Fluid exchange in a chamber on a microscope slide

Details Fluid exchange in a chamber on a microscope slide Fluid exchange in a chamber on a microscope slide

2000-04-14

2004-01-06

Warden, Jill (Department: 1743)

Chemistry: analytical and immunological testing

Automated chemical analysis

With sample on test slide

C436S174000, C436S180000, C422S091000, C422S105000, C422S105000, C422S105000, C359S398000

Reexamination Certificate

active

06673620

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to an instrument that is capable of performing incubations of small volumes of reagents on the surface of a microscope slide. A variety of assays are typically carried out on the surface of a microscope slide. These assays generally aim to determine if a suspected analyte is present in a patient biopsy. They include: (1) in situ hybridization, for the detection of nucleic acid targets in a tissue or cell sample, (2) immunohistochemistry, for the detection of specific proteins in a tissue or cell sample, (3) histochemical stains, for the detection of certain types of chemical compounds or classes of microorganisms in a tissue sample. In addition, there are two other types of assays that are often carried out on the surface of a glass slide. Rather than testing for the presence of an analyte in a tissue biopsy, these assays aim to detect specific molecules in a solution. They are (1) gene arrays, whereby an array of known nucleic acid targets are immobilized directly on the glass slide, and (2) protein arrays, hereby an array of known proteins are immobilized on the glass slide.
In each of these instances, a glass slide serves as the preferred support on which the assay is carried out. The reason a glass slide is used is that it is optically clear and flat. These physical properties facilitate the ability of an instrument, such as a microscope, to optically detect a fluorescent or colorimetric signal. In order to highlight certain desired features, the above assays require that the slides be treated with a series of reagent incubations. Each incubation needs to occur for a specific time (typically 15-60 minutes) and at a specified temperature (typically, from room temperature to 95° C.).
The optical advantages of a microscope slide are somewhat counterbalanced by certain difficulties in performing the assay. The treatment of the tissue sections on a microscope slide for the purpose of highlighting certain histologic features is often called “staining.” Since the surface of the slide is flat, reagent can easily drain off the edge of the microscope slide, especially if the slide is not perfectly level. Moreover, the large surface area to volume ratio of reagent spread over the slide surface promotes evaporation. Evaporation of reagent interferes with the performance of the assay. If the reagent evaporates, then it will not continuously contact the tissue sample. Drying artifacts may cause the assay result (“stain”) to not be accurate. Lastly, it is important to spread the reagent over the slide surface. Surfactants are sometimes used to promote reagent spreading. If the reagent does not spread, then the reaction may fail to occur over all of the tissue biopsy, or over all portions of the array. Therefore, the prior art comprises a great number of attempts to construct apparatus and devices that aim to facilitate or automate the sample preparation/treatment steps of a biological sample on a glass slide.
The general approach to solving these problems in the past has been to enclose an area of the slide surface, forming a chamber. Desirable features for such a chamber are:
a) Liquid spreading. Reagents must be evenly spread, without entrapped air bubbles.
b) Use of minimal reagent volume (ideally less than 100 microliters to cover the slide surface).
c) Prevent evaporation when the reagent is heated to 95° C.
d) Automatic reagent injection and removal. Namely, the apparatus needs to be compatible with an automated fluid transfer system.
e) Protection of the tissue section against physical damage.
One method of addressing at least some of the requirements described above is to entrap reagent under a coverslip. For in situ hybridization procedures, reagent is conventionally placed directly on top of the tissue section with a pipette and covered with a coverslip. The edges of the coverslip are then sealed with nail polish or rubber glue. The coverslip both spreads out the reagent into a relatively uniform layer and prevents evaporation. It is important to avoid entrapping air bubbles under the coverslip. Otherwise, there will be an area of the tissue section that does not contact the hybridization solution.
No existing technology is suited to automating coverslipping for in situ hybridization. Coverslips can be applied to slides in an automated fashion; several companies serving the histopathology market sell dedicated coverslipping machines. However, such coverslipping machines are not likely to be adaptable to this application, because (i) it will be difficult to automate sealing the coverslip edges, such as with glue, and (ii) it will be difficult to robotically remove the coverslips without damaging the tissue section.
An alternative method for spreading small amounts of reagents was described in 1988, by Unger, et.al. (Unger, E. R., D. J. Brigati, M. L. Chenggis, et.al. 1988. Automation of in situ hybridization: Application of the capillary action robotic workstation.
J. Histotechnology
11:253-258.) Specifically, they described a modification of the Code-On slide stainer for use with in situ hybridization. Instead of a coverslip, two slides were placed in close apposition to each other, forming a capillary gap. Liquids, such as a hybridization solution, could then be applied to the bottom of the gap. The reagents “wicked” in by capillary action. Placing the slides in a heated humidity chamber prevented evaporation. The Code-On worked well in the right hands, but was labor-intensive and required a great deal of experience and care each day in setting it up. Slight scratches or imperfections in the surface of the glass slide sometimes caused air bubbles to be entrapped in the capillary gap, resulting in areas of the tissue not being stained.
An early description of a slide chamber is disclosed in U.S. Pat. Nos. 4,847,208 and 5,073,504. A chamber was apposed to the surface of a slide, forming walls capable of preventing lateral spillover of reagent. Moreover, a hinged cover minimized the evaporative loss of reagent. Each chamber included a fluid inlet and outlet port.
WO99/34190 discloses a chamber formed by the insertion of a microscope slide into a cartridge device. Reagent is dispensed onto a portion of the slide that protrudes from the cartridge and is caused to flow into a capillary gap-type space by moving the slide inwards. The chamber is not sealed from the outside environment. Therefore, reagent will be expected to evaporate, especially if the samples are heated. Moreover, reagent can flow around and underneath the slide, thereby increasing the volume requirement to cover the slide surface. Lastly, it is unclear how often bubbles will be entrapped over the slide surface, since no specific mechanism in the cartridge prevents them.
A review of other methods of forming a chamber, and their drawbacks, are also described in the Background section of WO99/34190.
SUMMARY OF THE INVENTION
In embodiments of the present invention, a fluid handling apparatus is capable of spreading small amounts of liquid reagent over a flat surface, such as a microscope glass slide. The reagent may be sealed within a confined cavity, or “chamber”, so as to prevent evaporation even with heating of small amounts of reagent during an incubation period. One surface of this chamber is the flat slide surface. The remaining surfaces are formed by a cell. The cell is preferably a plastic disposable part that fits on top of the slide, over the area containing the tissue, biologic cells, or array mounted on the glass slide. The cell forms a fluid seal to the surface of the glass by means of a gasket. The gasket is mounted in a recess on the face of the cell that mates with the glass slide.
Each cell has two fluid ports. These ports are in fluid communication with the chamber. Therefore, when liquid reagent is inserted into a fluid port, it can travel into the chamber and contact the biologic sample or array mounted on the glass surface. The fluid ports on a cell are preferably positioned on opposite ends of the cell. This allows for liquid reagent or wash sol

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