Comparison of High Resolution Whole Slide Imaging (WSI)

vs Conventional Fluorescence Microscopy

for Viewing and Analyzing Multiplex Quantum Dot Immunostained

(MQDS) Microscopic Slides

No more lights off in the lab!No more microscope!

Kumiko Isse, M.D., Ph.D.Demetris Lab

Department of Pathology, Division of TransplantationThomas E. Starzl Transplantation InstituteUniversity of Pittsburgh Medical Center

What is Quantum Dots (Qdots)?

Traditional Fluorescence Markers vs Qdots (1)

Adapted from 25 SEPTEMBER 1998 VOL 281 SCIENCE Adapted from Invitrogen.com

Rhodamine

Qdot

Qdot

Rhodamine

1H

3min

Traditional Fluorescence Markers vs Qdots (2)

• No photo-bleaching• Wide Stokes’ shift•Narrow emission spectra •Permanent•Multiple staining

• Expensive• Special filter

Qdots

Enable pathologists to contribute to the molecular revolution in medicine by merging traditional morphologic examination with multiple markers to precisely characterize specific cell types and investigate intra-cellular signaling pathways

•Time consuming•Complicated protocol

Multiple Staining

• Panoramic overview of tissue at low magnification

• Distribution, localization and cell-cell interactions visible

• Can unlock decades of human biology/pathology from paraffin blocks

• Connect to conventional morphology (H&E)

• Immediate sample collection and triage

• Complicated to analyze• Artifacts (wrinkles, bubbles,

dust, scratches, etc.)

Tissue Staining, not Flow Cytometry

• Avidin Block• Biotin Block• Non Serum Protein Block• 1st Primary Antibody• 1st Biotinylated Secondary Antibody• 1st Streptavidin Qdot

• Extra Blocking for each segment

• Amplify Signals by 2-step immunohistochemical staining

• Avidin Block• Biotin Block• Non Serum Protein Block• 2nd Primary Antibody• 2nd Biotinylated Secondary Antibody• 2nd Streptavidin Qdot

• Avidin Block• Biotin Block• Non Serum Protein Block• 3rd Primary Antibody• 3rd Biotinylated Secondary Antibody• 3rd Streptavidin Qdot

• Repeat for additional stainings

• Before starting the panel staining, titration of antibodies will be done by immunohistochemistry to decide the staining order

• Requires the best antigen retrieval for all antibodies that you are going to use

• Antigen retrieval

Protocol for Multiplex Quantum Dot Immunostaining (MQDS)

CD3 Ab + Biotynilated anti-rabbit + streptavidin Qdot CD3 Ab + Anti-rabbit Qdot

Comparison of 2-Step and 3-Step IHC

2-Step 3-Step

Nuance420nm

720nm

unmix

420nm 720nm

Captured Image Pseudocolor Image

Unmixed Grayscale Individual Images

Subtract AF by program

Skeletal Muscle 2.36 Liver 1.84

Small Intestine 1.38Heart 1.51

Frozen Liver 1.21 Colon 1.03

Lung 1.0

Same AF pattern in different tissues

Autofluorescence (AF) in Different Tissue

+ =

+ =

LCA

CD68 CD3 CD68CD3

CD4 CD8 CD4CD8

LCACD68CD3CD4CD8

Physical problems:• Unpleasant usually isolated

environment• Limited availability

=

Data problems:• Multiple layered image with lower

opacity is unclear• Individual colors need to be saved

separately

Mechanical problems:• Repeated training often needed• Precise adjustment of settings needed for good quality images• Suboptimal at low magnification

Disadvantage of Traditional Fluorescent Microscopes

Qdot Filter

• Total 12 slides are scanned in one time• 10-20min by 20x lens, 20-40min by 40x lens

for biopsy size tissue• 2.2GB with 80% compression of JPG• Using filters specific for Qdots

Inside of the scannerZeiss/3D Histech scanner

What is Whole Slide Imaging (WSI)?

x40

Digital x20

Digital x10

Digital x100

12bit vs 8bit

•Permanent data•Share the same slide with many people at once

•Observe anytime, anywhere, portable.

•No need to reserve microscope•Easy surveillance and analysis•Preservation of context and detailed morphological information

•Saves space in your lab

•Large data•Mechanical problems•Requires lot of adjustment

•Cost

Advantage of WSI

Digitally Preserving and Sharing the World’s Cultural Heritage

WSI : HLADRCK19CD31 (3D HISTECH/ CRi Pannoramic Viewer)

5 fields from liver, all portal tracts in the biopsy using three different antibodies + DAPI = 4 colorsTotal 19 cases------over 400 images

Data Obtained From WSI

CD31 in x4 CD31 in x4 CD31 in x20 CD31 in x20Nuance Mirax Nuance Mirax

0

5

10

15

20

25

30

%CD31 signal

Microscope x4 Microscope x20

WSI Digital x4 WIS Digital x20

CD31

Adapted from Zeiss “Microscopy From The Very Beginning”

Microscope vs WSI

Unfocused Areabecause of hardware

Shifting Problem Mechanical adjustment Layer adjustment in the software

Digitally subtract AF

Multiple focus points Dark field condenser

DAPI DAPI + Q705

Disadvantage of WSI

Limited Abs numbers because of AF and DAPI

http://www.farsight-toolkit.org/wiki/Main_Page

FarSight__ Developed by Dr. Badri Roysam

FarSight__Nucleus Editor HLADRIL10TGFbDAPI

HLADR expression and HLADR +TGFβ+/- cell numbers

Data Obtained From FarSight

N=3 N=8 N=8 N=4 N=10 N=10

X40 magnification, unknown field size x50 magnification, 230x350μm2

Vδ1+CD3+Vδ2+CD3+Vδ1+2+CD3+

N=8 N=10

Problem of FarSight or Human??

H&E after Qdot multiple staining Fluorescence signal

H&E Qdot multiple staining

Combitnation of H&E and MQDS

• Eosin has wide spectrum( - - - - - - - Eosin)• Eosin is strong Acid• Hematoxylin is strong Base

Eosin emission spectrum on the top of Qdots

pH Ranges for Qdot® Nanocrystals

pH Recommendations >9 Not recommmended- Qdot® nanocrystals start to self-aggregate/clump.

(Qdot® nanocrystals are not degraded by basic pH. )

>6 to <9 Qdot® nanocrystals most optimal stability in this pH range.

>5 to <6 Marginal stability is shown in this range

<4 Not recommended- The polymer will dissociate; exposed core/shell will start to dissociate.

CD31CD34aSMA

MQDS H&EWSI

• Conventional Method

•Bottleneck because of limited resource (location and time)•Microscope often difficult to use•Not portable•Limited triage to automated image analysis •Looking at a tree and not the forest

•Easy to use, portable•Stronger signal at lower magnification•Combination of FL and Bright Field•Direct connection to automated image analysis

• Unfocused areas• Autofluorescence• Adjustments needed

Microscope WSI

• Analyze whole slide image Automated whole slide image analysis using a selected region of interest (ROI)

• Better performance Hardware – computer, scanner, filter Software – imaging, analysis

Future Plan

Thank you!Rensselaer Polytechnic InstituteRoysam Lab

Dr. Badri RoysamKedar Grama

Demetris Lab

Dr. DemetrisJohn Lunz IIISusan SpechtYoshiaki MizuguchiNatasha CorbittEnrico Pegolo

MIDI and system Consultant

Andrew Lesniak

RHS Lab

Lisa ChedwickLori PerezTrevor BenyackEleck WaltonTraci Ondik

ISH Lab

Kathy Cieply