BS en Iso 10993-4-2017

BS EN ISO 10993‑4:2017
BSI Standards Publication
Biological evaluation of medical devices
Part 4: Selection of tests for interactions with blood

BS EN ISO 10993‑4:2017 BRITISH STANDARD
National foreword
This British Standard is the UK implementation of EN ISO 10993‑4:2017.
It supersedes BS EN ISO 10993‑4:2009, which is withdrawn.
The UK participation in its preparation was entrusted to Technical
Committee CH/194, Biological evaluation of medical devices.
A list of organizations represented on this committee can be obtained on
request to its secretary.
This publication does not purport to include all the necessary provisions
of a contract. Users are responsible for its correct application.
© The British Standards Institution 2017
Published by BSI Standards Limited 2017
ISBN 978 0 580 84457 7
ICS 11.100.20
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 31 May 2017.
Amendments/corrigenda issued since publication

Date Text affected
EUROPEAN STANDARD EN ISO 10993‑4
NORME EUROPÉENNE
EUROPÄISCHE NORM May 2017
ICS 11.100.20 Supersedes EN ISO 10993‑4:2009
English Version
Biological evaluation of medical devices — Part 4:

Selection of tests for interactions with blood (ISO
10993-4:2017)
Évaluation biologique des dispositifs Biologische Beurteilung von Medizinprodukten —
médicaux — Partie 4: Choix des essais pour les Teil 4: Auswahl von Prüfungen zur
interactions avec le sang (ISO 10993-4:2017) Wechselwirkung mit Blut (ISO 10993-4:2017)
This European Standard was approved by CEN on 23 February 2017.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving
this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical
references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre
or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language
made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark,
Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain,
Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993‑4:2017: E
worldwide for CEN national Members
BS EN ISO 10993‑4:2017
EN ISO 10993‑4:2017 (E)
European foreword
This document (EN ISO 10993‑4:2017) has been prepared by Technical Committee ISO/TC 194
“Biological and clinical evaluation of medical devices” in collaboration with Technical Committee
CEN/TC 206 “Biological and clinical evaluation of medical devices” the secretariat of which is
held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by November 2017, and conflicting national standards
shall be withdrawn at the latest by November 2017.
Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such
patent rights.
This document supersedes EN ISO 10993‑4:2009.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA and Annex ZB, which is an integral part
of this document.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
the United Kingdom.
Endorsement notice
The text of ISO 10993‑4:2017 has been approved by CEN as EN ISO 10993‑4:2017 without any
modification.
ii
BS EN ISO 10993‑4:2017
EN ISO 10993‑4:2017 (E)
Annex ZA
(informative)
Relationship between this European Standard and the essential

requirements of Directive 93/42/EEC [OJ L 169] aimed
to be covered
This European Standard has been prepared under a Commission’s joint standardization request M/BC/
CEN/89/9 concerning harmonized standards relating to horizontal aspects in the field of medical
devices to provide one voluntary means of conforming to essential requirements of Council Directive
93/42/EEC of 14 June 1993 concerning medical devices [OJ L 169].
Once this standard is cited in the Official Journal of the European Union under that Directive, compliance
with the normative clauses of this standard given in Table ZA.1 confers, within the limits of the scope
of this standard, a presumption of conformity with the corresponding essential requirements of that
Directive and associated EFTA regulations.
NOTE 1 Where a reference from a clause of this standard to the risk management process is made, the risk
management process needs to be in compliance with Directive 93/42/EEC as amended by 2007/47/EC. This
means that risks have to be reduced ‘as far as possible’, ‘to a minimum’, ‘to the lowest possible level’, ‘minimized’
or ‘removed’, according to the wording of the corresponding essential requirement.
NOTE 2 The manufacturer’s policy for determining acceptable risk must be in compliance with Essential
Requirements 1, 2, 5, 6, 7, 8, 9, 11 and 12 of the Directive.
NOTE 3 This Annex ZA is based on normative references according to the table of references in the European
foreword, replacing the references in the core text.
NOTE 4 When an Essential Requirement does not appear in Table ZA.1, it means that it is not addressed by this
European Standard.
Table ZA.1 — Correspondence between this European Standard and Annex I of Directive 93/42/
EEC [OJ L 169]
Essential Requirements of Clause(s)/subclause(s) of Remarks/Notes
Directive 93/42/EEC this EN
ER 7.1 (first indent) is partly covered by
ISO 10993‑4, since the standard does
not provide requirements on design and
6.1 and A.1, B.1, C.1, D.1 and manufacture. However, this standard
7.1 (First indent)
E.1 provides a means to evaluate the inter‑
actions of medical devices with blood.
Other forms of toxicity and flammability
are not dealt with in this standard.
ER 7.1 (second indent) is partly covered
by ISO 10993‑4, since the standard does
not provide requirements on design and
manufacture. However, this standard
6.1 and A.1, B.1, C.1, D.1 and
7.1 (Second indent) provides a means to evaluate the inter‑
E.1
actions of medical devices with blood.
Other forms of toxicity are not dealt with
in this standard. This evaluation can be a
preliminary step for risk minimization.
3
BS EN ISO 10993‑4:2017
EN ISO 10993‑4:2017 (E)
Essential Requirements of Clause(s)/subclause(s) of Remarks/Notes

Directive 93/42/EEC this EN
ER 7.2 is partly covered by ISO 10993‑4,
since the standard does not provide
requirements on design, manufacture
6.1 and A.1, B.1, C.1, D.1 and and packaging. However, this standard
7.2
E.1 provides a means to assess the interac‑
tions of medical devices with blood to
contaminants and residues in medical
devices.
ER 7.5 is partly covered by ISO 10993‑4,
since the standard does not provide re‑
6.1 and A.1, B.1, C.1, D.1 and quirements on design and manufacture.
7.5
E.1 However, this standard provides a means
to evaluate interactions of substances
leaking from medical devices with blood.
General Note: Presumption of conformity depends on also complying with all relevant clauses/subclauses
of ISO 10993‑1.
WARNING 1 — Presumption of conformity stays valid only as long as a reference to this European
Standard is maintained in the list published in the Official Journal of the European Union. Users of this
standard should consult frequently the latest list published in the Official Journal of the European Union.
WARNING 2 — Other Union legislation may be applicable to the products falling within the scope of
this standard.
4
BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Contents Page
Foreword......................................................................................................................................................................................................................................... vi
Introduction............................................................................................................................................................................................................................. viii
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
© ISO 2017 – All rights reserved v

BS EN ISO 10993‑4:2017
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: www.iso.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;
vi © ISO 2017 – All rights reserved

BS EN ISO 10993‑4:2017
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.
© ISO 2017 – All rights reserved vii

BS EN ISO 10993‑4:2017
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
interactions, Report of the National Heart, Lung, and Blood Institute[14] chapters 9 and 10. This
publication was subsequently revised[15].
viii © ISO 2017 – All rights reserved

BS EN ISO 10993‑4:2017
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 10993‑12and 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
© ISO 2017 – All rights reserved 1

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

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
2 © ISO 2017 – All rights reserved

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

3.11
haemocompatible
<device or device material> 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
<material or device> 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
<material or device> due to its surface properties, having the tendency to promote or show increased
thrombin formation
blood contact.
3.19
thrombogenic
<material or device> due to its surface properties, having the tendency to form or promote
thrombus formation
blood contact.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

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

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

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;

BS EN ISO 10993‑4:2017
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.

BS EN ISO 10993‑4:2017
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
surface area used in the test, in cm2, 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-
characterized material[6].
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.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

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

Table 1 — Circulating blood-contacting devices or device components and the categories of appropriate testing for consideration —
External communicating devices and implant devices
Test category
Haemolysis Thrombosis
Device examples in vitro
In vivo/
Material- Mechanically-
Coagulation
Platelet
Complement d Haematology Ex vivoa
External communicating devices

induced induced activation
© ISO 2017 – All rights reserved

Blood monitors (temporary/ex vivo)b X X X X
Blood storage and administration equipment (e.g. infusion/transfusion sets), blood collec‑
X X X X
tion devices, extension sets
Catheters in place for less than 24 h (e.g. atherectomy devices,
intravascular ultrasound catheters, antegrade/retrograde coronary X Xc Xc Xc Xc
perfusion catheters, guide wires); cannulae
Catheters in place for more than 24 h (e.g. parenteral nutrition catheters, central venous
X Xc Xc Xc Xc
catheters); cannulae
Cell saversb X X X
Devices for adsorption of specific substances from bloodb X X X X X

Donor and therapeutic aphaeresis equipment and cell separation systemsb X X X X X
Cardiopulmonary bypass systemb X X Xc Xc X Xc Xc
Haemodialysis/haemofiltration equipmentb X X Xc Xc X Xc Xc
Leukocyte removal filterb X Xc Xc X Xc Xc
Implant devices
Percutaneous circulatory support devicesb X X Xc Xc X Xc Xc
Annuloplasty rings, mechanical heart valves X X X

Embolization devices X X
Endovascular grafts X X
Implantable defibrillator and cardioverter leads X X
Intra-aortic balloon pumpsb X X X
Pacemaker leads X X
Prosthetic (synthetic) vascular grafts and patches, including
X X
arteriovenous shunts
a Thrombosis is an in vivo or ex vivo phenomenon, but can be simulated with in vitro conditions. In vivo or ex vivo testing might not be necessary if clinically relevant in vitro thrombosis testing is performed.
b Direct or indirect blood-contacting components only. For components that have only indirect blood contact, in vivo thrombogenesis and mechanical haemolysis or complement activation might not be necessary.
c It is recognized that coagulation, platelet and leucocyte responses are primarily involved in the process of thrombosis. Therefore, it is up to the manufacturer to decide if specific testing in the coagulation, platelet and
haematology test categories is appropriate as an alternate to in vivo testing.
d
ISO 10993-4:2017(E)
9

BS EN ISO 10993‑4:2017
See also ISO/TS 10993‑20 for information on when complement activation should be considered for other end points such as anaphylaxis.

Table 1 (continued)
10
Test category
Haemolysis Thrombosis
Device examples in vitro
In vivo/
Material- Mechanically-
Coagulation
Platelet
Complement d Haematology Ex vivoa
induced induced activation
Stents (vascular) X X
ISO 10993-4:2017(E)
Tissue heart valves, vascular grafts and patches and AV shunts X X

BS EN ISO 10993‑4:2017
Total artificial hearts X X X

Vena cava filters X X
Ventricular-assist devicesb X X X
a Thrombosis is an in vivo or ex vivo phenomenon, but can be simulated with in vitro conditions. In vivo or ex vivo testing might not be necessary if clinically relevant in vitro thrombosis testing is performed.
b Direct or indirect blood-contacting components only. For components that have only indirect blood contact, in vivo thrombogenesis and mechanical haemolysis or complement activation might not be necessary.
c It is recognized that coagulation, platelet and leucocyte responses are primarily involved in the process of thrombosis. Therefore, it is up to the manufacturer to decide if specific testing in the coagulation, platelet and
haematology test categories is appropriate as an alternate to in vivo testing.
d See also ISO/TS 10993‑20 for information on when complement activation should be considered for other end points such as anaphylaxis.

© ISO 2017 – All rights reserved
BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

6.1.7 In vitro tests are regarded as useful in screening external communicating devices or implants and
potential early interactions between devices/materials with blood, but may not be accurate predictors
of blood/device interactions occurring upon prolonged or repeated exposure or permanent contact
(see 6.3.1).
NOTE For new devices or devices where there is a change in geometry, testing under physiologic flow can be
needed. For long-term catheters or permanent implants, in vitro test systems might not be sufficient due to blood
stability issues.
6.1.8 Devices or device components which come into very brief/transient contact with circulating
blood (e.g. lancets, hypodermic needles, capillary tubes that are used for less than 1 min) generally do
not require blood/device interaction testing.
NOTE 1 For products made with materials such as coatings that could be left in contact with blood after the
device is removed, blood/device interaction testing might be necessary.
NOTE 2 If some device components (e.g. syringe bodies) are in contact with fluids that will ultimately be
injected into the patient, and the storage time is unspecified or greater than 1 min, haemolysis testing of the
fluid-contacting component would be needed, even though the device itself would be in contact with circulating
blood for less than 1 min.
6.1.9 Disposable laboratory equipment used for the collection of blood and performance of in vitro
tests on blood shall be evaluated to ascertain that there is no significant interference with the test
being performed.
6.1.10 If tests are selected in the manner described and testing is conducted under conditions which
simulate clinical applications, the results of such testing have the greatest probability of predicting
clinical performance of devices. For devices that operate over a range of conditions, the extreme and the
average conditions should be considered. However, species differences and other factors may limit the
predictability of any test.
6.1.11 Because of species differences in blood reactivity, human blood should be used where possible
(with the exception of established test methods with animal blood, such as some haemolysis tests).
When animal models are necessary, for example for evaluation of devices used for prolonged or repeated
exposure or permanent contact, species differences in blood reactivity shall be considered.
Blood values and reactivity in humans and non-human primates are very similar[204]. The use of animals,
such as the rabbit, pig, calf, sheep or dog, can also be acceptable for a particular type of test. However,
since species differences may be significant (for example, platelet adhesion[148][150], thrombosis[44]
and haemolysis[47] tend to occur more readily in the canine than in the human), all results of animal
studies shall be interpreted with caution. The species selected and the number of animals used shall be
justified (see also ISO 10993‑2).
NOTE The use of non-human primates for in vivo blood compatibility and medical device testing is prohibited
by EU law (86/609/EEC) and some national laws.
6.1.12 The use of anticoagulants in in vivo and ex vivo tests should be avoided unless the device is
designed to perform in their presence. The type and concentration of anticoagulant used influence
blood/device interactions and their selection shall be justified. Devices that are used with anticoagulants
should be assessed using anticoagulants in the range of concentrations used clinically and/or described
in the product IFU or other appropriate literature. Species differences should also be considered when
determining the appropriate level of anticoagulation.
6.1.13 Modifications in a clinically accepted device shall be considered for their effect on blood/device
interactions and clinical functions. Examples of such modifications include changes in design, geometry,
changes in surface or bulk chemical composition of materials and changes in texture, porosity or
other properties. An in vitro flow model with application-consistent exposure conditions and relevant
measurements can be used to evaluate the effect of modifications to a clinically accepted device.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

6.1.14 A sufficient number of replications of a test including suitable controls should be performed to
permit statistical evaluation of the data. The variability in some test methods requires that those tests
be repeated a sufficient number of times to determine significance. In addition, repeated studies over an
extended period of blood/device contact provide information about the time-dependence of the blood-
device interactions[213]–[216]. Balance should be considered between statistical evaluation and animal
welfare when applying in vivo testing; see ISO 10993‑2.
6.1.15 The recommendations within 6.1, together with Figure 1 and Table 1, serve as a guide for the
selection of tests listed in Table 2. Further guidance on pre-clinical evaluations is given in Annexes A to G.
In summary, the following procedure shall be performed:
a) determine which potential blood interaction categories (see 6.2) are appropriate for consideration
to establish safety of the particular device (see examples in Table 1);
b) evaluate the existing information in each test category for the device;
c) where sufficient safety information exists, prepare an appropriate rationale to support this
conclusion and that further testing is not necessary;
NOTE Any difference in formulation, geometry, surface properties, fabrication methods, sterilization
technique and/or clinical use could limit the use of safety information on a similar product.
d) where insufficient information exists under a test category(ies), select appropriate tests, based upon
examples in Tables 1 and 2, to supply the additional safety information.
6.2 Categories of tests and blood interactions
6.2.1 Recommended tests for interactions of devices with blood
Recommended tests are organized on the basis of the type of device (see examples in Table 1). The tests
are divided into the following categories based on the primary process or system being measured:
— haemolysis
— material-induced
— mechanically-induced
— thrombosis
— in vitro
— coagulation
— platelet activation
— complement
— haematology
— in vivo/ex vivo
The principles and scientific bases for these tests are given in Annexes A to E.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Table 2 — Common tests used to assess interaction with blood

Tests by categories
Haemolysis Material-induced (e.g. ASTM[17], NIH[28],
MHLW[22])
Mechanical-induced
Thrombosis Gross analysisa, percentage occlusion, light
microscopy, SEM
(in vivo, ex vivo)
In vitro thrombosis
Coagulation Thrombin (e.g. TAT, F1.2), fibrin (e.g. FPA) assays,
PTT assay
Platelet activation Platelet count (% loss) and some indicator of
activation (e.g. release products or platelets
surface markers such as βTG, PF4, TxB2) or SEM
(platelet morphology)
Haematology Complete blood count (CBC), leucocyte activation
Complement system SC5b-9 (C3a optional)
a Included in all animal studies (see B.2.1 and ISO 10993‑6).
Not all tests are needed for each category and testing in each category might not be
equivalent.
6.2.2 Non-contact devices
These devices do not require blood/device interaction testing.
6.2.3 External communicating devices and implant devices
After using Table 1 to align a new device under investigation with similar existing devices and noting
the test categories for consideration, use Table 2, Annexes A and E to guide the selection of appropriate
tests for assessing blood interactions.
6.2.4 Limitations
Testing and study design parameters may present certain practical limitations/considerations based
upon science, technology and the particular application. For example:
a) materials/devices in a high blood flow (arterial) environment may interact with blood differently
in a low blood flow (venous) environment;
b) blood interactions may occur with all materials, i.e. the test materials/test devices and the non-test
materials (e.g. test system). Caution shall be taken to not confound blood interactions associated
with the test materials to those contributed by other factors;
c) studies that rely on just one type of test for blood interactions may be less predictive of the true
response than studies that include several different tests for blood interactions;
d) immunoassays for detecting protein indicators of haemocompatibility, e.g. TAT, C3a, etc., are often
available for human blood testing but are not generally available for use or functional with blood
from other species.
6.3 Types of tests
6.3.1 In vitro tests
In vitro testing (models) should consider designs to simulate the anticipated worst-case clinical use
conditions of each device application. Variables that shall be considered when using in vitro test methods

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

include haematocrit, anticoagulant (type and amount), test sample preparation, test sample age,
blood/blood component age, test sample storage, aeration and pH, temperature, proper randomization,
test sample surface area to blood volume ratio and for dynamic studies, fluid flow conditions, especially
flow rate, wall shear rate and pressure(s). Tests shall be started with minimal delay, usually within
4 h of blood draw, since some properties of blood change rapidly following collection. Alternatives to
the latter may be feasible if validated. In some cases, the resulting samples can also be frozen using
appropriate techniques for future analysis if the freeze/thaw process does not affect the analyte
being assessed.
NOTE Clinically relevant types and amounts of anticoagulant may or may not be appropriate, depending on
the test system and the ability to discern positive and negative responses.
When used to evaluate the haemocompatibility of device modifications, in vitro testing for haemolysis,
thrombus formation, platelet and coagulation responses may be assessed and compared between the
modified device and the clinically accepted device (see A.1.4).
6.3.2 Ex vivo tests
Ex vivo tests shall be performed when the intended use of the device is ex vivo, e.g. an external
communicating device. Ex vivo testing can also be useful when the intended use is in vivo, e.g. to assess
the acute response to an implant such as a vascular graft. Such use should not however substitute for an
implant test.
Ex vivo test systems are available for monitoring platelet adhesion, emboli generation, fibrinogen
deposition, thrombus mass, white-cell adhesion, platelet consumption and platelet activation[44][46]
[47][50][54][70][78][80]. Blood flow rates can be measured with either Doppler or electromagnetic flow
probes. Alterations in flow rates may indicate the extent and course of thrombus deposition and
embolization. Simple thrombus build-up can be assessed by gross and or microscopic visualization.
Other more advanced and technically demanding tools have also been used[53][69][73][74][79].
6.3.3 In vivo tests
In vivo testing involves implanting the material or device in animals. Vascular patches, vascular
catheters, vascular grafts, vascular stents, annuloplasty rings, heart valves and circulatory assist
devices are examples of devices tested in vivo. Given the diversity of blood-contacting medical device
applications, in vivo test models are expected to be equally diverse, in order to appropriately mimic
each clinical application.
“Patency of a conduit or device (i.e. the unimpeded flow of blood through the device)” is a common
measure of success or failure for some in vivo experiments. The percent occlusion and thrombus mass
are determined after the device is removed. The tendency of thrombi formed on a device to embolize
to distal organs should be assessed by careful gross as well as microscopic examination of organs
downstream from the device. In addition, histopathological evaluation of the surrounding tissue and
organs is useful. The kidneys are especially prone to trap thrombi which have embolized from devices
implanted upstream from the renal arteries (e.g. ventricular-assist devices, artificial hearts, aortic
prosthetic grafts)[184][187][236][237].
Methods to evaluate in vivo interactions without terminating the experiment are available. Arteriograms
or imaging from intravascular ultrasound (IVUS) catheters are used to determine patency or thrombus
deposition on devices. Radioimaging can be used to monitor platelet deposition at various time periods
in vivo; platelet survival and consumption can be used as indicators of blood/device interactions and
passivation due to neointima formation or protein adsorption[46][72][79].
In some in vivo test systems, the material’s properties may not be major determinants of the
blood/device interactions. Rather, flow parameters, compliance, porosity and implant design may be
more important than blood compatibility with the material itself. As an example, low flow rate systems
may give substantially different results when compared with the same material evaluated in a high
flow rate system. In such cases, test system performance in vivo should carry more importance than in
vitro test results.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

In vivo test protocols should contain precise and stand-alone sections stating how each test category
identified for testing, i.e. haemolysis, thrombosis, coagulation, platelets, haematology and complement
system, will be evaluated.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex A
(informative)
Preclinical evaluation of cardiovascular devices and prostheses
A.1 General considerations

A.1.1 Background
This annex provides background for selecting tests to evaluate the interactions of cardiovascular
devices with blood. Clause 6 contains guides to determine when testing is necessary, which blood
interaction categories might be appropriate for specific devices, and a list of tests for evaluating
blood/device interactions of non-contact-, external communicating- and implant devices. The
classification of blood/device interactions in A.1.2 is provided as background.
A.1.2 Classification
A.1.2.1 Interactions which mainly affect the device and which may or may not have an undesirable
effect on the animal or human are as follows:
a) adsorption of plasma proteins, lipids, calcium or other substances from the blood onto the surface
of the device; or absorption of such substances into the device;
b) adhesion of platelets, leukocytes or erythrocytes onto the surface of the device, or absorption of
their components into the device;
c) formation of pseudointima or neointima on the blood contacting surface and tissue capsule on the
surface of the device;
d) alterations in mechanical and other properties of the device.
A.1.2.2 Interactions which have a potentially undesirable effect on the animal or human are as follows:
a) activation of platelets, leukocytes or other cells, or activation of the coagulation, fibrinolytic, or

complement pathways;
b) formation of thrombus on the device surface;
c) embolization of thrombotic or other material from the device’s surface to another site within the
circulation;
d) injury to circulating blood cells resulting in anaemia, haemolysis, leucopoenia, thrombocytopenia
or altered function of blood cells;
e) injury to cells and tissues adjacent to the device;
f) intimal hyperplasia or accumulation of other tissue on or adjacent to the device, resulting in
reduced flow or affecting other functions of the device;
g) adhesion and growth of bacteria or other infectious agents on or near the device.
NOTE For items b), c) and d) above, some devices such as embolization coils require thrombus formation to
be functional.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

A.1.3 Advantages and limitations of animal models

Animal models permit the closest end use simulation of clinical devices prior to actual testing in
humans. They permit continuous device monitoring and a systematic controlled investigation of
important variables. However, the choice of an animal model may be restricted by size requirements,
the availability of certain species and cost. For example, a device may not be operated under the full
range of clinical use conditions in an animal model due to anatomical limitations. It is critical that the
investigators be mindful of the physiological differences and similarities of the species chosen with
those of the human, particularly those relating to coagulation, platelet functions and fibrinolysis,
and the response to pharmacological agents such as anaesthetics, anticoagulants, thrombolytic and
antiplatelet agents and antibiotics. Because of species differences in reactivity, subject differences in
reactivity and variable responses to different devices, data obtained from a single species should be
interpreted with caution. Non-human primates such as baboons exhibit a close similarity to the human
in haematological values, blood coagulation mechanism and cardiovascular system[50]. An additional
advantage of a non-human primate is that many of the immunological probes for thrombosis assays
developed for humans are suitable for use in primates. These probes include PF-4, β-TG, FPA, TAT
and F1.2. The dog is a commonly used species and has provided useful information; however, device-
related thrombosis in the dog tends to occur more readily than in the human, a difference which can
be viewed as an advantage (as a challenging or accelerated model) when evaluating this complication.
Pigs and sheep are generally regarded as suitable animal models because of their haematological and
cardiovascular similarities to the human[71][148][149][150]. The effect of the surgical implant procedure
on results should be kept in mind and appropriate controls included. The final decision on the use of an
animal or in vitro model ultimately involves consideration of the availability and ethical use of animals
(see ISO 10993‑2), the availability and limits of in vitro blood models and the applicability of proper
statistics for sound conclusions[213][214][215][216].
A.1.4 Advantages and limitations of in vitro models

In vitro blood-exposure models are attractive approaches to testing the haemocompatibility of medical
materials and cardiovascular devices because they allow
a) avoidance of costly animal models,
b) high replication testing of test objects alongside controls and reference materials using the same
batch of blood and at the same time,
c) use of human or animal blood where flow, temperature and anticoagulation is standardized,
d) worst case scenario testing, where activation products accumulate without clearance by kidneys
or liver or other organs and activation-inhibiting functions of endothelial cells are absent, and
e) isolation from confounding factors associated with device implantation/tissue injury associated
with in vivo usage.
Such testing of medical materials and devices should simulate as best as possible the range of clinical
conditions of blood exposure to the device, since testing on blood under clinically inapplicable conditions,
e.g. non-clinical anticoagulation (types or levels) and flow conditions, can make interpretation of results
difficult. Whenever possible, consult product IFU brochures or common medical practice literature
for applicable anticoagulant type(s) and amount(s). When appropriate, testing over the full range of
labelled use conditions for the device should be considered. For example, to evaluate mechanically-
induced haemolysis and platelet activation, testing is often performed at the highest blood flow rate.
For thrombosis testing, the minimum labelled blood flow rate may be important to characterize the
safety of the device. Since it has been shown that responses in blood may differ considerably between
various species[47][148][149][150], the use of human blood is more relevant to the interpretation of
results. Another advantage in using human blood is that it offers a more detailed array of test methods,
since most contemporary bioanalytical methods are based on human blood components/epitopes.
Conversely, there are certain limitations in the volume of blood that can be obtained from a single
human donor. Thus, the use of blood from a single large animal may be more practical in cases where
the model designed to simulate clinical-relevant conditions presents a large volume capacity.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

To test for general material/device haemocompatibility, the classical Chandler loop in vitro test model[43]
or modifications thereof [193][194][195][199][200][203] to impart physiological and/or quasi-physiological
flow have been used. Alternatively, blood-material (device) exposure using gentle agitation may also be
useful in some cases for evaluating the interactions of blood with materials. To gauge the impact of the
model on blood, haemolysis and general cell blood count can be monitored to check for blood normalcy.
These models appear effective for screening studies, in particular for those applications involving
short-term blood exposure.
A.1.5 Test protocols for animal testing

Thrombosis, thromboembolism, bleeding and infection are the major deterrents to the use and further
development of advanced cardiovascular prostheses. For devices with limited blood exposure (<24 h),
important measurements are related to the extent of acute variation of haematological, haemodynamic
and performance variables, gross thrombus formation and possible embolism. With prolonged or
repeated exposure or permanent contact (>24 h and >30 d, respectively), emphasis is placed on serial
measurement techniques that may yield information regarding the time course of thrombosis and
thromboembolism, the consumption of circulating blood components and the development of intimal
hyperplasia and infection. In both of these exposure and contact categories, assessment of haemolysis
and platelet function is important. Thrombus formation may be greatly influenced by surgical
technique, variable time-dependent thrombolytic and embolic phenomena, superimposed device
infections and possible alterations in exposed surfaces, e.g. intimal hyperplasia, fibrotic encapsulation
and endothelialization. Importantly, anticoagulation type(s) and amount(s) can have a profound
impact on results. For example, at clinically relevant levels, anticoagulation and antiplatelet drugs may
substantially reduce or abolish platelet, coagulation and thrombotic responses.
The consequences of the interaction of artificial surfaces with the blood can range from gross
thrombosis and embolization to subtle effects such as accelerated consumption of elements involved
in normal haemostasis. The latter may be clinically insignificant, e.g. platelet consumption by the
device could be so small that it does not affect the total platelet count. Alternatively, a device with a
large surface area could lead to depletion of platelets or plasma coagulation factors such that the total
platelet count may be significantly affected and normal haemostasis may become altered.
Regardless of the animal model used and the particular test category under evaluation, i.e. haemolysis,
thrombosis, coagulation, platelets, haematology and complement system, the in vivo study protocol
should provide sufficient detail in the methods and criteria to be used for evaluation for each test
category under investigation. A retrospective report on results for a particular test category, without
supporting original plans within the protocol, is often considered unacceptable as regulatory
submission documentation.
A.2 Cannulae used for direct vascular access and cannulae used for
indirect access
The term “cannulae” has been generally used in two rather different clinical applications. In one
application, cannulae are inserted directly through the skin and into one or more major blood vessels.
This is done to provide continuous and direct high-volume access to blood. For example, this type of
large-diameter cannulae is used during cardiopulmonary bypass surgery as a limited-exposure access
device that shunts blood to and from the body for blood oxygenation. Cannula testing, in this example,
should take place using exposure conditions that closely replicate clinical use, as such devices can
potentially induce some alteration in the levels of circulating blood cells as well as increase factors in
the coagulation or complement system. The particular response is often multifactorial as it depends on
a variety of factors such as implantation site, insertion technique, subject factors and anticoagulation
regimen. The term cannulae has also been used to describe much smaller diameter tubes that are
inserted only subcutaneously, and may be used for limited (<24 h) or prolonged (<30 d) indirect
exposure to blood. These cannulae, for example, are used for infusion of insulin from drug pumps and
in subcutaneous sensing for blood glucose levels. These later type of cannulae, like other indirect blood
path devices (see 5.2.2), generally require less testing than devices with direct contact with circulating
blood (see 5.2.3 and 5.3).

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

A.3 Catheters and guide wires

Most of the tests considered under blood-contacting cannulae are relevant to the study of blood-
contacting catheters and guide wires. The location or placement of catheters in the arterial or venous
system can have a major effect on blood/device interactions. It is advised that simultaneous control
studies, using a clinically approved device of similar dimensions and material(s), be performed using a
contralateral artery or vein. Care should be taken not to strip off thrombus upon catheter withdrawal.
Evaluation of the device in situ may permit assessment of the extent to which intimal or entrance
site injuries contribute to the thrombotic process. In general, Doppler blood flow measurements
are more informative than angiography. A venous or arterial implant model with anticoagulation
pertinent to the clinical application may be a useful tool for evaluating device blood-contact responses,
particularly when assessing a new device material or a coating developed to present anti-thrombogenic
properties[143][161][162][163]. See C.3. Alternatively, an appropriate in vitro model may be more sensitive
to detecting such material surface differences.
In cases where anticoagulation is called for, the rationale for the type and level of anticoagulation
used in testing should be based upon the clinical application, yet be able to provide sufficient evidence
that the test is able to distinguish between positive and negative responses. For example, following
simple dose-response kinetics, the thromboresistance of a medical device heparin coating can be
completely masked by normal (clinical) levels of solution heparin anticoagulant. However, under
a reduced/challenging level of solution heparin, the effectiveness of the heparin coating to reduce
thrombus formation becomes more apparent. In cases where the application may not involve use of
anticoagulation, testing should be conducted without anticoagulation.
Validation information for testing with a specific type and level of anticoagulation should demonstrate
the ability to discern between positive and negative responses.
A.4 Extracorporeal blood oxygenators, haemodialysis/haemofiltration devices,

donor and therapeutic apheresis equipment, devices for adsorption of specific
substances from blood
The blood responses to cardiopulmonary bypass can be significant and acute. Many variables such
as use of blood suction, composition of blood-pump priming fluid, hypothermia, blood contact
with air and time of exposure influence test values. Emboli in outflow lines may be detected by the
periodic placement of blood filters ex vivo or the use of ultrasound or other non-invasive techniques.
Thrombus accumulation can be directly assessed during bypass by monitoring performance factors
such as pressure drop across the oxygenator and oxygen transfer rate. An acquired transient platelet
dysfunction associated with selective alpha granule release has been observed in patients on
cardiopulmonary bypass[158]; other tests of platelet function and release are particularly useful.
Complement activation is caused by both haemodialysers and cardiopulmonary bypass equipment.
Clinically significant pulmonary leucostasis and lung injury with dysfunction can result[5][11][16][129]–
[147]. For these reasons, it is useful to quantify complement activation or leukopenia with these devices.
See also Annex E.
Therapeutic apheresis equipment and devices for adsorption of specific substances from the blood,
because of their high surface-to-volume ratio, can potentially activate complement, coagulation, platelet
and leukocyte pathways. Examination of blood/device interactions in these and any other high surface
area devices should follow the same principles as for extracorporeal oxygenators and haemodialysers.
A.5 Ventricular-assist devices and total artificial hearts

These devices can induce considerable alteration in various blood components. Factors contributing to
such effects include the large foreign surface area to which blood is exposed, the high flow regimes and
the regions of disturbed flow such as turbulence or separated flow. Tests of such devices may include
measurements of haemolysis, thrombus formation, fibrin formation, thromboembolization, thrombin
generation, platelet survival and activation, complement activation and close monitoring of liver,

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

renal, pulmonary and central nervous system effects. A detailed pathological examination at surgical
retrieval is an important component of the evaluation[236][237].
A.6 Heart valve prostheses

Invasive, non-invasive and in vitro hydrodynamic studies are important in the assessment of
prosthetic valves.
One of the most effective means of screening for prosthetic valve dysfunction is auscultation[186]. Two-
dimensional and M mode echocardiography makes use of ultrasonic radiation to form images of the
heart. Reflections from materials with different acoustic impedances are received and processed to
form an image. The structure of prosthetic valves can be examined. Mechanical prostheses emit strong
echo signals and the movement of the occluder can usually be clearly imaged. However, the quality
of the image may depend upon the particular valve being examined. Echocardiography can also be
useful in the assessment of function of tissue-derived valve prostheses. Vegetations, thrombus and
evidence of thickening of the valve leaflets are elucidated. Using conventional and colour flow Doppler
echocardiography, regurgitation can be identified and semi-quantified[2][185][186][187].
Measurements of platelet survival and aggregation, blood tests of thrombosis and haemolysis, pressure
and flow measurements, and autopsy of the valve and adjacent tissues are recommended[205][206].
A.7 Vascular grafts

Both porous and non-porous materials can be implanted at various locations in the arterial or venous
system. The choice of implantation site is determined largely by the anatomical considerations of
the model and the clinical site of use. Patency of a given graft is enhanced by larger diameter and
shorter length. Patency can be documented by palpation of distal pulses in some locations and by
periodic angiography. Ultrasound, MRI and PET may also be useful. Serial measurements of platelet
count, platelet release constituents, fibrinogen/fibrin degradation products and activated coagulation
proteins also are recommended. Autopsy of the graft and adjacent vascular segments for vascular
tissue responses can provide valuable information. A systematic evaluation of longitudinal and cross-
sectional sections of proximal and distal anastomoses and representative midgraft regions is necessary
for a thorough evaluation of the device[4][205]. As with many vascular devices, appropriate clinical
anticoagulation regimens are critical to device function and performance.
A.8 IVC filters, stents and stented grafts

These devices can be studied by angiography and ultrasonic radiation. Other techniques useful for
vascular graft evaluation (see A.7) are appropriate here as well[205].

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex B
(informative)
Recommended laboratory tests — Principles, scientific basis and

interpretation
B.1 General considerations

B.1.1 Background
The general principles and scientific bases of the more commonly used tests to evaluate the categories
of haemolysis, thrombosis, coagulation, platelets, haematology and complement system (see 6.2)
are described in B.1 to B.3. See Annexes C, D and E for further information on the test categories of
thrombosis, haemolysis and complement.
Additional, albeit less common methods, that may be of further value in the evaluation of particular
blood/device interactions are described in Annex F. Because of biological variability and technical
limitations, the accuracy and predictivity of many of these tests require careful attention to
methodology and caution in interpretation of results. Annex G lists tests which are not recommended.
B.4 presents methodology considerations for testing of plasma factors specific to coagulation, platelet
and leucocyte activation and complement activation using ELISA (or other similar) techniques.
All references in the bibliography describe in more detail and give examples of various standards, tests
and models for consideration.
B.1.2 In vitro versus ex vivo versus in vivo testing
B.1.2.1 A host of in vitro, ex vivo and in vivo models have been used extensively to estimate blood-
material interactions[1]–[30][42]–[147][157]–[237]. It is appropriate to select the model most suitable for the
device application and test objective and to consult ISO 10993‑12 regarding proper sample preparation
and control group considerations.
No single in vitro, ex vivo or in vivo model will be appropriate for all applications. Thus, the
appropriateness of the model to the application under consideration should be justified.
In vivo tests present a more realistic end-use simulation, yet are complicated by factors such as:
— choice of appropriate animal model;
— interspecies and intersubject variability in responses[47][71][148][149][150];
— scarcity of species-specific commercial test kits for common indicators of thrombosis and
coagulation[58][59][60];
— heightened costs and ethical and statistical concerns involved in using animal models.
B.1.2.2 Consult with vertical standards for preferred models[1]–[41]. See also Reference [186] for
the juvenile sheep as an accelerated model to study bio-prosthetic valve calcification, Reference [187]
for the adult pig or sheep to investigate transvascular-placed valves and surgically implanted valves,
References [217] to [231] for the adult canine and sheep femoral replacement models used in testing on
small and large diameter vascular graft model, and References [232] to [235] for the porcine coronary
model used extensively to investigate stent designs.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

B.1.2.3 As described in other parts of ISO 10993, carefully conducted in vitro tests offer valid screening
tools to assess the biological safety of medical devices and materials.
Important factors in an in vitro model that require specification are

— volume of whole blood in the basic test system, e.g. tube, loop, or other model,
— blood exposure time(s),
— blood temperature,
— blood flow condition,
— anticoagulant type and level,
— exposure ratio, i.e. ratio of material/device surface area (cm2) to volume of whole blood in the
system (ml), and
— blood-contacting surface area of the test system itself (cm2).
NOTE 1 The “blood exposure phase” of an in vitro investigation requires a precise definition of the exposure
conditions of the test material/device to blood. The closer the test conditions mimic the clinical application, the
greater the predictivity becomes of the model and the response(s) being evaluated.
NOTE 2 A “testing phase” follows the exposure phase where specific tests are conducted on the exposed
blood, blood plasma or the material/device itself. A test in the testing phase is usually targeted at one or more of
the general categories, i.e. haemolysis, thrombosis, coagulation, platelets, haematology and complement system.
B.2 and B.3 examine the common methods used to assess the main categories of blood-materials/device
interaction (see Table 2).
B.2 Thrombosis
B.2.1 Gross analysis — Retrieval and examination of device and autopsy of distal organs
Gross analysis should always be included as part of a basic device evaluation, as this segment of a device
evaluation is of central importance in evaluating the in vivo biological responses to implanted devices.
The distribution, visible size and nature of cellular and proteinaceous deposits, and any emboli, can
best be determined by a careful and detailed gross examination. Proposed procedures have been
published[7][205][206][207].
The rationale behind necropsy of distal organs is to examine for distal effects (such as emboli) of
implanted devices. The importance of this analysis varies with device application and is restricted
to applications where risk for thromboembolism or material/device embolization, for example with
mechanical heart valves and intra-aortic balloon pumps, is intermediate to high[206].
In this type of investigation, low- and/or high-magnification high-resolution colour film or digital
images at key points of interest (of the device, and the surrounding tissues, etc.) are taken and labelled
appropriately.
B.2.2 Percentage occlusion, surface area covered by thrombus and thrombus-free

surface area
Percentage occlusion may be quantitatively assessed during the in-life portion of the study using
contrast radiography and ultrasonography imaging techniques. Percentage occlusion can also be
visually assessed after an implanted device has been removed. The percentage of occlusion may be a
measure of the severity of the thrombotic process in a conduit. However, lack of occlusion does not
necessarily eliminate the existence of a thrombotic process, since thrombi may have embolized or been
dislodged before percentage occlusion is measured. Occlusion may be caused not only by thrombosis, but
also by intimal hyperplasia, especially at peri-anastomotic sites in vascular grafts. Thus, a supporting
microscopic examination is useful to identify the nature of the occlusive process. Determinations of

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

surface area covered by thrombus and thrombus-free surface area are semi-quantitative or quantitative
tests that can be used on a comparative basis with test and/or control devices.
B.2.3 Light microscopy

By this technique, information can be obtained regarding the density of cells, presence of cellular
aggregates, composition of encapsulating tissue, intensity of foreign body response and thrombus
or fibrin adherent to materials. The evaluation of geographic distribution of these deposits on the
materials or device is also possible. The method is semi-quantitative.
For polymeric or biologically-derived materials and devices, paraffin wax embedding methods and
special stains may be used to assess the device-biological interface.
For metal and ceramic materials and devices, more sophisticated hard plastic embedding and sectioning
techniques are useful to capture the intact material/device-biological interface[207]–[211].
B.2.4 Scanning electron microscopy (SEM)

For SEM, rationale and interpretation are the same as for light microscopy (see B.2.3). This method has
the advantage over light microscopy of providing greater detail about fine structure of components
being examined. Quantitative conclusions require sufficient replicate determinations to establish
degree of reproducibility. This type of microscopy best reflects what can be seen at surfaces. Cross-
sectional analyses can also be used to support the surface observations if additional details regarding
the cell and thrombus surface interactions are informative[70][71][143][205][206]. The morphological
evaluation of platelet and leukocyte activation, fibrin and thrombus formation after blood or blood
component (e.g. platelet-rich plasma) dynamic exposure with comparison to reference controls is
valuable[143][173].
B.3 In vitro haemocompatibility

B.3.1 Haemolysis — Methods for testing
Haemolysis is regarded as a significant screening test because an elevated in vivo plasma haemoglobin
level is abnormal and may be indicative of an underlying haemopathology or vascular problem. Properly
performed, an elevated plasma haemoglobin level indicates haemolysis, i.e. the release of contents
of red blood cells (RBCs), and may reflect erythrocyte membrane fragility or damage to RBCs. In the
assessment of blood-material/device interactions, haemolysis may result due to:
a) direct blood contact with the material(s)/device surface(s) (material-induced);
b) indirect contact from exposure to extractable chemicals from the device materials
(material induced);
c) exposure to turbulence and elevated (i.e. non-physiological) shear stresses from the device
operation (mechanically-induced).
See Annex D for more extensive details on testing for haemolysis.
B.3.2 Coagulation — Methods for testing
B.3.2.1 General
The coagulation cascade has two parallel pathways, the contact activation pathway (the intrinsic
pathway) and the tissue factor pathway (the extrinsic pathway) that merge to form a common pathway.
The latter includes the protein thrombin, which catalyses the formation of fibrin, a main component
of a thrombus. While it is known that the primary pathway for the initiation of blood coagulation is
the tissue factor pathway, coagulation associated with blood contacting devices and materials occurs
through the contact activation pathway. The pathways themselves are a series of reactions in which
successive inactive enzyme precursors (referred to as zymogens) interact with their glycoprotein

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

co-factors to become active components in a cascade of activation events. The reactions culminate in
the formation of active thrombin that then catalyses the formation of fibrin. Coagulation factors are
generally indicated by Roman numerals, with a lowercase “a” appended to indicate the active form.
See Figure B.1.
Assessment of coagulation activity, i.e. the degree of change in blood levels of proteins leading to
thrombin and fibrin formation (see Figure B.1), has long relied on clinical assays that measure plasma
levels of key proteins in the coagulation cascade. Normal resting (homeostasis) levels of coagulation
activity are well established, as are some elevated levels observed in various clinical coagulopathies.
The presumption for such testing with medical devices is that appropriate materials and device designs
should not be associated with excessive coagulation activity that could bring risk to the patient. High
levels of coagulation activity may be an indicator of a higher tendency for the material or device to
induce or be associated with acute thrombosis or thromboembolism. To measure coagulation activity,
clinical laboratories often rely upon test kits that use common enzyme-linked immunosorbent assay
technique. In a basic study, which may be either in vivo or in vitro and will depend upon availability
of appropriate antibodies to species-specific target coagulation protein epitopes, blood samples are
retrieved under defined conditions and prepared and analysed per assay instructions. Typical defined
conditions or factors important in an in vitro model are described in B.1.2.3. Comparison of results to
appropriate controls such as negative controls (e.g. baseline levels or no material/device exposure) and
results on a predicate device(s)/materials(s) is critical. Coagulation activity in blood that is statistically-
significantly and biologically-significantly higher than controls may be an indicator of a material/device
design that presents a higher risk of coagulation-related complications. Example coagulation activation
proteins for which commercially-available ELISA kits are available include TAT (thrombin-antithrombin
complexes), F.1.2 (protein fragment released from prothrombin upon formation of thrombin) and FPA
(protein fragment released from fibrinogen upon formation of fibrin).
Proteins indicating coagulation activation generally exhibits an initiation, propagation and termination
phase[56][57]. This reflects the initial formation reaction(s), a cascade/feedback amplification period
and a slowdown/deactivation period where critical precursors may be consumed or the measured
protein deactivated by negative control feedback proteins. Thus, order-of-magnitude differences in
levels of coagulation activation proteins are to be expected over time. Consequently, an important factor
to consider is when the activation phase actually occurs during the time of the material/device-blood
contact. For example, the impact of test materials when mixed with blood may be quite different at
each phase. In addition, as coagulation protein activation is generally proportional to blood-contacting
surface area, surface area (SA) of a device or device material can be very influential on results. For
this reason it is important to specify the test SA-to-blood-volume ratio (exposure ratio) in each study.
If possible, the exposure ratio may be treated as a variable to aid in understanding the specificity of
the material effect. Exposure ratios of 3,0 cm2 to 6,0 cm2/ml blood (based on device thickness) are
consistent with ISO 10993‑12. Other exposure ratios such as 1,5 and 2,0 times this ratio may be worth
considering as higher surface areas will theoretically increase the sensitivity of the coagulation
responses to the test material.
There will be a physical limitation on the amount of test material that can be tested due to the volume
of the test system, e.g. a test tube, and the target exposure ratio. In this case, it may be appropriate to
use cut sections of device materials. If the device contains more than one material, the proportion of
each in the complete device should be maintained. Care should also be made to avoid introducing cut
sections that result in exposure of significant amounts of non-blood contact surfaces.
Naturally, there are a number of mechanisms that have evolved to keep the coagulation cascade in
check. One of those mechanisms involves the protein antithrombin. Antithrombin is a serine protease
inhibitor that can bind to and deactivate the serine proteases thrombin, FIXa, FXa, FXIa and FXIIa.
While antithrombin is constantly active, its interaction with heparin alters its conformation, which
greatly accelerates its rate of inhibition of the proteases.
NOTE ELISA testing for blood coagulation factors represents the “testing phase” discussed in B.1.2, i.e.
for testing on the blood samples procured following in vitro or in vivo blood exposure to the medical device
or material.
Many standard coagulation assays are designed to detect clinical coagulation disorders which result
in delayed clotting or excessive bleeding, rather than conditions that enhance clotting/thrombosis.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Protocols for evaluating blood/device interactions shall be modified appropriately to evaluate

accelerated coagulation induced by biomaterials.
Interactions between the coagulation and complement systems are recognized[143]–[147].
Figure B.1 — Coagulation cascade
B.3.2.2 Thrombin-antithrombin (TAT), F1.2 and fibrin (FPA) ELISA assays
These ELISA assays that directly reflect thrombin (TAT, F1.2) and fibrin (FPA) formation are
commercially available. The output is a quantitative estimate of the amount of thrombin present and
the amount of fibrin being formed, both of which are reflective of the level of coagulation activity
taking place and may be reflective of thrombosis taking place. See B.4 for details on general ELISA
methodology.
B.3.2.3 Partial thromboplastin time (PTT)
The partial thromboplastin time is the clotting time of recalcified citrated plasma upon the addition of
partial thromboplastin which does not contain an activator. Partial thromboplastin is a phospholipid
suspension usually extracted from tissue thromboplastin, the homogenate from mammalian brain
or lung. Shortening of the PTT following contact with a material under standard conditions indicates
activation of the intrinsic coagulation pathway of blood coagulation. Heparin and other anticoagulants
cause a prolonged PTT. See Reference [23].
Reagents for tests based on the activated partial thromboplastin time (APTT) include an activator, such
as kaolin, celite or ellagic acid. Reagents with such activators should be avoided when assessing the
effects of blood-contacting devices or device materials because they mask the coagulation caused by
materials or devices.
In coagulation testing of medical materials and devices, it is the device or material itself that serves
as the activator of coagulation. Appropriate positive and negative control materials should be used
whenever available. A negative control, the blood plasma itself without the material/device, should
be included.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

B.3.3 Platelets — Methods for testing
B.3.3.1 General
Assessment of platelets and their state of activation has been described throughout the literature, e.g.
see References [69] to [94] as a partial list. However, for blood-contacting medical devices and materials,
the most commonly used methods have arguably been simple counting of platelets and measurement
of platelet degranulation proteins following controlled exposure of medical devices or materials to
blood. Normal resting (homeostasis) levels of platelets and degranulation proteins are well established
(see commercial ELISA kit product literature), as are some abnormal levels observed in various clinical
thrombocytopathies. The presumption for such testing with medical devices is that appropriate
materials and device designs should not be associated with excessive platelet consumption and/or
activation that could bring risk to the patient. High levels of platelet loss and/or degranulation may be
an indicator of a tendency for the material/device to induce or promote these conditions which can give
rise to complications of bleeding or thrombosis. To measure platelet counts and platelet degranulation,
counting is performed using a routine differential cell counter and the degranulation is assessed using
standard enzyme-linked immunosorbent assays (ELISAs) for well-recognized platelet alpha-granule
proteins. Clinical laboratories often rely upon test kits that use common enzyme-linked immunosorbent
assay technique to measure the alpha-granule proteins. In a basic study, which may be either in vivo or
in vitro and will depend upon availability of appropriate antibodies to species-specific target platelet
granule protein epitopes, blood samples are retrieved under defined conditions and prepared and
analysed per assay instructions. Typical defined conditions or factors important in an in vitro model
are described in B.1.2.3. Comparison of results to appropriate controls such as negative controls (e.g.
baseline levels or test system with no material/device exposure) and results on a predicate device(s)/
materials(s) is critical. Platelet count decreases and blood degranulation protein increases that are
statistically-significantly and biologically-significantly different than controls may be an indicator of
a material/device design that presents a higher risk for platelet consumption and activation. Example
alpha-granule proteins for which commercially-available ELISA kits are available include
— PF4 (platelet factor 4, a 70-amino acid protein that binds with high affinity to heparin; PF4’s major
physiologic role appears to be neutralization of heparin-like molecules on the endothelial surface of
blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation), and
— βTG (beta-thromboglobulin, a chemokine for fibroblasts and neutrophils).
NOTE Thrombin from the coagulation cascade is a potent platelet agonist that can readily cause platelet
degranulation. Thus, high levels of thrombin will correlate with high levels of platelet degranulation.
As in coagulation, platelet consumption (loss) is generally seen to be affected by blood-contacting

surface area. Thus, surface area (SA) of a device or device material can influence platelet count data.
For this reason, it is important to specify the SA-to-blood-volume ratio (exposure ratio) in each study.
If possible, the exposure ratio may be treated as a variable to aid in understanding the specificity of
the material effect. Exposure ratios of 3,0 cm2 to 6,0 cm2/ml blood (based on device thickness) are
consistent with ISO 10993‑12. Other exposure ratios such as 1,5 and 2,0 times this ratio may be worth
considering as higher surface areas will theoretically increase the sensitivity of the platelet responses
to the test material.
Platelet activation is a process that occurs over a time (minutes to hours) and is recognized to
be potentially reversible up to a point, or once initiated, it can progress non-reversibly to the point
of presenting significant shape deformation, loss of cytoplasmic constituents and shedding of
microparticles and complete destruction. This process is dependent on stimulus type and amount.
There are numerous known potent stimulants, called platelet agonists, examples of which are thrombin,
ADP and collagen. Foreign surfaces themselves, such as blood-contacting medical devices, can also
behave like agonists since they can cause thrombin generation. In addition, platelets can adhere to
these surfaces, remain adhered or detach in activated or non-activated states and go through shape
changes leading to platelet destruction. Thus, assessing the overall state of platelet activation at any

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

point in time may benefit from the use of agents that help to “arrest” the platelets in their physical and
biochemical state at a particular point in time. Consequently, if platelet assessment cannot be made
immediately after removing the test material or device from the test system, a number of agents have
been suggested to counteract further platelet activation and to stabilize platelets[88][89][90][91].
Examples of these agents are acid citrate dextrose (ACD), citrate, theophylline, adenosine, dipyridimol
(CTAD) and other platelet-stabilizing reagents, such as ThomboFix™1) 1 ThomboFixTM is an example of
a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute and endorsement by ISO of this product.
B.3.3.2 Platelet count
It is important to determine the platelet count[45][121] because of the key role platelets have in preventing
bleeding and in the general process of thrombosis. A significant drop in platelet count of blood exposed
to a device can be caused by platelet adhesion, platelet aggregation, platelet sequestration (for example
in the spleen) or thrombus formation on materials or devices. A reduction in platelet count during use
of an implanted device may also be caused by accelerated destruction or removal of platelets from the
circulation. Various anticoagulants may be suitable for enumerating platelets[151]–[156].
Blood collection techniques should be reproducible. Platelets can become hyperactive/activated under
a variety of conditions, including improper blood collection. Tests such as platelet aggregometry and
flow cytometry may be considered to verify normal platelet reactivity and activation.
B.3.3.3 Platelet activation: Platelet granule-release proteins beta-thromboglobulin (ß-TG) and

platelet factor 4 (PF4), thromboxane B2 (TxB2) and platelet morphological changes
The use of certain materials or devices may cause platelet activation, which can result in the following:
a) release of platelet granule substances, such as βTG, PF4, TxB2 and serotonin;
b) altered platelet morphology;
c) generation of platelet microparticles.
Activated platelets are pro-thrombogenic. Platelet activation can be evaluated by various means, such
as microscopic (light and electron microscopy) examination of morphology of platelets adherent to the
material or device and measurement of βTG, PF4 and TxB2 released from activated platelets.
βTG and PF4 are proteins that are stored in alpha-granules of platelets and released in large amounts
after platelet activation[85][86][87][106]. Both of these proteins can be assessed by commercially-available
ELISA assays. Increases in platelet activation can occur through multiple paths associated with medical
devices and materials. The device/material itself may be platelet activating, turbulence and excessive
shear forces can cause platelet activation and platelet activation can be caused by potent agonists such
as thrombin which may form as a result of thrombosis associated with the material/device or local
injury. High levels of TxB2, also measurable by ELISA, indicate high levels of its precursor compound
thromboxane A2, a potent platelet agonist thought to be produced by activated platelets; TxB2 is
also thought to be reliable species-independent marker of platelet activation. See B.3.3.1 and B.4 for
details on general ELISA methodology. It may also be valuable to assess platelet activation through
the evaluation of the morphological changes platelets undergo when activated on a material/device
surface[70][71][173].
B.3.4 Haematology — Methods for testing
B.3.4.1 Complete blood count (CBC)
The electronic complete blood count analysis (often referred to as CBC) is a vital test used every day
in hospital haematology laboratories. Its primary purpose is to quickly and accurately enumerate the
1) ThomboFixTM is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute and endorsement by ISO of this product.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

concentration of the various cell populations in the patient, where abnormal readings can provide early
and vital information on a host of potential disorders. The CBC is used to determine the number or
proportion of white and red blood cells in the body. The analysis includes platelet counting. In analyses
on blood-material/device interactions, CBC data provides basic information on the impact of the
device/material interaction with formed blood elements. Counts of platelets and leukocytes pre- and
post-blood exposure to material/device are valuable in deducing the loss of activated platelets and
leukocytes taken up in clot formation by thrombogenic surfaces and therefore provide an estimate of
the surface’s thrombogenic potential[24].
B.3.4.2 Leukocyte activation
Leukocyte activation can be determined by microscopic examination of the device surface for activated
leukocytes. A simple more quantitative method involves use of a commercial ELISA assay to determine
the amount of polymorphonuclear leukocyte (PMN) elastase released to plasma following activation
from interaction of a material or device with blood. Another approach based on the principle that
thrombi adhering to material will contain a large number of platelets and leukocytes involves assessing
the decrease in their counts in blood[24].
B.3.5 Complement system — Methods for testing for C3a and SC5b-9
The complement system resides in blood plasma in the form of a biochemical cascade that functions
as a defence mechanism designed to supplement or “complement” the ability of antibodies to clear
pathogens from the body. It is a part of the immune system referred to as the “innate immune system”.
Here, unlike antibody protection, the response activity is neither acquired nor adaptable over time.
The complement system forms a fundamental line of defence that works alongside and can mediate
specific antibody mechanisms. The complement system can, however, also be brought into action by the
surface(s) of materials foreign to the body, including blood-contacting medical devices[129][138].
The system consists of a number of proteins found in blood that normally circulate as inactive
precursors. The nomenclature for the complement proteins is “C” followed by a simple Arabic number
for the native protein, and if cleaved, a small “a”’ or “b”’ to indicate the fragment. In the presence of a low
level of spontaneously formed reactive C3b, the presence of a biomaterial can trigger an amplification
response whose end result is production of inflammatory mediators, e.g. C5a and cytotoxic protein
complexes, e.g. membrane attack complex (MAC), which can stimulate an array of inflammatory
responses including white blood cell (WBC) chemotaxis, reactive oxygen species (ROS) production and
cytokine expression[129][130]. See Figure B.2.
There are numerous proteins and protein fragments that make up the complement system and these can
be divided into three distinct activation pathways: the classical complement pathway, the alternative
complement pathway and the mannose-binding lectin pathway. It is the alternative pathway that is
most regarded as being affected by and reactive to the presence of medical materials.
A number of commercial ELISA assays are available to assess the amount of complement protein in
blood. As C3a is an ubiquitous fragment amplified during activation, this complement protein is
considered a good general indicator of complement activation. In addition, a soluble form of the terminal
MAC abbreviated SC5b-9 can also be assessed by ELISA assay. SC5b-9 is generally considered a more
important marker representative of the full extent of complement activation. Elevated levels of any of
complement components indicate activation of the complement system. High surface area devices such
as haemodialysis filters and cardiopulmonary bypass devices have been associated with high levels
of activated complement components[129]–[138][143] and this phenomenon has been linked to activate
leukocytes and leukocyte sequestration in the lungs[130][137].
Measurement of complement fragments has several disadvantages. First, ELISA kits only assay
complement components in the fluid phase (serum or plasma); they do not measure the complement
components which are activated and adhere to the device surface. Depending on the nature of the
device material, there could be significant amounts of activated complement on the device/material
surface which is undetected by commercial ELISA kits. Second, there is species-specificity for many
of the commercially-available kits and high baseline levels are observed in typical in vitro testing.
Thus, appropriate controls need to be included and compared. The classical CH-50 method appears

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

useful with human, bovine, porcine and rabbit serum. However, sensitivity of CH-50 for detecting
complement activation following contact with materials/devices is low, given the CH-50 test measures
residual complement activity and most often only a small portion of complement system is activated by
materials/devices. Another functional method of measurement of complement activation in vitro is the
generation of complement C3- or C5-convertase determined by substrate conversion. References [18]
and [19] also address complement activation. Annex E provides further information on considerations
for complement testing of medical materials and devices. See B.4 for details on general ELISA
methodology.
Key
biomaterial/device surface
NOTE Factors that are shown in red are measurable by commercially available assay kits.
Figure B.2 — Alternative complement pathway
B.4 Methodology considerations for testing of plasma factors specific to

coagulation, platelet and leucocyte activation and complement activation using
ELISA (or other similar) techniques
B.4.1 General
Assessment of coagulation, platelet, blood cell and complement activity has long relied on clinical
assays that measure plasma levels of key proteins formed in activation cascades or released from
activation or damage to various cells types. Here, clinical laboratories often rely upon test kits that
use common enzyme-linked immunosorbent assay technique. In a basic study, which may be either
in vivo or in vitro, implementation will depend upon availability of appropriate antibodies/test kits to
species-specific target protein epitopes. Additionally, blood samples will be retrieved under defined
conditions and prepared and analysed per assay instructions. Defined conditions may include blood
exposure time, anticoagulation and anticoagulant level, temperature, flow, haematocrit and other

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

factors. Comparison of results to appropriate controls, such as negative controls (e.g. baseline levels
or no material/device exposure) and a predicate LMCD, is critical. Activation markers in blood that
are statistically-significantly and biologically-significantly higher than controls may indicate a
material/device design that presents a higher level of coagulation-, platelet- or complement-mediated
risk to the patient. Example coagulation proteins for which ELISA kits are commercially available
include TAT (thrombin-antithrombin complexes), F1.2 (fragment released from prothrombin upon
formation of thrombin) and FPA (fibrinopeptide A released from fibrinogen upon formation of fibrin).
Similarly, commercial ELISA kits are available for assessing platelet activation (e.g. alpha granule
release of BTG and PF4) and complement activation (e.g. C3a and SC5b9 formation).
B.4.2 General assay methods and documentation
B.4.2.1 General
Include the following with any test reports.
B.4.2.1.1 Reference documents
Manufacturer ELISA protocol IFU in kit.
B.4.2.2 Storage and stability
Describe all storage and stability conditions of the kit reagents and the blood/blood plasma being used.
B.4.2.3 Procedure
B.4.2.3.1 Sample preparation
Provide general instructions.

Results of such testing are invariably dependent upon surface area (SA) of a device or device test
material being tested. For this reason, the SA-to-blood-volume ratio (exposure ratio) should be specified
in each study. Exposure ratios of 3,0 cm2 to 6,0 cm2/ml blood (based on device thickness) are consistent
with ISO 10993‑12. Other exposure ratios such as 1,5 and 2,0 times this ratio may be worth considering
as higher surface areas will theoretically increase the sensitivity of the response to the test surface.
It is important to specify the means of quenching the reaction following the incubation period, i.e. list
name and concentration(s) of the quenching agent(s).
B.4.2.3.2 Dilution factor (DF)
Provide details on dilution factors used, diluents used, etc.

WARNING — Considerable inter and intra donor-to-donor variability can be observed, making
a single ideal DF difficult to identify. A minimum of two test sample dilution factors is therefore
recommended to capture all sample values on the standard curve. The sample absorbances
shall be in the range defined by the lowest and the highest standards. If not, the samples should
be retested with a new DF so that results fall within the range of the standard curve.
B.4.2.3.3 Data selection in cases of testing with multiple DFs
If all or some samples are tested under conditions of no dilution, low dilution and high dilution, report
the values requiring the smallest DF that are on the standard curve. It is also acceptable that if the “no
dilution” samples contain some off-the-curve values and all “low dilution” samples are on the curve and
the “high dilution” samples contain some below-the-curve values, to simply report the “low dilution”
readings where all values are on the curve under the same DF.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

B.4.2.3.4 Preparation of standards
Describe in detail how the standard curve and controls are prepared.
B.4.2.3.5 Method
Describe in detail the overall methods in the order followed, including highlighting any deviations from
the ELISA kit instructions.
B.4.2.3.6 Evaluation
Describe in detail the calculations made to determine the plasma levels of the protein being measured.
B.4.2.3.7 Statistical analysis
Describe in detail the methods of statistical analysis.
B.4.2.3.8 Limitations and interferences
List all limitations or interfering factors such as incorrect blood collection technique, e.g. inadequate
mixing of the sample and citrate solution (or other anticoagulant such a heparin) may lead to falsely
elevated coagulation protein values; a wrong anticoagulant may lead to elevation of all background
values, etc.
B.4.2.3.9 Reference interval
List known normal and abnormal plasma levels of the protein being measured and expected values
of controls.
B.4.3 Attachments
Include suggested dilution factor information, ELISA assay data sheet forms, checklists for the ELISA
procedure, etc.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex C
(informative)
Thrombosis — Methods for in vivo testing
C.1 General considerations

Numerous approaches have been applied to assess the process and events of thrombosis. However,
for blood-contacting medical devices and materials, the in vivo nature of thrombosis has led to the
methods described in C.2 and C.3 being more commonly used to assess device-associated thrombosis.
These methods are used in evaluation of permanent- and temporary-contact devices. As pointed out in
this document, the diversity of blood-contacting medical device applications dictates that in vivo test
models will be necessarily diverse, in order to appropriately mimic each clinical application.
The method in C.2 is accepted to be the most relevant manner in which to assess devices for thrombosis,
as here the actual device is evaluated in its intended clinical implant configuration in an animal model.
The main elements in this work are alignment of the animal protocol according to ISO 10993‑2, accurate
simulation of the human clinical application and inclusion in the protocol of a detailed method(s) and
analyses to be used to assess the degree of thrombosis.
The method in C.3 is used less commonly and is not widely accepted throughout the world. However,
it may be required or requested by certain regulatory authorities. The method has been referred to
as the non-anticoagulated venous implant (NAVI) model (when anticoagulation is not used) and the
anticoagulated venous implant (AVI) model (when anticoagulation is included). The method itself
involves insertion of catheter-shaped devices, or device materials formed into catheter shapes,
into the veins of animals for up to 4 h followed by gross assessment of amount of thrombus on the
material/catheter surface. The caveats with the methodology are shown in Table C.3 along with noted
advantages provided in Table C.4. Because of these caveats, and to avoid the false labelling of materials
and devices as thrombogenic in nature, data generated using this model requires extreme caution in
interpretation[143]. See also A.3.
When the test device is a catheter-type device used in a venous application, the methods in C.2 and C.3
are equivalent.
C.2 In vivo implant study of final device in pre-clinical animal study

As stated in this document, in vivo testing should be performed on devices intended for in vivo/implant
applications (see 6.1.6, 6.3.2 and 6.3.3). Protocols should include detailed methods for appropriate
assessment of blood/device interactions, e.g. analyses on
a) the device itself,
b) blood samples from the test subject, and
c) susceptible tissues and end organs.
Such preclinical testing should use models that simulate actual in-use application conditions, e.g.
same implant site, geometry, flow, contact duration, temperature, sterility, etc. (see 6.1.2), and include
appropriate traceable controls, e.g. a predicate device (see 6.1.3). Importantly, testing should only
be done on actual complete (finished) devices or components (see 6.1.4), as testing on non-finished
products and testing in poorly simulated use conditions will not be highly predictive of performance in
clinical applications (see 6.1.6).
Accordingly, devices with ex vivo application and devices with in vivo application should be tested in
appropriate ex vivo and in vivo models, respectively. Anticoagulant use in ex vivo/in vivo models should

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

be consistent with the type and quantity used in the routine clinical device application and the product
IFU (see 6.1.12). Use of appropriate replication, statistical design and analysis methods should be
considered in animal studies (see 6.1.14 and References [213], [214], [215] and [216]).
Consider tests, as appropriate, in these categories: thrombosis, coagulation, platelets, haematology
and complement activation, as described in Table 2. By way of example, one analysis might consist of
gross examination and SEM on the device to assess degree of device-associated thrombosis. Inspection
of susceptible downstream organs, e.g. lung and kidney, for evidence of thromboemboli will aid in
assessing potential for device-associated thromboembolism. If appropriate antibodies are available,
coagulation may be assessed by measuring blood plasma levels of indicators of coagulation and fibrin
formation, e.g. TAT and FPA measured via ELISA technique. Likewise, simple platelet counting and or
measurement of platelet activation markers, e.g. βTG, can be used to assess the impact of the device
on platelets. Routine differential blood cell counting and plasma-free haemoglobin can be used for a
general assessment of haematology factors and assessment of blood cell physical damage. Finally, for
large surface area devices, providing availability of antibodies, plasma levels of various complement
factors may be used to assess activation of the alternative complement pathway.
Generally speaking, it is desired that the test device has no, low or equivalent impact on the factor being
measured relative to the results observed in a predicate device. Results higher than in the predicate
device may be justified based on a risk/benefit analysis. Devices without predicates should use an
appropriate control, with justification provided for selection of that control. Such evaluations are
preferred over the use of the method described in C.3, as this method most appropriately mimics the in
vivo application. As always, vertical standards should be consulted in the various device areas. For the
latter, numerous examples can be found among references.
NOTE 1 For catheter-shaped devices intended to be implanted in the venous environment under no
anticoagulation, or anticoagulation, the NAVI and AVI implant models, respectively as described in C.3, describe
the appropriate in vivo study approach for these devices.
NOTE 2 For catheter-shaped devices intended to be implanted in an arterial environment under no

anticoagulation, or anticoagulation, a non-anticoagulated arterial implant (NAAI) or anticoagulated arterial
implant (AAI) model, in analogous appropriate arterial implant positions to those described in C.3, describe the
ideal study approach for arterial implant devices.
See also 6.3.3 and Annexes A and B.
C.3 In vivo NAVI and AVI thrombogenicity tests

The NAVI and AVI test involves inserting a catheter (or other appropriately-shaped) device, or a device
material made into a catheter shape, into the vein of a large animal. It has been called for in situations,
for example, of characterizing a new catheter, evaluating a new device coating, characterizing a new
material and in circumstances of changing a vendor or material/device processing step. In the model
itself, a number of venous positions have been used (see Figures C.1 and C.2). In the absence (NAVI) or
presence (AVI) of anticoagulants, duplicate or triplicate implants are allowed to incubate in situ for a
period of up to 4 h. (Instances of less and more time may be justified, given the specific application.) The
implants are then removed and assessed for the amount of apparent thrombus on the surface. The canine
femoral or jugular vein model is most commonly used, where a test material/device is positioned in one
vein and a control material or predicate device is placed in the contralateral site. The test requires two
to three large animals, with alternating test and control implant locations to avoid bias, and assessment
of thrombus on the devices using a scoring method such as those shown in Tables C.1 and C.2. Results
may be supplemented with gravimetric analysis of the observed thrombus and observations of vessel
patency. Results of the test device should be equivalent to, or less thrombogenic than, an appropriate
LMCD, if available or unless otherwise justified. It is important to make sure that all clinically blood
contacting device components are evaluated. Various worldwide regulatory authorities have suggested
that up to 15 cm of the test and control be implanted in order to ensure adequate exposure during
testing. Depending on the device configuration, this may require that a custom device be created to
reduce the length of the components, while maintaining the same material ratios as the complete
device. As an alternative, a device can be subdivided into multiple test samples if necessary.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

For devices and materials intended for arterial implantation, methods may be adjusted accordingly to
use arterial implant positions.
CAUTION — Variation in results has been observed between
a) test facilities,
b) test evaluators,
c) replicates on the same material, and
d) scores obtained on controls[143].
Table C.3 gives a summary of the main controversies of the NAVI and AVI models. Table C.4 provides
some noted advantages of the NAVI and AVI models. The greatest utility of the model may be in
assessment of test materials intentionally modified to reduce acute thrombus formation, for example
in evaluating heparin coatings.
Key
1 femoral
2 jugular
Figure C.1 — Main implant positions used in the NAVI/AVI model

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Key
1 IVC-IVC
2 SVC-IVC
3 IVC-AA
AA abdominal aorta
IVC inferior vena cava
SVC superior vena cava
CAUTION — Use of these implant positions requires careful consideration for artefact due to device-device
interaction and/or bias for positional differences in thrombus formation.
Figure C.2 — Other less-frequently used NAVI and AVI implant positions
Table C.1 — NAVI/AVI scoring scheme A

Thrombus formation score description Score
No significant thrombosis
0
(a very small clot is acceptable at insertion).
Minimal thrombosis, one location. 1
Minimal thrombosis, multiple locations. 2
Significant thrombosis, > 1/4 to ≤ 1/2 the surface of the implant, vessel
3
patent.
Significant thrombosis, > 1/2 the surface of the implant, vessel patent. 4
Vessel completely occluded. 5
Table C.2 — NAVI/AVI scoring scheme B

Thrombus non-existent or minimal and, if present, appears to be
0
associated with implant venotomy site.
Thrombus minimal, observed to be covering 1 % to 25 % of material
1
surface.
Thrombus moderate, observed to be covering 26 % to 50 % of material
2
surface.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)


Thrombus severe, observed to be covering 51 % to 75 % of material
3
surface.
Thrombus extensive, covers 76 % to 100 % of material surface. 4

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Table C.3 — Main caveats in using the NAVI or AVI models[143]

Factor Description Concern
High flow environments lead to low levels of surface-associated thrombus and
vice versa. Therefore, slight differences in anatomical factors such as target vessel
diameters and/or venous valve positions can have potential impact on the amount
1 Implant position
of thrombus observed. Additionally, if the implant is placed at an angle or position
which alters blood flow to create eddy currents leading to stasis, thrombus
formation may occur unrelated to the material properties of the test device.
Each test and control should be identically and precisely inserted into the target
Implant tech‑
2 veins with each situated in an identical position (preferably centrally-located with
nique
no vessel wall contact).
This factor relates to vessel wall injury/endothelial denudation during the
Extent of implantation and incubation periods. The implant itself can contribute to
3 device-vessel thrombosis as a result of mechanical contact with the vessel wall, which induces
wall contact injury and tissue factor activation. Here, the material factor may play a minor role
in the extent of observed thrombus and device geometry can play a major role.
The main measured response of extent of surface-associated thrombus tends to be
Time/incubation intense within the first 1/2 h to 2 h. Sometime after this period, the thrombolytic
4
period (fibrinolytic) system can initiate and biochemically remove some of the associated
thrombus.
Depending on the makeup and the extent of the surface-associated thrombus on
the sample, the thrombus material being measured/scored can be fragile and
readily slough off during the device retrieval/exposure. Without special
precautions, this material, often referred to a “sleeve thrombus”, can be “squeegee
5 Explant technique
off” if the sample is retracted from or disturbed in its implant site. Some
investigators use of in situ perfusion fixation to flush away non-adherent blood
elements and concomitantly crosslink the device/thrombus/vessel into a tougher
(and more physiological) geometry.
This model has been used to assess the thrombogenic potential of new materials,
Material/
evaluate process changes on existing approved materials and to qualify new
6 material
vendors. Often a legally-marketed comparator device (LMCD) or material is used
surface
as a control, which yields equally variable results.
Non-thrombo Extensive work by experts in the field[177][178][179] has demonstrated that
adherent hydrophilic surfaces can be thrombogenic (and thromboembolic) yet not be
materials get thromboadherent. This test will give passing scores to devices and materials that
7
labelled are thrombogenic and non-thromboadherent.
non-thrombo‑
genic
Recipient/ Papers on this topic[56][57][180] indicate that test subjects can have significantly
subject different “thrombotic potentials”, i.e. differing capacity to form thrombus upon
8
thrombotic exposure to medical implants and other stimuli. This can result in significant
potential differences in scoring between test subjects.
In complying with ISO 10993‑2, combined with the variability in responses seen
9 Statistical power between predicate devices and materials, and between test subjects, obtaining a
statistically-meaningful conclusion is often not possible.
The training and skill of the evaluator assigning thrombus scores is extremely
Evaluator
10 important. Some may have difficulty differentiating between true in vivo thrombus
expertise
and false thrombus [post/ante-mortem (agonal) clot formations].

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)


Nearly all devices and materials tested in the AVI model receive scores of zero and
Anticoagulation pass the test. This brings into question the purpose and rationale of using the AVI
11
impact model. In the NAVI model, most hydrophobic materials show varying degrees of
thrombus while hydrophilic materials show minute levels of thrombus.
Besides potentially causing vessel wall damage, if the size of the implant relative to
Implant size to the vessel diameter is too large, blood flow can be affected (stagnation),
12
vessel diameter predisposing to thrombosis. In general, the cross-sectional area of the implant
should not occupy more than 50 % of the vessel lumen.
Table C.4 — Advantages in using the NAVI or AVI models

The NAVI model has been known to consistently show substantial in vivo
thrombus formation on hydrophobic polymers, particularly after 1 h or less
Studying thrombus
1 blood exposure. As such, the NAVI model is a good model to study thrombus
formation
formation. For example, it may be a useful tool to study the impact of
thrombus on devices, such as intravascular sensors[143].
Coatings applied to device surfaces to reduce thrombogenicity should show
Evaluating coatings measurably different and consistent responses, i.e. lower than non-modified
2 intended to reduce controls, when tested in the NAVI model.
thrombogenicity Caution: Testing should include steps to assess thromboembolic potential, as
non-thrombo-adherent surfaces may still be thrombogenic.
Coatings applied to device surfaces to enhance thrombogenicity should show
Evaluating
measurably different and consistent responses, i.e. higher than non-modified
coatings intended
3 controls, when tested in the NAVI or AVI model.
to enhance
Caution: Testing should include steps to assess thromboembolic potential, as
thrombogenicity
thrombogenic surfaces may shed emboli.
The NAVI and AVI models are most appropriate for testing catheter-type
Screening of
devices intended to be introduced into the vasculature for short periods of
materials for
4 time. Multiple materials and device designs can be tested in this model with
cardiovascular
the contralateral vessel being used to assess a control or non-modified
applications
version of test devices.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex D
(informative)
Haematology/haemolysis — Methods for testing — Evaluation

of haemolytic properties of medical devices and medical
device materials
D.1 General considerations

Extensive literature is available that describes blood/material interactions. Unfortunately, very few
methods exist which are reliable, reproducible and predict clinical performance. This annex will review
the known haemolysis test methods and discuss factors pertaining to their ability to characterize
medical materials and devices. Haemolysis is a function of blood-material exposure time and material
properties such as surface energy, surface morphology and surface chemistry. Haemolysis is also a
function of local mechanical forces and biochemical factors.
D.2 Causes of haemolysis

D.2.1 Osmotic pressure (osmotic pressure-induced haemolysis)
The erythrocyte membrane is a semi permeable membrane. A pressure differential will occur when two
solutions of different concentrations are separated by such a membrane. Osmotic pressure occurs when
the membrane is impermeable to passive solute movement, yet it allows passage of a pure solvent, such
as water. Such a pressure differential can cause erythrocyte swelling and cell membrane rupture with
release of free haemoglobin[175]. It should also be noted that the osmotic fragility between mammalian
red blood cells can vary[95]–[98].
D.2.2 Mechanical forces (mechanically-induced haemolysis)

Fluid dynamic factors such as blood flow rate, turbulence and non-physiological shear forces can
deform the erythrocyte membrane and potentially cause membrane rupture. The latter can potentially
be exacerbated by devices with mechanical operation and/or complicated flow paths. Examples of
such devices are
— apheresis and cell separation systems,
— arterial blood filters[11][36],
— blood pumps[29][30][124][157],
— cardiopulmonary bypass systems[5][10][11][13][37][41],
— cardiotomy/venous reservoir systems[10][11],
— circulatory support devices[9],
— haemodialysis systems[16][28][41],
— mechanical heart valves[2], and
— ventricular-assist devices[236][237].

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

D.2.3 Biochemical factors (material-induced haemolysis)

Changes to membrane structure on a molecular level can modify the strength and elastic properties of
the erythrocyte membrane. A deficiency of nutritional factors or metabolic energy (ATP) can result in
loss of the discoid shape and microvesiculation of haemoglobin. Other chemicals, e.g. extractables from
a medical device, bacterial toxins, pH and metabolic changes induced by temperature, can compromise
the erythrocyte membrane[95]. These changes can cause membrane rupture at lower than expected
osmotic pressures. A test to determine the pressure at which an erythrocyte membrane ruptures
(osmotic fragility) can be carried out.
D.3 Clinical significance of haemolysis

D.3.1 Toxic effects
Elevated levels of plasma-free haemoglobin can induce toxic effects or initiate processes which can stress
the kidneys or other organs[175]. The plasma-free haemoglobin concentration is a convenient measure
of injury to erythrocytes, but it is an indirect indicator of damage to other blood elements as well.
D.3.2 Thrombosis and anaemia

Intravascular haemolysis can promote thrombosis by a cascade of events involving liberated RBC ADP
and phospholipids[106] causing platelet activation and degranulation of prothrombotic agents. When
haemolysis causes a clinically significant drop in erythrocyte count, anaemia and compromised oxygen-
carrying capacity with its subsequent effects on the brain and other organs or tissues can result.
D.4 Determining a pass/fail assessment for haemolysis

Haemolysis is a function of exposure time and material properties such as surface energy, surface
morphology and surface chemistry. Haemolysis is also a function of shear stress, cell-wall interaction,
character of adsorbed protein layers, flow stability, air entrainment and variations of blood source,
age and chemistry[108][112][113]. These variables need to be adequately controlled for comparisons of
haemolytic potential among materials and medical devices. The spectrum of methods for evaluating
haemolysis varies from simplified to highly complicated models. Specific in vitro and in vivo models with
flowing blood have been published. Studies of haemolytic potential are relative comparisons against
materials or medical devices tested in the same model by a specific laboratory rather than absolute
measures. In vitro test methods are able to quantify small levels of plasma haemoglobin which may not
be measurable under in vivo conditions (e.g. due to binding of plasma haemoglobin to haptoglobin and
rapid removal from the body). Measurement of lactate dehydrogenase and haptoglobin, as indicators of
haemolysis in an in vivo test setting, may be applicable.
It is not possible to define a universal level for acceptable and unacceptable amounts of haemolysis for all
medical devices and applications. The effect of a device on haemolysis can be masked in the short-term
by the trauma of the surgical procedure. A device can cause a substantial amount of haemolysis, but
be the only treatment available in a life-threatening situation. Intuitively, a blood-compatible material
is non-haemolytic. In practice, many devices cause haemolysis, but their clinical benefit outweighs the
risk associated with the haemolysis. Therefore, when a device causes haemolysis, it is important to
confirm that the device provides a clinical benefit and that the haemolysis is within acceptable limits
clinically. Acceptance criteria may be justified based on some form of risk and benefit assessment. The
following questions are suggestions for developing such an assessment:
— What is the duration of exposure of the device to the patient?
— How much haemolysis does the material or device cause? Does the haemolysis continue for the entire
time the device is exposed to the patient? Does haemolysis continue after removal of the device?
— What are the relative risks and benefits of other available methods for treating the condition?

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

— What are the haemolytic properties of these known treatments? How does the device in question
compare to these other treatments?
— How effective is the test device compared with other forms of treatment? A more effective device
can cause more haemolysis during use but the additional effectiveness might increase the benefit to
the patient.
D.5 Haemolysis testing — General considerations

D.5.1 Methods
D.5.1.1 General
Haemolysis of red blood cells (erythrocytes) is assessed using in vitro tests. Direct methods determine
haemolysis due to physical and chemical interactions with erythrocytes. Indirect methods determine
haemolysis due to extractables from test articles. Reference [17] is one standard that is specific for
testing the haemolytic properties of materials (mainly due to chemical factors) and, depending on device
size and complexity, it may not be sufficient for testing whole intact medical devices. References [17],
[22] and [28] are examples of methodologies specifically developed for haemolysis testing of medical
devices and their component materials. Reference [20] was developed to assess haemolysis caused by
medical nanoparticles. In its simplest form, for highly diluted suspensions of erythrocytes in contact
with test materials, haemolysis is often reported as a percentage of haemoglobin which has been
liberated into the supernatant normalized by the total haemoglobin which was available at the beginning
of the test, i.e. (free haemoglobin concentration/total haemoglobin concentration) × 100 %. If all of the
erythrocytes present at the beginning of the experiment are destroyed, there is 100 % haemolysis.
In addition to material testing of devices, dynamic testing of whole medical devices under clinical use
conditions to evaluate the effects of the device structure, mechano-physical interactions of blood with
materials, range of clinically relevant use conditions (e.g. blood flow rate, rpm, pressure, exposure
time), intended use and haemodynamic factors on haemolysis should be considered. For many devices,
haemolysis caused by hydrodynamic forces and dynamic interaction with surfaces exceeds that caused
by the chemical effects of the material. To appropriately simulate the clinical use conditions, blood
haematocrit and other factors should be accounted for during the dynamic haemolysis testing[30][37][41]
[124]. To ascertain a worst case amount of haemolysis that may occur, in vitro testing is often conducted
at the highest blood flow rate for which the device is expected to be used. References that provide
protocols for mechanical haemolysis testing of devices include References [5], [37], [41] and [124].
The concentration of haemoglobin in plasma is significantly less than the total blood haemoglobin
concentration. The plasma-free haemoglobin concentration is normally 0 mg/dl to 10 mg/dl in vivo,
whereas the normal range of total blood haemoglobin concentration is 11 000 mg/dl to 18 000 mg/dl. For
this reason, different methods have been used to measure the great range of haemoglobin concentrations
which are encountered during haemolysis testing. A note of caution:
CAUTION — Some common haemolysis assays suggest a cut-off level for material-induced
haemolysis below which there is no/low risk of concern based on historically accepted but non-
validated values[14][17][28]. However, higher acceptable levels of material-induced haemolysis
may be justified based on a suitable risk/benefit analysis.
Researchers should be aware that haemolysis tests may be adversely affected by chemicals in medical
materials or solutions which may alter erythrocyte fragility (e.g. certain buffers and fixatives, such as
formaldehyde or glutaraldehyde), cause haemoglobin to precipitate (e.g. by copper or zinc ions) or alter
the absorption spectra of haemoglobin (e.g. by polyethylene glycol or ethanol)[115][170].
D.5.1.2 Total blood haemoglobin concentration measurements
Classically, the analytical methods outlined in D.5.1.2.1 and D.5.1.2.2 have been used to determine
total blood haemoglobin (Hb) concentrations[106]. Total blood haemoglobin concentration can also be
measured using calibrated complete blood cell counters and haemoglobinometers.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

D.5.1.2.1 Cyanmethaemoglobin method
The first classical method, cyanmethaemoglobin detection, was issued by the International Committee
for Standardization in Haematology[121]. The cyanmethaemoglobin (hemiglobincyanide; HiCN) analysis
has the advantage of convenience, ease of automation and the availability of a primary reference standard
(HiCN). The method is based on the oxidation of Hb and subsequent formation of haemoglobincyanide
which has a broad absorption maximum at 540 nm. Lysing agents such as detergents are used which,
in addition to releasing Hb from the erythrocyte, decrease the turbidity (a source of interference as
false absorbance at 540 nm) from protein precipitation. For the total haemoglobin concentration, the
spectral interference due to plasma is minimal and the sample absorbance can be compared with the
HiCN standard solution directly.
The broad absorption band of HiCN in this region enables the use of simple filter type photometers
as well as narrow band spectrophotometers for either manual or automated detection. The use of
the HiCN reference standard provides comparability among all laboratories employing this method.
The major disadvantage is the potential health risk in using the cyanide solutions. Cyano reagents
are themselves toxic by various routes of exposure, and additionally, release HCN upon acidification.
Disposal of reagents and products has also become a considerable concern and expense.
D.5.1.2.2 Iron method
The second classical method for determining the total haemoglobin concentration is based on
determining the haemoglobin iron concentration in solution. Iron is first separated from Hb, usually by
acid or by ashing. It is then titrated with TiCl3 or complexed with a reagent to develop colour that can be
measured photometrically. This method is too complex for routine work and is rarely used.
D.5.1.3 Plasma or supernatant haemoglobin concentration measurements
The following two methods have been used to measure plasma or supernatant haemoglobin
concentrations.
D.5.1.3.1 Direct optical and added chemical techniques
Due to many different factors (e.g. tradition, ease of use, disposal of waste chemicals, availability of
standard solutions), there have been a host of different assays used for measuring plasma haemoglobin
as an indicator of haemolysis, with no one method being widely accepted. The assays can be classified
into two broad categories: those which are direct optical techniques (i.e. based on quantifying the
oxyhaemoglobin absorbance peak at 415 nm, 541 nm or 577 nm, directly or through use of derivative
spectrophotometry) and those which are added chemical techniques (i.e. quantification of haemoglobin
based on a chemical reaction with reagents such as benzidine-like chromogens and hydrogen peroxide
or the formation of cyanmethaemoglobin)[109]. All of the assays can be performed manually or can
be automated.
A popular method for determining the concentration of haemoglobin is based on its catalytic effect on
the oxidation of a benzidine derivative, such as tetramethylbenzidene, by hydrogen peroxide. The rate
of formation of a coloured product (photometrically detected at 600 nm) is directly proportional to
the haemoglobin concentration. The advantages of this method are ease of automation (commercial
equipment), elimination of potentially toxic and environmentally unsafe cyano reagents and the
availability of Hb standard sets which are calibrated against the HiCN primary reference standards.
The detection limits of the assay (as low as 5,0 mg/dl) are comparable with the haemoglobin cyanide
method[106]. The major disadvantages are that there is still a potential health risk in using benzidine
dyes and an expense associated with disposal of reagents and products. Moreover, the reported
dynamic range of this method is low (5 mg/dl to 50 mg/dl)[116] and possible reaction inhibition (by as
much as 40 %)[117] may occur from calcium-chelating anticoagulants (e.g. citrates, oxalates, EDTA)[116],
albumin[104] or other non-specific plasma components[106] which may interfere with H2O2 oxidation.
For these reasons, direct optical methods, such as those by References [102], [105] or [118] with
comparable sensitivity and reproducibility may be substituted. However, as noted above, chemically
induced alterations to haemoglobin and its spectra can occur which may invalidate some of the

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

haemoglobin assays. Moreover, compensation needs to be made for endogenous plasma background
interference, since it can also alter the haemoglobin spectra[109]. The analyst should be aware of these
limitations in the plasma haemoglobin assays and ascertain whether they are using an appropriate
technique[104][109][115][170]. This includes evaluating the test supernatant for the presence of a
precipitate and comparing its optical spectra (e.g. 400 nm to 700 nm) to that of isolated oxyhaemoglobin.
D.5.1.3.2 Immunonephelometric method
The immunonephelometric method is based on determination of plasma haemoglobin by means of

nephelometry using a commercially available antibody. This method is for routine work. There is a
good correlation and comparability to the optical techniques[107].
D.5.2 Blood and blood component preservation
This subclause presents the best demonstrated practices for the preservation of human blood
components by the American Association of Blood Banks[99] and the Council of Europe[101]. In general,
materials and devices should be tested using blood whose chemical condition mimics that which
the device would experience clinically, e.g. proper choice of anticoagulant, minimal use of blood
preservatives and appropriate blood pH[151]–[156].
Anticoagulant solutions have been developed for use in blood collection that prevent coagulation and
permit storage of erythrocytes for a certain interval of time. These solutions all contain sodium citrate,
citric acid and glucose; additionally, some contain adenine, guanosine, mannitol, sucrose, sorbitol
and/or phosphate, among others[151]–[156]. Although heparin is not used for blood preservation, it is
often used for anticoagulation clinically with patients exposed to medical devices.
Blood clotting is prevented by citrate binding of calcium. Erythrocytes metabolize glucose during
storage. Two molecules of adenosine triphosphate (ATP) are generated by phosphorylation of adenosine
diphosphate (ADP) for each glucose molecule metabolized via the Embden-Myerhoff-Parnas anaerobic
glycolysis cycle. The ATP molecules support the energy requirements of the erythrocyte in maintenance
of membrane flexibility and certain membrane transport functions. Conversion of ATP to ADP releases
the energy necessary to support these functions. In order to prolong storage time, alkalinity should be
reduced by addition of citric acid to the anticoagulant solution. This provides a suitably high hydrogen
ion concentration at the beginning of erythrocyte storage at 4 °C. Increasing acidity during storage
reduces the rate of glycolysis. The adenosine nucleotides (ATP, ADP, AMP) are depleted during storage
and the addition of adenosine to the anticoagulant solution permits synthesis of replacement AMP,
ADP and ATP.
A considerable portion of glucose and adenine is removed with plasma when erythrocyte concentrates
are prepared. Sufficient viability of the erythrocytes can only be maintained after removal of plasma
if the cells are not over-concentrated. Normal citrate phosphate dextrose (CPD)-adenine erythrocyte
concentrates should not have an erythrocyte volume fraction greater than 0,80. Even if more than
90 % of the plasma is removed, erythrocyte viability can be maintained by addition of an additive or
suspension medium. Sodium chloride, adenine and glucose are necessary for viability while mannitol
or sucrose can be used to further stabilize the cell membrane and prevent haemolysis[99].
The suitability of containers for the storage of blood products is evaluated by various methods that
measure the quality of the blood product[103][106]. The container with blood product containing an
appropriate anticoagulant is stored upright at 1 °C to 6 °C under static conditions. At predetermined
intervals, the amount of cell-free plasma haemoglobin is measured to assess the viability and quality
of the stored product. The quality of the stored product can be enhanced by gentle mixing once a week.
Evaluation of storage in the container indirectly evaluates the permeability of the container to waste
carbon dioxide from erythrocyte metabolism in the absence of other confounding factors.
D.5.3 Protection of employees handling blood
Written procedures are necessary for protection of employees receiving, handling and working with
potentially contaminated human blood. Potentially contaminated materials include blood and other

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

body fluids and products, equipment which has been or may have been in contact with blood or other
body fluids and materials used in the culturing of organisms causing blood-borne infections[114].
D.5.4 Blood collection (phlebotomy)
While it is not possible to guarantee 100 % sterility of the skin surface for phlebotomy, a strict,
standardized procedure for preparation of the phlebotomy area should exist. It is especially important
to allow the antiseptic solution to dry on the skin surface prior to venipuncture and that no further
contact is made with the skin surface before the phlebotomy needle has been inserted[99].
A closed container system (i.e. one that does not contain room air) is preferred for blood collection
for the prevention of microbial contamination. Needle punctures in the rubber seal of the specimen
vial should be completely closed after withdrawal of the needles, otherwise the partial vacuum created
following cooling can draw in contaminated air[99].
NOTE Use of a vacuum tube has the potential to cause slight haemolysis[125][126][127].
Blood collected in an open system can be contaminated by exposure to room air and is not considered
sterile. Microbial contamination is a known cause of haemolysis.
D.5.5 Species selection
Ideally, haemolysis testing should be done with human erythrocytes. However, several factors can
make such a choice difficult or impossible. In some countries, human blood supplies are limited and
should be reserved for human transfusion. Health criteria for human and animal donors should also
be considered. All blood has a limited “shelf life” and it may be more difficult to obtain human blood
cells on a timely basis. If animal erythrocytes are used, attention should be paid to ensure 100 %
haemolysis to obtain total haemoglobin content due to differences in membrane stability among animal
species. Negative controls should cause minimal haemolysis so that the activity of the test material
is not masked. Rabbit and human erythrocytes are reported to have similar haemolytic properties
whereas monkey erythrocytes are more sensitive and guinea pig erythrocytes are less sensitive[95][96]
[97][98][123].
D.5.6 Evaluation of haemolysis — In vitro, ex vivo and in vivo exposure to blood or

blood components
Haemolysis can be evaluated by exposure of materials or devices under in vitro, in vivo and ex vivo
conditions. In vitro conditions are used to evaluate materials as well as devices. Ex vivo and in vivo
conditions are used to evaluate devices which may contain more than one material.
In vivo and ex vivo assessments in animal models or during clinical trials are possible. Justification can
be made for either of the following study designs. In the first case, the test device is compared with
reference control marketed devices with known acceptable levels of haemolysis. In the second case, the
test subject is evaluated for clinically significant consequences of haemolysis.
The purpose of in vivo or ex vivo tests is to characterize the haemolytic potential of a medical device. The
preliminary studies may be in vitro and may use fresh or outdated human blood or blood from a non-
human species. For medical devices indicated for ex vivo use, the general practice is to recirculate blood
through the device using conditions that simulate the most clinically relevant and intended worse-case
(e.g. highest blood flow rate) clinical usage. These investigations are followed by ex vivo simulations in
an animal model for some medical devices or by limited, controlled studies in humans. The size of the
medical device and the intended function influence the design of these studies.
D.5.7 Direct contact versus indirect methods
Extraction conditions to be used are outlined in ISO 10993‑12. Some test methods call for direct contact
of the device with erythrocytes, while other methods describe the preparation of an extract which is
then exposed to erythrocytes. Test selection should be based upon the device itself and the conditions in

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

which it will be used. Extraction conditions to be considered when elevated temperatures are used are
outlined in ISO 10993‑12.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex E
(informative)
Complement — Methods for testing
E.1 Background information

Complement activation has been implicated in certain adverse reactions during clinical extracorporeal
therapies, in particular in haemodialysis[129][130][131][132] and cardiopulmonary bypass applications[133]
[134][135][136]. These therapies notably involve devices with high blood contact surface areas and
relatively short contact times. Complement activation occurs usually in the early stage shortly after
blood contacts the device material surface; it has yet to be seen to continue for a longer period. Here,
activation is recognized to start with blood contact with the device material(s) and the deposition
of numerous plasma proteins, including the critical complement proteins C3 and C3b. The contact of
these specific proteins leads to the alternative pathway formation of the crucial C3 and C5 convertase
enzymes (C3b•Bb, C3b•C3b•Bb, respectively; see Figure B.2). The C5 convertase protein catalyses
the cleavage of C5 resulting in C5a and C5b generation. The C5a protein is a recognized effector
of receptor-mediated neutrophil and monocyte activation and the C5b fragment is the recognized
initial complement component that leads to formation of the complement membrane attach complex
(MAC) which binds to and activates and/or destroys bystander cells by causing lysis. WBCs can detect
material surface-bound C3 and C4 fragments, which results in their subsequent surface adhesion and
activation. Neutrophil and monocyte activation, MAC formation and activity, and WBC adhesion and
activation on materials account for the archetypical pathophysiology seen clinically in high surface
area device applications[136]. Importantly, Reference [138] have shown the critical role of the fragment
C5a in mediating many of these adverse reactions. Here, dose-dependent responses identical to those
seen under actual dialysis were observed in simulated haemodialysis and infusion of purified C5a.
Continued work in this field has shown that materials whose surfaces are highly nucleophilic (hydroxyl-
and amine-containing) present the highest complement activating potential and that various surface
modifications that reduce this type of surface chemistry greatly nullify the classic clinical sequelae.
This work is supported by other investigators studying the relationship of complement activation by
medical device materials and the biological response[136][137].
Fortunately, the discovery of hydroxyl- and amine-containing blood-contact materials as the source of
complement activation led to development of new materials that eliminated or masked these groups.
This material modification greatly reduced complement activation in the larger surface area/acute
contact applications. This attention to complement activation in these large device areas however has
driven such testing to become more common on all devices regardless of blood-contact surface area
and implant duration. To date, no scientific papers or clinical reports of complement-related adverse
events have been identified by this Working Group in other device applications, i.e. all medium to small
surface-area devices. Thus, appropriate references that link device-associated complement activation
to adverse events in humans, along with a threshold device surface area of concern are not available. It
may be noteworthy that classic anaphylactic reactions have occurred in association with use of medical
devices. Here, however, this reaction has been generally attributed to an agent being delivered, rather
than to a device or device material[139]–[142]. Some false associations of complement activation to the
devices involved may have arisen from such reports[143].
As in assessing coagulation, thrombosis and platelet activation, a number of molecular biology tools
(e.g. ELISA assays) exist to monitor the levels of complement pathway activation in blood. Example
complement proteins for which commercially-available ELISA kits are available include but are not
limited to C3a, C5a and SC5b9. Despite the strong link of complement-mediated pathophysiology to
the C5a fragment, classic complement testing has focused on assessing C3a and SC5b9 complex. In
conjunction with these tools, methods exist to assess related responses such as WBC adhesion (using
SEM) and WBC activation, for example, using bioassays for PMN elastase release.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

E.2 Complement activation tests and documentation (suggested to consider in

reporting complement test results for scientific or regulatory purposes)
The general methods and documentation used in complement testing using ELISA assays are
described in B.4.
Like other biological reactions, such as coagulation protein formation, complement formation generally
exhibits an initiation, propagation and termination phase[56][57][137]. This reflects the initial C3 and C5
convertase formation reactions, cascade/feedback amplification and a slowdown/deactivation period
where critical precursors may be consumed or the active proteins lose activity due to short half-life or
deactivation by negative control feedback proteins. Thus, order-of-magnitude differences in levels of
complement proteins are to be expected over time. Consequently, the phase of the complement cascade
in blood at the time the material/device-blood contact actually occurs is an important factor. That is,
the impact of test materials when mixed with blood may be quite different at each phase. In addition,
as complement activation is generally proportional to blood-contacting surface area, surface area
(SA) of a device or device material can be very influential on results. For this reason, the SA-to-blood
(whole, plasma or serum) volume ratio (exposure ratio) should be specified in each study. If possible,
the exposure ratio may be treated as a variable to aid in understanding the specificity of the material
effect. Exposure ratios of 3,0 cm2 to 6,0 cm2/ml blood (based on device thickness) are consistent with
ISO 10993‑12. Other exposure ratios such as 1,5 and 2,0 times this ratio may be worth considering as
higher surface areas will theoretically increase the sensitivity of the response to the test material.
NOTE There will be a physical limitation on the amount of test material that can be tested due to the volume
of the test system, e.g. a test tube, and the target exposure ratio.
E.3 Complement activation tests method considerations

A review of multiple test laboratory complement testing methodologies has shown that use of
commercially available test kits is common. However, a number of inconsistencies between laboratory
methods were observed. These were the following.
a) Blood preparation and anticoagulation use: the blood used for test material exposure
varied considerably between laboratories. For example, special commercially available human
serum, fresh and frozen citrated human plasma, fresh human serum and directly exposed
fresh heparinized whole human blood were used. The impact of these various preparations on
complement results has not been evaluated, with the exception of use of EDTA as anticoagulant.
For the latter, it is generally well known that complement activation through the classical pathway
is calcium-dependent and through the alternative pathway is magnesium-dependent. Thus, use of
potent calcium/magnesium chelators such as EDTA will bind up available calcium and magnesium
and shut off complement activation, making most measurements using these anticoagulants result
in close to background levels of complement proteins.
With the above said, there is no standard blood or blood preparation currently identified for
use in complement testing. However, when serum is used, serum should be functionally intact
and retain the capacity of showing complement activation. If whole blood or plasma is used, the
type of anticoagulant should be carefully selected to ensure that it does not inhibit or potentiate
complement activation caused by the test device itself.
b) Ratio of test article surface area to blood (whole, plasma or serum) volume: some laboratories
specify following ISO 10993‑12 ratios where the blood (whole, plasma or serum) volume is used in
place of the extract fluid. Some laboratories do not specify a ratio and/or a variable ratio is used.
As complement activation is influenced by surface area (SA), standardized and reported SAs are
important to inter- and intralaboratory interpretation of results.
c) Use of controls: use of a negative biomaterial control, such as polypropylene, and a positive
biomaterial control, such as latex or cellulose acetate, was somewhat consistent; use of a liquid
negative control, such as saline, and a positive liquid control, such as cobra venom factor, was
inconsistent; use of negative and positive controls provided in the commercially available kits was
fairly consistent.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

d) Complement activation on blood-contacting medical devices is recognized to be a surface

phenomenon. As such, testing with a device extract is not appropriate. Rather, complement testing on
devices or materials should always be conducted using a [blood or blood-constituent] direct-contact
method. A negative control material like polyethylene (PE) should be included in the assay along with a
positive control material such as non-modified cellulose, e.g. Cuprophan2) 2 Cuprophan is an example of
a suitable product available commercially. This information is given for the convenience of users of this
document and does not constitute and endorsement by ISO of this product.
e) Standard curve preparation/dilutions: laboratories used different dilutions of the standard
to generate a standard curve that captured most levels of test and control samples. For example,
dilutions of 1:100, 1:200, 1:1 000 and 1:10 000 on the highest standard were reported. The most
important factor here is that all samples ultimately lie between the low and high values on the
standard curve.
f) Incubation period for test articles and controls: incubation periods varied between
laboratories, with laboratories reporting use of 60 min, 90 min or 30 min and 60 min and 90 min
incubation periods.
g) Incubation time following addition of chromogenic substrate: 15 min to 30 min incubations
periods were reported.
h) Test sample evaluation: the method of assessing whether a test material gave a positive or negative
result was inconsistent between laboratories. Most laboratories made a statistical comparison of
test sample results to positive and negative biomaterial and/or liquid controls. Some laboratories
included a comparison with historical values, results on a predicate device and/or a special
mathematical formula involving negative and positive controls as part of the final assessment.
There are no established pass/fail criteria for a clinically acceptable level of complement activation.
Inclusion of a legally marketed comparator device in the complement activation testing would be helpful
for data interpretation. The comparator device data could be used to evaluate the clinical relevance of
the test device data.
If the comparator device is not legally marketed in the regulatory region where the device will be
submitted for marketing, regional regulatory authorities may request comparator testing for a device
already legally marketed in that region.
2) Cuprophan is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute and endorsement by ISO of this product.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex F
(informative)
Less common laboratory tests
F.1 General
This annex and Table F.1 describe tests that have been used primarily in research to assess
device/material interaction with blood. These tests have not, however, seen widespread use in
regulatory device submissions. The tests mentioned here are for informational purpose with the caveat
that they may not be standardized nor correlated for clinical relevance. As a medical device preclinical
biological evaluation strategy should focus on the most meaningful and widely accepted tests (see
Annex B), caution is advised in including any Annex F methods in device submissions. Laboratory tests
that are clearly not recommended can be found in Annex G.
Table F.1 — Less common tests used to assess interactions with blood
Tests by categoriesa
Thrombosis Flow reduction, gravimetric analysis, pressure drop
across device, adsorbed protein analysis, imaging
techniques
In vitro thromobosis
Coagulation thrombin generation assay using chromogenic
substrates, fibrinogen and fibrin degradation products
(FDP), D-dimer
Platelets platelet adhesion assessments, flow cytometry
analysis of platelet activation, platelet microparticle
formation, gamma imaging of radiolabelled platelets,
platelet aggregometry
Haematology Leucocyte activation by flow cytometry, blood cell
adhesion assessments, platelet-leucoyte complexes
(PLCs)
Complement system Bb, C3bBb, C5a
a Because of biological variability and technical limitations, the accuracy and predictivity
of many of these tests, which are most commonly used for research purposes, requires
careful attention to methodology and caution in interpretation of results.
F.2 Thrombosis
F.2.1 Flow reduction
Flow (rate or volume) is measured after a period of use. Measurements may be performed either during
use or before and after use. Rationale and interpretation are the same as for B.2.1.
F.2.2 Gravimetric analysis (thrombus mass)

This is conducted after removal of the device from the in-use position. Rationale and interpretation are
as described in B.2.1. Here, the difference in weight between the pre-implant weight of the device and
the weight immediately post-implant may reflect the amount of thrombus present. An important caveat
is that all tissue present may not be thrombus.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

F.2.3 Pressure drop across device

This is measured before and after a period of use.
F.2.4 Adsorbed protein analysis (via antibody binding)

The protein adsorption onto test materials or devices that occurs after contact with blood, e.g. the
first layer or the surface layer upon reaching equilibrium, is thought to potentially impact device
performance and/or clinical outcome. Next to qualitative microscopic judgement of fibrin and platelet
deposition on materials, a quantitative estimation of surface protein is possible by measuring the
amount of labelled antibody specific for proteins such as fibrinogen or platelet membrane receptors. For
this purpose, materials are first washed after exposure to blood to remove non-adherent proteins and
blood components. The surfaces or extracts of the surfaces are then combined with labelled antibody
binding for qualitative or quantitative analysis. Additionally, it is possible to measure the total amount
of adsorbed protein directly without using antibodies[164][181][182][183].
F.2.5 Imaging techniques — Angiography, intravascular ultrasound, Doppler

ultrasound, CT and MRI
Choices can be made among these methods to determine patency or degree of narrowing of a graft or
other conduit and to detect thrombus deposition on devices during their in vivo performance.
F.3 Coagulation
F.3.1 Thrombin generation assay using chromogenic substrates
Materials exposed to an intact coagulation system in the presence of phospholipids will generate
thrombin which can be measured by conversion of a chromogenic substrate[61][62][63].
F.3.2 Fibrinogen and fibrin degradation products (FDP)

Normal physiological fibrinolysis yields the FDPs X, Y, C, D and E in concentrations below 2 mg/ml of
plasma. The normally low level of FDPs is maintained by the low rate of the degradation reaction and
the high rate of clearance of FDPs from the circulation. Pathologic degradation of fibrin and fibrinogen,
a result of increased plasminogen activation, yields FDP of 2 mg/ml to 40 mg/ml or more[64]. The test is
mainly useful for evaluating implant devices. The use of commercially available methods such as ELISA
is recommended.
Dysfibrinogenaemia, afibrinogenaemia and hypofibrinogenaemia cause prolonged PT, PTT and TT
results[45].
F.3.3 D-dimer
An elevated level of D-dimer indicates activation of the coagulation mechanism. D-dimers are plasmin
digested degradation products of FXIII cross-linked fibrin (coagulation and fibrinolysis). The use of
ELISA and/or RIA assay is recommended for quantifying such proteins[65][66].
F.4 Platelets
F.4.1 Platelet adhesion assessments
Blood cell adhesion[167] is a measure of the blood compatibility of a material when considered in
conjunction with distal embolization or evidence of activation of one or more haematological factors.
Various methods have been designed to measure the adhesion of cells to surfaces, for example the
Kunicki K-score[75]. Most of these methods are based on the observation that a certain proportion of

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

platelets are removed from normal whole blood as a result of passage through a column of glass beads
under controlled conditions of flow or pressure.
An alternative method is the direct counting of platelets adherent to a test surface. Following exposure
to blood or platelet-rich plasma under standardized conditions, the test surface is rinsed to remove
non-adherent cells, fixed and prepared for either light or scanning electron microscopy. The number
of adherent platelets per unit area is directly counted and their morphology (e.g. amount of spreading,
degree of aggregate formation) is recorded. Alternatively, platelets pre-labelled with 51Cr or 111In
may be used[70][71][73]. An alternative non-isotope method, the LDH and the acid phosphatase methods,
which assess enzyme activity in the bulk after lysis of adhered platelets, has also been reported as a
useful tool to assess platelets on surfaces[83][84].
F.4.2 Flow cytometry analysis of platelet activation

The use of certain materials or devices may cause platelet activation and the expression of activation
markers at the platelet surface or the generation of platelet micro-particles[173]. Platelet surface
activation markers have been evaluated by flow cytometry for P-selectin (GMP-140) expression or
activated glycoprotein Ib and IIB/IIIa expression using monoclonal antibodies. Different epitopes of
activated platelets are recognized by flow cytometry using two antibodies: one specific for platelets
(i.e. GP Ib or GP IIb/IIIa) and one specific for platelet activation (P-Selectin)[69].
F.4.3 Gamma imaging of radiolabelled platelets

The high gamma emission of 111In enables it to be used for this purpose[46][47][50][72]. This method
enables the localization and quantification of platelets deposited on a device. The technique is useful for
external communicating as well as implant devices.
F.4.4 Platelet aggregometry

Platelet aggregation[74] is induced by adding aggregating agents (e.g. ADP, epinephrine, collagen and
thrombin) to platelet-rich plasma (PRP) that is being stirred continually. As the platelets aggregate, the
plasma becomes progressively clearer. An optical system (platelet aggregometer) is used to detect the
change in light transmission and a recorder graphically displays the variations in light transmission
from the baseline setting. Delayed or reduced platelet aggregation can be caused by platelet activation
and release of granular contents, increased FDP or certain drugs (e.g. aspirin, nonsteriod anti-
inflammatory drugs). It is important to bear in mind that platelet aggregation using some agents varies
or may be absent in some animal species. Spontaneous platelet aggregation, occurring in the absence of
added agonists, is an abnormal condition indicating activation of platelets. Platelet aggregates can also
be screened by the WU/HOAK method[79].
F.5 Haematology
F.5.1 Leucocyte state and morphology
Change in leukocyte state of activation can be determined by flow cytometry for the evaluation
of increased leukocyte markers, such as L-selectin and CD 11b, and by quantitative disturbances in
lymphocyte subpopulations. It is also possible to assess leukocyte activation through evaluation of
morphological changes leukocytes undergo when activated on a medical device surface. This is usually
performed via SEM[173].
F.5.2 Blood cell adhesion assessments

Blood cell adhesion[167] is a measure of the blood compatibility of a material when considered in
conjunction with distal embolization or evidence of activation of one or more haematological factors.
By such a method, it has been reported[167] that adhesion of canine species peripheral lymphocytes and
PMNs to beads coated with poly(hydroxyethyl methacrylate) (PHEMA) is lower than to beads coated
with polystyrene and certain other polymers. Isolated lymphocytes and PMNs were used in this study.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

F.5.3 Platelet leucocyte complexes (PLCs)

PLCs can be measured by flow cytometry and may be an indicator of white blood cell and platelet
activation following exposure to medical devices and materials[85].
F.6 Complement system

F.6.1 Complement activation assessment of Bb, C3bBb and C5a
Of these three complement proteins, the C5a fragment is considered one of the most critical complement
factors in blood-contacting device-related complement activation[145]. However, routine testing for C5a
is not required given the low sensitivity of commercially available ELISA kits for in vitro assessment of
this protein.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Annex G
(informative)
Tests which are not recommended
G.1 General
The tests described in Table G.1 and below are tests that are generally not used or accepted by
regulatory authorities as part of a preclinical assessment of a blood-contacting medical device safety
evaluation. These tests are considered either outdated or of insufficient/not applicable scientific merit
for such evaluations.
Table G.1 — Tests not used in preclinical assessment of medical device safety
Tests by categories
In vitro haemocompatibility
Coagulation APTT, PT and TT
Platelets template bleeding time, platelet lifespan (survival)
Haematology reticulocyte count
Complement system CH-50, C3 convertase, C5 convertase
G.2 Coagulation
G.2.1 Activated partial thromboplastin time (APTT), prothrombin time (PT) and
thrombin time (TT)
These tests generally measure coagulation disorders normally associated with abnormal levels of
patient clotting factors.
Partial thromboplastin reagents using various activating substances, such as kaolin or celite, are
commercially available. Using these reagents, the test is called the activated partial thromboplastin
time (APTT). The APTT is rarely useful in the in vitro evaluation of the thrombogenic properties of
blood-contacting devices/materials because the activating substances mask any activation caused by
the device or its component materials.
They are not in general used in the assessment of medical devices and/or materials that contact blood.
G.3 Platelets
G.3.1 Template bleeding time
The commercial availability of a sterile disposable device for producing a skin incision of standard
depth and length under standard conditions has significantly improved the reproducibility and value
of this test[67]. A prolonged result indicates reduced platelet function or reduced platelet count; the
latter can be determined separately. A prolonged bleeding time combined with a normal platelet count
has been observed in association with some external communicating devices with limited exposure
(e.g. cardiopulmonary bypass)[158]. The test is suitable for use with some experimental animals. In
vitro bleeding time measurements are also suitable. This test is not used in the assessment of medical
devices and/or materials that contact blood.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

G.3.2 Platelet lifespan

The high gamma emission of 111In platelets are obtained from the patient’s blood and are labelled with
51Cr or 111In[46][15][72][148]. Both these agents label platelets of all ages present in the sample, do not
elute excessively from the platelets and are not taken up by other cells or reused during thrombogenesis.
111In has the advantage of being a high gamma emitter, requiring the labelling of fewer platelets and
enabling surface body counting to assess localized platelet deposition to be combined with the lifespan
study. A reduced platelet lifespan indicates accelerated removal from the circulation by immune,
thrombotic or other processes. This test is not recommended in the non-clinical assessment of medical
devices and/or materials that contact blood.
G.4 Haematology
G.4.1 Reticulocyte count
An elevated reticulocyte count indicates increased production of erythrocytes in the bone marrow. This
may be in response to reduced erythrocyte mass caused by chronic blood loss (bleeding), haemolysis
or other mechanisms[55][77][111]. This test is not used in the assessment of medical devices and/or
materials that contact blood.
G.5 Complement system

G.5.1 Complement activation assessment of CH-50, C3 convertase, C5 convertase
A decrease in CH-50 is an indicator of total complement consumption. Elevated levels of any of these
complement components indicate activation of the complement system. Some materials activate
complement and activated complement components in turn activate leukocytes, causing them to
aggregate and be sequestered in the lungs[129][130][132][137].
Measurement of complement split products has the disadvantage of species specificity and high
baseline levels when performed after in vitro testing. The classical CH-50 method appears useful with
human, bovine, porcine and rabbit serum.
Another functional method of measurement of complement activation in vitro is the generation of
complement C3- or C5-convertase, determined by substrate conversion. Commercially available ELISA
kits for key complement components also exist.
These tests are in general not used in the assessment of complement activation of blood contacting
medical devices and/or materials.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Bibliography
International Standards
[1] ISO 3826‑3, Plastics collapsible containers for human blood and blood components — Part 3: Blood
bag systems with integrated features
[2] ISO 5840 (all parts), Cardiovascular implants — Cardiac valve prostheses
[3] ISO 5841‑3, Implants for surgery — Cardiac pacemakers — Part 3: Low-profile connectors (IS-1) for
implantable pacemakers
[4] ISO 7198, Cardiovascular implants and extracorporeal systems — Vascular prostheses — Tubular
vascular grafts and vascular patches
[5] ISO 7199, Cardiovascular implants and artificial organs — Blood-gas exchangers (oxygenators)
[6] ISO 10993 (all parts), Biological evaluation of medical devices
[7] ISO 12891‑1, Retrieval and analysis of surgical implants — Part 1: Retrieval and handling
[8] ISO 14708‑2, Implants for surgery — Active implantable medical devices — Part 2:
Cardiac pacemakers
[9] ISO 14708‑5, Implants for surgery — Active implantable medical devices — Part 5: Circulatory
support devices
[10] ISO 15674, Cardiovascular implants and artificial organs — Hard-shell cardiotomy/venous reservoir
systems (with/without filter) and soft venous reservoir bags
[11] ISO 15675, Cardiovascular implants and artificial organs — Cardiopulmonary bypass systems —
Arterial blood line filters
[12] ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories
[13] ISO 8637, Cardiovascular implants and extracorporeal systems — Haemodialysers, haemodiafilters,
haemofilters and haemoconcentrators
National standards
[14] Guidelines for blood/material interactions. Report of the National Heart, Lung, and Blood
Institute Working Group, U.S. Department of Health and Human Services, Public Health Service,
National Institutes of Health. NIH Publication No. 85-2185, revised September 1985. Available
from Biomaterials Program, Bioengineering Research Group, Division of Heart and Vascular
Diseases, NHLBI, Two Rockledge Center, 6701 Rockledge Drive, Bethesda, MD 20892, USA
[15] Harker L.A., Ratner B.D., Didisheim P. eds. Cardiovascular Biomaterials and Biocompatibility.
Supplement to Cardiovasc. Pathol. 2 (3). Suppl, Jul-Sept 1993, pp. 1S–224S.
[16] ANSI/AAMI RD16. Hemodialyzers, hemodiafilters, and hemoconcentrators. In: International
Organization for Standardization and Association for the Advancement of Medical
Instrumentation, editor. Cardiovascular implants and artificial organs. American National
Standards Institute; 2007. p. 30
[17] ASTM F756‑13, Standard practice for assessment of hemolytic properties of materials
[18] ASTM F1984‑99, Standard practice for testing whole blood complement activation in serum by
solid materials
[19] ASTM F2065‑00E1, Standard practice for testing for alternative pathway complement activation in
serum by solid materials

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[20] ASTM E2524‑08(2013), Standard test method for analysis of hemolytic properties of nanoparticles
[21] GB/T 16175, Biological evaluation test methods for medical organic silicon materials
[22] MHLW Notification by Director, OMDE, Yakushokuki-hatsu 0301 No. 20, March 1, 2012, Basic
Principles of Biological Safety Evaluation Required for Application for Approval to Market
Medical Devices
[23] ASTM 2382, Standard test method for assessment of intravascular medical device materials on
partial thromboplastin time (PTT)
[24] ASTM F2888‑13, Standard test method for platelet leukocyte count — An in-vitro measure for
haemocompatibility assessment of cardiovascular materials
[25] Anticoagulant Sodium Citrate Solution U.S.P. In: Larry Callahan, editor. USP 28: (USP Citric
Acid RS); Assay. U.S. Pharmacopeia; 2006d. p 168; PF 31(3), p. 731
[26] NIH. Public Health Service Policy on Humane Care and Use of Laboratory Animals; Health
Research Extension Act of 1985: Public Law 89-544. In: Office of Extramural Research; OLAW,
editor. Office of Laboratory Animal Welfare; 2002. (a/k/a OLAW, 2002)
[27] USDA. 9 CFR: Code of Federal Regulations: Chapter 1; Subchapter A - Animal Welfare. In: Animal
Welfare Information Center U, editor. Government Printing Office; 2004
[28] NIH. Evaluation of hemodialyzers and dialysis membranes. Report of a Study Group for the
Artificial Kidney-Chronic Uremia Program NIAMDD-1977. Chapter two. In vitro characterization
of hemodialyzers. Artif. Organs. 1977, 1 (2) pp. 59–77
[29] ASTM F1830‑97, Standard practice for selection of blood for in vitro evaluation of blood pumps
[30] ASTM F1841‑97, Standard practice for assessment of haemolysis in continuous flow blood pumps
US FDA guidance documents
[31] CDRH. Class II special controls guidance document for certain percutaneous transluminal
coronary angioplasty (PTCA) catheters—Document No. 1605. In: Office of Device Evaluation
(ODE); Division of Cardiovascular Devices; Interventional Cardiology Devices Branch, editor.
DRAFT guidance for industry and FDA staff: FDA; 2008. p. 34
[32] CDRH. Class II special controls guidance document: indwelling blood gas analyzers—Document
No. 1126. In: Office of Device Evaluation (ODE); Division of Cardiovascular and Respiratory
Devices; Anesthesiology and Respiratory Devices Branch, editor. Final guidance for industry
and FDA staff: FDA; 2001. p. 16
[33] CDRH. Coronary drug-eluting stents: nonclinical and clinical studies. In: Center for Devices and
Radiological Health (CDRH); Center for Drug Evaluation and Research (CDER); and Office of
Combination Products (OCP), editor. DRAFT guidance for industry: FDA; 2008. p. 89
[34] CDRH. Coronary drug-eluting stents: nonclinical and clinical studies—Companion Document
No. 6255. In: Center for Devices and Radiological Health (CDRH); Center for Drug Evaluation
and Research (CDER); and Office of Combination Products (OCP), editor. DRAFT guidance for
industry: FDA; 2008. p. 32
[35] CDRH. Guidance for annuloplasty rings 510(k) submissions—Document No. 1358. In: Office
of Device Evaluation (ODE); Division of Cardiovascular and Respiratory Devices; Circulatory
Support and Prosthetic Devices Branch, editor. Final guidance for industry and FDA staff:
FDA; 2001. p. 17
[36] CDRH. Guidance for cardiopulmonary bypass arterial line blood filter 510(k) submissions—
Document No. 1622. In: Office of Device Evaluation (ODE); Division of Cardiovascular
Respiratory and Neurology Devices; Circulatory Support and Prosthetic Devices Branch, editor.
Final guidance for industry and FDA staff: FDA; 2000. p. 8

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[37] CDRH. Guidance for cardiopulmonary bypass oxygenators 510(k) submissions—Document No.
1361. In: Office of Device Evaluation (ODE); Division of Cardiovascular and Respiratory Devices;
Circulatory Support and Prosthetic Devices Branch, editor. Final guidance for industry and FDA
staff: FDA; 2000. p. 23
[38] G95-1. 1997 Blue Book Memorandum: Use of International Standard ISO-10993, “Biological
Evaluation of Medical Devices Part 1: Evaluation and Testing” (Replaces #G87-1 #8294). FDA
<http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/
ucm080735.htm>. Accessed 04/15/ 2010
[39] Tripartite Subcommittee for Medical Devices. Tripartite Biocompatibility Guidance
for Medical Devices. In: Office of Device Evaluation (ODE) in FDA's Center for Devices and
Radiological Health (CDRH), editor. General Program Memorandum #G87-1. FDA; 1986
[40] FDA. 21 CFR 58(b) (3): Good Laboratory Practices (GLP) for Non-Clinical Laboratory
Studies, In: Department of Health and Human Services, editor. Volume 1, Code of Federal
Regulations. FDA; 2004
[41] Implanted Blood Access Devices for Hemodialysis - Draft Guidance for Industry
and Food and Drug Administration Staff. January 21, 2016, available at http://www.fda.
gov/ucm/groups/fdagov-public/@fdagov-meddev-gen/documents/document/ucm308598.pdf
Thrombosis
[42] Bosch T., Schmidt B., Blumenstein M., Gurland H.J. Thrombogenicity markers in clinical
and ex vivo assessment of membrane biocompatibility. Contrib. Nephrol. 1987, 59 pp. 90–98
[43] CHANDLER A.B.. In vitro thrombotic coagulation of the blood; a method for producing a
thrombus. Lab. Invest. 1958, 7 (2) pp. 110–114
[44] Cooper S.L., Fabrizius D.J., Grasel T.G. Methods of assessment of thrombosis ex vivo. Ann. N.
Y. Acad. Sci. 1987, 516 pp. 572–585
[45] Corriveau D.M., & Fritsma G.A. Hemostasis and thrombosis in the clinical laboratory.
Lippincott, Philadelphia, 1988, 443 p.
[46] Dewanjee M.K. Methods of assessment of Thrombosis in vivo. In: Blood in contact with natural
and artificial surfaces: Annals of the New York Academy of Sciences, (Leonard E.F., Turitto
V.T., Vroman L. eds.). New York Academy of Sciences, New York, N.Y., 1987, pp. 541–71.
[47] Dewanjee M.K., Kapadvanjwala M., Sanchez A., Elson R., Serafini A.N., Zilleruelo G.E.
Quantitation of comparative thrombogenicity of dog, pig, and human platelets in a hemodialyzer.
ASAIO J. 1992, 38 (2) pp. 88–90
[48] Didisheim P., Olsen D.B., Farrar D.J., Portner P.M., Griffith B.P., Pennington D.G.
Infections and thromboembolism with implantable cardiovascular devices. ASAIO Trans. 1989,
35 (1) pp. 54–70
[49] Grotemeyer K.H., Viand R., Beykirch K. [Thrombocyte function in vasomotor and migraine
headaches]. Dtsch. Med. Wochenschr. 1983, 108 (20) pp. 775–778
[50] Harker L.A., Kelly A.B., Hanson S.R. Experimental arterial thrombosis in nonhuman primates.
Circulation. 1991, 83 (6, Suppl) pp. IV41–IV55
[51] Hoch J.R., & Silver D. Hemostasis and Thrombosis. In: Moore WS, editor. Vascular Surgery: A
Comprehensive Review. 3rd ed. Philedelphia: W. B. Saunders; 1991. pp. 63-79
[52] Kay L. Essentials of Haemostasis and Thrombosis. Churchill Livingstone, Edinburgh,
1988, 290 p.

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[53] Lewis J., Sweeney J., Baldini L., Friedland G.H., Salzman E.W. Assessment of
thromboresistance of intravenous cannulae by 125I-fibrinogen scanning. J. Biomed. Mater. Res.
1985, 19 (2) pp. 99–113
[54] Zingg W., Ip W.F., Sefton M.V., Mancer K. A chronic arteriovenous shunt for the testing of
biomaterials and devices in dogs. Life Support Syst. 1986, 4 (3) pp. 221–229
Coagulation
[55] Henry J.B.Haematology and Coagulation. In: Clinical Diagnosis and Management By
Laboratory Methods. (Henry J.B. ed.). W. B. Saunders, Philadelphia, Eighteenth Edition, 1991,
pp. 556–603.
[56] Brummel-Ziedens K.E., Orfeo T., Rosendaal F.R., Undas A., Rivard G.E., Butenas S. Empirical
and theoretical phenotype discrimination. J. Thromb. Haemost. 2009, 7 (1) pp. 181–186
[57] Brummel-Ziedens K.E., Vossen C.Y., Rosendaal F.R., Umezaki K., Mann K.G. The plasma
hemostatic proteome: thrombin generation in healthy individuals. J. Thromb. Haemost. 2005, 3
pp. 1472–1481
[58] Boisclair M.D., Lane D.A., Wilde J.T., Ireland H., Preston F.E., Ofosu F.A A comparative
evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin-
antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen. Br. J. Haematol. 1990,
74 (4) pp. 471–479
[59] Pelzer H., Schwarz A., Heimburger N. Determination of human thrombin-antithrombin
III complex in plasma with an enzyme-linked immunosorbent assay. Thromb. Haemost. 1988,
59 (1) pp. 101–106
[60] Sommeijer D.W., Van Oerle R., Reitsma P.H., Timmerman J.J., Meijers J.C., Spronk H.M., Ten
, Cate H. Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a
home-made assay for thrombin-antithrombin complexes. Thromb. J. 2005, 3 p. 12
[61] Gatt A., van Veen J.J., Woolley A.M., Kitchen S., Cooper P., Makris M. Thrombin generation
assays are superior to traditional tests in assessing anticoagulation reversal in vitro. Thromb.
Haemost. 2008, 100 pp. 350–355
[62] Hemker H.C., Giesen P., Al Dieri R., Regnault V., de Smedt E., Wagenvoord R. Calibrated
automated thrombin generation measurement in clotting plasma. Pathophysiol. Haemost.
Thromb. 2003, 33 pp. 4–15
[63] Hemker H.C., Giesen P., AlDieri R., Regnault V., de Smed E., Wagenvoord R. The calibrated
automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability.
Pathophysiol. Haemost. Thromb. 2002, 32 pp. 249–253
[64] Gaffney P.J., Edgell T., Creighton-Kempsford L.J., Wheeler S., Tarelli E. Fibrin
degradation product (FnDP) assays: analysis of standardization issues and target antigens in
plasma. Br. J. Haematol. 1995 May, 90 (1) pp. 187–194
[65] Nieuwenhuizen W. A reference material for harmonisation of D-dimer assays. Fibrinogen
subcommittee of the Scientific and Standardization Committee of the International Society of
Thrombosis and Hemostasis. Thromb. Haemost. 1997, 77 pp. 1031–1033
[66] Spannagl M., Haverkate F., Reinauer H., Meijer P. The performance of quantitative D-dimer
assays in laboratory routine. Blood Coagul. Fibrinolysis. 2005, 16 pp. 439–443
[67] Lethagen S., & Kling S. New bleeding time devices with retractable blades evaluated in
children, healthy volunteers and patients with prolonged bleeding time. Thromb. Haemost.
1993 Oct 18, 70 (4) pp. 595–597
[68] Kottke-Marchant K. Performance and interpretation of routine coagulation assays. In:
Laboratory Haematology Practice. Wiley. Blackwell, Oxford, England, May 15, 2012, pp. 420–34

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Platelets
[69] Chignier E., Parise M., Mcgregor L., Delabre C., Faucompret S., Mcgregor J. A
P-selectin/CD62P monoclonal antibody (LYP-20), in tandem with flow cytometry, detects in
vivo activated circulating rat platelets in severe vascular trauma. Thromb. Haemost. 1994,
72 (5) pp. 745–749
[70] Goodman S.L., Lelah M.D., Lambrecht L.K., Cooper S.L., Albrecht R.M. In vitro vs. ex vivo
platelet deposition on polymer surfaces. Scan. Electron Microsc. 1984, (Pt 1) pp. 279–290
[71] Goodman S.L., Cooper S.L., Albrecht R.M. Activation of platelets from humans, canines, and
macaques on polymer surfaces. In: Nosé Y, Kjellstrand C.M., Ivanovich P., International Society
for Artificial Organs. World Congress, editors. Progress in artificial organs, 1985. Cleveland:
ISAO Press; 1986. p. xxi, 1204
[72] ICSH. Recommended method for indium-111 platelet survival studies. International Committee
for Standardization in Haematology. Panel on Diagnostic Applications of Radionuclides. J. Nucl.
Med. 1988, 29 (4) pp. 564–566
[73] Karwath R., Schurer M., Wolf H. Measurement of platelet adhesiveness onto artificial
surfaces using Cr-51 and In-111 labeled platelets. Studia Biophysica. 1989, 131 pp. 117–123
[74] Kundu S.K., Heilmann E.J., Sio R., Garcia C., Ostgaard R.A. Characterization of an In vitro
Platelet Function Analyzer, PFA-100TM. Clin. Appl. Thromb. Hemost. 1996, 2 (4) pp. 241–249
[75] Kunicki T.J., Tuccelli M., Becker G.A., Aster R.H. A study of variables affecting the quality of
platelets stored at “room temperature”. Transfusion. 1975, 15 (5) pp. 414–421
[76] Lewis S.M., Rowan R.M., Kubota F. Evaluation of a prototype for a reference platelet counter.
J. Clin. Pathol. 1990, 43 (11) pp. 932–936
[77] NCCLS. Methods for reticulocyte counting (Flow cytometry and supervitwal dyes); Approved
Guideline (H44-A, Vol. 17 No. 15). Volume H44-A, Vol. 17. Clinical and Laboratory Standards
Institute; 1997
[78] Palatianos G.M., Dewanjee M.K., Robinson R.P., Novak S., Dewanjee P.K., Kapadvanjwala
M., Hsu L.C., Sfakianakis G.N., Kaiser G.A. Quantitation of platelet loss with indium-111
labeled platelets in a hollow-fiber membrane oxygenator and arterial filter during extracorporeal
circulation in a pig model. ASAIO Trans. 1989, 35 (3) pp. 667–670
[79] Wu K.K., & Hoak J.C. A new method for the quantitative detection of platelet aggregates in
patients with arterial insufficiency. Lancet. 1974, 2 (7886) pp. 924–926
[80] Zingg W., Ip W.F., Sefton M.V., Mancer K. A chronic arteriovenous shunt for the testing of
biomaterials and devices in dogs. Life Support Syst. 1986, 4 (3) pp. 221–229
[81] Best Collaborative. Platelet radiolabelling procedure: The Biomedical Excellence for Safer
Transfusion (BEST) Collaborative. Transfusion. 2006, 46 (Suppl) pp. 59–66
[82] Holme S., Heaton A., Roodt J. Concurrent label method with 111In and 51Cr allows accurate
evaluation of platelet viability of stored concentrates. Br. J. Haematol. 1993, 84 pp. 717–723
[83] Fushimi F., Nakayama M., Nishimura K., Hiyoshi T. Platelet adhesion, contact phase
coagulation activation, and C5a generation of polyethylene glycol acid-grafted high flux cellulosic
membrane with varieties of grafting amounts. Artif. Organs. 1998 Oct, 22 (10) pp. 821–826
[84] Tamada Y., Kulik E., Ikada Y. Simple method for platelet counting, , in Biomaterials, Vol 16,
Issue 3, 1995, pp. 259-261

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[85] Gorbet M.B., & Sefton M.V. Biomaterial-associated thrombosis: roles of coagulation factors,
complement, platelets and leukocytes, Maud B. Gorbet, Michael V. Sefton, in Biomaterials 25
(2004), pp. 5681–5703
[86] Kottke-Marchant K. Clinical perspectives on platelet function testing. Medical Laboratory
Observer, May, 44(5): 8-14 (2012)
[87] Anderson J.M., & Kottke-Marchant K. Platelet interactions with biomaterials and artificial
devices. CRC Critical Reviews in Biocompatibility. 1985, 1 pp. 111–204
[88] Schmidt V., & Hilberg T. ThromboFix platelet stabilizer: advances in clinical platelet analyses
by flow cytometry? Platelets. 2006 Jun, 17 (4) pp. 266–273
[89] Hu H., Daleskog M., Li N. Influences of fixatives on flow cytometric measurements of platelet
P-selectin expression and fibrinogen binding. Thromb. Res. 2000 Nov 1, 100 (3) pp. 161–166
[90] Muriithi E.W., Belcher P.R., Menys V.C., Chaudhry M.A., Raco L., Day S.P. Quantitative
detection of platelet aggregates in whole blood without fixation. Platelets. 2000 Feb,
11 (1) pp. 33–37
[91] Macey M., Azam U., McCarthy D., Webb L., Chapman E.S., Okrongly D. Evaluation of
the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for
assessing platelet activation on the ADVIA 120 haematology system. Clin. Chem. 2002 Jun, 48 (6
Pt 1) pp. 891–899
[92] Dimitrievska S., Maire M., Diaz-Quijada G.A., Robitaille L., Ajji A., Yahia L. Low
thrombogenicity coating of nonwoven PET fiber structures for vascular grafts. Macromol.
Biosci. 2011 Apr 8, 11 (4) pp. 493–502
[93] De Somer F., Van Belleghem Y., Foubert L., François K., Dubrulle F., De Wolf D. In vivo
evaluation of a phosphorylcholine coated cardiopulmonary bypass circuit. J. Extra Corpor.
Technol. 1999 Jun, 31 (2) pp. 62–66
[94] Cenni E., Granchi D., Verri E., Cavedagna D., Gamberini S., Falsone G. CD62,thromboxane
B2, and beta-thromboglobulin: a comparison between different markers of platelet activation
after contact with biomaterials. J. Biomed. Mater. Res. 1997 Sep 5, 36 (3) pp. 289–294
Haematology (including haemolysis)
[95] Coldman M.F., Gent M., Good W. The identical effect of electrolyte and non-electrolyte on the
osmotic fragility of mammalian erythrocytes. Comp. Biochem. Physiol., 1970, Pergamon Press,
printed in Great Britain, Vol. 33, pp. 157-165
[96] Coldman M.F., Gent M., Good W. Relationships between osmotic fragility and other species-
specific variables of mammalian erythrocytes, Comp. Biochem. Physiol., 1970, Pergamon Press,
printed in Great Britain, Vol. 34, pp. 759-772
[97] Matsuzawa T., Ikarashi Y. Haemolysis of various mammalian erythrocytes in sodium chloride,
glucose and phosphate-buffer solutions. Lab. Anim. 1979, 13 pp. 329–331
[98] Coldman M.F., Gent M., Good W. The osmotic fragility of mammalian erythrocytes in hypotonic
solutions of sodium chloride. Comp. Biochem. Physiol., 1969, Pergamon Press, printed in Great
Britain, Vol. 31, pp. 605-609
[99] AABB. Standards for blood banks and transfusion services. In: AABB Committee on Standards,
editor. Washington, DC: American Association of Blood Banks 1994. p. v
[100] Bednar R., Bayer P.M. Correction: measurements of plasma hemoglobin. Clin. Chem. 1993,
39 (9) pp. 2027–2028

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[101] Council of Europe. Recommendation No. R (95) 15: Guide to the preparation, use and quality
assurance of blood components. In: Committee of Ministers to Member States, editor. 13th ed.
13th ed. Strasbourg: Council of Europe Pub.; 2007. p. 271
[102] Cripps C.M. Rapid method for the estimation of plasma haemoglobin levels. J. Clin. Pathol. 1968,
21 (1) pp. 110–112
[103] EDQM. Sterile plastic containers for human blood and blood components (3.2.3). European
Pharmacopoeia 5.0: European Directorate for the Quality of Medicines and Healthcare;
1997b. pp. 175-179
[104] Fairbanks V.F., Ziesmer S.C., O’Brien P.C. Methods for measuring plasma hemoglobin in
micromolar concentration compared. Clin. Chem. 1992, 38 (1) pp. 132–140
[105] Harboe M. A method for determination of hemoglobin in plasma by near-ultraviolet
spectrophotometry. Scand. J. Clin. Lab. Invest. 1959, 11 (1) pp. 66–70
[106] Henry J.B Haematology and Coagulation. In: Clinical Diagnosis and Management by
Laboratory Methods. (Henry J.B. ed.). W. B. Saunders, Philadelphia, Eighteenth Edition, 1991,
pp. 556–603.
[107] Lammers M., Gressner A.M. Immunonephelometric quantification of free haemoglobin. J. Clin.
Chem. Clin. Biochem. 1987, 25 (6) pp. 363–367
[108] Lampert R.H., Williams M.C. Effect of surface materials on shear-induced haemolysis. J.
Biomed. Mater. Res. 1972, 6 (6) pp. 499–532
[109] Malinauskas R.A. Plasma hemoglobin measurement techniques for the in vitro evaluation of
blood damage caused by medical devices. Artif. Organs. 1997, 21 (12) pp. 1255–1267
[110] Miale J.B. Laboratory medicine, haematology. St. Louis: Mosby; 1982. [64] p. of plates; xi, 1084 pp
[111] Mosby Year Book. A Color Atlas of Comparative, Diagnostic and Experimental Haematology.
Mosby-Year Book Europe Ltd, London, England, 1994
[112] Obeng E.K., Cadwallader D.E. In vitro dynamic method for evaluating the hemolytic potential
of intravenous solutions. J. Parenter. Sci. Technol. 1989, 43 (4) pp. 167–173
[113] Offeman R.D., Williams M.C. Material effects in shear-induced haemolysis. Biomater. Med.
Devices Artif. Organs. 1979, 7 (3) pp. 359–391
[114] OSHA. 29 CFR Regulation Part 1910: Occupational Safety and Health Standards—1910.1030:
Bloodborne pathogens. In: National Archives and Records Administration, editor. Washington,
DC: US Department of Labor; 2008
[115] Reed K.W., Yalkowsky S.H. Lysis of human red blood cells in the presence of various cosolvents.
J. Parenter. Sci. Technol. 1985, 39 (2) pp. 64–69
[116] SIGMA DIAGNOSTICS. Plasma Hemoglobin: Quantitative, colormetric determination in plasma at
600 nm (Procedure No. 527, April 1991). Sigma Diagnostics, St. Louis, MO, 1991
[117] Standefer J.C., Vanderjagt D. Use of tetramethylbenzidine in plasma hemoglobin assay. Clin.
Chem. 1977, 23 (4) pp. 749–751
[118] Taulier A., Levillain P., Lemonnier A. Value of derivative spectrophotometry for the
determination of plasma and urinary hemoglobin. Comparison with the method using Allen’s
correction]. Ann. Biol. Clin. (Paris). 1986, 44 (3) pp. 242–248
[119] Kottke-Marchant K. ed. Laboratory Haematology Practice. John Wiley & Sons, Ltd, Oxford,
England, 2012

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[120] Dudash L.A., Klingman F.L., Bastijanic J.M., Kottke-Marchant K., Marchant R.E. Cross-
reactivity of cell-selective CRETAWAC peptide with human and porcine endothelial cells. J.
Biomed. Mater. Res. A. 2014 Aug, 102 (8) pp. 2857–2863
[121] WHO. Recommended methods for the visual determination of white cells and platelet counts. In:
Expert panel of Cytometry of the International Committee for Standardization in Haematology,
editor. WHO/LAB/88.3: World Health Organization; 1988. p. 8
[122] Zwart A., Van Assendelft O.W., Bull B.S., England J.M., Lewis S.M., Zijlstra W.G.
Recommendations for reference method for haemoglobinometry in human blood (ICSH standard
1995) and specifications for international haemiglobinocyanide standard (4th edition). J Clin
Pathol 1996;49(4):271-4. (PREVIOUSLY CITED AS International Committee for Standardization
in Haematology)
[123] Wennberg A., Hensten-Pettersen A. Sensitivity of erythrocytes from various species to in
vitro hemolyzation. J. Biomed. Mater. Res. 1981, 15 (3) pp. 433–435
[124] Mueller M.R., & Schima H. In vitro hematological testing of rotary blood pumps: Remarks on
standardization and data interpretation. Artif. Organs. 1993, 17 (2) pp. 103–110
[125] Lippi G., Blanckaert N., Bonini P., Green S., Kitchen S., Palicka V. Haemolysis: an overview
of the leading cause of unsuitable specimens in clinical laboratories. Clin. Chem. Lab. Med. 2008,
46 pp. 764–772. Available at: http://dx.doi.org/10.1515/CCLM.2008.170
[126] Sharp M.K., & Mohammad S.F. Scaling of haemolysis in needles and catheters. Ann Biochem
Engineer. 1998, 26 pp. 788–797
[127] Savory J., & Bill J.G. Haemolysis of specimens drawn in the ER [Q&A]. Lab. Med. 1996, 27 p. 802
[128] Kennedy C., Angemuller S., King R. A comparison of haemolysis rates using intravenous
catheters versus venipuncture tubes for obtaining blood samples. J. Emerg. Nurs. 1996,
22 pp. 566–569
Complement
[129] Chenoweth D.E. Complement activation produced by biomaterials. ASAIO Trans. 1986,
32 (1) pp. 226–232
[130] Craddock P.R., Fehr J., Brigham K.L., Kronenberg R.S., Jacob H.S. Complement and leukocyte-
mediated pulmonary dysfunction in hemodialysis. N. Engl. J. Med. 1977, 296 pp. 769–774
[131] Chenoweth D.E. Complement activation during hemodialysis: clinical observations, proposed
mechanisms and theoretical implications. Artif. Organs. 1984, 8 pp. 231–287
[132] Hakim R.M., Breillatt J., Lazarus J.M., Port F.K. Complement activation and hypersensitivity
reactions to dialysis membranes. N. Engl. J. Med. 1984, 311 pp. 878–882
[133] Chenoweth D.E., Cooper S.W., Hugli T.E., Stewart R.W., Blackstone E.H., Kirlin J.W.
Complement activation during cardiopulmonary bypass: evidence for generation of C3a and C5a
anaphylatoxins. N. Engl. J. Med. 1981, 304 (9) pp. 497–503
[134] Velthuis H., Jansen P.G., Hack C.E., Eijsman L., Wildevuur C.R. Specific complement inhibition
with heparin-coated extracorporeal circuits. Ann. Thorac. Surg. 1996, 61 pp. 1153–1157
[135] Fitch J.C., Rollins S., Matis L., Alford B. Pharmacology and biological efficacy of a
recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing
coronary artery bypass surgery with cardiopulmonary bypass. Circulation. 1999, 100
pp. 2499–2506
[136] Hsu L.C. Heparin-coated cardiopulmonary bypass circuits: current status. Perfusion. 2001,
16 pp. 417–428

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[137] Johnson R.J. 2013 ‘The complement system’, in Ratner B, Hoffman A, Schoen F, and Lemons
J (eds), Biomaterials Science: An Introduction to Materials in Medicine, 3rd Ed., Oxford, UK,
Elsevier Academic Press, 533–545
[138] Johnson R.J., Burhop K.E., Van Epps D.E. Infusion of ovine C5a into sheep mimics the
inflammatory response of hemodialysis. J. Lab. Clin. Med. 1996, 127 pp. 456–469
[139] Neidhart P.P., Meier B., Polla B.S., Schifferli J.A., Morel D.R. Fatal anaphylactoid response
to protamine after percutaneous transluminal coronary angioplasty. Eur. Heart J. 1992,
13 (6) pp. 856–858
[140] Laroche D., Aimone-Gastin I., Dubois F. Mechanisms of severe, immediate reactions to
iodinated contrast material. Radiology. 1998, 209 (1) pp. 183–190
[141] Bergamaschini L., Mannucci P.M., Federici A.B., Coppola R., Guzzoni S., Agostini A.
Posttransfusion anaphylactic reactions in a patient with severe von Willebrand disease:
role of complement and alloantibodies to von Willebrand factor. J. Lab. Clin. Med. 1995,
125 (3) pp. 348–355
[142] Bergamaschini L., Santangelo T., Faricciotti A., Ciavarella N., Mannucci P.M., Agostoni
A. Study of complement-mediated anaphylaxis in humans. The role of IgG subclasses (IgG1
and/or IgG4) in the complement-activating capacity of immune complexes. J. Immunol. 1996,
156 (3) pp. 1256–1261
[143] Wolf M.F., & Anderson J.M. Practical approach to blood compatibility assessments: general
considerations and standards. In: Biocompatibility and performance of medical devices,
(Boutrand J.-P. ed.). Woodhead Publishing Ltd, 2012
[144] Blajchman M.A., & Ozge-Anwar A.H. The role of the complement system in hemostasis. Prog.
Hematol. 1986, XIV pp. 149–182
[145] Johnson R.J. Complement activation during extracorporeal therapy: biochemistry, cell biology
and clinical relevance. Nephrol. Dial. Transplant. 1994, 9 pp. 36–45
[146] Fiane A.E., Videm V., Lingaas P.S., Heggelund L., Nielsen E.W., Geiran O.R. Mechanism of
complement activation and its role in the inflammatory response after thoracoabdominal aortic
aneurysm repair. Circulation. 2003 Aug 19, 108 (7) pp. 849–856
[147] Speidl W.S., Katsaros K.M., Kastl S.P., Zorn G., Huber K., Maurer G. Coronary late
lumen loss of drug eluting stents is associated with increased serum levels of the complement
components C3a and C5a. Atherosclerosis. 2010 Jan, 208 (1) pp. 285–289
Animal models
[148] Dewanjee M.K., Kapadvanjwala M., Sanchez A., Elson R., Serafini A.N., Zilleruelo G.E.,
Sfakianakis G.N. Quantitation of comparative thrombogenicity of dog, pig, and human platelets
in a hemodialyzer. ASAIO J. 1992, 38 (2) pp. 88–90
[149] Didisheim P., Dewanjee M.K., Frisk C.S., Kaye M.P., Fass D.N. Animal Models for predicting
clinical performance of biomaterials for cardiovascular use. In: Boretos J.W, Eden M., National
Institutes of Health (U.S.), editors. Contemporary biomaterials: material and host response,
clinical applications, new technology, and legal aspects. Park Ridge, N.J., U.S.A.: Noyes
Publications; 1984a. pp. 132-179
[150] Didisheim P., Stropp J.Q., Borowick J.H., Grabowski E.F. Species differences in platelet
adhesion to biomaterials: Investigation by a two-stage technique. Trans. Am. Soc. Artif. Intern.
Organs. 1979, 2 pp. 124–132
Anticoagulation
[151] EDQM. Anticoagulant and Preservative solutions for human blood: Anticoagulant acid-citrate-
glucose solutions (ACD) and Anticoagulant citrate-phosphate-glucose solution (CPD). European

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Pharmacopoeia 5.0: European Directorate for the Quality of Medicines and Healthcare;
1997a. pp. 400-403
[152] Anticoagulant Citrate Dextrose Solution In U.S.P. Ian DeVeau, editor. USP 28: (USP Citric Acid
RS); Assay for total citrate. U.S. Pharmacopeia; 2006a. p 165; PF 31(3), p. 727
[153] Anticoagulant Citrate Phosphate Dextrose U.S.P. In: Larry Callahan, editor. USP 28: (USP Citric
Acid RS); Assay for total citrate and total phosphate. U.S. Pharmacopeia; 2006b. p 165; PF
31(3), p. 730
[154] Anticoagulant Citrate Phosphate Dextrose Adenine Solution U.S.P. In: Larry Callahan, editor. USP
28: (USP Citric Acid RS); Assay for total citrate and total phosphate. U.S. Pharmacopeia; 2006c.
p 166; PF 31(3), p. 728
[155] Anticoagulant Heparin Solution U.S.P. In: Biologics and Biotechnology-Blood and Blood Products
Committee, editor. USP 11: (USP Endotoxin RS) Assay for heparin sodium; Assay for sodium
chloride (RB 1-Oct-2009). U.S. Pharmacopeia; 2009
[156] Anticoagulant Sodium Citrate Solution U.S.P. In: Larry Callahan, editor. USP 28: (USP Citric Acid
RS); Assay. U.S. Pharmacopeia; 2006d. p 168; PF 31(3), p. 731
Blood pumps
[157] Mueller M.R., Schima H., Engelhardt H., Salat A., Olsen D.B., Losert U., Wolner E.
In vitro hematological testing of rotary blood pumps: remarks on standardization and data
interpretation. Artif. Organs. 1993, 17 (2) pp. 103–110
Cardiopulmonary bypass
[158] Harker L.A., Malpass T.W., Branson H.E., Hessel E.A 2NDSlichter S.J. Mechanism of
abnormal bleeding in patients undergoing cardiopulmonary bypass: acquired transient platelet
dysfunction associated with selective alpha-granule release. Blood. 1980, 56 (5) pp. 824–834
[159] Moen O., Fosse E., Dregelid E., Brockmeier V., Andersson C., Hogasen K., Venge P.,
Mollnes T.E., Kierulf P. Centrifugal pump and heparin coating improves cardiopulmonary
bypass biocompatibility. Ann. Thorac. Surg. 1996, 62 (4) pp. 1134–1140
[160] Palatianos G.M., Dewanjee M.K., Robinson R.P., Novak S., Dewanjee P.K., Kapadvanjwala
M., Hsu L.C., Sfakianakis G.N., Kaiser G.A. Quantitation of platelet loss with indium-111
labeled platelets in a hollow-fiber membrane oxygenator and arterial filter during extracorporeal
circulation in a pig model. ASAIO Trans. 1989, 35 (3) pp. 667–670
Catheters
[161] Leach K.R., Kurisu Y., Carlson J.E., Repa I., Epstein D.H., Rness M., Sahatjian R., Hunter
D.W., Casteneda-Zuniga W.R., Amplatz K. Thrombogenicity of hydrophilically coated guide
wires and catheters. Radiology. 1990, 175 (3) pp. 675–677
[162] Lee K.H., Han J.K., Byun Y, Moon H.T., Yoon C.J., Kim S.J., Choi B.I. Heparin-coated
angiographic catheters: an in vivo comparison of three coating methods with different heparin
release profiles. Cardiovasc. Intervent. Radiol. 2004, 27 (5) pp. 507–511
[163] Roberts G.M., Roberts E.E., Davies R.L., Lawrie B.W. Thrombogenicity of arterial catheters
and guidewires. Br. J. Radiol. 1977, 50 (594) pp. 415–418
General
[164] Cao L., Chang M., Lee C.Y., Castner D.G., Sukavaneshvar S., Ratner B.D., Horbett T.A.
Plasma-deposited tetraglyme surfaces greatly reduce total blood protein adsorption, contact
activation, platelet adhesion, platelet procoagulant activity, and in vitro thrombus deposition. J.
Biomed. Mater. Res. A. 2007, 81 (4) pp. 827–837

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[165] Dawids S.G. Test procedures for the blood compatibility of biomaterials. Dordrecht; Boston:
Kluwer Academic Publishers; 1993. xii, 684 p
[166] EMEA. Validation of Analytical Procedures: Text and Methodology–Q 2 (R1), (CPMP/ICH/381/95);
and Note for guidance on Validation of Analytical Procedures: Methodology–Q 2 B
(CPMP/ICH/281/95). In: ICH Harmonised Tripartite Guideline, (Unit H.M.E. ed.). European
Medicines Agency, 1995
[167] Kataoka KMaeda MNishimura TNitadori YTsuruta TAkaike TSakurai Y. Estimation of
cell adhesion on polymer surfaces with the use of “column-method”. J. Biomed. Mater. Res. 1980,
14 (6) pp. 817–823
[168] Levy R.JCardiovascular Biomaterials and Biocompatibility. In: Cardiovascular Pathology,
No. 3. (Ratner B.D., & Didisheim P. eds.). Suppl, Harker, LA, Vol. 2, 1993, pp. 1S–224S.
[169] NHLBI. Guidelines for blood/material interactions. Bethesda, MD: National Heart Lung and
Blood Institute; 1985 September 1985. Report nr NIH: 85-2185
[170] Northup S.J Hemocompatilbility: Not all devices are created equal. MDDI. 1997 January,
1997 pp. 145–150
[171] Ratner B.D., Hoffman A.S., Schoen F.J., Lemons J.E. Biomaterials Science, Third Edition: An
Introduction to Materials in Medicine. Elsevier Academic Press; 2012. 864 p
[172] Sefton M.V., Gemmell C.H., Gorbet M.B. What really is blood compatibility? J. Biomater. Sci.
Polym. Ed. 2000, 11 (11) pp. 1165–1182
[173] Sefton M.V., Sawyer A., Gorbet M., Black J.P., Cheng E., Gemmell C., Pottinger-Cooper E.
Does surface chemistry affect thrombogenicity of surface modified polymers? J. Biomed. Mater.
Res. 2001, 55 (4) pp. 447–459
[174] Seyfert U.T., Biehl V., Schenk J. In vitro haemocompatibility testing of biomaterials according
to the ISO 10993‑4. Biomol. Eng. 2002, 19 (2-6) pp. 91–96
[175] Taber C.W., & Thomas C.L. Taber's Cyclopedic Medical Dictionary. In: Thomas CL, M.D., editor.
Taber's Cyclopedic Medical Dictionary. 17th ed. Philadelphia: F.A. Davis Co.; 1993. p. 2600
[176] TRIPARTITE SUBCOMMITTEE FOR MEDICAL DEVICES. Tripartite Biocompatibility Guidance
for Medical Devices. In: Office of Device Evaluation (ODE) in FDA's Center for Devices and
Radiological Health (CDRH), editor. General Program Memorandum #G87-1. FDA; 1986
[177] Ratner B.D. Blood compatibility – a perspective. J. Biomater. Sci. Polym. Ed. 2000, 11
pp. 1107–1119
[178] Hoffman A.S., Horbett T.A., Ratner B.D., Hanson S.R., Harker L.A. 1982) ‘Thrombotic
events on grafted polyacrylamide-silastic surfaces as studied in a baboon’, in Cooper S L, Peppas
N A and Hoffman A S (eds), Biomaterials: interfacial Phenomena and Applications, 6, 59–80,
American Chemical Society
[179] Llanos G.R., & Sefton M.V. Immobilization of poly(ethylene glycol) onto poly(vinyl alcohol)
hydrogel: evaluation of thrombogenicity. J. Biomed. Mater. Res. 1993, 27 (11) pp. 1383–1391
[180] Kaplan S., Marcoe K.F., Sauvage L.R., Wu H.D., Mathesen S.R., Walker M.W. The effect of
predetermined thrombotic potential of the recipient on small-caliber graft performance. J. Vasc.
Surg. 1986, 3 (2) pp. 311–321
[181] Akizawa T., Kino K., Kinugasa E., Koshikawa S., Ikada Y., Kishida A., Hatanaka Y., Imamura
K. Clinical effects of a polyethylene glycol grafted cellulose membrane on thrombogenicity and
biocompatibility during hemodialysis. ASAIO Trans. 1990 Jul-Sep, 36 (3) pp. M640–M642

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[182] Ishihara K., Ziats N.P., Tierney B.P., Nakabayashi N., Anderson J.M. Protein adsorption
from human plasma is reduced on phospholipid polymers. J. Biomed. Mater. Res. 1991, 25
pp. 1397–1407
[183] Mulvihill J.N., Faradji A., Oberling F., Cazenave J.P. Surface passivation by human albumin of
plasmapheresis circuits reduces platelet accumulation and thrombus formation. Experimental
and clinical studies. J. Biomed. Mater. Res. 1990, 24 pp. 155–163
Heart valves
[184] Burns G.L., Pantalos G.M., Olsen D.B. The calf as a model for thromboembolic events with the
total artificial heart. ASAIO Trans. 1987, 33 (3) pp. 398–403
[185] Rosengart T.K., Lang S.J. Valvular Heart Disease. In: Surgical Intensive CareBarrie P.S., &
Shires G.T. eds.). Little, Brown and Company, Boston, 1993, pp. 577–612.
[186] Schoen F.J., Hirsch D., Bianco R.W., Levy R.J. Onset and progression of calcification in porcine
aortic bio prosthetic valves implanted as orthotopic mitral valve replacements in juvenile sheep.
J. Thorac. Cardiovasc. Surg. 1994 Nov, 108 (5) pp. 880–887
[187] Gallegos R.P., Nockel P.J., Rivard A.L., Bianco R.W. The current state of in-vivo pre-clinical
heart valve evaluation. J of Heart Valve Disease, May;14(3) 2005
Hemodialysis
[188] Kishida A., Akatsuka Y., Yanagi M., Aikou T, Maruyama I., Akashi M. In vivo and ex vivo
evaluation of the antithrombogenicity of human thrombomodulin immobilized biomaterials.
ASAIO J. 1995, 41 (3) pp. M369–M374
[189] Mahiout A., Meinhold H., Jorres A., Krieg R., Kessel M., Tretzel J., Baurmeister U. Ex
vivo model for pre-clinical evaluation of dialyzers containing new membranes. Life Support Syst.
1985, 3 (Suppl 1) pp. 448–452
[190] Spencer P.C., Schmidt B., Samtleben W., Bosch T., Gurland H.J. Ex vivo model of hemodialysis
membrane biocompatibility. Trans. Am. Soc. Artif. Intern. Organs. 1985, 31 pp. 495–498
[191] Ward R.A., Schmidt B., Blumenstein M., Gurland H.J. Evaluation of phagocytic cell function in an
ex vivo model of hemodialysis. Kidney Int. 1990, 37 (2) pp. 776–782
In vitro models
[192] Chandler A.B. In vitro thrombotic coagulation of the blood; a method for producing a thrombus.
Lab. Invest. 1958, 7 (2) pp. 110–114
[193] Munch K.,. Wolf M.F., Gruffaz P., Ottenwaelter C., Bergan M., SchroedeR P., Fogt E.J.
Use of simple and complex in vitro models for multiparameter characterization of human
blood-material/device interactions. J. Biomater. Sci. Polym. Ed. 2000, 11 (11) pp. 1147–1163
[194] Yoshizaki T.,. Tabuchi N., Van Oeveren W., Shibamiya A., Koyama T., Sunamori M. PMEA
polymer-coated PVC tubing maintains anti-thrombogenic properties during in vitro whole blood
circulation. Int. J. Artif. Organs. 2005, 28 (8) pp. 834–840
[195] Zimmermann A.K.,. Weber N., Aebert H., Ziemer G., Wendel H.P. Effect of biopassive and
bioactive surface-coatings on the haemocompatibility of membrane oxygenators. J. Biomed.
Mater. Res. B Appl. Biomater. 2007, 80 (2) pp. 433–439
[196] Tayama E.,. Ohtsubo S., Nakazawa T., Takami Y., Niimi Y., Makinouchi K., Glueck J.A., Nose
Y. The simple in vitro thrombogenic test: modified methods for same priming pumps. Artif.
Organs. 1997, 21 (12) pp. 1305–1308
[197] Amrani D.L., Lee C., Earle K., DiOrio J., Murphy M., Yang J. LiVecchi A. An In vitro Bovine
percardial Haemocompatibility Testing System. J. Heart Valve Dis. 1998, 7 pp. 268–272

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[198] Boswald M.. Lugauer S., Bechert T., Greil J., Regenfus A., Guggenbichler J.P. Thrombogenicity
testing of central venous catheters in vitro. Infection. 1999, 27 (Suppl 1) pp. S30–S33
[199] Van Oeveren W.,. Tielliu I.F., De Hart J. Comparison of modified chandler, roller pump,
and ball valve circulation models for in vitro testing in high flow conditions: application in
thrombogenicity testing of different materials for vascular applications. Int. J of Biomaterials
2012; 673163, 7 pages
[200] Kolandaivelu K., Edelman E.R., Background L. Pulsatile, In Vitro Flow Circuit for Modeling
Coronary Implant Thrombosis, in Transactions of the ASME, Vol. 124, Dec. 2002
[201] Kolandaivelu K., & Edelman E.R. Environmental influences on endovascular stent platelet
reactivity: An in vitro comparison of stainless steel and gold surfaces, 2004 Wiley Periodicals,
Inc. J. Biomed. Mater. Res. 2004, 70A pp. 186–193
[202] Kolandaivelu K., Leiden, Benjamin B., Edelman, Elazer R. Predicting response to
endovascular therapies: Dissecting the roles of local lesion complexity, systemic comorbidity,
and clinical uncertainty. J. Biomech. 2014, 47 pp. 908–921
[203] Stang K., Krajewski S., Neumann B., Kurz J., Post M., Stoppelkamp S. Haemocompatibility
testing according to ISO 10993‑4: Discrimination between pyrogen- and device-induced
hemostatic activation. Mater. Sci. Eng. C. 2014, 42 pp. 422–428
In vitro versus in vivo models
[204] Didisheim P.,. Dewanjee M.K., Kaye M.P., Frisk C.S., Fass D.N., Wahner H.W., Tirrell M.V.,
Zollman P.E. Nonpredictability of long-term in vivo response from short-term in vitro or ex vivo
blood-material interactions. Trans. Am. Soc. Artif. Intern. Organs. 1984b, 30 pp. 370–376
Pathology
[205] Anderson J.M., Cardiovascular Device Retrieval and Evaluation. In: Cardiovascular
Biomaterials and Biocompatibility. Cardiovascular Pathology, No. 3. (Ratner B.D., & Didisheim
P. eds.). Suppl, Harker, LA, Vol. 2, 1993, pp. 199S–208S.
[206] Schoen F.J.. Appendix: Pathology analysis of the cardiovascular system and prosthetic devices.
Interventional and surgical cardiovascular pathology: clinical correlations and basic principles.
Saunders, Philadelphia, 1989, pp. 369–96.
[207] Grewe P.H.,. Thomas D., Machraoui A., Barmeyer J., Muller K.M. Coronary morphologic
findings after stent implantation. Am. J. Cardiol. 2000, 85 (5) pp. 554–558
[208] Prado C.M.,. Viaro F., Baldo C.F., Augusto Vdos S., Rodrigues A.J., Evora P.R. Glycol
methacrylate-embedding medium to study morphological alterations of saphenous vein under
brief and crescent pressurizations. Acta Cir. Bras. 2008, 23 (Suppl 1) pp. 77–82, discussion 82
[209] Rippstein P.,. Black M.K., Boivin M., Veinot J.P., Ma X., Chen Y.X., Human P., Zilla P., O’Brien
E.R. Comparison of processing and sectioning methodologies for arteries containing metallic
stents. J. Histochem. Cytochem. 2006, 54 (6) pp. 673–681
[210] Singhrao S.K.,. Muller C.T., Gilbert S.J., Duance V.C., Archer C.W. An immunofluorescence
method for postembedded tissue in the acrylic resin Technovit 9100 New using fluorescein
isothiocyanate secondary detection. Microsc. Res. Tech. 2009, 72 (7) pp. 501–506
[211] Zhang Q., Wang J., Wu H., Zhang L., Zhou J., Ye Q., Shao X., Guan C., Xu J., Yang Y. et al. Low-
temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow
immunohistochemistry, PCR, and FISH analysis. Microsc Res Tech
[212] Mitrecić D. Cunko VF, Gajović S. Semi-thin sections of epoxy resin-embedded mouse
embryos in morphological analysis of whole mount in situ RNA hybridization. J. Microsc. 2008,
232 (3) pp. 504–507

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

Statistics
[213] Festing MF. The design and statistical analysis of animal experiments. ILAR J. 2002, 43 (4)
[214] Festing MF. Altman DG. Guidelines for the design and statistical analysis of experiments using
laboratory animals. ILAR J. 2002, 43 (4)
[215] Festing MF. Design and statistical methods in studies using animal models of development.
ILAR J. 2006, 47 (1)
[216] Design and Analysis of Animal Studies in Pharmaceutical Development. Shein-Chung
Chow and Jen-pei Liu, Editors, Chapman & Hall CRC Biostatistics Series, 1998
Vascular grafts
[217] Guidoin R., Douville Y. Basle M.F., King M., Marinov G.R., Traore A., Zhang Z., Guillemot
F., Dionne G., Sumanasinghe R. Biocompatibility studies of the Anaconda stent-graft and
observations of nitinol corrosion resistance. J. Endovasc. Ther. 2004, 11 (4) pp. 385–403
[218] Guidoin R., Gosselin C. Martin L., Marois M., Laroche F., King M., Gunasekera K., Domurado
D., Sigot-Luizard M.F., Blais P. Polyester prostheses as substitutes in the thoracic aorta of dogs.
I. Evaluation of commercial prostheses. J. Biomed. Mater. Res. 1983, 17 (6) pp. 1049–1077
[219] Hoch JR. SilVER D. Hemostasis and Thrombosis. In: Vascular Surgery: A Conprehensive Review,
(Moore W.S. ed.). W. B. Saunders, Philadelphia, Third Edition, 1991, pp. 63–79.
[220] Koskas F., Brocheriou I. Cluzel P., Singland J.D., Regnier B., Bonnot M., Kieffer E. Custom-
made stent-grafts for aortic aneurysm repair using gianturco Z stents and woven polyester:
healing in an animal model. Vasc. Endovascular Surg. 2005, 39 (1) pp. 55–65
[221] Toes G.J. Van Muiswinkel K.W., Van Oeveren W., Suurmeijer A.J., Timens W., Stokroos
I., Van Den Dungen J.J. Superhydrophobic modification fails to improve the performance
of small diameter expanded polytetrafluoroethylene vascular grafts. Biomaterials. 2002,
23 (1) pp. 255–262
[222] White R.A. Diagnosis and therapy of emergent vascular diseases. In: Textbook of critical care,
(Shoemaker W.C., Society of Critical Care Medicine, ed.). Saunders, Philadelphia, Second Edition,
1989, pp. 447–452
[223] Wilson G.J. Macgregor D.C., Bridgeman J., Weber B.A., Binnington A.G., Pinchuk L.
A Corethane/polyester composite vascular prosthesis for vascular access. Comparison
with expanded polytetrafluoroethylene grafts in a canine model. ASAIO J. 1995, 41 (3)
pp. M728–M734
[224] Yoneyama T., Ishihara K. Nakabayashi N., Ito M., Mishima Y. Short-term in vivo evaluation
of small-diameter vascular prosthesis composed of segmented poly(etherurethane)/2-
methacryloyloxyethyl phosphorylcholine polymer blend. J. Biomed. Mater. Res. 1998,
43 (1) pp. 15–20
[225] Zilla P.,. Greisler H.P. Tissue engineering of vascular prosthetic grafts (Tissue Engineering
Intelligence Unit). Landes Bioscience, 1999, 621 p.
[226] Dudash L.A., Klingman F., Sarett S.M., Kottke-Marchant K., Marchant R.E. Endothelial
cell attachment and shear response on biomimetic polymer-coated vascular grafts. J. Biomed.
Mater. Res. A. 2012 Aug, 100 (8) pp. 2204–2210
[227] Tang C., Klingman F., Larsen C.C., Kottke-Marchant K., Marchant R.E. Platelet and
endothelial adhesion on fluorosurfactant polymers designed for vascular graft modification. J.
Biomed. Mater. Res. A. 2009, 88 (2) pp. 348–358

BS EN ISO 10993‑4:2017
ISO 10993-4:2017(E)

[228] Kottke-Marchant K., Anderson J.M., Rabinovitch A., Huskey R.A., Herzig R. The effect
of heparin vs. citrate on the interaction of platelets with vascular graft materials. Thromb.
Haemost. 1985, 54 pp. 842–848
[229] Kottke-Marchant K., Anderson J.M., Rabinovitch A. The platelet reactivity of vascular graft
prostheses: An in vitro model to test the effect of preclotting. Biomaterials. 1986, 7 pp. 441–448
[230] Kottke-Marchant K., Anderson J.M., Miller K.M., Marchant R.E., Lazarus H. Vascular
graft associated complement activation and leukocyte adhesion in an artificial circulation. J.
Biomed. Mater. Res. 1987, 21 pp. 379–397
[231] Kottke-Marchant K., Anderson J.M., Umemura Y., Marchant R.E. The effect of albumin
coating on the in vitro blood compatibility of Dacron arterial prostheses. Biomaterials. 1989,
10 pp. 147–155
Vascular stents
[232] Grewe PH. Thomas D., Machraoui A., Barmeyer J., Muller K.M. Coronary morphologic
findings after stent implantation. Am. J. Cardiol. 2000, 85 (5) pp. 554–558
[233] Rippstein P., Black MK. Boivin M., Veinot J.P., Ma X., Chen Y.X., Human P., Zilla P., O’BRien
E.R. Comparison of processing and sectioning methodologies for arteries containing metallic
stents. J. Histochem. Cytochem. 2006, 54 (6) pp. 673–681
[234] SChwartz R.S. Edelman E.R., CarteR A., Chronos N.A., Rogers C., Robinson K.A., Waksman
R., Machan L., Weinberger J., Wilensky R.L. Preclinical evaluation of drug-eluting stents for
peripheral applications: recommendations from an expert consensus group. Circulation. 2004,
110 (16) pp. 2498–2505
[235] Kolandaivelu K., Leiden, Benjamin B., Edelman, Elazer R. Predicting response to
endovascular therapies: Dissecting the roles of local lesion complexity, systemic comorbidity,
and clinical uncertainty. J. Biomech. 2014, 47 pp. 908–921
Ventricular-assist devices
[236] Schoen F.J. Anderson J.M., Didisheim P., Dobbins J.J., Gristina A.G., Harasaki H., Simmons
R.L. Ventricular assist device (VAD) pathology analyses: guidelines for clinical studies. J. Appl.
Biomater. 1990, 1 pp. 49–56
[237] Wagner W.R. Schaub R.D., Sorensen E.N., Snyder T.A., Wilhelm C.R., Winowich S., Borovetz
H.S., Kormos R.L. Blood biocompatibility analysis in the setting of ventricular assist devices. J.
Biomater. Sci. Polym. Ed. 2000, 11 pp. 1239–1259

This page deliberately left blank
This page deliberately left blank
NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW
British Standards Institution (BSI)

BSI is the national body responsible for preparing British Standards and other
standards-related publications, information and services.
BSI is incorporated by Royal Charter. British Standards and other standardization
products are published by BSI Standards Limited.
About us Reproducing extracts

We bring together business, industry, government, consumers, innovators For permission to reproduce content from BSI publications contact the BSI
and others to shape their combined experience and expertise into standards Copyright & Licensing team.
-based solutions.
The knowledge embodied in our standards has been carefully assembled in Subscriptions
a dependable format and refined through our open consultation process. Our range of subscription services are designed to make using standards
Organizations of all sizes and across all sectors choose standards to help easier for you. For further information on our subscription products go to
them achieve their goals. bsigroup.com/subscriptions.
With British Standards Online (BSOL) you’ll have instant access to over 55,000
Information on standards British and adopted European and international standards from your desktop.
We can provide you with the knowledge that your organization needs It’s available 24/7 and is refreshed daily so you’ll always be up to date.
to succeed. Find out more about British Standards by visiting our website at You can keep in touch with standards developments and receive substantial
bsigroup.com/standards or contacting our Customer Services team or discounts on the purchase price of standards, both in single copy and subscription
Knowledge Centre. format, by becoming a BSI Subscribing Member.
PLUS is an updating service exclusive to BSI Subscribing Members. You will
Buying standards
automatically receive the latest hard copy of your standards when they’re
You can buy and download PDF versions of BSI publications, including British revised or replaced.
and adopted European and international standards, through our website at
To find out more about becoming a BSI Subscribing Member and the benefits
bsigroup.com/shop, where hard copies can also be purchased.
of membership, please visit bsigroup.com/shop.
If you need international and foreign standards from other Standards Development
With a Multi-User Network Licence (MUNL) you are able to host standards
Organizations, hard copies can be ordered from our Customer Services team.
publications on your intranet. Licences can cover as few or as many users as you
wish. With updates supplied as soon as they’re available, you can be sure your
Copyright in BSI publications
documentation is current. For further information, email subscriptions@bsigroup.com.
All the content in BSI publications, including British Standards, is the property
of and copyrighted by BSI or some person or entity that owns copyright in the Revisions
information used (such as the international standardization bodies) and has
Our British Standards and other publications are updated by amendment or revision.
formally licensed such information to BSI for commercial publication and use.
We continually improve the quality of our products and services to benefit your
Save for the provisions below, you may not transfer, share or disseminate any
business. If you find an inaccuracy or ambiguity within a British Standard or other
portion of the standard to any other person. You may not adapt, distribute,
BSI publication please inform the Knowledge Centre.
commercially exploit, or publicly display the standard or any portion thereof in any
manner whatsoever without BSI’s prior written consent.
Useful Contacts
Storing and using standards Customer Services
Standards purchased in soft copy format: Tel: +44 345 086 9001
Email (orders): orders@bsigroup.com
• A British Standard purchased in soft copy format is licensed to a sole named
user for personal or internal company use only. Email (enquiries): cservices@bsigroup.com
• The standard may be stored on more than 1 device provided that it is accessible Subscriptions
by the sole named user only and that only 1 copy is accessed at any one time. Tel: +44 345 086 9001
• A single paper copy may be printed for personal or internal company use only. Email: subscriptions@bsigroup.com
Standards purchased in hard copy format: Knowledge Centre

• A British Standard purchased in hard copy format is for personal or internal Tel: +44 20 8996 7004
company use only. Email: knowledgecentre@bsigroup.com
• It may not be further reproduced – in any format – to create an additional copy.
Copyright & Licensing
This includes scanning of the document.
Tel: +44 20 8996 7070
If you need more than 1 copy of the document, or if you wish to share the Email: copyright@bsigroup.com
document on an internal network, you can save money by choosing a subscription
product (see ‘Subscriptions’). BSI Group Headquarters
389 Chiswick High Road London W4 4AL UK

BS en Iso 10993-4-2017

Uploaded by

Copyright:

Available Formats

BS en Iso 10993-4-2017

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

BS en Iso 10993-4-2017

Uploaded by

Copyright:

Available Formats

BS EN ISO 10993‑4:2017

BSI Standards Publication

Biological evaluation of medical devices

Part 4: Selection of tests for interactions with blood

Amendments/corrigenda issued since publication

ICS 11.100.20 Supersedes EN ISO 10993‑4:2009

Biological evaluation of medical devices — Part 4:

This European Standard was approved by CEN on 23 February 2017.

EUROPEAN COMMITTEE FOR STANDARDIZATION

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

Relationship between this European Standard and the essential

Essential Requirements of Clause(s)/subclause(s) of Remarks/Notes

© ISO 2017 – All rights reserved ﻿ v

vi ﻿ © ISO 2017 – All rights reserved

i) subtle language refinements can be found throughout the revised document;

© ISO 2017 – All rights reserved ﻿ vii

viii ﻿ © ISO 2017 – All rights reserved

Biological evaluation of medical devices —

3 Terms and definitions

© ISO 2017 – All rights reserved ﻿ 1

2 ﻿ © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved ﻿ 3

Bb enzymatically active fragment of Factor B produced by cleavage (by Factor D) in the

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

ELISA enzyme-linked immunosorbent assay

FDP fibrin/fibrinogen degradation products

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

4 ﻿ © ISO 2017 – All rights reserved

PRP platelet-rich plasma

PTT partial thromboplastin time

SC5b-9 product of terminal pathway complement activation

SEM scanning electron microscopy

TAT thrombin-antithrombin complexes

5 Types of devices in contact with blood (as categorized in ISO 10993‑1)

5.1 Non-blood-contact devices

5.2 External communicating devices

5.2.2 External communicating devices that serve as an indirect blood path

— blood collection devices;

5.2.3 External communicating devices directly contacting circulating blood

© ISO 2017 – All rights reserved ﻿ 5

— donor and therapeutic apheresis equipment;

5.3 Implant devices

6 Characterization of blood interactions

6.1 General requirements

6 ﻿ © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved ﻿ 7

8 ﻿ © ISO 2017 – All rights reserved

External communicating devices

© ISO 2017 – All rights reserved

Annuloplasty rings, mechanical heart valves X X X

Tissue heart valves, vascular grafts and patches and AV shunts X X

© ISO 2017 – All rights reserved v

vi © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved vii

viii © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 1

2 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 3

4 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 5

6 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 7

8 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 11

12 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 13

14 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 15

16 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 17

18 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 19

20 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 21

22 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 23

24 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 25

26 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 27

28 © ISO 2017 – All rights reserved

© ISO 2017 – All rights reserved 29