EN 30993-4:1993

SLOVENSKI STANDARD
01-januar-2000
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Biological evaluation of medical devices - Part 4: Selection of tests for interactions with
blood (ISO 10993-4:1992)
Biologische Beurteilung von Medizinprodukten - Teil 4: Auswahl von Prüfungen zur
Wechselwirkung von Blut mit Fremdoberflächen (ISO 10993-4:1992)
Evaluation biologique des dispositifs médicaux - Partie 4: Choix des essais concernant
les actions avec le sang (ISO 10993-4:1992)
Ta slovenski standard je istoveten z: EN 30993-4:1993
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.

INTERNATIONAL IS0
STANDARD 10993-4
First edition
1992-l 2-15
Biological evaluation of medical devices -
Part 4:
Selection of tests for interactions with blood
gvalua tion biologique des dispositifs mgdicaux -
Par-tie 4: Choix des essais concernant /es interactions avec le sang
Reference number
IS0 10993-4:1992(E)
IS0 10993=4:1992(E)
Contents
Page
. . . . . . . . . . . . . . . . . . . . . . .~.~.
1 Scope
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 Normative reference
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
3 Definitions
.,,,.~.,.,,,.,,. 1
4 Abbreviations
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Devices contacting blood
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
5.1 Non-contact devices
............................................ 2
5.2 External communicating devices
5.3 Implant devices .
..........................................................................................
6 Tests
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1 General recommendations
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Test methods
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3 Types of tests
Annexes
A Evaluation of cardiovascular devices and prostheses during in vivo
function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.................... 13
B Laboratory tests: principles and scientific basis
............................................................................
C Bibliography
63 IS0 1992
All rights reserved. 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 per-
mission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
IS0 10993=4:1992(E)
Foreword
IS0 (the International Organization for Standardization) is a worldwide
federation of national standards bodies (IS0 member bodies). The work
of preparing International Standards is normally carried out through IS0
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. IS0
collaborates closely with the International Electrotechnical Commission
(I EC) on all matters of electrotechnical standardization.
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.
International Standard IS0 10993-4 was prepared by Technical Committee
lSO/TC 194, Biological evaluation of medical devices.
IS0 10993 consists of the following parts, under the general title Biological
evaluation of medical devices:
- Part 1: Guidance on selection of tests
- 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 cytotoxicity: in vitro methods
- Part 6: Tests for local effects after implantation
- Part 7: Ethylene oxide sterilization residuals
- Part 8: Clinical investigation
- Part 9: Degradation of materials related to biological testing
- Part 10: Tests for irritation and sensitization
- Part 11: Tests for systemic toxicity
- Part 12: Sample preparation and reference materials
Future parts will deal with other relevant aspects of biological testing.
Annexes A, B and C of this part of IS0 10993 are for information only.
. . .
III
IS0 10993=4:1992(E)
Introduction
The initial source for developing this part of IS0 10993 was the publi-
cation, Guidelines for blood/material interactions: Report of the National
Heart, Lung, and Blood Institute working group [26]; chapters 9 and 10.
This publication is being revised [29].

INTERNATIONAL STANDARD IS0 10993=4:1992(E)
Biological evaluation of medical devices -
Part 4:
Selection of tests for interactions with blood
the standard indicated below. Members of IEC and
1 Scope
IS0 maintain registers of currently valid International
Standards.
This part of IS0 10993 gives guidance to agencies,
manufacturers, research laboratories and others for
IS0 10993-I : 1992, Biological evaluation of medical
evaluating the interactions of medical devices with
devices - Part 1: Guidance on selection of tests.
blood.
It describes:
a) a classification of medical and dental devices that
3 Definitions
are intended for use in contact with blood, based
on the intended use and duration of contact as
For the purposes of this part of IS0 10993, the defi-
defined in IS0 10993-l;
nitions given in IS0 10993-l and the following defi-
nitions apply.
b) the fundamental principles governing the evalu-
ation of the interaction of devices with blood;
3.1 blood/device interaction: Any interaction be-
tween blood or any component of blood and a device
c) the rationale for structured selection of tests, to-
resulting in effects on the blood, or on any organ or
gether with the principles and scientific basis of
tissue or on the device. Such effects may or may not
these tests.
have clinically significant or undesirable conse-
quences.
Detailed requirements for testing cannot be specified
because of the limitations in knowledge and precision
3.2 ex vivo: Term applied to test systems that shunt
of tests for interactions of devices with blood.
blood directly from a human subject or test animal
into a test chamber. If using an animal model, the
blood may be shunted directly back into the animal
(recirculating) or collected into test tubes for evalu-
ation (single pass). In either case, the test chamber is
2 Normative reference
located outside the body.
The following standard contains provisions which,
through reference in this text, constitute provisions
of this part of IS0 10993. At the time of publication,
4 Abbreviations
the edition indicated was valid. All standards are sub-
ject to revision, and parties to agreements based on
this part of IS0 10993 are encouraged to investigate Table 1 provides a list of abbreviations used in the
the possibility of applying the most recent edition of context of this part of IS0 10993.

IS0 10993=4:1992(E)
Table 1 - Abbreviations
Meaning
Abbreviation
Bb Product of alternate pathway complement activation
Beta-thromboglobulin
B TG
&d Product of classical pathway complement activation
C3a, C5a (active) complement split products from C3 and C5
D-Dimer Specific fibrin degradation products (F XIII cross-linked fibrin)
ECMO Extracorporeal membrane oxygenator
E.M. Electron microscopy
FDP Fibrin/fibrinogen degradation products
FPA Fibrinopeptide A
Prothrombin activation fragment 1 + 2
h+2
Product of central C complement activation
iC3b
Interleukin-1
IL-l
Inferior vena cava
IVC
Magnetic resonance imaging
MRI
PAC-1 Monoclonal antibody which recognizes the activated form of platelet surface glycoprotein llb/llla
PET Positron emission tomography
PF-4 Platelet factor 4
PT Prothrombin time
PTT Partial thromboplastin time
RIA Radioimmunoassay
Monoclonal antibody which recognizes the alpha granule membrane component GMP140 exposed
S-l 2
during the platelet release reaction
Product of terminal pathway complement activation
SC5b-9
Thrombin-antithrombin complex
TAT
Terminal complement complex
TCC
TT Thrombin time
von Willebrand factor
VWF
devices for the collection of blood,
5 Devices contacting blood
devices for the storage and administration of blood
Devices contacting blood are categorized in
and blood products (e.g. tubing, needles and
IS0 10993-l :1992, clause 5.
bags).
5.1 Non-contact devices
(See subclause 5.1.1 of IS0 10993-1:1992.)
5.2.2 External communicating devices in contact
An example is in vitro diagnostic devices.
with circulating blood [see 5.1.3c) of
IS0 10993-I :I9921 include but are not limited to
5.2 External communicating devices
cardiopulmonary bypass,
(See subclause 5.1.3 of IS0 10993-l :1992.)
extracorporeal membrane oxygenators,
These are devices that contact the circulating blood
haemodialysis equipment,
and serve as a conduit into the vascular system. Ex-
amples include but are not limited to those in 5.2.1
donor and therapeutic apheresis equipment,
and 5.2.2.
devices for absorption of specific substances from
5.2.1 External communicating devices that serve as
blood,
an indirect blood path [see subclause 5.1.3 a) of
IS0 10993-I :I 9921 include but are not limited to interventional cardiology and vascular devices,
cannulae,
percutaneous circulatory support systems,
extension sets, temporary pacemaker electrodes.

IS0 10993=4:1992(E)
6.1.3 Testing of materials which are candidates to
5.3 Implant devices
be components of a device should be conducted for
screening purposes. However, such tests do not
These are devices (see 5.1.4 of IS0 10993-I :I 992)
serve as a substitute for the requirement that the
that are placed largely or entirely within the vascular
complete device be tested under conditions which
system. Examples include but are not limited to
simulate clinical application.
mechanical or tissue heart valves,
6.1.4 Tests which do not simulate the conditions of
a device during use may not predict accurately the
prosthetic or tissue vascular grafts,
nature of the blood/device interactions which may
occur during clinical applications. For example, some
circulatory support devices (ventricular-assist de-
short-term in vitro or ex viva tests are poor predictors
vices, artificial hearts, intra-aortic balloon pumps),
of long-term in vivo blood/device interactions [22],
inferior vena cava filters, .
cm
stents,
6.1.5 It follows from the above that devices whose
intended use is ex v;vo (external communicating)
arteriovenous shunts,
should be tested ex viva and devices whose intended
use is in viva (implants) should be tested in vivo in an
blood monitors, animal model under conditions simulating where
possible clinical use.
internal drug delivery catheters,
6.1.6 In vitro tests are regarded as useful in screen-
pacemaker electrodes,
ing external communicating devices or implants but
may not be accurate predictors of blood/device inter-
intravascular membrane oxygenators (artificial
actions occurring upon prolonged or repeated ex-
lungs).
posure or permanent contact (see 6.3.2). 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
6 Tests
(e.g. lancets, hypodermic needles, capillary tubes)
generally do not require blood/device interaction test-
ing.
6.1 General recommendations
6.1.7 The two recommendations in 6.1.5 and 6.1.6,
together with clause 5, serve as a guide for the
6.1.1 Where possible, tests should use an appropri-
selection of tests listed in 6.2.1.
ate model or system which simulates the geometry
and conditions of contact of the device with blood
6.1.8 Disposable laboratory equipment used for the
during clinical applications, including duration of con-
collection of blood and performance of in vitro tests
tact, temperature, sterile condition and flow con-
on blood should be validated to ascertain that there is
ditions. For devices of defined geometry such as
no significant interference with the test being per-
vascular grafts of varying lengths, the relation of sur-
formed. This can be conducted by performing tests
face area (length) to test results should be evaluated.
on reference standards and comparing results with
those obtained by a clinically approved technique.
paramete rs should be in
The selected methods and
state of t he art.
acco rdance with the current
6.1.9 If tests are selected in the manner described
and testing is conducted under conditions which
NOTE 1 Only blood-contacting parts should be tested.
simulate clinical applications, the results of such test-
ing have the greatest probability of predicting clinical
performance of devices. However, species differ-
6.1.2 Controls shall be used unless their omission
ences and other factors may limit the predictability of
can be justified. Where possible, testing should in-
any test.
clude a device already in clinical use or well-
characterized reference materials. Several materials
6.1.10 Because of species differences in blood re-
and configurations are available (see IS0 10993-I 2
activity, human blood should be used where possible.
.
VI)
When animal models are necessary, for example for
Reference materials used should include negative and
evaluation of devices used for prolonged or repeated
positive controls. All materials tested should meet all
exposure or permanent contact, species differences
quality control and quality assurance procedures of
in blood reactivity should be considered. Blood values
the manufacturer and test laboratory and should be and reactivity between humans and non-human
identified as to source, manufacturer, grade and type. primates are very similar [23].

IS0 10993=4:1992(E)
NOTE 2 The use of non-human primates for blood com-
Table 5:
Implant devices - Level 1
patibility and medical device testing is prohibited by EC law
(86/906/EEC) and some national laws. Table 6: Implant devices - Level 2 - optional
Level 1 and level 2 tests are classified into five cat-
However, the use of species other than non-human
egories based on the primary process or system being
primates such as the pig, calf or sheep may also yield
measured:
satisfactory results. The canine model has been found
useful in the pre-clinical evaluation of prosthetic
a) thrombosis,
vascular grafts [43]. Because species differences
may be significant (for example platelet adhesion,
b) coagulation,
thrombosis, [17] and haemolysis tends to occur more
readily in the canine species than in the human), all
c) platelets and platelet functions,
results of animal studies should be interpreted with
caution.
d) haematology,
6.1.11 The use of anticoagulants should be avoided
e) immunology.
unless the device is designed to perform in their
presence. The choice and concentration of anticoagu-
Two levels of tests are presented. Select one or more
lant used influence blood/device interactions. Devices
test method(s) from each category of level 1 tests
that are used with anticoagulants shall be assessed
(tables 2, 3 and 5), in order to obtain the maximum
using anticoagulants in the range of concentrations
information about the spectrum of reactions initiated
used clinically.
when a device contacts blood.
Additional testing from level 2 (tables 4 and 6) is op-
6.1.12 Minor modifications in a clinically accepted
tional. The principles and scientific basis for these
device may cause significant changes in its clinical
tests are presented in annex B.
functions. Examples of such modifications include
changes in design, changes in surface or bulk chemi-
6.2.1 .I Non-contact devices
cal composition of materials and changes in texture,
porosity or other properties of vascular grafts. There-
These devices generally do not require blood/device
fore the effect of such changes on blood/device
interaction testing. Disposable test kits should be
interactions shall be considered for clinical signifi-
validated to rule out interference of materials with test
cance.
accuracy.
6.1.13 A sufficient number of tests including suit-
6.2.1.2 External communicating devices
able controls shall be performed to permit statistical
evaluation of the data. The variability in some test
The external communicating devices and their test
methods requires that those tests be repeated a suf-
methods are listed in tables 2 to 4. These test meth-
ficient number of times to determine significance. In
ods are recommended for devices intended for Iim-
addition repeated studies over an extended period of
ited (LI, < 24 h) and prolonged or repeated (PR, 24 h
blood/device contact provide information about the
to 30 days) exposure. See also 6.1.6.
time-dependence of the interactions. A statistician
should be consulted in the early stages of exper-
6.2.1.3 Implant devices
imental design.
The implant devices and their test methods are listed
in tables 5 and 6. These test methods are rec-
ommended for devices intended for prolonged or re-
6.2 Test methods
peated (PR, 24 h to 30 days) exposure or permanent
contact (PC, > 30 days).
6.2.1 Recommended tests for interactions of
with
devices and materials blood
6.2.2 Indications and limitations
Recommended tests are organized on the basis of the
Table 7 gives a list of commercially available assays
type of device:
validated for use with human blood and tables 2 to 6
present a list of tests. Level 1 tests, which shall be
Table 2: External communicating devices -
considered, are relatively simple for general use in
Level 1 - blood path, indirect
evaluating the interactions of materials and devices
with blood. Level 2 tests are more sophisticated, re-
Table 3: Externa communicating
devices -
quire special expertise in performance and interpret-
Level 1 - circulating blood
ation, and are regarded as optional. In both categories,
Externa communicating
Table 4: devices - strict attention to technical detail is required. RIAs are
Level 2 - optional
available for human blood testing but are not generally

IS0 10993=4:1992(E)
of conditions, including improper blood collection.
available for other species. The human test kits usu-
ally do not cross-react with other species except for Tests to verify normal platelet reactivity are usually
some non-human primates. Care should be taken performed with an aggregometer. Platelet prep-
when designing test systems to ensure that one is arations with reduced reactivity are easily detected
actually measuring activation due to the test material using this method, but hyperreactive platelets are not
and not an artifact of the system. normally detected. Platelet aggregation tests can be
modified (by appropriately reducing the concentration
Discrepancies in evaluating blood/device interactions
of platelets or aggregating agents) to determine if
may occur because of inadequate materials charac-
platelets become hyperreactive following exposure to
terization or inappropriate handling before blood tests
a material or device.
are performed. For example the studies may have re-
lied on only one type of test or may have permitted
the introduction of foreign material unrelated to the
6.3.1.2 Coagulation tests
material or device under test. Materials to be used in
a low flow (venous) environment may interact with
Coagulation methods are based on the use of native
blood quite differently when used in high flow (ar-
(fresh, non-anticoagulated) whole blood, anticoagu-
terial) situations. Changes in design and/or flow con-
lated whole blood (usually titrated), platelet-rich
ditions can alter the apparent in vivo haemo-
plasma or platelet-poor plasma. Since most of the
compatibility of a material.
standard coagulation assays are designed to detect
clinical coagulation disorders which result in delayed
6.3 Types of tests
clotting or excessive bleeding, the protocols for eval-
uating blood/device interactions should be modified
6.3.1 In vitro tests appropriately to evaluate accelerated coagulation in-
duced by biomaterials. Reagents for tests based on
Variables that should be considered when using in
the activated partial thromboplastin time include an
vitro test methods include haematocrit, anticoagu-
activator such as kaolin, celite, or ellagic acid. Re-
lants, sample collection, sample age, aeration and pH,
agents with such activators should be avoided be-
temperature, sequence of test versus control studies,
cause they tend to mask the acceleration of
surface-to-volume ratio, and fluid dynamic conditions
coagulation which materials and devices cause. The
(especially wall shear rate). Tests should be per-
material to be tested serves as the activator; controls
formed with minimal delay, usually within 4 h, since
(without the material) should be included.
some properties of blood change rapidly following
collection. Blood is exposed to test materials either in a static
chamber such as a parallel plate cell or in a closed-
loop system where the inner surface of the tubing is
6.3.1.1 Platelet tests
the test material. After a predetermined contact time
Blood collection techniques should be reproducible. with the test surface, tests of the surface and blood
Platelets can become hyperreactive under a variety can be conducted.
Table 2 - External communicating devices - Level 1 - Blood path, indirect (see 5.2.1)
Test category Method Comments
I
I
Thrombosis Light microscopy (adhered platelets, leucocytes, Light microscopy can be replaced by scanning E.M.
aggregates, erythrocytes, fibrin, etc.) if the nature of the material presents technical
problems for light microscopy.
PTT (non-activated)
Coagulation 1
I
Platelet count
Platelets
Leucocyte count and differential; Haemolysis is regarded as an especially significant
Haematology
screening test to perform in this category because
haemolysis (plasma haemoglobin)
of its measurement of red blood cell membrane
fragility in contact with materials and devices. The
method used should be one of the normative
standard test methods for haemolysis.
Immunology C3a, C5a, TCC, Bb, iC3b, C4d, SC5b-9 A panel including the last four tests encompasses
the various complement activation pathways.

IS0 10993=4:1992(E)
Table 3 - External communicating devices - Level 1 - Circulating blood (see 5.2.2)
Method Comments
Test category
Thrombosis Per cent occlusion; Light microscopy can be replaced by scanning E.M.
if the nature of the material presents technical
flow reduction;
problems for light microscopy.
gravimetric analysis (thrombus mass);
Pressure drop not recommended for devices in-
tended for PR (see 6.212).
light microscopy (adhered platelets, leucocytes, ag-
gregates, erythrocytes, fibrin, etc.);
pressure drop across device
PTT (non-activated)
Coagulation
Platelet count;
Platelets
platelet aggregation;
template bleeding time
Haematology Leucocyte count and differential; Haemolysis is regarded as an especially significant
screening test to perform in this category because
haemolysis (plasma haemoglobin)
of its measurement of red blood cell membrane
fragility in contact with materials and devices. The
method used should be one of the normative
standard test methods for haemolysis.
Immunology C3a, C5a, TCC, Bb, iC3b, C4d, SC5b-9 A panel including the last four tests encompass the
various complement activation pathways.
Table 4 - External communicating devices - Level 2 - optional
Method Comments
Test category
Scanning E.M. (platelet adhesion and aggregation;
Thrombosis
platelet and leucocyte morphology; fibrin)
Coagulation Specific coagulation factor assays;
FPA, D-dimer, F1 + 2, PAC-1, S-12, TAT
PF-4, j3-TG; thromboxane B2; Wn-labelling is recommended for PR only (see
Platelets
6.2.1.2).
gamma imaging of radiolabelled platelets Wn-
labelled platelet survival
Haematology Reticulocyte count; activation specific release pro- Recommended for PR only (see 6.2.1.2).
ducts of peripheral blood cells (i.e. granulocytes)
Immunology IL-I and other cytokines; detection of
messenger-RNA specific for cytokines

IS0 10993=4:1992(E)
Table 5 - Implant devices - Level 1 (see 6.3)
Method
Test category Comments
Thrombosis Per cent occlusion;
flow reduction;
autopsy of device (gross and microscopic);
autopsy of distal organs (gross and microscopic)
Coagulation PTT (non-activated), PT, TT;
plasma fibrinogen, FDP
Platelet count;
Platelets
platelet aggregation
Haematology 1 Leucocyte count and differential; 1 Haemolysis is regarded as an especially significant
screening test to perform in this category because
haemolysis (plasma haemoglobin)
of its measurement of red blood cell membrane
I
fragility in contact with materials and devices. The
method used should be one of the normative
standard test methods for haemolysis.
Immunology C3a, C5a, TCC, Bb, iC3b, C4b, SC5b-9 A panel including the last four tests encompassses
the various complement activation pathways.
Table 6
- Implant devices - Level 2 - optional
Method
Test category Comments
r
Scannning E.M.;
Thrombosis
angiography
Coagulation Specific coagulation factor assays;
FPA, D-dimer, F1 + 2, PAC-1, S-l 2, TAT
Platelets Yn-labelled platelet survival PF-4, j%TG
thromboxane B2;
gamma imaging of radiolabelled platelets
Reticulocyte count; activation specific release pro-
Haematology
ducts of peripheral blood cells (i.e. granulocytes)
Immunology IL-I and other cytokines; detection of
messenger-RNA specific for cytokines
6.3.2 Ex viva tests Ex vko test systems are available for monitoring
platelet adhesion, emboli generation, fibrinogen de-
position, thrombus mass, white cell adhesion, platelet
consumption, and platelet activation [I 71, [27], [43].
Blood flow-rates can be measured with either Doppler
Ex viva tests should be performed when the intended
or electromagnetic flow probes. Alterations in flow-
use of the device is ex viva, for example an external
rates may indicate the extent and course of thrombus
communicating device. Ex viva testing may also be
deposition and embolization.
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.
IS0 10993=4:1992(E)
Many ex vivo test systems use radiolabelled blood per cent occlusion and thrombus mass are deter-
components to monitor blood/device interactions. mined after the device is removed. The tendency of
Radiolabelled platelets and fibrinogen are the most thrombi formed on a device to embolize to distal or-
commonly labelled components of blood. Alteration gans should be assessed by a careful gross as well
as microscopic examination of organs downstream
of platelet reactivity by the labelling procedure can be
minimized by strict attention to technical detail [20], from the device. The kidneys are especially prone to
trap thrombi which have embolized from devices im-
[21], c321.
planted upstream from the renal arteries (for example
Advantages of ex viva tests over in vitro tests are that
ventricular-assist devices, artificial hearts, aortic pros-
flowing native blood is used (thus eliminating artifacts
thetic grafts) [I 61.
caused by anticoagulants as well as providing physio-
logical flow conditions), several materials can be
Methods to evaluate in viva performance without ter-
evaluated since the chambers can be changed, and it
minating the experiment are available. Arteriograms
is possible to monitor some events in real time. Some
are used to determine graft patency or thrombus de-
disadvantages include variability in blood flow from
position on devices. Radioimaging can be used to
one experiment to another, variable blood reactivity
monitor platelet deposition at various time periods in
from one animal to the other, and the usually relatively
viva; platelet survival and consumption can be used
short time intervals that can be evaluated. Positive
interactions and
as indicators of blood/device
and negative controls using the same animal are rec-
passivation due to neointima formation or protein
ommended in this regard.
adsorption.
6.3.3 ln vivo tests
In some in viva test systems the material’s properties
may not be major determinants of the blood/device
In viva testing involves implanting the material or de-
interactions. Flow parameters, compliance, porosity
vice in animals. Vascular patches, vascular grafts,
and implant design may be more important than blood
prosthetic rings, heart valves and circulatory-assist
compatibility of the material itself. As an example, low
devices are examples of configurations used in in vivo
flow-rate systems may give substantially different re-
testing. In viva tests are usually designed to examine
sults when compared to the same material evaluated
haemocompatibility over a period longer than 24 h.
in a high flow-rate system. In such cases, test system
performance in vivo should carry more importance
Patency (of a conduit) is the most common measure
than in vitro test results.
of success or failure for most in viva experiments. The

IS0 10993-k 1992(E)
Table 7 - Commercially available assays for platelet,
coagulation, fibrinolytic and complement factors
Factor Types of assay
Plasminogen Calorimetric
Fluorogenic
Chromogenic, fluorogenic
Antithrombin III
Protein C Chromogenic
Chromogenic
Protein S
Antiplasmin Chromogenic
Prekallikrein Chromogenic
Kallikrein Fluorogenic
RIA, ELISA
PF-4
Chromogenic
$-TG RIA, ELISA
Thromboxane B2 RIA, ELISA
Chromogenic; clotting time
Factor VI II-VWF
Factor IX Chromogenic; clotting time
Factor IXa Fluorogenic
Chromogenic; clotting time
Factor X
Fluorogenic
Factor Xa
Chromogenic; clotting time
Factor XI I
Factor Xl la Fluorogenic
FPA RIA, ELISA
RIA
C3,, C5,
Bb, iC3b, C4d, SC5b-9 ELISA
TCC ELISA
TAT RIA, ELISA
IL-I RIA, ELISA
RIA, ELISA
h+2
RIA, ELISA
D-dimer
NOTE - These assays have been validated for human use. Vali-
dation of their accuracy for other species shall be determined prior
to use.
IS0 10993=4:1992(E)
Annex A
(informative)
Evaluation of cardiovascular devices and prostheses during in I&O function
e) injury to cells and tissues adjacent to the device;
A.1 General considerations
f) intimal hyperplasia or accumulation of other tissue
A.1.1 This annex provides background for selecting
on or adjacent to the device, resulting in reduced
tests to evaluate the interactions of cardiovascular flow or affecting other functions of the device;
devices with blood. Subclause 6.2.1 contains a com-
plete list of recommended (level 1) and optional (level
g) adhesion and growth of bacteria or other infectious
2) tests for evaluating blood/device interactions of the
agents to or near the device.
three categories of devices: non-contact, external
communicating, and implant.
A.l.3 Procedures used to evaluate cardiovascular
devices in animals are essentially the same as those
A.l.2 The following classification of blood/device
employed in the clinical setting. However, animal
interactions is provided as background.
models permit continuous device monitoring and
systematic controlled study of important variables.
A.1.2.1 Interactions which mainly affect the device
and which may or may not have an undesirable effect
A.1.4 The test protocols recommended follow cer-
on the subject are as follows:
tain general guidelines. Thrombosis, thromboembol-
ism, bleeding and infection are the major deterrents
adsorption of plasma proteins, lipids, calcium or
a)
to the use and further development of advanced car-
other substances from the blood onto the surface
diovascular prostheses. For devices with limited blood
of the device; or absorption of such substances
exposure (< 24 h), important measurements are re-
into the device;
lated to the acute extent of variation of haematologic,
haemodynamic and performance variables, gross
adhesion of platelets, leucocytes or erythrocytes
b)
thrombus formation and possible embolism. With
onto the surface of the device, or absorption of
prolonged or repeated exposure, or permanent con-
their components into the device;
tact (> 24 h), emphasis is placed on serial measure-
ment techniques that may yield information regarding
formation of pseudointima or tissue capsule on the
d
the time course of thrombosis and thromboembolism,
surface of the device;
the consumption of circulating blood components, the
development of intimal hyperplasia and infection. In
alterations in mechanical and other properties of
d)
both of the above exposure and contact categories,
the device.
assessment of haemolysis is important. Thrombus
formation may be greatly influenced by surgical tech-
A.l.2.2 Interactions which have a potentially unde-
nique, variable time-dependent thrombolytic and
sirable effect on the animal or human are as follows:
embolic phenomena, superimposed device infections
and possible alterations in exposed surfaces, for ex-
a) activation of platelets, leucocytes or other cells,
ample intimal hyperplasia and endothelialization.
or activation of the coagulation, fibrinolytic, com-
plement, or other pathways, including immuno-
The consequences of the interaction of artificial sur-
toxicity (immunosuppression, immunopoten-
faces with the blood may range from gross throm-
tiation, immunomodulation);
bosis and embolization to subtle effects such as
accelerated consumption of haemostatic elements;
b) formation of thrombi on the device surface;
the latter may be compensated or lead to depletion
of platelets or plasma coagulation factors.
c) embolization of thrombotic or other material from
the device’s luminal surface to another site within
the circulation;
A.1.5 Disturbances of organ function may occur due
to blood/device interaction. For instance kidney func-
d) injury to circulating blood cells resulting in anae-
tion and pulmonary function may be affected by acti-
mia, haemolysis, leucopenia, thrombocytopenia or
vated blood coagulation and platelet/leucocyte/
altered function of blood cells;
complement interactions.
IS0 1099s4:1992(E)
Platelet survival and plasma levels of the platelet-
cells or clotting factors. Cannulae, like other indirect
specific proteins PF-4 and P-TG may reflect the extent
blood path devices (5.2.1), generally require less test-
of platelet activation in viva (and perhaps risk of
ing than devices in circulating blood (6.2.1.2).
thromboembolism) even in the absence of signifi-
cantly elevated rates of platelet consumption. The
A.3 Catheters and guidewires
template bleeding time is an index of in viva platelet
function; a prolonged value suggests thrombocyto-
Most of the tests considered under cannulae are rel-
penia or a qualitative platelet disorder, such as may
evant to the study of catheters and guidewires. The
occur during cardiopulmonary bypass [28]. Measure-
location or placement of catheters in the arterial or
ments of FPA may indicate activation of intrinsic co-
venous system can have a major effect on
agulation.
blood/device interactions. It is advised that simul-
Localization of thrombotic material by radionuclide
taneous control studies be performed using a
imaging techniques using radiolabelled platelets has
contralateral artery or vein. Care should be taken not
been demonstrated in studies of vascular grafts, valve to strip off thrombus upon catheter withdrawal. In situ
prostheses, and other devices, both implants and ex- evaluation may permit assessment of the extent to
ternally communicating devices [20], [21], [36]. In which intimal or entrance site injuries contributed to
addition, duplex scanning and a careful examination the thrombotic process.
Kinetic studies with
of the explanted device can provide very useful infor- radiolabelled blood constituents are recommended
mation [37], [38]. only with chronic catheters, but may be useful for
imaging thrombus accumulation in vivo. Angiography
and Doppler blood flow measurements may also be
Al.6 The choice of an animal model may be re-
useful.
stricted by size requirements, the availability of certain
species and cost. It is critical that the investigators be
mindful of the physiological differences and simi-
A.4 Extracorporeal oxygenators,
larities of the species chosen with those of the hu-
haemodialyzers, therapeutic apheresis
man, particularly those relating to coagulation, platelet
equipment, and devices for absorption of
functions and fibrinolysis, and the response to
specific substances from blood
pharmacological agents such as anaesthetics, anti-
coagulants, thrombolytic and antiplatelet agents, and
The haemostatic response to cardiopulmonary bypass
antibiotics. Because of species differences in reactiv-
may be significant and acute. Many variables such as
ity and variable responses to different devices, data
use of blood suction, composition of blood pump
obtained from a single species should be interpreted
priming fluid, hypothermia, blood contact with air and
with caution. Non-human primates such as baboons
time of exposure influence test values. Emboli in
bear a close similarity in haematologic values, blood
outflow lines may be detected by the periodic place-
coagulation mechanism and cardiovascular system to
ment of blood filters ex vivo, or the use of ultrasonic
the human [27]. An additional advantage of a non-
radiation or other non-invasive techniques. Thrombus
human primate is that many of the immunologic
accumulation can be indirectly assessed during by-
probes for thrombosis developed for humans are
pass by monitoring performance factors such as
suitable for use in other primates. These probes in-
pressure drop across the oxygenator and oxygen
clude PF-4, P-TG, FPA, TAT, and F, +2. The dog is a
transfer rate. An acquired transient platelet
commonly used species and has provided useful in-
dysfunction associated with selective alpha granule
formation; however, device-related thrombosis in the
release has been observed in patients on cardio-
dog tends to occur more readily than in the human, a
pulmonary bypass [28]; the template bleeding time
difference which can be viewed as an advantage
and other tests of platelet function and release are
when evaluating this complication. The pig is gener-
particularly useful.
ally regarded as a suitable animal model because of
its haematologic and cardiovascular similarities to the
Complement activation is caused by both haemodia-
human. The effect of the surgical implant procedure
lyzers and cardiopulmonary bypass apparatus. Clini-
on results should be kept in mind and appropriate
cally significant pulmonary leucostasis and lung injury
controls included.
with dysfunction may result. For these reasons, it is
useful to quantify complement activation with these
devices.
A.2 Cannulae
Therapeutic apheresis equipment and devices for ab-
Cannulae are typically inserted into one or more major sorption of specific substances from the blood, be-
blood vessels to provide repeated blood access. They cause of their high surface-to-volume ratio, can
are also used during cardiopulmonary bypass and potentially activate complement, coagulation, platelet
other procedures. They may be tested acutely or and leucocyte
pathways. Examination of
chronically and are commonly studied as arterio- blood/device interactions should follow the same
venous (AV) shunts. The use of cannulae appears to principles as for extracorporeal oxygenators and hae-
induce little alteration in the levels of circulating blood modialyzers.

IS0 10993=4:1992(E)
Measurements of platelet survival and aggregation,
A.5 Ventricular-assist devices
blood tests of thrombosis and haemolysis, pressure
and flow measurements, and autopsy of the valve and
These devices may induce considerable alteration in
adjacent tissues are also recommended.
various blood components. Factors contributing to
such effects include the large foreign surface area to
which blood is exposed, the high flow regimes and
A.7 Vascular grafts
the regions of disturbed flow such as turbulence or
separated flow. Tests of such devices may include
Both porous and non-porous materials can be im-
measurements of haemolysis, platelet and fibrinogen
planted at various locations in the arterial or venous
concentration, platelet survival, complement acti-
system. The choice of implantation site is determined
vation, and close monitoring of liver, renal, pulmonary
largely by the intended use for the prosthesis.
and central nervous system function. A detailed
Patency of a given graft is enhanced by larger diam-
pathologic examination at surgical retrieval is an im-
eter and shorter length. A rule of thumb for grafts less
portant component of the evaluation [37], [38].
than 4 mm ID is that the length should exceed the
diameter by a factor of 10 (i.e., 40 mm for a 4 mm
graft) for a valid model. Patency can be documented
A.6 Heart valve prostheses
by palpation of distal pulses in some locations and by
periodic angiography. Ultrasonic radiation, MRI, and
Invasive, non-invasive and in vitro hydrodynamic
PET may also be useful. Results of serial radiolabelled
studies are important in the assessment of prosthetic
platelet imaging studies correlate with the area of
valves.
non-endothelialized graft surface in baboons [27].
Radiolabelled platelets facilitate non-invasive imaging
2D and M mode echocardiography makes use of
of mural thrombotic accumulations. Serial measure-
ultrasonic radiation to form images of the heart. Re-
ments of platelet count, platelet release constituents,
flections from materials with different acoustic im-
fibrinogen/fibrin degradation products and activated
pedances are received and processed to form an
coagulation species also are re
...