WO1993011419A1

WO1993011419A1 - Method and device for preparing cell and tissue samples

Info

Publication number: WO1993011419A1
Application number: PCT/SE1992/000813
Authority: WO
Inventors: Christer Busch; Jan SÄLLSTRÖM
Original assignee: Individual
Current assignee: Individual
Priority date: 1991-11-25
Filing date: 1992-11-25
Publication date: 1993-06-10
Anticipated expiration: 1994-05-25
Also published as: SE9103483L; SE9103483D0; SE469148B

Abstract

The present invention relates to a method to prepare cell or tissue samples for analysis, comprising applying of said samples to slides or other supports; attaching said slides in a holder; and subjecting said samples to buffer solutions in relatively large volumes and reagent solutions in small volumes. The method is characterized in alternately arranging said slides in a straight form, and in a staircase form, in which different lengths of each slide is protruding out of said holder to provide individual access to said samples. The invention also relates to a device and system for use in said method.

Description

METHOD AND DEVICE FOR PREPARING CELL AND TISSUE SAMPLES

The present invention relates to a method and a device for preparing cell and tissue samples for subsequent analysis.

Histological samples are prepared by embedding tissue samples in paraffin, cutting in thin sections and, after fixating, arranging on slides. Alternatively, sections on slides can be made from frozen tissue. Thereafter, the sample is prepared for analysis which starts with removing the paraffin and converting the sample to an aqueous phase. This preparation, or 'staining', continues with incubating of the samples with different reagents in a specific order prior to final analysis.

Some types of staining methods, such as immunohistochemistry and in situ hybridization, the purpose of which is to specifically stain single biochemical components in the preparation, are performed by a large number of different preparation steps and with small volumes of expensive reagents.

Devices for holding slides having tissue sections thereon have been known for several years, for example as described in US A 2 058 128. When preparing samples using these holders, it is necessary to remove the slides out of the holders for individual reagent treatments which is very time consuming.

From eg. EP A2 414 405, slide holding devices are known in which the slides are arranged in pairs with the sample provided sides of each slide in a pair facing each other to form a chamber being filled with reagents by capillary action. The drawback with this approach is that it requires relatively large volumes and high concentrations of expensive reagents.

Manual preparation of, for example, immunohistochemical tissue samples, is, therefore, commonly performed as in the above mentioned US A 2 058 128 from 1934. During preparation steps in¬ volving large volumes, the slides are batchwise soaked into different baths all consisting of non-expensive chemicals. When small volumes of expensive reagents are applied to the tissue sections, the slides are individually removed from the holder and the reagents are pipetted onto the samples. After completion of this step, the slides are put back into the holders and further bath steps are performed. These insertions and removals into and out of the holder are done a required number of times until the procedure is completed and the tissue sections are ready to be analyzed, for example by microscopy. This method has the drawback that it is elaborate and also expensive and time consuming.

Therefore, it was the object of the present invention to provide a method and a device for preparation of cell or tissue samples, being reliable, non-expensive and time saving.

The present invention solves this by the characterizing portions of claims 1 and 2, respectively.

In order to describe the advantages of the invention, first a conventional method for immunohistochemical staining and thereafter a method according to the present invention will be described.

After paraffin embedding of the tissue sample, sectioning and attachment on slides, in the conventional method, first a number of baths are employed to remove the paraffin, which contain in the named order: xylene, absolute alcohol, 96 % alcohol, 80 % alcohol, 50 % alcohol, destilled water. All these steps are performed in a closed hood, the slides being arranged in a holder and batchwise soaked in the baths according to a predetermined timecycle. Thereafter, two additional bath steps follow: 0,3 % H₂0₂ in PBS (Phosphate buffered saline) and 1 % BSA (bovine serum albumine) in PBS. After these steps, the primary antibody is added to the samples by pipetting. For this purpose, the slides have to be taken out of the holder in a specific order to avoid mix up.

Following a specific incubation period, the slides are carefully put back into the holder one by one to avoid the risk of mixing up the slides. The next step is usually a PBS wash. Thereafter, the slides are taken out of the holder, carefully as above, and the samples provided with biotinylated antibody being pipetted on the sample. Following a further PBS wash, the slides are again taken out of the holder, this time to be provided with small amounts of ABC (avidin, biotin) complex, and then inserted into the holder for a repeated PBS wash.

Finally, the enzyme development occurs in a closed hood. First, the slides are taken out of the holders to provide the samples with an enzyme substrate, thereafter into the holders again for PBS wash. The three last steps: counterstaining, PBS bluestai- ning, PBS wash, are also performed with the slides inserted in the holders. Then the samples are ready for analysis.

In the conventional method, the slides have to be removed from and inserted into the holders in a total of three to four times. Besides the time and labour demanding aspects of this, there is also the risk of mixing ,up the slides.

In the method according to the present invention a specially designed device is used. The device is shown in the attached drawings, in which

Fig. 1 is a schematic view of a device according to the in¬ vention;

Fig. 2 is a section view along line II-II in Fig. 1 of the holder shown in Fig 1; and

Fig. 3 is a schematic view of a preparation system comprising the device according to Fig. 1.

In Fig. 1 there is shown a device, generally shown at the reference numeral 1, comprising a holder 2 with inserted slides 3 each provided with sectioned samples on the upper side. The samples are arranged at the same location on every slide, usually close to one of the shorter side edges. The holder 2 is open at both ends and the slides 3 extend a distance through both ends of the holder.

The device 1 also comprises a staircase formation unit 4 having, for example, six steps as shown. The holder is gripped and the slides are pressed against the steps of the unit 4 in the- direction of the arrow in Fig. 1 to obtain the shown staircase form. This form is used for pipetting small volumes of reagents on each sample individually.

Fig. 2 shows a section view of the holder 2. The slides 3 are securely held by resilience of tongues 5 forming grooves into which the slides 3 are inserted.

In Fig. 3 a system for preparing tissue or cell samples is shown. In the system the holder 2 and unit 4 are releasably arranged on a bottom plate 6. During the wash steps the holder is removed from the plate and the slides are soaked in different solution contained in cuvettes 7. The holder 1 can optionally be connected with one or more other holders. In this case, the protrusions 8 on the holder 9 are inserted into suitably dimensioned holes of another holder for connection. These holders are then placed cuvettes 9 being dimensioned for more than one cuvette. An incubation chamber 10 is provided to cover the holder 2 and slides 3 during incubation. To absorbe any moist on the slides following incubation, there is provided an absorbtion chamber 11 with absorbating material in the bottom. The positions on plate 6 can be varied enabling mounting of an optional reaction and wash chain.

In the method according to the invention, the slides 3 are alternately arranged in a straight form, ie. uniform length of each slide protrudes out of the holder, and in the staircase form according to Fig. 1, ie. different lengths of each slide is protruding out of the holder, the straight form being used for dipping of the slides into different baths and staircase form, in which the holder is rotated 90° in relation to the straight form, is used to apply small amounts of reagents to the samples on the slides.

The device 1 can be used either in a manual or in an automatic system.

From the above, it is seen that the present invention does not require removing of the slides from the holder at any time during the preparation steps and, thus, the mix up risk is avoided. The staircase form enables access to every individual slide sample. Having completed the addition of small amounts of ragents, the slides are again arranged to the initial position, in which the slides are arranged in a straight form. Because the insertion and removal steps into and out of the holders are eliminated, the method furthermore becomes time and labour saving and requires a minimum of reagents.

The holder and its tongues are preferably made of plastic with deformation zones or rubber or other materials with ability to take up different tolerances of the slides or other sample supports.

Besides the above described immunohistochemical staining the device and method according to the invention are suited for all types of stainings, for example immunocytochemical staining, enzyme histochemical staining and in situ hybridization.

Claims

1. Method to prepare cell or tissue samples for analysis, comprising applying of said samples to slides or other supports; attaching said slides in a holder; and subjecting said samples to buffer solutions in relatively large volumes and reagent solutions in small volumes, c h a r a c t e r i z e d in- alternately arranging said slides in a straight form, and in a staircase form, in which different lengths of each slide is protruding out of said holder to provide individual access to said samples.

2. Device for preparing cell or tissue samples for analysis, comprising a holder (2) for slides (3) or other sample supports, provided with inner tongues (5) forming grooves for inserting and attachment of individual slides, c h a r a c t e r i z e d in that it comprises a staircase forming unit (4) for arranging said slides (3) in a staircase form.

3. Device according to claim 2, c h a r a c t e r i z e d in that said holder (2) has two open ends.

4. System for preparing cell or tissue samples for analysis, cha r a c t e r i z e d in that it comprises a base plate (6), one or more holders (2) for slides (3), one or more cuvettes (7; 9), a staircase forming unit (4), an incubation chamber (10 and an absorbing chamber (11).