WO2001040760A1 - Systeme et procede de traitement d'echantillons de coupes de tissus - Google Patents

Systeme et procede de traitement d'echantillons de coupes de tissus Download PDF

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Publication number
WO2001040760A1
WO2001040760A1 PCT/SE2000/002359 SE0002359W WO0140760A1 WO 2001040760 A1 WO2001040760 A1 WO 2001040760A1 SE 0002359 W SE0002359 W SE 0002359W WO 0140760 A1 WO0140760 A1 WO 0140760A1
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WO
WIPO (PCT)
Prior art keywords
tray
samples
guiding means
nozzles
slides
Prior art date
Application number
PCT/SE2000/002359
Other languages
English (en)
Inventor
Thomas N. Brodin
Lennart G. Ohlsson
Pia J. KARLSTRÖM
Elisabeth T. Folkesson
Original Assignee
Active Biotech Ab
Brodin Thomas N
Ohlsson Lennart G
Karlstroem Pia J
Folkesson Elisabeth T
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Active Biotech Ab, Brodin Thomas N, Ohlsson Lennart G, Karlstroem Pia J, Folkesson Elisabeth T filed Critical Active Biotech Ab
Priority to AU17499/01A priority Critical patent/AU1749901A/en
Priority to EP00980205A priority patent/EP1255973A1/fr
Publication of WO2001040760A1 publication Critical patent/WO2001040760A1/fr

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Classifications

    • 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

Definitions

  • the present invention relates to histochemistry, including immunohistochemistry .
  • the invention concerns a system for handling and processing tissue section samples and a method for processing tissue sections using the system according to the invention.
  • the invention also concerns a tray for handling and incubating tissue sections on microscopic slides.
  • Histochemistry is a branch of biochemistry devoted to the study of the chemical composition and structure of human, animal and plant tissues.
  • One of the methods used for studying tissues is microscopy. To obtain contrast between individual cells and other tissue components, the tissue sections are normally stained before the microscopic examination.
  • immunohistochemistry labelled an- tibodies and other ligands including nucleic acid probes are used for directly viewing of the cellular and tissue distribution of a molecule.
  • Cadenza tissue sections are stained on a vertically standing slide.
  • these automated staining equipments have not solved all the problems.
  • the Cadenza has the disadvantages that only one reagent can be applied per slide and that a limited number of slides can be stained each day, namely a maximum of 20 slides a day. Tissue sections on different areas of the slide may sometimes be stained differently resulting in unevenly stained preparations.
  • US-4 274 359 discloses a biological slide staining apparatus comprising a tray having predetermined positions for slides.
  • the tray is arranged for horisontal insertion in the apparatus for staining of the slides. Removal of liquid from the slides is performed by blowing air onto the slides and by tilting of the slides. With this method of removing liquid from the slides, the liquid will be spread over a major part of the slide surface .
  • WO 99/44031 discloses a slide staining apparatus having slides mounted on a rotary horisontal carousel.
  • Rinse liquids are removed by an aspiration head having a flattened bottom with eight holes.
  • the aspiration head is lowered vertically down into a cavity formed by a slide frame, surrounding the staining surface.
  • the aspiration head is within this cavity moved into contact with the liquid covering the surface, so as to spread the liquid over the surface and create a capillary gap during aspiration of liquid.
  • this aspirator head ensures an accurate removal of liquid. For removal of liquid from consecutive samples it is necessary to: a) rotate the carousel, b) lower the aspirator head into the cavity, c) aspirate, and d) rise the aspirator head.
  • a mutual problem with the prior art devices is that it is not possible to process more than one reagent sample on each slide.
  • An object of the present invention is to overcome problems related to previous staining methods by providing a new and improved system and method for processing tissue section samples for use in the staining procedure.
  • a specific object of the invention is to provide for a higher efficiency in the wash procedure in the staining process related to microscopic examination of tissue samples .
  • Another specific object of the invention is to provide an apparatus and a method for staining tissue samples, wherein the staining of different tissue samples and with different reagents on the same surface, e.g. a slide surface, is enabled, thus enabling a higher efficiency.
  • the system according to the invention comprises a tray for carrying microscopic slides at predetermined positions on the tray.
  • the tray is connectable to an aspirating device for sliding engagement therewith and thereby co-ordinating nozzle positions of said device with the positions of the microscopic slides.
  • a multiple number of samples may be prepared and placed on treatment surfaces, such as on microscopic slides. Liquid is added to the samples. The samples are positioned at a predetermined distance below the aspirating nozzles and solution is aspirated from the samples. The samples are positioned below the nozzles with a sliding horizontal motion.
  • the tray is capable of carrying a number of tissue samples at the same time.
  • tissue samples can be prepared in advance and thereafter be processed parallelly, which leads to drastically altered efficiency.
  • the relative sliding motions has also proven to be an efficient way of positioning a plurality of samples at predetermined locations below a number of nozzles such that consecutive liquid removals from consecutive samples can be performed quickly.
  • the sliding horizontal motion is relative and could be performed with the tray or the aspiration device.
  • High efficiency can with the method and system of the invention be reached either by placing a number of samples transversely for parallel liquid removal by a plurality of nozzles at the same time, or by placing a number of samples in a longitudinal row for liquid to be removed by a number of consecutive aspirating steps. By using both directions, placing samples both transversely and longitudinally an even higher efficiency can be reached.
  • each tray has a first longitudinal guiding means for cooperation with a longitudinal guiding means on the aspirating device.
  • the nozzles on the aspiration device are preferably arranged essentially equidistant to and perpendicular above the centre of samples on the microscopic slides placed on a tray, when liquid is aspirated.
  • the distance between samples, on slides at predetermined positions on the tray, and the tips of the nozzles on the aspirating device connected to the tray is 0,1-1,5 mm, preferably 0,3- 1,0 mm, and most preferably 0,5-0,8 mm.
  • the negative pressure used for the aspiration device is at least 1 mbar, preferably at least 20 mbar and most preferably at least 30 mbar and is less than 100 mbar, preferably less than 80 mbar and most preferably less than 50 mbar. A preferred range of the negative pressure is 30-50 mbar.
  • the system further comprises a dispensing device having a plurality of nozzles for dispensing small accurate volumes of liquid solution to the tissue section samples.
  • a dispensing device having a plurality of nozzles for dispensing small accurate volumes of liquid solution to the tissue section samples.
  • the step of adding solution to samples is performed in a very efficient way, thus also rendering the staining process more efficient.
  • each tray preferably has a second longitudinal guiding means for co-operation with a longitudinal guiding means on the dispensing device.
  • said first guiding means for the aspirating device and the second guiding means for the dispensing device might in specific embodiments be incorporated as one single guiding means.
  • the dispensing device is preferably stationary which gives the structure more stability when liquid is dis-claimedd.
  • the tray is moved to shift the relative positions between samples and nozzles.
  • the diameter of the tips of the nozzles on the dispensing device is 0,5-2,0 mm, preferably 0,5-0,8 mm, giving the dispensed water droplets a preferred size.
  • the slides are placed on the tray at predetermined positions.
  • the slides can as mentioned above optionally be placed in a row in a longitudinal direction and/or in a transverse direction on the tray.
  • tissue sections must be washed efficiently, normally with a buffer solution, between the application of reagents.
  • the liquid wash solution is dispensed by dispensing nozzles and aspirated with aspirating nozzles of the dispensing device and the aspirating device respectively.
  • the microscopic slides have got defined surfaces, preferably cir- cular surfaces, for the tissue section samples and the defined surfaces are surrounded by a hydrophobic surface surface. Since the defined surfaces on each slide are surrounded by a hydrophobic surface, the surface tension can be used to retain a large volume of the wash solution on the defined surfaces.
  • the surface tension is used to aspirate a majority of the wash solution from the tissue section sample and its defined surface on the slide.
  • the surface tension is finally used to retain a small amount of liquid on the tissue section sample. This small amount of liquid prevents the section from drying in between reagent treatments and helps spreading the reagent evenly across the surface of the tissue section leading to good staining results.
  • Fig. la is a front view of an aspirating device and a tray connected to each other and forming part of a system for staining tissue samples.
  • Fig. lb is a detail view of Fig. la.
  • Fig. 2 is a front view of a dispensing device and a tray connected to each other and forming part of a system for staining tissue samples.
  • Fig. 3 is a plan view of the tray according to Fig la.
  • Fig. 5 is a plan view of a removable closure.
  • Fig. 6 is a sectional view taken along line VI-VI in Fig. 5.
  • Fig. 7 is a plan view of a microscopic slide forming part of a system for staining tissue samples.
  • FIG. 8 is a flow chart of a method for processing tissue section samples.
  • a presently preferred embodiment of a system for processing tissue section samples in accordance with the invention is illustrated in Figure la and b.
  • the system comprises a tray 1 and an aspirating device 20 slidably connected with each other.
  • the aspirating device 20 comprises a body 21 formed by a transversely extending central beam 22 and two support legs 23 extending down from the beam 22.
  • the beam 22 carries a number of aspirating nozzles 24 as will be explained below.
  • the support legs 23 each has a guiding means 25 for co-operation with a corresponding first guiding means 5 on the tray 1.
  • the beam 22 comprises an eight-channel intake manifold with eight tubular nozzles 24 of stainless steel connected to one output tubing (not shown) .
  • the nozzles 24 are equipped with a plastic tubing 28 for easy adjustment of the length of the nozzles 24.
  • the diameter of the tips of the nozzles 24 is in the range of 0,8-2,0 mm.
  • the guiding means 25 on the support legs 23 are longitudinal, i.e. directed perpendicularly to the drawing, and connectable to corresponding first longitudinal guiding means 5 on the tray 1.
  • the guiding means 25 of the aspirating device 20 comprises downwards facing sides 26 and opposed inwards facing sides 27 co-operating slid- ingly with upper sides 6 and outwards facing sides 7, respectively on the tray 1.
  • the downwards and upwards facing sides 26; 6 co-operate to control the vertical dis- tance between the nozzle tips 29 and the tray 1.
  • the outwards and inwards facing sides 7; 27 co-operate to decide the relative position between the tray 1 and the aspirating device 20 in a transverse direction B.
  • the guiding means may of course be embodied alternatively, still pro- viding co-operating longitudinal sliding areas that decide and preferably fix relative positions vertically and transversely between the aspirating device 20 and the tray 1.
  • the guiding means 25 of the aspirating device 20 and the first guiding means 5 of the tray 1 are prefera- bly made of acrylic plastic or might alternatively have another suitable low-friction material surface that enables a sliding movement between the tray 1 and the aspirating device 20.
  • the aspirating device 20 is when in use connected to a laboratory vacuum pump (not shown) capable of generating a vacuum in the range 1-100 mbar.
  • a stationary dispensing device 30, suitable for use with the system according to the invention is shown in Fig. 2.
  • the dispensing device 30 is formed similarly to the aspirating device 20 and has a body 31 comprising a transversely extending central beam 32 and two support legs 33 extending down from the beam 32.
  • the support legs 33 are in their lower ends connected by a bottom plate 40.
  • the beam 32 carries a number of dispensing nozzles 34 as will be explained below.
  • the support legs 33 and the bottom plate 40 together forms longitudinal guiding means 35 for co-operation with a corresponding second longitudinal guiding means 5a on the tray 1.
  • the dispensing device 30 has an eight -channel mani- fold with eight dispensing nozzles 34 of stainless steel.
  • the diameter of the tip of the nozzles 34 is in the range 0,5-0,8 mm.
  • the distance between the tips of the nozzles 34 on the dispensing device 30 and samples on slides 50 at predetermined positions on the tray 1, the tray 1 being connected to the dispensing device 30, is 5-20 mm.
  • the dispensing device 30 is connected to a glass syringe and a reservoir for liquid solution (not shown) for dispensing small accurate volumes of wash solution to samples placed on microscopic slides 50 on the tray.
  • the tray 1, illustrated in Figs. 3 and 4, is an elongated flat plate of acrylic plastic having an upper surface 10 four longitudinal grooves 11 and carrier surfaces 12 for microscopic slides.
  • the grooves 11 start and end at a distance from the short sides of the tray 1.
  • On the longitudinal sides of each groove 11 the carrier surfaces are provided in the form of flat elevations 12.
  • the first and second guiding means 5 and 5b of the tray are as described above formed by elevated longitudinal sides 7, adjacent longitudinal upper surfaces 6 and a lower surface 8.
  • the tray 1 has two thinner longitudinal partitions 13 and a thicker central partition in the form of a longitudinal middle section 14 of the same height as the surrounding upper surface 10 of the tray.
  • Microscopic slides 50 are schematically shown in a left groove 11, and placed at predetermined positions on the tray 1 resting on the carrier surfaces 12.
  • the slides 50 are placed with one of their short ends pointing in the longitudinal direction A of the tray 1.
  • the tray can carry 12 slides 50, four slides 50 in the transverse direction B and three slides 50 in the longitudinal direc- tion A.
  • the positions of the slides 50 are fixed in the transverse and longitudinal directions.
  • the slides 50 are fixed in the transverse direction by one of the longitudinal inner walls 17 of the grooves 11.
  • the slides 50 on the tray 1 are fixed in a longitudinal direction between the end walls of the grooves 11.
  • the tray may also be stackable and in this way be used as a lid for the tray below in a pile.
  • the microscopic slides 50 preferable used with the system of the invention are of standard size (75 x 25 mm) with 12 defined circular surfaces treatment 51 (two rows with six surfaces each) , the surfaces 51 being surrounded by a hydrophobic stained surface 52.
  • the treatment surfaces have a diameter of 8 mm.
  • Slides 50 of this type are commercially available from ICN. With reference to the flow chart of Fig. 8 a preferred method for processing tissue section samples will be described below.
  • step 101 tissue section samples are positioned and fixed to the circular treatment surfaces on the microscopic slides 50, see fig 7.
  • the tissues are first air dried on the circular surfaces on the slides and then fixed thereto with ice cold acetone in a cold cuvette.
  • the samples are incubated on the slides at -20°C for 10 minutes.
  • a circle is drawn around each circular surface with a wax pen to increase the hydrofobic barrier between the treatment surfaces .
  • step 104 to a first (along direction B) transverse row (not shown) of circu- lar treatment surfaces formed by a first transverse row of microscopic slides 50 on the tray 1.
  • Liquid is dispensed with a glass syringe (not shown) into the manifold of the dispensing device 30 and equal amounts of liquid are pressed out of each nozzle 34 onto the circular sur- faces on the slides 50. Normally 100 ⁇ l of liquid wash solution is used for each circular surface.
  • the samples are thereafter often incubated, step 107, on the tray 1 for twenty minutes at room temperature.
  • the tray 1 is sealed by a removable closure 60, see fig 5, and the water in the grooves 11 of the tray causes a moist atmosphere and prevents the samples from drying.
  • the aspirating device 20 is connected to the tray 1, step 108.
  • the aspirating device 20 and the tray 1 are connected by the guiding means 25 on the aspirating device 20 and the guiding means 5 on the tray 1. This can be done optionally when the tray is still connected to the dispensing device 30.
  • the aspirating device 20 is then moved to the first transverse row of treatment surfaces 51 formed by the first transverse row of microscopic slides 50 on the tray 1 with a substantially horisontal sliding movement in a longitudinal direction.
  • the solution is then aspirated from the treatment surfaces, step 109, with the aspirating device 2 con- nected to a vacuum pump.
  • the negative pressure used is preferably 50 mbar or lower.
  • the solution is removed almost instantaneously leaving only a small amount of liquid on the tissue section samples. This remaining liquid is retained by the surface tension.
  • the distance between the nozzles 24 of the aspiration device 20 and the tissue section samples is in the range 0,5-0,8 mm during the aspiration.
  • step 110 the aspirating device 20 is thereafter moved with a sliding movement, step 111, in a longitudinal direction (see fig 3) along the tray 1 so that the second row of treatment surfaces on the slides 50 are positioned under the noz- zles 24 and solution is thereafter aspirated, step 109, from the treatment surfaces .
  • step 110 the aspirating device 20 is moved again, step 111, and solution is aspirated from the new row of circular surfaces, step 109, and so on until all the surfaces on the slides on the tray 1 are processed.
  • a staining reagent can now be added to the tissue section samples with a separate standard multiwell pi- pette.
  • the positions of the surfaces on the slides are adapted after the distance between the tips on the multi- well pipette. Normally a volume of 50 ⁇ l of reagent solution is added to each circular surface.
  • the samples are incubated on the tray 1. During the incubation the tray is sealed by a removable closure and the water in the grooves 11 of the tray 1 gives a moist atmosphere and prevents the tissue section samples from drying.
  • the reagent is aspirated with the aspirating device 20 and a wash procedure according to the above described steps 104-111 is started by adding liquid wash solution to the samples with the dispensing device 30. The wash procedure is performed three times between each reagent treatment .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne un système de manipulation et de traitement d'échantillons de coupe de tissus et un procédé de traitement des coupes de tissus avec ce système. Ce système comprend un plateau destiné à porter les microplaquettes dans des positions prédéterminées sur le plateau. Ce plateau peut être relié à un dispositif d'aspiration en vue d'un contact coulissant, permettant ainsi de coordonner les positions des buses dudit dispositif avec celles des microplaquettes. Selon le procédé de l'invention, un nombre multiple d'échantillons peut être préparé et placé sur des surfaces de traitement, notamment des microplaquettes. Un liquide est ajouté aux échantillons. Les échantillons sont placés à une distance prédéterminée en-dessous des buses d'aspiration, la solution étant aspirée des échantillons. Les échantillons sont placés sous les buses avec un mouvement horizontal coulissant.
PCT/SE2000/002359 1999-11-30 2000-11-29 Systeme et procede de traitement d'echantillons de coupes de tissus WO2001040760A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU17499/01A AU1749901A (en) 1999-11-30 2000-11-29 System and method for processing tissue section samples
EP00980205A EP1255973A1 (fr) 1999-11-30 2000-11-29 Systeme et procede de traitement d'echantillons de coupes de tissus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9904349A SE9904349D0 (sv) 1999-11-30 1999-11-30 Novel device
SE9904349-9 1999-11-30

Publications (1)

Publication Number Publication Date
WO2001040760A1 true WO2001040760A1 (fr) 2001-06-07

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PCT/SE2000/002359 WO2001040760A1 (fr) 1999-11-30 2000-11-29 Systeme et procede de traitement d'echantillons de coupes de tissus

Country Status (4)

Country Link
EP (1) EP1255973A1 (fr)
AU (1) AU1749901A (fr)
SE (1) SE9904349D0 (fr)
WO (1) WO2001040760A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004001390A1 (fr) * 2002-06-20 2003-12-31 Vision Biosystems Limited Reacteur biologique a messianisme de drainage
US9346053B2 (en) 2003-03-31 2016-05-24 Leica Biosystems Melbourne Pty Ltd Method and apparatus for fluid dispensation, preparation and dilution
CN113082483A (zh) * 2021-03-31 2021-07-09 上海市东方医院（同济大学附属东方医院） 一种食道癌病变位置判定用注射装置及其判定方法
CN115308176A (zh) * 2022-07-26 2022-11-08 山东大学 一种癌变细胞判别用口腔黏膜多频荧光成像系统及方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4046927A (en) * 1976-11-29 1977-09-06 John Melnyk Process for semi-automated production of mammalian cell slides
US4274359A (en) * 1978-04-19 1981-06-23 A.J.P. Scientific, Inc. Biological slide staining apparatus
EP0255057A2 (fr) * 1986-07-30 1988-02-03 Hoechst Aktiengesellschaft Dispositif permettant d'imbiber des bandes test
US5338358A (en) * 1992-04-06 1994-08-16 Kabushiki Kaisha Tiyoda Seisakusho Apparatus for dyeing tissues
WO1999044031A1 (fr) * 1998-02-27 1999-09-02 Cytologix Corporation Appareil de coloration de lames de microscope a acces aleatoire avec separation de dechets liquides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4046927A (en) * 1976-11-29 1977-09-06 John Melnyk Process for semi-automated production of mammalian cell slides
US4274359A (en) * 1978-04-19 1981-06-23 A.J.P. Scientific, Inc. Biological slide staining apparatus
EP0255057A2 (fr) * 1986-07-30 1988-02-03 Hoechst Aktiengesellschaft Dispositif permettant d'imbiber des bandes test
US5338358A (en) * 1992-04-06 1994-08-16 Kabushiki Kaisha Tiyoda Seisakusho Apparatus for dyeing tissues
WO1999044031A1 (fr) * 1998-02-27 1999-09-02 Cytologix Corporation Appareil de coloration de lames de microscope a acces aleatoire avec separation de dechets liquides

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004001390A1 (fr) * 2002-06-20 2003-12-31 Vision Biosystems Limited Reacteur biologique a messianisme de drainage
US9029154B2 (en) 2002-06-20 2015-05-12 Leica Biosystems Melbourne Pty Ltd Fill fluid for biological reaction apparatus with draining mechanism
US10011015B2 (en) 2002-06-20 2018-07-03 Leica Biosystems Melbourne Pty Ltd Biological reaction apparatus with draining mechanism
US11345038B2 (en) 2002-06-20 2022-05-31 Leica Biosystems Melbourne Pty Ltd Biological reaction apparatus with draining mechanism
US9346053B2 (en) 2003-03-31 2016-05-24 Leica Biosystems Melbourne Pty Ltd Method and apparatus for fluid dispensation, preparation and dilution
CN113082483A (zh) * 2021-03-31 2021-07-09 上海市东方医院（同济大学附属东方医院） 一种食道癌病变位置判定用注射装置及其判定方法
CN113082483B (zh) * 2021-03-31 2023-05-16 上海市东方医院（同济大学附属东方医院） 一种食道癌病变位置判定用注射装置
CN115308176A (zh) * 2022-07-26 2022-11-08 山东大学 一种癌变细胞判别用口腔黏膜多频荧光成像系统及方法

Also Published As

Publication number Publication date
AU1749901A (en) 2001-06-12
EP1255973A1 (fr) 2002-11-13
SE9904349D0 (sv) 1999-11-30

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