Wei Huang, MDPathology TRIP Laboratory

Histology Tissue processing and embedding Cutting tissue sections

Unstained FFPE tissue sections Unstained fresh tissue sections

Hematoxylin and eosin staining of tissue sections (fresh or FFPE)

Histology• Special stains• Special Stain I Special Stain II Special Stain III • Amyloid Ziehl-Nielsen GMS • Bile AFB Reticulin• Elastic PAS Grimelius• Giemsa Calcium Dieterle• TolBlu Mast Cell Gram Lester King/Bielchowsky• PBR Mucicarmine Col-Iron • PTAH Trichrome Shikata• Iron Hematoxylin MGP Fontana-Masson • Alcian Blue pH 2.5 HPS Grocott-Methenamine

AgNO3 LFB Fraser-Lendrum Fibrin Oil red O

Tissue Microarray (TMA) Construction

TMAs from formalin-fixed, paraffin-embedded tissue blocks constructed to research needs. Requests for TMA construction require consultation with TRIP and may involve TSB-BioBank if tissue acquisition is needed and an appropriate IRB is not in place

Immunohistochemistry

Assays Chromogenic immunohistochemical assays Immunofluorescent assays

Antibody optimization Target detection in tissue or TMA

sections Single antibody staining Multiplex IHC Opal assay

In situ hybridization Conventional automated ISH Have potential to perform RNAscope

(Advanced Cell Diagnostics, Inc., Hayward, CA) RNAscope™ is a novel multiplex nucleic acid in situ hybridization technology This technology has overcome the pitfalls of the conventional ISH/FISH-based in situ RNA

detection techniques, such as lack robustness and sensitivity to reliably detect the expression of most human genes

The assay consists of a set of target probes and a signal amplification system composed of preamplifiers (PreAMP), amplifiers (AMP), and label probes

On average, each set of target probes spans an approximately 1kb region of the target RNA and hybridizes to 20 preamplifiers. Each preamplifier can hybridize to 20 amplifiers and each amplifier can hybridize to 20 label probes. This results in over 8,000 fluorescent molecules spanning just 1kb of RNA, which is readily visible using a standard fluorescent microscope

To increase the signal intensity further the label probes can be conjugated with an AP or a HRP moiety instead of the fluorescent molecule. This allows for a colorimetric reaction that leaves colored dots at the enzymatic reaction site.

Furthermore, multiple distinct amplification systems have been built that do not cross-react with each other and recognize unique sequences on different sets of target probes allowing for the simultaneous detection of multiple RNA targets

Tissue Imaging• Nuance System (Perkin Elmer)

– A manual multispectral imaging system (one slide capacity)

– It enables imaging of multiple molecular markers in tissue sections for both fluorescence and brightfield, even when they are co-localized

– Nuance imaging software can eliminate autofluorescence, unmixed co-localized signals for quantification and make weak signals visible and quantitatable by using a spectral library

– It also enables quantifying co-localized signals (e.g., percentage of double positivity, etc) and selecting regions of interest (ROI) for analysis

Tissue Imaging • Vectra System(Perkin Elmer)

– Is the most advanced instrument for extracting proteomic and morphometric information from tissue microarray or intact tissue sections

– Vectra merges automated slide-handling, multispectral imaging technology, and unique pattern-recognition-based image analysis (inForm software) into a powerful system for biomarker discovery and clinical studies

– This system accurately measures protein expressions and morphometric characteristics in distinct tissue regions of interest or on whole slides

– Sections can be labeled with either immunofluorescent (IF) or immunohistochemical (IHC) stains, or in situ hybridization (ISH or FISH) or with conventional stains such as H&E and trichrome

– With IF or IHC, single or multiple proteins or molecular markers (mRNA or DNA) can be measured on a per-tissue, per-cell, and per-cell-compartment (eg. nuclear, cytoplasmic) basis, even if those signals are spectrally similar, are in the same cell compartment or are obscured by autofluorescence

– Objects or structures of interest on H&E sections can be identified and counted with inForm software

– Vectra™ processes up to 200 slides in a single run or analyzes every core in a tissue microarray (TMA)

Tissue Imaging • AQUA System (HistoRx)

– Is a fluorescence-based, automated platform (PT-2000) with single slide capacity

– Images acquired with AQUAsition software are used to localize and quantify various protein biomarker concentrations

– AQUAnalysis software uses proprietary algorithms to identify and localize cellular and sub-cellular (e.g. nucleus, cytoplasm, membrane) compartments of protein biomarkers

– This software allows the user to accurately identify and image individual tissue fields at multiple wavelengths in both tissue microarrays and whole tissue sections.

– Signal resolution rivals confocal microscopy while eliminating the visual subjectivity associated with conventional IHC.

– The software’s algorithms and compartmentalization combine to provide an AQUA score reflecting protein concentration in a molecularly defined area—true biomarker localization

– Data is presented with significant data stratification, identifying subpopulations not seen with traditional IHC

Instrument (Software) Key Features

NuanceTM (Perkin Elmer) VectraTM (Perkin Elmer) AQUATM (HistoRx)

Brightfield √ √

Fluorescence √ √ √

TMA slide scanning √ manual √ automated √ automated

Whole section slide scanning √manual, single slide capacity

√ automated, 200 slide capacity

√ manual, single slide capacity

Multiplexing analysis √ up to 10 channels √ up to 10 channels √ up to 4 channels

Autofluorescence removal √ √

Spectral library tool √ √

Software for analysis Nuance and inForm Nuance and InForm AQUA algorithm

Project application:

Biomarker quantification

co-localization quantification

Per-tissue analysis (epithelium vs. stroma)

Subcellular quantification

Per-cell data

Tissue structure (vessel, glomerulus, etc) counting

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√ (by manual drawing)

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√ (automated)

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√ (automated)

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Data format continuous Both ordinal and continuous

continuous

Workflow for Biomarker Quantification using Vectra Imaging System

1. Decide how many markers to be stained on a

single slide

Multiple

Bright field (IHC): up to 4 markers

Dark field (IF): up to 6 markers

Single Bright field (IHC) or dark field (IF)

2. Optimize target

antibodies

Use vendor suggested tissue first

Test on the intended tissue(s) (breast, prostate, skin, etc)

Run on intended

experimental slides

(TMAs)

3. Build a spectral library (SL): use a common working

antibody, e.g., AE1/AE3, vimentin, Ki-67, etc.One dye per slide

IF-SL: tissue specific (due to autofluorescence)

# of slides = # of intended markers + 1 nuclear

counterstain + 1 unstained slide (without any

counterstain)

IHC-SL: not tissue specific# of slides = # of intended

markers + nuclear counterstain (hematoxylin)

4.Scan the SL and Experiment Slides

(TMA/Whole Sections) with Vectra Scanner

IHC slides: no specific requirement

IF slides

The SL slides and the experiment slides are to

be scanned with the same channels

5. Biomarker Analysis Using inForm/Nuance

Software

Make sure the staining protocol,

scanned images are in the PI’s folder(s)

Notify researcher(s) and reserve the

station

Laser Capture Microscopy

Laser microdissection of tissue specimens to isolate cells/DNA/RNA of interest

Training of individuals with subsequent access to equipment through reservation process