EP1101143A1

EP1101143A1 - Slides with reaction zones defined by hydrophobic barriers

Info

Publication number: EP1101143A1
Application number: EP99928640A
Authority: EP
Inventors: Maria Fu; B. J. Kerns
Original assignee: Biogenex Laboratories Inc
Current assignee: Biogenex Laboratories Inc
Priority date: 1998-08-07
Filing date: 1999-06-14
Publication date: 2001-05-23
Also published as: WO2000008511A1; JP2002526785A

Abstract

Slides for the immobilization and subsequent analytical processing of biological specimens having at least one reaction zone defined by a hydrophobic barrier circumscribing substantially the entire boundary of said zone.

Description

Slides with Reaction Zones Defined by Hydrophobic Barriers

Technical Field

The present invention relates to microscope slides and, more particularly, to microscope slides with at least one reaction zone which is bounded by a hydrophobic border and which has an affinity for the samples to be analyzed.

Background of the Invention

Cell antigens have been recognized as important markers indicating the presence of pathological conditions in patients. Methods have been described in the literature for the detection of such antigen markers on cells, based on the visualization of the reaction of the antigens with the corresponding antibodies by fluorescence, gold particles or enzymatic development of visible dyes. Such methods are generally referred to as immunostaining techniques. The reaction steps can be carried out on suspended cells, where evaluation takes place by means of transmission measuring devices, or by means of a microscope after transfer of the stained cells to a microscope slide.

Immunostaining techniques are also performed on tissue specimens take from a patient, for example by means of a biopsy. Such tissue specimens are routinely mounted on slides for ease of handling and analysis. Because of the manner in which slides with tissue specimens are prepared (see Elias, J., "Immunohistopathology: A Practical Approach to Diagnosis" ASCO Press, 1990, pp. 3-4, for examples of such preparation), the size and/or location of a tissue sample on a microscope slide can vary considerably within a relatively large area of the slide. In order to apply a stain to the correct location on a slide and to provide rinsing and other manipulation steps at appropriate times and in proper amounts, until recently all such staining operations were carried out by hand.

However, modern immunostaining techniques often require multi-step staining techniques, and laboratories that examine large numbers of tissue specimens or conduct other diagnostic tests find it desirable to automate the staining or diagnostic processes. Accordingly, a number of manufacturers have developed equipment for automated staining of tissue samples on slides, as well as devices which automate other aspects of diagnostic procedures utilizing various reagents.

In an attempt to facilitate the handling and analysis of microscope slides in automated processing devices, it is considered desirable to delimit the area of the slide into discrete reaction zones, and to prepare these zones in a manner to ease the mounting and fixation of the tissue specimens.

The literature contains descriptions of techniques for fixing the cells employed for such detection methods on the slide surface, for example by producing delimited reaction fields, which are in a position to fix the preparations, such as cells, to be examined. Foe example, it is known to cover the surface of a slide with a plastic film, in which one or more open regions have been created. The slide surface exposed by these apertures in the plastic film is then coated with a substance, for example with poly-L-lysine (Transplantation 25(6):331-334 (1978)), which is able to adhere the cell preparation to the slide. It is also known to produce slides provided with such regions by coating the entire slide surface with a water-repellent agent, and then eliminating the agent in the desired region by using concentrated sulphuric acid and then sodium hydroxide solution (Transplantation 28(3):257-259 (1979)). In this example as well, the exposed glass surface of the region is coated with poly-L-lysine, in order to facilitate the adhesion of the cells.

Additional developments in such slides, and in the manner of their preparation, are disclosed in U.S. Patent No. 4,705,705, in which a region of one surface of the slide is covered with a removable material which adheres to the surface. The entire surface of the slide is then coated with a hydrophobic material, the region is exposed by removal of the covering material, and the exposed region is coated with a substance having an affinity for the tissue preparation.

All of these techniques permit the anchoring of the cells to be treated onto microscope slides, which represents a considerable advance as compared with the previously known methods with suspended cells. In particular, simpler handling is achieved in numerous investigations, so that incubations and washing operations can be undertaken in an automated mariner. In addition, the automated processing device will be more readily able to ensure adequate treatment with reagents, and reduce the amount of waste by not requiring the entire slide to be covered.

However, the previously known slides with such treated regions are not able to provide complete satisfaction, on one hand in view of the liquid-repellent action of the regions bounding the reaction fields and on the other hand with regard to the fixing of the preparation, in particular cells, to be examined, on the actual surface of the reaction fields. Thus, it is considered desirable to provide both slides and methods for their preparation which facilitate their use for frozen tissue sections, formalin-fixed paraffin sections, and cytology preparations.

It is also considered desirable to provide a straightforward method for producing such slides which facilitates their use in automated processing devices with preprogrammed, repetitive motions and processing steps.

Disclosure of the Invention

The present invention provides a slide for the immobilization and subsequent analytical processing of biological specimens. In one aspect, the present invention provides a slide comprising a slide base defining a substantially rectangular planar upper surface and at least one reaction zone defined on the upper surface comprising a hydrophobic barrier circumscribing substantially the entire boundary of the reaction zone. The barrier by virtue of being applied to the slide base will project upwardly from the upper surface of the slide to a height sufficient to prevent a predetermined amount of an aqueous solution deposited on the reaction zone from crossing the boundary of the zone. In addition, the slide provides means for increasing the binding between the upper surface of the slide within the reaction zone and a biological specimen which is placed in contact with the upper surface. This binding will be sufficient to permit the specimen to be subjected to analytical processing steps as desired by the user.

The present invention is particularly adapted to the use of automated slide processing apparatus which utilize typically preprogrammed, repetitive motions and processing steps.

Brief Description of the Drawings

Figure 1 presents a top plan view of a slide in accordance with the present invention wherein the reaction zone consists of a single zone covering approximately

2/3 of the upper surface of the rectangular slide;

Figure 2 depicts a top plan view of a slide wherein the reaction zone covers approximately 50% of the slide; and

Figure 3 depicts a top plan view of a slide in accordance with the invention which comprises a plurality of reaction zones provided by including two additional intermediate hydrophobic barriers in the slide of Figure 1 which subdivide the reaction zone into three separate zones of approximately equal dimensions. Detailed Description of the Invention

The present invention provides a slide for the immobilization and subsequent analytical processing of biological specimens. In one aspect, the present invention provides a slide comprising a slide base defining a substantially rectangular planar upper surface and at least one reaction zone defined on the upper surface comprising a hydrophobic barrier circumscribing substantially the entire boundary of the reaction zone. The barrier by virtue of being applied to the slide base will project upwardly from the upper surface of the slide to a height sufficient to prevent a predetermined amount of an aqueous solution deposited on the reaction zone from crossing the boundary of the zone. In addition, the slide provides means for increasing the binding between the upper surface of the slide within the reaction zone and a biological specimen which is placed in contact with the upper surface. This binding will be sufficient to permit the specimen to be subjected to analytical processing steps as desired by the user. Although it is possible to implement the present invention with slides of virtually any dimensions and constructed of virtually any materials, in order to facilitate acceptance and use in automated processing apparatus a high degree of standardization is considered to be desirable. In its first aspect, microscope slides are typically used in analytical processing of cell and tissue specimens and the dimensions of such slides have largely been standardized as approximately one inch wide by three inches long or, in metric units, 25 mm wide by 75 mm long by approximately 1 mm thick. Slides have been constructed from numerous different materials including glass and various plastics but glass slides have proven to be the most readily accepted for various properties including their strength, transparency, and resistence to scratching or other defects. Particularly preferred glass includes soda lime glass.

Thus, in part due to the use of a standardized glass slide, the present invention comprises in part a slide base defining a substantially rectangular planar upper surface. By substantially rectangular is meant that a series of slides of the present invention utilizing standardized dimensions will be perceived as rectangular and will very typically only within the confineds of the standard variations inherent in modern manufacturing techniques. For purposes of automated slide processing, it is important that the slides do not vary substantially one from another or from lot to lot, but the use of rectangular slides can be viewed as largely a matter of accepted convention. Although the upper and lower surface of a conventional slide will largely be interchangeable, once the various aspects of the present invention begin to manifest on the slide, the interchangeability of the upper and lower surface is lost and the upper surface of the slide is clearly defined.

In a slide constructed in accordance with the present invention, the upper surface of the slide can be generally recognized as comprising two distinct regions: the reaction region and the labeling region.

The labeling region will be identified on the present slide as consisting of a region of up to approximately 25% of the planar upper surface of the slide and located at one end of the rectangle. Again, for purposes of ease in the use of automation, the labeling region can be viewed as an index region identifying a certain portion of the slide which is not directly involved in the processing of the biological specimen and the remaining portion of the slide will be standardized for recognition by the automated processing apparatus as a region potentially to be treated during the course of analytical processing.

The labeling region of the slide will typically consist of a labeling zone intended for identification of the biological specimen, its origin, the analytical processes to be conducted thereupon, and any additional information which the user considers important to identify in close conjunction with the specimen on the slide. This labeling can be provided by a roughened surface treatment, termed "frosted glass" which enables the user to write information onto the slide as desired. Alternatively, or in addition, such information can be provided by means of a label containing either verbal or machine readable information, such as, e.g., bar code encoded information. Thus, for automated processing apparatus which are capable of discerning and acting upon encoded information, the labeling region provides a means for unique identification of the slide and its contents. Optionally, the labeling region will also include sufficient area to identify, for example, the manufacturer of the slide or the processing apparatus whose operation the slide is intended to facilitate.

In the reaction region of the slide, the present invention provides at least one reaction zone which will be defined on the upper surface of the slide. Again, in order to facilitate automated processing, and to conserve the expenditure of valuable and expensive analytical reagents, it is considered desirable to standardize the size of the reaction zones and their location on the slide surface. By providing a limited number of alternative selections of size and location of reaction zones, the selection from this limited number of slides and the use of, for example, machine readable labels in the labeling zone will facilitate the operation of the automated processing apparatus and the aforementioned conservation in the use of valuable reagents. In the present invention, it is considered desirable to provide at least three alternative configurations for reaction zones. In each case, the outer edges of at least a portion of the reaction zone will be substantially coterminous with the edges of the slide base providing the aforementioned upper surface of the slide. For example, in the first instance, a reaction zone is provided which covers approximately 2/3 of the upper surface of the slide leaving from 1/4 to 1/3 of the upper surface as definin the labeling region. In this embodiment, the reaction zone boundary will be substantially coterminous with the edges of the slide base on three sides and parallel with the fourth side of the slide base. As shown in Figure 1 , utilizing a standard slide in which the slide base is approximately 75 mm long and approximately 25 mm wide, the reaction zone of this embodiment will be approximately 50 mm long and approximately 25 mm wide, assuming that the barrier member which defines the reaction zone will be substantially negligible in width. By substantially negligible is intended to mean on the order of 1 to 2 mm or less. Thus, the hydrophobic barrier member will substantially circumscribe the entire boundary of the reaction zone, desirably allowing only such gaps in continuity as are consistent with automated manufacturing processes. In the ideal case, no gaps will exist and the barrier member will be seen to completely circumscribe the boundary of the reaction zone. By virtue of its application to the slide base, the barrier member will have a certain height in that it will project upwardly from the upper surface of the slide base. However, the magnitude of upward projection is considered to be relative insubstantial in that it is the properties of the barrier member itself rather any mechanical attributes which are intended to define the reaction zone and contain within its boundary any materials applied during the course of processing.

A further embodiment of the present invention is as depicted in Figure 2 in which the reaction zone comprises approximately 50% of the entire upper surface of the slide base. That is, again utilizing a standard slide with the aforementioned dimensions, the reaction zone in this embodiment will be approximately 30 mm long and approximately 25 mm wide. In this regard, a somewhat smaller region of the upper surface of the slide will be utilized for the immobilization of the biological specimen and any subsequent processing steps in automated analytical processing will require application of reagents and other solutions to a decreased area on the slide. In this manner, smaller biological specimens may be mounted in the more constrained region and less reagent coverage will be required in order to insure that the biological specimen has been treated.

Yet another embodiment of the present invention is as depicted in Figure 3. This embodiment comprises a plurality of reaction zones which are defined by the inclusion of two intermediate barrier members subdividing the reaction zone depicted in

Figure 1 into three separate zones of approximately equal dimension. That is, utilizing the standardized slide mentioned previously, each of the three separate zones will comprise a region approximately 15 mm long and approximately 25 mm wide. In this manner, a plurality of biological specimens can be mounted on the same slide and subject to the same, or different, analytical processing steps for purposes of direct comparison. Alternatively, it may simply be desirable to include three specimens of somewhat lesser size in order to increase the throughput of the automated processing apparatus. Although perhaps somewhat more cumbersome, it is contemplated that the information included in the labeling region, typically by resort to machine readable data, will be able to identify the three reaction zones separately by resort to the indexing function of the labeling region and in this regard, separate biological specimens and separate processing steps can be utilized and the information can be tracked appropriately.

The barrier member of the present invention which defines and circumscribes substantially the entire boundary of the reaction zone, will desirably have various properties which inhibit the migration of various materials applied to the reaction zone and substantially prevent cross contamination beyond the reaction zone boundaries. One feature mentioned previously which could be expected to play a modest role in this barrier property would be the height of the barrier member projecting upwardly from the upper surface of the slide base. However, it is anticipated that this contribution would be relatively modest although difficult to gauge in any absolute sense. More importantly, the barrier member will comprise a hydrophobic material and the hydrophobic properties of the barrier member will be expected to perform the majority of the barrier function, particularly in view of the use of aqueous-based reagents and solutions in virtually all automated processing protocols. Use of a hydrophobic compound to form the barrier member, in conjunction with the rather substantial cohesive force as an inherent property in aqueous solutions, provides a substantial physical barrier to the migration of aqueous solutions from the reaction zone. The use of such hydrophobic and hydrophilic interactions thus enables the design of a reaction zone on the slide base which permits a predetermined quantity of reagent to be applied systematically throughout the reaction zone in anticipation that the reagent and/or aqueous solution will remain in a desired location and will not result in loss of reagent nor in cross-contamination between reaction zones on a single slide or between reaction zones on adjacent slides. Although numerous hydrophobic compounds are well known in the art, it is presently considered desirable to make use of a hydrophobic ink to permit the reaction zone to be defined on the upper surface of the slide base by the use of various mechanical manufacturing techniques. Among such techniques include, for example, silk screen application or computerized control of a precise barrier member by utilization of for example, a computer controlled ink jet printer. A desirable component of the hydrophobic ink will be a material such as Teflon^® a well known material having very substantial hydrophobic properties. In this regard, the ink will contain various non- volatile components, such as the Teflon^® and any pigments or other materials intended to promote uniformity and homogeneity in the applied barrier member. Also included will be various volatile components, such as solvents, drying agents, and the like which facilitate the flowing of the ink and its rapid drying once applied to the slide. Thus, the barrier member constructed in accordance with this preferred embodiment will, on the slide, consist of the non- volatile residue of the hydrophobic ink which is utilized in the creation of the barrier member. Although the precise formulation is unknown, a presently preferred embodiment of such ink is termed S-902 gray, a hydrophobic ink including Teflon^® available from Erie Scientific Company, Portsmouth, New Hampshire.

It should also be noted that the various analytic protocols intended to be practiced upon the present slides typically call for at least one "washing" step, in which reagents or solutions previously deposited upon the reaction zone of the slide will be swept clear of the zone, often by resort to a washing solution or removal by other mechanical means. Thus, the resistence of the present reaction zone to the migration of applied reagents or solutions beyond the zone is intended to be overcome by such a washing step, using either a washing solution or other means, such as a blast of a gas such as air.

Yet another desirable feature of the present invention is the use of a means for increasing the binding between the upper surface of the slide, primarily within the reaction zone defined on the upper surface, and a biological specimen which is brought into contact with the upper surface and thus immobilized in the reaction zone for subsequent analytical processing. Clearly it is desirable that the binding between the biological specimen and the upper surface of the slide within the reaction zone be sufficient to permit the specimen to be subjected to reasonable treatments commensurate with the accepted protocols for analytical processing. In this regard, the specimen should be prevented from detaching from the slide once it has been immobilized and should remain substantially permanently attached during subsequent processing, analysis, and storage.

The means for increasing the binding between the surface of the slide and the biological specimen can be included as a feature of the entire slide or can be restricted or limited to the reaction zone proper. The selection of the appropriate technique for application will depend largely on considerations of efficiency of manufacture of the present slides and, possibly, the cost of the material used in providing the increased binding.

Although it is possible to employ slides of plastic materials, such as polymethyl methacrylate or polycarbonate, which have substantial binding affinity for the most commonly encountered biological specimens, it has been noted previously that glass slides are particularly preferred in spite of the fact that it is desirable to increase the binding between the surface of the glass slide and the biological specimen.

Typically, the region of the upper surface of the slide base within the reaction zone can be treated with an alkaline agent, such as with an alkali metal hydroxide or alkaline earth metal hydroxide, such as sodium hydroxide, or an alkaline detergent, which would render the treated surface of the slide base alkaline and thus provide a net positive charge. This net positive charge can have the effect of increasing the binding between the glass surface and the biological specimen or further agents can be employed to enhance or otherwise modify this binding effect. Substances which have been shown to be suitable for this purpose are those which will adhere to the glass surface and bind to cells or proteins characteristic of the biological specimens both basic proteins and basic dyes such as poly-L-lysine, spermidine, polyarginine, and the like will bind to various components of biological cells or tissues, or isolated biological proteins as a result of the net negative charge on the cell or protein surface. As an alternative, numerous substances are known which are capable of reacting with specific molecules including molecules located on the surface of cells and tissues, such as, for example, antibodies and including those which are specific to specified cell membrane antigens. This technique can be utilized for the isolation of specific proteins and cells where the slide of the invention is to be utilized not only for the immobilization of the biological specimen but for its identification and selection as well.

Although numerous alternatives are well known in the art for enhancing the binding between glass and biological specimens, it is presently preferred to utilize a proprietary process developed by Erie Scientific Company, Portsmouth, New

Hampshire, known as the ADCELL advanced cell adhesive technology which has been shown to be particularly advantageous for attracting biological cells and tissue sections to the glass surface and binding the biological specimens securely to the glass. This technique is claimed to result in better establishment of monolayer specimens which improves the biological information density per square millimeter of the reaction zone.

This technology is claimed to work well for adhering fresh cells, frozen tissue sections, and formalin fixed tissue specimens for various analytical processing protocols including immunoperoxidase, immunoalkalinephosphatase, color imetric and radiolabeled protocols for in situ DNA hybridization procedures. It has also been shown to be useful for use with frozen tissue sections, and other cytology preparations.

The specimens will bind to the slide and remain in place during staining procedures, microwave and chemical antigen retrieval procedures (subject of U.S. Patent Nos. 5,244,787 and 5,578,542 the entire contents of which are incorporated herein by this reference) as well as enzyme digestion, DNA denaturation and RNA hybridization procedures. It has however been noted that once the ADCELL-treated glass slides have been exposed to environmental agents, the net positive charge and thus the binding enhancement may dissipate over time.

The following examples are provided in order to illustrate various aspects of the present invention and should not be construed as limiting the scope thereof.

Examples

The following examples illustrate the preparation of slides in accordance with the present invention for the immobilization and subsequent analytical processing of biological specimens. The slides will have at least one reaction zone defined by hydrophobic barrier circumscribing substantially the entire boundary of the zone. Example 1

A soda lime glass slide having the dimension 75 mm by 25 mm by 1 mm plus or minus 0.015 inches is provided. The soda lime glass will consist of approximately 72.2% silicon dioxide, 14.3 % sodium oxide, 1.2% potassium oxide, 6.4% calcium oxide, 4.3% magnesium oxide, 1.2% aluminum oxide and trace amounts of ferric oxide and sulfur trioxide. The slide will desirably be free of embedded foreign material such as bubbles, blisters and internal cloudiness and will also be free of rough or sharp cutting surfaces and edges. Optionally, one end of the slide as depicted in Figure 1 may be frosted to provide a labeling region for verbal or machine readable information. Upon the surface of the slide is screen printed a hydrophobic barrier region in the dimensions depicted in Figure 1 utilizing S902 gray hydrophobic ink containing Teflon^® (Erie Scientific Company, Portsmouth, NH). The barrier member circumscribing the reaction zone will have a width of approximately 1 mm in the members defining the length of the reaction zone and approximately 2 mm in the members defining the width of the reaction zone.

The slide will have been treated with ADCELL adhesive cell treatment (Erie Scientific Company, Portsmouth, NH) to enhance its specimen binding characteristics.

Example 2

A soda lime glass slide having the dimension 75 mm by 25 mm by 1 mm plus or minus 0.015 inches is provided. The soda lime glass will consist of approximately

72.2% silicon dioxide, 14.3 % sodium oxide, 1.2% potassium oxide, 6.4% calcium oxide, 4.3% magnesium oxide, 1.2% aluminum oxide and trace amounts of ferric oxide and sulfur trioxide. The slide will desirably be free of embedded foreign material such as bubbles, blisters and internal cloudiness and will also be free of rough or sharp cutting surfaces and edges. Optionally, one end of the slide as depicted in Figure 2 may be frosted to provide a labeling region for verbal or machine readable information.

Upon the surface of the slide is screen printed a hydrophobic barrier region in the dimensions depicted in Figure 2 utilizing S902 gray hydrophobic ink containing Teflon^® (Erie Scientific Company, Portsmouth, NH). The barrier member circumscribing the reaction zone will have a width of approximately 1 mm in the members defining the length of the reaction zone and approximately 2 mm in the members defining the width of the reaction zone.

Example 3

A soda lime glass slide having the dimension 75 mm by 25 mm by 1 mm plus or minus 0.015 inches is provided. The soda lime glass will consist of approximately 72.2% silicon dioxide, 14.3% sodium oxide, 1.2% potassium oxide, 6.4% calcium oxide, 4.3 % magnesium oxide, 1.2% aluminum oxide and trace amounts of ferric oxide and sulfur trioxide. The slide will desirably be free of embedded foreign material such as bubbles, blisters and internal cloudiness and will also be free of rough or sharp cutting surfaces and edges. Optionally, one end of the slide as depicted in Figure 3 may be frosted to provide a labeling region for verbal or machine readable information. Upon the surface of the slide, is screen printed a hydrophobic barrier region in the dimensions depicted in Figure 3 utilizing S902 gray hydrophobic ink containing Teflon^® (Erie Scientific Company, Portsmouth, NH). The barrier member circumscribing the reaction zone will have a width of approximately 1 mm in the members defining the length of the reaction zone and approximately 2 mm in the members defining the width of the reaction zone. The inclusion of two intermediate barrier members thus defining three substantially identical reaction zones is also depicted in Figure 3.

Example 4

Slide prepared in accordance with Examples 1 through 3 are thereafter employed in numerous analytical processing protocols by utilizing the slides in conjunction with the optimax automated immunostainer device (BioGenex Laboratories, San Ramon, California) in accordance with the instructions of the manufacturer. This automated immunostainer is the subject of U.S. Patent No. 5,439,649, the entire contents of which are specially incorporated by this reference.

In this manner, a uniform system of slides is provided for performing numerous analytical protocols as described previously while enabling the conservation of various costly reagents and preventing cross-contamination on the slides and between slides all by resort to the practice of the present invention.

All patents and patent applications cited in this specification are hereby incorporated by reference as if they had been specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those of ordinary skill in the art in light of the disclosure that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

Claims

1. A slide for the immobilization and subsequent analytical processing of biological specimens comprising: a. a slide base defining a substantially rectangular planar upper surface: b. at least one reaction zone defined on said upper surface comprising a hydrophobic barrier member circumscribing substantially the entire boundary of said reaction zone, which barrier member projects upwardly from said upper surface to a height sufficient to prevent a predetermined amount of an aqueous solution deposited on said reaction zone from crossing the boundary of the zone; and c. means for increasing the binding between the upper surface of said slide within said reaction zone and a biological specimen which is placed in contact with said upper surface; which binding is sufficient to permit said specimen to be subjected to analytical processing.

2. A slide as recited in Claim 1, wherein said reaction zone boundary is substantially continuous with the edges of said slide base on three sides, and is parallel with the fourth side of said slide base.

3. A slide as recited in Claim 2, wherein said slide base is approximately 75 mm long and approximately 25 mm wide.

4. A slide as recited in Claim 3, wherein said reaction zone is approximately 50 mm long and approximately 25 mm wide.

5. A slide as recited in Claim 3, wherein said reaction zone is approximately 30 mm long and approximately 25 mm wide.

6. A slide as recited in Claim 4, wherein said reaction zone further comprises two intermediate barrier members which subdivide said reaction zone into three separate zones which are each approximately 15 mm long and approximately 25 mm wide.

7. A slide as recited in Claim 1, wherein said boundary member comprises a hydrophobic compound affixed to said slide base.

8. A slide as recited in Claim 7, wherein said hydrophobic compound comprises Teflon^┬«.

9. A slide as recited in Claim 8, wherein said hydrophobic compound is the non- volatile residue of a hydrophobic ink.

10. A slide as recited in Claim 1 wherein said means for increasing binding comprises a material which provides a bond between said slide upper surface and said biological specimen.

11. A slide as recited in Claim 10 wherein said bond is an ionic bound.

12. A slide as recited in Claim 11 wherein said binding means provides a net positive charge to the upper surface of said slide base within said reaction zone.