Title of Invention	SYSTEM AND METHOD FOR FULLY AUTOMATED ROBOTIC-ASSISTED IMAGE ANALYSIS FOR IN VITRO AND IN VIVO GENOTOXICITY TESTING
Abstract	A system (100) and method for performing genotoxicity screening utilize: (1) one or more computers (110); (2) a frame, grabber (120) connected to the one or more computers (110); (3) a camera (140) connected to the frame grabber (120); (4) a microscope (150) connected to the one or more computers (110); (5) a slide feeder (160) connected to the one or more computers (110); and (6) a program operating on the one or more computers (110). The program facilitates the screening of a second batch of biological material using a second genotoxicity testing method after screening a first batch of biological material using a first genotoxicity testing method. The screening operates substantially free of any manipulation of the camera (140), the microscope (150) or the slide feeder (160).

Title of Invention

SYSTEM AND METHOD FOR FULLY AUTOMATED ROBOTIC-ASSISTED IMAGE ANALYSIS FOR IN VITRO AND IN VIVO GENOTOXICITY TESTING

Abstract

A system (100) and method for performing genotoxicity screening utilize: (1) one or more computers (110); (2) a frame, grabber (120) connected to the one or more computers (110); (3) a camera (140) connected to the frame grabber (120); (4) a microscope (150) connected to the one or more computers (110); (5) a slide feeder (160) connected to the one or more computers (110); and (6) a program operating on the one or more computers (110). The program facilitates the screening of a second batch of biological material using a second genotoxicity testing method after screening a first batch of biological material using a first genotoxicity testing method. The screening operates substantially free of any manipulation of the camera (140), the microscope (150) or the slide feeder (160).

Full Text	SYSTEM AND METHOD FOR FULLY AUTOMATED ROBOTIC- ASSISTED IMAGE ANALYSIS FOR IN VITRO AND IN VIVO GENOTOXICITY TESTING REFERENCE TO COMPUTER PROGRAM LISTING APPENDIX A Computer Program Listing Appendix to this document has been submitted to the U.S. Patent and Trademark Office in accordance with 37 C.F.R. §§ 1.52 and 1.96 on the filing date of this document and is hereby incorporated herein by reference in its entirety. The Computer Program Listing Appendix is contained on one (1) CD-ROM, two copies of which have been filed with the U.S. Patent and. Trademark Office and each of which are labeled with the name of the inventor of the present invention, the title of the invention, the attorney's docket number and the creation date of the CD-ROMs. FIELD OF THE INVENTION The present invention is directed to genotoxicity testing and, more particularly, to a metho.l and system for utilizing, in conjunction, an automated robotic slide feeder or equivalent device, an electronically driven microscope, a microprocessor-based computer and additional components and software to facilitate high-throughput in vitro and in vivo genotoxicily testing. BACKGROUND OF THE INVENTION Toxicologicial testing is used in various technologies, industries and disciplines for assessing the effect of drugs and other chemical compounds on the nature and properties of oiological natter. Genotoxicity testing is particularly useful for analyzing the effect of certain chemicals on the DNA structure of the cells of humans, animals and other life forms, including he analysis of the potential for induction of hereditary diseases and mutations. Genotoxicity testing generally includes screening of either in vivo or in vitro biological matter. Well known in vitro test systems include, but are not limited to: (1) the comet assay, which is used for detecting primary DNA damage, DNA to DNA crosslinks, and DNA-protein interactions. A specific version of the comet assay is the alkaline Comet Assay which is described in a publication titled "A simple technique for quantiation of low levels of DNA damage in individual cells," Singh et al., Experimental ( ellular Research, vol. 175, pp. 184-191 (1988). The Alkaline Comet Assay is also described in a publication titled "Modification of the Comet Assay for the detection of DNA strand breaks in extremely small tissue samples," Tebbs et al., Mutagenesis, vol. 14, pp. 437 -438 ( 999); (2) the micronucleus test in cell lines (V79 cells, Mouse Lymphome cells, TK6 colls) or hurian lymphocytes, which are all known to be useful in the early screening of new impounds n industrial toxicology; and (3) the chromosome aberration test, which is required by certain regulatory authorities,such as the Organization for Economic Co-Operation and Development and the I nited States Food and Drug Administration, for approval of new drugs. For this in vitro test, ie assessment of chromosomal aberrations is done on the basis of metaphases which must be detected for analysis. In vivo genotoxicity test systems include, but are not limited to: (1) the in vivo micronucleus test in bone marrow for clastogenic or aneugenic petential of test compound administered to rodents. This test is described in a publication titled "A Rapid in vivo test for chromosomal damage," Heddle, JA., Mutual Res., vol. 18, pp. [i 7-90 (197:0; (2) the in vivo comet assay, which under certain circumstances may be accepted as a regulatory assay in addition to the micronucleus test in vivo, to verify in vitro test results. The in vivo comet assay is described in a publication titled "Recommendations for conducting th : in vivo alkaline Comet assay", Hartmanr. et al., Mutagenesis vol. 18, no. 1, pp. 45-51 (2 )03). Other in vivo and in vitro testing methods are also well known in the art. Limited automated methods for facilitating genotoxicity screening ("screening" being understood to refer to the analysis of biological material samples previously treated with the test compound) of both in vivo and in vitro materials have also been attempted. As an example, art automated in vivo micronucleus assay analysis of mouse bone marrow used in the pharmaceutical industry to test the genotoxicity potential of new compounds is described in a publication co-authored by the inventor of the present invention which is titled "Technical aspects of automatic micronucleus analysis in rodent bone," Cell Biology and Toxicology, vol. 10, pp. 283 -289 (1994). Automated forms of analysis for in vitro micronucleus tests are also known. The inventor of the present invention authored an article titled "Automatic analysis of t le in vitro micronucleus test on V79 cells" in Mutation Research, vol. 413, pp. 57-68 (1998), cescribing an automated in vitro micronucleus test for V79 cells. The techniques for automated genotoxicity screening for both in vivo and in Nitro biological material that were noted above utilize image analysis software and techniques mat are individually designed for the specific type of test and the specific type of material that i being screened. Automation of genotoxicity testing that utilizes image analysis simplifies the process of compound screening, eliminates the tedium of manual scoring and significantly Ficreases the overall number of genotoxicity screenings which can be performed in any given period of time. Generally, an automated electronically driven microscope with image capturing capabilities and a micro-processor based computer running microscope control and inage analysis software, each specifically designed, calibrated and programmed for the rarticular screening being performed, is used to operate and facilitate the image analysis-based automated screening process. To provide still further increases in the throughput of genotoxicity sample screening, prior art devices are known to have incorporated robotic arm assemblies and equivalent devices to facilitate sample slide feeding, thus freeing the user from the tedium of nanually loading slides for image analysis and further increasing screening throughput rates. Known prior art systems do not, however, allow for both in vivo and in vitro cenotoxicity screening using a single platform to perform automatically all manners of in vitro and in vivo genotoxicity testing such as the micronucleus test, the comet assay and metaphase detection for chromosome analysis, nor do any known prior art system provide for utilization of a rot otic slide feeder or equivalent device for all manner of in vitro and in vivo testing without the tedium of extensive user intervention. Summary An embodiment of a genotoxicity screening system of the present invention includes (1) one or more computers; (2) a frame grabber connected to the one or more computers; (3) a camera connected to the frame grabber; (4) a microscope connected to the one or more computers; (5) a slide feeder connected to the one or more computers; and (6) a program operating on the one or more computers. The program facilitates the screening a second batch of biological material using a second genotoxicity testing method after screening a first batch of biological material using a first genotoxicity testing method. The genotoxicity methods are performed substantially free of any manual manipulation of the camera, the microscope or the slide feeder. In another embodiment of the present invention, software is provided that controls the operation of a genotoxicity analysis system. The software provides automatic onfiguration of configurable components of the genotoxicity analysis system and allows the enotoxicity analysis system to perform a plurality of genotoxicity tests on respective luralities cf biological samples by way of the automatic configuration. In another embodiment of the present invention, genotoxicity testing of b ological materials is performed using a genotoxicity analysis system. The genotoxicity system includes hardware components that are operated with software controls. The genotoxicity analysis system is capable of performing a multiplicity of genotoxicity tests. Use o) the genotoixicity analysis system performs as follows: (1) preparing a first batch of samples of biological materials for processing using a first genotoxicity test; (2) utilizing the genotoxicity analysis system to perform a first genotoxicity test on the samples of the first tach of biological materials; (3) preparing a second batch of samples of biological materials or processing using a second genotoxicity test; and (4) utilizing the genotoxicity analysis System to perform a second genotoxicity test on the samples of the second batch of biological materials. The software controls manipulate the configuration of the hardware components during the time period between performance of the first and second genotoxicity tests to allow the first and second genotoxicity tests to be performed using the same hardware components. Yet another embodiment of the present invention includes a method for performing various types of genotoxicity tests on respective batches of biological samples using a genotoxicity analysis system. The method including the steps of: (1) receiving a command from a user of the genotoxicity analysis system, the command specifying the type of genotoxicity test to be performed; (2) performing an automatic configuration of the component of the genotoxicity analysis system to thereby allow the genotoxicity analysis system to perform the genotoxicity test specified in step 1; (3) performing the specified genotoxicity test ot a batch of biological samples; (4) recording results of the genotoxicity test; (5) repeating stops 1 through 4. In yet another embodiment of a method for performing genotoxicity screening in i c ;ordance vith the present invention, the following steps are performed: (1) preparing a batch of slides for genotoxicity screening; (2) selecting a genotoxicity test; (3) automatically reprieving the first of a plurality of slides containing biological samples from a slide retaining device; (4) automatically delivering the slide to an electronically driven microscope; (5) au omatically focusing on the material contained on the slide; (6) automatically recording a visual representation of the focused image; (7) automatically delivering the focused image to a mecroprocessor-based computer; (8) automatically performing image analysis on the recorded imge using image analysis software appropriate for the genotoxicity test selected in step 2; (9) automatically recording the data resulting from the analysis of the image; (10) automatically returning the slide retrieved in step 3 to the slide retaining device; (11) automatically retrieving the next slide for analysis; (12) automatically repeating steps 3 through 11 for successive slides in he batch until all of the slides in the batch have been analyzed; and (13) repeating steps 1 through 12 urtil all desired slides have been processed. Brief Description of the Drawings The foregoing and other features of the present invention will be more readily apparent from the following detailed description and drawings of illustrative embodiments of the invention, in which: Figure 1 illustrates, in block diagram form, an embodiment of the automated genotoxicity analysis system; Figure 2 illustrates, in logical block diagram form, an embodiment of application software to control the operation of a genotoxicity analysis system and files which s ore the results of the genotoxicity analysis; Figure 3 illustrates a flow chart describing the operation of an embodiment of a genotoxicity analysis system; Figure 4 illustrates an embodiment of a user interface screen for entering information relating to a slide that is to be analyzed using a genotoxicity analysis system; Figure 5 illustrates an embodiment of a user interface screen for entering data identifying particular slides to be processed using a genotoxicity analysis system; Figure 6 illustrates an embodiment of a user interface screen for adjusting the parameters for a particular slide to be analyzed using a genotoxitiy analysis system; Figure 7 illustrates an embodiment of a user interface screen that allows a user to adjust the threshold settings for the particular slide that is to be analyzed using a genotoxicitv analysis system; Figure 8 illustrates an embodiment of a user interface form for adjusting nicroscope parameters for the a particular slide using a genotoxicity analysis system; Figure 8a illustrates an embodiment of a user interface form for use in selecting genotoxicity test that to be processed using a genotoxicity analysis system; Figure 9 illustrates an embodiment of a user interface screen for a user to select scanning options for a genotxicity analysis system; Figure 10 illustrates an embodiment of a user interface screen for selecting results of a genotoxicity test for review using a genotoxicity analysis system; Figure 11 illustrates an embodiment of a user interface screen for specifying a particular study containing a slide desired to be reviewed by a user of a genotoxicity analysis system; Figure 12 illustrates an embodiment of a user interface screen for identifying the particular slide for review in the study selected in the user interface screen of Figure 11; Figure 13 illustrates an embodiment of a user interface screen for displaying the results of screening of a particular slide using a particular genotoxicity test; Figure 14 illustrates an embodiment of a user interface screen that allows a user to retrieve objects that have been detected during an automatic scanning process using an automated genotoxicity testing system; and Figures 15a through 15e present a listing of computer files for use in creating an enbodiment of an automated genotoxicity testing system. Detailed Description of an Embodiment of the Invention Described herein is a single automated platform for genotoxicity screening which can accommodate both in vivo and in vitro micronucleus testing, comet assay screening and in, vitro metaphase finding, but requires minimal user monitoring and/or user interaction. As will be more fully described, the present invention is an automated system and method for performing sample analysis for genotoxicity testing. An embodiment of the invenire system Includes: (1) a robotic slide feeder, (2) an electronically driven microscope, (3) an image capturing apparatus, (4) a microprocessor-based computer running program contro software, and (5) required communication cables and interface apparatus for interconnecting the various components, The invention is embodied in a system and method as exempt t ed in the embodiments described below, but is not limited to the details of those embodimentsnts. One skilled in the art will readily appreciate that the invention may include and utilize equivalent components and processes that fall within the scope of the invention, which invention is defined solely by the claims that will accompany this disclosure. Moreover, the inventior an comprise aspects of the foregoing components and their interrelationship to one another, rehiding, without limitation, programmed control of such components. Using the inventive automated genotoxicity analysis system and method, a laboratory technician or other user may optimally process, for analysis, successive batches of slic es containing biological material using different tests and different types of biological maerial for each batch, without the need to manually adjust any hardware and with only rminiimal user interaction. As will be more fully described below, the method of operation of the automated genotoxicity analysis system of the present invention proceeds as follows. A lab bratory technician or other user prepares a batch of slides for genotoxicity screening. These slices may include in vivo or in vitro biological materials and may be prepared for screening by any of the following (or additional) tests: (1) in vivo micronucleus test, (2) in vitro moni ronucleus test, (3) in vitro or in vivo comet assay and (4) in vitro metaphase finding. Once tt e slides are prepared for testing, the user selects the appropriate genotoxicity test system (from the described list of possibilities) from a menu or equivalent user interface displayed on the screen of he microprocessor based computer. The robotic slide feeder then automatically ret ieves the first of the slides from the batch prepared by the user and delivers the slide to the electronically driven microscope, which then automatically and appropriately focuses on the material contained on the slide. Next, the image capturing apparatus records a visual resresentatior of the focused image and delivers it to the microprocessor-based computer. The mmicroprocessor-based computer then performs image analysis on the recorded image using the aproropriate image analysis software preloaded on the computer. The computer then records the data resuling from the analysis of the image until either the given delimiting number of ce1 s have been counted or the maximum number of image fields to be analyzed has been reached for the slide currently under analysis. Once the analysis of the slide is complete, the robotic slide feeder returns the slide to the slide rack and retrieves the next slide for analysis. This process continues until all of the slides in the batch have been analyzed. The user may then prepare ,tnew batch of slides of any type of in vivo or in vitro material and initiate animated screening of the material using any of the genotoxicity assays described above wi hout the need to manually change or modify any of the system equipment. Figure 1 illustrates, in block diagram form, an embodiment of the automated ge lotoxicity analysis system 100 of the present invention. Genotoxicity analysis system 100 i lcludes a microprocessor-based computer 110 having a frame grabber board 120, two color display monitors 130 and 132, a charge coupled device (CCD) camera 140, an electronically driven microscope 150 and a robotic slide feeder 160. Computer 110 of Figure 1 may be any of the many known IBM-compatible personal or server computers running any known operating system for such computers, e.g., Windows XP, Windows NT Server or UNIX. In the preferred embodiment, a Transtec 1300 OBM compatible PC, operating at 1.3 GHz., having at least 128 Mbyte of internal RAM Tiemory and tunning the Windows NT 4, Service Pack 5 operating system or Windows 2000 is utilized. Computer 110 executesall operating, control and image processing software which w 11 be described more fully below, for genotoxicity analysis system 100 and is connected to and controls the operation of all other components of gentoxicity analysis system 100. Computer 110 is connected to electronic miscroscope 150 and robotic slide feeder 160 via RS- 232 serial interfaces. Computer 110 includes a frame grabber board 120 which is preferably a Meteor-II frame grabber utilizing Matrox MIL 6.1 or later version driver software available from Matrox Imaging of Dorval, Quebec. Computer 110 stores all program software and generated data on a local harddrive. Alternately, computer 110 may be connected to a local area network (LAN 200) to support data on a networked data base (not illustrated) or to allow axess, retrieval and storage of parameter data files and other program software located on a separate networked computer server (not illustrated). In the preferred embodiment, the executable programs, compiled from the Visual Basic and C/C+ + source code and the generated measurement data results files are stored on a networked database and server while the C-language DLLs and related files reside locally on the hard drive of computer 110. Robotic slide feeder 160 is preferably an ES-553S robot with an SRC-320 driver avallable from Seiko Epson Corporation of Japan. Robotic slide feeder 160 is controlled and operated by electronic commands received from computer 110 via a serial cable 170. Robotic side feeder 160 functions primarily to remove a current slide from a slide rack (not illustrated) conaining a multiplicitly of slides, then place the slide onto the stage of electronically driven m c-oscope 150 and then return the slide to the slide rack after the analysis of the slide is complete. Under the embodiment of the invention described herein, the slide rack may include as many as 130 glass slides containing biological material or "samples." Electronically driven microscope 150 of genotoxicity analysis system 100 is preferably a Leica DM RXA/2 electronic microscope running Leica SDK driver software, which is manufactured and sold by Leica Microsystems AG of Wetzlar, Germany. Electronically driven microscope 150 preferably includes the following modules: stage, focus drive, illumination, objectives, fluorescence cubes, diaphragms for aperture and field, additional magnification changer and fluorescence shutter, all of which components are software driven and controlable. Electronically driven microcospe 150 is controlled and operated by electronic commands received from computer 110 via two serial cables 180 and 182, one each for the stage controller and for the microscope stand of electronically driven microscope 150. Camera 140 is preferably an XC-003 or DXC-390 CCD camera sold by Sony Corporation of America. Camera 140 is mounted on electronically driven microscope 150 in the known manner using a C-mount adapter and is utilized to grab the current image from electronically driven microscope 150 and send the image in analog format to frame grabber board 120/via seriarcable 190. Camera 140 is under operational control of computer 110 via frame grabber 120. The analog fomatted image received from camera 140 is digitized by computer 110. analysis system 100 also includes color display modules 130 and 132 connected to computer 110. Preferably, color display module 130 provides the user interface to the user of the genotoxicity analysis system 100 while color display module 132 displays the current image provided by electronically driven microscope 150 or, alternatively, the result of the image processing analysis. Computer 110 executes software which controls the operation of genotoxicitiy analysis system 100. .Computer 110, and any networked server that may also be utilized to control and operate genotoxicity analysis system 100, preferably runs Microsoft NT version 4 or Windows 2000 operating system software. The software executed by computer 110 to control genotoxicity analysis system 110 is created using Microsoft Visual Basic version 6 as well as Microsoft Visual C/C++ version 6. Annotated source code that may be utilized to create executable code as well as additional software and data files are attached as the Computer Program Listing Appendix for this documents and are described in greater detail below. One sk lied in the art can implement the presently-described embodiment of the claimed ge lotoxicity analysis system, in part, by utilizing the software source code and related files in the Computer Program Listing Appendix and software available from third party providers. Figure 2 illustrates, in logical block diagram form, a preferred embodiment of the application software 200 which resides in computer 110 and in a networked control server, to control the operation of genotoxicity analysis system 100 and also illustrates the files which stone the resulis of the genotoxicity analysis. Application software 200 includes main executable programs 210, library link and DLL files 220, parameter files 230 and data results files 240, all of which will be described in greater detail below. Robot control program 250 is prterably control software provided by the manufacturer of robotic slide feeder 160. Main executable programs 210 include DataInput.exe 252, AutoScan.exe 254 and Relocation exe 256. Datalnput.exe 252 allows a user to enter information particular to each slide that s to be analyzed as shown, e.g., in Figure 5 which will be explained below. Autc Scan.exe 254 is used to initiate and provide fully automated selected genotoxicity scres ning of the slides that are identified using Datalnput.exe 252. Relocation.exe 256 is a utility which allows a user to retrieve and manually view slides that have been processed using AutoScan.exe 254 in order to allow the user to visually inspect features of the biological material contained on the slide if necessary. Executable programs 210 are each preferably compiled and linked to library link and I LL files 220 using Microsoft Visual Basic version 6.0. The source code for each of executable progiams 210 references a respective file named "Globals.bas," each version of whiel contains the respective "main" function for each of executable programs 210, and futher induces other modules and necessary Visual Basic forms and code to create the various user interface window,?. Also, as explained further below, executable programs 210 and the modass and forms associated with executable programs 210 operate by calling library link and DLL lies 220 during operation. The Computer Program Listing Appendix for this document includes the source code for creating each of executable programs 210 using Microsoft Visual Basic version 6.0. More particularly. the Computer Program Listing Appendix includes a folder named "VB6" which coniains various subfolders. The subfolders named "DATAINPUT," 'AUTOMATICSCAN" and "RELOCATION" contain the source code for creating Datalnput.exe 252, AutoScan.exe 254 and Relocation.exe 256, respectively. The remaining subfolders in the folder labeled "VB6" in the Computer Listing Appendix contain source code for providing additional functionality for genotoxicity analysis s/stem 100. These subfolders include "SUPERUSER" which stores source code for creating v ;er interfaces that allow for manual adjustment of system parameters when necessary, " TOOLFORMS" which stores source code for user interface modules that may be used by pecutable programs 210, "PASSWORD," which stores source code for providing password- protected access to genotoxicity analysis system 100 and "MODULES," which includes source code for calling necessary library link and DLL files 220 during operation of genotoxicity analysis system 100. In addition to the source code for creating executable programs 210, the Computer Program Listing Appendix for this document also includes source code for creating library link and DLL files 220 using Microsoft Visual C/C + + version 6.0. More particularly, the source code for generating library link and DLL files 220 is found in the subtifolder labled "VC6" on the Computer Program Listing Appendix. The subfolder labeled "AUTOO" in the folder named "VC6" contains source cod : for generating a C library called "autoO" 262 which provides functionality for facilitating the lutomatic lunctioning of genotoxicity analysts system 100, including autofocus control and auxmatic lamp adjustment, among others. The functionality provided by autoO 262 is based on the related lunctionality provided by the "microO" 264 and "improcO" 266 DLLs which are described in greater detail below. The subfolder labeled "COMET" in the folder named "VC6" contains source code for generating a C library called "comet" 268 which provdes functionality required for performing image analysis on slides being analyzed for the comet assay. The subfolder labeled "GENERAL0" in the folder named "VC6" contains soun e code for generating a C library called "generalO" 270 which provides functionality for general purpose tools, including input and output functionality and graphic display routines. The subfolder labeled "IMPROCO" contains source code for generating a C library called "improcO" 266 which provides interface functionality for the library of functions associated with the Matrox driver software of frame grabber board 120. These include fucactions relating to general image processing. The subfolder labeled "METFIN" contains source code for generating a C library callec "metfin" 272 which provides functionality required for performing image analysis on slides that are being analyzed for the metaphase finding application. The subfolder labeled "MICRO0" contains source code for generating a C 1 brary called "microO" 264 which provides interface and control functionality associated with the Leica SDK driver software for electronically driven microscope 150. The subfolder labeled "MNTINVIVO" contains source code for generating a C libary called "MNTinvivo" 274 which provides functionality required for performing image analysis on slides that are being analyzed for the micronucleus test in vivo application. The subfolder labeled "NNETO" contains source code for generating a C library called "nnetO' 276 which provides functionality required for pattern classification through prediction using neural networks, e.g., the backpropagation algorithm, for the micronucleus tes in vitro. The subfolder labeled "RELOC0" contains source code for generating a C limary called ' relocO" 278 which provides functionality for object retrieval within Retocation.exe 254, e.g., data input and output functionality and retrieval of analysis results. The subfolder labeled "ROBO0" contains source code for generating a C library callied "roboO' 280 which provides functionality required for communicating with robotic slide feeder 160. The subfolder labeled "SCANO" contains source code for generating a C library called "scanO" 282 which provides functionality required for facilitating an automatic scanning process, e.g., handling scanning mode settings, triggering the sequential analysis of the batch of Slides to be processed and interfacing to specific application DLLs. Additional libraries may also be included with library link and DLL files 220, incluiding necessary library files provided by third party vendors for controlling operation of the electronicaily driven microscope 150 and frame grabber board 120. The source code for certain of the above-described library link and DLL files 220 defefne the algorithm and image analysis processing that is conducted for the various screenings. The image analysis processing for the micronucleus test in vivo uses red and blue canera channel information and thresholding techniques for discrimination between polychromatic and normochromatic erythrocytes. Thereafter, gradient and watershed transformation for segmentation of micronucleus candidates is utilized. Individual analysis of segmented objects uses supervised training of patterns on the basis of morphometric features, as well as structural features such as "periphery percentage," "focus deviation" and "gray deviation." Reference may be made to the applicable source code described above for further de a 1. Metaph use finding utilizes differences of spectral images as the gray image basis and thereafter utilizes a combination of watershed transformation and "top-hat" segmentation for nucleus candidate segmentation. That is followed by restriction of metaphase range on non- nuclar regions which is followed thereafter with another application of top-hat and watershed segmentation. Finally, feature base metaphase candidate classification, involving individual parameters for chromsomal structuring, is applied. Reference may be made to the applicable source code described above for further detail. Comet assay analysis involves red channel uses of fluorescence image on a first run to detect valid nuclei, including classification on morphometric features. Automatic relocation of detected nuclei for tail moment measurement and use of a sequentially degrading thresholding tecluique which involves a gradient for the pixel sum change in the image is also utilized. Refrenence may be made to the applicable source code described above for further detail. The micronucleus test in vitro uses all three color channel images. The image algorithms attenpt segmentation of valid nuclei and cytoplasm range, and then detect micronucleus candidates using a combination of gradient, top-hat and thresholding segmentation. Final classification uses an off-line trained backpropagational neural network for predicting the probability of a true micronucleus. Reference may be made to the applicable source code described above for further detail. Continuing with Figure 2, application software 200 further includes parameter libs 230 which store information about the proper settings for the operation of electronically craven microscope 150 and the image analysis software operating on genotoxicity analysis system 100, depending upon the particular analysis being conducted. Each of the parameters and adjustments varies depending on the genotoxicity test to be conducted and is set automatically by designating the particular test. Parameter files 230 include the following files: - "cometpar.txt" 290 - contains parameters for the configuration of the image analysts algorithms used for the Comet assay application; - "metfinpar.txt" 292 - contains parameters for the configuration of the irr age analysis algorithms used for the metaphase finding application; - "mntinvivopar.txt" 294, contains parameters for the configuration of the image analysis algorithms used for the micronucleus test in vivo application; and - "molymntpar.txt" 295 contains parameters for the configuration of the image analysis algorithms used for the micronucleus test in vitro application. Parameter files 230 further include a file called "focus std.txt" 284 which to itains parameter data that controls the automatic focus features of electronically driven microscope 150 in connection with the autofocus execution for Datainput.exe 252 and AutoScan.exe; 256. Parameter file 230 called "focus_reloc.txt" 286 generally contains the same parameter definitions as "focus_std.txt" 284, but is more refined to allow for autofocus performance that is better suited for operation under Relocation.exe 254. Parameter file 230 labeled "scantef.txt" 288 contains parameter data that is used for the configuration of electronically operated microscope 150 depending on the selected application. Such configuration includes automatic adjustment of optical modules of the microscope, and setting general parameters referring to the scanning process of the application. Also, the parameter file 230 called "roboplace.txt" 296 contains parameter data to control the initialization and placement of robotic slide feeder 160. These parameters include x,y positioning and speed. Each of "focus_std.txt" and "focus_reloc.txt," are particularized for the sceening tes being performed, i.e., there exists a "focus_std.txt" and "focus_reloc.txt" for sach of the in vivo micronucleus test, in vitro micronucleus test, comet assay or in vitro netaphase finding. Computer Program Listing Appendix stores the parameter files 230 for :ach screening type in respective file folders. More particularly, Computer Program Listing Appendix includes a folder lamed "Applications" which includes subfolders labeled "COMETASSAY" containing the above described parameter files 230 used for comet assay analysis. Similarly, the subfolder called "METFIN" contains the above described parameter files for metaphase finding analysis, "he subfolcer called "MNTINVIVO" contains the above described parameter files for in vivo iuecronucleus test analysis. The subfolder called "MOLYMNT" contains the above described parameter tiles for in vitro micronucleus test analysis. The "MOLMNT" subfolder further includes a file called "p21h9.net" and includes parameters for the neural network pattern prediction and cassification utilized for the in vitro micronucleus test analysis. In a preferred embodiment, "robias.txt," which holds system specific information for the application in general for genotoxiciy analysis system 100 and " oboplace.txt" 296, which contains parameters for use by robotic slide feeder 160 during initialization, reside locally on the hard drive of computer 110 while the remaining programs and files reside on a networked server connected to computer 110. In addition to the above-described parameter data files, calibration files conntaining "shadimages", including "shadref_black" and "shadref_whitbl", are referenced by the executable programs 210. One skilled in the art may generate these files to provide colibration for shading correction. Calibration files are particular to each screening publication. The calibration files are preferably stored in a subdirectory that is parallel to the respective subdirectories containing the parameter data. Application software 200 of Figure 2 further includes data results files 240 which are generated and modified by executable programs 210. There are three types of data results files 240 having the following forms: (1) > scanresults/// .txt; (2) > individualdata/// and (3) >slidedata/slidedata .txt In the above-listed file formats for data results files 240, > indicates the preliminary file path of the directory containing the file at issue. This part of the path may vary depending upon how the file structure of the overall operational software is configured, "scanresults," individualdata" and "slidedata" represent respective subfolder names for the files. represents a placeholder for the study name coding the toxicological testing of i certain test compound and is correlated with a unique "study name", represents a placeholder for a particular experiment in the context of the selected study. Experiments belonging to a specific study can vary with respect to treatment time or the aosence or presence of the metabolic activation of cells, or sampling time after treatment of aiimals. Generally, it specifies the "experimental" conditions for the same test compound of interest. represents a placeholder for the identity of a particular slide and represents a placeholder for a particular position of a slide in a rack. The operation of genotoxicity analysis system 100 will now be described with reference to (he flow chart of Figure 3 and the exemplary screen interfaces of genotoxicitiy analysis system 100 illustrated in Figures 4 through 14. At step 302 of the process of Figure 3, the user selects one of Datalnput.exe, Auto Scan.exe and Relocation.exe for execution from the main display screen of color display monitor 130. Bach application is preferably represented as an application or shortcut icon on the main display screen of the Windows NT platform. The user may select the desired program by double clicking the corresponding icon in the known manner. If the user desires to enter information for each slide that is to be analyzed, the uss, selects the icon representing Datalnput.exe for execution at step 302. As a result, the process proceeds to step 304 where the form illustrated in Figure 4 is displayed to the user, thing the fonr of Figure 4, the user specifies the current application, i.e., the analysis that is to be performed, by selecting a unique path to which the slide data will be written. Thus, selection of the path also designates the analysis that will be performed, i.e., comet assay, micronucleus test in vivo, micronucleus test in vitro or metaphase finding. The form of Figure 4 is seated from the source code found in the file called "frolmite" in the VB6/T00 LFORMS subdirectory of the Computer Program Listing Appendix for this document. The process then moves to step 306 where the form illustrated in Figure 5 is displayed to the user. Using this form, the user enters data for identifying each slide that is to the processed. The identification string for each slide consists of a study name (col. 501), tollowed b> experiment name (col. 502) and a slide code (col, 503), each of which may utilize Lumerals ot characters. It is noted that the exemplary slide codes 503 presented in figure 5 are appended by "a" and "b." In the presently disclosed embodiment of the present invention, two simples of biological material may be included on each slide, one designated by "a", the other by "b." The precision provided by the components of genotoxicity analysis system 100 in combination with application software 200 allows for this efficient use of slide space which effectively doubles slide capacity for screening. For slides sharing the same study and experiment code, a common folder for resulting storage will be created. The form of Figure 5 is created from source code found in the; file called "frmSlides.frm" in the VB6/DATAINPUT subdirectory of the Computer Program Lisiitig Appendix for this document. At step 308, the user accepts the settings entered at step 306 by pressing the "accept settings" button 506 of the form of Figure 5, at which point the system ends operation of Datalnput exe, creates all necessary folders (for studies and experiments) and data files and returns to step 302 of Figure 3. Alternately, at step 310, the user may select any of the respective detail buttons (see column 504 of the form of Figure 5) for each slide to adjust specific parameters relating to each slide. Figure 6 represents the form presented to the user for adjusting the parameters for a particular slide. The form of Figure 6 is created from the source code found in the file called fire Slideparam frm in the VB6/DATAINPUT subdirectory of the Computer Program Listing A poendix for this document. Among the various parameters that the form of Figure 6 allows a user to control is threshold adjustment (button 602) and microscope adjustment (button 604). The form for providing the user the ability to adjust the threshold settings for the particular slide is illustrated in Figure 7. This form is created from the source code found i the file called "frmlnterThresh.frm" in the VB6/T00LF0RMS subdirectory of the Computer Program Listing Appendix for this document. The form for providing the user the ability to adjust microscope parameters for the particular slide at issue is illustrated in Fig. 8. This form is created from source code found in the file called "frmAdjustMicro.frm" in the VB6/TOOLFORMS subdirectory of the Computer Program Listing Appendix for this document. Once the user is satisfied with the adjustments made to the particular slides, the user may select the "acc. Settings for ALL slides" button 606 of the form of Figure 6 which will set these parameters for all previously identified slides that have valid slide code entries in the form of Figure 5. Alternatively, the user may select the "ace. settings for CURRENT slide" (button 608) which saves parameter settings only for the currently selected slide. Control is then returned to the form of Figure 5 (step 306). Returning now to step 302 of the process illustrated in Figure 3, if the user selects the icon to initiate execution of AutoScan.exe, the process moves to step 312 where the f rom illustrated in Figure 8a is presented to the user. Here, the user selects the genotoxicity tos. that will he processed, i.e., one of the comet assay, micronucleus in vivo, micronucleus in vitro or metaphase finding analyis, by selecting the respective subdirectory illustrated in window 802 in the form of Figure 8a. The form of Figure 8a is created from source code found in the file called "frmlnit.from" in the VB6/TOOLFORMS subdirectory of the Computer Pro gram Listing Appendix for this document. The process then proceeds to step 314 where the form of Figure 9 is presented to the user. The form of Figure 9 allows the user to select the scanning options for the genotxicity analysis to be performed as was specified at step 312 using the form of Figure 8. The options presented by the form of Figure 9 include: (1) scanning the slides without display (button 902), meaning that no intermediate image display will be presented to the user during analysis of the slides; (2) scanning the slides with display (button 904), meaning that the most important intermediate image processing results will be displayed during analysis without req liring user interaction to continue analysis; (3) scanning the slides with Testl level (button 9(), meaning that several intermediate image processing steps are performed and the process is t: en halted until the user presses a key to continue automatic analysis; and (4) scanning the slides wit a Test2 level (button 908), which results in operation similar to that of button 906 except that detection results are not displayed. This last mode is utilized to validate operation of the application where a user performs manual analysis of a slide in parallel with automated analysis in the same image fields. Finally, the user may press button 910 for scanning the slides with autofocus test, which processes the slides while presenting a graphical display of the autofocus results, e.g., contrast curve, for each slide. The user may also abort running the analysis by pressing the exit button 912. If the user does not abort the automatic scanning, the process proceeds to step 116 and the automatic scanning is executed by referencing the applicable library link and DLL tiles 220 and parameter files 230 of application software 200 for the specific type of analysis teing performed. The form of Figure 9 is created from source code found in the file called frmMain.frm" in the VB6/AUTOMATICSCAN subdirectory of the Computer Program I isting Appendix for this document. When the automatic scanning is complete and all results data has been written and stored, the process returns to step 302 of Figure 3. If at step 302, the user executes Relocations.exe, the process of Figure 3 proceeds to step 318 where the form of Figure 10 is presented to the user. Using the form of Figure 10, the user selects the genotoxicity test for which results are to be reviewed, i.e., one of the comet issay, micronucleus in vivo, micronudeus in vitro or metaphase finding analyis, by selecting the respective subdirectory illustrated in window 1002 in the form of Figure 10. The form of Figure 10 is created from the source code found in the file called "frmlnit.frm" in the VB6/TOCLFORMS subdirectory of the Computer Program Listing Appendix for this document. The process then proceeds to step 320 where the user is presented with forms to select a specif c slide to be reviewed. More particularly, the user is presented with the forms the trated in F igures 11 and 12. In the form of Figure 11, the user selects the particular study con aining the slide by selecting the appropriate subdirectory labeled with the appropriate study and experimem name (see window 1102). Using the form of Figure 12, the user identifies the palcular slide by selecting the file containing the slide data (see window 1202). The form of Figure 11 is created from source code found in the file called "frmMain.frm" in the VI 6/RELOCATION subdirectory of the Computer Program Listing Appendix for this document. The form of Figure 12 is created using a standard Visual Basic CommonDialog USER interface object. Next, at step 322, the user is presented with the form of Figure 13 which includes a display (window 1302) of the scanning results associated with the slide specified using the form of Figure 12. The form of Figure 13 is similar to that of Figure 11 except that it row includes, in window 1302, the most relevant data that had been acquired during automatic slile analysis, such as the number of detected objects, number of scanned fields, error codes, and other application specific information for the slide under review. The user may exit Relocation.exe by eliciting button 1304 of the form of Figure 3 (step 324) at which point the process of Figure 3 returns to step 302. Alternatively, the user may select button 1306 of the form of Figure 13 causing the process of Figure 3 to move to step 326 at which point the user is presented with the form or Figure 14 The form of Figure 14 allows a user to retrieve the objects that had been detected during the automatic scanning process. For this purpose, one can move from one object to another (and then back again) using the arrow buttons 1402 and 1404. Using the additional controls presented in the form of Figure 14, each object's coordinates, which had been stored during scanning, and the current live image showing the object are displayed on color display screens 130 and 132 for visual inspection. The user may operate the right or left n ouse button to flag an object under observation and discard an object as a valid micronucleus (by using the left mouse button) or accept an object as a valid micronucleus (using the right mouse button). By moving from the first detected object to the last for each slide, the user can assign the proper label (i.e., "accept" or "reject") to each object and, therefore, adjust the result of automatic scanning through supervised visual inspection. The corrected result for the current slide, i.e. the number of micronuclei for micronucleus application, or number of metaphases for metaphase finding application, will be stored after exiting the form of Figure 14. The othir options present in the form in Figure 14 support the adjustment of the current image, e.g. microscope and focus, and support image analysis for other objects of interest in c rder to confirm proper performance of the the algorithms utilized for image analysis. The form of Figure 14 is created from source code found in the file called "FrmRelocation.frm" in the VB6/RELOCATION subdirectory of the Computer Program L sting Appendix for this document. Thus, it is seen by the above, that by creating software code which can facilitate different types of genotoxicity screening and which references parameter data files respectively configured for each of various genotoxicity tests, the genotoxicity analysis system of the present inversion provides a flexible and easy to use platform for performing various genotoxicity screenings with minimal user interaction. Depending upon the type of screenings being performed, no manual microscope module adaptation is necessary between screening runs for different analysis testing. In the case of comet assay screening, a manual change to iuident illumination to support fluorescence staining in comet assay analysis and then back to transmitted light illumination for other genotoxicity screenings may be necessary. Moreover, as described above, the genotoxicity analysis system of the present invention allows interactive pattern control to permit a user to manually perform artifact rejection for objects wrongly c a isified dur ng automatic scanning. In accordance with 37 C.F.R. 1.52 (e), the name, respective creation date and sizi (in bytes), of each file contained on the CD-ROM of the Computer Program Listing Apendix are listed in Figures 15a - 15. For ease of reference, the file names are listed as they appear in the directory structure of the Computer Program Listing Appendix. WE CLAIM; A system for providing in vivo and / or in vitro genotoxicity screening of samples of biological material, the system comprising: a. one or more computers; b. a frame grabber connected to and being controlled by the one or more computers to create electronic images; c. a camera connected to the frame grabber and providing images to the frame grabber under the control of the one or more computers; d. a microscope connected to and being controlled by the one or more computers, said microscope providing images of samples to be screened to the camera; e. a slide feeder connected to and being controlled by the one or more computers to move the samples to positions where the microscope can image them; and f. a program operating on the one or more computers operative to perform genotoxicity screening by analyzing the electronic images in order to facilitate the screening a second batch of biological material using a second genotoxicity testing method after a first batch of biological material using a first genotoxicity testing method, substantially free of any manual manipulation of the camera, the microscope or the slide feeder 2. The system as claimed in claim 1, having a user interface presented on a display monitor connected to the one or more computers, for allowing a user of the genotoxicity screening system to select the gentoxicity screening method to be performed on a given batch of biological material. 3. The system as claimed in claim 1, the camera, the microscope and the slide feeder including physical connections that receive electronic signals from the one or more computers which control the operations of the camera, the microscope and the slide feeder. 4. A genotoxicity analysis system for providing in vivo and / or in vitro genotoxicity screening of samples of biological material, the system comprising: at least one computer including a software application that is configured to automatically configure components of the system and allow the genotoxicity analysis system to perform a plurality of genotoxicity tests on respective pluralities of biological samples by way of the automatic configuration. 5 The system as claimed in claim 4, wherein the software is configured to allow a user to specify the genotoxicity test to be performed on a given group of biological samples. 6 The system as claimed in claim 5, wherein after the user has specified the geotoxicity test to be performed, the software is configured to automatically generate signals which are sent to the configurable components of the genotoxicity analysis system in accordance with the specified genotoxicity test. 7. The system as claimed in claim 6, wherein the sent signals cause the configurable components of the genotoxicity analysis system to be configured in a manner conducive to the selected genotoxicity test. 8. The system as claimed in claim 4, wherein the software provides a user of the genotoxicity analysis system with the ability to provide identifying information for each biological sample. 9. The system as claimed in claim 4, wherein the software is configured to record the results of the genotoxicity testing for each analyzed sample and provide lhe manual inspection of the recorded results of the genotoxicity testing. 10. The system as claimed in claim 4, wherein the software is configured to have respective files containing data defining configurable parameters of the configurable components for each of the plurality of genotoxicity test. 11. The system as claimed in claim 4, wherein the software is configured to contain software code defining respective image analysis techniques for use by each of the plurality of genotoxicity tests. 12 A method for performing in vivo and / or in vitro genotoxicity testing of samples of biological materials by utilizing a genotoxicity analysis system, including hardware components that are operated with software controls, the genotoxicity analysis system being capable of performing a multiplicity of genotoxicity tests in which electronic images of the samples are analyzed by software, the method comprising the steps of: -a. preparing a first batch of samples of biological materials for processing using a first genotoxicity test; b. utilizing the genotoxicity analysis system to perform a first genotoxicity test on the samples of the first batch of biological materials: c. preparing a second batch of samples of biological materials for processing using a second genotoxicity test; d utilizing the genotoxicity analysis system to perform a second genotoxicity test on the samples of the second batch of biological materials, and e. manipulating through software the configuration of the hardware components during the time period between performance of the first and second genotoxicity tests to thereby allow the first and second genotoxicity tests to be performed using the same hareware components. 13. A method for performing various types of in vivo and / or in vitro genotoxicity tests on respective batches of biological samples using a genotoxicity analysis system that analyzes electronic images of the samples, the method including the steps of: a. receiving a command from a user of the genotoxicity analysis system, the command specifying the type of genotoxicity test to be performed: b. performing an automatic configuration of the component of the genotoxicity analysis system to thereby allow the genotoxicity analysis system to perform the genotoxicity test specified in step a; c. performing the specified genotoxicity test on a batch of biological samples; d. recording results of the genotoxicity test; e. repeating steps a through d. 14. The method as claimed in claim 13, wherein the types of genotoxicity tests are selected from the group consisting of one or more of the following: the micronucleus test in vivo, the micronucleus test in vitro, the comet assay and metaphase finding. 15. A method for performing genotoxicity screening comprising the steps of: a. preparing a batch of slides for genotoxicity screening; b. selecting a genotoxicity test; c. automatically retrieving the first of a plurality of slides containing biological samples from a slide retaining device; d. automatically delivering the slide to an electronically driven microscope: e. automatically focusing on the material contained on the slide; f. automatically recording a visual representation of the focused image; g. automatically delivering the focused image to a microprocessor-based computer; h. automatically performing image analysis on the recorded image using image analysis software appropriate for the genotoxicity test selected in step b. i. automatically recording the data resulting from the analysis of the image; j. automatically returning the slide retrieved in step c to the slide retaining device; k. automatically retrieving the next slide for analysis; 1. automatically repeating steps c through k for successive slides in the batch until all of the slides in the batch have been analyzed; and m. repeating steps a through 1 until all desired slides have been processed. 16. The method as claimed in claim 15, having the step of manually verifying the recorded data. 17. The method as claimed in claim 15, wherein the batch of slides is prepared in accordance with the genotoxicity test to be performed, the genotoxicity test being selected from the group consisting of: the micronucleus test in vivo, the micronucleus test in vitro, the comet assay and metaphase finding. 18. The method as claimed in claim 15, wherein the selecting step is performed by choosing the appropriate test from a menu displayed on a video monitor. 19. The method as claimed in claim 15, wherein the steps of automatically retrieving and automatically returning is performed by a robotic slide feeder. 20. The method as claimed in claim 15, wherein the step of automatically recording the data resulting from the analysis of the image is continuously performed until either a given delimiting number of cells have been counted or the maximum number of image fields to be analyzed has been reached for the slide currently under analysis. A system (100) and method for performing genotoxicity screening utilize: (1) one or more computers (110); (2) a frame, grabber (120) connected to the one or more computers (110); (3) a camera (140) connected to the frame grabber (120); (4) a microscope (150) connected to the one or more computers (110); (5) a slide feeder (160) connected to the one or more computers (110); and (6) a program operating on the one or more computers (110). The program facilitates the screening of a second batch of biological material using a second genotoxicity testing method after screening a first batch of biological material using a first genotoxicity testing method. The screening operates substantially free of any manipulation of the camera (140), the microscope (150) or the slide feeder (160).

Full Text

SYSTEM AND METHOD FOR FULLY AUTOMATED ROBOTIC-
ASSISTED IMAGE ANALYSIS FOR IN VITRO AND IN VIVO GENOTOXICITY
TESTING
REFERENCE TO COMPUTER PROGRAM LISTING APPENDIX
A Computer Program Listing Appendix to this document has been submitted to
the U.S. Patent and Trademark Office in accordance with 37 C.F.R. §§ 1.52 and 1.96 on the
filing date of this document and is hereby incorporated herein by reference in its entirety. The
Computer Program Listing Appendix is contained on one (1) CD-ROM, two copies of which
have been filed with the U.S. Patent and. Trademark Office and each of which are labeled with
the name of the inventor of the present invention, the title of the invention, the attorney's
docket number and the creation date of the CD-ROMs.
FIELD OF THE INVENTION
The present invention is directed to genotoxicity testing and, more particularly,
to a metho.l and system for utilizing, in conjunction, an automated robotic slide feeder or
equivalent device, an electronically driven microscope, a microprocessor-based computer and
additional components and software to facilitate high-throughput in vitro and in vivo
genotoxicily testing.
BACKGROUND OF THE INVENTION
Toxicologicial testing is used in various technologies, industries and disciplines
for assessing the effect of drugs and other chemical compounds on the nature and properties of
oiological natter. Genotoxicity testing is particularly useful for analyzing the effect of certain

chemicals on the DNA structure of the cells of humans, animals and other life forms, including
he analysis of the potential for induction of hereditary diseases and mutations. Genotoxicity
testing generally includes screening of either in vivo or in vitro biological matter.
Well known in vitro test systems include, but are not limited to:
(1) the comet assay, which is used for detecting primary DNA damage, DNA to
DNA crosslinks, and DNA-protein interactions. A specific version of the comet assay is the
alkaline Comet Assay which is described in a publication titled "A simple technique for
quantiation of low levels of DNA damage in individual cells," Singh et al., Experimental
( ellular Research, vol. 175, pp. 184-191 (1988). The Alkaline Comet Assay is also described
in a publication titled "Modification of the Comet Assay for the detection of DNA strand
breaks in extremely small tissue samples," Tebbs et al., Mutagenesis, vol. 14, pp. 437 -438
( 999);
(2) the micronucleus test in cell lines (V79 cells, Mouse Lymphome cells, TK6
colls) or hurian lymphocytes, which are all known to be useful in the early screening of new
impounds n industrial toxicology; and
(3) the chromosome aberration test, which is required by certain regulatory
authorities,such as the Organization for Economic Co-Operation and Development and the
I nited States Food and Drug Administration, for approval of new drugs. For this in vitro test,
ie assessment of chromosomal aberrations is done on the basis of metaphases which must be
detected for analysis.
In vivo genotoxicity test systems include, but are not limited to:
(1) the in vivo micronucleus test in bone marrow for clastogenic or aneugenic
petential of test compound administered to rodents. This test is described in a publication
titled "A Rapid in vivo test for chromosomal damage," Heddle, JA., Mutual Res., vol. 18, pp.
[i 7-90 (197:0;
(2) the in vivo comet assay, which under certain circumstances may be accepted
as a regulatory assay in addition to the micronucleus test in vivo, to verify in vitro test results.
The in vivo comet assay is described in a publication titled "Recommendations for conducting
th : in vivo alkaline Comet assay", Hartmanr. et al., Mutagenesis vol. 18, no. 1, pp. 45-51
(2 )03).

Other in vivo and in vitro testing methods are also well known in the art.
Limited automated methods for facilitating genotoxicity screening ("screening"
being understood to refer to the analysis of biological material samples previously treated with
the test compound) of both in vivo and in vitro materials have also been attempted. As an
example, art automated in vivo micronucleus assay analysis of mouse bone marrow used in the
pharmaceutical industry to test the genotoxicity potential of new compounds is described in a
publication co-authored by the inventor of the present invention which is titled "Technical
aspects of automatic micronucleus analysis in rodent bone," Cell Biology and Toxicology, vol.
10, pp. 283 -289 (1994). Automated forms of analysis for in vitro micronucleus tests are also
known. The inventor of the present invention authored an article titled "Automatic analysis of
t le in vitro micronucleus test on V79 cells" in Mutation Research, vol. 413, pp. 57-68 (1998),
cescribing an automated in vitro micronucleus test for V79 cells.
The techniques for automated genotoxicity screening for both in vivo and in
Nitro biological material that were noted above utilize image analysis software and techniques
mat are individually designed for the specific type of test and the specific type of material that
i being screened. Automation of genotoxicity testing that utilizes image analysis simplifies the
process of compound screening, eliminates the tedium of manual scoring and significantly
Ficreases the overall number of genotoxicity screenings which can be performed in any given
period of time. Generally, an automated electronically driven microscope with image
capturing capabilities and a micro-processor based computer running microscope control and
inage analysis software, each specifically designed, calibrated and programmed for the
rarticular screening being performed, is used to operate and facilitate the image analysis-based
automated screening process.
To provide still further increases in the throughput of genotoxicity sample
screening, prior art devices are known to have incorporated robotic arm assemblies and
equivalent devices to facilitate sample slide feeding, thus freeing the user from the tedium of
nanually loading slides for image analysis and further increasing screening throughput rates.
Known prior art systems do not, however, allow for both in vivo and in vitro
cenotoxicity screening using a single platform to perform automatically all manners of in vitro
and in vivo genotoxicity testing such as the micronucleus test, the comet assay and metaphase

detection for chromosome analysis, nor do any known prior art system provide for utilization
of a rot otic slide feeder or equivalent device for all manner of in vitro and in vivo testing
without the tedium of extensive user intervention.
Summary
An embodiment of a genotoxicity screening system of the present invention
includes (1) one or more computers; (2) a frame grabber connected to the one or more
computers; (3) a camera connected to the frame grabber; (4) a microscope connected to the one
or more computers; (5) a slide feeder connected to the one or more computers; and (6) a
program operating on the one or more computers. The program facilitates the screening a
second batch of biological material using a second genotoxicity testing method after screening
a first batch of biological material using a first genotoxicity testing method. The genotoxicity
methods are performed substantially free of any manual manipulation of the camera, the
microscope or the slide feeder.
In another embodiment of the present invention, software is provided that
controls the operation of a genotoxicity analysis system. The software provides automatic
onfiguration of configurable components of the genotoxicity analysis system and allows the
enotoxicity analysis system to perform a plurality of genotoxicity tests on respective
luralities cf biological samples by way of the automatic configuration.
In another embodiment of the present invention, genotoxicity testing of
b ological materials is performed using a genotoxicity analysis system. The genotoxicity
system includes hardware components that are operated with software controls. The
genotoxicity analysis system is capable of performing a multiplicity of genotoxicity tests. Use
o) the genotoixicity analysis system performs as follows: (1) preparing a first batch of samples
of biological materials for processing using a first genotoxicity test; (2) utilizing the
genotoxicity analysis system to perform a first genotoxicity test on the samples of the first
tach of biological materials; (3) preparing a second batch of samples of biological materials

or processing using a second genotoxicity test; and (4) utilizing the genotoxicity analysis
System to perform a second genotoxicity test on the samples of the second batch of biological
materials. The software controls manipulate the configuration of the hardware components
during the time period between performance of the first and second genotoxicity tests to allow
the first and second genotoxicity tests to be performed using the same hardware components.
Yet another embodiment of the present invention includes a method for
performing various types of genotoxicity tests on respective batches of biological samples using
a genotoxicity analysis system. The method including the steps of: (1) receiving a command
from a user of the genotoxicity analysis system, the command specifying the type of
genotoxicity test to be performed; (2) performing an automatic configuration of the component
of the genotoxicity analysis system to thereby allow the genotoxicity analysis system to
perform the genotoxicity test specified in step 1; (3) performing the specified genotoxicity test
ot a batch of biological samples; (4) recording results of the genotoxicity test; (5) repeating
stops 1 through 4.
In yet another embodiment of a method for performing genotoxicity screening in
i c ;ordance vith the present invention, the following steps are performed: (1) preparing a batch
of slides for genotoxicity screening; (2) selecting a genotoxicity test; (3) automatically
reprieving the first of a plurality of slides containing biological samples from a slide retaining
device; (4) automatically delivering the slide to an electronically driven microscope; (5)
au omatically focusing on the material contained on the slide; (6) automatically recording a
visual representation of the focused image; (7) automatically delivering the focused image to a
mecroprocessor-based computer; (8) automatically performing image analysis on the recorded
imge using image analysis software appropriate for the genotoxicity test selected in step 2; (9)
automatically recording the data resulting from the analysis of the image; (10) automatically
returning the slide retrieved in step 3 to the slide retaining device; (11) automatically retrieving
the next slide for analysis; (12) automatically repeating steps 3 through 11 for successive slides
in he batch until all of the slides in the batch have been analyzed; and (13) repeating steps 1
through 12 urtil all desired slides have been processed.

Brief Description of the Drawings
The foregoing and other features of the present invention will be more readily
apparent from the following detailed description and drawings of illustrative embodiments of
the invention, in which:
Figure 1 illustrates, in block diagram form, an embodiment of the automated
genotoxicity analysis system;
Figure 2 illustrates, in logical block diagram form, an embodiment of
application software to control the operation of a genotoxicity analysis system and files which
s ore the results of the genotoxicity analysis;
Figure 3 illustrates a flow chart describing the operation of an embodiment of a
genotoxicity analysis system;
Figure 4 illustrates an embodiment of a user interface screen for entering
information relating to a slide that is to be analyzed using a genotoxicity analysis system;
Figure 5 illustrates an embodiment of a user interface screen for entering data
identifying particular slides to be processed using a genotoxicity analysis system;
Figure 6 illustrates an embodiment of a user interface screen for adjusting the
parameters for a particular slide to be analyzed using a genotoxitiy analysis system;
Figure 7 illustrates an embodiment of a user interface screen that allows a user
to adjust the threshold settings for the particular slide that is to be analyzed using a
genotoxicitv analysis system;
Figure 8 illustrates an embodiment of a user interface form for adjusting
nicroscope parameters for the a particular slide using a genotoxicity analysis system;
Figure 8a illustrates an embodiment of a user interface form for use in selecting
genotoxicity test that to be processed using a genotoxicity analysis system;
Figure 9 illustrates an embodiment of a user interface screen for a user to select
scanning options for a genotxicity analysis system;
Figure 10 illustrates an embodiment of a user interface screen for selecting
results of a genotoxicity test for review using a genotoxicity analysis system;

Figure 11 illustrates an embodiment of a user interface screen for specifying a
particular study containing a slide desired to be reviewed by a user of a genotoxicity analysis
system;
Figure 12 illustrates an embodiment of a user interface screen for identifying the
particular slide for review in the study selected in the user interface screen of Figure 11;
Figure 13 illustrates an embodiment of a user interface screen for displaying the
results of screening of a particular slide using a particular genotoxicity test;
Figure 14 illustrates an embodiment of a user interface screen that allows a user
to retrieve objects that have been detected during an automatic scanning process using an
automated genotoxicity testing system; and
Figures 15a through 15e present a listing of computer files for use in creating an
enbodiment of an automated genotoxicity testing system.
Detailed Description of an Embodiment of the Invention
Described herein is a single automated platform for genotoxicity screening
which can accommodate both in vivo and in vitro micronucleus testing, comet assay screening
and in, vitro metaphase finding, but requires minimal user monitoring and/or user interaction.
As will be more fully described, the present invention is an automated system
and method for performing sample analysis for genotoxicity testing. An embodiment of the
invenire system Includes: (1) a robotic slide feeder, (2) an electronically driven microscope,
(3) an image capturing apparatus, (4) a microprocessor-based computer running program
contro software, and (5) required communication cables and interface apparatus for
interconnecting the various components, The invention is embodied in a system and method as
exempt t ed in the embodiments described below, but is not limited to the details of those
embodimentsnts. One skilled in the art will readily appreciate that the invention may include and
utilize equivalent components and processes that fall within the scope of the invention, which
invention is defined solely by the claims that will accompany this disclosure. Moreover, the
inventior an comprise aspects of the foregoing components and their interrelationship to one
another, rehiding, without limitation, programmed control of such components.

Using the inventive automated genotoxicity analysis system and method, a
laboratory technician or other user may optimally process, for analysis, successive batches of
slic es containing biological material using different tests and different types of biological
maerial for each batch, without the need to manually adjust any hardware and with only
rminiimal user interaction.
As will be more fully described below, the method of operation of the
automated genotoxicity analysis system of the present invention proceeds as follows. A
lab bratory technician or other user prepares a batch of slides for genotoxicity screening. These
slices may include in vivo or in vitro biological materials and may be prepared for screening
by any of the following (or additional) tests: (1) in vivo micronucleus test, (2) in vitro
moni ronucleus test, (3) in vitro or in vivo comet assay and (4) in vitro metaphase finding. Once
tt e slides are prepared for testing, the user selects the appropriate genotoxicity test system
(from the described list of possibilities) from a menu or equivalent user interface displayed on
the screen of he microprocessor based computer. The robotic slide feeder then automatically
ret ieves the first of the slides from the batch prepared by the user and delivers the slide to the
electronically driven microscope, which then automatically and appropriately focuses on the
material contained on the slide. Next, the image capturing apparatus records a visual
resresentatior of the focused image and delivers it to the microprocessor-based computer. The
mmicroprocessor-based computer then performs image analysis on the recorded image using the
aproropriate image analysis software preloaded on the computer. The computer then records
the data resuling from the analysis of the image until either the given delimiting number of
ce1 s have been counted or the maximum number of image fields to be analyzed has been
reached for the slide currently under analysis. Once the analysis of the slide is complete, the
robotic slide feeder returns the slide to the slide rack and retrieves the next slide for analysis.
This process continues until all of the slides in the batch have been analyzed. The user may
then prepare ,tnew batch of slides of any type of in vivo or in vitro material and initiate
animated screening of the material using any of the genotoxicity assays described above
wi hout the need to manually change or modify any of the system equipment.
Figure 1 illustrates, in block diagram form, an embodiment of the automated
ge lotoxicity analysis system 100 of the present invention. Genotoxicity analysis system 100

i lcludes a microprocessor-based computer 110 having a frame grabber board 120, two color
display monitors 130 and 132, a charge coupled device (CCD) camera 140, an electronically
driven microscope 150 and a robotic slide feeder 160.
Computer 110 of Figure 1 may be any of the many known IBM-compatible
personal or server computers running any known operating system for such computers, e.g.,
Windows XP, Windows NT Server or UNIX. In the preferred embodiment, a Transtec 1300
OBM compatible PC, operating at 1.3 GHz., having at least 128 Mbyte of internal RAM
Tiemory and tunning the Windows NT 4, Service Pack 5 operating system or Windows 2000 is
utilized. Computer 110 executesall operating, control and image processing software which
w 11 be described more fully below, for genotoxicity analysis system 100 and is connected to
and controls the operation of all other components of gentoxicity analysis system 100.
Computer 110 is connected to electronic miscroscope 150 and robotic slide feeder 160 via RS-
232 serial interfaces. Computer 110 includes a frame grabber board 120 which is preferably a
Meteor-II frame grabber utilizing Matrox MIL 6.1 or later version driver software available
from Matrox Imaging of Dorval, Quebec. Computer 110 stores all program software and
generated data on a local harddrive. Alternately, computer 110 may be connected to a local
area network (LAN 200) to support data on a networked data base (not illustrated) or to allow
axess, retrieval and storage of parameter data files and other program software located on a
separate networked computer server (not illustrated). In the preferred embodiment, the
executable programs, compiled from the Visual Basic and C/C+ + source code and the
generated measurement data results files are stored on a networked database and server while
the C-language DLLs and related files reside locally on the hard drive of computer 110.
Robotic slide feeder 160 is preferably an ES-553S robot with an SRC-320 driver
avallable from Seiko Epson Corporation of Japan. Robotic slide feeder 160 is controlled and
operated by electronic commands received from computer 110 via a serial cable 170. Robotic
side feeder 160 functions primarily to remove a current slide from a slide rack (not illustrated)
conaining a multiplicitly of slides, then place the slide onto the stage of electronically driven
m c-oscope 150 and then return the slide to the slide rack after the analysis of the slide is
complete. Under the embodiment of the invention described herein, the slide rack may include
as many as 130 glass slides containing biological material or "samples."

Electronically driven microscope 150 of genotoxicity analysis system 100 is
preferably a Leica DM RXA/2 electronic microscope running Leica SDK driver software,
which is manufactured and sold by Leica Microsystems AG of Wetzlar, Germany.
Electronically driven microscope 150 preferably includes the following modules: stage, focus
drive, illumination, objectives, fluorescence cubes, diaphragms for aperture and field,
additional magnification changer and fluorescence shutter, all of which components are
software driven and controlable. Electronically driven microcospe 150 is controlled and
operated by electronic commands received from computer 110 via two serial cables 180 and
182, one each for the stage controller and for the microscope stand of electronically driven
microscope 150.
Camera 140 is preferably an XC-003 or DXC-390 CCD camera sold by Sony
Corporation of America. Camera 140 is mounted on electronically driven microscope 150 in
the known manner using a C-mount adapter and is utilized to grab the current image from
electronically driven microscope 150 and send the image in analog format to frame grabber
board 120/via seriarcable 190. Camera 140 is under operational control of computer 110 via
frame grabber 120. The analog fomatted image received from camera 140 is digitized by
computer 110.
analysis system 100 also includes color display modules 130 and
132 connected to computer 110. Preferably, color display module 130 provides the user
interface to the user of the genotoxicity analysis system 100 while color display module 132
displays the current image provided by electronically driven microscope 150 or, alternatively,
the result of the image processing analysis.
Computer 110 executes software which controls the operation of genotoxicitiy
analysis system 100.
.Computer 110, and any networked server that may also be utilized to control
and operate genotoxicity analysis system 100, preferably runs Microsoft NT version 4 or
Windows 2000 operating system software. The software executed by computer 110 to control
genotoxicity analysis system 110 is created using Microsoft Visual Basic version 6 as well as
Microsoft Visual C/C++ version 6. Annotated source code that may be utilized to create
executable code as well as additional software and data files are attached as the Computer

Program Listing Appendix for this documents and are described in greater detail below. One
sk lied in the art can implement the presently-described embodiment of the claimed
ge lotoxicity analysis system, in part, by utilizing the software source code and related files in
the Computer Program Listing Appendix and software available from third party providers.
Figure 2 illustrates, in logical block diagram form, a preferred embodiment of
the application software 200 which resides in computer 110 and in a networked control server,
to control the operation of genotoxicity analysis system 100 and also illustrates the files which
stone the resulis of the genotoxicity analysis. Application software 200 includes main
executable programs 210, library link and DLL files 220, parameter files 230 and data results
files 240, all of which will be described in greater detail below. Robot control program 250 is
prterably control software provided by the manufacturer of robotic slide feeder 160.
Main executable programs 210 include DataInput.exe 252, AutoScan.exe 254
and Relocation exe 256. Datalnput.exe 252 allows a user to enter information particular to
each slide that s to be analyzed as shown, e.g., in Figure 5 which will be explained below.
Autc Scan.exe 254 is used to initiate and provide fully automated selected genotoxicity
scres ning of the slides that are identified using Datalnput.exe 252. Relocation.exe 256 is a
utility which allows a user to retrieve and manually view slides that have been processed using
AutoScan.exe 254 in order to allow the user to visually inspect features of the biological
material contained on the slide if necessary.
Executable programs 210 are each preferably compiled and linked to library link
and I LL files 220 using Microsoft Visual Basic version 6.0. The source code for each of
executable progiams 210 references a respective file named "Globals.bas," each version of
whiel contains the respective "main" function for each of executable programs 210, and futher
induces other modules and necessary Visual Basic forms and code to create the various user
interface window,?. Also, as explained further below, executable programs 210 and the
modass and forms associated with executable programs 210 operate by calling library link and
DLL lies 220 during operation.
The Computer Program Listing Appendix for this document includes the source
code for creating each of executable programs 210 using Microsoft Visual Basic version 6.0.
More particularly. the Computer Program Listing Appendix includes a folder named "VB6"

which coniains various subfolders. The subfolders named "DATAINPUT,"
'AUTOMATICSCAN" and "RELOCATION" contain the source code for creating
Datalnput.exe 252, AutoScan.exe 254 and Relocation.exe 256, respectively.
The remaining subfolders in the folder labeled "VB6" in the Computer Listing
Appendix contain source code for providing additional functionality for genotoxicity analysis
s/stem 100. These subfolders include "SUPERUSER" which stores source code for creating
v ;er interfaces that allow for manual adjustment of system parameters when necessary,
" TOOLFORMS" which stores source code for user interface modules that may be used by
pecutable programs 210, "PASSWORD," which stores source code for providing password-
protected access to genotoxicity analysis system 100 and "MODULES," which includes source
code for calling necessary library link and DLL files 220 during operation of genotoxicity
analysis system 100.
In addition to the source code for creating executable programs 210, the
Computer Program Listing Appendix for this document also includes source code for creating
library link and DLL files 220 using Microsoft Visual C/C + + version 6.0. More
particularly, the source code for generating library link and DLL files 220 is found in the
subtifolder labled "VC6" on the Computer Program Listing Appendix.
The subfolder labeled "AUTOO" in the folder named "VC6" contains source
cod : for generating a C library called "autoO" 262 which provides functionality for facilitating
the lutomatic lunctioning of genotoxicity analysts system 100, including autofocus control and
auxmatic lamp adjustment, among others. The functionality provided by autoO 262 is based
on the related lunctionality provided by the "microO" 264 and "improcO" 266 DLLs which are
described in greater detail below.
The subfolder labeled "COMET" in the folder named "VC6" contains source
code for generating a C library called "comet" 268 which provdes functionality required for
performing image analysis on slides being analyzed for the comet assay.
The subfolder labeled "GENERAL0" in the folder named "VC6" contains
soun e code for generating a C library called "generalO" 270 which provides functionality for
general purpose tools, including input and output functionality and graphic display routines.

The subfolder labeled "IMPROCO" contains source code for generating a C
library called "improcO" 266 which provides interface functionality for the library of functions
associated with the Matrox driver software of frame grabber board 120. These include
fucactions relating to general image processing.
The subfolder labeled "METFIN" contains source code for generating a C
library callec "metfin" 272 which provides functionality required for performing image
analysis on slides that are being analyzed for the metaphase finding application.
The subfolder labeled "MICRO0" contains source code for generating a C
1 brary called "microO" 264 which provides interface and control functionality associated with
the Leica SDK driver software for electronically driven microscope 150.
The subfolder labeled "MNTINVIVO" contains source code for generating a C
libary called "MNTinvivo" 274 which provides functionality required for performing image
analysis on slides that are being analyzed for the micronucleus test in vivo application.
The subfolder labeled "NNETO" contains source code for generating a C library
called "nnetO' 276 which provides functionality required for pattern classification through
prediction using neural networks, e.g., the backpropagation algorithm, for the micronucleus
tes in vitro.
The subfolder labeled "RELOC0" contains source code for generating a C
limary called ' relocO" 278 which provides functionality for object retrieval within
Retocation.exe 254, e.g., data input and output functionality and retrieval of analysis results.
The subfolder labeled "ROBO0" contains source code for generating a C library
callied "roboO' 280 which provides functionality required for communicating with robotic slide
feeder 160.
The subfolder labeled "SCANO" contains source code for generating a C library
called "scanO" 282 which provides functionality required for facilitating an automatic scanning
process, e.g., handling scanning mode settings, triggering the sequential analysis of the batch
of Slides to be processed and interfacing to specific application DLLs.
Additional libraries may also be included with library link and DLL files 220,
incluiding necessary library files provided by third party vendors for controlling operation of
the electronicaily driven microscope 150 and frame grabber board 120.

The source code for certain of the above-described library link and DLL files 220
defefne the algorithm and image analysis processing that is conducted for the various
screenings.
The image analysis processing for the micronucleus test in vivo uses red and blue
canera channel information and thresholding techniques for discrimination
between polychromatic and normochromatic erythrocytes. Thereafter, gradient and watershed
transformation for segmentation of micronucleus candidates is utilized. Individual analysis of
segmented objects uses supervised training of patterns on the basis of morphometric features,
as well as structural features such as "periphery percentage," "focus deviation" and "gray
deviation." Reference may be made to the applicable source code described above for further
de a 1.
Metaph use finding utilizes differences of spectral images as the gray image basis and
thereafter utilizes a combination of watershed transformation and "top-hat" segmentation for
nucleus candidate segmentation. That is followed by restriction of metaphase range on non-
nuclar regions which is followed thereafter with another application of top-hat and watershed
segmentation. Finally, feature base metaphase candidate classification, involving individual
parameters for chromsomal structuring, is applied. Reference may be made to the applicable
source code described above for further detail.
Comet assay analysis involves red channel uses of fluorescence image on a first run to
detect valid nuclei, including classification on morphometric features. Automatic relocation of
detected nuclei for tail moment measurement and use of a sequentially degrading thresholding
tecluique which involves a gradient for the pixel sum change in the image is also utilized.
Refrenence may be made to the applicable source code described above for further detail.
The micronucleus test in vitro uses all three color channel images. The image
algorithms attenpt segmentation of valid nuclei and cytoplasm range, and then detect
micronucleus candidates using a combination of gradient, top-hat and thresholding
segmentation. Final classification uses an off-line trained backpropagational neural network
for predicting the probability of a true micronucleus. Reference may be made to the applicable
source code described above for further detail.

Continuing with Figure 2, application software 200 further includes parameter
libs 230 which store information about the proper settings for the operation of electronically
craven microscope 150 and the image analysis software operating on genotoxicity analysis
system 100, depending upon the particular analysis being conducted. Each of the parameters
and adjustments varies depending on the genotoxicity test to be conducted and is set
automatically by designating the particular test.
Parameter files 230 include the following files:
- "cometpar.txt" 290 - contains parameters for the configuration of the
image analysts algorithms used for the Comet assay application;
- "metfinpar.txt" 292 - contains parameters for the configuration of the
irr age analysis algorithms used for the metaphase finding application;
- "mntinvivopar.txt" 294, contains parameters for the configuration of
the image analysis algorithms used for the micronucleus test in vivo application; and
- "molymntpar.txt" 295 contains parameters for the configuration of the
image analysis algorithms used for the micronucleus test in vitro application.
Parameter files 230 further include a file called "focus std.txt" 284 which
to itains parameter data that controls the automatic focus features of electronically driven
microscope 150 in connection with the autofocus execution for Datainput.exe 252 and
AutoScan.exe; 256. Parameter file 230 called "focus_reloc.txt" 286 generally contains the
same parameter definitions as "focus_std.txt" 284, but is more refined to allow for autofocus
performance that is better suited for operation under Relocation.exe 254. Parameter file 230
labeled "scantef.txt" 288 contains parameter data that is used for the configuration of
electronically operated microscope 150 depending on the selected application. Such
configuration includes automatic adjustment of optical modules of the microscope, and setting
general parameters referring to the scanning process of the application.
Also, the parameter file 230 called "roboplace.txt" 296 contains parameter data
to control the initialization and placement of robotic slide feeder 160. These parameters
include x,y positioning and speed.
Each of "focus_std.txt" and "focus_reloc.txt," are particularized for the
sceening tes being performed, i.e., there exists a "focus_std.txt" and "focus_reloc.txt" for

sach of the in vivo micronucleus test, in vitro micronucleus test, comet assay or in vitro
netaphase finding. Computer Program Listing Appendix stores the parameter files 230 for
:ach screening type in respective file folders.
More particularly, Computer Program Listing Appendix includes a folder
lamed "Applications" which includes subfolders labeled "COMETASSAY" containing the
above described parameter files 230 used for comet assay analysis. Similarly, the subfolder
called "METFIN" contains the above described parameter files for metaphase finding analysis,
"he subfolcer called "MNTINVIVO" contains the above described parameter files for in vivo
iuecronucleus test analysis.
The subfolder called "MOLYMNT" contains the above described parameter
tiles for in vitro micronucleus test analysis. The "MOLMNT" subfolder further includes a file
called "p21h9.net" and includes parameters for the neural network pattern prediction and
cassification utilized for the in vitro micronucleus test analysis.
In a preferred embodiment, "robias.txt," which holds system specific
information for the application in general for genotoxiciy analysis system 100 and
" oboplace.txt" 296, which contains parameters for use by robotic slide feeder 160 during
initialization, reside locally on the hard drive of computer 110 while the remaining programs
and files reside on a networked server connected to computer 110.
In addition to the above-described parameter data files, calibration files
conntaining "shadimages", including "shadref_black" and "shadref_whitbl", are referenced by
the executable programs 210. One skilled in the art may generate these files to provide
colibration for shading correction. Calibration files are particular to each screening
publication. The calibration files are preferably stored in a subdirectory that is parallel to the
respective subdirectories containing the parameter data.
Application software 200 of Figure 2 further includes data results files 240
which are generated and modified by executable programs 210.
There are three types of data results files 240 having the following forms:
(1) > scanresults/// .txt;
(2) > individualdata/// and

(3) >slidedata/slidedata .txt
In the above-listed file formats for data results files 240, > indicates the
preliminary file path of the directory containing the file at issue. This part of the path may
vary depending upon how the file structure of the overall operational software is configured,
"scanresults," individualdata" and "slidedata" represent respective subfolder names for the
files. represents a placeholder for the study name coding the toxicological testing of
i certain test compound and is correlated with a unique "study name",
represents a placeholder for a particular experiment in the context of the selected study.
Experiments belonging to a specific study can vary with respect to treatment time or the
aosence or presence of the metabolic activation of cells, or sampling time after treatment of
aiimals. Generally, it specifies the "experimental" conditions for the same test compound of
interest. represents a placeholder for the identity of a particular slide and
represents a placeholder for a particular position of a slide in a rack.
The operation of genotoxicity analysis system 100 will now be described with
reference to (he flow chart of Figure 3 and the exemplary screen interfaces of genotoxicitiy
analysis system 100 illustrated in Figures 4 through 14.
At step 302 of the process of Figure 3, the user selects one of Datalnput.exe,
Auto Scan.exe and Relocation.exe for execution from the main display screen of color display
monitor 130. Bach application is preferably represented as an application or shortcut icon on
the main display screen of the Windows NT platform. The user may select the desired
program by double clicking the corresponding icon in the known manner.
If the user desires to enter information for each slide that is to be analyzed, the
uss, selects the icon representing Datalnput.exe for execution at step 302. As a result, the
process proceeds to step 304 where the form illustrated in Figure 4 is displayed to the user,
thing the fonr of Figure 4, the user specifies the current application, i.e., the analysis that is
to be performed, by selecting a unique path to which the slide data will be written. Thus,
selection of the path also designates the analysis that will be performed, i.e., comet assay,
micronucleus test in vivo, micronucleus test in vitro or metaphase finding. The form of Figure
4 is seated from the source code found in the file called "frolmite" in the

VB6/T00 LFORMS subdirectory of the Computer Program Listing Appendix for this
document.
The process then moves to step 306 where the form illustrated in Figure 5 is
displayed to the user. Using this form, the user enters data for identifying each slide that is to
the processed. The identification string for each slide consists of a study name (col. 501),
tollowed b> experiment name (col. 502) and a slide code (col, 503), each of which may utilize
Lumerals ot characters. It is noted that the exemplary slide codes 503 presented in figure 5 are
appended by "a" and "b." In the presently disclosed embodiment of the present invention, two
simples of biological material may be included on each slide, one designated by "a", the other
by "b." The precision provided by the components of genotoxicity analysis system 100 in
combination with application software 200 allows for this efficient use of slide space which
effectively doubles slide capacity for screening.
For slides sharing the same study and experiment code, a common folder for
resulting storage will be created. The form of Figure 5 is created from source code found in
the; file called "frmSlides.frm" in the VB6/DATAINPUT subdirectory of the Computer
Program Lisiitig Appendix for this document.
At step 308, the user accepts the settings entered at step 306 by pressing the
"accept settings" button 506 of the form of Figure 5, at which point the system ends operation
of Datalnput exe, creates all necessary folders (for studies and experiments) and data files and
returns to step 302 of Figure 3.
Alternately, at step 310, the user may select any of the respective detail buttons
(see column 504 of the form of Figure 5) for each slide to adjust specific parameters relating to
each slide. Figure 6 represents the form presented to the user for adjusting the parameters for
a particular slide. The form of Figure 6 is created from the source code found in the file called
fire Slideparam frm in the VB6/DATAINPUT subdirectory of the Computer Program Listing
A poendix for this document.
Among the various parameters that the form of Figure 6 allows a user to control
is threshold adjustment (button 602) and microscope adjustment (button 604).
The form for providing the user the ability to adjust the threshold settings for
the particular slide is illustrated in Figure 7. This form is created from the source code found

i the file called "frmlnterThresh.frm" in the VB6/T00LF0RMS subdirectory of the
Computer Program Listing Appendix for this document. The form for providing the user the
ability to adjust microscope parameters for the particular slide at issue is illustrated in Fig. 8.
This form is created from source code found in the file called "frmAdjustMicro.frm" in the
VB6/TOOLFORMS subdirectory of the Computer Program Listing Appendix for this
document.
Once the user is satisfied with the adjustments made to the particular slides, the
user may select the "acc. Settings for ALL slides" button 606 of the form of Figure 6 which
will set these parameters for all previously identified slides that have valid slide code entries in
the form of Figure 5. Alternatively, the user may select the "ace. settings for CURRENT
slide" (button 608) which saves parameter settings only for the currently selected slide.
Control is then returned to the form of Figure 5 (step 306).
Returning now to step 302 of the process illustrated in Figure 3, if the user
selects the icon to initiate execution of AutoScan.exe, the process moves to step 312 where the
f rom illustrated in Figure 8a is presented to the user. Here, the user selects the genotoxicity
tos. that will he processed, i.e., one of the comet assay, micronucleus in vivo, micronucleus in
vitro or metaphase finding analyis, by selecting the respective subdirectory illustrated in
window 802 in the form of Figure 8a. The form of Figure 8a is created from source code
found in the file called "frmlnit.from" in the VB6/TOOLFORMS subdirectory of the Computer
Pro gram Listing Appendix for this document.
The process then proceeds to step 314 where the form of Figure 9 is presented
to the user. The form of Figure 9 allows the user to select the scanning options for the
genotxicity analysis to be performed as was specified at step 312 using the form of Figure 8.
The options presented by the form of Figure 9 include: (1) scanning the slides without display
(button 902), meaning that no intermediate image display will be presented to the user during
analysis of the slides; (2) scanning the slides with display (button 904), meaning that the most
important intermediate image processing results will be displayed during analysis without
req liring user interaction to continue analysis; (3) scanning the slides with Testl level (button
9(), meaning that several intermediate image processing steps are performed and the process
is t: en halted until the user presses a key to continue automatic analysis; and (4) scanning the

slides wit a Test2 level (button 908), which results in operation similar to that of button 906
except that detection results are not displayed. This last mode is utilized to validate operation
of the application where a user performs manual analysis of a slide in parallel with automated
analysis in the same image fields. Finally, the user may press button 910 for scanning the
slides with autofocus test, which processes the slides while presenting a graphical display of
the autofocus results, e.g., contrast curve, for each slide.
The user may also abort running the analysis by pressing the exit button 912.
If the user does not abort the automatic scanning, the process proceeds to step
116 and the automatic scanning is executed by referencing the applicable library link and DLL
tiles 220 and parameter files 230 of application software 200 for the specific type of analysis
teing performed. The form of Figure 9 is created from source code found in the file called
frmMain.frm" in the VB6/AUTOMATICSCAN subdirectory of the Computer Program
I isting Appendix for this document.
When the automatic scanning is complete and all results data has been written
and stored, the process returns to step 302 of Figure 3.
If at step 302, the user executes Relocations.exe, the process of Figure 3
proceeds to step 318 where the form of Figure 10 is presented to the user. Using the form of
Figure 10, the user selects the genotoxicity test for which results are to be reviewed, i.e., one
of the comet issay, micronucleus in vivo, micronudeus in vitro or metaphase finding analyis,
by selecting the respective subdirectory illustrated in window 1002 in the form of Figure 10.
The form of Figure 10 is created from the source code found in the file called "frmlnit.frm" in
the VB6/TOCLFORMS subdirectory of the Computer Program Listing Appendix for this
document.
The process then proceeds to step 320 where the user is presented with forms to
select a specif c slide to be reviewed. More particularly, the user is presented with the forms
the trated in F igures 11 and 12. In the form of Figure 11, the user selects the particular study
con aining the slide by selecting the appropriate subdirectory labeled with the appropriate study
and experimem name (see window 1102). Using the form of Figure 12, the user identifies the
palcular slide by selecting the file containing the slide data (see window 1202). The form of
Figure 11 is created from source code found in the file called "frmMain.frm" in the

VI 6/RELOCATION subdirectory of the Computer Program Listing Appendix for this
document. The form of Figure 12 is created using a standard Visual Basic CommonDialog
USER interface object.
Next, at step 322, the user is presented with the form of Figure 13 which
includes a display (window 1302) of the scanning results associated with the slide specified
using the form of Figure 12. The form of Figure 13 is similar to that of Figure 11 except that it
row includes, in window 1302, the most relevant data that had been acquired during automatic
slile analysis, such as the number of detected objects, number of scanned fields, error codes,
and other application specific information for the slide under review.
The user may exit Relocation.exe by eliciting button 1304 of the form of Figure
3 (step 324) at which point the process of Figure 3 returns to step 302.
Alternatively, the user may select button 1306 of the form of Figure 13 causing
the process of Figure 3 to move to step 326 at which point the user is presented with the form
or Figure 14 The form of Figure 14 allows a user to retrieve the objects that had been
detected during the automatic scanning process. For this purpose, one can move from one
object to another (and then back again) using the arrow buttons 1402 and 1404. Using the
additional controls presented in the form of Figure 14, each object's coordinates, which had
been stored during scanning, and the current live image showing the object are displayed on
color display screens 130 and 132 for visual inspection. The user may operate the right or left
n ouse button to flag an object under observation and discard an object as a valid micronucleus
(by using the left mouse button) or accept an object as a valid micronucleus (using the right
mouse button). By moving from the first detected object to the last for each slide, the user can
assign the proper label (i.e., "accept" or "reject") to each object and, therefore, adjust the
result of automatic scanning through supervised visual inspection. The corrected result for the
current slide, i.e. the number of micronuclei for micronucleus application, or number of
metaphases for metaphase finding application, will be stored after exiting the form of Figure
14. The othir options present in the form in Figure 14 support the adjustment of the current
image, e.g. microscope and focus, and support image analysis for other objects of interest in
c rder to confirm proper performance of the the algorithms utilized for image analysis.

The form of Figure 14 is created from source code found in the file called
"FrmRelocation.frm" in the VB6/RELOCATION subdirectory of the Computer Program
L sting Appendix for this document.
Thus, it is seen by the above, that by creating software code which can facilitate
different types of genotoxicity screening and which references parameter data files respectively
configured for each of various genotoxicity tests, the genotoxicity analysis system of the
present inversion provides a flexible and easy to use platform for performing various
genotoxicity screenings with minimal user interaction. Depending upon the type of screenings
being performed, no manual microscope module adaptation is necessary between screening
runs for different analysis testing. In the case of comet assay screening, a manual change to
iuident illumination to support fluorescence staining in comet assay analysis and then back to
transmitted light illumination for other genotoxicity screenings may be necessary. Moreover,
as described above, the genotoxicity analysis system of the present invention allows interactive
pattern control to permit a user to manually perform artifact rejection for objects wrongly
c a isified dur ng automatic scanning.
In accordance with 37 C.F.R. 1.52 (e), the name, respective creation date and
sizi (in bytes), of each file contained on the CD-ROM of the Computer Program Listing
Apendix are listed in Figures 15a - 15. For ease of reference, the file names are listed as they
appear in the directory structure of the Computer Program Listing Appendix.

WE CLAIM;
A system for providing in vivo and / or in vitro genotoxicity screening of
samples of biological material, the system comprising:
a. one or more computers;
b. a frame grabber connected to and being controlled by the one or more
computers to create electronic images;
c. a camera connected to the frame grabber and providing images to the frame
grabber under the control of the one or more computers;
d. a microscope connected to and being controlled by the one or more
computers, said microscope providing images of samples to be screened to the
camera;
e. a slide feeder connected to and being controlled by the one or more
computers to move the samples to positions where the microscope can image them;
and
f. a program operating on the one or more computers operative to perform
genotoxicity screening by analyzing the electronic images in order to facilitate the
screening a second batch of biological material using a second genotoxicity testing
method after a first batch of biological material using a first genotoxicity testing
method, substantially free of any manual manipulation of the camera, the microscope
or the slide feeder
2. The system as claimed in claim 1, having a user interface presented on a
display monitor connected to the one or more computers, for allowing a user of the
genotoxicity screening system to select the gentoxicity screening method to be
performed on a given batch of biological material.
3. The system as claimed in claim 1, the camera, the microscope and the slide
feeder including physical connections that receive electronic signals from the one or
more computers which control the operations of the camera, the microscope and the
slide feeder.
4. A genotoxicity analysis system for providing in vivo and / or in vitro

genotoxicity screening of samples of biological material, the system comprising:
at least one computer including a software application that is configured to
automatically configure components of the system and allow the genotoxicity
analysis system to perform a plurality of genotoxicity tests on respective
pluralities of biological samples by way of the automatic configuration.
5 The system as claimed in claim 4, wherein the software is configured to allow
a user to specify the genotoxicity test to be performed on a given group of biological
samples.
6 The system as claimed in claim 5, wherein after the user has specified the
geotoxicity test to be performed, the software is configured to automatically
generate signals which are sent to the configurable components of the genotoxicity
analysis system in accordance with the specified genotoxicity test.

7. The system as claimed in claim 6, wherein the sent signals cause the
configurable components of the genotoxicity analysis system to be configured in a
manner conducive to the selected genotoxicity test.
8. The system as claimed in claim 4, wherein the software provides a user of the
genotoxicity analysis system with the ability to provide identifying information for
each biological sample.
9. The system as claimed in claim 4, wherein the software is configured to
record the results of the genotoxicity testing for each analyzed sample and provide lhe
manual inspection of the recorded results of the genotoxicity testing.
10. The system as claimed in claim 4, wherein the software is configured to have
respective files containing data defining configurable parameters of the configurable
components for each of the plurality of genotoxicity test.
11. The system as claimed in claim 4, wherein the software is configured to
contain software code defining respective image analysis techniques for use by each
of the plurality of genotoxicity tests.

12 A method for performing in vivo and / or in vitro genotoxicity testing of
samples of biological materials by utilizing a genotoxicity analysis system, including
hardware components that are operated with software controls, the genotoxicity
analysis system being capable of performing a multiplicity of genotoxicity tests in
which electronic images of the samples are analyzed by software, the method
comprising the steps of:
-a. preparing a first batch of samples of biological materials for
processing using a first genotoxicity test;
b. utilizing the genotoxicity analysis system to perform a first genotoxicity
test on the samples of the first batch of biological materials:
c. preparing a second batch of samples of biological materials for processing
using a second genotoxicity test;
d utilizing the genotoxicity analysis system to perform a second genotoxicity
test on the samples of the second batch of biological materials, and
e. manipulating through software the configuration of the hardware
components during the time period between performance of the first and second
genotoxicity tests to thereby allow the first and second genotoxicity tests to be
performed using the same hareware components.
13. A method for performing various types of in vivo and / or in vitro genotoxicity
tests on respective batches of biological samples using a genotoxicity analysis
system that analyzes electronic images of the samples, the method including the
steps of:
a. receiving a command from a user of the genotoxicity analysis system, the
command specifying the type of genotoxicity test to be performed:
b. performing an automatic configuration of the component of the
genotoxicity analysis system to thereby allow the genotoxicity analysis system to
perform the genotoxicity test specified in step a;
c. performing the specified genotoxicity test on a batch of biological samples;
d. recording results of the genotoxicity test;
e. repeating steps a through d.
14. The method as claimed in claim 13, wherein the types of genotoxicity tests

are selected from the group consisting of one or more of the following: the
micronucleus test in vivo, the micronucleus test in vitro, the comet assay and
metaphase finding.
15. A method for performing genotoxicity screening comprising the steps of:
a. preparing a batch of slides for genotoxicity screening;
b. selecting a genotoxicity test;
c. automatically retrieving the first of a plurality of slides containing
biological samples from a slide retaining device;
d. automatically delivering the slide to an electronically driven microscope:
e. automatically focusing on the material contained on the slide;
f. automatically recording a visual representation of the focused image;
g. automatically delivering the focused image to a microprocessor-based
computer;
h. automatically performing image analysis on the recorded image using
image analysis software appropriate for the genotoxicity test selected in step b.
i. automatically recording the data resulting from the analysis of the
image;
j. automatically returning the slide retrieved in step c to the slide retaining
device;
k. automatically retrieving the next slide for analysis;
1. automatically repeating steps c through k for successive slides in the batch
until all of the slides in the batch have been analyzed; and
m. repeating steps a through 1 until all desired slides have been processed.
16. The method as claimed in claim 15, having the step of manually verifying
the recorded data.
17. The method as claimed in claim 15, wherein the batch of slides is prepared in
accordance with the genotoxicity test to be performed, the genotoxicity test being
selected from the group consisting of: the micronucleus test in vivo, the
micronucleus test in vitro, the comet assay and metaphase finding.
18. The method as claimed in claim 15, wherein the selecting step is performed by

choosing the appropriate test from a menu displayed on a video monitor.
19. The method as claimed in claim 15, wherein the steps of automatically
retrieving and automatically returning is performed by a robotic slide feeder.
20. The method as claimed in claim 15, wherein the step of automatically
recording the data resulting from the analysis of the image is continuously performed
until either a given delimiting number of cells have been counted or the maximum
number of image fields to be analyzed has been reached for the slide currently under
analysis.

A system (100) and method for performing genotoxicity screening
utilize: (1) one or more computers (110); (2) a frame, grabber (120) connected
to the one or more computers (110); (3) a camera (140) connected to the frame
grabber (120); (4) a microscope (150) connected to the one or more computers
(110); (5) a slide feeder (160) connected to the one or more computers (110);
and (6) a program operating on the one or more computers (110). The program
facilitates the screening of a second batch of biological material using a
second genotoxicity testing method after screening a first batch of biological
material using a first genotoxicity testing method. The screening operates
substantially free of any manipulation of the camera (140), the microscope
(150) or the slide feeder (160).

Documents:

2376-kolnp-2005-granted-abstract.pdf

2376-kolnp-2005-granted-assignment.pdf

2376-kolnp-2005-granted-claims.pdf

2376-kolnp-2005-granted-correspondence.pdf

2376-kolnp-2005-granted-description (complete).pdf

2376-kolnp-2005-granted-drawings.pdf

2376-kolnp-2005-granted-examination report.pdf

2376-kolnp-2005-granted-form 1.pdf

2376-kolnp-2005-granted-form 18.pdf

2376-kolnp-2005-granted-form 3.pdf

2376-kolnp-2005-granted-form 5.pdf

2376-kolnp-2005-granted-gpa.pdf

2376-kolnp-2005-granted-reply to examination report.pdf

2376-kolnp-2005-granted-specification.pdf

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Patent Number

226761

Indian Patent Application Number

2376/KOLNP/2005

PG Journal Number

52/2008

Publication Date

26-Dec-2008

Grant Date

24-Dec-2008

Date of Filing

24-Nov-2005

Name of Patentee

NOVARTIS AG

Applicant Address

LICHTSTRASSE 35, CH-4056 BASEL

Inventors:

#	Inventor's Name	Inventor's Address
1	FRIEAUFF WILFRIED	MOENDENWEG 21, 79594 INZLINGEN

PCT International Classification Number

G06F 19/00

PCT International Application Number

PCT/IB2004/000623

PCT International Filing date

2004-03-06

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/465,564	2003-04-25	U.S.A.