Bridging the DivideUsing Digital Pathology to Guide
Ultrastructural Pathology
Evaluations.
GD Gagne, JH Decker and JA Fagerland.
Preclinical Safety, Abbott Laboratories
Overview
• Digital pathology at Abbott
• Ultrastructural pathology: Identification of
pigment in liver and adrenal tissues
• Evaluating biomarkers of glomerular injury
using TEM, laser capture microdissection
• Image analysis applications
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Digital Pathology at Abbott
• 2008- Digital pathology system (120-slide) installed in Pathology
Department, Lake County, IL (LC)
• 2009
– 120-slide system installed in Ludwigshafen, Germany
Pathology Department
– 6-slide scanner installed in LC Ultrastructural
Pathology/Investigative Toxicology facility
– Added whole-slide image analysis capability, including pattern
recognition software
• Applications: multisite slide sharing, global pathology
teleconferencing, pathologist-to-pathologist consultation
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Ultrastructural Pathology
• Single global EM facility
(LC)
• Transmission electron
microscopy (TEM)
evaluation used to further
characterize histopathology
findings
• TEM sections:1-2 mm2
LM sections: ≤8 cm2
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Bridging the divide between LM and TEM
• Digital pathology system allows:
– consultation between electron microscopist and
pathologist to assure changes seen by histopath are
present in sections taken for TEM
– comparison of H&E-stained histological sections for LM
with toluidine blue-stained semithin sections for TEM
– interactively review by personnel at different sites
– example: Identification of pigment in liver and adrenal
gland in a 12-month toxicity study in cynomolgus
monkeys
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Evaluation of Pigment in Liver and Adrenal Tissues
• Twelve month toxicity study in cynomolgus monkeys
• Accumulation of pigment noted by LM in liver and adrenal gland
from monkeys administered high dose of test compound (had
not been seen in previous monkey tox studies)
• Liver and adrenal tissue was retrieved from formalin and sent to
Abbott for EM evaluation
• Questions:
– Nature of the pigment
– Similarity to pigment seen in rats dosed with same compound
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Liver, high dose monkey
Images from digital slides of H&E and semithin sections
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Liver, high dose monkey, Kupffer cell,TEM images
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Liver, high dose monkey, endothelial cells
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Adrenal gland, high dose monkey.
Semithin section image from digital slide.
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Adrenal gland, zona reticularis, high dose monkey
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Evaluating biomarkers of glomerular injury
• Purpose of Study- To determine if urinary biomarkers can
provide a sensitive and accurate measurement of receptor
tyrosine kinase (RTK) inhibitor-induced glomerular injury
• RTK inhibitors have been shown to cause proteinuria and
glomerular injury (visible by EM) in experimental animals
and humans in clinical trials
• TEM used to
– characterize glomerular changes
– correlate changes to biomarker measurements
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Biomarker Characteristics
Protein
Mechanism of Release into Urine
Injury Site
Albumin
Freely filtered through the glomerular
basement membrane, reabsorbed (and a
fraction degraded) by the proximal tubule
Glomerular and
tubular injury
Lipocalin-2
(NGAL)
Expressed and released in response to injury
Glomerular and
tubular injury
Osteopontin
Expressed and released in response to injury
Glomerular and
tubular injury
KIM-1 (Kidney
Injury Molecule-1)
Expressed and released in response to injury
Tubular injury
Courtesy Y. Yang
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Individual Animal Biomarker Results(specimens examined by TEM)
Total Albumin
(ug)
Total
Osteopontin
(ug)
Total Lipocalin
(ng)
Total KIM-1
(ug)
1001a
87
0.6
1404.8
8.5
1003a
4001b
4005b
175.6
3247.2
4456.9
18.1
201.6
73.1
1896.1
15389.9
4506.2
9
20.4
11.6
a: Vehicle
b: RTK Inhibitor, dosed for 7 days at 10 mg base/kg/day
Increase in albumin and lipocalin, but not KIM-1 suggests
glomerular injury
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Compare H&E to semithin section at identical mags
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Locate features on EM monitor while viewing semithin
section slide image
(High dose kidney: Glomerulus)
Semithin section
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
TEM Image
High Dose kidney: Glomerulus
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Conclusion
• TEM evaluation revealed glomerular changes
consistent with RTK inhibition:
– Damage to capillary endothelium, loss of fenestrations
– Subendothelial electron-dense deposits
– Accumulation of electron-dense granules (protein) in
podocytes
• TEM supported the biomarker results that indicated
glomerular injury
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Laser capture microdissection (LCM) of glomeruli
• Collect glomeruli for gene expression analysis to
correlate with biomarker changes
• Use LCM to collect tissue
• Use digital pathology system to document efficiency of
laser capture
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Laser Capture Microdissection
Prepare stained tissue section
(H&E, immunostains, etc.)
Fire laser pulse to embed
cells in thermoplastic film
of cap
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Align cap (with
thermoplastic transfer film)
over area of interest
Lift cap to remove
microdissected cells
Under microscope,
locate cells to be
dissected
Place cap in microfuge
tube with appropriate
extraction reagents for
molecular analysis
Documentation of Laser Capture Microdissection of
Glomeruli
Before LCM
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
After LCM
Image Analysis
Quantification of hepatic lipid on liver sections
• Select 5 random fields (5x magnification) from each
specimen, using whole slide image
• Detect lipid droplets with MetaMorph® software, using
color, size, and shape discriminators to detect lipid
droplets as white, round objects. Report total detected
area.
• Measure total liver area.
• Calculate “Area fraction lipid” using a spreadsheet.
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Lipid Area Measurement
Original Image
Feature extraction-lipid
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Thresholded
Tissue area threshold
Hepatocyte Lipid Quantification
Dose
Area
Animal # fraction
0
1001
0.010
1003
0.005
1005
0.007
( mg / kg / d ay)
2001
2003
2005
0.018
0.021
0.012
100
3001
3003
3005
0.045
0.025
0.015
200
4001
4003
0.097
0.085
0.100
Area Fraction "Lipid"
50
0.120
0.080
0.060
0.040
0.12
0.020
Lipid Area Fraction
0.10
0.08
0.000
1001 1003 1005
0.06
0.04
0.02
0.00
0
50
100
200
Dose
(mg/kg/day)
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
2001 2003 2005
3001 3003 3005
4001 4003 4005
Next Step
• Apply pattern recognition software to identify
and measure areas of vacuolated hepatocytes
on whole slide images
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Quantitation of percent human hepatocytes in
sections of chimeric liver
Human
hepatocytes
Mouse
hepatocytes
Anti-human mitochondria antibody used to identify human hepatocytes in
chimeric mouse liver [PXB-mouse, PhoenixBio]
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Morphometric Thresholding on Whole Slides
Control
Treated
Blue = Mouse origin
Yellow = Human origin
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Liver Chimerism by Morphometry
Area Fraction Positive Pixels (anti-human mitochondria)
0.9
Control
0.8
Treated
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Left Median Lobe
Right Median Lobe
Left Lateral Lobe
Treatment caused an unexpected reduction in percentage of human
hepatocytes in chimeric liver
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Summary
• Applications of digital pathology and whole slide imaging
presented:
– Ultrastructural pathology
• Correlation of light microscopy and electron microscopy observations
• Use of TEM and LCM for biomarker validation
– Image analysis
• Hepatic lipid quantitation
• Quantitation of human hepatocyte fraction in whole-slide images of
chimeric livers
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
© 2009 Abbott
Acknowledgements
• Cellular Molecular and
Experimental Toxicology
– Yi Yang
– Wayne Buck
– Andrew Lisowski
– Rita Ciurlionis
– Eric Blomme
– Tami Pilot-Matias
– Christine Collins
– Susan Lacy
– Teresa Ng
• Investigative Toxicology
• Pathology
– Carmen Nasarre
– George Foley
Bridging the Divide
Pathology Visions 2009
Jerry Gagne
• Abbott Antiviral Research
© 2009 Abbott
– David Cugier