WO2014130576A1

WO2014130576A1 - Automated fish analysis of tissue and cell samples using an isolating barrier for precise dispensing of probe and other reagents on regions of interest

Info

Publication number: WO2014130576A1
Application number: PCT/US2014/017204
Authority: WO
Inventors: Brian L. KIRK; Anthony V. Lemmo; Thomas C. Tisone; Gary R. SAMS
Original assignee: Biodot, Inc.
Priority date: 2013-02-19
Filing date: 2014-02-19
Publication date: 2014-08-28

Abstract

Devices and methods for performing FISH (Fluorescent/Fluorescence In Situ Hybridization) analysis of biological and/or chemical samples are described, such as, on a substrate or slide. Some of these relate to FFPE (Formalin-Fixed, Paraffin-Embedded tissue and circulating tumor cell sample(s) FISH probe addition and hybridization. Automated systems and methods for performing analysis of FFPE tissue and circulating tumor cell sample(s) utilizing FISH probe addition and hybridization are advantageously incorporated. Some desirable advantages and benefits include novel and inventive methods and technologies for precisely identifying, locating and isolating regions or areas of interest for FISH by providing a barrier to isolate said regions or areas such that expensive probe, and other reagents, can be utilized in an efficient and economical manner, to maximize or enhance process efficiency.

Description

AUTOMATED FISH ANALYSIS OF TISSUE AND CELL SAMPLES USING AN ISOLATING BARRIER FOR PRECISE DISPENSING OF PROBE AND OTHER REAGENTS ON REGIONS OF INTEREST

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/766,583, filed February 19, 2013, and U.S. Provisional Patent Application Serial No. 61/934, 185, filed January 31, 2014, the entirety of each one of which is hereby incorporated by reference herein, and comprises a part of the instant disclosure.

FIELD

[0002] The invention relates generally to FISH (Fluorescent/Fluorescence In Situ Hybridization) probe addition and hybridization of biological and/or chemical tissues and cell samples on precisely located region(s) or area(s) of interest, and more particularly, in some examples, of FFPE (Formalin-Fixed, Paraffin-Embedded) tissue and circulating tumor cells FISH probe addition and hybridization, and further to automated systems and methods for performing the FISH probe addition and hybridization with an isolating barrier for precise and cost-effective dispensing of probe, and other, reagents on regions or areas of interest on a substrate such as a slide.

BACKGROUND INFORMATION

[0003] In order to perform a fluorescent in situ hybridization (FISH) process on tissue or cell samples, tissue or cell sample(s) is/are applied to a microscope slide, and a scientist/technician determines the location of a tumor (cancerous) section of the sample relative to the normal area or areas of the one or more tissue or cell samples. Once the scientist/technician has determined the size and location of the cancerous section(s) relative to the normal section(s) within the sample, the scientist then applies a "probe" reagent that contains the molecular material selected to stain the sample and produce the appropriate signals for analysis that will indicate the presence and/or condition of a particular gene.

[0004] After the probe has been applied to the slide, the probe is sealed to the slide by applying a glass coverslip to the slide. Then a bead of sealant material (often the material is rubber cement) is applied around the border of the coverslip to produce a barrier between the "probe" reagent and the environment. The slide is heated to expose the sample DNA to the probe DNA molecules and then brought to a temperature appropriate for "hybridization" in which the probe or labeled DNA can bind to the gene of interest.

[0005] Disadvantageously, the slide is generally "flooded" with the probe reagent(s), which are expensive, due to the lack of isolating the regions of interest. Moreover, due to the complexity of the reagent, "probe reagent" is expensive. Undesirably, once again, since the current process is performed manually the scientist must flood the entire tissue area (including the normal tissue sections) with probe in order to ensure that the tumor section is covered. Disadvantageously, much of the expensive probe is wasted during this process since the primary or main region of interest is the tumor sections. Excessive probe reagents also end up being used because it is not possible or feasible to have an air/probe interface under the coverslip during hybridization as undesirably, such an interface will cause the probe to dry out during the hybridization process and this will compromise the ultimate signal quality.

[0006] Currently, scientists typically try to either select special coverslips that only cover the tumor section or create custom coverslips that cover only the tumor section of the tissue in order to minimize the amount of probe reagent utilized for the assay. Disadvantageously, this current manual process is tedious, error prone, and wasteful with respect to the expensive probing reagents.

SUMMARY

[0007] Devices and methods for performing FISH (Fluorescent/Fluorescence In Situ Hybridization) analysis of biological and/or chemical samples are provided in accordance with some embodiments. Some embodiments relate to FFPE (Formalin-Fixed, Paraffin- Embedded) tissue and circulating tumor cell sample(s) FISH probe addition and hybridization. Desirably, automated systems and methods for performing FFPE tissue and circulating tumor cell sample(s) FISH probe addition and hybridization are utilized and incorporated in accordance with some embodiments. Advantages, in accordance with at least some embodiments, include novel and inventive methods and technologies for isolating precisely located and identified region(s) or area(s) of interest for FISH by providing a barrier to isolate the at least one regions or areas such that expensive probe, and other reagents, can be utilized in an efficient and economical manner, for maximizing or enhancing process efficiency. [0008] In accordance with some embodiments, a method of FISH analysis of one or more tissue or cell samples is provided. The method comprises identifying at least one region or location of interest on a slide or substrate of the tissue or cell sample. The coordinates of the at least one region of interest on the slide or substrate are located. An immiscible material is dispensed to circumscribe said at least one region of interest so as to form an isolated barrier over said at least one region of interest. One or more probes are precisely dispensed within the identified one or more isolated regions of interest to perform FISH analysis of the one or more samples.

[0009] In accordance with some embodiments, a method of FISH analysis of one or more circulating tumor cell samples is provided. The method comprises identifying at least one region or location of interest on a slide or substrate of the cell sample. The coordinates of the at least one region of interest on the slide or substrate are located. An immiscible material is dispensed to circumscribe the at least one region of interest so as to form an isolated barrier over the at least one region of interest. One or more probes are precisely dispensed within the identified one or more isolated regions of interest to perform FISH analysis of the one or more samples.

[0010] Certain embodiments of the invention disclosed herein reduce, mitigate or overcome the disadvantages of the currently used FISH technologies as applied to FFPE tissue samples and circulating tumor cell samples.

[0011] In accordance with some embodiments, advantageously, automated systems, devices and methods are utilized for FFPE tissue sample, including circulating tumor cell samples, FISH probe addition and hybridization. In some embodiments, desirably, an immiscible material such an oil barrier or rubber cement can be used for miniaturizing or minimizing the amount of expensive probe that is utilized for the hybridization process, by selectively isolating the regions(s) of interest on the slide or substrate.

[0012] It is one advantage of certain embodiments the invention to provide and use an immiscible and non-reactive fluid around a designated tumor section (either on isolated tumor section or on the complete normal tissue/tumor tissue embodiment). Desirably, the immiscible fluid acts as a barrier to air and evaporation during the hybridization process. The probe reagent is dispensed inside the pattern made by the immiscible fluid which substantially circumscribes the tumor section of the tissue sample. The fluid can be oil such as mineral oil, rubber cement or the like, among others. In some embodiments, a one or more cell samples, such as circulating tumor cell samples, may be the regions(s) or area(s) of interest.

[0013] Certain embodiments of the invention provide the following systems, devices, mechanisms and methods for performing FFPE tissue sample(s) (and circulating tumor cell sample(s)) FISH probe addition and hybridization, individually, or in any combination thereof. Moreover, particular aspects of the disclosure and/or non-limiting embodiments of the invention(s) are set out in at least the following numbered items, any of which may be combined as appropriate:

1. An automated system or device that comprises or utilizes a camera system or another sensor to determine a scribed or visibly marked section on the slide or substrate that defines the area and position of a tumor section.

2. A mechanism, method or process of applying an immiscible and non- reactive fluid around the designated tumor section (either on isolated tumor section or on the complete normal tissue/tumor tissue embodiment). The immiscible fluid acts as a barrier to air and evaporation during the hybridization process. The probe reagent is dispensed inside the pattern made by the immiscible fluid which circumscribes the tumor section of the tissue sample.

3. An automated system or device that is adapted and configured to dispense the immiscible, non-reactive reagent around both regularly and irregularly shaped tumor sections, single or multiple tumor sections, or tumor sections of various sizes on a single or multiple slides.

4. The probe reagent may be dispensed as a single dispense that effectively covers the tumor section or sections or the probe reagent may be dispensed as a smaller dispenses that effectively cover the tumor section. The smaller dispenses may range from about 10 nl (nanoliters) to about 100 nl, about 101 nl to about 200 nl, about 201 nl to about 300 nl, about 301 nl to about 400 nl, about 401 nl to about 500 nl, about 501 nl to about 600 nl, about 601 nl to about 700 nl, about 701nl to about 800 nl, about 801 nl to about 900 nl, about 901nl to about 1,000 nl, about 1,001 nl to about 2,000 nl, about 2,001 nl to about 3,000 nl, about 3,001nl to about 4,000 nl, about 4,001 nl to about 5,000 nl, about 5,001 nl to about 6,000 nl, about 6,001 nl to about 7,000 nl, about 7,001 nl to about 8,000 nl, about 8,001 nl to about 9,000 nl, and about 9,001nl to about 10,000 nl, including all values and sub-ranges therebetween. The smaller dispenses may or may not be offset spatially from one another. The spot spacing (if the smaller drops are offset spatially) may be spaced about 0.01 millimeters (mm) to about 0.1 mm apart, about 0.11 mm to about 1 mm apart, about 1.01 mm to about 10 mm apart, and about 10.01 mm to about 100 mm apart, including all values and sub-ranges therebetween.

5. An automated device or system that is configured and adapted to apply individual coverslips to slides.

6. An automated device or system that is configured and adapted to take advantage of the immiscible material barrier to cover one or more than one tumor section on a slide with a single cover slip that covers substantially the entire slide.

7. An automated device or system that is configured and adapted to dispense sealant material around the perimeter of the coverslip. The sealant material may be an adhesive (such as rubber cement).

8. An automated device or system that is configured and adapted to heat slides to the temperatures that are appropriate or suitable for hybridization and maintain the relative humidity in the local environment in which the slide is located during the hybridization process.

9. An automated dispensing system or device that is configured and adapted to print counterstain reagents such as DAPI (Nuclear Counterstain - diamidino-2-phenylindole dye for fixed-cell, fluorescent staining of DNA content and nuclei for cellular imaging techniques - this can be a blue nuclear counterstain for fluorescence microscopy and cellular imaging) to the slide as a preparation step for the final or next analysis.

10. A mechanism, method or process that dissects, then removes the normal tissue from the slide for the purpose of isolating only tumor tissue on the slide during probe addition. 11. A mechanism, method or process that dissects and places the tumor tissue onto a separate slide for the purpose of isolating only tumor tissue on a slide during probe addition.

12. The mechanism for dissecting tissue (either normal or tumor tissue) can be either laser microdissection or laser capture microdissection.

13. In accordance with certain FISH based embodiments, circulating tumor cells are the target rather than tissue samples. These tumor cells can be identified with regards to their location on the slide relative to normal cells (also on the slide). This can be similar to the tissue application where the tumor cells are in specific areas within the total tissue sample (a mixture of normal tissue and tumor tissue). This patent disclosure does specifically contemplate circulating tumor cell, along with FFPE (tissue) sample(s) analysis and the like. Embodiments of the invention include any means of performing FISH where tumor cells have been identified on the slide among a population of normal cells, and, for example, the barrier technology in accordance with at least some embodiments disclosed herein, is used to FISH/analyze only the cells of interest (tumor or normal).

[0014] Undesirably, probe reagent application, coverslip application, and counterstain application (DAPI) are for the most part currently done manually. Automated systems that are currently available in the market, disadvantageously, simply flood the slide with probe reagent covering both the normal tissue section(s) and the tumor tissue section(s). Even if the region of interest is identified, the dispensing of the probe results in undesirable and uncontrolled spreading of the probe once the cover slip is applied.

[0015] Embodiments of the invented methods and systems disclosed herein only substantially use the amount or quantity of probe that is appropriate, suitable or required for the process, thereby desirably reducing the overall cost of the assay significantly. Standardizing these methods with desirably with a single piece of automation (or combined system or equipment) brings much more consistency to the methods (as compared to either the manual method or other automated stations that do not perform the complete list of process steps). Moreover and advantageously, operator efficiency increases significantly since the automated system can perform each of the selected or desired process steps. This consolidation of each of the selected or desired process steps to be performed by one piece of automation (or combined system or equipment) desirably also makes the operators more efficient since they do not need to move slides from one piece of equipment to the next.

[0016] Currently, if a lab wishes to minimize the probe reagent volume used in the assay, they must either select a standard coverslip that covers only the tumor section or fabricate a custom coverslip to apply to the slide after the probe reagent has been dispensed. In the case where multiple and discrete tumor sections are present on a single tissue sample, the lab must apply more than one standard or customized coverslip as a part of the process. These multiple steps and procedures significantly, and undesirably, complicate the process and are time consuming.

[0017] By creating a barrier with the immiscible and non-reactive material and isolating the regions of interest, in accordance with certain embodiments, enables the use of a single coverslip that covers substantially the entire slide or tissue section. Advantageously, this greatly simplifies the process and reduces the time required for set up, and efficient use of the probe reagent.

[0018] Some systems, methods and techniques for dispensing droplets, in accordance with certain embodiments of the invention, are disclosed in U.S. Patent No. 6,063,339 to Tisone et al, U.S. Patent No. 5,743,960 to Tisone, and U.S. Patent Application Publication No. 2012/0304929 Al to Ivri the entirety of each one of which is hereby incorporated by reference herein. Some dispensing systems, in accordance with certain embodiments of the invention, are available from BioDot, Inc. of Irvine, California, U.S.A. such as. Bio Jet, Bio Jet Plus, Bio Jet Quanti HR and Bio Jet Ultra Piezo ( www, biodot . com ) .

[0019] Advantageously, the novel and unique printing of the isolating barrier to define region(s) of interest on a slide or substrate for FISH analysis is enhanced by the automated techniques disclosed herein. Conventional technologies suffer from an extreme disadvantage of flooding a whole slide with expensive probe reagents, which adds to not just cost but overall process inefficiency. Even when the regions of interest are provided with probe reagents, conventional technologies have no precise measure of dispensing the probe reagents which inefficiently spread out when the cover slip is applied and cause expensive wastage of the probe reagents. [0020] In certain other embodiments, one or more circulating tumor cell samples are subject to FISH analysis. Once again, by creating an isolating barrier, in accordance with some embodiments, the cell samples of interest - either tumorous or normal - can be isolated for efficient use of the probe reagents. Conventionally, and disadvantageously, the typical approach once again involves flooding of the slide or uncontrolled dispensing of probe reagents on the slide wherein spreading of the same -when the coverslip is applied - leads to process inefficiency and wastage of expensive probes.

[0021] For purposes of summarizing the invention, certain aspects, advantages and novel features of the invention have been described herein above. Of course, it is to be understood that not necessarily all such advantages may be achieved in accordance with any particular embodiment of the invention. Thus, the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught or suggested herein without necessarily achieving other advantages as may be taught or suggested herein.

[0022] All of these embodiments are intended to be within the scope of the invention herein disclosed. These and other embodiments of the invention will become readily apparent to those skilled in the art from the following detailed description of the preferred embodiments having reference to the attached figures, the invention not being limited to any particular preferred embodiment(s) disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Having thus summarized the general nature of the invention and some of its features and advantages, certain preferred embodiments and modifications thereof will become apparent to those skilled in the art from the detailed description herein having reference to the figures that follow, of which:

[0024] FIG. 1 is a schematic drawing illustrating a general overview of an overall FISH process in accordance with some embodiments.

[0025] FIG. 2 provides a general overview of a FISH analysis process in accordance with some embodiments.

[0026] FIGS. 3 and 4 illustrate an example of a typical vision application or system for locating areas of interest during a FISH process [0027] FIG. 5 shows one sealant dispenser used in the FISH process in accordance with some embodiments.

[0028] FIGS. 6, 7 and 8 illustrate a dynamic determination of the size and location of each region of interest on a slide for a FISH process in accordance with some embodiments.

[0029] FIG. 9 is an image of CytoBond rectangles to create sealant "wells" that have been printed for probe dispensing in accordance with some embodiments and FIG. 10 shows the well with sealant barrier walls in accordance with some embodiments.

[0030] FIG. 11 shows probe dispensing to distribute probe to each area of interest or created "well" in accordance with some embodiments.

[0031] FIGS. 12 and 13 illustrate an example of a coverslip applicator and its operation in accordance with some embodiments.

[0032] FIG. 14 is a simplified machine process diagram or flow chart illustrating a FISH method in accordance with some embodiments.

[0033] FIG. 15 is a simplified flow chart illustrating a FISH method in accordance with some embodiments.

[0034] FIG. 16 is a simplified flow chart directed to another embodiment wherein circulating tumor cell samples are subject to FISH analysis.

DETAILED DESCRIPTION

[0035] Some preferred embodiments of the invention described herein relate generally to FISH (Fluorescent/Fluorescence In Situ Hybridization) analysis of biological and/or chemical samples, such as on a substrate or slide. In certain embodiments, these involve FFPE (Formalin-Fixed, Paraffin-Embedded) tissue sample(s) and circulating tumor cell sample(s) FISH probe addition and hybridization and, more particularly in some embodiments, to automated systems and methods for performing FFPE and circulating tumor cell sample(s) FISH probe addition and hybridization, including the advantages associated with isolating region(s) or area(s) of interest for at least FISH by providing a barrier to isolate said region(s) or (area(s) such that expensive probe, and other reagents, can be utilized in an efficient and economical manner. [0036] While the description sets forth various embodiment specific details, it will be appreciated that the description is illustrative only and should not be construed in any way as limiting the invention. Furthermore, various applications of the invention, and modifications thereto, which may occur to those who are skilled in the art, are also encompassed by the general concepts described herein.

[0037] Some embodiments provide for an automated substrate or slide processing system for, e.g., FFPE tissue. One major aim for the systems and technologies disclosed herein is to understand the location and size of the tissue sample, or other, sample that is going to be analyzed. While only the tumor areas within each tissue section (or cell sample) are meant to be analyzed, current conventional protocols disadvantageously coat an entire tissue sample (and often times an entire slide) with probe because there are no automated means for precisely distinguishing 'areas of interest' from the rest of the tissue or sample.

[0038] Undesirably, the laboratory must either then coat the entire slide (thereby consuming considerable amounts of probe and counterstain) or they must systematically select cover slips that are appropriate for a given sample and carefully apply them to the 'area of interest' (thereby consuming considerable amounts of technician time). In addition to automating the current steps involved in FFPE, and other biological and chemical sample, processing, some embodiments of systems and methods provide and bring a novel and unique approach to at least the probe dispensing technique. This, in some embodiments, comprises implementation of a dynamically defined size of each 'area of interest' and use that desirably conserve probe, DAPI counterstain, and scan time on a microscope used for analysis.

[0039] In accordance with certain embodiments, a barcode reading system, device or apparatus is integrated into the overall system so that the identification of each substrate or slide can be automatically read once the substrates slides are placed on the system.

[0040] Some embodiments for location of a dynamic 'Area of Interest' location uses, utilizes or employs an integrated vision system. Once an H&E section has been performed on a biological or chemical sample, such as, a tissue or cell sample, it is possible to distinguish the tumor cells (or area of interest) from the normal cells within the tissue section. Pathology or lab technician marks the 'area of interest' on the H&E section and then transposes the mark(s) onto a FISH slide by circumscribing the 'area (or areas) of interest' on the FISH slide with a diamond pen, knife or the like.

[0041] This etched circle or defined area (or circles or defined areas) can be seen by the technicians who process the substrates or slides and ultimately by the person who analyzes the substrates or slides using a microscope or other viewing system or device.

[0042] Conventionally, when technicians cover the tissue sections with probe for FISH, they undesirably try to select a unique coverslip or coverslips for each case that fully covers the 'area of interest' in order to properly cover the tumor cells with probe without wasting too much probe (the area of tissue sample where coverslips extend over into normal cell areas represent waste with regards to probe consumption).

[0043] Turning now to the figures, FIG. 1 is a schematic drawing illustrating a general overview of an overall FISH process in accordance with some embodiments. A doctor or medical practitioner D obtains a biological or chemical sample 11, such as a tissue sample, from a patient P.

[0044] A laboratory typically using a medical technician T or the like then places the tissue sample onto one or more substrates or slides for further analysis. The sample can be processed, in some embodiments, to comprise an FFPE (Formalin-Fixed, Paraffin-Embedded) tissue sample. This process can be employed at an earlier or later stage, as needed or desired.

[0045] A laboratory typically using a medical technician T then places the tissue sample onto one or more substrates or slides for further analysis. For purposes of this disclosure, at least a portion of the extracted sample 1 is placed on a substrate or slide 10 for FISH analysis.

[0046] Hematoxylin and eosin stain (H&E stain or HE stain) is one of the principal stains in histology, and is typically utilized at this stage to stain at least the extracted sample 1. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed "H&E section", "H+E section", or "HE section".

[0047] Once the lab or medical technician T has the stained sample it is put through a an analysis, typically a microscopic study, to identify one or more regions or areas of interest 2 that may be indicative of cancer or tumors. In some cases, for example for comparison, even normal regions or areas may be tagged or targeted. This identification, in some embodiments, involves marking or etching of the back portion of the slide or substrate 10 by a knife, etching pen or the like.

[0048] The one or more slides or substrates are then fed downstream for further analysis, e.g., FISH and hybridization. This downstream analysis may be performed by the same technician or another entity down the chain of the process line, in accordance with certain embodiments of the invention.

[0049] FIG. 2 provides a general overview of the analysis process in accordance with some embodiments. Once the one or more slides or substrates 10 have been categorized, one next step involves identifying the slides with a particular marker, such as a barcode identifier. Any necessary H& E and FFPE issues should have either been handled at this stage, or will be done so downstream, as desired or required.

[0050] The system 110 generally comprises a barcode identification system 112, a camera system 114 for identifying areas of interest on the slides, a sealant dispenser 120, a probe reagent dispenser 130, a cover-slip applicator 140, a hybridization unit 150, a DAPI counterstain dispenser 160 and microscope 170 or the like for analysis and then sending the results to a doctor or medical practioner.

[0051] In accordance with certain embodiment, an integrated vision system 114 is used where the location of a printed material is critical relative to features on the device. FIGS. 3 and 4 illustrate an example of a typical vision application for locating areas of interest.

[0052] The sealant dispenser 120, in some embodiments, is used to dispense sealant such as rubber cement or an immiscible liquid such as mineral oil to form the barrier. FIG. 5 shows one sealant dispenser 120 as available from AdvanJet of Escondido, California, USA (www. advanj et.com). This dispenser (model PDJ- 1 100) is particularly suited for dispensing liquids of high viscosity e.g. 1-200 mPas with drops as small as 25 nanoliters. Other Advanjet dispensers (e.g. model HV-2000) or similar dispensers may efficaciously be utilized, as needed or desired.

[0053] In accordance with some embodiments, and referring in particular to FIGS. 6, 7 and 8 the automated FFPE processing system, or a subset of the overall system 110, dynamically determines the size and location of each region of interest 2 on every slide 10. The system accomplishes this by integrating a CCD camera system 114 into the automated workstation. The camera images each slide 10 and determine the location and size of each etched circle (since these circles represent the area(s) of interest).

[0054] The system typically first scans in the barcode label on each slide 10 that is loaded onto the instrument and check the sample (or case) identity with a daily order list. Once the instrument has confirmed the number of slides that have been loaded onto the instrument, the system inspect each slide 10 to determine the coordinates associated with each 'area of interest' 2 and dispense pattern 4 for sealant dispensing.

Rubber Cement/Immiscible Fluid Border Dispensing

[0055] Once the size and location of each area of interest has been determined, the system then transfers these coordinates to the 'rubber cement (or immiscible fluid) dispenser' and this information will be used to print rectangle boarders around each individual area of interest on every slide. By doing this, the instrument creates custom 'wells' 100 that define and contain probe within the areas of interest. FIG. 9 is an image of CytoBond rectangles to create wells 100 that have been printed in accordance with certain embodiments. FIG. 10 shows a well 100 with sealant barrier walls 102.

Probe Dispensing

[0056] Some systems, methods and techniques for dispensing probe droplets, in accordance with certain embodiments of the invention, are disclosed in U.S. Patent No. 6,063,339 to Tisone et al. and U.S. Patent No. 7,754,439 B2 to Moore et al, the entirety of each one of which is hereby incorporated by reference herein. Some dispensing systems, in accordance with certain embodiments of the invention, are available from BioDot, Inc. of Irvine, California, U.S.A. as can be found on the www.biodot.com website, without limitation.

[0057] Those skilled in the art will recognize that other types of dispensers and associated actuation devices exist and may be used with efficacy. These may include, for example, but are not limited to piezoelectric dispensers, fluid impulse dispensers, heat actuated dispensers, air brush dispensers, and the like, as desired or required. Some systems, methods and techniques for dispensing droplets, in accordance with certain embodiments of the invention, are disclosed in U.S. Patent Application Publication No. 2008/0227663 Al to Tisone et al, the entirety of which is hereby incorporated by reference herein.

[0058] Referring in particular to FIG. 11, the probe dispenser 130 is used to distribute probe to each area of interest according to the information that has been provided by the lab order list. The volume of probe dispensed to each area of interest will be determined by the size of each printed well 100. The system can transfer the probe from a source such as but not limited to 96 well plates or the like.

Automated Coverslip Application

[0059] The automated coverslip applicator 140 applies coverslips 142 to each slide once the probes have been dispensed to each slide. A single, e.g., 20 mm x 60 mm coverslip can be used since a coverslip that covers the entire slide will be capable of covering all potential areas of interest on slides and therefore the system will not have to choose from a variety of coverslips based on each individual area of interest. The adhesive properties of the rubber cement (or immiscible fluid) will ensure that the coverslip is properly sealed during hybridization.

[0060] Referring in particular to FIGS. 12 and 13, these drawings for purposes of illustration a series of screen shots from a BioDot instrument 140 that 'pick-and-placed' 16 round coverslips simultaneously on top of 96 well plates for a protein crystallization application. A similar coverslip applicator 140 is incorporated into the system 110.

Denaturing and Hybridization

[0061] The nest that holds the slides is capable of heating the slides to temperatures required for denaturing the samples (e.g. about 70 to about 90 degrees Celsius or Centigrade in some cases). Once the probe and coverslips have been applied to each slide, the slides are ready for hybridization. The operator applies a cover over the slide nest in order to maintain elevated humidity levels throughout the hybridization process. The nest temperature target temperature and hold time can be programmed via a system software interface and once the hold time has been reached for the denaturing process, the instrument quickly achieve the about 37 degrees Celsius or Centigrade, in some cases, temperature required or desired for the hybridization process. DAPI Dispense and "Coversliping"

[0062] The slides 1 come off of the instrument or system 110 after hybridization so that the coverslips can be removed and the slides can go through the post hybridization wash procedure. After the slides have gone through the wash procedure, the slides are loaded onto the instrument. The barcode scanner will rescan each slide so that the instrument can determine the proper volume and location in which to dispense DAPI counterstain. After applying DAPI to each slide, the system automatically applies the final coverslip as the slides are now ready for analysis.

Flowchart Depiction of Certain Embodiments

[0063] FIG. 14 is a simplified machine process diagram or flow chart illustrating a method in accordance with certain embodiments.

[0064] FIG. 15 is a simplified flow chart illustrating features and advantages in accordance with certain embodiments of the invention. Advantageously, the novel and unique printing of the isolating barrier to define region(s) of interest on a slide or substrate for FISH analysis is enhanced by the automated techniques disclosed herein. Conventional technologies suffer from an extreme disadvantage of flooding a whole slide with expensive probe reagents, which adds to not just cost but overall process inefficiency. Even when the regions of interest are provided with probe reagents, conventional technologies have no precise measure of dispensing the probe reagents which inefficiently spread out when the cover slip is applied and cause expensive wastage of the probe reagents.

[0065] The flowchart of FIG. 16 is directed to another embodiment wherein circulating tumor cell samples are subject to FISH analysis. Once again, by creating an isolating barrier, in accordance with some embodiments, the cell samples of interest - either tumorous or normal - can be isolated for efficient use of the probe reagents. Conventionally, and disadvantageously, the typical approach once again involves flooding of the slide or uncontrolled dispensing of probe reagents on the slide wherein spreading of the same -when the cover-slip is applied - leads to process inefficiency and wastage of expensive probes. Clauses

[0066] Particular aspects of the disclosure and/or embodiments of the invention(s) are set out in at least the following numbered clauses, which can be efficaciously be combined in any form or manner, as needed or desired:

[0067] 1. A method of FISH analysis of one or more tissue or cell samples, comprising: identifying at least one region or location of interest on a slide or substrate of the tissue or cell sample; locating the coordinates of the at least one region of interest on the slide or substrate; dispensing an immiscible material to circumscribe said at least one region of interest so as to form an isolated barrier over said at least one region of interest; and precisely dispensing one or more probes within the identified one or more isolated regions of interest to perform FISH analysis of the one or more samples.

[0068] 2. The method of Clause 1, wherein the tissue sample comprises FFPE (Formalin-Fixed, Paraffin-Embedded (tissue)).

[0069] 3. The method of Clause 1, wherein the immiscible material comprises an immiscible liquid.

[0070] 4. The method of Clause 1, wherein the immiscible material comprises a liquid with a predetermined viscosity.

[0071] 5. The method of Clause 1, wherein the immiscible material comprises rubber cement.

[0072] 6. The method of Clause 1, wherein the immiscible material comprises a mineral oil.

[0073] 7. The method of Clause 1, wherein locating the coordinates of the at least one region of interest on the slide or substrate comprises using a camera, imaging or viewing system.

[0074] 8. The method of Clause 1, wherein an automated system or device that comprises or utilizes a camera system or another sensor to determine a scribed or visibly marked section on the slide or substrate that defines the area and position of a tumor section.

[0075] 9. The method of Clause 1, wherein the immiscible material comprises a non-reactive fluid. [0076] 10. The method of Clause 1, wherein the immiscible material acts as a barrier to air and evaporation during the FISH/hybridization process, and wherein the probe reagent is dispensed inside a pattern made by the immiscible material which circumscribes a tumor section of the tissue sample.

[0077] 11. The method of Clause 1, wherein an automated system or device is adapted and configured to dispense the immiscible material around both regularly and irregularly shaped tumor sections, single or multiple tumor sections, or tumor sections of various sizes on a single slide or substrate.

[0078] 12. The method of Clause 1, wherein the probe may be dispensed as a single dispense that effectively covers a tumor section or sections or the probe may be dispensed as a smaller dispenses that effectively cover the tumor section, wherein the smaller dispenses may range from about 10 nl (nanoliters) to about 100 nl, about 101 nl to about 200 nl, about 201 nl to about 300 nl, about 301 nl to about 400 nl, about 401 nl to about 500 nl, about 501 nl to about 600 nl, about 601 nl to about 700 nl, about 701nl to about 800 nl, about 801 nl to about 900 nl, about 901nl to about 1,000 nl, about 1,001 nl to about 2,000 nl, about 2,001 nl to about 3,000 nl, about 3,001nl to about 4,000 nl, about 4,001 nl to about 5,000 nl, about 5,001 nl to about 6,000 nl, about 6,001 nl to about 7,000 nl, about 7,001 nl to about 8,000 nl, about 8,001 nl to about 9,000 nl, and about 9,001nl to about 10,000 nl, including all values and sub-ranges therebetween, wherein the smaller dispenses may or may not be offset spatially from one another, and wherein the spot spacing (if the smaller drops are offset spatially) may be spaced about 0.01 millimeters (mm) to about 0.1 mm apart, about 0.11 mm to about 1 mm apart, about 1.01 mm to about 10 mm apart, and about 10.01 mm to about 100 mm apart, including all values and sub-ranges therebetween.

[0079] 13. The method of Clause 1, wherein an automated device or system is configured and adapted to apply individual covers-slip(s) to one or more slides or substrates.

[0080] 14. The method of Clause 1, wherein an automated device or system is configured and adapted to take advantage of the immiscible material barrier to cover one or more than one tumor section on the slide or substrate with a single cover- slip that covers substantially the entire slide or substrate. [0081] 15. The method of Clause 1, wherein an automated device or system is configured and adapted to dispense sealant material around the perimeter of a cover-slip dispose over the slide or substrate, and wherein, the sealant material comprises an adhesive such as rubber cement.

[0082] 16. The method of Clause 1, wherein an automated device or system is configured and adapted to heat the slide or substrate to temperatures that are appropriate or suitable for hybridization (FISH) and maintain a relative humidity in the local environment in which the slide or substrate is located during the hybridization (FISH) process.

[0083] 17. The method of Claim 1, wherein an automated dispensing system or device is configured and adapted to print counter-stain reagents such as DAPI to the slide or substrate as a preparation step for a final or next analysis.

[0084] 18. The method of Clause 1, wherein the method further comprises dissection, then removal of normal tissue from the slide or substrate for the purpose of isolating only tumor tissue on the slide or substrate during probe addition.

[0085] 19. The method of Clause 18, wherein dissecting tissue (either normal or tumor tissue) is one of laser micro-dissection and laser capture micro-dissection.

[0086] 20. The method of any one of of Clauses 1 to 19, wherein the one or more cell samples are circulating tumor cell samples.

[0087] 21. A method of FISH analysis of one or more circulating tumor cell samples, comprising: identifying at least one region or location of interest on a slide or substrate of the cell sample; locating the coordinates of the at least one region of interest on the slide or substrate; dispensing an immiscible material to circumscribe said at least one region of interest so as to form an isolated barrier over said at least one region of interest; and precisely dispensing one or more probes within the identified one or more isolated regions of interest to perform FISH analysis of the one or more samples.

[0088] 22. The method of Clause 21, wherein the tumor cells are identified with regards to their location on the slide or substrate relative to normal cells which may also on the slide or substrate. [0089] 23. The method of any one of Clauses 1 to 19, wherein the method(s) are applicable, as appropriate, to the FISH analysis of circulating tumor cell samples and/or normal cells on the slide or substrate.

Conclusion

[0090] Any methods which are described and illustrated herein are not limited to the sequence of acts described, nor are they necessarily limited to the practice of all of the acts set forth. Other sequences of acts, or less than all of the acts, or simultaneous occurrence of the acts, may be utilized in practicing embodiments of the invention.

[0091] It is to be understood that any range of values disclosed, taught or suggested herein comprises all values and sub-ranges therebetween. For example, a range from 5 to 10 will comprise all numerical values between 5 and 10 and all sub-ranges between 5 and 10.

[0092] From the foregoing description, it will be appreciated that a novel approach for FISH analysis of biological and/or chemical samples has been disclosed - including but not limited to FFPE tissue (and circulating tumor cell) samples. This disclosure can be extended to many other applications wherein regions of interest for FISH are isolated by providing a barrier to target the dispensing of probe, or other reagent, material. While the components, techniques and aspects of the invention have been described with a certain degree of particularity, it is manifest that many changes may be made in the specific designs, constructions and methodology herein above described without departing from the spirit and scope of this disclosure.

[0093] While a number of preferred embodiments of the invention and variations thereof have been described in detail, other modifications and methods of using and applications for the same will be apparent to those of skill in the art, such as, but not limited to: (i) diagnostics applications, including without limitation medical, veterinary, human, agricultural, food and/or clinical usage; (ii) life sciences and pharmaceutical research and applications, including without limitation genomic and proteomic related research and drug discovery and development, combinatorial chemistry, high throughput screening and/or crystallography; and (iii) material sciences applications, including without limitation combinatorial investigations for the development of new materials which may include combinatorial methods in drug discovery and development. Accordingly, it should be understood that various applications, modifications, and substitutions may be made of equivalents without departing from the spirit of the invention or the scope of any claims.

[0094] Various modifications and applications of the invention may occur to those who are skilled in the art, without departing from the true spirit or scope of the invention. It should be understood that the invention is not limited to the embodiments set forth herein for purposes of exemplification, but is to be defined only by a fair reading of any claims, including the full range of equivalency to which each element thereof is entitled.

Claims

WHAT IS CLAIMED IS:

1. A method of FISH analysis of one or more tissue or cell samples, comprising: identifying at least one region or location of interest on a slide or substrate of the tissue or cell sample;

locating the coordinates of the at least one region of interest on the slide or substrate;

dispensing an immiscible material to circumscribe said at least one region of interest so as to form an isolated barrier over said at least one region of interest; and precisely dispensing one or more probes within the identified one or more isolated regions of interest to perform FISH analysis of the one or more samples.

2. The method of Claim 1, wherein the tissue sample comprises FFPE (Formalin- Fixed, Paraffin-Embedded (tissue)).

3. The method of Claim 1, wherein the immiscible material comprises an immiscible liquid.

4. The method of Claim 1, wherein the immiscible material comprises a liquid with a predetermined viscosity.

5. The method of Claim 1, wherein the immiscible material comprises rubber cement.

6. The method of Claim 1, wherein the immiscible material comprises a mineral oil.

7. The method of Claim 1, wherein locating the coordinates of the at least one region of interest on the slide or substrate comprises using a camera, imaging or viewing system.

8. The method of Claim 1, wherein an automated system or device that comprises or utilizes a camera system or another sensor to determine a scribed or visibly marked section on the slide or substrate that defines the area and position of a tumor section.

9. The method of Claim 1, wherein the immiscible material comprises a non- reactive fluid.

10. The method of Claim 1, wherein the immiscible material acts as a barrier to air and evaporation during the FISH/hybridization process, and wherein the probe reagent is dispensed inside a pattern made by the immiscible material which circumscribes a tumor section of the tissue sample.

11. The method of Claim 1, wherein an automated system or device is adapted and configured to dispense the immiscible material around both regularly and irregularly shaped tumor sections, single or multiple tumor sections, or tumor sections of various sizes on a single slide or substrate.

12. The method of Claim 1, wherein the probe may be dispensed as a single dispense that effectively covers a tumor section or sections or the probe may be dispensed as a smaller dispenses that effectively cover the tumor section, wherein the smaller dispenses may range from about 10 nl (nanoliters) to about 100 nl, about 101 nl to about 200 nl, about 201 nl to about 300 nl, about 301 nl to about 400 nl, about 401 nl to about 500 nl, about 501 nl to about 600 nl, about 601 nl to about 700 nl, about 701nl to about 800 nl, about 801 nl to about 900 nl, about 901nl to about 1,000 nl, about 1,001 nl to about 2,000 nl, about 2,001 nl to about 3,000 nl, about 3,001nl to about 4,000 nl, about 4,001 nl to about 5,000 nl, about 5,001 nl to about 6,000 nl, about 6,001 nl to about 7,000 nl, about 7,001 nl to about 8,000 nl, about 8,001 nl to about 9,000 nl, and about 9,001nl to about 10,000 nl, including all values and subranges therebetween, wherein the smaller dispenses may or may not be offset spatially from one another, and wherein the spot spacing (if the smaller drops are offset spatially) may be spaced about 0.01 millimeters (mm) to about 0.1 mm apart, about 0.11 mm to about 1 mm apart, about 1.01 mm to about 10 mm apart, and about 10.01 mm to about 100 mm apart, including all values and sub-ranges therebetween.

13. The method of Claim 1, wherein an automated device or system is configured and adapted to apply individual covers-slip(s) to one or more slides or substrates.

14. The method of Claim 1, wherein an automated device or system is configured and adapted to take advantage of the immiscible material barrier to cover one or more than one tumor section on the slide or substrate with a single cover- slip that covers substantially the entire slide or substrate.

15. The method of Claim 1, wherein an automated device or system is configured and adapted to dispense sealant material around the perimeter of a cover-slip dispose over the slide or substrate, and wherein, the sealant material comprises an adhesive such as rubber cement.

16. The method of Claim 1, wherein an automated device or system is configured and adapted to heat the slide or substrate to temperatures that are appropriate or suitable for hybridization (FISH) and maintain a relative humidity in the local environment in which the slide or substrate is located during the hybridization (FISH) process.

17. The method of Claim 1, wherein an automated dispensing system or device is configured and adapted to print counter-stain reagents such as DAPI to the slide or substrate as a preparation step for a final or next analysis.

18. The method of Claim 1, wherein the method further comprises dissection, then removal of normal tissue from the slide or substrate for the purpose of isolating only tumor tissue on the slide or substrate during probe addition.

19. The method of Claim 18, wherein dissecting tissue (either normal or tumor tissue) is one of laser micro-dissection and laser capture micro-dissection.

20. The method of any one of Claims 1 to 19, wherein the one or more cell samples are circulating tumor cell samples.

21. A method of FISH analysis of one or more circulating tumor cell samples, comprising:

identifying at least one region or location of interest on a slide or substrate of the cell sample;

22. The method of Claim 21, wherein the tumor cells are identified with regards to their location on the slide or substrate relative to normal cells which may also on the slide or substrate.

23. The method of any of Claims 1 to 19, wherein the method(s) are applicable, as appropriate, to the FISH analysis of circulating tumor cell samples and/or normal cells on the slide or substrate.