Multi-Specimen Slides for Immunohistologic Procedures

This invention relates to multi-specimen slides useful in immunohistologic procedures. More particularly, the invention relates to slides bearing a plurality of specimens in spaced array appropriate for automated image analysis and to technology germane to such slides.

Various multi-specimen slides are known. Paraffin block sections each containing multiple tissue specimens are described in Lillie, Histopathologic Technic and Practical Histochemistry, McGraw-Hill, Inc., New York, N.Y. (1965) pp. 74-77. Composite snap-frozen tissue sections mounted on a slide for use in diagnostic autoimmunology are described in Nairn, Fluorescent Protein Tracing, 4th Ed., Churchill Livingstone, London (1976) pp. 131-138. Johnson, et al. Handbook of Experimental Immunology, 3rd Ed., Blackwell Scientific Publications, Oxford, England (1978) refers to composite frozen tissues useful for autoantibody testing with the admonition that "To get satisfactory sections the tissue pieces must be frozen together without leaving spaces between them . . . " (p. 154). Mason, et al. in Bullock, et al. Techniques in Immunocytochemistry, Vol. 2, Academic Press, London (1983) pp. 175-216 states that tissue culture supernatants may be tested against either paraffin embedded sections or cryostat sections of snap-frozen tissue. Cryostat sections may be placed in the wells of multitest slides (pp. 192-193). Mason also states that hybridoma supernatants may be tested on air dried cell smears (p. 192). Battifora describes a multitissue tumor block useful for immunohistochemical antibody testing in Laboratory Investigation 55:244-248 (1986). Various multitissue slides are described in Stocker U.S. Pat. No. 4,647,543.

Computer controlled automatic image analysis instruments useful with appropriate software to analyze the spaced specimen array of slides of this invention are commercially available. Typical instruments include Recognition Concepts, Inc, Gould DeAnza, Inc. and Megabesion, Inc.

This invention provides slides bearing a plurality of specimen fragments in spaced array appropriate for automated computer-controlled image analysis. The specimen fragments may be of any kind. Fixed or frozen unfixed tissue specimens and cell culture specimens are preferred. The invention also subsumes technology germane to the production and use of such slides.

FIG. 1 is a schematic representation of a slide in accordance with the invention.

FIG. 2 is a perspective view of a multiblade device for cutting specimens into strips.

FIG. 3 is a perspective view of a mold provided with parallel grooves to receive specimen strips.

FIG. 4 is a perspective view of an embedding medium structure having specimen strips containing ridges of a type formed from the mold of FIG. 3.

FIG. 5 is a perspective view of a stack of structures as shown in FIG. 4.

FIG. 6 is a perspective view of a container having perforated walls for receiving a stack of structures as depicted by FIG. 5.

FIG. 7 is a perspective view of a section as produced by a microtome or the like of a block as depicted in FIG. 6.

Slides pursuant to the invention bear a plurality of specimen fragments in a spaced array. The pattern of the array may be selected to accommodate computer controlled image analysis. Quadrangular, i.e., square or rectangular patterns are preferred.

The invention is particularly concerned with slides useful in immunohistologic procedures. Such slides typically have tissues or cell culture specimen fragments mounted thereon. Either fixed or unfixed, frozen tissue specimens may be used. For many purposes, frozen tissue slides are preferred to insure the preservation of substantially unmodified tissue components such as antigens. The tissue specimens may be stained in known manner.

FIG. 1 illustrates a slide 10 bearing a plurality of tissue specimen fragments 11 in a substantially equally spaced rectangular array. In practice, the spacing may be arranged to accommodate automated image analysis. For example, a minimum of 3 pixels or about 75 to 100 microns space between specimens at a magnification of 25 times with a 512.times.512 array is appropriate.

Slides in accordance with the invention are appropriately provided with fragments from a plurality of different relatively large tissue or cell culture specimens. Each relatively large specimen is cut into narrow strips in any appropriate manner, for example, with a multiblade cutting device as illustrated by FIG. 2. Referring to the figure, the device comprises a series of blades 12 separated by spacer means 13 of an appropriate dimension to provide specimen strips of a desired narrow width. The cutting device knives and spacers are mounted on support means 14, each of which includes a removable retention means 15.

The relatively large tissue specimens for subdivision into narrow strips may be obtained from any available source such as autopsies or operations. Cell culture samples may, for example, be suspended in a gel, and the gel poured over a plate and dried to provide a layer of appropriate thickness, preferably about 0.5 to about 1.5 mm, and the layer thereafter removed from the plate and cut into narrow strips with a device as shown in FIG. 2. Cell culture smears formed in known manner, see Mason, supra at page 193, comprise another source of specimen strips.

Strips 16 of fixed or of unfixed frozen tissue or of cell culture are placed in the parallel grooves 17 of a mold such as the mold 18 illustrated by FIG. 3. An appropriate embedding medium, e.g., agar gel, is added to the mold containing the specimen strips and allowed to solidify thus producing a solidified embedding medium structure 19 as illustrated by FIG. 4 upon removal from the mold 18.

The structure 19 comprises embedding medium in the form of a base member 20 having a substantially planar surface 21 and an opposed surface 22 having a plurality of spaced ridges 23 extending therefrom. Each ridge 23 includes a specimen strip 16.

A plurality of structures 19 are stacked as shown by FIG. 5. In the stack, the terminal surfaces 24 of each ridge 23 abut the planar surface of the adjacent lower structure. The spaces between ridges provide channels 25 for access of fluids such as fixatives to the specimen strips in the ridges.

The stack of structures is placed in a container 26 as shown by FIG. 6. The container walls include perforations 27 to permit the ingress and egress of fluids such as clearing and dehydrating agents.

A fixative may be introduced into and passed through the channels 25 to condition the specimen strips for further processing.

After fixing, the stack of structures 19 is removed from the container 26 and placed in a deep mold for final embedding to form a multispecimen block. The final embedding medium may be conventional, for example, paraffin or another wax, a high molecular weight polyethylene glycol or polyvinyl alcohol, nitrocellulose, a methacrylate resin, or an epoxy resin.

The block is sectioned by a microtome or like device to provide a plurality of sections 28, each containing a spaced array of specimen sections as shown in FIG. 7. In the spaced array the channels 25 are filled by the final embedding material.

The block sections are mounted in known manner to provide slides of the kind indicated generally by the slide 10 of FIG. 1.

To produce slides of the invention bearing fragments of unfixed frozen tissue or of frozen cell cultures, snap-frozen unfixed, preferably different, specimens are cut into narrow strips, placed while frozen in the parallel grooves 18 of a mold such as the mold 18, and embedded in an embedding medium such as OCT appropriate for use in freeze drying procedures to produce frozen structures 19 of the kind illustrated by FIG. 4. Such structures, while frozen are stacked and the stack is embedded in a final embedding medium to provide a frozen block containing a plurality of spaced, parallel specimen strips as shown generally by FIG. 7. The block is sectioned, e.g., by a cryostat to provide sections containing a plurality of specimen fragments in spaced array also as shown by FIG. 7. The sections are mounted, in known manner, while frozen on slides and may thereafter be freeze dried.

Specimen fragments on the slides of this invention may be arranged in defined segments in which related specimen fragments are grouped together or associated in a manner to facilitate automated image processing. For example, one run of specimens, each of different, but known characteristics, may be positioned across a slide, e.g., a top run, to provide standards. Columns of unknown specimens may be provided above or below each standard included.