Wei Huang, MD Pathology TRIP Laboratory Histology Tissue - - PowerPoint PPT Presentation

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

• f 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)

• r with conventional stains such as H&E and trichrome

–

With IF or IHC, single or multiple proteins or molecular markers (mRNA

• r 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 √ √ √ (by manual drawing) √ √ √ (automated) √ √ √ √ √ √ (automated) √ Data format continuous Both ordinal and continuous continuous

Workflow for Bioma marker er Quantification using g Vectra I Imagi ging S g System m

• 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 C Capture Mi Micros

• scopy

 Laser microdissection of tissue

specimens to isolate cells/DNA/RNA of interest

 Training of individuals with subsequent

access to equipment through reservation process