ISO 10993-4:2017

INTERNATIONAL ISO
STANDARD 10993-4
Third edition
2017-04
Biological evaluation of medical
devices —
Part 4:
Selection of tests for interactions
with blood
Évaluation biologique des dispositifs médicaux —
Partie 4: Choix des essais pour les interactions avec le sang
Reference number
ISO 10993-4:2017(E)
©
ISO 2017

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ISO 10993-4:2017(E)

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ISO 10993-4:2017(E)

Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 4
5 Types of devices in contact with blood (as categorized in ISO 10993-1) .5
5.1 Non-blood-contact devices . 5
5.2 External communicating devices . 5
5.2.1 General. 5
5.2.2 External communicating devices that serve as an indirect blood path . 5
5.2.3 External communicating devices directly contacting circulating blood . 5
5.3 Implant devices . 6
6 Characterization of blood interactions . 6
6.1 General requirements . 6
6.2 Categories of tests and blood interactions .12
6.2.1 Recommended tests for interactions of devices with blood .12
6.2.2 Non-contact devices .13
6.2.3 External communicating devices and implant devices.13
6.2.4 Limitations .13
6.3 Types of tests .13
6.3.1 In vitro tests .13
6.3.2 Ex vivo tests .14
6.3.3 In vivo tests .14
Annex A (informative) Preclinical evaluation of cardiovascular devices and prostheses .16
Annex B (informative) Recommended laboratory tests — Principles, scientific basis
and interpretation .21
Annex C (informative) Thrombosis — Methods for in vivo testing .32
Annex D (informative) Haematology/haemolysis — Methods for testing — Evaluation of
haemolytic properties of medical devices and medical device materials .39
Annex E (informative) Complement — Methods for testing .46
Annex F (informative) Less common laboratory tests .49
Annex G (informative) Tests which are not recommended .53
Bibliography .55
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ISO 10993-4:2017(E)

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 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
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 194, Biological and clinical evaluation of
medical devices.
This third edition cancels and replaces the second edition (ISO 10993-4:2002), which has been
technically revised.
It also incorporates the Amendment ISO 10993-4:2002/Amd 1:2006.
The following changes were made:
a) some definitions have been revised and new definitions have been added;
b) Tables 1 and 2 have been consolidated into a single new Table 1 with test categories and headers
reorganized to emphasize and include material and mechanical-induced haemolysis testing and in
vitro and in vivo testing for assessment of risk for thrombosis;
c) Tables 3 and 4 have been consolidated into a single new Table 2 with a simplified list of suggested
and most common tests;
d) Annex B has been updated to cover only the most common practiced tests for assessing blood
interactions;
e) Annex C has been added to cover the topic of in vivo thrombosis and methods for testing;
f) Annex D, which was Annex C in the previous edition, has been updated and now includes added
information on mechanically-induced haemolysis;
g) Annex E has been added to cover the topic of complement testing and best test method practices;
h) Annexes F and G have been added to present the less common tests used to assess interactions with
blood and the tests that are not recommended for preclinical assessment of medical device blood
interaction, respectively. Many of these methods were previously included in Annex B;
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ISO 10993-4:2017(E)

i) subtle language refinements can be found throughout the revised document;
j) the Bibliography has been reorganized by common subjects of interest and updated with additional
and more current references.
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ISO 10993-4:2017(E)

Introduction
The selection and design of test methods for the interactions of medical devices with blood should take
into consideration device design, materials, clinical utility, usage environment and risk benefit. This
level of specificity can only be covered in vertical standards.
The initial source for developing this document was the publication, Guidelines for blood/material
[14]
interactions, Report of the National Heart, Lung, and Blood Institute chapters 9 and 10. This
[15]
publication was subsequently revised .
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INTERNATIONAL STANDARD ISO 10993-4:2017(E)
Biological evaluation of medical devices —
Part 4:
Selection of tests for interactions with blood
1 Scope
This document specifies general requirements for evaluating the interactions of medical devices
with blood.
It describes
a) a classification of medical devices that are intended for use in contact with blood, based on the
intended use and duration of contact as defined in ISO 10993-1,
b) the fundamental principles governing the evaluation of the interaction of devices with blood,
c) the rationale for structured selection of tests according to specific categories, together with the
principles and scientific basis of these tests.
Detailed requirements for testing cannot be specified because of limitations in the knowledge and
precision of tests for evaluating interactions of devices with blood. This document describes biological
evaluation in general terms and may not necessarily provide sufficient guidance for test methods for a
specific device.
The changes in this document do not indicate that testing conducted according to prior versions of
this document is invalid. For marketed devices with a history of safe clinical use, additional testing
according to this revision is not recommended.
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 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-12 and
the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
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3.1
anticoagulant
agent which prevents or delays blood coagulation
EXAMPLE Heparin, ethylenediaminetetraacetic acid (EDTA), sodium citrate.
3.2
blood/device interaction
interaction between blood or a blood component and a device
3.3
coagulation
phenomenon that results from activation of the clotting (coagulation) factor cascade
Note 1 to entry: Factors of the coagulation cascade and fibrinolytic systems can be measured following exposure
to devices either in vitro or in vivo.
3.4
complement system
part of the innate immune system consisting of over 30 distinct plasma proteins, including enzymes,
cofactors, and cellular receptors which may be involved in the promotion of thrombosis
Note 1 to entry: Effector molecules produced from complement components are possible components in the
phenomena of inflammation, phagocytosis and cell lysis. Complement activation related to immunotoxicity,
hypersensitivity and generation of anaphylatoxins is not covered in this document. (See ISO/TR 10993-20.)
Note 2 to entry: The focus in this document is complement activation as it can promote and accelerate haemolysis,
platelet and leukocyte activation and thrombosis on device material surfaces. (See also Annex E on complement
activation.)
3.5
direct blood contact
term used when the device or device material comes into physical contact with blood or blood
constituents
3.6
embolization
process whereby a blood thrombus, or foreign object, is carried in the bloodstream and which may
become lodged and cause obstructed blood flow downstream
3.7
ex vivo test system
term applied to a test system that shunts blood directly from a human subject or test animal into a test
chamber located outside the body
Note 1 to entry: If using an animal model, the blood may be shunted directly back into the animal (recirculating)
or collected in test tubes for evaluation (single pass). In either case, the test chamber is located outside the body.
3.8
haematology
study of blood that includes quantification of cellular and plasma components of the blood
3.9
haematocrit
ratio of the volume of erythrocytes to that of whole blood in a given sample
3.10
haemolysis
liberation of haemoglobin from erythrocytes, either by destruction or through a partially damaged but
intact cell membrane
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3.11
haemocompatible
able to come into contact with blood without any appreciable clinically-
significant adverse reactions such as thrombosis, haemolysis (3.10), platelet, leukocyte, and complement
activation, and/or other blood-associated adverse event occurring
3.12
indirect blood contact
nature of devices that contact the patient’s blood path at one point and serve as a conduit for entry into
the vascular system
EXAMPLE Drug and parenteral nutrition solution delivery devices.
3.13
legally-marketed comparator device
LMCD
approved, or cleared long-established, and recognized-to-be-safe medical device used as a reference
control in an in vitro or in vivo safety evaluation of a test device of similar design, material(s), and
clinical use
Note 1 to entry: It may be necessary that the LMCD be legally marketed in the same region as the regulatory
submission for the test device.
3.14
non-blood-contact
nature of the device or material contact with the patient’s body where the device or potentially
extracted material does not have direct or indirect contact with blood
3.15
colloidal osmotic pressure
total influence of the proteins or other large molecular mass substances on the osmotic activity of plasma
3.16
platelets
anuclear, cellular bodies that are present in blood and contribute to the process of thrombosis by
adhering to surfaces, releasing factors, and/or aggregating to form a haemostatic plug
3.17
platelet adherent
having the tendency to allow or promote platelets (3.16) to attach to its surface
Note 1 to entry: This is often characterized relative to a negative control, positive control, and/or LMCD upon
blood contact due to its surface properties.
Note 2 to entry: Platelet adherent does not necessarily mean platelet activating, i.e. platelets on a surface may or
may not be activated.
3.18
thrombin generating
due to its surface properties, having the tendency to promote or show increased
thrombin formation
Note 1 to entry: This is often characterized relative to a negative control, positive control, and/or LMCD upon
blood contact.
3.19
thrombogenic
due to its surface properties, having the tendency to form or promote thrombus
formation
Note 1 to entry: This is often characterized relative to a negative control, positive control, and/or LMCD upon
blood contact.
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3.20
thromboembolization
process where a dislodged thrombus (3.21) is carried downstream, where it may cause subsequent
vascular blockage or occlusion
3.21
thrombus
coagulated mixture of red blood cells, aggregated platelets (3.16), fibrin and other cellular elements
3.22
thrombosis
formation of a thrombus (3.21) under in vivo, ex vivo, or in vitro simulated conditions, caused by
activation of the coagulation system and platelets (3.16) in flowing whole blood
Note 1 to entry: Thrombosis can also occur in regions of a blood vessel or device where there is stasis.
3.23
whole blood
unfractionated blood drawn from a human donor or test animal
Note 1 to entry: The blood may be non-anticoagulated or anticoagulated, e.g. contain sodium citrate or heparin
as an anticoagulant.
4 Abbreviated terms
Bb enzymatically active fragment of Factor B produced by cleavage (by Factor D) in the
activation of the alternative pathway
β-TG beta-thromboglobulin
C4d degradation product of C4 by classical pathway complement activation
C3a, C5a complement split products from C3 and C5
CH-50 amount of complement required to lyse 50 % of a RBC suspension
D-Dimer specific fibrin degradation products (F XIII cross-linked fibrin) consisting of
D-fragment dimer
ELISA enzyme-linked immunosorbent assay
FDP fibrin/fibrinogen degradation products
FPA fibrinopeptide A
F1.2 the non-catalytic fragment split off from prothrombin in its conversion to thrombin (also
referred to as F1+2)
iC3b inactive form of C3b, a sub-fragment of C3
IFU instruction for use
IVC inferior vena cava
MRI magnetic resonance imaging
PET positron emission tomography
PF-4 platelet factor 4
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PRP platelet-rich plasma
PT prothrombin time
PTT partial thromboplastin time
SC5b-9 product of terminal pathway complement activation
SEM scanning electron microscopy
TAT thrombin-antithrombin complexes
TCC terminal complement complex; also called membrane attack complex (MAC); estimated
by measuring SC5b-9
TT thrombin time
TxB2 thromboxane B2
5 Types of devices in contact with blood (as categorized in ISO 10993-1)
5.1 Non-blood-contact devices
Non-blood-contact devices are devices that do not have direct or indirect contact with either blood or
blood constituents that reside in the body or that are returned to the body. An in vitro diagnostic device
and a blood-collection tube are examples of non-blood-contact devices. Some devices, such as introducer
systems for implants, may contain both blood-contacting and non-blood-contacting components.
5.2 External communicating devices
5.2.1 General
These are devices that contact the circulating blood and serve as a conduit into the vascular system.
Some devices may have components or portions with different types of contact (direct and indirect).
Examples include but are not limited to the following.
5.2.2 External communicating devices that serve as an indirect blood path
— blood collection devices;
— cannulae;
— cell savers;
— devices for the storage and administration of blood and blood products (e.g. tubing and bags);
— extension sets;
— intravascular catheters.
5.2.3 External communicating devices directly contacting circulating blood
— atherectomy devices;
— blood monitoring devices with direct or indirect blood contact;
— cardiopulmonary bypass circuitry;
— devices for adsorption of specific substances from blood;
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ISO 10993-4:2017(E)

— donor and therapeutic apheresis equipment;
— extracorporeal membrane oxygenators;
— haemodialysis/haemofiltration devices;
— interventional cardiology and vascular devices;
— intravascular catheters (balloon, imaging, laser, ultrasound);
— leukocyte removal filters;
— percutaneous circulatory support devices;
— retrograde coronary perfusion catheters;
— vascular guide wires.
5.3 Implant devices
Implant devices are placed largely or entirely within the vascular system. Examples include but are not
limited to the following:
— annuloplasty rings;
— arteriovenous shunts;
— blood monitors (implantable);
— circulatory support devices (ventricular-assist devices, artificial hearts, intra-aortic balloon pumps);
— embolization devices;
— endovascular synthetic vascular grafts;
— implantable defibrillator and cardioverter leads;
— inferior vena cava filters;
— internal drug delivery catheters;
— intravascular oxygenators (artificial lungs);
— mechanical or tissue heart valves;
— pacemaker leads;
— surgical synthetic or tissue vascular grafts;
— vascular stents.
6 Characterization of blood interactions
6.1 General requirements
IMPORTANT — Since this is a horizontal International Standard, sound rationales can be
supplied to justify the choice of test category(ies) based on the device being characterized. For
example, in vivo testing for evidence of thrombosis is frequently the preferred method for device
characterization in the thrombosis category. However, in some cases, written rationales that
include a combination of tests from the categories of coagulation, platelets, haematology and
complement can be used as a substitute for thrombosis testing.
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ISO 10993-4:2017(E)

6.1.1 Figure 1 illustrates a decision tree that can be used to determine whether testing for interaction
with blood is necessary. Blood interactions can be divided into several categories based on the primary
process or system being measured. Table 1 lists examples of devices which contact circulating blood and
the categories of testing appropriate to each device. The list is not all inclusive and sound judgement
shall be applied to devices not listed in the tables.
For medical devices where a specific International Standard (vertical standard) exists, the biological
evaluation requirements and test methods set forth in that vertical standard shall take precedence
over the general requirements suggested in this document.
6.1.2 Where possible, tests shall use an appropriate model or system which simulates the geometry
and conditions of contact of the device with blood during clinical applications. The simulation should
include an appropriate duration of contact, temperature, sterile condition, anticoagulant (and level; see
6.1.12) and flow conditions. For example, for devices of defined geometry such as a vascular stent, the
2
surface area used in the test, in cm , shall be given consideration relative to the fluid volume of the in vitro
test system. For devices with undefined or complicated geometry (such as a dispersion of PVA particles
used as an embolization agent), mass should be used instead of surface area to determine the amount of
sample used in test system.
Only direct or indirect blood-contacting parts should be tested. The selected test methods and
parameters should be in accordance with the current state of the art.
Appropriate type and level of anticoagulant may be case specific depending on both the device
use indication and the type of test conducted. Include information on the specific type and level of
anticoagulation used and provide a discussion on the ability to discern positive and negative responses.
For further information, see 6.1.6 and C.2 for animal studies, 6.1.12 for in vivo and ex vivo tests, 6.3.1 for
in vitro tests and A.3 for catheters and guide wires.
As many tests for haemocompatibility are recognized to be strictly surface-contact dependent, such
tests (e.g. complement activation) will not apply to indirect contact applications.
6.1.3 Controls (positive and negative) shall be used unless their omission can be justified. Where
possible, testing should include a relevant predicate device already in clinical use (i.e. a LMCD) or a well-
[6]
characterized material .
Controls should include negative and positive reference materials. All materials and LMCDs tested shall
meet all quality control and quality assurance specifications of the manufacturer and test laboratory.
All materials and devices tested shall be identified as to source, manufacturer, grade and type.
6.1.4 Testing of materials which are candidates for components of a device may be conducted for
screening purposes. However, such preliminary tests do not serve as a substitute for the requirement that
the complete sterilized device or device component should be tested under conditions which simulate or
exaggerate clinical application.
NOTE 1 Changes in manufacturing process (including use of manufacturing aids) that could affect the surface
properties, or chemistry of the complete sterilized device, could also impact haemocompatibility.
NOTE 2 Where aging could impact the final device properties, use of aged samples can also be necessary. (For
example, the properties of biologically active coatings such as heparin could change over time.)
6.1.5 Tests which do not simulate the conditions of a device during use may not predict accurately
the nature of the blood/device interactions which can occur during clinical applications. In addition, the
capacity of short-term in vitro or ex vivo tests to predict performance in actual clinical applications is
thought to be higher when the clinical application involves limited exposure rather than prolonged or
permanent exposure.
NOTE Simplified testing of candidate device materials (e.g. surface geometric and functional chemical
modifications) can serve as a crucial step in device material identification, optimization and selection.
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6.1.6 If an animal study is to be conducted, devices whose intended use is ex vivo (external
communication) should be tested ex vivo and devices whose intended use is in vivo (implants) should be
tested in vivo in an animal model simulating as closely as possible conditions of clinical use. Protocols in
such investigations should specifically call out each test category (see 6.2.1) being evaluated and describe
the specific method(s) of assessment.
a
For direct and indirect contact devices, the necessity for haemocompatibility testing should be con-
sidered based upon appropriate risk analysis, including prior haemocompatibility testing, clinical data,
extractable/leachable data, and/or information on surface characteristics. For example, for devices
with direct contact, extractable/leachable testing may not be sufficient if the surface morphology is
changed, even if the extractable/leachable chemistry is the same (see ISO 10993-1).
Figure 1 — Decision tree to help determine whether testing for interaction with blood is
necessary
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Table 1 — Circulating blood-contacting devices or device components and the categories of appropriate testing for consideration —
External communicati
...