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