Proof of principle: Colocalization of pan cytokeratin(AE1/AE3), pan cytokeratin (PCK26), and cytokeratin 8/18 using an Integrated Sequential Staining and Imaging Device

Judit Zubovits1, Kashan Shaikh3, Alex Corwin3, Dan Wang4, Sean Dinn2, Gina Clarke4

Chris Peressotti4 , Zhengyu Pang2, Robert Filkins2, Martin J. Yaffe4

1 Department of Anatomic Pathology, Sunnybrook Health Sciences Centre2 Diagnostic and Biomedical Technologies, GE Global Research Center3 Electrical Technologies & Systems, GE Global Research Center4 Ontario Institute for Cancer Research and Sunnybrook Research Institute

International Academy Of Digital PathologyAugust 3, 2011Quebec City, Quebec, Canada

Multiplexing technology – Standard Approach

- primary and secondary Abs

- limited by secondary Ab

New approach to multiplexing technology:

Dye Cycling

Step 1DigitalImage

InactivateDye

Step 3DigitalImage

InactivateDye

Step 2

Step 4

Image registration using the DAPI channel

DAPI channel:

-reference channel

-not bleached by our

process.

Courtesy of Musodiq Bello and Ali Can

Image registration using the DAPI channel

DAPI channel:

-reference channel

-not bleached by our

process.

Courtesy of Musodiq Bello and Ali Can

Autofluorescence removal using image subtraction

Step 1:AF only Step 2: AF + p53-Cy3

P53 staining on a breast TMA.Images from D. Hollman-Hewgley

Autofluorescence can

be successfully

removed

Automated cycling: StainingScanning/ImagingDye Inactivation

Sample Automated Platform Image/Data

Flow Cell

Automated labeling & Image acquisition

Benefits of an Automated System

• Greatly reduced sample handling � Reduced tissue damage

• Quick turnaround

• Much easier to track the sample

Flow Cell

•~100µl flow cell is rigidly fixed to stage to maintain sampleregistration

• Tighter process control

• Microfluidics allow faster staining by maintaining a higherconcentration of stains close to tissue sample

Three types of cytokeratin are widely used in breast pathology

CK(#) AE1 AE3 PCK26 CK8/18 Type

1 1 (68) 1 (68) suprabasal layer Neutral to

2 2(67) basic

3 3(64) pI 6 and

4 4(59) above

5 5(58) 5(58) stratified epithelial

6 6(56) 6(56) hyperproliferating Type II

7 7(54)

8 8(52) 8(52) 8(52) simple epithelial

9

10 10(56.5) Acidic

11 pI < 5.5

12 Type I

13 13(54)

14 14(50)

15 15(50)

16 16(48)

17(46)

18 18(45)

19 19(40)

20 (46)

Note

Multiplexing using the Autosys

Step 2: AE1/AE3

Step 1 Step 3

Step 4: CK 8/18 Step 4: PCK26

Step 5

JACoP ImageJ plugin is used for co-localization

-1: Negatively correlated

0: No correlation

1: Positively correlatedJ Microsc. 2006 Dec;224(Pt 3):213-32. A guided tour into subcellular

colocalization analysis in light microscopy. Bolte S, Cordelières FP.

Analysis was performed on Regions of Interest on registered image

Co-localization of cytokeratins AE1/AE3, PCK26, and CK8/18

AE1/AE3 PCK26 CK8/18

ImageJ JACoP output for

pixel based analysis

All cytokeratins show good correlation (r) ranging from 0.896 to 0.959

Highest correlation is between:1.CK8/18 and PCK262.AE1/AE3 and PCK263.AE1/AE3 and CK8/18

Correlation between AE1/AE3 and CK8/18

Position AFR Raw

P002 0.906 0.887

P003 0.871 0.897

P004 0.951 0.929

P005 0.905 0.937

P006 0.810 0.916

P007 0.927 0.928

P008 0.886 0.920

P009 0.871 0.934

P010 0.882 0.900

P011 0.879 0.845

P012 0.925 0.889

P013 0.935 0.850

P014 0.879 0.903

P015 0.917 0.916

Grand Total 0.896 0.904

Standard deviation 0.036 0.029

Correlation between AE1/AE3 and PCK26

Position AFR Raw

P002 0.912 0.901

P003 0.941 0.937

P004 0.974 0.944

P005 0.928 0.933

P006 0.840 0.934

P007 0.940 0.946

P008 0.950 0.941

P009 0.922 0.946

P010 0.956 0.927

P011 0.929 0.855

P012 0.947 0.904

P013 0.946 0.859

P014 0.938 0.918

P015 0.968 0.910

Grand Total 0.935 0.918

Standard deviation 0.032 0.030

Correlation between PCK26 and CK8/18

Position AFR Raw

P002 0.975 0.978

P003 0.944 0.956

P004 0.970 0.980

P005 0.922 0.970

P006 0.957 0.974

P007 0.976 0.968

P008 0.938 0.970

P009 0.905 0.961

P010 0.914 0.955

P011 0.931 0.960

P012 0.925 0.946

P013 0.938 0.923

P014 0.875 0.936

P015 0.934 0.949

Grand Total 0.936 0.959

Standard deviation 0.028 0.016

Conclusion

Our work demonstrates a novel and versatile

system for co-localization and co-expression

studies that can be easily adapted to work

with existing commercially available

antibodies in a translational research setting.

More studies can be done using this

automated system to address challenging

questions in digital pathology

JACoP ImageJ plugin is used for co-localization

-1: Negatively correlated

0: No correlation

1: Positively correlated