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

1. Applications of Histology in Personalized Medicine
ALS 320
Juan Jose Aponte
Kathleen Coquia
Michael Davis
Samira Khodai
Stephen Kim
Debleena Sinha

2. We will develop a NSCLC-specific automated
histological device that provides precise and
accurate computer-assisted diagnosis to
pathologists and physicians. The Simul-Histo
Analyzer allows fully automated staining and
quantitative assessment of multiple stains and
slides. This comprehensive diagnostic tool is easy
to use and functions well with validated biomarkers
of targeted therapies. Generated reports can be
easily accessed in a digital format and readily
transferred to approved devices.

3. Immunohistochemistry and Detection

4. Immunohistochemistry Staining
Antigen Retrieval
Retrieval buffer Heat 121°C- 30 sec Cell or Tissue
Cooling 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. 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 TRIM29
DSG3 + Napsin A TTF-1 + Ck5 P63+TRIM29
Skin Lung Lung Prostate Prostate Prostate
Tonsil Bladder Placenta

7. 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 capture
Image Capture

8. Image Capture – Image Acquisition (Auto-Focus)

9. Image Capture – Image Acquisition (Auto-Focus)

10. Image Capture

11. Image Capture

12. Image Capture – Photo Stitch (Quantification)
Image Analysis
Edge Detection

13. Imaging Storyboard
http://library.wolfram.com/examples/edgedetection/

17. Inside view
4
1. Reagents storage
7 2. Mechanical arm
3. Slides holder
2 4. Peltier
Imaging area Staining area 1 heating/cooling
9 system
5. Waste container
4 6. Reagents big
3 containers
8
7. Imaging system
8. Base with light
Reagents storage area source
9. Tip storage
5
6

18. 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

19. 1. Slide holder
Metal-made holder
Capacity: 6 slides
One metal frame per slide
Lateral aspiration system -> remaining reagent volume
Lateral channel for fluids

20. 2. Heating / cooling system

21. 3. Small storages for reagents and tips
Big and small tips for both types of pipettes.
Storages: polypropylene containers under temperature control
Small storages keep the reagent ready for use.
The device sends a notification when either tips or reagents storages are empty .

22. 4. Holder entrance and
scanning
TRAY 1

23. 5. Pipette system

24. 6. Image capture

25. Bright Field Microscopy
• Light source
– Light-emitting diode (LED)
Camera • Diffusion filter
– Scatters light source
Magnification lens
• Condenser Lens:
Objective lens
– focuses light onto
specimen
• Specimen on slide holder
• Objective lens:
Specimen
– Determines level of
magnification
Condenser lens • Magnification lens
Diffusion Filter – 10x
• Camera
Light source:
LED
– RGB Progressive Scan
Camera

26. • 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

28. WELCOME TO SIMUL-HISTO
ANALYZER
User ID:
Password:
Hospital:

29. Home Page
ID # Last First Date Location Doctor
Touch & Scroll
scroll Btwn
up/down Letters
Keyboard

30. Home Page
ID # Last First Date Location Doctor A
B
C
D
E
F
Touch & G Scroll
scroll H Btwn
up/down Letters
I
J
K
L
M
N
O
P
Keyboard

31. Patient Report DOE, John
Back 11/30/2011 9:12 AM
Whole Tissue Image Biomarker Data
Red-boxed areas are
areas of interest
Notes on patient, etc.
Home
Keyboard

32. "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

33. DOE, John 11/30/2011 9:12 AM
Back
Biomarker Readout
Images in
Grid
Swipe Between Images
Legend
Home P63-----Brown % cells
TRIM 29----Red % cells

34. 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 k
DSG3 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 TRIM29
P63 Expression
TRIM 29 Expression
Home

36. 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
biomarkers
Hardware
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

37. Product Requirement Justification
Software
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.

38. Failure Mode Effect Analysis
Potential Failure Potential Effects of
Device / Function Mode Failure(s) (S) Potential Cause of Failure(s) (O) Current Controls (D) RPN
Assay / 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)

39. Failure Mode Effect Analysis
Potential Failure Potential Effects of
Device / 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.

41. • 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