EP1255973A1 - System and method for processing tissue section samples - Google Patents

System and method for processing tissue section samples

Info

Publication number
EP1255973A1
EP1255973A1 EP00980205A EP00980205A EP1255973A1 EP 1255973 A1 EP1255973 A1 EP 1255973A1 EP 00980205 A EP00980205 A EP 00980205A EP 00980205 A EP00980205 A EP 00980205A EP 1255973 A1 EP1255973 A1 EP 1255973A1
Authority
EP
European Patent Office
Prior art keywords
tray
samples
guiding means
nozzles
slides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00980205A
Other languages
German (de)
French (fr)
Inventor
Thomas N. Brodin
Lennart G. Ohlsson
Pia J. Karlström
Elisabeth T. Folkesson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Brodin Thomas N
Folkesson Elisabeth T
KARLSTROEM, PIA J.
OHLSSON, LENNART G.
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP1255973A1 publication Critical patent/EP1255973A1/en
Withdrawn legal-status Critical Current

Links

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.
  • the tray may also be used for incubating the sam- pies.
  • the tray has for this purpose a reservoir for water or the like to give the tissue samples a moist atmosphere during incubation and prevent them from drying.
  • the tray may be provided with a removable closure to help creating a suitable incubation environment for the samples. It is of great advantage that the same tray can be used for washing and for incubation of the samples. Thereby a high efficiency in the processing of the samples can be achieved.
  • each slide on the tray can form incubation vessels for a number of separate samples.
  • 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. 4 is a sectional view taken along line III-III in Fig. 3.
  • 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 guiding means 35 of the dispensing device 30 is connectable to a corresponding second guiding means 5a on the tray 1 for a sliding movement of the tray 1 relative the stationary dispensing device 30.
  • the second guiding means 5a of the tray 1 comprises the outwards facing sides 7 and the downwards facing side 8 of the tray 1.
  • the guiding means 35 of the dispensing device 30 comprises opposed inwards facing sides 37 on the support legs 33 and an upwards facing side 41 on the bottom plate 40.
  • the guiding means 5b and 35 co-operate to decide the relative positions between the tray 1 and the dispensing nozzles 34.
  • the guiding means 35 on the dispensing device 30 and the second guiding means 5b on the tray 1 are made of acrylic plastic or another suitable material that makes a sliding movement possible.
  • 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 grooves 11 on the tray 1 comprises a central reservoir 19 that can be filled with water in order to give samples on the slides 50 a moist atmosphere during incubation.
  • the tray 1 might also be equipped with a removable closure 60, see Figs. 5 and 6, which can be placed on the tray 1 during incubation.
  • the removable closure 60 is slightly bigger than the tray 1 and is resting on the upper surface 10 of the tray 1 and in this way sealingly closes the tray 1.
  • the removable closure 60 is a flat plate 61 of acrylic plastic with four border strips 62, 63, for keeping the closure 60 in place.
  • 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 102 entails adding water to the longitudinal grooves 11 on a tray 1 and positioning the slides 50 at predetermined positions on the carrier surfaces 12 on the tray 1.
  • the tray 1 is connected to the stationary dispensing device 30, step 103, by inserting the tray 1 on top of the bottom plate 40 guided by the second guiding means 5a on the tray 1 and the guiding means 35 of the dispensing device 30.
  • 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.
  • step 105 the tray 1 thereafter is moved with a sliding movement, step 106, in a longitudinal direction A so that the second transverse row of treatment surfaces 51 on the slides 50 are positioned below the nozzles 34 and solution is thereafter dispensed, step 104, to the surfaces 51. If there are further samples to be processed, step 105, the tray 1 is moved again, step 106, and solution is dispensed to the new row of circular surfaces, step 104, and so on until all the surfaces 51 on the slides 50 on the tray 1 are processed, step 105.
  • 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 .

Abstract

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. Thus 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. According to the method of the invention 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.

Description

System and method for processing tissue section samples.
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. In 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.
Most of these staining methods involve a large number of incubations for various time periods, separated by washing steps to remove reagents in excess. For a long time all operations were carried out by hand. This procedure involved adding and removing reagent solutions to each tissue section sample with a pipette, washing the samples by immersing the slides in a large buffer volume and changing the buffer a couple of times and finally wiping around the tissue sections by hand using a paper tissue before incubating with the next reagent solution. This sequence is then repeated for several steps of the procedure. For hospital- and research laboratories hand- ling large amounts of tissue samples, this tedious staining procedure limited the amount of samples that could be examined every day. In accordance to this manual procedure one person can process about 15 tissue sections a day. Some years ago, manufacturers started to develop equipment for automated staining of tissue sections on microscopic slides and a number of different types are now on the market, one of which is "Cadenza" from Shandon Scientific (U.K.). In the Cadenza system tissue sections are stained on a vertically standing slide. However, these automated staining equipments have not solved all the problems. For example, 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. Furthermore, it is not possible to work in the presence of detergents, as is the case when using intracellular stains. 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. For removal of liquid 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. In WO 99/44031 it is also pointed out that 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.
These objects, as well as other objects that will be apparent from the description below, have now been ob- tained according to the present invention by providing a system and a method for processing tissue section samples according to claim 1 and 9.
The objects are also achieved by a tray according to claim 8. Thus 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.
According to the method of the invention 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.
In accordance with the invention the tray is capable of carrying a number of tissue samples at the same time. Thus a greater number of tissue samples can be prepared in advance and thereafter be processed parallelly, which leads to drastically altered efficiency.
Thanks to the inventive way of removing liquid solution from the tissue samples by aspiration of the solution, a high reproducability and a very precise and fast removing of the liquid is accomplished. As will be ex- plained below, by aspiration, physical phenomena related to the surface tension of the solution can be used to reach very accurate results.
By connecting a plurality of nozzles with a plurality of samples and permitting a relative displacement by a sliding longitudinal motion, a high number of consecutive samples can be treated efficiently in consecutive steps. Also, the distance between nozzles and samples can be controlled and kept fixed in a simple fashion, thus rendering the aspirating step even more precise and effi- cient. 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.
Thanks to the inventive way of removing liquid solution from tissue samples by aspiration with a plurality of nozzles and a sliding relative motion for co-ordinating the positions of the nozzles and positions of the slides, it has proven possible to process a plurality of samples on the same slide surface, without the need of walls between different samples. A specific reason there- fore is that the liquid to be removed is in no way spread out horisontally but only aspirated upwards.
According to a preferred embodiment of the invention the treatment surfaces for the samples are microscopic slides or specific treatment surfaces on such slides. According to a specifically preferred embodiment each tray has a first longitudinal guiding means for cooperation with a longitudinal guiding means on the aspirating device. By the longitudinal guiding means of the tray and the aspirating device respectively, the relative positions of the nozzles are controlled in a transverse direction and in a direction perpendicular to the tray.
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. In a preferred embodiment of the invention 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.
Preferably 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. Thereby also the step of adding solution to samples is performed in a very efficient way, thus also rendering the staining process more efficient. When the system comprises a dispensing device with a plurality of nozzles each tray preferably has a second longitudinal guiding means for co-operation with a longitudinal guiding means on the dispensing device. It should be noted that 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- pensed. In this embodiment 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.
The tray may also be used for incubating the sam- pies. According to one specific embodiment the tray has for this purpose a reservoir for water or the like to give the tissue samples a moist atmosphere during incubation and prevent them from drying. During incubation the tray may be provided with a removable closure to help creating a suitable incubation environment for the samples. It is of great advantage that the same tray can be used for washing and for incubation of the samples. Thereby a high efficiency in the processing of the samples can be achieved. Of specific importance for the ef- ficiency is that each slide on the tray can form incubation vessels for a number of separate samples. It is generally accepted that in order to obtain optimal results from a staining procedure, tissue sections must be washed efficiently, normally with a buffer solution, between the application of reagents. In a preferred embodiment of the present invention the liquid wash solution is dispensed by dispensing nozzles and aspirated with aspirating nozzles of the dispensing device and the aspirating device respectively. This accurate dispensation and aspiration of liquid leads to a reproducable wash resulting in high quality staining results of the tissue section samples. The physical relations behind the wash process is explained below.
According to one aspect of the invention 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.
When aspirating solution, 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. When the majority of the wash solution is aspi- rated, 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.
The invention will now be further described below with reference to the appended drawings showing a presently preferred embodiment .
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. 4 is a sectional view taken along line III-III in Fig. 3.
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 guiding means 35 of the dispensing device 30 is connectable to a corresponding second guiding means 5a on the tray 1 for a sliding movement of the tray 1 relative the stationary dispensing device 30. The second guiding means 5a of the tray 1 comprises the outwards facing sides 7 and the downwards facing side 8 of the tray 1. The guiding means 35 of the dispensing device 30 comprises opposed inwards facing sides 37 on the support legs 33 and an upwards facing side 41 on the bottom plate 40. In correspondence with the aspirating device 20, the guiding means 5b and 35 co-operate to decide the relative positions between the tray 1 and the dispensing nozzles 34.
The guiding means 35 on the dispensing device 30 and the second guiding means 5b on the tray 1 are made of acrylic plastic or another suitable material that makes a sliding movement possible.
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 grooves 11 on the tray 1 comprises a central reservoir 19 that can be filled with water in order to give samples on the slides 50 a moist atmosphere during incubation. The tray 1 might also be equipped with a removable closure 60, see Figs. 5 and 6, which can be placed on the tray 1 during incubation. The removable closure 60 is slightly bigger than the tray 1 and is resting on the upper surface 10 of the tray 1 and in this way sealingly closes the tray 1. The removable closure 60 is a flat plate 61 of acrylic plastic with four border strips 62, 63, for keeping the closure 60 in place.
The tray may also be stackable and in this way be used as a lid for the tray below in a pile. Now turning to fig 7, 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.
In the first step, 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 .
The next step, step 102, entails adding water to the longitudinal grooves 11 on a tray 1 and positioning the slides 50 at predetermined positions on the carrier surfaces 12 on the tray 1.
Reference is now being made to fig 2 and 3. The tray 1 is connected to the stationary dispensing device 30, step 103, by inserting the tray 1 on top of the bottom plate 40 guided by the second guiding means 5a on the tray 1 and the guiding means 35 of the dispensing device 30.
Thereafter solution is added, 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 μl of liquid wash solution is used for each circular surface.
If more samples shall be processed, step 105, the tray 1 thereafter is moved with a sliding movement, step 106, in a longitudinal direction A so that the second transverse row of treatment surfaces 51 on the slides 50 are positioned below the nozzles 34 and solution is thereafter dispensed, step 104, to the surfaces 51. If there are further samples to be processed, step 105, the tray 1 is moved again, step 106, and solution is dispensed to the new row of circular surfaces, step 104, and so on until all the surfaces 51 on the slides 50 on the tray 1 are processed, step 105.
The samples are thereafter often incubated, step 107, on the tray 1 for twenty minutes at room temperature. During the incubation 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.
Turning now to fig la and lb 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.
If more samples shall be processed, 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 .
If further samples need to be processed, 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 μl 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. After incubating the samples 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 .

Claims

1. A system for processing a plurality of tissue section samples comprising: at least one tray (1) for carrying at least one slide (50) at a predetermined position on said tray (1) ; an aspirating device (20) with a plurality of nozzles (24) and means for supplying negative pressure; each tray (1) having guiding means (35) for co-operation with corresponding guiding means (25) on the aspirating device (20), the guiding means being connectable for sliding movement in a longitudinal direction (A) and for co-ordinating the position of the nozzles (24) with the positions for the microscopic slides (50) on the tray (1) .
2. A system according to claim 1, wherein the guiding means (5) of each tray (1) comprises a first longitudinal guiding means (6) and the guiding means of the aspirating device (20) comprises a corresponding longitudi- nal guiding means for co-operation with said first guiding means (5) .
3. A system according to claim 1 or 2 , further comprising a dispensing device (30) with a plurality of nozzles (34) and means for dispensing small accurate volumes of liquid.
4. A system according to claim 3, wherein each tray (1) has a second longitudinal guiding means (7) for cooperation with a corresponding guiding means (37) on the dispensing device (30) , said second guiding means (7) and guiding means of the dispensing device (37) movement in a longitudinal direction (A) and for co-ordinating the position of the nozzles (34) with the positions of the microscopic slides (50) on the tray (1) .
5. A system according to any of the claims 1-4, wherein the nozzles (24) of the aspirating device (20) are arranged in a transverse direction at predetermined distances from the guiding (26) means of the aspiration device (20) and, when the device (20) is connected to the tray (1) , at a predetermined height perpendicular above the tray (1) .
6. A system according to any of the claims 1-5, fur- ther comprising microscopic slides (50) on the tray (1) , the slides (50) having defined surfaces (51) for samples.
7. A system according to claim 6, wherein the defined surfaces (51) are surrounded by a hydrophobic surface (52) .
8. A tray (1) for handling and incubating tissue section samples on microscopic slides (50) comprising: an essentially elongated plate having predetermined positions for the microscopic slides (50) and at least one longitudinal groove (11) , the tray (1) having longitudi- nal guiding (6) means for co-operation with corresponding guiding means (26) on an aspirating device (20) , the guiding means being connectable for sliding movement in a longitudinal direction (A) and for co-ordinating the position of nozzles (24) on the aspiration (20) device with the positions for the microscopic slides (50) on the tray (1) •
9. Method for processing a plurality of tissue section samples comprising the steps of : a) positioning and fixation of the samples to at least one treatment surface; b) adding a liquid solution to the samples; c) fixing the samples in a plane at a predetermined distance below aspirating nozzles (24); d) positioning the samples in predetermined positions be- low the aspirating nozzles (24) by a relative sliding motion in a substantially horizontal direction; and e) aspirating solution from the samples.
10. Method according claim 9, wherein the step of positioning and fixation of the samples to at least one treatment surface entails positioning and fixation of samples to microscopic slides (50) and positioning the slides (50) at predetermined positions on a tray (1) .
11. Method according to claim 9 or 10, wherein the distance between the samples on the treatment surfaces (50) and the aspirating nozzles is 0,1-1,5 mm, preferably 0,3-1,0 mm and most preferably 0,5-0,8 mm.
12. Method according to claim 10, wherein the microscopic slides (50) have defined surfaces (51) for samples, the surfaces (51) being surrounded by a hydrophobic surface (52) .
13. Method according to any of the claims 9-12, wherein the negative pressure used for the aspiration of liquid solution 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.
EP00980205A 1999-11-30 2000-11-29 System and method for processing tissue section samples Withdrawn EP1255973A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE9904349A SE9904349D0 (en) 1999-11-30 1999-11-30 Novel device
SE9904349 1999-11-30
PCT/SE2000/002359 WO2001040760A1 (en) 1999-11-30 2000-11-29 System and method for processing tissue section samples

Publications (1)

Publication Number Publication Date
EP1255973A1 true EP1255973A1 (en) 2002-11-13

Family

ID=20417918

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00980205A Withdrawn EP1255973A1 (en) 1999-11-30 2000-11-29 System and method for processing tissue section samples

Country Status (4)

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

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003901871A0 (en) 2003-03-31 2003-05-08 Vision Biosystems Limited A method and apparatus for fluid dispensation, preparation and dilation
WO2004001390A1 (en) * 2002-06-20 2003-12-31 Vision Biosystems Limited Biological reaction apparatus with draining mechanism
CN113082483B (en) * 2021-03-31 2023-05-16 上海市东方医院（同济大学附属东方医院） Injection device for determining esophagus cancer position

Family Cites Families (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
DE3625683A1 (en) * 1986-07-30 1988-02-11 Hoechst Ag DEVICE FOR WETING TEST STRIPS
JP3320444B2 (en) * 1992-04-06 2002-09-03 株式会社千代田製作所 Nozzle cleaning equipment for dyeing equipment
US6096271A (en) * 1998-02-27 2000-08-01 Cytologix Corporation Random access slide stainer with liquid waste segregation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0140760A1 *

Also Published As

Publication number Publication date
WO2001040760A1 (en) 2001-06-07
SE9904349D0 (en) 1999-11-30
AU1749901A (en) 2001-06-12

Similar Documents

Publication Publication Date Title
US5985669A (en) Method and apparatus for treatment of human or animal cell samples
US5439649A (en) Automated staining apparatus
US5948359A (en) Automated staining apparatus
AU762808B2 (en) Automated staining apparatus
CA2720326C (en) Cartridge device for processing a sample mounted on a surface of a support member
CA2529886C (en) Apparatus and method for handling fluids for analysis
WO1997039328A1 (en) Method and apparatus for treatment of human or animal cell samples
GB2265981A (en) Dyeing tissues
US11579053B2 (en) Specimen processing systems, pipette assemblies and methods for preparing reagents
WO2001068259A1 (en) Automated specimen processing apparatus with fluid detection
JP2883294B2 (en) Biological tissue processing device and reagent processing unit
WO2001040760A1 (en) System and method for processing tissue section samples
US4200056A (en) Segmented platen for applying liquids to a flat surface
JP7337815B2 (en) Apparatus and method for staining organic material on slides
EP1681570B1 (en) Apparatus and method for handling fluids for analysis
CN112710527B (en) Automatic dyeing device for biological tissue sample slices
AU718761B2 (en) Method and apparatus for treatment of human or animal cell samples
AU689013B2 (en) Method and apparatus for treatment of human or animal cell samples

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020524

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: KARLSTROEM, PIA J.

Owner name: OHLSSON, LENNART G.

Owner name: FOLKESSON, ELISABETH T.

Owner name: BRODIN, THOMAS N.

RIN1 Information on inventor provided before grant (corrected)

Inventor name: KARLSTROEM, PIA J.

Inventor name: BRODIN, THOMAS N.

Inventor name: OHLSSON, LENNART G.

Inventor name: FOLKESSON, ELISABETH T.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040602