sop for immunohistochemistry procedure on ffpe tissue · 2020. 6. 11. · sop for...

Funded by European Union Horizon 2020 Grant No 825771

Translational studies of HEAD and neck cancer in

South America and Europe

SOP for immunohistochemistry procedure on FFPE tissue

Version 1.0 – May 2020

Deliverable No. D3.1 Part 2

Deliverable title SOPs for FFPE tissue accession sectioning, tissue array processing, and storage

Responsible partner University El Bosque (UnBosque)

Contributors Hospital Universitario Fundación Santa Fe de Bogotá (FSFB), Catalan Institute of Oncology (ICO), University of Turin (UNITO), Hospital Santa Rita de Cassia (AFECC), University of Bristol (UBRIS)

D3.1_P2_SOPs_IHC_FFPE_tissue.docx

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Contents 1. Introduction ........................................................................................................................................... 3

2. Objective ................................................................................................................................................ 3

3. Required equipment/material .............................................................................................................. 3

4. Sample conditions ................................................................................................................................. 3

5. General Procedure ................................................................................................................................ 3

5.1 Antibody CD8 ................................................................................................................................. 4

5.2 Antibody p16 ................................................................................................................................. 5

5.3 Antibody PDL-1 22C3 ..................................................................................................................... 6

5.4 Antibody MLH-1............................................................................................................................. 7

5.5 Antibody MSH2 .............................................................................................................................. 8

5.6 Antibody MSH6 .............................................................................................................................. 9

5.7 Antibody PMS2 ............................................................................................................................ 10

5.8 Antibody CD4 ............................................................................................................................... 11

5.9 Antibody CD3 ............................................................................................................................... 12

6. Process for immunohistochemistry done automatized by VENTANA BenchMark ULTRA IHC/ISH System ......................................................................................................................................................... 13

7. Process for immunohistochemistry done automatized by DAKO Autostainer Link 48 ....................... 13

8. References ........................................................................................................................................... 14


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1. Introduction The purpose of this SOP is to describe the instructions for the analysis of formalin-fixed paraffin-embedded (FFPE) samples using immunohistochemical (IHC) assays for the HEADSpAcE project. WP3.

2. Objective To define the general procedure and establish the basic quality guidelines for immunohistochemistry studies in formalin-fixed paraffin-embedded (FFPE) tissue samples

3. Required equipment/material • Microtome • Water bath • Slides • Automated system Ventana BenchMark ULTRA or Dako Autostainer Link 48. • H2O • 80%, 90%, 100% Ethanol • Xylene • Resin • Anti p16, P-L1, MSH2, MSH6, PMS2, MLH1, CD4, CD8 antibodies. • Microscope

4. Sample conditions • Formalin-fixed paraffin-embedded (FFPE) samples

5. General Procedure 1) Cut thin slices (3 or 4 μm) of the paraffin-embedded tissue on a microtome and put the slices

to float in a water bath. 2) Mount slices on charged slides for easier adhesion to the slide and let them dry overnight.

Mount the slides with positive control. Let the slide dry for 30 minutes at 60ºC. 3) Process is automatized using Ventana BenchMark ULTRA or Dako Autostainer Link 48. 4) Remove the slides from the automated slide stainer and wash with dishwater to remove the

coverslide solution. Rinse the slide with H2O 5) Dehydrate, clear and cover the slide:

a. 80% Ethanol, 3 minutes b. 90% Ethanol, 3 minutes c. 100% Ethanol, 3 minutes d. Xylene, two times for 3 minutes each e. Resin

6) Analyze, identify and verify quality control tissue staining pattern 7) For validation follow the recommendations of College of American Pathologist (Am J Clin

Pathol. 2010 Mar;133(3):354-65. doi: 10.1309) After you have optimized the protocol, identify the tissues required for test validation. An assay accuracy of a 95% concordance rate is recommended for test validation for positive and negative categories.

8) Apply the suggested protocol according to your validation process and available platform (see validation) these are only guidelines.


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5.1 Antibody CD8 Antibody CD8 Ventana

Clone SP57 Reagent

Provided Rabbit Monoclonal Primary Antibody

Solutions Ready to use

Platform BenchMArk ULTRA instrument

External control recommended

Tonsil recommended as positive and negative tissue control for CD8

Control Assessment

In tonsil strong membranous staining reaction of virtually all suppressor/cytotoxic T-cells. No staining reaction must be seen in other cells including B-cells, stromal cells or squamous epithelial cells of the tonsil.

Procedure Method Indications

Deparaffinization EZ PREP for 4 minutes at 72ºC

Antigen Unmasking Cell Conditioning 1 (CC1)

Heating time at max. temp: 64 min

Heating temp: 99º C

Primary Antibody Incubation antibody ready to

use

Time / Tº:20 min

Temp / 36º C

Amplification / Detection ultraView Universal DAB Detection Kit

Default

Counterstain Hematoxylin II Time/ T° 16 min

Post Counterstain Bluing reagent Time/ T° 16 min


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5.2 Antibody p16 Antibody CINtec® p16 Histology VENTANA

Clone E6H4

Reagent Provided

Mouse Monoclonal

Solutions

Ready to use



Squamous cell carcinoma positive for p16 and normal tonsil for negative control

Control Assessment

squamous cell carcinoma with moderate to strong nuclear and cytoplasmic, en bloc staining in at least 70% of the tumor and normal tonsil epithelium with none or weak cytoplasmic, discontinuos reactivity




Heating time at max. temp: 92 min Heating temp: 95º C


use

Time / Tº:20 min Temp / 36º C


Default




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5.3 Antibody PDL-1 22C3 Antibody PD-L1 DAKO

Clone 22C3

Reagent Provided

Monoclonal Primary Antibody

Solutions

Ready to use

Platform Dako Autostainer Link 48+

External control recommendd

Tonsil is recommended

Control Assessment

No staining reaction in the vast majority of lymphocytes including mantle zone and germinal centre B-cells, no staining reaction in superficial epithelial cells, a weak to moderate, typically punctuated membranous staining reaction of the majority of germinal center macrophages and finally a moderate to strong staining reaction of the majority of epithelial crypt cells.



Antigen Unmasking EnVision™ Flex Target Retrieval

Solution (TRS) low pH 6.1 (SK006)

(PT Link)



use

Time / Tº:30 min

Amplification / Detection EnVision Flex+ Time / Tº:30 min




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5.4 Antibody MLH-1 Antibody MLH-1 VENTANA

Clone M1

Reagent Provided

Mouse Monoclonal Primary Antibody

Solutions

Ready to use



Normal Tonsil with lymphoepithelial interface

Control Assessment

Virtually all mantle zone B-cells must show at an-at least weak to moderate nuclear staining reaction, while a moderate to strong nuclear staining reaction must be seen in the proliferating germinal centre B-cells.






use

Time / Tº: 1 hora 20 min Temp / 36º C


Default




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5.5 Antibody MSH2 Antibody MSH2 VENTANA

Clone G219-1129

Reagent Provided


Solutions

Ready to use




Control Assessment

Virtually all mantle zone B-cells must show at an-at least weak to moderate nuclear staining reaction Moderate to strong nuclear staining reaction must be seen in the proliferating germinal centre B-cells.







use

Time / Tº: 16 min Temp / 37º C


Default




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5.6 Antibody MSH6 Antibody MSH6 VENTANA

Clone SP93

Reagent Provided


Solutions

Ready to use



Tonsil is recommended

Control Assessment

Virtually all mantle zone B-cells must show at an-at least weak to moderate nuclear staining reaction. Moderate to strong nuclear staining reaction must be seen in the proliferating germinal centre B-cells.






use

Time / Tº: 1 hora 20 min

Temp / 37º C


Default




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5.7 Antibody PMS2 Antibody PMS2 VENTANA

Clone A16-4

Reagent Provided


Solutions

Ready to use




Control Assessment

Virtually all mantle zone B-cells must show at an at least weak to moderate nuclear staining reaction. Moderate to strong nuclear staining reaction must be seen in the proliferating germinal center B-cells.






use


Amplification / Detection OptiView DAB IHC Detection Kit

Default - Incubation time linker: T°8 min/ Temp

/ 36º C - Incubation time multimer: 8 min

Temp / 36º C Counterstain Hematoxylin II Time/ T° 16 min



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5.8 Antibody CD4 Antibody CD4 DAKO

Clone 4B12

Reagent Provided


Solutions

Ready to use

Platform Autostainer Link 48



Control Assessment

Tonsil is recommended as positive and negative tissue controls for CD4. a distinct and strong membranous staining reaction in all helper/inducer T-cells. Germinal center macrophages should at least display a moderate and distinct staining reaction. No staining reaction must be seen in other cells, including B-cells and epithelial cells of the tonsil.


Antigen Unmasking (HIER/target retrieval) pH 9



Rinse Station Wash Buffer Time / Tº:5 min Temp / room temperature


use


Amplification / Detection Envision Flex + mouse

Default - Incubation time linker mouse: T°15

min/ Temp / 20º C - Incubation time polymer: 20 min

Temp / 20º C Chromogen:

Counterstain Hematoxylin Time/ T° 10 seg

Post Counterstain Ammoniacal Water

Time/ T° 3 seg


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5.9 Antibody CD3 Antibody CD3 Ventana

Clone 2GV6 Reagent

Provided

Rabbit Monoclonal Primary Antibody

Solutions

Ready to use



Tonsil is an appropriate control for CD3.

Control Assessment

Both T-cells in the interfollicular areas and dispersed T-cells in the mantle zone and within the germinal centers must show a moderate to strong distinct membranous staining reaction, while B-cells must be negative.






use

Time / Tº:16 min

Temp / 37º C


Default




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6. Process for immunohistochemistry done automatized by VENTANA BenchMark ULTRA IHC/ISH System

1. Register on the Ventana’s Benchmark Ultra computer the slide bar code label which corresponds to the protocol to be performed and adhere the bar code label to the slide.

2. Load the Benchmark Ultra equipment with the primary antibody, detection kit dispensers, and additional reagents onto the reagent tray and place them on the automated slide stainer.

3. Check bulk fluids and empty waste. 4. Load the slides onto the automated slide stainer. 5. Apply the suggested protocol according to your validation process and platform (see

validation) these are guidelines only.

7. Process for immunohistochemistry done automatized by DAKO Autostainer Link 48

Reagent Preparation. The following reagents must be prepared prior to staining:

• EnVision FLEX Target Retrieval Solution, Dilute Target Retrieval Solution (50x) 1:50 using distilled or deionized water (reagent-quality water)

• EnVision FLEX Wash Buffer (20x), Dilute Wash Buffer (20x) 1:20 using distilled or deionized water (reagent-quality water) for the wash steps.

• DAB+ Substrate-Chromogen Solution, to prepare DAB+ Substrate-Chromogen Solution, add 1 drop of Liquid DAB+ Chromogen per mL of DAB+ Substrate Buffer and mix.

Prepared Substrate-Chromogen is stable for 5 days if stored in the dark at 2-8 °C.

1. Load the Autostainer Link 48 Solution and select the specific protocol for the antibody that’s being studied.

2. Apply the suggested protocol according to your validation process and platform (see validation) these are guidelines only.


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8. References • Agilent. (2018). PD-L1 IHC 22C3 pharmDx. Retrieved from https://www.agilent.com • Agilent. (2017). Monoclonal Mouse Anti-Human CD4 Clone 4B12, (33), 44–45. • Biocare Medical. (s/f). MSH6. • Biologicals, N. (n.d.). CHROMOGENIC IMMUNOHISTOCHEMISTRY STAINING OF PARAFFIN-

EMBEDDED TISSUE, 3–5. • Cell Marque. (2018). MSH2 (G219-1129) Mouse Monoclonal Antibody. • NEQAS, U. (2015). Immunocytochemistry, (November). • Cell Marque. (2017). PMS2 (EPR3947) Rabbit Monoclonal Antibody. • Recommended protocol for MSH2 Obtained in run 50. (2017), 9100. • Recommended protocol for PD-L1 (lung) Obtained in run C3. (2017), 1, 9100. • Recommended protocol for PMS2 Obtained in run 53. (2018), 9100. • Recommended protocol for CD4 Obtained in run. (2015), 9100. • Roche Diagnostics GmbH. (2012). CINtec® p16 Histology. Roche Diagnostics GmbH, 3. Retrieved

from www.ventana.com • Ventana Medical Systems Inc. (2017). VENTANA PD-L1 (SP263) Rabbit Monoclonal Primary

Antibody, 1, 8–11. • Ventana Medical Systems Inc. (2011). Anti-MLH-1 (M1) Mouse Monoclonal Primary Antibody. • Ventana Medical Systems Inc. (2011). OptiView DAB IHC Detection Kit, 1–5. • Ventana Medical Systems Inc. (2011). UltraView Universal DAB Detection Kit, 1, 5. • Ventana Medical Systems Inc. (2009). CONFIRM anti-CD8 (SP57) Rabbit Monoclonal Primary

Antibody, 8. • Ventana Medical Systems Inc. (2009). CONFIRM anti-MSH6 (44) Mouse Monoclonal Primary

Antibody.

1. Introduction2. Objective3. Required equipment/material4. Sample conditions5. General Procedure5.1 Antibody CD85.2 Antibody p165.3 Antibody PDL-1 22C35.4 Antibody MLH-15.5 Antibody MSH25.6 Antibody MSH65.7 Antibody PMS25.8 Antibody CD45.9 Antibody CD3

6. Process for immunohistochemistry done automatized by VENTANA BenchMark ULTRA IHC/ISH System7. Process for immunohistochemistry done automatized by DAKO Autostainer Link 488. References

sop for immunohistochemistry procedure on ffpe tissue · 2020. 6. 11. · sop for...

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