Informatics, digital & computational pathology

Fluorescence microscopy

Multiplex immunofluorescence



Topic Completed: 29 November 2021

Minor changes: 30 November 2021

Copyright: 2021, PathologyOutlines.com, Inc.

PubMed Search: Multiplex immunofluorescence [TI]

Harsh Batra, M.B.B.S., D.C.P., D.N.B.
Anil Parwani, M.D., Ph.D., M.B.A.
Page views in 2021 to date: 233
Cite this page: Batra H, Parwani A. Multiplex immunofluorescence. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/informaticsmultiplex.html. Accessed December 8th, 2021.
Definition / general
  • Multiplex immunofluorescence (mIF) is a high throughput immunofluorescence technology based on tyramide signal amplification and multispectral imaging, which allows the simultaneous detection of multiple markers on a single tissue section without compromising the tissue architecture
  • Multiplex refers to many elements in an interrelated landscape
  • Can analyze 2 - 50 markers at one time, expressed on a single cell level with high precision (J Immunother Cancer 2015;3:47, Methods 2014;70:46, Cell 2016;165:780)
  • Powerful tool for immune profiling to achieve a targeted therapy
Essential features
  • Multiplex immunofluorescence is a technology that is based on enzyme based tyramide signal amplification and uses multispectral imaging to generate images for analysis
  • For multiplex immunofluorescence, it is necessary to excite all the fluorophores contained in a single slide after the staining but the emission spectral range cannot overlap with the excitation range
  • Basic principle of analysis of the generated data is linear unmixing of images, segmentation of tissue into tumor and stroma, segmenting individual cells and then phenotyping markers based on marker of interest
  • Subsequent data can be used to generate multiple datasets elaborating cell phenotypes, nearest neighbor distances, which can be used for spatial position of single cells in a tissue
Terminology
  • Tyramide signal amplification (TSA): a technique for detection of low abundance targets in fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC) and in situ hybridization (FISH) applications
    • mIF is based on the principle of TSA, wherein fluorescent opal dyes are conjugated with tyramide molecules to produce enzymatic (alkaline phosphatase or horseradish peroxidase) amplification; the primary and secondary antibodies can be sequentially applied and washed but tyrinamide signal remains (Mol Oncol 2020;14:2384)
  • Multispectral imaging:
    • Spectral imaging combines spectroscopy and imaging (Cell 2016;165:780)
    • Can acquire 2 dimensional, spatial (X and Y) and temporal data information from objects depending on parameters like spatial resolution, field of view, dynamic range and lowest detectable signal based on their emitted wavelength (Cell 2016;165:780)
    • Multispectral imaging can capture approximately 10 - 30 wavelengths at a time at each pixel of an image, providing the intensity wavelength spectrum at every pixel of the image, in addition to the typical 2 dimensional position of every pixel
    • This 3D generation of an image is done via specially designed cameras
    • This technique forms the basis of image generation in multiplex immunofluorescence
Diagrams / tables

Contributed by Harsh Batra, M.B.B.S., D.C.P., D.N.B., Maria Gabriela Raso, M.D. and Edwin Roger Parra Cuentas, M.D., Ph.D.
Procedure

Procedure

Fundamentals and procedure
  • Panel design and selection: select (a) epitopes and markers with lowest redundancy; (b) optimal combination of colocalizing markers based on clinical implications; (c) other antibodies to answer specific scientific or research questions, such as immune checkpoint (Methods Mol Biol 2020;2055:467)
  • For purposes of colocalizing, the convention is to pair the brightest opal fluorophores with the weaker expressing proteins and vice versa
  • Optimization of primary antibodies by chromogenic IHC: refers to antibodies that have been tested using multiple titers and antigen retrieval conditions on control tissues to screen for markers which produce the best staining pattern
  • Use of automated staining is preferred
  • Careful selection of positive and negative control to be done; a serial of at least 10 different tissues must be tested to determine the assay's operating characteristics (sensitivity and specificity) for each antibody
  • Uniplex immunofluorescence and optimization of primary antibodies: each target to be assessed by a uniplex immunofluorescence assay using single fluorophore to optimize the primary antibodies starting with the same primary antibody concentration as in IHC validation
  • mIF staining consists of following steps:
    • Slide preparation
    • Epitope retrieval
    • Blocking
    • Primary antibody incubation
    • Incubation with secondary antibody conjugated with horseradish peroxidase (HRP), which catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to tyrosine moieties close to the epitope of interest on formalin fixed paraffin embedded (FFPE) tissue
    • Tyramide signal amplification (TSA) with fluorophore incubation: combination of tyramide and fluorophore is water insoluble and when attached to antigen, the assembly is very stable (Methods Mol Biol 2020;2055:467)
    • Primary and secondary antibody washout by microwave treatment
    • Repeating the above steps for each primary antibody and corresponding TSA fluorophore
    • Counterstain with DAPI and mount
  • Spectral library and linear unmixing
    • Spectral library: collection of spectral references matching the intensities contained in the multispectral image used to decode the information in the image at each pixel
    • Made by staining single samples with one fluorescent dye at a time with a primary antibody directed against a well known and highly prevalent antigen (e.g. CD20, CD3, etc.)
    • Once the library is created, the emitted spectra from each dye can be extracted and registered using a software
    • Linear unmixing: for the purpose of analysis of a multispectral image, it needs to be unmixed into its component fluorochromes, which is done using a software constructed algorithm which keeps spectral library and tissue autofluorescence as reference
  • Other fluorescence based multiplex staining methods without signal amplification
    • Multiepitope ligand cartography (MELC): technology using samples subjected to cycles of fluorescent staining, imaging and photobleaching, with different antibody in each cycle, thus creating analytical images with different antigen localization on single sample
    • Sequential immunoperoxidase labeling and erasing (SIMPLE): can visualize 5 - 12 markers using the alcohol soluble peroxidase substrate 3 amino 9 ethylcarbazole with a fast, stable antibody antigen separation method
    • MultiOmyx™ staining or hyperplexed immunofluorescence assay: involves a pair of directly conjugated cyanine dye labeled (Cy3, Cy5) antibodies per round of staining, with imaging at end of each round and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation for up to 60 protein biomarkers
      • Advantageous particularly in hematopathology studies
    • Codetection by indexing or fluorescent immunohistohybridization (CODEX): this technology uses an antibody conjugated to barcoded unique oligonucleotide sequence
      • CODEX assay targets specific barcodes with a dye labeled reporter for highly specific detection
      • Tissue sample is stained with the antibody panel in a single step and the reporters bind to the complementary barcodes through repeated cycles of imaging and removal
Application Images

Contributed by Harsh Batra, M.B.B.S., D.C.P., D.N.B., Maria Gabriela Raso, M.D. and Edwin Roger Parra Cuentas, M.D., Ph.D.
Caption

Equipment

Library building and unmixing

Library building and unmixing

Composite image after linear unmixing

After linear unmixing

Colocalization of 3 markers

Colocalization of 3 markers

Colocalization's markers

Colocalization's markers

Software analysis

Software analysis

Image analysis
  • Image analysis: multiplex immunofluorescence images are read digitally on specially built software, which needs expertise to build an algorithm to analyze each case
  • Slides with region of interest are scanned based on pathologists' expertise and disease in question
  • Basic workflow of an algorithm is image preparation by linear unmixing → tissue segmentation → individual cell segmentation → phenotyping
  • Data generated can be read using computer programming languages viz R, Python and C to generate meaningful results
  • Software and platforms to analyze multiplex data (J Cancer Treatment Diagn 2018;2:1):
    • Commercial:
      • InForm® (Akoya / PerkinElmer)
      • MultiOmyx™ Quantification Program (NeoGenomics)
      • Aperio eSlide Manager analysis (Leica Biosystems)
      • Tissue Studio / Image Developer (Definiens)
      • AQUAnalaysis (HistoRx)
      • SlidePath's tissue image analysis (SlidePath)
      • HALO® (Indica Labs)
      • Visimoph Tissuemorph (Visiopharm)
      • Image Pro (Media Cybernetics)
      • iCyte / iBroser / iNovator (CompuCyte)
      • HistoQuest / TissueQuest / StrataQuest (TissueGnostics)
    • Open source:
Advantages & disadvantages
  • Advantages:
    • Can study the cell biology through capturing multidimensional data related to tissue architecture, spatial distribution of multiple cell phenotypes and coexpression of signaling and cell cycle markers at the same time (J Cancer Treatment Diagn 2018;2:1)
    • Can assess multiple antibodies on a single tissue section
  • Disadvantages:
    • Requires a significantly longer time to acquire, process and analyze (Cytometry A 2006;69:735)
    • Needs costlier equipment and special expertise, which presently lacks trained individuals
Clinical & pathological applications
  • To study the tumor microenvironment in detail, including spatial configuration (Lung Cancer 2018;117:73)
  • To discover new therapeutic biomarkers and possible drug targets, thus helping to discover new immunotherapies (J Thorac Oncol 2018;13:1884)
  • Immunophenotypic findings of the tumor associated immune infiltrate determined by multiplex immunofluorescence can further be correlated with genomic or transcriptomic data from the same patients (Lab Invest 2014;94:107)
Board review style question #1

Which of the following steps does a raw multispectral image undergo before analysis on a digital software?

  1. Linear unmixing using spectral libraries
  2. Segmenting
  3. Uncoating and pixelation
  4. Unrevealing
Board review style answer #1
A. Linear unmixing using spectral libraries, where the raw multispectral image is unmixed into its component fluorochromes

Comment Here

Reference: Multiplex immunofluorescence
Board review style question #2
The principle of multiplex immunofluorescence is based on which of the following phenomena?

  1. Confocal imaging
  2. Transmission of electrons
  3. Tyramide signal amplification
  4. Tyrosine signal amplification
Board review style answer #2
C. Tyramide signal amplification (TSA), wherein fluorescent opal dyes are conjugated with tyramide molecules to produce enzymatic (alkaline phosphatase or horseradish peroxidase) amplification

Comment Here

Reference: Multiplex immunofluorescence
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