Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1999-03-02
2001-04-17
Beisner, William H. (Department: 1744)
Chemistry: analytical and immunological testing
Biological cellular material tested
C435S040500, C435S040510, C435S288300, C435S286300, C435S287300, C436S046000, C422S063000, C422S105000
Reexamination Certificate
active
06218191
ABSTRACT:
The present invention relates to a method and apparatus for treatment of human or animal cell samples. In particular, the invention relates to treatment of the samples to enable diagnosis of clinical conditions. A sample is fixed on a flat surface such as a microscope slide and chemically treated with liquid for the purpose of sample hydration or dehydration, or sample staining, or in chemical analysis such as detection of antigens or nucleic acid sequences, for example. The liquids used to treat such sample include:
1. Organic solvents.
2. Antibodies.
3. DNA and RNA probes.
4. Chemical solutions.
5.Washing solutions.
Conventionally the chemical treatment and the chemical analysis of the samples is done by immersing the glass slides on which the samples are fixed into beakers that contain the treatment solutions. Certain treatment liquids are very expensive and are therefore dispensed onto a slide using a pipette with the slide in a horizontal orientation and a glass coverslip is placed on top of the slide to provide spread of the solution and to slow the rate of evaporation of the expensive treatment liquid. The conventional process is labour intensive, exposes workers to reagent fumes and possibly to contact with the chemicals. Accurate timing of the processing steps can also be difficult to achieve. The amount of liquid waste generated is often large, which may be a problem, since the waste that needs to be disposed can contain aggressive solvents or biohazards such as infectious viruses. To overcome some of these problems a number of inventions have been previously proposed for automating the process.
In U.S. Pat. Nos. 4,731,335 and 4,777,020 and 5,002,736 Brigati, D. et al there is described a system where two flat surfaces such as microscope slides are placed face to face with sample sides facing inward. Abutting coating portions of the slides define a capillary gap between the samples. This slide pair can be placed so that the lower edge of the slide pair connects with the treating liquid which will then migrate into the capillary gap. Liquid can then be removed from the gap by placing the slide pair on top of and in contact with absorbent material which will drain and absorb the liquid.
Shandon Scientific Limited U.S. Pat. No. 4,985,206 describes an apparatus for processing tissue. The core of the invention is a channel-defining element. This element is joined together with a slide holding the sample with the sample side facing towards the element. The element forms a channel between its main wall and the slide. When the channel is substantially vertical the upper part of the element forms a liquid dispensing reservoir. An operator or a liquid handling robot is can then fill the reservoir with appropriate reagent. Gravity and capillary action will cause the reagent to migrate into the channel. Once the channel is filled with liquid and the reservoir is empty, the liquid will stay in the gap due to surface tension of the liquid. The liquid in the gap can be replaced by placing new reagent in the reservoir.
Toya, M. et al in U.S. Pat. No. 5,068,091 and UK patent 2,265,981 describes a substantially horizontal wedge shaped capillary gap between a microscopic slide and lower plateau. Liquids can be dispensed to an exposed end of the plateau and capillary action will cause them to migrate to the wedge shaped gap. The gap can then be cleared of the reagent by using suction. Surface tension of the liquid will keep the liquid volume together during the removal process.
The aforementioned prior art apparatus all suffer a disadvantage in that they can in some instances fail to provide an even treatment of the sample with the treating liquid. This is caused by air becoming trapped in the capillary gap. In the case of the Brigati inventions, capillary forces can only lift the liquid a certain distance upwardly from the lower edge of the slide pair and this can lead to a reduced treatment area on the slide. The speed of liquid removal cannot be controlled in the Brigati inventions. The capillary gap also needs to be drained before a new liquid can be applied. These form a disadvantage, because in certain cases it is desirable that the samples are not exposed to air at all when replacing liquids. This is desirable especially when using volatile liquids such as organic solvents that evaporate easily and may let samples dry out during liquid replacement. Sample drying can lead to reduced processing quality such as high non-specific staining. In other cases a film of liquid should be left on the sample to keep it moist during liquid replacement. In the remaining cases it is desirable that the samples are dried completely before applying a new liquid to ensure maximum concentration of the applied liquid.
The apparatus of Shandon has the additional problem that no provision is made for clearing the gap (filling it with air) between different liquid treatments and therefore any air voids trapped in the gap are likely to remain through the process. Also the apparatus of Shandon cannot provide capability to expose the sample to air during processing while liquids are replaced. In the apparatus of Toya M. et al the suction to clear the capillary gap can lead to a breaking-up of the liquid into two or more sections with only one section being sucked into the waste containment system and such an incomplete clearing of the liquid can cause unacceptable treatment of the sample.
Further, the abovementioned prior art all suffer a disadvantage in that the treatment liquid is exposed to the atmosphere and will consequently quickly evaporate, particularly when subjected to heating which is often required for incubation to occur. If the treatment liquid evaporates too quickly the sample will dry out which results in a poor quality treatment or erroneous results.
It has been previously proposed to reduce the rate of evaporation by controlling the humidity of the surrounding atmosphere. This has been most commonly achieved by the use of an electrically heated water bath. However, the level of humidity is difficult to control. Too little humidity can result in the sample drying out and excess humidity can result in condensation forming on the glass slides. This condensation can result in the treatment liquid becoming diluted which lengthens the incubation and can lead to erroneous results. In addition, the condensation forms on surrounding apparatus and chamber walls which make it difficult to observe the process, as well as leading to degradation and corrosion of machine parts. In addition, the warm water in the water bath is a potential source of bacteria and contaminant build up.
Other methods have been proposed for minimizing evaporation, including nail polish or rubber cement to seal coverslip edges onto slides. However, these methods are laborious and difficult to automate. Also, the coverslips are difficult to remove after incubation has been completed since in the first example, acetone is required to remove the nail polish and in the second, the rubber cement must be peeled off manually.
PCT/US91/01108 discloses yet another method of reducing evaporation of a treatment liquid in which an aqueous treatment liquid covering a sample on a glass slide is itself covered by an evaporation inhibitor liquid. This method has the disadvantage that it is not suitable for use with a coverslip since mixing of the treatment liquid and evaporation inhibitor liquid will occur when the coverslip is applied. Further, excessive treatment liquid is required to be applied with this method to ensure an even coverage of the sample by the treatment liquid.
Therefore, it is an object of the present invention to provide a method and apparatus for treating a human or animal cell sample with treatment liquid, in a manner which avoids or at least minimizes evaporation of the treatment liquid.
Accordingly, the invention provides a method of treating a human or animal cell sample with treatment liquid comprising the steps of applying the treatment liquid to a cell sample in a cavity formed between a slide on which the sample i
Beisner William H.
Pillsbury & Winthrop LLP
Vision Instruments Limited
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