The cutting of larger tissue blocks for pathological examination has normally been performed by hand. This technique involves a special pathology knife that is used for cutting slices of parenchymateous organs such as brain, liver, kidney and heart. This cutting technique is quick and sufficient for the daily qualitative examinations on a pathological institute. The technique, however, result in tissue sections with highly variable form and thickness, just as the hand cutting does not prevent deformation of the organ.

From US 5,148,729 a biological tissue slicer is known that can produce thin slices of live tissue for biochemical, pharmacological or toxicological studies. With this machine a thin slice of tissue can be peeled off the tissue block one at the time by a reciprocally cutting blade. The slices obtained hereby are completely inadequate for pathological examination purposes.

US 4,820,504 discloses a method of preparing a multi-specimen tissue block and sections thereof, where a plurality of different anitigenically reactive tissue specimens are formed into a rods and embedded in a medium and then sliced off into sections which each contain a cross-section of the rod. With this technique, the resulting tissue slices are inaccurate in form due to possible deformation during the formation and during the slicing of a rod. Moreover, these sections are only usable as check samples of the different tissues in the multi-specimen tissue block from which the section is sliced off.

The present techniques for preparing tissue sections for pathological examination are in accurate and does prevent deformation of the tissue block to an acceptable degree.

It is the object by the present invention to circumvent the problems that lead to bias when quantitative organ or tissue examinations are desired. Another object of the invention is to provide for new quantitative unbiased stereological techniques which require cutting of the tissue in question in sections with equal thickness and orientation.

These objects are achieved by a method for cutting of a tissue block in slices with a predetermined orientation in the tissue block preferably corresponding to orientation of the plane of a scanning, such as a CT, MR or PET scanning, wherein the tissue block, such as an internal organ or other internal anatomical structures, is placed with a predetermined position and then simultaneously sliced into a multiple of sections.

In a second aspect, the invention involves an apparatus for cutting of a tissue block in slices with a predetermined orientation in the tissue block for obtaining a direct correlation of CT, MR or PET images for pathological examination, said apparatus comprising a support surface for receiving a tissue block, sectioning means comprising a multiple of cutting members, and driving means for moving the sectioning means towards the support surface for slicing a tissue block into sections.

The invention circumvents the problems that lead to bias when quantitative organ or tissue examinations are desired. The invention is ideally suited for the new quantitative unbiased stereological techniques which require cutting of a tissue block in sections with equal thickness and orientation. The invention also allows the resulting organ or tissue sections to be directly correlated to corresponding scanning planes from imaging modalities such as e. g. computerised tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET).

In a third and fourth aspects, the invention involves a method and an apparatus for preparing a tissue block for sectioning in the slicing machine. By this preparation, the organ or tissue is embedded in an alginate plastic polymer mould that together with

the embedded tissue subsequently can be sectioned in the tissue slicing machine. finally the invention also involves a tissue embedding prepared by the use of such method.

In a first embodiment of the method and apparatus for cutting of tissue blocks invention, the sectioning means comprise a multiple of parallel cutting members arranged in a cutting frame. Hereby, the sections obtained by the simultaneous sectioning of the tissue block can be easily produced by lowering a frame with cutting members down to and through the underlying tissue block.

In a preferred embodiment the distance between the cutting members can be adjusted. Hereby sections of a predetermined thickness can be obtained.

The tension of the cutting members can preferably also be adjusted. Hereby, the risk of causing a deformation of the tissue during the cutting action.

In a first embodiment the cutting members are razor blades. This ensures a sharp and accurate cut without deforming the tissue block during the slicing.

In an alternative embodiment the cutting members can be wires. Hereby, a more simple and less expensive solution can be provided where appropriate.

In the preferred embodiment of the invention, the support surface is provided with positioning means for allowing accurate positioning of a tissue block, preferably embedded in an embedding having predetermined reference surfaces. This ensures that the tissue can be positioned relative to the cutting members in such a way that the resulting sections correspond to scanning planes used in a scanning.

In the preferred embodiment, the support surface is provided with vacuum supply means for retaining the tissue block in a predetermined position. Hereby, a simple and hygienic and stable retention means is provided.

In the preferred embodiment centring means with a laser pointer are provided for accurate positioning of the tissue block on the support surface. The laser can be used for accurate position of the tissue block relative to the cutting members by assisting the positioning of the tissue block in the centre of the support surface.

This positioning could also comprise concentric centring marking circles in the support surface and possibly supplemented with an aiming crossing lines. This could e. g. be in the form of concentric recesses in the support surface.

In particular, concentric circular suction rings are provided that can be supplied with vacuum from the vacuum supply means for retaining the tissue block. This is particularly advantageous since the vacuum can be used not only for the retention but also for the aligning or centring of the tissue block.

The cutting members are preferably connected to vibration means for vibration during the slicing action, in order to facilitate the cutting action and prevent deformation of the tissue during the cutting action.

The vibration means could advantageously comprise a pneumatic vibrator that is connected to pneumatic supply means.

The vacuum in the vacuum supply means could preferably be generated by vacuum generating means connected to the pneumatic supply means. Hereby, only the number of control or supply systems needed can be reduced.

In a preferred embodiment, the driving means comprise pillar guiding means provided on the support surface and linear actuation means for linear movement of the sectioning means towards the support surface along the path defined by the pillar guiding means. This allows an accurate and smooth linear movement of the cutting frame up and down relative to the support surface for cutting the tissue. By the use of a die set for the guiding means, the travel of the cutting frame can be carried out virtually without slack whereby an accuracy in the sectioning is achieved. The linear

actuation means preferably comprise a threaded driving spindle parallel to the guide means and a corresponding threading in the cutting frame.

In a first embodiment, the threaded driving spindle is provided with a handle for manual operation. This offers a simple apparatus for carrying out the sectioning.

However, in an alternative embodiment, the driving spindle can be pneumatically or electrically driven.

In order to ensure a good positioning of the tissue block in the apparatus and to prevent deformation during the cutting, the invention also relates to method and an apparatus for preparing a tissue block. This method comprises the steps of filling a moulding form with an appropriate amount of non-toxic, biologically inert polymer moulding material, said form having at least one reference surface, and positioning a tissue block in said polymer moulding material in a predetermined position relative to said at least one reference surface, while the polymer moulding material is in its soft state.

By this method, the tissue block is provided with regular outer surfaces. that due to the form of the mould can adapted to the support surface of the sectioning apparatus.

The tissue block is in a preferred embodiment positioned in the polymer material with an orientation that corresponds to the orientation of the tissue block in vivo.

Hereby, a correlation between scanning images and the sections can be ensured.

The tissue block is embedded in a bottom mould part and a top mould is formed in a top moulding form that is filled with polymer moulding material and placed on the top of the lower moulding part with a partly encased tissue block, so that the tissue block is completely encased by in the moulding. This provides an effective insurance against the otherwise free top part of the tissue to be deformed by the cutting members.

The tissue block is in a preferred method of preparation fixed to a reference moulding of predetermined dimensioned and that said reference moulding is pivoted into a predetermined position in one or more directions, and then moulded into at least a bottom moulding. Hereby, the orientation of the tissue block can be vary accurately embedded relative to the reference surfaces.

The polymer material that is preferably used, is a cold polymerisate that polymerises by addition of water, such as an algino plastic polymer.

The apparatus and the details of the functions of the apparatus can be appreciated in the dependent claims 33 to 36.

Finally, the invention also relates to a tissue embedding comprising a tissue block made by this preparation method and apparatus. This tissue embedding providing the tissue block with regular reference surfaces ensures an accurate cutting of slices of the block for pathological and other purposes. It is realised that this technique of embedding the tissue block in an alginate or similar suitable moulding material can advantageously be used prior to any cutting action, whether a slice at the time is cut or-as it is the case in the first aspect of the invention-that the slices are cut simultaneously.

The invention will be described more detailed below with reference to the accompanying drawings, in which Fig. 1 is a perspective view of an apparatus for sectioning a tissue block according to the invention, fig. 2 shows a cutting frame of said apparatus, fig. 3a and 3b show the tissue block embedded in an alginate bottom and with an alginate top mould, fig. 4a to 4g show the embedding apparatus and the steps in the oriented alginate embedding procedure, and fig. 5 is a top view of the embedding apparatus of figs. 4a-4g.

Referring to fig. 1, a preferred embodiment of the tissue slicing machine is shown.

The tissue slicing machine stand on an aluminium or steel base plate 1 that preferably rest on a rubber pad or rubber knobs attached to the base plate 1. The base plate 1 is connected to the aluminium or steel top plate 2 by pillar guiding means comprising two the pillars 3 which through operation of the crank and spindle 8,9 allow lowering and elevation of the top plate 2 in relation to the base plate 1. In the centre of the top plate 2 a rectangular hole leaves room for attachment of the cutting frame 12. The cutting frame 12 is fixed in place by screws or a handle on the side of the cutting frame 12. The cutting frame 12 comprises a number of cutting members 14 (see fig. 2), preferably in the form of thin razor blades of hardened steel. The razor blades 14 are spaced by spacing blocks 38 that can be made of metal or plastic.

In the preferred embodiment the cutting frame 12 is exchangeable as a whole when the razor blades 12 are worn out and has lost their sharpness. In another embodiment the knife frame 12 allow changing or removal of individual blades 14, just as spacing blocks 38 of different thickness can be used. On the side of the top plate 2 a pneumatic or electric operated vibrator 4 is placed. It will when activated set the sectioning means comprising the top plate 2 and knife frame 12 into vibrations along the long axis of the razor blades 14. This facilitates the cutting procedure by lowering of the friction as the knives 14 pass through the tissue 20 and alginate block 25 (see figs. 3a and 3b). On the side of the top plate 2 there is also placed a pneumatic valve 7 for pressurised air to operate the embodiment with the pneumatic vibrator 4. The pneumatic vibrator 4 is connected to the pneumatic air valve 7 through a pneumatic hose 5. The pneumatic air valve 7 is connected to a pressurised air source at the pneumatic intake 6. On the other side of the top plate 2 or on the base plate 1 in another embodiment a valve 13 for vacuum with vacuum outtake 10 is placed. In one embodiment the vacuum outtake 10 is connected to a vacuum pump (not shown). In a second embodiment the vacuum is produced by a second pneumatic air flow valve (not shown). The vacuum hose 15 connects the vacuum valve 13 to a recess and associated apertures 16 for retention and vacuum fixation of alginate and tissue block 25. In the support surface of the base plate 1 concentric circles 17 and a cross hair cut allow centring of alginate and tissue block 25. To further aid the centring of the

tissue and alginate block 25 a laser pointer 11, that point to the central vacuum hole of the vacuum apertures 16, is provided on the top plate.

Fig. 2 show an embodiment of the cutting frame 12 consisting of knives 14 that are angled in relation to the horizontal plane of the base plate 1. This embodiment will reduce friction and deformation during the cutting of the tissue alginate block 25. In one embodiment the knife frame 12 consist of razor blades 14 that cannot be replaced, and the whole frame 12 must be changed when the blades become dull. In another embodiment the knife frame allow exchanging of individual blades and use of spacing blocks 38 with different thickness.

The bearing construction of the tissue slicing machine comprises a pillar guided base and top plate 1 and 2. The top plate 2 contains a set of parallel oriented knives 14 positioned in a frame 12. The knives 14 are mounted in a"knife frame set"and the distance between the knives 14 are spaced by high tolerance spacing blocks 38 with an equal thickness. Changing between different knife frame sets can vary the knives distance. The frame of the tissue slicing machine is equipped with a pneumatic vibrator 4 that make the knife frame set vibrate along its longitudinal axis, i. e. along the cutting edge of the knives. The vibration 4 of the knife frame 12 diminishes friction as it moves through alginate and tissue block 25. The knife frame with vibrator is mounted on a columnar lead equipped with a crank 19 that by turning allow movement of the knife frame 12 in the vertical plane. For fixation of alginate and tissue block 25 the base plate 1 of the tissue slicing machine is equipped with a suction pad that is activated by opening a vacuum valve after placement of the alginate tissue block. The concentric rings of the suction pad also serve to centre the alginate tissue block, just as a laser pointer identifies the centre.

Referring to figure 3a and 3b, any tissue block 20 or organ can be embedded into an alginate plastic mould 25. In one embodiment of this invention the tissue 20 is first embedded in an alginate bottom mould 22. This can be done by pouring the mixture of alginate powder and water in to a moulding form 21, such as a plastic jar, followed by placement of the tissue 20 into the still soft alginate-water mixture in the

mould 22. When the alginate bottom 22 has hardened an alginate top mould 23 can be cast in a similar fashion by placing a second moulding form 24, such as a second plastic jar 24. This top mould 23 can subsequently be removed for better placement of the alginate bottom 22 in the tissue slicing machine as shown in fig. 1 by then use of anatomical landmarks. In a second embodiment the tissue 20 can be cast entirely into alginate followed by CT or MRI scanning of the tissue and alginate block. When placed in the tissue slicing machine in same way as in the CT or MRI scanner the resulting tissue sections will correspond to the scanning planes. In a third embodiment of the embedding procedure, alginate embedded tissue can be cut on prior art tissue sectioning machines, such as cryostats, vibratomes and microtomes.

For tissue embedding alginate plastic polymer from Bayer Dental was used. The alginate is a non-toxic cold polymerisate that polymerises after addition of water.

The alginate powder is stirred into the water and then poured into a plastic jar or other moulding form 21 of appropriate size for the tissue block in question. The organ or tissue block 20, such as a pig brain, is then placed in the still soft polymer and hold in place until the alginate hardens. The embedding is the crucial step in the process and care must be taken to orient the tissue 20 in the alginate as it is oriented in vivo. For less accuracy this can achieved by the use of an angle protractor 34-36 and anatomical landmarks on the tissue 20 in question. For high accuracy the tissue embedder must be used. A further option is to cast another alginate mould 24on top of the tissue and alginate bottom 22. This done in order to support the tissue 20 during the cutting procedure and avoid tissue deformation. In the following this will be described as a tissue and alginate bottom 22 and an alginate lid 24.

An alternative strategy that can be used, if no scanning is needed before pathological extraction of the organ, is to embed the organ 20 in alginate and then perform the desired computer assisted scanning modality on tissue and alginate block 25 followed by the sectioning as described by the first aspects of the present invention.

This strategy abolishes the need for orientation of the tissue block 20 as the resulting digital image scanning planes will correspond to the histological sections provided

that the tissue and alginate block 25 is placed in the cutting machine in the same fashion as in the CT, MRI or PET scanner.

In the apparatus for cutting of tissue blocks, the tissue block 25 with the embedded tissue 20, is placed on the suction pads 16 of the support surface and centred in relation to the cutting frame 12 by use of the concentric circles 17 of the tissue slicing machine base plate 1 and the laser pointer 11. Following the centring the alginate tissue block 25 is fixed by activation of the vacuum valve 13 and is now ready for the cutting. This can be done with or without the alginate lid 24. An opening of the pneumatic valve 7 activates the pneumatic vibrator 4 and the cutting frame 12 starts vibrating. By a steady rotating movement, the crank 19 of the columnar lead is turned and the cutting frame 12 is lowered through the alginate and tissue block 25. The cutting results in a set of alginate and tissue slabs (not shown) that are of equal thickness and oriented corresponding to the scanning plane of the given computerised scanning modality.

Referring to figures 4a to 4g a method and an apparatus for preparing a tissue block 20 by embedding the tissue block 20 in alginate plastic polymer. This can be done in such a way that the tissue 20 is oriented to existing CT, MRI or PET scanning planes.

Fig. 4a shows the embedding apparatus that comprises of a central plastic rod 28 with a spheric top end 28a and two concentric plastic cylinders, i. e. an inner cylinder 29 and an outer cylinder 30. The rod 28 is fixed to a plastic base plate 31, where upon also the cylinders 29,30 rest in their retracted positions. On top of the outer cylinder 30 four plastic pins 33 with or without a screw thread are placed at 90 degrees interval and orthogonal to the long axis of the cylinder 30 (see fig. 5).

A reference moulding form 27 is placed on plate means 32 comprising two half parts placed on the inner cylinder 29 on each side of the rod 28. Hereby, a reference moulding form is defined. This form is filled with polymer moulding material 26 in which the tissue block 20 is placed. This means that the tissue block 20 is embedded

in a reference mould 26, such as shown in fig. 4b, where the plate means 32 and the form 27 is removed.

Fig. 4c shows a tissue and alginate reference mould 26 placed in the embedding apparatus. The alginate mould 26 is first fixed with two plastic pins 33 facing each other at 180 degrees. This plane can be defined as the X plane. The tilt angle of the tissue and alginate reference mould 26 in relation to the horizontal Z plane, as determined from the desired CT, MRI or PET scanning, is determined by an angle protractor 36 (see fig. 4d) in one embodiment. In a second embodiment the angle protractor 36 is equipped with a laser guide 34 directing a beam 35 at the mould for a measure of the angle of inclination. When the tissue and alginate reference mould 26 is fixed in the desired angle in the X plane, it is then fixed in a similar way by the two plastic pins 33 placed orthogonally in the Y plane.

Fig. 4e shows the casting of the second alginate bottom 22. The shape of the bottom mould 22 is adapted to fit into the tissue slicing machine of fig. 1. First, the outer cylinder 30 is raised and a second base plate 37 is slid into a corresponding horizontal opening in the outer plastic cylinder 30, followed by casting of the second alginate bottom 22, as the outer cylinder 30 forms the side part of the moulding form.

Fig. 4f shows the placement of a top form 21 on the outer cylinder 30 followed by casting of an alginate top mould 24 on the top of the bottom mould 22 and the tissue block 20.

Fig. 4g shows the embedded tissue block 20 with its alginate moulding 25-i. e. the reference mould 26, bottom mould 22 and top mould 24-free of the embedding apparatus after the outer cylinder 30 has been slid back towards the base plate 31.

Fig. 5 shows a top view of the outer cylinder 30, inner cylinder 29, centre rod 28 and plastic pins 33 for pivoting the reference mould 26 of the tissue block.

This embedding apparatus allows accurate three dimensional orientation of a tissue reference moulding 26 in relation to CT, MRI or PET scans. Following the accurate orientation of the tissue reference moulding 26, a second moulding is performed to produce a second alginate bottom 22 with an outer surface that will fit into the tissue slicing machine. If desired a final alginate lid 24 can be cast on top of the tissue 20 and alginate bottom 22 in order to avoid tissue deformations during the cutting procedure. The embedding apparatus comprises a circular rod 29 of transparent plastic, such as plexi-glass or similar material, two outer concentric plastic cylinders 29 and 30, fixation pins 33, semicircular plastic plates32 and an insertable base plate 37 that preferably also is made from a plastic material.