EN ISO 10993-4:2002

SLOVENSKI STANDARD
01-marec-2003
1DGRPHãþD
SIST EN 30993-4:2000
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO,]ELUDSUHVNXVRY]D
XJRWDYOMDQMHLQWHUDNFLMVNUYMR,62
Biological evaluation of medical devices - Part 4: Selection of tests for interactions with
blood (ISO 10993-4:2002)
Biologische Beurteilung von Medizinprodukten - Teil 4: Auswahl von Prüfungen zur
Wechselwirkung mit Blut (ISO 10993-4:2002)
Evaluation biologique des dispositifs médicaux - Partie 4: Choix des essais concernant
les interactions avec le sang (ISO 10993-4:2002)
Ta slovenski standard je istoveten z: EN ISO 10993-4:2002
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 10993-4
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2002
ICS 11.100 Supersedes EN 30993-4:1993
English version
Biological evaluation of medical devices - Part 4: Selection of
tests for interactions with blood (ISO 10993-4:2002)
Evaluation biologique des dispositifs médicaux - Partie 4: Biologische Beurteilung von Medizinprodukten - Teil 4:
Choix des essais concernant les interactions avec le sang Auswahl von Prüfungen zur Wechselwirkung mit Blut (ISO
(ISO 10993-4:2002) 10993-4:2002)
This European Standard was approved by CEN on 19 August 2002.
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 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 Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-4:2002 E
worldwide for CEN national Members.

CORRECTED  2002-12-11
Foreword
This document (EN ISO 10993-4:2002) has been prepared by Technical Committee ISO/TC 194
"Biological evaluation of medical devices" in collaboration with Technical Committee CEN/TC
206 "Biocompatibility of medical and dental materials and devices", the secretariat of which is
held by NEN.
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 April 2003, and conflicting national
standards shall be withdrawn at the latest by April 2003.
This document supersedes EN 30993-4:1993.
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 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, Czech
Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg,
Malta, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 10993-4:2002 has been approved by CEN as EN ISO 10993-4:2002 without any
modifications.
NOTE Normative references to International Standards are listed in Annex ZA (normative).
Annex ZA
(normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions of
any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications, indicated
by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 10993-1 1997 Biological evaluation of medical EN ISO 10993-1 1997
devices - Part 1: Evaluation and
testing
ISO 10993-2 1998 Biological evaluation of medical EN ISO 10993-2 1992
devices - Part 2: Animal welfare
requirements
Annex ZB
(informative)
Clauses of this European Standard addressing essential requirements or
other provisions of EU Directives
This European Standard 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 93/42/EEC, EU Directive 90/385/EEC and EU Directive 86/609/EEC.
WARNING Other requirements and other EU Directives may be applicable to the product(s)
falling within the scope of this standard.
The following clauses of this standard are likely to support requirements of Directive 93/42/EEC,
EU Directive 90/385/EEC and EU Directive 86/609/EEC.
Compliance with these clauses of this standard provides one means of conforming with the
specific essential requirements of the Directive concerned and associated EFTA regulations.
Table ZB.1— Correspondence between this European Standard and EU Directive 93/42/EEC,
EU Directive 90/385/EEC and EU Directive 86/609/EEC
Clause/subclause of this Corresponding Essential Comments
European Standard Requirement of Directive
93/42/EEC, EU Directive
90/385/EEC and EU Directive
86/609/EEC.
6 7.1 of Annex I and Annex II of
93/42/EEC
I.9 of Annex I of 90/385/EEC
6.1.10 18 of 86/609/EEC
A.1 7.1 of Annex I and Annex II of
93/42/EEC
I.9 of Annex I of 90/385/EEC
INTERNATIONAL ISO
STANDARD 10993-4
Second edition
2002-10-15
Biological evaluation of medical devices —
Part 4:
Selection of tests for interactions with
blood
Évaluation biologique des dispositifs médicaux —
Partie 4: Choix des essais concernant les interactions avec le sang

Reference number
ISO 10993-4:2002(E)
©
ISO 2002
ISO 10993-4:2002(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not
be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this
file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this
area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters
were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event
that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2002
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body
in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.ch
Web www.iso.ch
Printed in Switzerland
ii © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
Contents Page
Foreword . iv
Introduction. vi
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Abbreviated terms. 2
5 Types of device in contact with blood (as categorized in ISO 10993-1). 3
5.1 Non-contact devices. 3
5.2 External communicating devices. 3
5.3 Implant devices. 4
6 Characterization of blood interactions . 5
6.1 General requirements. 5
6.2 Categories of tests and blood interactions . 8
6.3 Types of test . 11
Annex A (informative) Preclinical evaluation of cardiovascular devices and prostheses. 13
Annex B (informative) Laboratory tests — Principles, scientific basis and interpretation. 17
Annex C (informative) Evaluation of haemolytic properties of medical devices and their components . 23
Bibliography. 30

ISO 10993-4:2002(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
The main task of technical committees is to prepare International Standards. Draft International Standards adopted
by the technical committees are circulated to the member bodies for voting. Publication as an International
Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this part of ISO 10993 may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 10993-4 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This second edition cancels and replaces the first edition (ISO 10993-4:1992), which has been technically revised.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
— Part 1: Evaluation and testing
— Part 2: Animal welfare requirements
— Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
— Part 4: Selection of tests for interactions with blood
— Part 5: Tests for in-vitro cytotoxicity
— Part 6: Tests for local effects after implantation
— Part 7: Ethylene oxide sterilization residuals
— Part 8: Selection and qualification of reference materials for biological tests
— Part 9: Framework for identification and quantification of potential degradation products
— Part 10: Tests for irritation and sensitization
— Part 11: Tests for systemic toxicity
— Part 12: Sample preparation and reference materials
— Part 13: Identification and quantification of degradation products from polymeric medical devices
— Part 14: Identification and quantification of degradation products from ceramics
— Part 15: Identification and quantification of degradation products from metals and alloys
iv © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
— Part 16: Toxicokinetic study design for degradation products and leachables
— Part 17: Establishment of allowable limits for leachable substances
— Part 18: Chemical characterization of materials
Future parts will deal with other relevant aspects of biological testing.
Annexes A, B and C of this part of ISO 10993 are for information only.
ISO 10993-4:2002(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 part of ISO 10993 was the publication, Guidelines for blood/material
[29]
interactions, Report of the National Heart, Lung, and Blood Institute ; chapters 9 and 10. This publication has
[32]
since been revised .
vi © ISO 2002 – All rights reserved

INTERNATIONAL STANDARD ISO 10993-4:2002(E)

Biological evaluation of medical devices —
Part 4:
Selection of tests for interactions with blood
1 Scope
This part of ISO 10993 provides general requirements for evaluating the interactions of medical devices with blood.
It describes
a) a classification of medical and dental 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 interactions of devices with blood. This part of ISO 10993 describes biological evaluation in general terms and
may not necessarily provide sufficient guidance for test methods for a specific device.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 10993. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 10993 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated
references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO 10993-1:1997, Biological evaluation of medical devices — Part 1: Evaluation and testing
ISO 10993-2:1992, Biological evaluation of medical devices — Part 2: Animal welfare requirements
3 Terms and definitions
For the purposes of this part of ISO 10993, the terms and definitions given in ISO 10993-1 and the following apply.
3.1
blood/device interaction
any interaction between blood or any component of blood and a device resulting in effects on the blood, or on any
organ or tissue, or on the device
NOTE Such effects may or may not have clinically significant or undesirable consequences. Annex A contains further
information on these interactions.
ISO 10993-4:2002(E)
3.2
ex vivo
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 If using an animal model, the blood may be shunted directly back into the animal (recirculating) or collected into test
tubes for evaluation (single pass).
3.3
thrombosis
in vivo phenomenon resulting in the partial or complete occlusion of a vessel or device by a thrombus
NOTE 1 Characterization of thrombosis includes ex vivo and in vivo methods, in either animals or the clinical setting.
NOTE 2 A thrombus is composed of a mixture of red cells, aggregated platelets, fibrin and other cellular elements.
3.4
coagulation
phenomenon that results from activation of the clotting factor cascade
NOTE Factors of the coagulation cascade and fibrinolytic systems can be measured following exposure to devices either in
vitro or in vivo.
3.5
platelet
anuclear, cellular body that is present in the circulation which adheres to surfaces and aggregates to form a
hemostatic plug to minimize bleeding
NOTE Platelet testing includes quantification of platelet numbers as well as analysis of their structure and function. The
testing can include analysis of platelet factors, or components on the platelet surface which are released from platelets or
adherent to the device surface.
3.6
haematology
study of blood, including quantification of cellular and plasma components of the blood
3.7
complement system
part of the innate immune system, consisting of several plasma proteins, including enzymes and cellular receptors
NOTE Effector molecules produced from complement components are involved in inflammation, phagocytosis and cell
lysis.
4 Abbreviated terms
Bb product of alternative pathway complement activation
β-TG beta-thromboglobulin
C4d product of classical pathway complement activation
C3a, C5a (active) complement split products from C3 and C5
CD62L L-selectin
CH-50 50% total haemolytic complement
CT computerized tomography
D-Dimer specific fibrin degradation products (F XIII cross-linked fibrin)
ECMO extracorporeal membrane oxygenator
2 © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
ELISA enzyme/linked immunosorbent assay
EM electron microscopy
FDP fibrin/fibrinogen degradation products
FPA fibrinopeptide A
F prothrombin activation fragment 1 + 2
1+2
iC3b product of central C complement activation
IVC inferior vena cava
MRI magnetic resonance imaging
PAC-1 monoclonal antibody which recognizes the activated form of platelet surface glycoprotein IIb/IIIa
PET positron emission tomography
PF-4 platelet factor 4
PRP platelet-rich plasma
PT prothrombin time
PTT partial thromboplastin time
P-selectin receptor exposed during either platelet or endothelial cell release reaction
RIA radioimmunoassay
S-12 monoclonal antibody, which recognizes the alpha-granule membrane component P-selectin exposed
during the platelet release reaction
SC5b-9 product of terminal pathway complement activation
TAT thrombin-antithrombin complex
TCC terminal complement complex
TT thrombin time
VWF von Willebrand factor
5 Types of device in contact with blood (as categorized in ISO 10993-1)
5.1 Non-contact devices
An in vitro diagnostic device is an example of a non-contact device.
5.2 External communicating devices
These are devices that contact the circulating blood and serve as a conduit into the vascular system. Examples
include but are not limited to those in ISO 10993-1.
a) External communicating devices that serve as an indirect blood path include but are not limited to
 cannulae,
 extension sets,
 blood collection devices,
 devices for the storage and administration of blood and blood products (e.g. tubing, needles and bags),
 cell savers.
ISO 10993-4:2002(E)
b) External communicating devices in contact with circulating blood include but are not limited to
 atherectomy devices,
 blood monitors,
 catheters,
 guidewires,
 intravascular endoscopes,
 intravascular ultrasound,
 intravascular laser systems,
 retrograde coronary perfusion catheters,
 cardiopulmonary bypass circuitry,
 extracorporeal membrane oxygenators,
 haemodialysis/haemofiltration equipment,
 donor and therapeutic apheresis equipment,
 devices for absorption of specific substances from blood,
 interventional cardiology and vascular devices,
 percutaneous circulatory support systems.
5.3 Implant devices
Implant devices are placed largely or entirely within the vascular system. Examples include but are not limited to
 annuloplasty rings,
 mechanical or tissue heart valves,
 prosthetic or tissue vascular grafts,
 circulatory support devices (ventricular-assist devices, artificial hearts, intra-aortic balloon pumps),
 inferior vena cava filters,
 embolization devices,
 endovascular grafts,
 implantable defibrillators and cardioverters,
 stents,
 arteriovenous shunts,
 blood monitors,
4 © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
 internal drug delivery catheters,
 pacemaker leads,
 intravascular membrane oxygenators (artificial lungs),
 leukocyte-removal filters.
6 Characterization of blood interactions
6.1 General requirements
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 classified into five categories based on the primary process or system being measured.
Tables 1 and 2 list examples of devices which contact circulating blood and the categories of testing appropriate to
the device.
NOTE Since this is a horizontal International Standard, good rationales can be developed to justify the choice of category
based on the device being characterized. Thrombosis testing is frequently the preferred method for device characterization. In
many cases, rationales can be used to substitute some combination of coagulation, platelets, haematology and complement
system testing for thrombosis testing.
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 part of ISO 10993.
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, including duration of contact, temperature,
sterile condition and flow conditions. For devices of defined geometry, the ratio of test parameter (concentration per
unit volume) to exposed surface area (cm ) shall be evaluated.
Only blood-contacting parts should be tested. The selected test methods and parameters should be in accordance
with the current state of the art.
6.1.3 Controls shall be used unless their omission can be justified. Where possible, testing should include a
[7]
relevant device already in clinical use or a well-characterized reference material .
Reference materials used should include negative and positive controls. All materials and devices 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
device or device component be tested under conditions which simulate or exaggerate clinical application.
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. For example, some short-term in vitro or
[25], [26]
ex vivo tests are poor predictors of long-term in vivo blood/device interactions .
6.1.6 It follows from the above that 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.
ISO 10993-4:2002(E)
Figure 1 — Decision tree to determine whether testing for interaction with blood is necessary

6 © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
Table 1 — Devices or device components which contact circulating blood
and the categories of appropriate testing — External communicating devices
Device examples Test category
Complement
Thrombosis Coagulation Platelets Haematology
system
a
Atherectomy devices  x
a
Blood monitors x  x
a
Blood storage and administration equipment, x x x
blood collection devices, extension sets
Extracorporeal membrane oxygenator systems,
haemodialysis/haemofiltration equipment, x x x x x
percutaneous circulatory support devices
Catheters, guidewires, intravascular endoscopes,
a
intravascular ultrasound, laser systems, x x x
retrograde coronary perfusion catheters,
a
Cell savers x x x
Devices for absorption of specific substances from x x x x
blood
Donor and therapeutic apheresis equipment x x x x
a
Haemolysis testing only.
Table 2 — Devices or device components which contact circulating blood
and the categories of appropriate testing — Implant devices
Test category
Device examples
Complement
Thrombosis Coagulation Platelets Haematology
system
a
Annuloplasty rings, mechanical heart valves x  x
Intra-aortic balloon pumps x x x x x
Total artificial hearts, ventricular-assist devices x  x
a
Embolization devices  x
a
Endovascular grafts x  x
a
Implantable defibrillators and cardioverters x  x
a
Pacemaker leads x  x
a
Leukocyte removal filter x x x
a
Prosthetic (synthetic) vascular grafts and patches, x  x
including arteriovenous shunts
a
Stents x  x
a
Tissue heart valves x  x
a
Tissue vascular grafts and patches, x  x
including arteriovenous shunts
a
Vena cava filters x  x
a
Haemolysis testing only.
ISO 10993-4:2002(E)
6.1.7 In vitro tests are regarded as useful in screening external communicating devices or implants, but may not
be accurate predictors of blood/device interactions occurring upon prolonged or repeated exposure or permanent
contact (see 6.3.1). Devices intended for non-contact use only do not require evaluation of blood/device
interactions. Devices which come into very brief contact with circulating blood (e.g. lancets, hypodermic needles,
capillary tubes) generally do not require blood/device interaction testing.
6.1.8 The recommendations in 6.1.6 and 6.1.7, together with clause 5, Figure 1 and Table 2, serve as a guide for
the selection of tests listed in 6.2.1.
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. 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. 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.
[26]
Blood values and reactivity in humans and non-human primates are very similar . The use of animals such as
the rabbit, pig, calf, sheep, or dog may also yield satisfactory results. Because species differences may be
[20]
significant (for example platelet adhesion, thrombosis and haemolysis tend to occur more readily in the canine
species than in the human), all results of animal studies shall be interpreted with caution. The species used and the
number of species used shall be justified (see 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 choice 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.
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.
6.1.14 A sufficient number of replications of a test including suitable controls shall 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 interactions.
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 according to Tables 3 and 4.
The tests are classified into the following five categories based on the primary process or system being measured:
a) thrombosis (see 3.3);
b) coagulation (see 3.4);
c) platelets (see 3.5);
d) haematology (see 3.6);
e) complement system (see 3.7).
The principles and scientific bases for these tests are presented in annex B.
8 © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
Table 3 — Test methods for external communicating devices
Test category Evaluation method Comment
Thrombosis Percent occlusion
Flow reduction
Gravimetric analysis (thrombus mass)
Light microscopy (adhered platelets,
leukocytes, aggregates, erythrocytes,
fibrin, etc. )
Pressure drop across device
Labelled antibodies to thrombotic
components
Scanning EM (platelet adhesion and
aggregation; platelet and leukocyte
morphology, fibrin)
Coagulation PTT (non-activated)
Thrombin generation: Specific
coagulation factor assays; FPA, D-dimer,
F , TAT
1+2
Platelets Platelet count/adhesion
Platelet aggregation
Template bleeding time
Platelet function analysis
PF-4, β-TG; thromboxane B2
Platelet activation markers
Platelet microparticles
Gamma imaging of radiolabelled In-labelling is recommended for
platelets, In-labelled platelet survival prolonged or repeated use (>24 h to
30 days) and permanent contact
(>30 days)
Haematology Leukocyte count with or without
differential
Leukocyte activation
Haemolysis
Reticulocyte count; activation-specific
release products of peripheral blood cells
(i.e. granulocytes)
Complement system C3a, C5a, TCC, Bb, iC3b, C4d, SC5b-9,
CH50, C3 convertase, C5 convertase
ISO 10993-4:2002(E)
Table 4 — Test methods for implant devices
Test category Method Comments
Thrombosis Scanning EM (platelet adhesion and
aggregation); platelet and leukocyte
morphology; fibrin
Percent occlusion
Flow reduction
Labelled antibodies to thrombotic
components
Autopsy of devices (gross and
microscopic); histopathology
Autopsy of distal organs (gross and
microscopic): histopathology
Coagulation Specific coagulation factor assay; FPA,
D-dimer, F , PAC-1, S-12, TAT
1+2
PTT( non-activated), PT, TT; plasma
fibrinogen; FDP
Platelets PF-4, β-TG, thromboxane B2,
Platelet activation markers
Platelet microparticles
Gamma imaging of radiolabelled
platelets; -In labelled platelet survival
Platelet function analysis
Platelet count/adhesion
Platelet aggregation
Haematology Leukocyte count with or without
differential;
Leukocyte activation
Haemolysis
Reticulocyte count; activation-specific
release products of peripheral blood cells
i.e. granulocytes)
Complement system C3a, C5a, TCC, Bb, iC3b, C4b, SC5b-9,
CH 50, C3 convertase, C5 convertase

6.2.2 Non-contact devices
These devices do not require blood/device interaction testing. Disposable test kits should be validated to rule out
interference of materials with test accuracy.
6.2.3 External communicating devices
After using Tables 1 and 2 to determine the relevant blood interaction category for a specific device type, Table 3
can be used as a guide to select the appropriate tests for external communicating devices as a function of the
blood interactions appropriate for evaluation (see also 6.1.6). Test selection criteria depend on the specific device
evaluated.
10 © ISO 2002 – All rights reserved

ISO 10993-4:2002(E)
6.2.4 Implant devices
After using Tables 1 and 2 to determine the relevant blood interaction category for a specific device type, Table 4
can be used as a guide to select the appropriate tests for implant devices as a function of the blood interactions
appropriate for evaluation (see also 6.1.6). Test selection criteria depend on the specific device evaluated.
6.2.5 Indications and limitations
Immunoassays are available for human blood testing but are not generally available for other species. The human
test kits usually do not cross-react with other species except for some non-human primates. Care should be taken
when designing test systems to ensure that one is actually measuring activation due to the test material and not an
artifact of the system. In vitro and ex vivo simulations with human blood often produce plasma levels of analytes
that require a low, medium or high level of dilution, depending on experimental conditions, for measurement in the
valid range of the immunoassay. Care should be taken to report only those results measured within the valid
ranges of the assays. Care should also be taken to ensure that a range of dilutions of the sample tested is
measured.
Discrepancies in evaluating blood/device interactions may occur because of inadequate materials characterization
or inappropriate handling before blood tests are performed. For example, the studies may have relied on only one
type of test or may have permitted the introduction of foreign material unrelated to the material or device under test.
Materials to be used in a low-flow (venous) environment may interact with blood quite differently when used in high-
flow (arterial) situations. Changes in design and/or flow conditions can alter the apparent in vivo
haemocompatibility of a material.
6.3 Types of test
6.3.1 In vitro tests
Variables that shall be considered when using in vitro test methods include haematocrit, anticoagulants, sample
collection, sample age, sample storage, aeration and pH, temperature, sequence of test versus control studies,
surface-to-volume ratio, and fluid dynamic conditions (especially wall shear rate). Tests shall be performed with
minimal delay, usually within 4 h, since some properties of blood change rapidly following collection.
6.3.2 Ex vivo tests
Ex vivo tests shall be performed when the intended use of the device is ex vivo, for example an external
communicating device. Ex vivo testing can also be useful when the intended use is in vivo, for example 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,
[20], [30], [48]
thrombus mass, white-cell adhesion, platelet consumption, and platelet activation . Blood flowrates can
be measured with either Doppler or electromagnetic flow probes. Alterations in flowrates may indicate the extent
and course of thrombus deposition and embolization.
Many ex vivo test systems use radiolabelled blood components to monitor blood/device interactions. Platelets and
fibrinogen are the components of blood which are most commonly radiolabelled . Alteration of platelet reactivity by
[23], [24], [25]
the labelling procedure can be minimized by strict attention to technical detail .
The advantages of ex vivo tests over in vitro tests are that flowing native blood is used (providing physiological flow
conditions), several materials can be evaluated since the chambers can be changed, and it is possible to monitor
some events in real time. Some disadvantages include variability in blood flowrate from one experiment to another,
variable blood reactivity from one animal to the other, and the usually relatively short time intervals that can be
evaluated. Positive and negative controls using the same animal are recommended in this regard.
6.3.3 In vivo tests
In vivo testing involves implanting the material or device in animals. Vascular patches, vascular grafts, prosthetic
rings, heart valves and circulatory assist devices are examples of configurations used in in vivo testing.
ISO 10993-4:2002(E)
Patency (of a conduit) is the most common measure of success or failure for most 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 (for example ventricular-assist devices, artificial hearts, aortic prosthetic grafts) [19].
Methods to evaluate in vivo interactions without terminating the experiment are available. Arteriograms are used to
determine graft 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.
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 flowrate systems may give substantially different results
when compared to the same material evaluated in a high flowrate system. In such cases, test system performance
in vivo should carry more importance than in vitro test results.
12 © ISO 2002 – All rights reserved

ISO 10993-4:2002(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 of this part of ISO 10993 contains guidance on when testing is necessary, what 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.
A.1.2 Classification
The following classification of blood/device interactions is provided as background.
a) Interactions which mainly affect the device and which may or may not have an undesirable effect on the
subject are as follows:
1) 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;
2) adhesion of platelets, leukocytes or erythrocytes onto the surface of the device, or absorption of their
components into the device;
3) formation of pseudointima or tissue capsule on the surface of the device;
4) alterations in mechanical and other properties of the device.
b) Interactions which have a potentially undesirable effect on the animal or human are as follows:
1) activation of platelets, leukocytes or other cells, or activation of the coagulation, fibrinolytic, or complement
pathways;
2) formation of thrombi on the device surface;
3) embolization of thrombotic or other material from the device's luminal surface to another site within the
circulation;
4) injury to circulating blood cells resulting in anemia, haemolysis, leucopenia, thrombocytopenia or altered
function of blood cells;
5) injury to cells and tissues adjacent to the device;
6) intimal hyperplasia or accumulation of other tissue on or adjacent to the device, resulting in reduced flow
or affecting other functions of the device;
7) adhesion and growth of bacteria or other infectious agents on or near the device.
ISO 10993-4:2002(E)
A.1.3 Advantages and limitations of animal and in vitro testing
Animal models permit continuous device monitoring and systematic controlled study of important variables.
However the choice of an animal model may be restricted by size requirements, the availability of certain species
and cost. 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 anesthetics, 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 bear a close sim
...