bearing the respective liquid reagents, or syringes (in said case the pumping and storing functionality is in the same device).
  • the storage means can be wells, or cavities provided on the flow through device, or microreaction vessels (e.g., of the Eppendorf type).
  • the storage means can also comprise dried reagents which are solubilized prior to use.
  • the device comprises one pumping means connected by suitable valves and/or manifolds, to at least two storage means comprising different liquid reagents, or their dried counterparts.
  • the device comprises at least two pumping means which are directly connected to respective storage means comprising different liquid reagents. In this case, the at least two pumping means are connected to one another by means of at least one suitable valve and/or manifold.
  • the device further comprises a control unit for controlling the at least one pumping means, at least one valve and/or at least one manifold.
  • the controlling means can thus be used to establish a given staining protocol, namely by controlling pumping speed, and pumping duration of the different liquid reagents, as e.g. shown in Fig. 5.
  • the at least one liquid reagent is selected from the group consisting of buffers, stains, dyes, washing solutions, and/or fixatives.
  • the at least one liquid reagent is selected from the group consisting of a nuclear stain, a first counterstain, and/or a second counterstain.
  • the nuclear stain comprises at least one selected from the group of Carmine, Methylene blue, Neutral/To luylene red, Haematoxylin, Safranin and/or Nile blue
  • the said counterstains are preferably, cytoplasmic stains.
  • the cytoplasm stain comprises at least one selected from the group of Eosin, Alician blue, Xylidine Ponceau, Biebrich scarlet, Tartazine, Van Gieson's stain (Picric Acid and Acid Fuchsin) and/or Wright stain.
  • stain combinations can be used, as for example comprised in the so called Pap staining mixture, and which comprises a combination of haematoxylin, Orange G, Eosin Y, Light Green SF yellowish, and sometimes Bismarck Brown Y.
  • Masson's trichrome which comprises Weigert's hematoxylin, acid fuchsin, Xylidine Ponceau, phosphomolybdic acid, and Light Green SF yellowish, or alternatively Fast Green FCF, methyl blue, water blue or aniline blue.
  • Lillie's trichrome which is similar to Masson's trichrome, but uses Biebrich scarlet instead of acid fuchsin and/or Xylidine Ponceau.
  • fluorescent stains like DAPI (4',6-diamidino-2-phenylindole) can also be used.
  • stain combinations which are commercially available can also be used, like the HCS cell maskTM staining kits provided by Invitrogen.
  • the nuclear stain serves for staining the nuclei of cells comprised in the sample when the sample is deposited on said first sample support means, or covered by said first sample support means.
  • the cytoplasm stain serves for staining the cytoplasm of cells comprised in the sample when the sample is deposited on said second sample support means, or covered by said second sample support means.
  • the first counterstain is OG-6 counterstain, in which
  • Orange G and phosphotungstic acid are used ("-6" denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8).
  • the second counterstain is EA (Eosin Azure) counterstain, which comprises three dyes, namely Eosin Y (stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells), Light Green SF yellowish (stains the cytoplasm of other cells, including non-keratinized squamous cells and is sometimes replaced by Fast Green FCF) and Bismarck brown Y.
  • EA Eosin Azure counterstain
  • Eosin Azure comprises three dyes, namely Eosin Y (stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells), Light Green SF yellowish (stains the cytoplasm of other cells, including non-keratinized squamous cells and is sometimes replaced by Fast Green FCF) and Bismarck brown Y.
  • Other dyes which can be used include, but are not restricted to Giemsa, Mallory, Bouein, Van de Gieson, Ziehl-Neelsen, Dieterle's Stain, Diff-Quik Stain, Grocott's Methenamine Silver (GMS) Stain, Mayer's Mucicarmine Stain, Sayeed's Stain, Warthin- Starry Stain, Gomori's One-Step Trichrome Stain, Masson's Trichrome Stain, Russel-Movat Pentachrome Stain, Oil Red O and Sudan Black B Stains, Lendrum's Method (Picro-Mallory Stain), Perls' Prussian Blue Stain, the dyes used in the Gram staining protocol, the Ziehl- Neelsen staining procedure, the Masson's trichrome procedure and Leishman stains.
  • Giemsa Mallory, Bouein, Van de Gieson, Zi
  • the support for the biological sample is a slide that can be disposed on a carrier providing the conduit to which at least one liquid reagent can be delivered.
  • the biological sample can be deposited on said slide, which in turn is then disposed on said carrier.
  • the conduit is in fluid communication with the support, the sample can be treated, in a sequential manner, by fixing, staining, washing and the like in flow through mode.
  • the carrier providing the conduit to which at least one liquid reagent can be delivered is a microfluidic device.
  • a microfluidic device is a device in which fluids are manipulated under the rules of microfluidics, i.e. geometrically constrained to a small, typically sub-millimeter, scale, with small volumes (nl or pi scale) and small geometric sizes of conduits and channels ( ⁇ to mm scale).
  • microfluidic devices are manufactured with technologies taken from the semiconductor industry, like photolithographic etching and the like; microfluidic devices may also be produced using polymer processing based methods such as injection molding or hot embossing.
  • microfluidic device transportation of liquids can be carried out by capillary forces alone, or by micropumps, e.g., piezoelectronic pumps, pumps based on pneumatically actuated valves, or pumps based on magnetic microbeads. Valves in such devices can for example adopt the type of a hydrophobic valve.
  • Microfluidic devices have a range of advantages, among which are small volumes of reagents, small overall size, ease of transportation, easy manufacture in high numbers and low costs, small laboratory size (if at all) required and so forth. Therefore, the microfluidic device according to the preferred embodiment can represent a "laboratory on a chip". In such case, the flow through device according to the present invention is independent of periphery and/or local infrastructure.
  • the support for the biological sample and/or the carrier providing the conduit are arranged in such way that they can be placed on the stage of an optical magnifying device.
  • the device further comprises an optical magnifying device.
  • optical magnifying device which allows optical inspection of the sample by a skilled person, or automatic image analysis by means of a dedicated image capturing/processing device and method, is preferably a microscope capable of creating a magnification of between > 20 and ⁇ 1000 x. Further, such magnifying device is preferably capable of using epi-illumination, transmitted illumination or fluorescence.
  • the support and/or the carrier are made from glass or transparent plastic, which is particularly useful in case transmitted illumination is used.
  • the device further comprises an image acquisition/processing device.
  • an image acquisition/processing device is preferably a CCD (linear or two dimensional) or a CMOS (linear or two dimensional) image acquisition device combined with an image processor comprising the respective software and hardware.
  • the analysis of the sample e.g., for absence or presence of abnormal cells, can be carried our automatically, e.g., without intervention of medical or trained personnel.
  • the latter embodiment thus allows a complete automation of the sample treatment and analysis process, further, this embodiment can increase reproducibility and comparability of the analytic results.
  • the image acquisition/processing device is preferably mounted on a magnifying device, e.g., a microscope. Such image acquisition/processing device can, for example, carry out the following steps:
  • step el selecting a new field of view and carrying on with step c) if, in step d), it turns out that, in said field of view, the determination of cell aggregates is negative, or
  • step e2) carrying out further process steps if, in step d), it turns out that, in said field of view, the determination of cell aggregates is affirmative.
  • the system comprises a data storage (e.g., a hard disk, or a flash storage) and/or electronic communication means (e.g., a modem, or a Bluetooth, WiFi, GSM, GPRS or 3G interface).
  • a data storage e.g., a hard disk, or a flash storage
  • electronic communication means e.g., a modem, or a Bluetooth, WiFi, GSM, GPRS or 3G interface.
  • the analysis results can be stored on said data storage for later use, e.g., by medical personnel, or the results can even be sent, electronically, to a service unit for later use, e.g., by medical personnel.
  • the device can even be remotely operated, or controlled in real time, by medical personnel using the electronic communication means.
  • the biological sample is selected from the group consisting of smear sample, tissue slice, liquid sample, and/or other cytology samples.
  • a smear sample is for example similar or identical to those samples commonly used a pap test, cervical smear, or smear test.
  • Such sample can be obtained, e.g., by inserting a speculum into a body cavity, e.g., the woman's vagina to obtain access to the cervix.
  • a sample of cells is then collected, e.g., from the outer opening or of the cervix by scraping it with a spatula, which comprises an endocervical brush which is rotated in the central opening of the cervix.
  • the cells obtained are then placed on a glass slide by "smearing" them over the slide with help of said brush.
  • a tissue slice is for example provided by a microtome.
  • a liquid sample can preferably consist of a suspension of cells, e.g., obtained by a smear.
  • FNAC fine needle aspiration cytology
  • abrasive cytology samples and/or exfoliated samples.
  • a sample is indeed placed on a slide to make it available for investigation, e.g. a tissue slice, or a smear.
  • abnormalities relates to cells which are in a process of becoming cancerous, or malignant ("premalignant"), or are cancerous, or malignant, already.
  • Such cells can differ from normal cells, e.g., in one of the following features cell nucleus size, or area regularity of shape of a cell, and/or a cell nucleus, size ratio of cytoplasm to nucleus, in a given cell, and/or ratio of areas of cytoplasm and nucleus, in a given cell.
  • the method further comprises the step of disposing a biological sample on the support.
  • the method further comprises the step of treating the sample with at least one liquid reagent by means of delivering said liquid reagent to the sample through the conduit.
  • the method further comprises carrying out a histological staining protocol with at least two liquid reagents delivered to the sample in a sequential manner.
  • the method further comprises carrying out a PAP test staining protocol.
  • the method further comprises the analysis of the treated sample by means of optical inspection by a skilled person and/or automatic image analysis by means of a dedicated image processing device and method. Either at the end of the staining process, or in different process steps, e.g., to carry out a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dye (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counterstain like a cytoplasm dye).
  • the optical inspection is preferably carried out by means of a magnifying device, e.g., a microscope.
  • the automatic image analysis is preferably carried out by an image acquisition/processing device as described above, the latter being preferably mounted on a magnifying device, e.g., a microscope.
  • the method further comprises carrying out, or recommending further investigation by, at least one step selected from the group consisting of
  • Colposcopy is a medical diagnostic procedure to examine an illuminated, magnified view of the cervix and the tissues of the vagina and vulva. Primarily in order to detect premalignant lesions and malignant lesions which may result in cancer. Colposcopy is done using a colposcope, which provides an enlarged view of the areas, allowing the colposcopist to visually distinguish normal from abnormal appearing tissue and take directed biopsies for further pathological examination. The main goal of colposcopy is to prevent cervical cancer by detecting precancerous lesions early and treating them.
  • a system for optical examination of the cervix is used for said purpose, said system comprising optical magnification means, illumination means, dispensing means for administration of at least one stimulation and/or contrasting agent, imaging means and image processing means.
  • HPV DNA test detects cervical infection with human papilloma virus (HPV), which is one of the most important infectious causes of cervical cancer. 84% of new cervical cancers were in the developing world, compared with about 50% of all new cancers. HPV DNA test kits are today commercially available. Such test may be carried out during a routine smear test, as described above (in which case part of the smear sample is taken for the HPV DNA test, while another part is taken for the method according to the invention), or with a newly taken sample with comparable properties, and can be used to improve, confirm or falsify the diagnostic significance of the method according to the invention.
  • Biomarker tests have been developed to investigate whether or not a patient suspected to be predisposed for cervical cancer, or a patient who is suspected for having cervical cancer, or in which cervical cancer has already been diagnosed, has, in its genome or proteome, an abnormality which coincides with increased or decreased likelihood of getting a given cancer, or which coincides with increased or decreased responsiveness towards a given therapy.
  • abnormality is, for example, a mutation in a given gene, an abnormality in an epigenomic feature, like DNA methylation, or an abnormality with respect to expression of a given gene.
  • the method further comprises a step of readjusting at least one method step under optical inspection and/or automatic image analysis.
  • the staining process can be observed in real time, in order to either modify or adapt the process, if needed (e.g., extend exposure to a given stain in case it turns out, under real time control, that a particular sample has unexpectedly poor staining behavior), or to carry a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dy (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counterstain like a cytoplasm dye).
  • a first dy e.g., a nuclear dye
  • a second dye e.g., a counterstain like a cytoplasm dye
  • Fig. 1 shows a flow through device according to the invention.
  • Fig. 2 shows said flow through device connected to a pumping means and combined with a magnifying device.
  • Figs. 3 and 4 show results of staining protocols carried out with the flow through device according to the invention.
  • Fig. 5 shows said flow through device connected to another pumping means and combined with a magnifying device.
  • Fig. 1 shows a flow through device 10 for staining and/or analyzing a biological sample 11 being deposited on a support 12.
  • the flow through device comprises a conduit 13 in fluid communication with the support to which at least one liquid reagent can be delivered in such way that the biological sample provided on the support engages and/or interacts with the at least one liquid reagent.
  • the device further comprises two connectors 14, preferably of the Luer type, which act as inlet and/or outlet, and to which tubes 15 for delivery and/or removal of liquid reagents can be connected.
  • the biological sample is loaded to the support , which adopts the shape of a glass slide, and the support is then disposed on the flow through device in such way that the biological sample engages and/or interacts with the liquid reagents delivered there through by means of the tubes.
  • the biological sample can comprise cells of a cytology sample obtained either directly from a brush/swab or from a liquid based cytology (LBC) sample loaded to a support, e.g., a slide.
  • LBC liquid based cytology
  • a LBC sample with cells is loaded into the cartridge and the liquid is subsequently dried, leading to cells attached to the slide. Liquid reagents can then be delivered to the conduit in a sequential manner.
  • the device shown in Fig. 1 can have different variations.
  • the device can have two or more inlets and/or outlets.
  • the support and the flow through device define parts of one and the same integrated item.
  • Fig. 2 shows a flow through device 20 which is placed on a magnifying device comprising a light source 21 and a magnifying objective 22.
  • the magnifying device shown here is using trans-illumination, but epi-illumination and/or fluorescence can also be used.
  • a series of pumping and storage means 23 - 25 are connected to the inlet of the flow through device by means of manifolds 26 - 28.
  • the pumping and storage means adopt the shape of syringe pumps comprising different liquid reagents each. Said syringe pumps are thus combined pumping and storage means.
  • Both the syringe pumps and the manifolds which can be replaced, or complemented, by suitable valves, too, are under control of a control unit (not shown), in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, and/or opening and closing of valves of manifolds, and the like.
  • the outlet of the flow through device is connected to a canister 29, which collects the waste liquids.
  • Fig. 3 shows images taken during PAP staining steps of buccal cells as set forth in example 1 in a device according to the Invention.
  • Fig. 3A Buccal cells in preservative solution
  • Fig. 3B Air dried cells
  • Fig. 3C In flow of tap water
  • Fig. 3D In Hematoxylin
  • Fig. 3E Wash in tap water
  • Fig. 3F In 0.5% acidic EtOH
  • Fig. 3G Wash in tap water
  • Fig. 3H In 90% EtOH
  • Fig. 31 In EA50/OG6 (50:50)
  • Fig. 3J In 90% EtOH
  • Fig. 4 shows buccal cells after staining with the semi-automated protocol using Hematoxylin and Cytostain, and 0.1% acetic acid according to the protocol set forth in example 2.
  • Fig. 5 shows a flow through device 50 which is placed on a magnifying device comprising a light source 51 and a magnifying objective 52.
  • the magnifying device shown here is using trans-illumination, but epi-illumination and/or fluorescence can also be used.
  • a pumping means 53 is provided which is, on one side, connected to storage means 54 - 56 which contain the different liquid reagents by means of manifolds 27 - 29, and, on the other side, connected to the inlet of the flow through device.
  • the pumping means is a peristaltic pump, but other suitable pumping means can also be used.
  • Both the peristaltic pump and the manifolds which can be replaced, or complemented by suitable valves, too, are under control of a control unit (not shown), in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, and/or opening and closing of valves of manifolds, and the like.
  • the outlet of the flow through device is connected to a canister, which collects the waste liquids.
  • Fig. 6 shows a schematic example of a sample treatment protocol using four different liquid reagents (e.g., dyes, stains, buffers, fixatives, and/or washing solutions) which are delivered to the conduit which is in fluid communication with the support to which at least one liquid reagent can be delivered.
  • liquid reagents e.g., dyes, stains, buffers, fixatives, and/or washing solutions
  • a biological sample provided on the support engages and/or interacts with the liquid reagents.
  • the liquid reagents are delivered to the conduit with a pumping means, suitable tubes, and manifolds and/or valves.
  • Both the pumping means and the manifolds and/or valves are under control of a control unit, which in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, or opening and closing of valves of manifolds, and the like.
  • a control unit which in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, or opening and closing of valves of manifolds, and the like.
  • Example 1 Pap stain of buccal cells in a flow through device, driven by syringe pump (longer protocol using Harris' Hematoxylin, EtOH, EA50/OG6)
  • a buccal swab was taken and the cells were transferred in 150 ⁇ preservative solution (buccal cells were taken as model system for cervical cells). Subsequently, 100 ⁇ were transferred to a flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1). Once the specimen was air dried it was connected to prefilled syringes (containing the dyes and washing solutions) by means of surgical tubing.
  • the different fluids were pumped over the specimen and images were taken during every step of the staining.
  • the procedure was performed as described below; the flow was set to 0.5 ml/min.
  • Flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1) 10 ml syringes, BD
  • Buccal swab Flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1), 10 ml syringes, BD
  • thermo scientific 7501R thermo scientific 7501R
  • the liquid reagents can be stored either outside the flow through device, e.g., in separated storing means, or in storage means integrated to the flow through device. In either case, the liquid reagents can either be stored in liquid form, or in dried form which is then solubilized on the fly prior to use. The latter is particularly beneficial for microfluidic devices, or lab-on-a-chip-devices.

Abstract

The present invention is related to a flow through device for staining and/or analyzing a biological sample, said device comprising a support for the biological sample, a conduit in fluid communication with the support to which at least one liquid reagent can be delivered in such way that a biological sample provided on the support engages and/or interacts with the at least one liquid reagent.

Description

FLOW THROUGH DEVICE FOR STAINING AND/OR ANALYZING A BIOLOGICAL SAMPLE
FIELD OF THE INVENTION
The invention relates to a flow through device for staining and/or analyzing a biological sample.
BACKGROUND OF THE INVENTION
Cancer arising from cervix is the number one cancer in women in many developing countries. About 30% of cancers in women are due to cervical cancer with more than 100,000 new cases diagnosed every year, e.g., in India. The estimated compounded annual growth rate (CAGR) for cervical cancer cases is 2.56% and at this growth rate 174,001 new cases of cervical cancer will be detected in the year 2012.
One of the recommended tools for screening for cervical cancer is to detect cyto logical precursors of cancer in Papanicolaou tests (also called PAP smear, PAP test, cervical smear, or smear test), which is a screening test used in gynecology to detect premalignant and malignant processes in the endo cervical canal.
In taking a Pap smear, a speculum is used to gather cells from the outer opening of the cervix of the uterus and the endocervix. The cells are examined under a microscope to look for abnormalities. The test aims to detect potentially pre-cancerous changes, which are usually caused by sexually transmitted human papillomaviruses. The test remains an effective, widely used method for early detection of pre-cancer and cervical cancer. The test may also detect infections and abnormalities in the endocervix and endometrium.
This procedure has been effective in bringing down the incidence of cervical cancer in the developed countries. However, pap-smear has a false negative rate of 10-29%. Currently, in manual methods, the staining process is performed by dipping glass slides in various containers containing dyes and washing solutions. These solutions tend to worsen in quality over time due to various reasons (evaporation of solvent, dye aggregation, spill over from one container to the next, consumption of dye, dye degradation and/or over-oxidation), thus the quality of each sample is different, or prone to artifacts. This applies not only to manual approaches, but also for automated analysis, which use essentially the same process. SUMMARY OF THE INVENTION
According to a first aspect of the invention, a flow through device for staining and/or analyzing a biological sample is provided, said device comprising a support for the biological sample, and a conduit in fluid communication with the support to which at least one liquid reagent can be delivered in such way that a biological sample provided on the support engages and/or interacts with the at least one liquid reagent.
As used herein, the term, "liquid reagent" relates to a reagent which is either liquid as such, or solved in a liquid solvent to obtain the liquid form. The latter can apply both for gaseous and solid reagents, which are thus brought into the liquid form. The reagents can also be provided in dried form which is then solubilized on the fly prior to use, thus avoiding the oxidation and light degradation.
As used herein, the term "biological sample" is defined as a biological or body fluid sample or a biological tissue sample. Examples of biological or body fluid samples include lymph, blood, saliva, cervical fluid, cervicovaginal fluid, vaginal fluid, synovial fluid, semen, stool, sputum, mucus, lung sputum and lavage or samples derived therefrom (e.g., reagent-modified and/or fractionated samples). Further, the term encompasses samples which consist of cells taken up in a liquid, e.g., a buffer and/or preservation liquid, in order to make a cell suspension.
Fluid samples can be collected with a swab. Exemplary swab samples include cervicovaginal swab samples, including, but not limited to swab of the point of a possible cervicovaginal lesion, the cervical canal, the cervical os, the ectocervix, the transition zone on the cervix between squamous and columnar cells (i.e., the squamo columnar junction), the vagina, the posterior fornix, the portion of the vagina below the posterior fornix such as the lower third of the vagina, the labia, or combinations thereof. Biological tissue samples are samples containing an aggregate of cells, usually of a particular kind, together with intercellular substances that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissue samples also include organs (e.g., breasts), tumors, lymph nodes, arteries and individual cells. For example, the sample can be a tissue sample suspected of being cancerous.
The device according to the invention has particular advantages over the prior art. First, it avoids the artifacts caused by subsequent dipping of the sample into different reagents, as it is currently performed both in manual and automated processes of, e.g., PAP staining. Further, it avoids the manual handling of liquid reagents, which can be toxic, and thus reduces the risk of intoxication and/or environmental pollution.
Furthermore, as the device can be built up as a closed system, it can work independently of environmental conditions (humidity, pollution), and thus provides reproducible results under different environmental conditions, e.g. in points of care where there is no air conditioning, or no clean laboratory space. Another advantage is that the system can be built up as a self sustained system, and thus work independent of laboratory periphery and/or infrastructure. This makes the device suitable for use in regions with poor infrastructure, where access to laboratories and/or access to qualified personnel is absent. Another advantage is that, in the device according to the invention, the staining process can by highly automated, in order to avoid artifacts caused by faulty operation through poorly trained and/or overworked personnel. Further, the staining process can be highly standardized, in order to make analytic results more reproducible and comparable.
Yet another advantage is that the device can be integrated, e.g., combined with a magnifying unit (e.g., a microscope), in order to increase its independence from local periphery and/or infrastructure. Further, the device can be combined with an automatic image processing and analysis device, in order to increase reproducibility and comparability of the analytic results.
Another advantage is that the staining process can be observed in real time, in order to either modify or adapt the process, if needed (e.g., extend exposure to a given stain in case it turns out, under real time control, that a particular sample has unexpectedly poor staining behavior), or to carry a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dye (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counter stain like a cytoplasmic dye).
In a preferred embodiment, the device further comprises at least one pumping means for the delivery of at least one liquid reagent to the conduit, at least one valve and/or one manifold, and/or at least one storage means for providing at least one liquid reagent.
Pumping means can be, e.g., Syringe pumps (also called syringe drivers), i.e. small infusion pumps comprising a syringe as storage and delivery means. Harvard Apparatus, 11 plus, or World Precision Instruments (SP1200PZ). Another option is a peristaltic pump, which is a type of positive displacement pump used for pumping a variety of fluids. Such pump is, e.g., a syringe pump or a peristaltic pump that either pulls or pushes liquid reagents through the conduit. Storage means can be, e.g., canisters bearing the respective liquid reagents, or syringes (in said case the pumping and storing functionality is in the same device). In another embodiment, the storage means can be wells, or cavities provided on the flow through device, or microreaction vessels (e.g., of the Eppendorf type). The storage means can also comprise dried reagents which are solubilized prior to use. In another preferred embodiment, the device comprises one pumping means connected by suitable valves and/or manifolds, to at least two storage means comprising different liquid reagents, or their dried counterparts. Alternatively, the device comprises at least two pumping means which are directly connected to respective storage means comprising different liquid reagents. In this case, the at least two pumping means are connected to one another by means of at least one suitable valve and/or manifold.
In yet another preferred embodiment, the device further comprises a control unit for controlling the at least one pumping means, at least one valve and/or at least one manifold. The controlling means can thus be used to establish a given staining protocol, namely by controlling pumping speed, and pumping duration of the different liquid reagents, as e.g. shown in Fig. 5.
In yet another preferred embodiment the at least one liquid reagent is selected from the group consisting of buffers, stains, dyes, washing solutions, and/or fixatives.
Further, it is preferred that the at least one liquid reagent is selected from the group consisting of a nuclear stain, a first counterstain, and/or a second counterstain.
It is preferred that the nuclear stain comprises at least one selected from the group of Carmine, Methylene blue, Neutral/To luylene red, Haematoxylin, Safranin and/or Nile blue
The said counterstains, are preferably, cytoplasmic stains. Likewise, it is preferred that the cytoplasm stain comprises at least one selected from the group of Eosin, Alician blue, Xylidine Ponceau, Biebrich scarlet, Tartazine, Van Gieson's stain (Picric Acid and Acid Fuchsin) and/or Wright stain.
Even more preferred, stain combinations can be used, as for example comprised in the so called Pap staining mixture, and which comprises a combination of haematoxylin, Orange G, Eosin Y, Light Green SF yellowish, and sometimes Bismarck Brown Y.
Another suitable stain combination is used in Masson's trichrome, which comprises Weigert's hematoxylin, acid fuchsin, Xylidine Ponceau, phosphomolybdic acid, and Light Green SF yellowish, or alternatively Fast Green FCF, methyl blue, water blue or aniline blue.
Yet another suitable stain combination is used in Lillie's trichrome, which is similar to Masson's trichrome, but uses Biebrich scarlet instead of acid fuchsin and/or Xylidine Ponceau.
Further, fluorescent stains like DAPI (4',6-diamidino-2-phenylindole) can also be used.
Further, particular stain combinations which are commercially available can also be used, like the HCS cell mask™ staining kits provided by Invitrogen.
The nuclear stain serves for staining the nuclei of cells comprised in the sample when the sample is deposited on said first sample support means, or covered by said first sample support means. The cytoplasm stain serves for staining the cytoplasm of cells comprised in the sample when the sample is deposited on said second sample support means, or covered by said second sample support means.
Particularly preferred, the first counterstain is OG-6 counterstain, in which
Orange G and phosphotungstic acid are used ("-6" denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8).
Likewise preferred, the second counterstain is EA (Eosin Azure) counterstain, which comprises three dyes, namely Eosin Y (stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells), Light Green SF yellowish (stains the cytoplasm of other cells, including non-keratinized squamous cells and is sometimes replaced by Fast Green FCF) and Bismarck brown Y.
Other dyes which can be used include, but are not restricted to Giemsa, Mallory, Bouein, Van de Gieson, Ziehl-Neelsen, Dieterle's Stain, Diff-Quik Stain, Grocott's Methenamine Silver (GMS) Stain, Mayer's Mucicarmine Stain, Sayeed's Stain, Warthin- Starry Stain, Gomori's One-Step Trichrome Stain, Masson's Trichrome Stain, Russel-Movat Pentachrome Stain, Oil Red O and Sudan Black B Stains, Lendrum's Method (Picro-Mallory Stain), Perls' Prussian Blue Stain, the dyes used in the Gram staining protocol, the Ziehl- Neelsen staining procedure, the Masson's trichrome procedure and Leishman stains.
In yet another preferred embodiment the support for the biological sample is a slide that can be disposed on a carrier providing the conduit to which at least one liquid reagent can be delivered. In such way, the biological sample can be deposited on said slide, which in turn is then disposed on said carrier. Because the conduit is in fluid communication with the support, the sample can be treated, in a sequential manner, by fixing, staining, washing and the like in flow through mode.
In another preferred embodiment, the carrier providing the conduit to which at least one liquid reagent can be delivered is a microfluidic device.
A microfluidic device is a device in which fluids are manipulated under the rules of microfluidics, i.e. geometrically constrained to a small, typically sub-millimeter, scale, with small volumes (nl or pi scale) and small geometric sizes of conduits and channels (μιη to mm scale). Typically, microfluidic devices are manufactured with technologies taken from the semiconductor industry, like photolithographic etching and the like; microfluidic devices may also be produced using polymer processing based methods such as injection molding or hot embossing. In said microfluidic device, transportation of liquids can be carried out by capillary forces alone, or by micropumps, e.g., piezoelectronic pumps, pumps based on pneumatically actuated valves, or pumps based on magnetic microbeads. Valves in such devices can for example adopt the type of a hydrophobic valve. Microfluidic devices have a range of advantages, among which are small volumes of reagents, small overall size, ease of transportation, easy manufacture in high numbers and low costs, small laboratory size (if at all) required and so forth. Therefore, the microfluidic device according to the preferred embodiment can represent a "laboratory on a chip". In such case, the flow through device according to the present invention is independent of periphery and/or local infrastructure.
According to another preferred embodiment, the support for the biological sample and/or the carrier providing the conduit are arranged in such way that they can be placed on the stage of an optical magnifying device. In another preferred embodiment, it can be preferred that the device further comprises an optical magnifying device.
In either case, such optical magnifying device, which allows optical inspection of the sample by a skilled person, or automatic image analysis by means of a dedicated image capturing/processing device and method, is preferably a microscope capable of creating a magnification of between > 20 and < 1000 x. Further, such magnifying device is preferably capable of using epi-illumination, transmitted illumination or fluorescence. Preferably, the support and/or the carrier are made from glass or transparent plastic, which is particularly useful in case transmitted illumination is used.
According to another preferred embodiment, the device further comprises an image acquisition/processing device. Such device is preferably a CCD (linear or two dimensional) or a CMOS (linear or two dimensional) image acquisition device combined with an image processor comprising the respective software and hardware. In such embodiment, the analysis of the sample, e.g., for absence or presence of abnormal cells, can be carried our automatically, e.g., without intervention of medical or trained personnel. The latter embodiment thus allows a complete automation of the sample treatment and analysis process, further, this embodiment can increase reproducibility and comparability of the analytic results. The image acquisition/processing device is preferably mounted on a magnifying device, e.g., a microscope. Such image acquisition/processing device can, for example, carry out the following steps:
a) acquiring an image of the sample by digital image acquisition b) optionally, carrying out digital image processing
c) selecting a field of view
d) determining, by digital image processing, whether or not, in said field of view, cell aggregates exist, and
el) selecting a new field of view and carrying on with step c) if, in step d), it turns out that, in said field of view, the determination of cell aggregates is negative, or
e2) carrying out further process steps if, in step d), it turns out that, in said field of view, the determination of cell aggregates is affirmative.
In yet another preferred embodiment, the system comprises a data storage (e.g., a hard disk, or a flash storage) and/or electronic communication means (e.g., a modem, or a Bluetooth, WiFi, GSM, GPRS or 3G interface). In such case, the analysis results can be stored on said data storage for later use, e.g., by medical personnel, or the results can even be sent, electronically, to a service unit for later use, e.g., by medical personnel. Further, the device can even be remotely operated, or controlled in real time, by medical personnel using the electronic communication means.
According to another preferred embodiment the biological sample is selected from the group consisting of smear sample, tissue slice, liquid sample, and/or other cytology samples.
A smear sample is for example similar or identical to those samples commonly used a pap test, cervical smear, or smear test. Such sample can be obtained, e.g., by inserting a speculum into a body cavity, e.g., the woman's vagina to obtain access to the cervix. A sample of cells is then collected, e.g., from the outer opening or of the cervix by scraping it with a spatula, which comprises an endocervical brush which is rotated in the central opening of the cervix. The cells obtained are then placed on a glass slide by "smearing" them over the slide with help of said brush. A tissue slice is for example provided by a microtome. A liquid sample can preferably consist of a suspension of cells, e.g., obtained by a smear.
Other suitable samples comprise, but are not restricted to, fine needle aspiration cytology (FNAC) samples, abrasive cytology samples and/or exfoliated samples.
In many cases a sample is indeed placed on a slide to make it available for investigation, e.g. a tissue slice, or a smear.
According to another aspect of the invention, a method for the detection of abnormalities in a biological sample, in which method a device according to any of the aforementioned claims is used.
As used herein, the term "abnormalities" relates to cells which are in a process of becoming cancerous, or malignant ("premalignant"), or are cancerous, or malignant, already. Such cells can differ from normal cells, e.g., in one of the following features cell nucleus size, or area regularity of shape of a cell, and/or a cell nucleus, size ratio of cytoplasm to nucleus, in a given cell, and/or ratio of areas of cytoplasm and nucleus, in a given cell.
According to a preferred embodiment, the method further comprises the step of disposing a biological sample on the support.
According to another preferred embodiment, the method further comprises the step of treating the sample with at least one liquid reagent by means of delivering said liquid reagent to the sample through the conduit.
According to yet another preferred embodiment, the method further comprises carrying out a histological staining protocol with at least two liquid reagents delivered to the sample in a sequential manner. Preferably, the method further comprises carrying out a PAP test staining protocol.
According to yet another preferred embodiment, the method further comprises the analysis of the treated sample by means of optical inspection by a skilled person and/or automatic image analysis by means of a dedicated image processing device and method. Either at the end of the staining process, or in different process steps, e.g., to carry out a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dye (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counterstain like a cytoplasm dye). The optical inspection is preferably carried out by means of a magnifying device, e.g., a microscope. The automatic image analysis is preferably carried out by an image acquisition/processing device as described above, the latter being preferably mounted on a magnifying device, e.g., a microscope.
According to a preferred embodiment, the method further comprises carrying out, or recommending further investigation by, at least one step selected from the group consisting of
a) detailed inspection of the cervix by colposcopy;
b) carrying out a HPV DNA test in said biological sample, or in new sample with comparable properties;
c) carrying out a Biomarker test in said biological sample, or in new sample with comparable properties; and/or
d) visual inspection of said biological sample, or of a new sample with comparable properties, by a qualified pathologist.
Colposcopy is a medical diagnostic procedure to examine an illuminated, magnified view of the cervix and the tissues of the vagina and vulva. Primarily in order to detect premalignant lesions and malignant lesions which may result in cancer. Colposcopy is done using a colposcope, which provides an enlarged view of the areas, allowing the colposcopist to visually distinguish normal from abnormal appearing tissue and take directed biopsies for further pathological examination. The main goal of colposcopy is to prevent cervical cancer by detecting precancerous lesions early and treating them. Preferably, a system for optical examination of the cervix is used for said purpose, said system comprising optical magnification means, illumination means, dispensing means for administration of at least one stimulation and/or contrasting agent, imaging means and image processing means.
A HPV DNA test detects cervical infection with human papilloma virus (HPV), which is one of the most important infectious causes of cervical cancer. 84% of new cervical cancers were in the developing world, compared with about 50% of all new cancers. HPV DNA test kits are today commercially available. Such test may be carried out during a routine smear test, as described above (in which case part of the smear sample is taken for the HPV DNA test, while another part is taken for the method according to the invention), or with a newly taken sample with comparable properties, and can be used to improve, confirm or falsify the diagnostic significance of the method according to the invention.
Biomarker tests have been developed to investigate whether or not a patient suspected to be predisposed for cervical cancer, or a patient who is suspected for having cervical cancer, or in which cervical cancer has already been diagnosed, has, in its genome or proteome, an abnormality which coincides with increased or decreased likelihood of getting a given cancer, or which coincides with increased or decreased responsiveness towards a given therapy. Such abnormality is, for example, a mutation in a given gene, an abnormality in an epigenomic feature, like DNA methylation, or an abnormality with respect to expression of a given gene.
According to another preferred embodiment, the method further comprises a step of readjusting at least one method step under optical inspection and/or automatic image analysis.
Thereby, the staining process can be observed in real time, in order to either modify or adapt the process, if needed (e.g., extend exposure to a given stain in case it turns out, under real time control, that a particular sample has unexpectedly poor staining behavior), or to carry a differential analysis of the sample in different process steps (e.g., an image is taken after staining the sample with a first dy (e.g., a nuclear dye), and a second picture is taken after the sample has been stained with a second dye (e.g., a counterstain like a cytoplasm dye).
According to another aspect of the invention, the use of a system, kit or method according for at least one purpose selected from the group of
• cancer screening
• cancer diagnosis
• prediction with respect to a given therapy, and/or
• concomitant monitoring of a given cancer therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. In the drawings:
Fig. 1 shows a flow through device according to the invention.
Fig. 2 shows said flow through device connected to a pumping means and combined with a magnifying device.
Figs. 3 and 4 show results of staining protocols carried out with the flow through device according to the invention.
Fig. 5 shows said flow through device connected to another pumping means and combined with a magnifying device.
DETAILED DESCRIPTION OF EMBODIMENTS While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.
Fig. 1 shows a flow through device 10 for staining and/or analyzing a biological sample 11 being deposited on a support 12. The flow through device comprises a conduit 13 in fluid communication with the support to which at least one liquid reagent can be delivered in such way that the biological sample provided on the support engages and/or interacts with the at least one liquid reagent. The device further comprises two connectors 14, preferably of the Luer type, which act as inlet and/or outlet, and to which tubes 15 for delivery and/or removal of liquid reagents can be connected. In the shown embodiment, the biological sample is loaded to the support , which adopts the shape of a glass slide, and the support is then disposed on the flow through device in such way that the biological sample engages and/or interacts with the liquid reagents delivered there through by means of the tubes. In particular, the biological sample can comprise cells of a cytology sample obtained either directly from a brush/swab or from a liquid based cytology (LBC) sample loaded to a support, e.g., a slide. Alternatively, a LBC sample with cells is loaded into the cartridge and the liquid is subsequently dried, leading to cells attached to the slide. Liquid reagents can then be delivered to the conduit in a sequential manner.
The device shown in Fig. 1 can have different variations. According to one variation, the device can have two or more inlets and/or outlets. According to another variation, the support and the flow through device define parts of one and the same integrated item.
Fig. 2 shows a flow through device 20 which is placed on a magnifying device comprising a light source 21 and a magnifying objective 22. The magnifying device shown here is using trans-illumination, but epi-illumination and/or fluorescence can also be used. Further, a series of pumping and storage means 23 - 25 are connected to the inlet of the flow through device by means of manifolds 26 - 28. The pumping and storage means adopt the shape of syringe pumps comprising different liquid reagents each. Said syringe pumps are thus combined pumping and storage means. Both the syringe pumps and the manifolds, which can be replaced, or complemented, by suitable valves, too, are under control of a control unit (not shown), in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, and/or opening and closing of valves of manifolds, and the like. The outlet of the flow through device is connected to a canister 29, which collects the waste liquids.
Fig. 3 shows images taken during PAP staining steps of buccal cells as set forth in example 1 in a device according to the Invention. Fig. 3A: Buccal cells in preservative solution; Fig. 3B: Air dried cells; Fig. 3C: In flow of tap water; Fig. 3D: In Hematoxylin; Fig. 3E: Wash in tap water; Fig. 3F: In 0.5% acidic EtOH; Fig. 3G: Wash in tap water Fig. 3H: In 90% EtOH Fig. 31: In EA50/OG6 (50:50), and Fig. 3J: In 90% EtOH
Fig. 4 shows buccal cells after staining with the semi-automated protocol using Hematoxylin and Cytostain, and 0.1% acetic acid according to the protocol set forth in example 2.
Fig. 5 shows a flow through device 50 which is placed on a magnifying device comprising a light source 51 and a magnifying objective 52. The magnifying device shown here is using trans-illumination, but epi-illumination and/or fluorescence can also be used. Further, a pumping means 53 is provided which is, on one side, connected to storage means 54 - 56 which contain the different liquid reagents by means of manifolds 27 - 29, and, on the other side, connected to the inlet of the flow through device. In the shown embodiment, the pumping means is a peristaltic pump, but other suitable pumping means can also be used.
Both the peristaltic pump and the manifolds, which can be replaced, or complemented by suitable valves, too, are under control of a control unit (not shown), in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, and/or opening and closing of valves of manifolds, and the like. The outlet of the flow through device is connected to a canister, which collects the waste liquids.
Fig. 6 shows a schematic example of a sample treatment protocol using four different liquid reagents (e.g., dyes, stains, buffers, fixatives, and/or washing solutions) which are delivered to the conduit which is in fluid communication with the support to which at least one liquid reagent can be delivered. In such way, a biological sample provided on the support engages and/or interacts with the liquid reagents. The liquid reagents are delivered to the conduit with a pumping means, suitable tubes, and manifolds and/or valves. Both the pumping means and the manifolds and/or valves are under control of a control unit, which in order to establish a given staining protocol, namely by controlling pumping speed and pumping duration of the different liquid reagents, or opening and closing of valves of manifolds, and the like. Thus, the biological sample can be treated, in a sequential manner, by fixing, staining, washing and the like in flow through mode.
EXAMPLES
Example 1 : Pap stain of buccal cells in a flow through device, driven by syringe pump (longer protocol using Harris' Hematoxylin, EtOH, EA50/OG6)
Briefly, a buccal swab was taken and the cells were transferred in 150 μΐ preservative solution (buccal cells were taken as model system for cervical cells). Subsequently, 100 μΐ were transferred to a flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1). Once the specimen was air dried it was connected to prefilled syringes (containing the dyes and washing solutions) by means of surgical tubing.
Then, the different fluids were pumped over the specimen and images were taken during every step of the staining. The procedure was performed as described below; the flow was set to 0.5 ml/min.
Materials
Buccal cells taken up in preservative solution (MeOH:H20, 40:60)
- Flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1) 10 ml syringes, BD
Syringe pump, Harvard Apparatus, 1 lplus
Leica DME microscope with a μeye camera
- Tap water
la Harris Haemotoxylin solution, Merck, 1.09253.0500
2a Orange G solution, Merck, 1.06888.0500
3b EA50 solution, Merck, 1.09072.0500
0.5% acidic ethanol solution 90% ethanol
Methods
1. A swab with buccal cells was taken and cells transferred into a vial containing 150 ul preservative solution
2. 100 ul of this solution were transferred into an ibidi slide and left to dry.
3. After connecting the different tubes and pre filled syringes a flow was applied 0.5 ml/min.
4. Wash with tap water
5. Haematoxylin 5 min.
6. Wash with tap water 5 min.
7. 0.5% acidic EtOH 10 sec.
8. Wash with tap water 5 min.
9. 90% ethanol
10. Stain with OG6 and EA50 (mixed in a 1 : 1 ratio) 1 min
11. Wash with 90% ethanol until clear
The results of these experiments are shown in Fig. 3.
Example 2: Pap stain of buccal cells in a flow through device, driven by syringe pump (shorter protocol than example 1 , using acetic acid, RA Hematoxylin (= Richard Allan Hematoxylin) and RA Cytostain (= Richard Allan Cytostain).
For preparation of the sample the same steps were followed as in example 1. The difference was in the staining procedure where in this case 0.1 % acetic acid was used instead of EtOH, and the staining solutions were from a different supplier, namely Richard Allan, having slightly different compositions. The procedure was performed as described below; the flow was set to 0.5 ml/min. The actual staining procedure here was performed in less than 4 minutes.
Materials
Buccal swab Flow through device consisting of a glass slide (26 x 76 mm, 2.5 mm thickness) having an inlet and an outlet and a conduit disposed between (see Fig. 1), 10 ml syringes, BD
Syringes, BD
Syringe pump, Harvard Apparatus
Leica DME microscope with a ueye camera
Tap water
RA Hematoxylin, thermo scientific 7231
RA Cytostain, thermo scientific 7501R
0.1% Acetic acid ( AA)
Methods
1. A swab with buccal cells was taken, cells were transferred to a microscope slide, the sample was dried
2. The glue layer from the Ibidi sticky-slide was removed using 96% EtOH
3. Microscope and ibidi slide were assembled with 3M double sided tape
4. Tubing was attached
5. The order and speed of staining was programmed in the syringe pump (pulling), therefore the only actuation was switching the valves:
6. Tubing pre filled with EtOH
7. First valve open, RA Hematoxylin 30" at 0.5 ml/min. (total 250 ul dye)
8. Switch valve to wash with AA for 60" at 2 ml/min. (total 2 ml)
9. Switch valve to stain with cytostain 30" at 0.5 ml/min. (total 250 ul dye)
10. Switch valve to wash with AA 60" at 2 ml/min. (total 2 ml)
The results of these experiments are shown in Fig. 4.
Many different protocols for PAP staining, but also for a variety of other diagnostic staining methods can be applied in the flow through device according to the present invention. The delivery of liquid reagents can either be done by applying external pressure (= pumping) or suction, or by actuation within the conduit. The liquid reagents can be stored either outside the flow through device, e.g., in separated storing means, or in storage means integrated to the flow through device. In either case, the liquid reagents can either be stored in liquid form, or in dried form which is then solubilized on the fly prior to use. The latter is particularly beneficial for microfluidic devices, or lab-on-a-chip-devices.

Claims

CLAIMS:
1. A flow through device for staining and/or analyzing a biological sample, said device comprising a support for the biological sample, a conduit in fluid communication with the support to which at least one liquid reagent can be delivered in such way that a biological sample provided on the support engages and/or interacts with the at least one liquid reagent.
2. The device according to claim 1, which device further comprises:
• at least one pumping means for the delivery of at least one liquid reagent to the conduit,
• at least one valve and/or one manifold, and/or
• at least one storage means for providing at least one liquid reagent.
3. The device according to any of the aforementioned claims, wherein the at least one liquid reagent is selected from the group consisting of:
• buffers
• stains
• dyes
• washing solutions, and/or
• fixatives.
4. The device according to any of the aforementioned claims, wherein the at least one liquid reagent is selected from the group consisting of:
• a nuclear stain
• a first counterstain, and/or
• a second counterstain.
5. The device according to any of the aforementioned claims, wherein the support for the biological sample is a slide that can be disposed on a carrier providing the conduit to which at least one liquid reagent can be delivered.
6. The device according to any of the aforementioned claims, wherein the carrier providing the conduit to which at least one liquid reagent can be delivered is a micro fluidic device.
7. The device according to any of the aforementioned claims, wherein the support for the biological sample and/or the carrier providing the conduit are arranged in such way that they can be placed on the stage of an optical magnifying device.
8. The device according to any of the aforementioned claims, wherein the biological sample is selected from the group consisting of
• smear sample
• tissue slice
• liquid sample, and/or
• other cytology samples.
9. A method for the detection of abnormalities in a biological sample, in which method a device according to any of the aforementioned claims is used.
10. The method according to claim 9, which method further comprises the step of treating the sample with at least one liquid reagent by means of delivering said liquid reagent to the sample through the conduit.
11. The method according to any of claims 9 - 10, which method further comprises carrying out a histological staining protocol with at least two liquid reagents delivered to the sample in a sequential manner.
12. The method according to any of claims 9 - 11, which method further comprises carrying out a PAP test staining protocol.
13. The method according to any of claims 12-17, which method further comprises carrying out, or recommending further investigation by, at least one step selected from the group consisting of
i. detailed inspection of the cervix by colposcopy; ii. carrying out a HPV DNA test in said biological sample, or in new sample with comparable properties;
iii. carrying out a Biomarker test in said biological sample, or in new sample with comparable properties, and/or;
iv. visual inspection of said biological sample, or of a new sample with comparable properties, by a qualified pathologist.
14. The method according to any of claims 9 - 13, which method further comprises a step of readjusting at least one method step under optical inspection and/or automatic image analysis.
15. Use of a device or method according for at least one purpose selected from the group of
cancer screening
cancer diagnosis
prediction with respect to a given therapy, and/or
concomitant monitoring of a given cancer therapy.
PCT/IB2013/050142 2012-01-24 2013-01-08 Flow through device for staining and/or analyzing a biological sample WO2013111025A1 (en)

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