oSIST prEN ISO 13366-2:2026

SLOVENSKI STANDARD
01-september-2026
Mleko - Ugotavljanje števila somatskih celic - 2. del: Metoda pretočne citometrije
(ISO/DIS 13366-2:2026)
Milk - Enumeration of somatic cells - Part 2: Flow cytometry method (ISO/DIS 13366-
2:2026)
Milch - Zählung somatischer Zellen - Teil 2: Leitfaden zum Betrieb
fluoreszenzoptoelektronischer Zählgeräte (ISO/DIS 13366-2:2026)
Lait - Dénombrement des cellules somatiques - Partie 2: Méthode de cytométrie en flux
(ISO/DIS 13366-2:2026)
Ta slovenski standard je istoveten z: prEN ISO 13366-2
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 13366-2
IDF 148-2
ISO/TC 34/SC 5
Milk — Enumeration of somatic
Secretariat: NEN
cells —
Voting begins on:
Part 2:
Flow cytometry method
Voting terminates on:
ICS: 67.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
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NOTIFICATION OF ANY RELEVANT PATENT
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PROVIDE SUPPORTING DOCUMENTATION.
Reference numbers
ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
© ISO and IDF 2026
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
ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Factors affecting the results of measurements. 2
5.1 Sample bottles . .2
5.2 Sampling .2
5.2.1 General .2
5.2.2 Bulk milk samples .2
5.2.3 Milk samples from individual animals .2
5.3 Preservation .3
5.4 Sample storage and transport .3
5.5 Interfering substances . .3
5.6 Sample quality at analysis .4
5.7 Chemicals .4
5.8 Instrument condition . .4
6 Calibration . 4
6.1 Reference materials .4
6.1.1 General .4
6.1.2 Preparation of calibration samples .5
6.1.3 Assignment of reference values .5
6.1.4 Storage conditions and keeping time .5
6.2 Calibration procedure .6
6.2.1 Calibration . . .6
6.2.2 Check on linearity .6
7 Sampling . 7
8 Determination . 7
9 Checks on performance in routine operation . 7
9.1 Blank checks .7
9.2 Carry-over effect .7
9.3 Pilot checks .8
9.3.1 General .8
9.3.2 Pilot milk .8
9.3.3 Assignment of pilot milk values .8
9.3.4 Use of pilot milk samples .8
9.4 Additional instrument surveillance .9
9.5 Repeatability .9
9.6 Intralaboratory reproducibility .9
9.7 External comparisons .9
10 Specific remarks for the use with milk from different species . 9
10.1 General .9
10.2 Cow milk .10
10.3 Goat milk .10
10.4 Sheep milk .10
10.5 Buffalo milk .10
11 Precision . 10
11.1 Repeatability .10
11.2 Intralaboratory reproducibility .11
11.3 Reproducibility .11

iii
ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
12 Test report .12
Annex A (informative) Direct labelling (DNA stain) for the total cell counting in milk using flow
cytometry in milk .13
Bibliography .18

iv
ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
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 through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of 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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products – Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement). It is being published jointly by ISO and IDF.
This third edition cancels and replaces the second edition (ISO 13366-2 | IDF 148-2:2006), which has been
technically revised.
The main changes are as follows:
— Outdated method has been deleted as rotating disk technique;
— Indications to build a robust metrological system as the use of a certified reference material (CRM) and
the procedure to assign the value to the secondary reference material;
— The control of ratio/reagent has been eliminated;
— Addition of method for total somatic cell counts (dSCC) in milk, using a non-dedicated flow cytometer
and commercially available non-proprietary chemicals as Annex.
A list of all parts in the ISO 13366 | IDF 148 series can be found on the ISO website.
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.
IDF (the International Dairy Federation) is a non-profit private sector organization representing the
interests of various stakeholders in dairying at the global level. IDF members are organized in National
Committees, which are national associations composed of representatives of dairy-related national interest
groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/
food control authorities.
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ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and
sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards
using the logos and reference numbers of both organizations.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, IDF had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. IDF shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
This document was prepared by the IDF Standing Committee on Laboratory Statistics and Quality Assurance
and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is
being published jointly by ISO and IDF.
The work was carried out by the IDF/ISO Action Team S18 of the Standing Committee on Laboratory Statistics
and Quality Assurance under the aegis of its project leaders Mrs S. Orlandini (IT) and Mr P. Trossat (FR).

vi
ISO/DIS 13366-2:2026(en)
DRAFT International Standard
IDF 148-2:2026(en)
Milk — Enumeration of somatic cells —
Part 2:
Flow cytometry method
1 Scope
This document gives guidance on the operating conditions for counting the total number of somatic cells, in
both raw and chemically preserved milk, using flow cytometers.
The guidance is applicable to the counting of total somatic cells in raw cow milk. The guidance is also
applicable to raw milk of other species, such as goat, sheep and buffalo, if the specified prerequisites are met.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 8196-1, | IDF 128-1, Milk — Definition and evaluation of the overall accuracy of alternative methods of milk
analysis — Part 1: Analytical attributes of alternative methods
ISO 8196-2, | IDF 128-2, Milk — Definition and evaluation of the overall accuracy of alternative methods of milk
analysis — Part 2: Calibration and quality control in the dairy laboratory
ISO 13366-1, | IDF 148-1, Milk — Enumeration of somatic cells — Part 1: Microscopic method (Reference method)
ISO 17034, General requirements for the competence of reference material producers
ISO/IEC 17043:2023, Conformity assessment — General requirements for the competence of proficiency testing
providers
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
reference method
method described in ISO 13366-1 | IDF 148-1 for the counting of somatic cells
3.2
somatic cells
those cells that show more than a threshold intensity of fluorescence due to the staining of DNA in their
nuclei
Note 1 to entry: The number of somatic cells is expressed in cells per millilitre.

ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
4 Principle
Flow cytometers designed for automatically counting the total number of somatic cells in milk have functions
for the uptake of reagents and test sample, a mixing section and a counting section. In the mixing section,
the test sample is mixed with a buffer and a stain solution. Part of the resulting mixture is transferred to the
counting section.
For other flow cytometers, the steps described above can follow the procedure described in (see Annex A).
In the mixing section, closely controlled volumes of test sample and buffer/stain solutions are dosed and
mixed. Mixing can take place in a cup, a mixing chamber, or in the tubing leading to a flow cell.
In the counting section, part of the mixture is placed in the high-speed flow of a surrounding sheath liquid
in a capillary flow cell. Through acceleration, the mixture forms a thin string in which the somatic cells are
dynamically focused and aligned. This string then passes the objective of a fluorescence microscope.
Each stained particle produces an electrical pulse that is filtered, amplified and recorded. The resulting pulse
height distribution is electronically processed, whereby discrimination is made between noise signals and
pulses that are attributed to stained somatic cells. The discriminator level can either be fixed or dynamic.
Some instruments contain two channels in the counting section. In terms of analytical quality assurance,
such a situation should be considered equivalent to working with two separate units, so the performance
should be evaluated separately for each channel.
5 Factors affecting the results of measurements
5.1 Sample bottles
Sample bottles should be fit for use; i.e. to transfer test samples from the point of sampling to the laboratory
without loss or damage.
Care is to be exercised that sample bottles are leak-proof and that a proper empty volume is left. Too large an
empty volume can facilitate churning; too small an empty volume can cause problems with mixing.
5.2 Sampling
5.2.1 General
Sampling materials (i.e. sample bottles, beakers and sampling devices) should be clean and dry. Where
automatic samplers are used, these should have been properly validated.
Test samples should preferably be cooled immediately after sampling at 4 °C ± 2 °C and be kept at that
temperature until counting (see 5.4) rather than being preserved. Freezing should be avoided. If preservation
is necessary, suitable means for chemical preservation of test samples are described in 5.3.
5.2.2 Bulk milk samples
Thorough mixing of the raw bulk milk to be sampled is essential. Somatic cells will concentrate in the upper
layer in the case of insufficient stirring.
5.2.3 Milk samples from individual animals
The release of somatic cells in the milk during milking is uneven. When the aim is to produce a representative
counting result for a whole milking, it is essential that a representative sample of the whole milking is
obtained. For diagnostic purposes, a sample of a partial milking may suffice.

ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
5.3 Preservation
If chemical preservation is considered necessary, the test sample (5.2.1) should be preserved as soon
as possible, but in any case within 24 h after sampling. In all cases, the test sample should be kept cool
(4 °C ± 2 °C) until the addition of the preservative.
WARNING — Sodium azide and potassium dichromate are highly hazardous reagents. Handle with
extreme care. Prepare and use the reagents only in a properly equipped laboratory, in a fume hood,
and always wear appropriate personal protective equipment (PPE), including gloves, lab coat an eye
protection.
Suitable preservatives are the following.
a) Boric acid: its final concentration in the test sample should not exceed 0,6 g/100 ml. Such preserved
samples may be stored at between 6 °C and 12 °C for up to a further 24 h.
b) Sodium azide: its final concentration in the test sample should not exceed 0,024 g/100 ml. Such preserved
samples may be stored at between 2 °C and 10 °C for up to a further 72 h.
c) Bronopol (2-bromo-2-nitro-1,3-propanediol): its final concentration in the test sample should not exceed
0,05 g/100 ml. Such preserved samples may be stored at between 2 °C and 12 °C for up to a further 6 d.
d) Potassium dichromate: its final concentration in the test sample should not exceed 0,2 g/100 ml. Such
preserved samples may be stored at between 2 °C and 12 °C for up to a further 6 d.
Accompanying colour tracers found to be suitable are:
— Patent Blue V with a final concentration in the test sample of up to 0,15 mg/100 ml;
— Yellow Orange S (E110) with a final concentration in the test sample of up to 1 mg/100 ml;
— a mixture of Patent Blue V and Eosin B with a final concentration in the test sample of up to 0,03 mg/100 ml
and 0,45 mg/100 ml, respectively;
— Methylene blue with a final concentration in the test sample of up to 0,5 mg/100 ml.
Other preservatives and colour tracers may be used provided that their effectiveness and conditions for use
have been soundly validated.
In all cases, properly validate the absence of interference for the counting equipment concerned before
application.
In cases where flow cytometers are combined with milk analysers for the measurement of other test
sample components, care should be taken that the applied preservatives and colour tracers do not affect the
counting result.
5.4 Sample storage and transport
Unpreserved test samples should be stored at 4 °C ± 2 °C and should be counted within 96 h after the
completion of sampling. Avoid freezing the test samples. Storage at higher temperatures and/or over longer
time scales may result in considerable loss of countable cells and therefore non-representative values. The
measurement of samples after freezing and thawing may result in lower counts (by 10 % to 20 %). The age
of the samples at freezing and the type of thawing process can influence the counting result.
5.5 Interfering substances
The use of substances that interfere in the counting should be avoided. Substances known to influence the
instrument read-out are preservatives and colour tracers at higher concentrations than specified 5.3.

ISO/DIS 13366-2:2026(en)
IDF 148-2:2026(en)
5.6 Sample quality at analysis
Breakdown of somatic cells (lysis) will result in an increase of smaller cell fragments. The lower intensity of
fluorescence after staining of these particles causes a shift in the pulse height distribution to the left. This
will hamper proper differentiation from noise pulses and therefore result in a lower count.
NOTE In several types of instruments, features are available for evaluating the position and the shape of the pulse
height distribution. See the relevant instructions and guidance from the instrument manufacturer.
After the processing of problematic samples, the flow path should be checked and possible cleaned. The
proper functioning of the instrument should be tested before further use. Possible problematic test samples
are
a) milk samples from severely infected udders, i.e. with clots;
b) milk samples with impurities;
c) milk samples with high numbers of erythrocytes;
d) colostrum;
e) late lactation milk;
f) sour milk.
Where possible, analysis of problematic test samples should be avoided.
5.7 Chemicals
All reagents used should be of recognized analytical and bacteriological quality. Water used should be
demineralized (remaining conductivity < 10 µS/cm) or water of at least equivalent purity. Manufacturer's
instructions for the preparation of the working solutions, the maximum storage time and storage
requirements should be followed.
Local conditions regarding the use and discharge of applied chemicals and effluents should be observed.
5.8 Instrument condition
5.8.1 General points of attention are:
a) the functioning of the mixing device and the stirrer,
b) possible disturbances at sample intake and in the flow system due to blocking by impurities, clots or
...