Chemistry: analytical and immunological testing – Including sample preparation – Volumetric liquid transfer
Reexamination Certificate
1998-12-23
2004-03-09
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Volumetric liquid transfer
C422S051000, C422S091000, C422S105000, C422S105000, C435S287200, C436S046000
Reexamination Certificate
active
06703247
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to an apparatus for processing biological samples on slides for a wide variety of purposes. Biological samples are analyzed for many purposes using a variety of different assays. Pathologists often use histochemistry or immunocytochemistry for analyzing biological samples, molecular biologists may perform in situ hybridization or in situ polymerase chain reactions on biological samples, etc. Often the sample to be analyzed will be embedded in paraffin and mounted on a microscope slide.
The assays usually involve the use of antibodies, enzymes and other expensive reagents and it is desirable to keep reagent volume use to a minimum to lower costs. These assays are also quite labor intensive although there are now some automated systems (e.g., the Ventana ESIHC Staining System, the Shandon Lipshaw Cadenza Automated Immunostainer; also see Brigati et al. (1988)). The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References. Most automated systems can only perform 40 to 48 slides per run. Fisher automated systems can perform 120 slides per run. Most automated systems which only perform immunocytochemistry do not perform deparaffinizing, histochemistry (such as hematoxylin and eosin staining) and coverslipping steps and these consequently must be done separately by hand which is time and labor intensive. The automated systems perform only a small part of the overall process of preparing and analyzing slides. Steps which are still manually performed-prior to the automated portion include sorting of cases and slides, labeling slides, programming the automated equipment, daily antibody and reagent preparation, preparing control tissue which is mounted on slides, and microwave antigen retrieval. Procedures still performed manually after the automated steps are dehydration, coverslipping, slide labeling and sorting of slides and cases. Furthermore, most commercial ready-to-use reagents are not suitable for automated systems which are required to use specially designed reagents. Laboratories which process large numbers of samples are likely to be willing to pay the high cost associated with buying these automated systems as well as the high cost of using the disposable accessories and reagents to perform the assays, but small to intermediate sized laboratories find it more cost effective to continue to process samples manually.
A typical immunocytochemistry assay requires a series of many steps. These include: obtaining a biological sample such as from a biopsy, fixing the sample in formalin, processing the sample overnight, embedding the sample in paraffin, cutting serial sections and mounting on microscope slides and drying. These steps are followed by steps to deparaffinize (treatments in xylene, ethanol and water), and finally the reaction can be performed on the sample which has been mounted on the slide. Typically a series of solutions including reagents such as enzymes, primary antibody, secondary antibody, detection reagent, chromogen, counterstain, etc. is dropped onto the slide, incubated, and washed off. Finally the sample may be viewed under the microscope. Clearly there are many individual steps involved and each sample on a slide must be processed individually. Besides being very labor intensive, there are drawbacks associated with the commonly used method of simply dropping solutions on top of the mounted sample on the microscope slide. The solution is not restricted simply to the area of the biological sample itself and the solution may be relatively deep rather than being a thin layer. These features require use of extra reagents which are quite expensive. Leaving the solutions open to the air as they sit on the slide also may lead to evaporation if the samples must incubate for a long period of time. Evaporation leads to concentration or drying out of the reagents and high concentrations may lead to increased background levels which are clearly undesirable. If the solutions evaporate totally the assay will fail. Incubating samples in humidity chambers with covers may prevent evaporation problems, but water droplets which condense onto the humidity chamber cover may fall onto the slides and this will ruin the assay.
Improved methods for more rapidly assaying several samples at once, but without the high cost of automated systems, will be welcomed by small to intermediate sized laboratories. Furthermore, methods which will allow use of smaller amounts of reagents and overcome the drawbacks of processing samples on slides open to the atmosphere will be a welcome advance.
SUMMARY OF THE INVENTION
The present invention is an apparatus for performing manual assays on biological samples mounted on microscope slides. One aspect of the invention is a multislide slideholder capable of holding multiple standard microscope slides, preferably 3-10 slides and more preferably 3 or 6 slides, thereby allowing for the processing of multiple samples at one time. A second aspect of the invention is a multiwell tray containing multiple shallow wells, preferably 3-10 and more preferably 3 or 6 such wells, into which reagents are placed and upon which the slideholder plus slides is placed. A third aspect of the invention is a set of prealigned and prespaced coverslips for rapidly placing said coverslips onto the processed slides. Another aspect of the invention is a second type of multiwell tray which is useful in automating several of the steps of the procedures.
Besides this new design of a slideholder and corresponding tray and coverslips, other aspects of the invention are set out which aid in making assays more rapid and convenient. One such aspect is the use of reagents which are predried in the wells of the tray thereby simply necessitating the addition of water or buffer to the well without having to add the reagents at the time of assay. The well is then covered with a slide with a biological sample premounted on the slide. The different wells of a multiwell tray can be pretreated with different reagents dried in each well. Multistep assays can be performed by moving a slideholder with attached slides from one multiwell tray to the next, with each well of a multiwell tray having the desired reagents predried on it. A variation of this is to employ a multilayer coating of reagents in each well such that the first set of reagents dissolves quickly and acts upon the biological sample, the second layer then dissolves releasing the reagents for the second step, etc., thereby requiring the use of fewer trays, possibly only a single tray.
Another aspect of the invention is to have built in controls on each slide. This is a portion of the slide to which are attached positive and negative controls. These controls allow one to determine whether the assay has worked properly for each individual slide since each slide has its own set of controls and which simultaneously act as labels for each slide.
The slideholder is designed in conjunction with the tray. The purpose of the slideholder is to have multiple slides, preferably up to six slides, all attached to a single holder so that all the attached slides may be processed simultaneously throughout all of the steps of the staining procedure from deparaffinizing to coverslipping without ever separating the slides from the slideholder. This is a labor intensive step and the ability to process multiple slides at once rather than processing slides individually is an important aspect of the invention. Since one technician typically is capable of easily processing about 40-50 individual slides without mistakes, using a slideholder with six slides per slideholder will allow a single technician easily to perform approximately 240-300 slides for routine histochemistry and immunochemistry staining. This is about 2-6 times as many slides as handled by automated systems per each run.
One useful aspect of the pre
American Registry of Pathology
Rothwell Figg Ernst & Manbeck
Soderquist Arlen
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