Wei Huang, MD Pathology 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 M Micr croar array ( (TMA) A) C Construct ctio ion n 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 s situ u hybrid idiz izat atio ion 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 Nuance TM (Perkin Elmer) Vectra TM (Perkin Elmer) AQUA TM (HistoRx) √ √ Brightfield √ √ √ Fluorescence √ manual √ automated √ automated TMA slide scanning √manual, single slide √ automated, 200 slide √ manual, single slide Whole section slide scanning capacity capacity capacity √ up to 10 channels √ up to 10 channels √ up to 4 channels Multiplexing analysis √ √ Autofluorescence removal √ √ Spectral library tool Software for analysis Nuance and inForm Nuance and InForm AQUA algorithm Project application: √ √ √ Biomarker quantification √ √ √ co-localization quantification √ (by manual drawing) √ (automated) √ (automated) Per-tissue analysis (epithelium vs. stroma) √ √ Subcellular quantification √ Per-cell data √ Tissue structure (vessel, glomerulus, etc) counting Data format continuous Both ordinal and continuous continuous
Workflow for Bioma marker er Quantification using g Vectra I Imagi ging S g System m Bright field (IHC): up to 4 markers Multiple Dark field (IF): up 1. Decide how to 6 markers many markers to be stained on a single slide Bright field (IHC) or Single dark field (IF)
Test on the Run on intended 2. Optimize Use vendor intended tissue(s) target suggested experimental (breast, antibodies tissue first slides prostate, (TMAs) skin, etc)
# of slides = # of intended markers + 1 nuclear IF-SL: tissue specific (due to counterstain + 1 unstained autofluorescence) slide (without any 3. Build a spectral library (SL): counterstain) use a common working antibody, e.g., AE1/AE3, vimentin, Ki-67, etc. One dye per slide # of slides = # of intended IHC-SL: not tissue specific markers + nuclear counterstain (hematoxylin)
IHC slides: no specific requirement 4.Scan the SL and Experiment Slides (TMA/Whole Sections) with Vectra Scanner The SL slides and the experiment slides are to IF slides be scanned with the same channels
Make sure the staining protocol, scanned images are in the PI’s folder(s) 5. Biomarker Analysis Using inForm/Nuance Software Notify researcher(s) and reserve the station
Laser C Capture Mi Micros oscopy Laser microdissection of tissue specimens to isolate cells/DNA/RNA of interest Training of individuals with subsequent access to equipment through reservation process
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