Conventional immunohistochemistry (IHC) is commonly used as a diagnostic technique in the field of tissue pathology but suffers from certain limitations. The most critical of these is that this technique only permits the labelling of a single marker per tissue section. This results in missed opportunities to gain important prognostic and diagnostic information from patient samples. Multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) technologies, which allow the simultaneous detection of multiple markers on a single tissue section, have been introduced and adopted in both research and clinical settings in response to increased demand for improved techniques. A number of highly multiplexed tissue imaging technologies have also emerged, permitting comprehensive studies of cell composition, functional state and cell-cell interactions which suggest improved diagnostic benefit.

1. Exploring The Concept Of
Multiplexing In
Immunohistochemistry:
An Advanced Technology
BY
OSAYANDE CHELSEA IMUETINYANOSA
(B.Mls, AMLS)
Medical Laboratory Science Department, Histopathology Unit
National Hospital, Abuja
29th APRIL, 2022

2. OUTLINE
 INTRODUCTION
 IMMUNOHISTOCHEMISTRY
 PRINCIPLE OF STANDARD CHROMOGENIC IHC
 LIMITATIONS OF IHC
 MULTIPLEXING
 METHODS OF MULTIPLEXING
 TECHNOLOGIES OF MULTIPLEXED IHC
 CASE STUDY
 APPLICATIONS
 CONCLUSION
 RECOMMENDATION
 REFERENCES

3. INTRODUCTION
 Conventional immunohistochemistry (IHC) is commonly used as a diagnostic technique in the field of tissue pathology but
suffers from certain limitations. The most critical of these is that this technique only permits the labelling of a single
marker per tissue section. This results in missed opportunities to gain important prognostic and diagnostic information
from patient samples (Tan et al., 2020).
 Multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) technologies, which allow the simultaneous detection of
multiple markers on a single tissue section, have been introduced and adopted in both research and clinical settings in
response to increased demand for improved techniques.
 A number of highly multiplexed tissue imaging technologies have also emerged, permitting comprehensive studies of cell
composition, functional state and cell-cell interactions which suggest improved diagnostic benefit (Lu et al., 2019).
Source: National Hospital Abuja

4. IMMUNOLOGY HISTOLOGY CHEMISTRY
WHAT IS IMMUNOHISTOCHEMISTRY?
Source: National Hospital Abuja, Author’s Illustration

5. IMMUNOHISTOCHEMISTRY
 Immunohistochemistry (IHC) represents a powerful tool used for the identification of target proteins in the
context of the cell or tissue analysed. It is used to identify tumours, classify tumours, utilize prognostic markers,
predict response to therapy and identify infectious and biological behaviours (Dixon et al., 2015).
 This type of in situ analysis can be used to identify sub-populations of cells based on the expression of specific
markers. It is an important tool in the post-genomic era, necessary for uncovering protein function (Schubert et
al., 2006).
 Samples such as mammalian tissues are rapidly fixed (most commonly in formaldehyde) and embedded in
paraffin wax - or frozen where this is not possible - to maintain their morphology. Samples are then thinly sliced
into sections and mounted onto glass slides, and then dried. It is then analyzed following the standard operating
procedure of immunohistochemistry (Dixon et al., 2015).
Source: National Hospital Abuja

6. Figure 1: Principle of Standard Chromogenic IHC (Taube et al., 2020).
Colored Product
Substrate
Peroxidase or
Alkaline Phosphatase
Secondary Antibody
Primary Antibody
Proteins
Fixed Tissue
Source: National Hospital Abuja, Author’s Illustration, Taube et al., 2020

7. PROTOCOL OF STANDARD CHROMOGENIC IHC
Epitope
retrieval
Blocking
The primary
antibody
Visualization
Amplification
Detection
system
Counterstaining
Cover slipping
Microscopy
(Taube et al., 2020)
Source: National Hospital Abuja

8. Figure 2: Hematotoxylin and eosin staining (H&E) and immunohistochemical staining for estrogen receptor (ER), progesterone
receptor (PR) and HER2/neu proteins (400X).(a) Ductal carcinoma. (b)Lobular carcinoma (Barekati et al., 2012).
Source: National Hospital Abuja

9. LIMITATIONS OF IHC
 As aforementioned, the inability to label more than one marker per tissue section is the most important limitation
of IHC. For example, tumor-infiltrating CD8+ T cells can be identified through CD8, CD3, forkhead box P3
(FOXP3) and CD20 expression (Mazzaschi et al., 2018).
 Another drawback of IHC-based biomarker assessment is high inter-observer variability. For instance, Ki-67 is a
widely endorsed marker for a range of cancers (Tan et al., 2020).
Source: National Hospital Abuja

10. MULTIPLEXING
Source: National Hospital Abuja

11. MULTIPLEXED IHC
 In the last decade, various adaptations of IHC and related technologies have been developed through which several
targets can be stained and visualised in one sample. Together, these applications are often referred to as “multiplexed
IHC” (Dixon et al., 2015).
 These applications are hugely useful for detecting multiple biomarkers in various cell types in complex tissues such as the
tumour microenvironment, neural tissues, and lung biopsies.
 Some initial examples of so-called “low level” multiplexed IHC closely follow the principals of standard IHC (Dixon et al.,
2015).
 Such technologies involve the use of carefully designed antibody cocktails that localise to distinct cellular locations. These
approaches help to identify patterns in the distribution of groups of proteins in different cell types, where previously only
one or two proteins could be assessed simultaneously (Yanagita et al.,2011).
Source: National Hospital Abuja

12. How does multiplex IHC work?
Step 1
Add primary antibodies
Step 2
Add conjugated secondary antibodies
Step 3
Add chromogen substrates
Antigen Primary antibody Goat anti-mouse HRP Goat anti-rabbit AP Chromogen
Mouse Rabbit DAB FastRed
Source: National Hospital Abuja, (Lee et al., 2019)

13. Multiplexed staining
methods
Brightfield Fluorescence
Source: National Hospital Abuja

14. BRIGHTFIELD MULTIPLEXING
 In order to perform mIHC on FFPE tissues in brightfield microscopy, chromogenic deposition of various
chromogens/enzyme pairs is used. While this is useful when distinguishing different cell types, it is more challenging to
assess when trying to co-localize targets within cells (Chris, 2010).
 Some of the specific chromogens available for brightfield mIHC include: 3,3′-diaminiobenzidine (DAB) and nickel
enhanced DAB (DAB-Ni), 3-amino-9-ethylcarbazole (AEC), and Vector VIP which produces an insoluble purple
precipitate
 These chromogens can be visualized with either horseradish peroxidase (HRP; DAB, DAB + Ni, AEC and VIP)
or alkaline phosphatase (AP; NBT/BCIP). In addition, several counterstaining dyes can enhance brightfield
multiplexing, such as methyl green or hematoxylin, which stain nuclei green or blue, respectively (Stack et al., 2014).
Source: National Hospital Abuja

15. Figure 3: Light microscopy multiplex chromogenic immunohistochemistry staining. Representative image of triplex
FOXP3/CD8/KRT staining with purple (Discovery HRP, Ventana, Roche Tissue Diagnostics), yellow (Discovery AP, Ventana, Roche
Tissue Diagnostics) and teal (Discovery HRP, Ventana, Roche Tissue Diagnostics) chromogens and a hematoxylin counterstain
(Taube et al., 2020).
Source: National Hospital Abuja

16.  It is a relatively easy, inexpensive
 Multiple reagents and automated
platforms are available
 The stains are most often read
using light microscopy, which
allows for easier quality oversight
 High- throughput brightfield digital
image acquisition platforms are
available (Taube et al., 2020)
 Only a few existing
chromogens that are very
effective in allowing for the
study of marker co- expression.
 The dynamic range of marker
intensity is also limited
 Often used to simply assess
expression as positive versus
negative, or a semi-quantitative
H-score (Remark et al., 2016).
ADVANTAGES
DISADVANTAGES
Source: National Hospital Abuja

17. MULTIPLEX IMMUNOFLUORESCENCE
 The basic principle of mIF is that multiple protein targets can each be stained by specific antibodies labeled with
distinct fluorophores. When excited, the fluorophores emit at a characteristic wavelength (Stack et al., 2014).
 One of the most widely used approaches for mIF is an indirect approach that employs Tyramide Signal
Amplication (TSA). This method provides signal amplification through a polymer-HRP detection system combined
with activation of tyramide fluorophores (Taube et al., 2020).
 Using this approach, it is possible to create a protocol that can allow researchers to use antibodies raised in the
same species and create panels that can accommodate simultaneous detection of up to six to eight individual
targets (Stack et al., 2014).
Source: National Hospital Abuja

18. Figure 5: Multiplex immunofluorescence (IF) using tyramide signal amplification (TSA)-based detection methods and multispectral
imaging (Taube et al., 2020).
Source: National Hospital Abuja

19.  Hundreds of commercially available
purified fluorophores
 Four to five different carefully
selected fluorophores may be
applied
 When multispectral microscopes are
used, the number of fluorophores
applied to a single slide can be
increased up to eight
 Tissue auto fluorescence can also
be subtracted from the image
 Imaging approaches that do not
use multispectral technologies
may be limited in their
quantitative ability in some
circumstances by tissue
autofluorescence,
 Those that require multispectral
technologies are expensive.
 Risk of overactive tyramide
deposition, potentially
contributing to an umbrella effect
and/or signal bleed-through
ADVANTAGES
DISADVANTAGES
Source: National Hospital Abuja, (Taube et al., 2020).

20. TECHNOLOGIES OF MULTIPLEXED IHC
1
Stain Removal Technology
2
Fluorophore Inactivation
Technology
3
Multiplexed Signal
Amplification
4
DNA Barcoding Technology
5
Mass Cytometry
Source: National Hospital Abuja

21. STAIN REMOVAL TECHNOLOGY
 One of the most commonly used approaches of multiplexing IHC is stain removal. Stain removal technologies
(sometimes called dye cycling) refer to a class of protocols that rely on a cycle of antibody staining, image capture,
removal of stain, and re-staining to greatly increase the number of markers that can be tested in a single experiment.
(Kim et al., 2016).
 There are a variety of ways in which a stain or signal can be removed; two examples include:
 multiepitope-ligand cartography (MELC) and
 sequential immunoperoxidase labeling and erasing (SIMPLE).
 Others include multiplexed immunohistochemical consecutive staining on the same slide (MICSSS)
(Aktürk et al., 2020).
Source: National Hospital Abuja

22. Figure 6: (A) Simultaneous visualization of five antigens in mouse cerebellum. (B) The images were individually pseudocolored and
overlaid. (C) The resultant image reveals the morphology of different cell types and fine details of interactions of Purkinje cells,
Bergmann glia, astrocytes, and basket cell terminals that would not be obvious with single or dual labeling
Source: National Hospital Abuja

23. FLUOROPHORE INACTIVATION TECHNOLOGY
 The technology aimed to overcome the limitations associated with standard immunofluorescence or
immunohistochemistry techniques.
 The principle of the technique is similar to the stain removal techniques where pH, denaturation, or photo-bleaching is
used to remove a stain, but instead, this technique relies on the inactivation of the fluorophores.
 The fluorophores are inactivated through alkaline oxidation chemistry, which quickly eliminates cyanine-based dye
fluorescence
 Multiplexed fluorescence microscopy method
 Cyclic immunofluorescence (Gerdes et al., 2013).
Source: National Hospital Abuja

24. Figure 7: Tyramide signal amplification: The HRP-conjugated secondary antibody binds to an unconjugated primary antibody specific to
the target/antigen of interest. Detection is ultimately achieved with a fluorophore-conjugated tyramide molecule that serves as the
substrate for HRP. Activated tyramide forms covalent bonds with tyrosine residues on or neighboring the protein of interest and is
permanently deposited upon the site of the antigen. The method allows for serial stripping of the primary/secondary antibody pairs, while
preserving the antigen-associated fluorescence signal, making this process amenable to multiple rounds of staining in a sequential fashion
{Parra, 2017).
Slide
Identification
Ag
Slide
Identification
Ag
Slide
Identification
Ag
HRP
HRP
Ag
F
T
F
T
F
T
F
T
F
T
F
T
F
T
F
T
HRP
Ag
First primary
Antibody
incubation
Introduce
HRP
TSA incubation and HRP
Catalyzes with TSA to free
Radicals formation
Covalent bonds formation with
TSA residuals next to HRP
Second primary
Antibody incubation
(repeated the cycle)
H2O2 H2O
Source: National Hospital Abuja, Author’s Illustration
MULTIPLEXED SIGNAL AMPLIFICATION

25. Figure 8: Fluorescent nucleotides are added along the first indexing nucleotide G in the antibodies (Ab) 1 and 2. Cells are washed of free
nucleotides and the slide is imaged. A clearing step is performed using tris (2-carboxyethyl) phosphine which cleaves the disulfide linkers to
release the fluorophores and then a new indexing cycle 2 is doing in T nucleotide (Ab3 and Ab4) for fluorescent nucleotides U and C to be
incorporated onto Abs 3 and 4. The cycle is repeated, using the index by the position G in the Ab5 and Ab6 with fluorescent nucleotides to start
another cycle (Parra, 2017).
Slide
Identification
Ab1
AB2
ACG
TGCGA
ACG
TGCCG
Ab3
AB4
ACGA
TGCTGA
ACG
TGCCGT
Ab5
AB6
ACGC
TGCGAT
ACGT
TGCAGC
INDEX 1
Staining
Slide
Identification
F
F
F
F
F
F
Multiplexed
Image
Imaging / Bleaching Imaging / Bleaching Imaging / Bleaching
Ab1
AB2
ACG
TGCGA
ACG
TGCCG
Ab3
AB4
ACGA
TGCTGA
ACG
TGCCGT
Ab5
AB6
ACGC
TGCGAT
ACGT
TGCAGC
F
F
INDEX 1
Cycle 1
Index 1/Render
Ab1
AB2
ACG
TGCGA
ACG
TGCCG
Ab3
AB4
ACGA
TGCTGA
ACG
TGCCGT
Ab5
AB6
ACGC
TGCGAT
ACGT
TGCAGC
F
F
INDEX 2
Cycle 2
Index 2/Render
Ab1
AB2
ACG
TGCGA
ACG
TGCCG
Ab3
AB4
ACGAU
TGCTGAC
ACG
TGCCGT
Ab5
AB6
ACGC
TGCGAT
ACGT
TGCAGC
F
F
INDEX 3
Cycle 3
Index 3/Render
Source: National Hospital Abuja
DNA BARCODING TECHNOLOGIES

26. Figure 9: Different samples can be barcoded with unique combinations of heavy metal tags, enabling them to be pooled together prior to
staining to minimize technical variability at this step. The samples are incubated with antibodies targeted against proteins of interest. The
cells are nebulized into droplets as they are introduced into the mass cytometer. They then travel into an inductively-coupled argon
plasma, in which covalent bonds are broken and ions are liberated. The ion cloud is filtered to remove common biological elements and
enrich the heavy metal reporter ions to be quantified by time-of-flight mass spectrometry. Ion signals are integrated on a per-cell basis,
resulting in single-cell measurements for downstream analysis (Parra, 2017).
Slide
Identification
Slide
Identification
Antibodies labeled
With elemental isotopes
Stain cell samples
With metal-labelled
antibodies
Cells are nebulized
into droplets
Mass filter to remove
Common biological
elements
Mass
Time of Flight
Ion cells in plasma flame
(Argon Plasma
Source: National Hospital Abuja
MASS CYTOMETRY

27. CASE STUDY:
Diagnostic differentiation of non-small cell lung cancer
National Hospital Abuja

28. Figure 10: DAB IHC of independent markers can be used to identify subtypes of NSCLC, but overconsumes tumor material. Serial 4 um
sections were stained for TTF-1, Napsin A, p40 and CK5/ 6 using DAB IHC. Shown are examples of adenocarcinoma (A), squamous cell
carcinoma (B) and Adenosquamous carcinoma (C) stained for all four markers. Objective = 40×. Scale bar = 20 μm (Robert et al., 2020).
Source: National Hospital Abuja

29. Figure 11: Chromogenic multiplexing is able to identify mixed NSCLC subtype morphology. The quadruplex assay was applied to
all cases in the cohort. Shown are examples of staining in adenocarcinoma (A), squamous cell carcinoma (B) and adenosquamous
carcinoma (C). TTF-1 = yellow; napsin A = pink/purple; p40 = teal; CK5/ 6 = DAB. Objective = 40× (Robert et al., 2020).
Source: National Hospital Abuja

30. APPLICATIONS
ONCOLOGY
 mIHC on biopsied tissues can aid the rapid classification of tumor subtypes, without using up too much sample. For
instance, breast cancer is classified based on the presence or absence of estrogen, progesterone, and Her2 receptors.
In one study on breast cancer samples.
 mIHC may be used to gain insights into the tumor microenvironment and quantify predictive biomarkers to stratify
patients based on their therapeutic response (Tsujikawa et al., 2017).
NEUROLOGY
 mIHC can be used to label brain cells, neurotransmitters, the blood-brain barrier, and peripheral players such as immune
cells.
 mIHC can also be used to identify and monitor biomarkers of neurodegenerative disease progression. One study used
mIHC to assess neuroinflammation and neural tissue damage in a rat model of traumatic brain injury (Bogoslovsky et al.,
2018).
Source: National Hospital Abuja

31. CONCLUSION
Scientists’ understanding of molecular biomarkers that define normal and disease states is constantly evolving. To accurately characterize
cell populations in research and diagnosis in a high-throughput and high-resolution manner, novel imaging platforms, assays, and
technologies are required. Multiplexing immunohistochemistry is one avenue increasing the capabilities of clinical research and diagnostics,
providing researchers with the tools to investigate complex and heterogeneous tissues in a powerful manner.
Source: National Hospital Abuja

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