ISO/TS 23366:2023

Nanotechnologies - Performance evaluation requirements for quantifying biomolecules using fluorescent nanoparticles in immunohistochemistry

This document describes minimum requirements for performance evaluation of applying fluorescent nanoparticles in quantitative immuno-histochemistry.

Nanotechnologies — Exigences d'évaluation des performances pour la quantification de biomolécules en immunohistochimie à l'aide de nanoparticules fluorescentes

General Information

• Status: Published
• Publication Date: 02-May-2023
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229/WG 5 - Products and Applications
• Current Stage: 6060 - International Standard published
• Start Date: 03-May-2023
• Due Date: 16-Dec-2023
• Completion Date: 03-May-2023

Overview - ISO/TS 23366:2023 (Nanotechnologies)

ISO/TS 23366:2023 specifies minimum requirements for performance evaluation when using fluorescent nanoparticles to quantify biomolecules by immunohistochemistry (IHC). Published by ISO/TC 229 (Nanotechnologies), the technical specification addresses the full quantification workflow - from nanoparticle selection and staining through microscopy, image processing, validation and reporting - to promote reliable, comparable relative quantification results across products and systems.

Key topics and technical requirements

The document outlines practical requirements for performance evaluation, including:

• Selection of fluorescent nanoparticles
  • Characteristics to consider (brightness, photostability, aggregation/agglomeration behaviour, quantum yield).
• Quantification system design (section 6)
  • Components: antibodies, staining protocols, imaging instrumentation and image processing software.
  • Antibody attributes: type, specificity, titre, immunoglobulin class, species applicability.
• Staining and imaging
  • Staining conditions, robustness of procedures, light-source adjustment and image quality factors.
• Performance characteristics (section 8)
  • Background issues (autofluorescence, nonspecific adsorption, photobleaching), and reference-material dependent indices such as limit of detection (LOD), limit of quantification (LOQ), linearity and dynamic range.
• Comparability and reference materials (section 7)
  • Use of calibrated reference material and light-source standardization to achieve comparable results.
• Validation and verification (section 9)
  • Single-lab precision, reproducibility and robustness testing before routine use.
• Reporting requirements (section 10)
  • Minimum information to document system performance and enable data comparability.
• Informative annexes provide examples for reference material and nanoparticle aggregation/agglomeration evaluation.

Applications and users

ISO/TS 23366:2023 is intended for organizations and professionals involved in quantitative fluorescence IHC using nanoparticles, including:

• Research laboratories performing biomarker quantification
• Clinical and diagnostic laboratories validating quantitative assays
• Biotech and reagent manufacturers developing nanoparticle-labelled IHC kits
• Quality teams and regulatory specialists assessing method performance

Practical uses include method development, product performance claims, inter-lab comparison studies, and establishing standardized reporting for nanoparticle-based IHC assays.

Related standards

While ISO/TS 23366:2023 focuses on nanoparticle-specific performance evaluation, users may also consult existing industry-specific and imaging standards (e.g., terminology and fluorescence measurement guidance) to complement method-specific criteria and regulatory requirements.

Keywords: ISO/TS 23366:2023, fluorescent nanoparticles, immunohistochemistry, performance evaluation, quantifying biomolecules, LOD, LOQ, photobleaching, aggregation, validation.

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 23366
First edition
2023-05
Nanotechnologies — Performance
evaluation requirements for
quantifying biomolecules using
fluorescent nanoparticles in
immunohistochemistry
Nanotechnologies — Exigences d'évaluation des performances pour
la quantification de biomolécules en immunohistochimie à l'aide de
nanoparticules fluorescentes
Reference number
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Selection of fluorescent nanoparticles . 3
5.1 General . 3
5.2 Characteristics of nanoparticle . 3
6 Quantification system .4
6.1 Overall design . 4
6.2 Antibody . 5
6.2.1 General . 5
6.2.2 Antibody type . 5
6.2.3 Target antigen . 5
6.2.4 Applicability of antibody to method . 6
6.2.5 Animal species . 6
6.2.6 Immunoglobulin class . 6
6.2.7 Titre . 6
6.2.8 Specificity . 7
6.3 Staining procedure . 7
6.3.1 Staining conditions . . 7
6.3.2 Robustness of staining procedure . 7
6.4 Image processing . 8
6.4.1 Image quality and relevant factors . 8
6.4.2 Selection of image processing software . 8
7 Comparability of results . 9
7.1 Light source adjustment. 9
7.2 Reference material . 9
8 Performance characteristics . 9
8.1 Background . 9
8.1.1 General . 9
8.1.2 Autofluorescence . 10
8.1.3 Nonspecific absorption of nanoparticles . 10
8.1.4 Nonspecific absorption of antibodies . 10
8.1.5 Loss of intensity and interferences . 10
8.2 Reference material dependent indices . 11
8.2.1 General . 11
8.2.2 Limit of detection (LOD) . 11
8.2.3 Limit of quantification (LOQ) . 11
8.2.4 Linearity and dynamic range . 11
8.3 Robustness .12
9 Validation and verification .12
9.1 General .12
9.2 Single lab precision .12
9.3 Reproducibility . 13
10 Reporting .13
Annex A (informative) Example of the reference material .14
iii
Annex B (informative) Example of the nanoparticle aggregation/agglomeration evaluation
method .16
Bibliography .18
iv
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
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electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
Introduction
Fluorescent nanoparticles are expanding their market into bio-technological and research fields as a
labelling material to be used for immunohistochemical staining.
Conventionally, various fluorescent dyes, including FITC (fluorescent isothiocyanate), rhodamine-
isothiocyanate and sulforhodamine 101 acid chloride, have been used for immunohistochemical
staining. They are still powerful tools for identifying localization of target biomolecules, for example,
proteins and sugar chains, mainly for qualitative analyses. They are also applied to quantitative analysis
in combination with various algorithms for calculating signal intensity related to the quantity of the
target biomolecules. The quantification system generally consists of sample preparation, staining,
microscopic observation and photography, and image processing for obtaining quantification results
as shown in Figure 1. For reliable measurement results of quantification, fluorescent dyes that are
brighter and more photostable by exposure to the excitation light are more appropriate.
Large number of fluorescent nanoparticles are available in the market. Generally, they show higher
brightness and are more resistant to photobleaching, compared to the conventional fluorescent dyes.
The characteristics of fluorescent nanoparticles can be an advantage for the quantification of target
biomolecules by immunohistochemical methods also combining with the same algorithm employed for
[1][2][3][4]
the quantification with conventional fluorescent dyes.
In this context, various staining kits with fluorescent nanoparticles and various quantification systems
[5]
have been developed and are available in the market. Thus, the needs to realise the compatibility of
various systems are expanding in the research and industrial fields.
In this document the minimum requirements for performance evaluation of products and application
using fluorescent nanoparticles is addressed. This document provides information ensuring the
comparability of the results of relative quantification by using fluorescent nanoparticles.
This document does not provide industry segment specific performance criteria for the workflow of
measuring biomolecules. When applicable, users can also additionally consult existing industry specific
standards.
Figure 1 — Target quantification process by using of fluorescent nanoparticles
vi
TECHNICAL SPECIFICATION ISO/TS 23366:2023(E)
Nanotechnologies — Performance evaluation
requirements for quantifying biomolecules using
fluorescent nanoparticles in immunohistochemistry
1 Scope
This document describes minimum requirements for performance evaluation of applying fluorescent
nanoparticles in quantitative immuno-histochemistry.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
fluorescent nanoparticle
nanoparticle emitting fluorescence excited by light of specific wavelength
3.2
cell block array
paraffin block re-embedded with plural cylinders gouged out from paraffin embedded cell suspension
for histopathology
3.3
cell block array section
thin slice of cell block array obtained by cutting cell block array using microtome, and mounted onto a
glass slide
3.4
agglomerate
collection of weakly or medium strongly bound particles where the resulting external surface area is
similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.2]
3.5
agglomeration
process through which agglomerates form
3.6
aggregate
particle comprising strongly bonded or fused particles where the resulting external surface area is
significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example, covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO 26824:2022, 3.1.3, modified — Note 1 to entry has been adapted.]
3.7
aggregation
process through which aggregates form
3.8
quantum yield
number of quanta emitted per quantum absorbed
[SOURCE: ISO 22493:2014, 6.6.9]
3.9
molar extinction coefficient
optical density of 1 M fluorescent substance per 1 cm of optical path of absorption cell
3.10
photobleaching
destruction of fluorescing properties of molecules by light, resulting in reduced fluorescence of the
sample
[SOURCE: ISO 10934:2020, 3.2.31]
4 Principle
Immunofluorescence methods are based on staining of thin tissue sections with specific antibodies
recognizing intended molecules, e.g. proteins and sugars. The specific antibodies are prepared by
immunizing animals, e.g. goat, rabbit or mouse, with target molecules. While monoclonal and polyclonal
antibodies are used for immunofluorescence methods, antibodies need to be labelled with fluorescent
substances, e.g. dyes or nanoparticles. For investigating localization and quantity of the target molecules,
tissue sections are stained with fluorescence labelled antibodies. There are two basic methods for
staining with labelled antibodies. One is the direct immunofluorescence method, the other is an indirect
immunofluorescence method. The direct immunofluorescence method is performed by using a single
fluorescence-labelled antibody recognizing the target molecules. The indirect immunofluorescence
method is performed with a non-labelled antibody recognizing the target molecules (first antibody)
and antibody recognizing the first antibody (second antibody). When the first antibody (e.g. IgG) is
produced in mouse, anti-mouse IgG antibody should be selected for the second antibody. In addition,
other molecular systems enhancing fluorescence signal are also available in market. For example, the
avidin-biotin system is well known for this application. In the avidin-biotin system, the second antibody
can be labelled with biotin and reacted with avidin conjugated with fluorescent materials including
nanoparticles, e.g. quantum dots (QD), i.e. semiconductor particles with a few nanometres in size. The
first antibody-second antibody-avidin-QD complex emits a strong fluorescence signal with less photo
bleaching. It is preferred for the quantification of the target molecules by using nanoparticles including
QD.
Fluorescence signal from the stained specimen is measured by fluorescence microscopy, which is
an optical imaging technique that detects simultaneously the emitted fluorescence from the field of
view using a camera. Fluorescence intensity, namely the emitted fluorescence, can be measured as
an intensity value in fluorescence microscopy, which is computed by summing together the intensity
values from a group of individual pixels in a digital image acquired using a digital camera. In addition
to the measurement of integrated fluorescence intensity, counting bright spots is used to quantify the
target biomolecules.
Quantitative comparison of the intensity data or the numbers of the bright spots requires sound
experimental design and appropriate operation of the whole quantitative photometric system
including a digital camera e.g. a charge coupled device (CCD) or a scientific complementary metal
oxide semiconductor (sCMOS). Issues of the quantitative comparison of intensity data involving the
digital camera and controller software settings, including collection of dark count images to estimate
the offset, flat-field correction, background correction, benchmarking of the excitation lamp and the
fluorescent collection optics are described in Reference [15].
For quantitative analysis by immunofluorescence microscopy, fluorescence intensity or the number of
bright spots can be compared to the measured value by enzyme-linked immuno-sorbent assay (ELISA)
or fluorescence activated cell sorter (FACS) and a calibration curve drawn (see A.3). Fluorescence
microscopy measures the fluorescence intensity or the number of bright spots from immunostained
thin slice of tissues and cells, however, it does not measure the biomolecule number per cell. It measures
the fluorescence intensity or the number of bright spots from a slice of a population of the cells although
it is corelated to the number of biomolecules in cells when fluorescence intensity or the number of the
bright spots is measured from a sufficient number of the cells in a field of view. In this sense, microscopic
measurement of fluorescence intensity or the number of bright spots is a relative measurement.
Fluorescence intensity does not in itself have an associated SI unit, because it is a relative measurement.
The number of the bright spots has “unit one” but it is also a relative measurement in principle. A relative
intensity measurement (RIM) is determined as the ratio of one intensity measurement or bright spot
count to another. The fluorescence intensity measurement is an accurate estimate of the ratio of the
irradiance from part or all of a specimen, to the irradiance from part or all of the same or another
specimen. For the counting of bright spots, the results can be interpreted in a similar way with the
integrated fluorescence intensity measurements.
Indices for performance evaluation of quantitative values and application of CBA (cell block array) for
realizing comparability among values that form various quantification systems are described in the
following clauses.
5 Selection of fluorescent nanoparticles
5.1 General
Nanoparticles shall be selected to fit the purpose of the quantification system. Required performance of
nanoparticles varies with respect to quantity of target biomolecules, performance of imaging systems
including sensitivity, available excitation wavelengths, colours for staining including multicolour
staining. For the selection of nanoparticle-labelled antibodies, including commercially available labelled
antibodies, the performance of nanoparticles and the titre of the antibodies should be evaluated (see
5.2).
5.2 Characteristics of nanoparticle
When nanoparticle labelling is performed in the laboratory, the nanoparticle shall be selected based on
the characteristics of nanoparticle. The characteristics of nanoparticles to be evaluated shall include
but not limited to the following.
a) Brightness.
Brightness is important for the quantification of biomolecules by immunohistochemistry. Initial
brightness can be used as a characteristic for the selection of nanoparticles. It is a relative
brightness that is proportional to molar extinction coefficient and quantum yield. For the selection
of nanoparticles, these parameters, i.e. molar extinction coefficient and quantum yield, should be
evaluated.
Molar extinction coefficient is defined as the optical density of 1 M fluorescent substance per 1 cm
of optical path of absorption cell, measured by absorption photometry. Quantum yield is defined as
the number of quanta emitted per quantum absorbed and can be measured with a fluorospectro-
photometer.
b) Photobleaching time.
When fluorescent dyes or nanoparticles are continuously irradiated by excitation light, their
emission output decreases and is eventually bleached. It can be characterized by the half-time of
bleaching when the number of emitted photons per hour is halved.
c) Particle size and size distribution.
Average particle size should be analysed with coefficient of variation (CV). Dynamic light scattering
(DLS) and scanning electron microscopy (SEM) can be used for this analysis. For SEM analysis
[16]
some standards are helpful.
d) Aggregation and agglomeration.
Agglomeration is the process or degree to form agglomerates that are a collection of weakly or
medium strongly bound particles. In agglomeration, the resulting external surface area is similar
to the sum of the surface areas of the individual particles. Aggregation is the process or degree to
form aggregates that are particles comprising strongly bonded or fused particles. In aggregation,
the resulting external surface area is significantly smaller than the sum of the surface areas of the
individual particles (see Clause 3).
An example of an evaluation method of aggregation is shown in Annex B.
[6]
NOTE That includes QD aggregate and agglomerate in biological media as well as the other types of
[7][8][9][10] [11]
nanoparticles. The excess aggregated QDs can be separated and removed.
e) Non-specific absorption of nanoparticles.
Nonspecific absorption of fluorescent nanoparticles can be a part of the background noise,
diminish the quality of fluoromicroscopic images, and hinder the relative-quantification analysis.
Nonspecific absorption of nanoparticles should be evaluated (see 7.1).
f) Uniformity of nanoparticle(s).
When nanoparticles are used as fluorescent substances, variation in particle size can reduce the
level of correlation between quantity of biomolecules and fluorescence intensity. Particle size shall
be evaluated along with CV and reported.
6 Quantification system
6.1 Overall design
When designing a quantification system for selected fluorescent nanoparticles, the intended use shall
be defined and documented. The quantification system described in this document uses technology
of immunohistochemistry that is an application staining thin sections of tissues with an antibody
specifically recognizing a target biomolecule. The thin section can be prepared from formalin fixed
paraffin embedded and frozen tissues. They are subsequently stained with antibodies linking to
nanoparticles. In some cases, another molecular system including biotin-avidin conjugation can be used
to detect the antibody binding to the target molecules (see 6.2.1). For the quantification, stained slides
are observed with fluorescence microscope and images of tissues are captured with a digital camera,
followed by quantification of the fluorescence signals from the tissue image with the image analysis
software. The design of the quantification system should cover all the steps of the process, from the
prepared tissue sections and the experimentally measured values, to the image processing and final
quantification of the data with the processing software.
The design description shall contain but not be limited to the following specifications: the fluorescent
nanoparticle to be used and its performance, antibodies including nanoparticle labelled and non-
labelled antibodies when used, staining conditions, microscope system with imaging equipment,
spectral characteristics of the light source used, and image analysis software. The design shall be
reviewed to ensure conformity to the requirements of the system.
For selecting image processing software, requirements of the software shall be described. Minimum
exposure time for imaging is critical for the quality of image processing by the software as well as other
instrument configurations stated in 6.4. Staining procedure, imaging and image processing software
are not independent of each other. When quantification analysis method is newly developed or changed,
the design description shall be reviewed.
6.2 Antibody
6.2.1 General
Various antibodies are used in the immunohistochemistry. For example, a single labelled antibody
for direct immunofluorescence methods and a set of two antibodies comprising primary antibody
recognizing a target biomolecule and labelled second antibody recognizing the first antibody molecule.
The labelled antibody is conjugated with a nanoparticle directly or molecules attached to the other
molecules, for example biotin attached to avidin. By any method, the antibody recognizing the target
biomolecule and nanoparticle are linked after staining to mark the target biomolecules.
NOTE Various labelling designs can be employed for immunohistochemistry. Typical labelling schemes are
as follows.
a) Primary antibody - Biotinylated secondary antibody -avidinated nanoparticles.
b) Biotinylated antibody -avidinated nanoparticles.
c) Nanoparticles with primary antibody bound to the surface.
Antibodies shall be selected based on various characteristics including antibody type, target antigen,
applicability to method, animal species, immunoglobulin type, titre and affinity. The specific issues
described below should be considered.
6.2.2 Antibody type
Antibody type, i.e. monoclonal vs polyclonal should be considered for the choice of the quantification
method.
Monoclonal antibodies generally bind to a single recognition site (single epitope), leading to 1:1 ratio
with respect to biomarker molecules, and allowing direct quantification (e.g. by fluorescence intensity).
On the contrary, polyclonal antibodies can bind to multiple epitopes within the same biomolecule,
providing some advantages in terms of tagging efficiency but precluding any intensity-based
quantification. Depending on spatial resolution, polyclonal antibodies can still be used for quantification
of biomolecules by using the method of counting the number of fluorescent points in the image (see
6.4.2).
6.2.3 Target antigen
The target antigen is selected according to the role of the antibody used in the staining process.
For antibody to be used for recognizing biomolecules, e.g. antibodies for the direct immunofluorescence
method and primary antibodies for the indirect immunofluorescence method, the antigen shall be
confirmed as the target biomolecule.
A monoclonal antibody recognizes the epitope that is a specific part of the antigen. The epitope can
[13][14]
be determined by various epitope mapping technologies, when appropriate. Information on the
epitope can be helpful for quantification of biomolecules.
On the other hand, the antigen of the second antibody are the primary antibodies recognizing the target
biomolecules. The antigenicity of the primary antibodies is determined mainly by the animal species
(see 6.2.5) and immunoglobulin type (see 6.2.6).
6.2.4 Applicability of antibody to met
...