Bridging the Divide- Using Digital Pathology to Guide Ultrastructural Pathology Evaluations.

GD Gagne, JH Decker and JA Fagerland. Preclinical Safety, Abbott Laboratories

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

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 DividePathology 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 DividePathology 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 DividePathology 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 DividePathology 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Liver, high dose monkeyImages from digital slides of H&E and semithin sections

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Liver, high dose monkey, Kupffer cell,TEM images

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Liver, high dose monkey, endothelial cells

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Adrenal gland, high dose monkey. Semithin section image from digital slide.

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Adrenal gland, zona reticularis, high dose monkey

Bridging the DividePathology 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Protein Mechanism of Release into Urine Injury Site

AlbuminFreely 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

OsteopontinExpressed 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

Biomarker Characteristics

Courtesy Y. Yang

Bridging the DividePathology 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 175.6 18.1 1896.1 9

4001b 3247.2 201.6 15389.9 20.4

4005b 4456.9 73.1 4506.2 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Compare H&E to semithin section at identical mags

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Locate features on EM monitor while viewing semithin section slide image (High dose kidney: Glomerulus)

Semithin section TEM Image

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

High Dose kidney: Glomerulus

Bridging the DividePathology 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 DividePathology 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Prepare stained tissue section (H&E, immunostains, etc.)

Align cap (with thermoplastic transfer film) over area of interest

Under microscope, locate cells to be dissected

Fire laser pulse to embed cells in thermoplastic film of cap

Lift cap to remove microdissected cells

Place cap in microfuge tube with appropriate extraction reagents for molecular analysis

Laser Capture Microdissection

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Documentation of Laser Capture Microdissection of Glomeruli

Before LCM After LCM

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Image AnalysisQuantification 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Feature extraction-lipid Tissue area threshold

Original Image Thresholded

Lipid Area Measurement

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Dose(mg/kg/day) Animal #

Area fraction

0 1001 0.0101003 0.0051005 0.007

50 2001 0.0182003 0.0212005 0.012

100 3001 0.0453003 0.0253005 0.015

200 4001 0.0974003 0.085

0.000

0.020

0.040

0.060

0.080

0.100

0.120

1001 1003 1005 2001 2003 2005 3001 3003 3005 4001 4003 4005

Are

a F

rac

tio

n "

Lip

id"

Hepatocyte Lipid Quantification

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0 50 100 200

Dose(mg/kg/day)

Lip

id A

rea

Fra

ctio

n

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Next Step

• Apply pattern recognition software to identify and measure areas of vacuolated hepatocytes on whole slide images

Bridging the DividePathology 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Morphometric Thresholding on Whole SlidesControl Treated

Blue = Mouse originYellow = Human origin

Bridging the DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Liver Chimerism by Morphometry

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Left Median Lobe Right Median Lobe Left Lateral Lobe

Are

a F

ract

ion

Po

siti

ve P

ixel

s (a

nti

-hu

man

mit

och

on

dri

a)

Control

Treated

Treatment caused an unexpected reduction in percentage of human hepatocytes in chimeric liver

Bridging the DividePathology 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 DividePathology Visions 2009 Jerry Gagne

© 2009 Abbott

Acknowledgements

• Cellular Molecular and Experimental Toxicology

– Yi Yang

– Wayne Buck

– Andrew Lisowski

– Rita Ciurlionis

– Eric Blomme

• Pathology

– Carmen Nasarre

– George Foley

• Abbott Antiviral Research

– Tami Pilot-Matias

– Christine Collins

– Susan Lacy

– Teresa Ng

• Investigative Toxicology

– David Cugier