TITLE: 812.1 Lung FFPE OMAP Multiplexed Immunofluorescence Phenocycler-Fusion® Antibody Validation Protocol
AUTHORS: Jeffrey Purkerson, Gloria S Pryhuber, Heidie Huyck, gail.deutsch

[DESCRIPTION]
This protocol describes validation of a 34-antibody panel used for multiplexed immunofluorescent (MxF) staining and imaging of FFPE lung tissue sections utilizing the Phenocycler-Fusion® platform (Akoya Biosciences).  
The validation strategy involves staining two serial lung sections, one with cocktail of custom barcode-conjugated antibodies plus antibody-barcoded conjugates sourced from Akoya Biosciences and the second with the respective unconjugated, carrier-free control antibodies and omitting the Akoya antibodies.  
In addition, a tonsil section was exposed to the all-conjugated antibody cocktail to confirm specificity of antibodies directed against lung specific markers, and specificity of pan-epithelial, endothelial, megakaryocyte, and immune cell markers in 2 different tissues (e.g. lung, tonsil).  
The protocol includes sections describing 1) Tissue sections, 2) Labeling with barcode-conjugated or unconjugated Ab, 3) Reporter Plate and Experimental Design, 4) Multiplexed Imaging and Image Upload to OMERO for review, and 5) Review criteria. Detailed descriptions of staining and imaging procedures can be found in a companion protocol dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2. 


[BEFORE_START]
Review HubMAP guidelines for Ab validation in the attached SOP for OMAP construction

[GUIDELINES]
In order to validate both commercial and custom conjugated antibody specificity, control tissue sections were selected for the expected presence of specific tissue cells and structures expected to be specifically marked by the panel of antibodies to be tested.   In multiplexed Ab panels, inclusion of antibodies directed against general or pan-markers of epithelial, endothelial, and immune cells is recommended, as they will serve as positive controls for tissue section quality and effectiveness of the staining protocol when assessing the presence or absence of staining by tissue specific markers.  No primary antibody, in this case, Reporters-only, tests are also recommended to assess non-specific background.

[STEPS]
SECTION: Tissue Sections
1. Lung serial sections, and a tonsil section were prepared in FFPE as described in dx.doi.org/10.17504/protocols.io.kxygxejwdv8j/v2 

SECTION: Labeling of Tissue Sections with barcode conjugated Ab or unconjugated Ab
2. One of two serial healthy Lung sections (D016-RLL-11B2-6), a known diseased Lung section (D115-RLL-11A2-19) and a Tonsil Section (Tonsil-11-5) was labeled with a current panel (Table 1) of barcode antibodies as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2.  The other lung serial section (D016-RLL-11B2-5) was labeled with a panel of carrier free, unconjugated antibodies used for custom conjugations from step 18.1 in the above protocol and are listed in Table 1.

Table 1.  Antibodies used for custom conjugations.
 


Table 2.  Antibodies commercially conjugated (Akoya Biosciences).
 

SECTION: Reporter Plate and Experiment Design
3. Reporter plate design and Phenocycler-Fusion run protocols were developed using the PhenoCycler Experiment Designer Software (Akoya Biosciences) as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2.  The Reporter Plate design and exposures used for validation of the Lung FFPE OMAP-34 Panel is shown in Table 3.  Note that all 3 slides included in the validation were exposed to the same reporter panel.

Table 3.  Lung FFPE OMAP-34 Reporter Plate Design and exposures
Cycle #, reporters and exposure times
 

SECTION: Multiplexed Imaging and Upload to OMERO for review
4. Tonsil and Lung Sections were imaged as described in dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v2.


SECTION: Multiplexed Imaging and Upload to OMERO for review
4.1. OMAP-34 Validation consisted of the following slide runs.

1. Tonsil 11-5 (OMAP-34 Antibody Panel)
2. D016-RLL-11B2-5 (Unconjugated Ab panel)
3. D016-RLL-11B2-6 (OMAP-34 Antibody Panel)
4. D115-RLL-11A2-19 (OMAP-34)



SECTION: Multiplexed Imaging and Upload to OMERO for review
4.2. The resulting .qptiff images are converted to ome.tiffs utilizing command line: C:\>bfconvert -no-sas -series 0 -compression LZW -pyramid-resolutions 5 -pyramid-scale 2 INPUT.qptiff OUTPUT_ome.tiff.
 
SECTION: Review Criteria
5. Validation of marker signals is reviewed by a 3-person panel that includes the protocol author (MxIF Research Scientist), Dr. Gloria Pryhuber (Principal investigator), and Dr. Gail Deutsch (Board Certified Pathologist, Pulmonary specialist).
SECTION: Multiplexed Imaging and Upload to OMERO for review
4.3.  Utilizing Omero importer, ome.tiff files are uploaded to the URMC Omero-CODEX folder and the respective validation subfolder (i.e. OMAP34validation) for review.
SECTION: Review Criteria
5.2. Slides labeled with unconjugated Ab are assessed for the contribution, if any, of nonspecific reporter binding to generate fluorescent signal. 
 

SECTION: Review Criteria
5.3.  The tonsil section(s) was reviewed for any nonspecific interactions of respective Ab-barcode conjugates expected to uniquely target lung specific markers (e.g. RAGE, SFTPC, SCGB1A1, SCGB3A2, MUC5AC).


 

SECTION: Review Criteria
5.1. Lung and tonsil sections are reviewed for marker fluorescence signal localization to appropriate anatomical structures (e.g. airway, vasculature) and cell types within anatomical structures (e.g. alveolar epithelia, vascular endothelium, immune cell clusters), colocalization with appropriate pan-markers (e.g. CD4 with CD3/CD45, CD11c with HLADR/CD45; SCEL with RAGE and pan-cytokeratin (pan-CK), etc.).