Applications of Histology in Personalized Medicine                                               ALS 320                  ...
We will develop a NSCLC-specific automatedhistological device that provides precise andaccurate      computer-assisted    ...
Immunohistochemistry and Detection
Immunohistochemistry Staining    Antigen RetrievalRetrieval buffer Heat 121°C- 30 sec                                     ...
Positive and Negative Controls                                                       S    L   P     P      S   L   P   P  ...
Imaging Calculations     Estimated Area of      20mm x 20mm        400mm2     Tissue samples     Magnification          10...
Image Capture – Image Acquisition (Auto-Focus)
Image Capture – Image Acquisition (Auto-Focus)
Image Capture
Image Capture
Image Capture – Photo Stitch (Quantification)                Image Analysis                Edge Detection
Imaging Storyboardhttp://library.wolfram.com/examples/edgedetection/
Inside view                                                        4                                                      ...
Top view                       4                   1                       2                           1. Mechanical arm  ...
1. Slide holderMetal-made holderCapacity: 6 slidesOne metal frame per slideLateral aspiration system -> remaining reagent ...
2. Heating / cooling system
3. Small storages for reagents and tipsBig and small tips for both types of pipettes.Storages: polypropylene containers un...
4. Holder entrance andscanning                         TRAY 1
5. Pipette system
6. Image capture
Bright Field Microscopy                     • Light source                         – Light-emitting diode (LED)     Camera...
•   20” Touch-enabled widescreen                  •   Report Storage Servers    BrightView LCD Monitor with glass         ...
WELCOME TO SIMUL-HISTO      ANALYZER   User ID: Password:  Hospital:
Home Page          ID #   Last   First   Date Location DoctorTouch &                                                Scroll...
Home Page          ID #   Last   First Date Location   Doctor   A                                                       B ...
Patient Report                  DOE, John     Back                                                        11/30/2011 9:12 ...
"Patient Screen"                              DOE, John                                 Touch-screen                      ...
DOE, John       11/30/2011 9:12 AMBack          Biomarker Readout                                                        I...
Back                 Biomarker Readout (Grid View)                                 S     L   P    P   S   L   P   P   S   ...
Product Requirement                                  Justification Assay • WillThe device will distinguish between NSCLCJu...
Product Requirement                                   JustificationSoftware PR01        The device will distinguish betwee...
Failure Mode Effect Analysis                     Potential Failure    Potential Effects ofDevice / Function    Mode       ...
Failure Mode Effect Analysis                     Potential Failure      Potential Effects ofDevice / Function    Mode     ...
•   1. [Webpage] Compound light microscope: How it works - bright field microscope .    Retrieved on: 11/29/2011. Availabl...
Poster Presentation Slideshow
Poster Presentation Slideshow
Poster Presentation Slideshow
Poster Presentation Slideshow
Poster Presentation Slideshow
Poster Presentation Slideshow
Poster Presentation Slideshow
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Poster Presentation Slideshow

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Slideshow for Keck Graduate Institute Medical Diagnostics team projects poster presentation.

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Poster Presentation Slideshow

  1. 1. Applications of Histology in Personalized Medicine ALS 320 Juan Jose Aponte Kathleen Coquia Michael Davis Samira Khodai Stephen Kim Debleena Sinha
  2. 2. We will develop a NSCLC-specific automatedhistological device that provides precise andaccurate computer-assisted diagnosis topathologists and physicians. The Simul-HistoAnalyzer allows fully automated staining andquantitative assessment of multiple stains andslides. This comprehensive diagnostic tool is easyto use and functions well with validated biomarkersof targeted therapies. Generated reports can beeasily accessed in a digital format and readilytransferred to approved devices.
  3. 3. Immunohistochemistry and Detection
  4. 4. Immunohistochemistry Staining Antigen RetrievalRetrieval buffer Heat 121°C- 30 sec Cell or TissueCooling 90°C-10 min Antigen (Biomarker) TBS wash Primary Antibody Peroxidase Block TBS wash Secondary Antibody Protein Block Conjugated with HRP Secondary Antibody Primary Antibody Conjugated with AP TBS wash Incubation 30 min RT TBS wash DAB Substrate Fast Red Secondary Antibody conjugated with HRP and AP TBS wash Substrate to colored product Incubation 30 min RT TBS wash Image transfer and observation Chromogen substrate Incubation 5 min RT
  5. 5. Positive and Negative Controls S L P P S L P P S L P P r l r l r l B B B B T B B B T B B T k k k k k k T B B P T B B P T B B P k l k l k l DSG3 TTF-1 P63 P L S B P L S B P L S B Napsin A CK5 TRIM29 r k r k r k Skin Lung Prostate Placenta Bladder Blank Tonsil Blank Tonsil Blank Bladder Placenta Prostate Lung Skin Blank DSG3 TTF-1 P63 Napsin A CK5 TRIM29DSG3 + Napsin A TTF-1 + Ck5 P63+TRIM29Skin Lung Lung Prostate Prostate ProstateTonsil Bladder Placenta
  6. 6. Imaging Calculations Estimated Area of 20mm x 20mm 400mm2 Tissue samples Magnification 10x * 40x = 400x Field of View 625μm x 625μm 0.3906mm2 Maximum Grid 32 x 32 1024 images Maximum time / image 1.5 seconds 25.6 min / slide Grid pattern of Image captureImage Capture
  7. 7. Image Capture – Image Acquisition (Auto-Focus)
  8. 8. Image Capture – Image Acquisition (Auto-Focus)
  9. 9. Image Capture
  10. 10. Image Capture
  11. 11. Image Capture – Photo Stitch (Quantification) Image Analysis Edge Detection
  12. 12. Imaging Storyboardhttp://library.wolfram.com/examples/edgedetection/
  13. 13. Inside view 4 1. Reagents storage 7 2. Mechanical arm 3. Slides holder 2 4. PeltierImaging area Staining area 1 heating/cooling 9 system 5. Waste container 4 6. Reagents big 3 containers 8 7. Imaging system 8. Base with lightReagents storage area source 9. Tip storage 5 6
  14. 14. Top view 4 1 2 1. Mechanical arm 2. Reagents and tips storage 3. Slide holder 5 4. Peltier heating /cooling system 5. Imaging system 3 4
  15. 15. 1. Slide holderMetal-made holderCapacity: 6 slidesOne metal frame per slideLateral aspiration system -> remaining reagent volumeLateral channel for fluids
  16. 16. 2. Heating / cooling system
  17. 17. 3. Small storages for reagents and tipsBig and small tips for both types of pipettes.Storages: polypropylene containers under temperature controlSmall storages keep the reagent ready for use.The device sends a notification when either tips or reagents storages are empty .
  18. 18. 4. Holder entrance andscanning TRAY 1
  19. 19. 5. Pipette system
  20. 20. 6. Image capture
  21. 21. Bright Field Microscopy • Light source – Light-emitting diode (LED) Camera • Diffusion filter – Scatters light sourceMagnification lens • Condenser Lens:Objective lens – focuses light onto specimen • Specimen on slide holder • Objective lens: Specimen – Determines level of magnificationCondenser lens • Magnification lensDiffusion Filter – 10x • CameraLight source:LED – RGB Progressive Scan Camera
  22. 22. • 20” Touch-enabled widescreen • Report Storage Servers BrightView LCD Monitor with glass – Store patient reports from Simul-Histo covering and anti-reflective coating Analyzer – Display Resolution 1920 x 1080 – Accessible from personal computer, resolution (16:9) tablet, smart-phone (User clearance – Adjustable tilt for on-screen typing (0- required) 90 degrees) – Available to physicians, laboratory – 360 degree rotation for easy viewing technicians, and patients – Where software will process images
  23. 23. WELCOME TO SIMUL-HISTO ANALYZER User ID: Password: Hospital:
  24. 24. Home Page ID # Last First Date Location DoctorTouch & Scrollscroll Btwnup/down Letters Keyboard
  25. 25. Home Page ID # Last First Date Location Doctor A B C D E FTouch & G Scrollscroll H Btwnup/down Letters I J K L M N O P Keyboard
  26. 26. Patient Report DOE, John Back 11/30/2011 9:12 AM Whole Tissue Image Biomarker DataRed-boxed areas areareas of interest Notes on patient, etc. Home Keyboard
  27. 27. "Patient Screen" DOE, John Touch-screen Red-boxed areas are Back Whole Tissue Image areas of interest Biomarker Data Notes on patient, etc.Home Keyboard
  28. 28. DOE, John 11/30/2011 9:12 AMBack Biomarker Readout Images in Grid Swipe Between Images LegendHome P63-----Brown % cells TRIM 29----Red % cells
  29. 29. Back Biomarker Readout (Grid View) S L P P S L P P S L P P r l r l r l B B B B T B B B T B B T k k k kDSG3 Expression k k T B B P T B B P T B B P k l k l k l P L S B P L S B P L S B r k r k r k CK 5 Expression DSG3 TTF-1 P63 Napsin A CK5 TRIM29P63 ExpressionTRIM 29 ExpressionHome
  30. 30. Product Requirement Justification Assay • WillThe device will distinguish between NSCLCJustify with table will help PR01 system meet goals? 3 pairs of biomarkers that • stephen cell carcinoma subtypes of adenocarcinoma and squamous differentiate between NSCLC subtypes PR02 The device will test 6 NSCLC biomarkers. 3 pairs of antibody cocktails for NSCLC biomarkersHardware PR02 The device will test 6 NSCLC biomarkers. IHC kits with antibody cocktails in containers for use with automatic pipettors PR03 The device will be fully automated during User only places slides in trays, inserts trays staining and image analysis, with option of into device and acknowledges IDs; device user correction. will receive the samples and begin staining process and finishes with image capture and analysis PR04 The device will automatically upload the Internet connectivity will allow secure reports (with images) to a central location for connection to off-site servers which will immediate and secure remote access. process the images and make them available for viewing and download
  31. 31. Product Requirement JustificationSoftware PR01 The device will distinguish between NSCLC Image analysis software algorithms will subtypes of adenocarcinoma and squamous calculate intensity of stains, correlated to a cell carcinoma panel of specific biomarkers for distinguishing between adenocarcinoma and squamous cell carcinoma subtypes. PR03 The device will be fully automated during Software will guide the automated steps; user staining and image analysis, with option of will be able to correct image analysis -- user correction. specifying new regions of interest or adjusting levels. PR04 The device will automatically upload the The files will be accessible through approved reports (with images) to a central location for devices with installed software immediate and secure remote access. PR05 The device will automatically identify Software, using edge detection on composite landmarks and reference points on the images, will make links between composite composite tissue images for comparison, images of the different stain pairings to orient relation and orientation of multiple images. the images. The software will allow for user correction by highlighting regions for compatibility.
  32. 32. Failure Mode Effect Analysis Potential Failure Potential Effects ofDevice / Function Mode Failure(s) (S) Potential Cause of Failure(s) (O) Current Controls (D) RPNAssay / Barcode scan No barcode / barcode Samples cannot be 3 Barcode is faded or 1 Barcode scanner at point of entry; 2 6 unreadable linked properly to damaged; samples not database linked to patient records and patient and identified; placed on barcoded slides order; barcoded slides are packaged with will be unable to track device and available for purchase - the which slides receive slides will be all unique; unique code each pair of generated for unattended and poorly biomarker stains scanned/unlabeled slides.Assay / Load Samples Tissue slides and Tissue not stained; User incorrectly places slides Controls will be clearly labeled (colored & Controls control slides lose samples (if into holder (upside down); and text); controls will have barcodes that incorrectly loaded loaded in control tray) user places samples in are recognized as controls, order of (wrong orientation, control tray or controls in controls placed in control tray is irrelevant wrong tray samples tray. as software will recognize position and placement) stain accordingly; slides will have writing on labels for correct orientation (the text should be visible and readable).Assay / Load Reagents Reagents incorrectly Assay will not work 7 User places reagents in 1 Reagent bottles are color-coded, loaded into machine wrong position, user uses barcodes on bottles to be read for correct non-authentic reagents placement and positioning. (wrong container dimensions) Assay / Crossover Contamination of Reagents will not 7 Reagent bottles placed in 2 Control slides should determine whether 2 28 Contamination reagents, such as work wrong position, cross or not assay ran successfully; reagent secondary antibody in contamination via pipettor... bottles will be color-coded substrate container. Assay / Control Temperature control Slides will not be 7 Power failure, device failure, Device runs diagnostics periodically (once Temperature too low or too high incubated properly, software control failure daily/ weekly/ monthly) will not properly stain (less stain or no stain)
  33. 33. Failure Mode Effect Analysis Potential Failure Potential Effects ofDevice / Function Mode Failure(s) (S) Potential Cause of Failure(s) (O) Current Controls (D) RPN Assay / Reagents, antibodies The assay will not run Surfaces or circulated air Disposable tips; air filtration system for air Contamination of and buffers properly or non- carry contaminants heating elements; reagents are sealed reagents (microbial) contaminated with specific binding or no until use, barcodes linked with lot microbes binding events may numbers are traceable -- known occur problematic lots will automatically be flagged, and all network connected devices will be updated with notification of problem lot. Assay / Reagent Reagents, antibodies, The assay may yield User loading old lots Database will contain information of expiry buffers are expired less than optimal expiration dates and check barcodes and results lot information against expiration dates, notify user of expired lots Image Analysis/ Alignment is off or The images will not Bumping the machine, the Periodic and scheduled calibration checks. Image Capture the camera provide accurate data pieces become loose Diagnostic tests can be run remotely (Focus) positioning is from image analysis through the persistent connection to the malfunctioning network system. Image Analysis / Images are poorly lit Images may be Lamps lose power or burn Periodic and scheduled calibration checks. Image Capture difficult to process out, lamp and/or condenser Diagnostic tests can be run remotely (Lighting) and of low quality for is out of place through the persistent connection to the comparison and network system. Technician will be presentation alerted to service instrument. Image Analysis / Images may not be of Overlap of some Vibrations from other Diagnostics and calibration checks camera bumped correct position areas or no data from machinery or processes between runs, software will detect during operation some areas, occurring on the same excessive overlap in images, sensor information may be bench, physical impact (accelerometer) can detect for impact missed (bumping), malfunction or events and correlate to image time disfiguration of the track stamps and re-take images mechanism. Report / server Unable to Images will not be Power outage, server Images can be stored locally and ready for malfunction communicate with analyzed and reports outage, network connection upload to server when communication re- server cannot be generated outage established.
  34. 34. • 1. [Webpage] Compound light microscope: How it works - bright field microscope . Retrieved on: 11/29/2011. Available from: http://www.indepthinfo.com/microscopes/compound.htm• 2. [Webpage] Olympus microscopy resource center | anatomy of the MIC-D digital microscope - brightfield illumination . Retrieved on: 11/29/2011. Available from: http://www.olympusmicro.com/micd/anatomy/micdbrightfield.html• 3. [Webpage] Ventana medical systems, inc. - ventana medical systems - VIAS . Retrieved on: 11/29/2011. Available from: http://www.ventana.com/product/page?view=vias• 4. [Chapter] Bloom, K. J. (2009) Virtual Microscopy and Image Analysis, in Immunohistochemical Staining Methods (J. Schmid, and M. Verardo, Eds.) 5th ed., pp 131-135, Dako North America, Carpinteria, California• 5. [Article] Gurcan, M. N., Boucheron, L., Can, A., Madabhushi, A., Rajpoot, N., and Yener, B. (2009) Histopathological Image Analysis: A Review IEEE Rev. Biomed. Eng. 2, 147-171, http://dx.doi.org/10.1109/RBME.2009.2034865• 6. [Article] He, L., Long, L. R., Antani, S., and Thoma, G. (2011) Distribution Fitting- Based Pixel Labeling for Histology Image Segmentation , 79633D <last_page> 79633D, http://dx.doi.org/10.1117/12.877726

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