COMMITTEE DRAFT ISO/CD 10993-4
Date 1999-02-01
Reference number ISO/TC 194 /SC N318
ISO/TC 194/sc
Title
Biological evaluation of medical devices
Secretariat DIN
Circulated to P- and O-members, and to technical
committees and organizations in liaison for:
x discussion at next WG 9 meeting on 99-05-19
[Venue/date of meeting]
x comments by 1999-05-01 [date]
x approval for registration as a DIS in accordance
with 2.5.6 of part 1 of the ISO/IEC Directives,
by 1999-05-01 [date]
(P-members vote only: ballot form attached)
P-members of the technical committee or subcommittee
concerned have an obligation to vote.
Title (English)
Biological evaluation of medical devices
- Part 4: Selection of tests for interactions
with blood
Reference language version: English
Introductory note
ISO/TC 194 decided at the Plenary meeting
held on 1998 05-14 in Washington by resolution
number 214 to distribute the enclosed document
as Committee Draft.
Contents Page
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.
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 ISO 10993-4 was prepared
by Technical Committee ISO/TC 194, Biological
evaluation of medical devices.
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 the 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: Guidance on the selection and qualification
of reference materials for biological tests
- Part 9: Degradation of materials related
to biological testing [Technical Report]
- 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 polymers
- Part 14: Identification and quantification
of degradation products from ceramics
- Part 15: Identification and quantification
of degradation products from metals and alloys
- Part 16: Toxicokinetic study design for degradation
products and leachables
- Part 18: Chemical characterization
Future parts will deal with other relevant
aspects of biological testing. Annexes A,
B and C, and Annex D of this part of ISO
10993 are for information only.
The initial source for developing this part
of ISO 10993 was the publication, 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].
This part of ISO 10993 gives guidance to
agencies, manufacturers, research laboratories
and others 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 the limitations in
knowledge and precision of tests for interactions
of devices with blood. Further, 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. Such test methods
must take into consideration device design,
materials, clinical utility, usage environment
and risk benefit. This level of specificity
can only be covered in vertical standards.
Therefore, for medical devices where a device
specific 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.
The following standard contains provisions
which, through reference in this text, constitute
provisions of this part of ISO 10993. At
the time of publication, the edition indicated
was valid. All standards are subject to revision,
and parties to agreements based on this part
of ISO 10993 are encouraged to investigate
the possibility of applying the most recent
edition of the standard indicated below.
Members of IEC and ISO maintain registers
of currently valid International Standards.
ISO 10993 - 1: 1997, Biological evaluation of medical devices-Part
1 :Evaluation and testing
For the purposes of this part of ISO 10993,
the definitions given in ISO 10993-1 and
the following definitions 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. Such effects may or may not
have clinically significant or undesirable
consequences.
3.2 ex vivo: Term applied to test systems that shunt
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 evaluation (single pass).
In either case, the test chamber is located
outside the body.
3.3 thrombosis/thromboembolism: An in vivo phenomenon resulting in the
partial or complete occlusion of the vessel
or device. Therefore, characterization of
thrombosis/thromboembolism includes ex vivo,
in vitro circulation systems and in vivo
methods, in either animals or the clinical
setting. A thrombus is composed of a mixture
of red cells, aggregated platelets, fibrin
and other cellular elements.
3.4 coagulation: A measurement of factors of the coagulation
and fibrinolytic systems following exposure
to devices either in vitro or in vivo.
3.5 platelets and platelet function: Platelet testing includes quantitation
of platelet numbers as well as analysis of
their struction and function. The testing
can include analysis of platelet factors,
or components on the platelet surface, released
from platelets or adherent to the device
surface.
3.6 hematology: Includes quantitation of other factors in
the blood. including cellular and soluble
components of the blood.
3.7 complement system: Includes the quantitation and characterization
of any components of the complement system.
Table 1 provides a list of abbreviations
used in the context of this part of ISO 10993.
| Table 1 - Abbreviations |
| Abbreviation |
Meaning |
| Bb |
Product of alternate pathway complement activation
|
| b-TG |
Beta-thromboglobulin |
| C4d |
Product of classical pathway complement activation
|
| C3a, C5a |
(active) complement split products from C3
and C5 |
| CH 50- |
50% total hemolytic complement |
| CT |
Computerised Tomography |
| D-Dimer |
Specific fibrin degradation products (F XIII
cross-1inked fibrin) |
| ECMO |
Extracorporeal membrane oxygenator |
| E.M. |
Electron microscopy |
| FDP |
Fibrin/Fibrinogen degradation products |
| FPA |
Fibrinopeptide A |
| F1+2 |
Prothrombin activation fragment 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 |
| PT |
Prothrombin time |
| PTT |
Partial thromboplastin time |
| RIA |
Radioimmunoassay |
| S-12 |
Monoclonal antibody which recognizes the
alpha granule membrane
component GMP 140 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 |
Devices contacting blood are categorised
in ISO 10993-1.
See ISO 10993- 1.
An example is in vitro diagnostic 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.
5.2.1 External communicating devices that
serve as an indirect blood path (see ISO
10993-1) 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
5.2.2 External communicating devices in contact
with circulating blood (see ISO 10993-1)
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,
- extracorporeal membrane oxygenators,
- intravascular membrane oxygenators,
- hemodialysis/hemofiltration equipment,
- donor and therapeutic apheresis equipment,
- devices for absorption of specific substances
from blood,
- interventional cardiology and vascular devices,
- percutaneous circulatory support systems,
- temporary pacemaker electrodes.
These are devices (see ISO 10993-1) that
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 cave filters,
- embolization devices
- endovascular grafts (covered stems)
- implantable defibrillators and cardioverters
- stents
- arteriovenous shunts
- blood monitors
- internal drug delivery catheters
- pacemaker electrodes
- intravascular membrane oxygenators (artificial
lungs)
- leukocyte removal filters
Figure 1 is a decision tree that provides
guidance on whether testing for interaction
with blood is necessary.
Characterisation of blood interactions may
be classified into five categories based
on the primary process or system being measured.
Table 2 is a listing of blood contacting
devices and the categories of testing appropriate
to the device. These are recommendations,
good rationales can always be developed to
justify different selections.
6.1.1 Where possible, tests should 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 relation
of surface area (length) to test results
should be evaluated.
The selected methods and parameters should
be in accordance with the current state of
the art.
NOTE - Only blood-contacting parts should
be tested.
6.1.2 Controls shall be used unless their
omission can be justified. Where possible,
testing should include a device already in
clinical use or well-characterised reference
materials. Several materials and configurations
are available (see ISO 10993-12 [7]).
Reference materials used should include negative
and positive controls. All materials tested
should meet all quality control and quality
assurance procedures of the manufacturer
and test laboratory and should be identified
as to source, manufacturer, grade and type.
6.1.3 Testing of materials which are candidates
to be components of a device should be conducted
for screening purposes. However, such tests
do not serve as a substitute for the requirement
that the complete device be tested under
conditions which simulate clinical application.
Figure 1 - Decision tree that provides guidance
on whether testing for interaction with blood
is necessary
Table 2 - Blood contacting devices and the
categories of testing appropriate *Hemolysis
testing only
| Device / Test Category |
T
h
r
o
m
b
o
s
i
s
|
C
o
a
g
u
l
a
t
i
o
n
|
P
l
a
t
e
l
e
t
s
|
H
e
m
a
t
o
l
o
g
y
|
Complement
System
|
| Annuloplasy rings, mechanical heart valves
|
x |
|
|
x* |
|
| Atherectomy devices |
|
|
|
x* |
|
| Blood monitors |
x |
|
|
x* |
|
Blood storage and administration equipment,
Blood collection devices, Extension
sets
|
|
x |
x |
x* |
|
Cardiopulmonary bypass system,
Extracorporeal membrane oxygenators
system
Intravascular oxygenators,
Hemodialysis/hemofiltration equipment
percutaneous circulatory support devices
|
x |
x |
x |
x |
x |
| intra-aortic balloon pumps, |
x |
x |
x |
x |
x |
Catheters, Guidewires, Intravascular endoscopes,
Intravascular ultrasound, Lasers systems,
retrograde coronary perfusion catheters,
|
x |
x |
|
x* |
|
| Cell Savers |
|
x |
x |
x* |
|
| total artificial hearts, ventricular-assist
devices, |
x |
|
|
x |
|
| Devices for absoption of specific substances
from blood |
|
x |
x |
x* |
x |
| Donor and therapeutic apheresis equipment
|
|
x |
x |
x* |
x |
| Embolization devices |
|
|
|
x* |
|
| Endovascular grafts (covered stents) |
x |
|
|
x* |
|
| Implantable defibrillators and cardioverters,
|
x |
|
|
x* |
|
Intravascular temporary or permanent pacemaker
electrodes,
pacemakers lead insulators, |
x |
|
|
x* |
|
| Leukocyte removal filter |
|
x |
x |
x* |
|
Prosthetic (synthetic) vascular grafts and
patches,
including arteriovenous shunts |
x |
|
|
x* |
|
| Stents |
x |
|
|
x* |
|
| Tissue heart valves |
x |
|
|
x* |
|
Tissue vascular grafts and patches,
including arteriovenous shunts |
x |
|
|
x* |
|
| Vena cava filters |
x |
|
|
x* |
|
6.1.4 Tests which do not simulate the conditions
of a device during use may not predict accurately
the nature of the blood/device interactions
which may occur during clinical applications.
For example, some short-term in vitro or
ex vivo tests are poor predictors of long-term
in vivo blood/device interactions [22], [23].
6.1.5 It follows from the above that devices
whose intended use is ex vivo (external communicating)
should be tested ex vivo and devices whose
intended use is in vivo (implants) should
be tested in vivo in an animal model under
conditions simulating where possible clinical
use.
6.1.6 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 6.3.2). 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 (e.g. Iancets, hypodermic needles,
capillary tubes) generally do not require
blood/device interaction testing.
6.1.7 The two recommendations in 6.1.5 and
6.1.6, together with clause 5, Figure 1 and
Table 2 serve as a guide for the selection
of tests listed 6.2.1.
6.1.8 Disposable laboratory equipment used
for the collection of blood and performance
of in vitro tests on blood should be validated
to ascertain that there is no significant
interference with the test being performed.
6.1.9 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.
6.1.10 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 should
be considered.
Blood values and reactivity between humans
and non-human primates are very similar [23].
NOTE - The use of non-human primates for
blood compatibility and medical device testing
is prohibited by EU law (86/906/EEC) and
some national laws.
However, the use of species other than non-human
primates such as the rabbit, pig, calf, sheep,
or dog may also yield satisfactory results.
Because species differences may be significant
(for example platelet adhesion, thrombosis,
[17] and hemolysis tends to occur more readily
in the canine species than in the human),
all results of animal studies should be interpreted
with caution.
6.1.11 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. Devices that are used with
anticoagulants shall be assessed using anticoagulants
in the range of concentrations used clinically.
6.1.12 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.
6.1.13 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.
6.2.1 Recommended tests for interactions
of devices with blood
Recommended tests are organised on the basis
of the type of device:
Table 3 - External communicating devices
Table 4 - Implant devices
The tests are classified into five categories
based on the primary process or system being
measured:
d) thrombosis/thromboembolism,
Thrombosis/thromboembolism is an in vivo
phenomenon resulting in the partial or complete
occlusion of the vessel or device. Therefore,
characterization of thrombosis/thromboembolism
includes ex vivo, in vitro circulation systems
and in vivo methods, in either animals or
the clinical setting. A thrombus is composed
of a mixture of red cells, aggregated platelets,
fibrin and other cellular elements.
d) coagulation,
Coagulation is a measurement of factors of
the coagulation and fibrinolytic systems
following exposure to devices either in vitro
or in vivo.
d) platelets and platelet functions,
Platelets and platelet function - Platelet
testing includes quantitation 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, released from platelets
or adherent to the device surface.
e) haematology,
Hematology includes quantitation of other
factors in the blood. Including cellular
and soluble components of the blood.
e) complement system
Complement system - Includes the quantitation
and characterization of any components of
the
complement system.
The principles and scientific basis for these
tests are presented in annex B.
6.2.1.1 Non-contact devices
These devices generally do not require blood/device
interaction testing. Disposable test kits
should be validated to rule out interference
of materials with test accuracy.
6.2.1.2 External communicating devices
After using Table 2 to determine relevant
blood interaction categories for a specific
device type, blood interactions appropriate
to evaluate for external communicating devices
are listed in Table 3. Testing is recommended
for devices intended for limited (LI, <
24 hours) and prolonged or repeated (PR,
24 hours to 30 days) exposure. See also 6.1.6.
6.2.1.3 Implant devices
After using Table 2 to determine relevant
blood interaction categories for a specific
device type, blood interactions appropriate
to evaluate for implant devices are listed
in Tables 4. Testing is recommended for devices
intended for prolonged or repeated (PR, 24
hours to 30 days) exposure or permanent contact
(PC, > 30 days).
6.2.2 Indications and limitations
RIAs 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.
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 hemocompatibility
of a material.
Table 3 - External communicating devices
| Test Category |
Evaluation Method |
Comment |
| Thrombosis |
Percent occlusion |
Pressure drop is not recommended
for devices intended for PR
( see 6.2.1.2) |
| |
flow reduction |
|
| |
gravimetric analysis (thrombus mass) |
|
| |
light microscopy ( adhered platelets,
leukocytes, aggregates, erythrocytes,
fibrin, etc.) |
|
| |
pressure drop across device |
|
| |
labeled antibodies to thrombotic
components |
|
| |
Scanning E.M.(platelet adhesion and
aggregation; platelet and leukocyte
morphology, fibrin) |
|
| Coagulation |
PTT (non-activated) |
|
| |
Thrombin Generation |
|
| |
Specific coagulation factor assays; FPA,
D-dimer, F1+2, PAC-1, S-12, TAT |
|
| Platelets |
Platelet count / adhesion |
|
| |
platelet aggregation |
|
| |
template bleeding time |
|
| |
platelet function analysis |
|
| |
PF-4, Beta-TG;thromboxane B2 |
|
| |
platelet activation markers |
|
| |
platelet microparticles |
|
| |
gamma imaging of radiolabelled platelets
111In-1abeled platelet survival |
In-labeling is recommended for PR
only (see 6.2.1.2) |
| Hematology |
Leucocyte count with or without differential;
|
|
| |
Leucocyte activation; |
|
| |
Hemolysis |
|
| |
reticulocyte count; activation specific
release products of peripheral blood
cells(i.e. granulocytes) |
recommended for PR only
( see 6.2.1.2) |
| Complement system |
C3a; C5a; TCC; Bb; iC3b; C4d; SC5b-9;
CH50; C3 Convertase; C5 Convertase
|
|
Table 4 - Implant devices
| Test category |
Method |
Comments |
| Thrombosis |
Scanning E.M.(platelet adhesion and
aggregation; platelet and leukocyte
morphology ; fibrin |
|
| |
Percent occlusion |
|
| |
flow reduction |
|
| |
autopsy of devices (gross and
microscopic) |
|
| |
autopsy of distal organs (gross and
microscopic) |
|
| Coagulation |
Specific coagulation factor assay; FPA,
D-dimer, F1+2, PAC-1, S-12, TAT |
|
| |
PTT( non activated), PT, TT; Plasma fibrinogen;
FDP |
|
| Platelets |
PF-4, p-TG, thromboxane B2, |
|
| |
platelet activation |
|
| |
platelet microparticles |
|
| |
gamma imaging ofradiolabelled platelets
lll-In labeled platelet survival |
|
| |
platelet count / adhesion |
|
| |
platelet aggregation |
|
| Hematology |
Leukocyte count with or without differential;
|
|
| |
Leukocyte activation |
|
| |
hemolysis |
|
| |
Reticulocyte count; activation specific
release products of peripheral blood
cells
(i.e., granulocyes) |
|
Complement
system |
C3a, C5a, TCC, Bb, iC3b, C4b, SC5b-9,
CH 50, C3 Convertase, C5 Convertase
|
|
Table 5 gives a list of commercially available
assays validated for use with human blood.
6.3.1 In vitro tests
Variables that should be considered when
using in vitro test methods include hematocrit,
anticoagulants, sample collection, sample
age, aeration and pH, temperature, sequence
of test versus control studies, surface-to-volume
ratio, and fluid dynamic conditions (especially
wall shear rate). Tests should be performed
with minimal delay, usually within 4 hours,
since some properties of blood change rapidly
following collection.
6.3.2 Ex vivo tests
Ex vivo tests should be performed when the
intended use of the device is ex vivo, for
example an external communicating device.
Ex vivo testing may 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, thrombus mass, white cell adhesion,
platelet consumption, and platelet activation
[17], [27], [43]. 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.
May ex vivo test systems use radiolabelled
blood components to monitor blood/device
interactions. Radiolabelled platelets and
fibrinogen are the most commonly labelled
components of blood. Alteration of platelet
reactivity by the labelling procedure can
be minimized by strict attention to technical
detail [20], [21], [32].
Advantages of ex vivo tests over in vitro
tests are that flowing native blood is used
(thus eliminating artifacts caused by anticoagulants
as well as 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
flow 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. In
vivo tests are usually designed to examine
hemocompatibility over a period longer than
24 hours.
Patency (of a conduit) is the most common
measure of success or failure for most in
vivo experiments. The per cent 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 a careful gross
as well as microscopic examination of organs
downstream from the device. 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) [16].
Methods to evaluate in vivo performance 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. Flow parameters,
compliance, porosity and implant design may
be more important than blood compatibility
of the material itself As an example, low
flow-rate systems may give substantially
different results when compared to 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.
Table 5 - Commercially available assays for
platelet, coagulation, fibrinolytic and complement
factors
| Factor |
Type of assay |
| Plasminogen |
Colorimetric; Fluorogenic |
| Antithrombin III |
Chromogenic, fluorogenic |
| Protein C |
Chromogenic |
| Protein S |
Chromogenic |
| Antiplasmin |
Chromogenic |
| Prekallikrein |
Chromogenic |
| Kallikrein |
Fluorogenic |
| PF-4 |
RIA, ELISA, Chromogenic |
| b-TG |
RIA, ELISA |
| Thromboxane B2 |
RIA, ELISA |
| Factor VIII-VWF |
Chromogenic; clotting time |
| Factor IX |
Chromogenic; clotting time |
| Factor Ixa |
Fluorogenic |
| Factor X |
Chromogenic; clotting time |
| Factor Xa |
Fluorogenic |
| Factor XII |
Chromogenic; clotting time |
| Factor XIIa |
Fluorogenic |
| FPA |
ELISA |
| C3a, C5a |
RIA |
| Bb, iC3b, C4d, SC5b-9 |
ELISA |
| TCC |
ELISA |
| TAT |
RIA, ELISA |
| IL-l |
RIA, ELISA |
| F1+2 |
RIA, ELISA |
| D-dimer |
RIA, ELISA |
| P-selectin |
ELISA |
| L-selectin |
ELISA |
NOTE -These assays have been validated for
human use. Validation of their accuracy for
other species shall be determined prior to
use.
A.1 General considerations
A.1.1 This annex provides background for
selecting tests to evaluate the interactions
of cardiovascular devices with blood. Subclause
6 contains guidance on when testing is necessary,
what blood interaction categories might be
appropriate for specific devices, and a complete
list of tests for evaluating blood/device
interactions of non-contact-, external communicating-,
and implant-devices. A.1.2 The following
classification of blood/device interactions
is provided as background.
A.1.2.1 Interactions which mainly affect
the device and which may or may not have
an undesirable effect on the subject 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 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 thrombi on the device surface;
- c) embolization of thrombotic or other material
from the device's luminal surface to another
site within the circulation;
- d) injury to circulating blood cells resulting
in anemia, hemolysis, leucopenia, 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 to or near the device.
A.1.3 Advantages and limitations of animal
and in vitro testing
A.1.3.1 Animal 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
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 similarity in
hematologic values, blood coagulation mechanism
and cardiovascular system to the human [27].
An additional advantage of a non-human primate
is that many of the immunologic probes for
thrombosis developed for humans are suitable
for use in other primates. These probes include
PF-4, b-TG, FPA, TAT, and F. + 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 when evaluating
this complication. The pig is generally regarded
as a suitable animal model because of its
hematologic and cardiovascular similarities
to the human. The effect of the surgical
implant procedure on results should be kept
in mind and appropriate controls included
A.1.3.2 In vitro testing
Because of species differences in hemostatic
and hematologic factors and activities it
is preferable to use human blood. Thrombus
formation is a dynamic process. Therefore
in vitro testing is advisable to simulate
as much as possible the dynamic conditions
(for example shear forces of the blood -
material interface) in which thrombosis occurs.
Since patients with cardiovascular devices
may be receiving anticoagulant or antithrombotic
drugs, it is important to simulate these
conditions in vitro. Static tests are useful
for evaluating the interactions of blood
with materials.
A.1.4 Test protocols for animal testing
The test protocols recommended follow certain
general guidelines. 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 acute extent
of variation of hematologic, hemodynamic
and performance variables, gross thrombus
formation and possible embolism. With prolonged
or repeated exposure, or permanent contact
(> 24 h), 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, the development of intimal
hyperplasia and infection. In both of the
above exposure and contact categories, assessment
of hemolysis assessment 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, for example
intimal hyperplasia and endothelialization.
The consequences of the interaction of artificial
surfaces with the blood may range from gross
thrombosis and embolization to subtle effects
such as accelerated consumption of hemostatic
elements; the latter may be compensated or
lead to depletion of platelets or plasma
coagulation factors.
A.1.5 Recommended test protocols for in vitro
testing
In vitro testing allows for the performance
of sufficient number of tests for statistical
evaluation without the sacrifice of animals
and with relatively low costs. Measurements
are related to the acute extent of variation
of hematologic, hemodynamic and performance
variables, gross thrombus formation and complement
activation. The in vitro approach permits
the study of the kinetics of various factors
and activities, by varying the duration of
exposure of material or devices to blood.
A.2 Cannulae
Cannulae are typically inserted into one
or more major blood vessels to provide repeated
blood access. They are also used during cardiopulmonary
bypass and other procedures. They may be
tested acutely or chronically and are commonly
studied as arteriovenous (AV) shunts. The
use of Cannulae induces little alteration
in the levels of circulating blood cells
or factors in the coagulation or complement
system. Cannulae, like other indirect blood
path devices (5.2.1), generally require less
testing than devices in direct contact with
circulating blood (5.2.2, 5.3).
A.3 Catheters and guidewires
Most of the test considered under Cannulae
are relevant to the study of catheters and
guidewires. 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
be performed using a contralateral artery
or vein. Care should be taken not to strip
off thrombus upon catheter withdrawal. In
situ evaluation may permit assessment of
the extent to which intimal or entrance site
injuries contributed to the thrombotic process.
In general Doppler blood flow measurements
are more informative than angiography. Kinetic
studies with radiolabelled blood constituents
are recommended only with chronic catheters.
A.4 Extracorporeal oxygenators, hemodialyzers,
therapeutic apheresis equipment, and devices
for absorption of specific substances from
blood
The hemostatic response to cardiopulmonary
bypass may 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 ultrasonic
radiation 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 [28];
the template bleeding time and other tests
of platelet function and release are particularly
useful.
Complement activation is caused by both hemodialyzers
and cardiopulmonary bypass apparatus. Clinically
significant pulmonary leucostasis and lung
injury with dysfunction may result. For these
reasons, it is useful to quantify complement
activation with these devices.
Therapeutic apheresis equipment and devices
for absorption 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
should follow the same principles as for
extracorporeal oxygenators and hemodialyzers.
A.5 Ventricular-assist devices and total
artificial hearts
These devices may 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 hemolysis, fibrinogen concentration, thrombin
generation, platelet survival and activation,
complement activation, and close monitoring
of liver, renal, pulmonary and central nervous
system function. A detailed pathologic examination
at surgical retrieval is an important component
of the evaluation [37], [38].
A.6 Heart valve prostheses
Invasive, non-invasive and in vitro hydrodynamic
studies are important in the assessment of
prosthetic valves.
2D 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 may also be useful
in the assessment of function of tissue-derived
valve prostheses. Vegetations, clots and
evidence of thickening of the valve leaflets
are elucidated. Using conventional and color
flow Doppler echocardiography, regurgitation
can be identified and semi-quantified.
Measurements of platelet survival and aggregation,
blood tests of thrombosis and hemolysis,
pressure and flow measurements, and autopsy
of the valve and adjacent tissues are also
recommended.
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 intended use for the prosthesis. Patency
of a given graft is enhanced by larger diameter
and shorter length. A rule of thumb for grafts
less 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 by palpation of
distal pulses in some locations and by periodic
angiography. Ultrasonic radiation, MRI, and
PET may also be useful. Results of serial
radiolabelled platelet imaging studies correlate
with the area of nonendothelialized graft
surface in baboons [27]. Radiolabelled platelets
facilitate non-invasive imaging of mural
thrombotic accumulations. Serial measurements
of platelet count, platelet release constituents,
fibrinogen/fibrin degradation products and
activated coagulation species also are recommended.
Autopsy of the graft and adjacent vascular
segments for morphometric studies of endothelial
integrity and proliferative response can
provide valuable information.
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.
B.1 General
B.1.1 The principles and scientific basis
of the tests listed in 6.2.1 are described
here. Detailed methods are found in standard
texts of laboratory medicine and clinical
pathology. References [14] to [39] and [41]
to [44] describe tests which may be useful
in the evaluation of blood/device interactions.
Because of both biological variability and
technical limitations, the accuracy of many
of these tests is limited. When possible,
the tests should be repeated a sufficient
number of times to determine the significance
of the results.
(Ref. ICH)*** Note to WG-9 Group - Does anyone recall what
this reference is supposed to be? I can't
find it)
B.1.2 Principles for in vitro testing
Static systems and dynamic systems i. e.
Chandler and other circulatory loop and centrifugation
systems are used (Ref.***).
B. 1.3 Test conditions
In order for tests to be of use in the in
vitro evaluation of blood/device interactions,
anticoagulated blood or plasma collected
from normal human subjects or experimental
animals should first be exposed to the material
or device under standardized conditions including
time, temperature and flow. An aliquot of
the exposed blood or plasma is then tested
shortly after exposure. Conditions of exposure
should be based on the intended use of the
device. One proposed set of conditions is
as follows:
- a) time of exposure: not less than 15 min;
- b) temperature: 37_C;
- c) flow: to simulate intended use of the
device;
- d) start of test: within 15 min following
exposure.
In the preparation of the test specimens
it is essential to avoid activation or release
of any component from blood before testing.
However, the appropriate conditions depend
on the device or material being tested and
its intended end-use.
B.1.4
When evaluating externally communicating
devices and implant devices while in their
in-use position, blood is collected into
an anticoagulant and the test is performed
as described without a prior exposure stage.
The tests are classified into five categories,
as defined in 6.2.1, according to the process
or system being tested: thrombosis, coagulation,
platelets and platelet functions, hematology
and complement system.
B.2 Thrombosis
B.2.1 Percentage occlusion
Percentage occlusion is visually quantified
after a device has been in use and has been
removed. This is a measure of the severity
of the thrombotic process in a conduit. 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 perianastomotic sites in vascular grafts;
microscopic examination is required to identify
the nature of the occlusive process. Surface
area covered by thrombus and thrombus free
surface area are semi-quantitative tests
that can be used on a comparative basis.
B.2.2 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 B.2.1.
B.2.3 Gravimetric analysis (thrombus mass)
This is conducted after removal of the device
from the in-use position. Rationale and interpretation
are as for B.2.1.
B.2.4 Light Microscopy
By this technique, information can be obtained
regarding the density of cells, cellular
aggregates and fibrin adherent to materials,
as well as the geographic distribution of
these deposits on the materials or device.
The method is semiquantitative.
B.2.5 Pressure drop across device
This is measured before and after a period
of use. Rationale and interpretation are
as for B.2.1.
B.2.6 Scanning E.M.
Rationale and interpretation are the same
as B.2.4. This method has the advantage over
B.2.4 of providing greater detail about fine
structure of components being examined. Quantitative
conclusions require sufficient replicate
determinations to establish degree of reproducibility.
B.2.7 Antibody binding
Next to qualitative microscopic judgment
of fibrin and platelet deposition on materials,
a quantitative estimation is possible by
measuring the amount of labeled antibody
specific for fibrin(ogen) or platelet membrane
receptors. For this purpose materials are
washed after exposure to blood to delete
nonadherent blood components prior to labeled
antibody binding.
B.2.8 Autopsy of device
This method is of great importance in evaluating
the biological responses to implanted devices.
The distribution, size and microscopic nature
of cellular deposits can best be determined
by a careful and detailed autopsy. Proposed
procedures have been published [37], [38].
B.2.9 Autopsy of distal organs
The rational is to examine for distal effects
of implanted devices. These effects include
thromboembolism, infection and embolization
of components of the device.
B.2.10 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.
B.3 Coagulation
Coagulation methods are based on the use
of native (fresh, non-anticoagulated) whole
blood, anticoagulated whole blood (usually
citrated), platelet-rich plasma or platelet-poor
plasma. Since most of the standard coagulation
assays are designed to detect clinical coagulation
disorders which result in delayed clotting
or excessive bleeding, the protocols for
evaluating blood/device interactions should
be modified appropriately to evaluate accelerated
coagulation induced by biomaterials. Reagents
for tests based on the activated partial
thromboplastin time include an activator
such as kaolin, celite, or ellagic acid.
Reagents with such activators should be avoided
because they tend to mask the acceleration
of coagulation which materials and devices
cause. The material to be tested serves as
the activator; controls (without the material)
should be included.
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 the test material.
After a predetermined contact time with the
test surface, tests of the surface and blood
can be conducted.
B.3.1 Partial Thromboplastin Time (PTT)
The partial thromboplastin time [35] is the
clotting time of recalcified citrated plasma
on the addition of partial thromboplastin.
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 contact phase
of blood coagulation. A prolonged PTT suggests
a deficiency in any of the plasma coagulation
factors I (fibrinogen), II (prothrombin),
V, VIII, IX, X, XI, or XII, but not VII or
XIII. Heparin and other anticoagulants also
cause a prolonged PTT.
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 of
no value in the in vitro evaluation of blood/device
interactions because the activating substances
mask any activation caused by the device
or its component materials.
B.3.2 Prothrombin Time (PT)
Blood is mixed with a measured amount of
citrate and the plasma is obtained by centrifugation
[35]. This test is based on having an optimum
concentration of calcium ions and an excess
of thromboplastin, the only variable being
the concentration of prothrombin and accessory
factors in a carefully measured volume of
plasma. Oxalate is not recommended as the
anticoagulant because factor V is less stable
in this solution and the PT may become prolonged
as a result.
This test measures prothrombin and accessory
factors. In the presence of tissue thromboplastin,
the clotting time depends on the concentrations
of prothrombin, factor V, factor VII and
factor X (assuming fibrinogen, fibrinolytic
and anticoagulant activity to be normal).
A prolonged prothrombin time generally indicates
a deficiency of prothrombin or factor V,
VII, X or fibrinogen.
B.3.3 Thrombin Time (TT)
The thrombin time [35] is the time required
for plasma to clot when a solution of thrombin
is added. The thrombin time is prolonged
with a deficiency in fibrinogen (below 100
mg/dl), qualitative abnormalities in fibrinogen
and elevated levels of FDP or heparin. The
test is useful for evaluating implant devices
only.
B.3.4 Thrombin generation
Materials exposed to an intact coagulation
system in the presence of phospholipids (see
B.3.1) will generate thrombin which can be
measured by conversion of a chromogenic substrate.
This method has a much lower variability
than the conventional coagulation tests.
B.3.5 Fibrinogen
Dysfibrinogenemia, afibrinogenemia and hypofibrinogenemia
cause prolonged PT, PTT and TT results [18].
The screening test most sensitive to fibrinogen
deficiency is the TT. If the exact level
of fibrinogen is needed, a commercially available
modified TT is recommended. The test is useful
for evaluating implant devices only.
B.3.6 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.
The test is useful for evaluating implant
devices only. The use of ELISA is recommended.
B.3.7 Specific coagulation factor assays
Significant reduction (e.g. to less than
50% of the normal or control level) of coagulation
factors following exposure of blood to a
material or device under standard conditions
suggests accelerated consumption of those
factors by adsorption, coagulation or other
mechanisms.
B.3.8 FPA, D-dimer, F1+2, TAT
Elevated levels of FPA, D-dimer, or F1 +
2 indicate activation of the coagulation
mechanism. FPA and F1+2 indicate an activation
of Prothrombin to thrombin. Elevated TAT
complexes indicate activation of blood coagulation
and formation of a complex between thrombin
being generated and circulating antithrombin.
Dimers are plasmin digested degradation products
of F XIII cross linked fibrin (coagulation
and fibrinolysis). The use of ELISA and RIA
is recommended.
B.4 Platelets and platelet functions
It is essential to avoid activation in the
preparation of platelet suspensions.
B.4.1 Platelet count
It is important to determine the platelet
count [18], [44] because of the key role
of platelets in preventing bleeding. A significant
drop in platelet count of blood exposed to
a device may be caused by platelet adhesion,
platelet aggregation, platelet sequestration
(for example in the spleen), or blood coagulation
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.
Platelet count is performed using an EDTA
suspension medium.
Blood collection techniques should be reproducible.
Platelets can become hyperactive under a
variety of conditions, including improper
blood collection. Tests to verify normal
platelet reactivity are usually performed
with an aggregometer. Platelet preparations
with reduced reactivity are easily detected
using this method, but hyperactive platelets
are not normally detected. Platelet aggregation
tests can be modified (by appropriately reducing
the concentration of platelets or aggregating
agents) to determine if platelets become
hyperactive following exposure to a material
or device.
B.4.1.1 Manual platelet count
Platelet counts are performed manually in
some clinical laboratories despite the wide
availability of highly accurate platelet
counting instruments (B.4. 1.2).
B.4.1.2 Automated platelet count using whole
blood or platelet-rich plasma
Automated platelet counts may be performed
using either well mixed whole blood or platelet-rich
plasma (PRP). The use of whole blood is preferred
because using PRP may yield inaccurate results.
Instrument counts are made on a greater number
of particles than manual counts. Consequently,
the coefficient of variation for automated
counts may be as small as 4%, whereas the
best achievable for the phase microscope
manual count is 11%. Most instruments also
contain circuitry that distinguishes between
platelets and small nonbiological particles,
eliminating the need for visual recognition
and differentiation of debris from platelets.
B.4.2 Platelet aggregation
Platelet aggregation [35] is induced by adding
aggregating agents to PRP that is being stirred
continually (e.g. ADP, epinephrine, collagen,
thrombin, etc.). As the platelets aggregate,
the plasma becomes progressively clearer.
An optical system (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 may
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
automatically by the WU/HOAK method (Ref***).
Note: Need WG-9 members help in determining
what this reference should be.
B.4.3 Blood cell adhesion
Blood cell adhesion [31] 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
hematological factors.
Various methods have been designed to measure
the adhesion of cells to surfaces. Most of
these methods are based on the observation
that a certain proportion of 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.
This principle has been adapted to the quantification
of the adhesion of other blood cells to polymers
coated on glass beads. By such a method it
has been reported [31] that adhesion of canine
species peripheral lymphocytes and polymorphonuclear
leukocytes (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.
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 nonadherent
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 prelabeled
with 51Cr or 111In may be used [30].
B.4.4 Platelet activation
The use of certain materials or devices may
cause platelet activation, which can result
in; 1) the release of platelet granule substances,
such as BTG ( Beta thromboglobulin), platelet
factor 4 (PF 4), and serotonin, 2) altered
platelet morphology and 3) cause the generation
of platelet microparticles. Activated platelets
are pro-thrombotic. Platelet activation can
be evaluated by various means: microscopic
(light and electron microscopy) examination
of platelet morphology of platelets adherent
to the material or device, measurement of
BTG, PF4 and serotonin, and the evaluation
of platelet activation by flow cytometry
(for microparticle generation, Pselectin
(GMP-140) expression, or activated glycoprotein
Ib and JIB/ IIIa expression using monoclonal
antibodies. Different epitopes of activated
platelets are recognized by flow cytometry
using 2 antibodies: one specific for platelets
(i.e. GP Ib or GP IIb/IIIa) and one specific
for platelet activation (P-Selectin).
B 4.5 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. A
prolonged result indicates reduced platelet
function or reduced platelet count, the latter
can be determined separately (B.4.1). 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)
[28]. The test is suitable for use with some
experimental animals. In vitro bleeding time
measurements are also suitable.
B.4.6 Platelet function analysis
The classical template bleeding time has
been used as the principle for an automated
method. Whole blood is aspirated through
a collagen filter with a 150 um aperture.
Platelets adhere and aggregate until the
aperture is closed. Blood pressure and temperature
are standardized, anticoagulation does not
affect this test. The test is suitable for
animal blood.
B.4.7 Gamma imaging of radiolabelled platelets
The high gamma emission of 111In enables
it to be used for this purpose [20], [27].
This method enables the localization and
quantification of platelets deposited in
a device. The technique is useful for external
communicating as well as implant devices.
B.4.8 Platelet lifespan (survival)
Platelets are obtained from the patient's
blood and are labeled with 51Cr or 111In
[20], [21],[32]. 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 thrombopoiesis. Idium-111 has the
advantage of being a high gamma emitter,
requiring the labeling 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.
B.5 Hematology
B.5.1 Leukocytes
Leukocyte activation can be determined by
the microscopic examination of the device
surface or activated leukocytes and the use
of flow cytometry for the evaluation of increased
leukocyte markers such as L- selectin and
CD 1 lb and quantitative disturbances in
lymphocyte subpopulations.
B.5.2 Hemolysis
This is regarded as an especially significant
screening test because an elevated plasma
hemoglobin level, which if properly performed
indicates hemolysis, reflects red blood cell
membrane fragility in contact with materials
and devices (see annex C).
B.5.3 Reticulocyte count
An elevated reticulocyte count indicates
increased production of red blood cells in
the bone marrow. This may be in response
to reduced red blood cell mass caused by
chronic blood loss (bleeding), hemolysis
or other mechanisms.
B.6 Complement System - CH 50 and C3a,C5a,
TCC, Bb, iC3b, C4d, SC5b-9
CH 50 decrease 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.
Measurement of complement split products
have the disadvantage if species-specificity
and high baseline levels when performed after
in vitro testing. The classical CH-50 method
appears useful with human, bovine, porcine,
and lupine serum.
Another functional method of measurement
of complement activation in vitro is the
generation of complement C3- or C5-convertase,
determined by substrate conversion.
Extensive literature exists describing blood/material
interactions. Unfortunately, very few methods
exist which are reliable, reproducible, and
predict clinical performance. This annex
will review the known hemolysis test methods
and discuss factors pertaining to their ability
to characterize medical and dental materials
and devices.
C.1 Definitions
C.1.1 anticoagulant: An agent which prevents
or delays blood coagulation [45]. For example,
heparin and citrate.
C.1.2 oncotic pressure (colloidal osmotic
pressure): The total influence of the proteins
or other large molecular weight substances
on the osmotic activity of plasma [46].
C.1.3 hematocrit: The ratio of the volume
of erythrocytes to that of whole blood.
C.1.4 hemolysis: The rupture of the erythrocyte
membrane with the liberation of hemoglobin,
which diffuses into the fluid surrounding
the erythrocytes, or the premature destruction
of, erythrocytes.
C.1.5 negative reference material: High Density
Polyethylene, or similar validated alternative.
(see ISO 10993-12).
C.1.6 packed erythrocytes: Component obtained
by centrifugation from a unit of human blood
following removal of plasma supernatant.
NOTE - Properties of human erythrocytes for
transfusion: The erythrocyte volume fraction
of the component is 0.65 to 0.80. The unit
contains all of the original unit's erythrocytes,
the greater part of its leukocytes (about
2.5 to 3.0 x 109 cells) and a varying content
of platelets depending on the method of centrifugation.
C.1.7 washed erythrocytes: An erythrocyte
suspension obtained from whole blood after
removal of plasma and washing in an isotonic
solution.
NOTE - Properties: This component is an erythrocyte
suspension from which most of the plasma,
leukocytes and platelets have been removed.
The amount of residual plasma will depend
upon the washing protocol. Storage time should
be as short as possible after washing and
certainly not longer than 24 hours at 1_C
to 6_C.
C.1.8 whole blood: Whole blood is unfractionated
blood drawn from a selected donor and contains
citrate or heparin as an anticoagulant.
C.2 Causes of hemolysis
C.2.1 Mechanical forces - pressure
The red blood cell membrane is a semipermeable
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. These pressure
differentials can cause erythrocyte swelling
and cell membrane rupture with release of
free hemoglobin [45].
C.2.2 Mechanical forces - rheologic
Factors which influence velocity of blood
flow, shear forces and other forces that
can deform the red blood cell membrane can
cause membrane rupture.
C.2.3 Biochemical factors
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 hemoglobin.
Other chemicals, bacterial toxins, pH and
metabolic changes induced by temperature
can compromise the erythrocyte membrane [47].
These changes may cause membrane rupture
at lower than expected osmotic pressures.
A test to determine the pressure at which
an erythrocyte membrane ruptures is termed
osmotic fragility.
C.3 Clinical significance of hemolysis
C.3.1 Toxic effects
Elevated levels of free plasma hemoglobin
may induce toxic effects or initiate processes
which may stress the kidneys or other organs
[45].
C.3.2 Thrombosis
Intravascular hemolysis may promote thrombosis
by liberating phospholipids [48]. When hemolysis
causes a clinically significant drop in red
blood cell count, anemia and compromised
oxygen carrying capacity with its subsequent
effects on the brain and other organs or
tissues may result.
C.4 Determining a Pass/Fail assessment for
hemolysis
Hemolysis is a function of time and material
properties such as surface energy, surface
morphology, and surface chemistry. Hemolysis
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
[49], [50], [51]. These variables need to
be adequately controlled for comparisons
of hemolytic potential among materials and
medical devices. The spectrum of methods
for evaluating hemolysis varies from simplified
to highly complicated models. Specific in
vitro and in vivo models with flowing blood
have been published. Studies of hemolytic
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 plasma
hemoglobin which may not be measurable under
in vivo conditions (e.g. due to binding of
plasma hemoglobin to heptoglobin and rapid
removal from the body). Hence, the measurement
of in vivo plasma hemoglobin levels as an
indicator of overall blood damage is not
as sensitive as in vitro models, which therefore
are possibly of no clinical consequence.
It is not possible to define a universal
level for acceptable and unacceptable amounts
of hemolysis for all medical devices and
applications. The effect of a device on hemolysis
may be masked in the short term by the trauma
of the surgical procedure. A device may cause
a substantial amount of hemolysis, but be
the only treatment available in a life-threatening
situation. Intuitively, a blood compatible
material is non-hemolytic. In practice, many
devices cause hemolysis, but their clinical
benefit outweighs the risk associated with
the hemolysis. Therefore when a device causes
hemolysis, it is important to confirm that
the device provides a clinical benefit and
that the hemolysis is within acceptable limits
clinically. Acceptance criteria should be
justified based on some form of Risk and
Benefit assessment. The following questions
are suggestions for developing such an assessment:
- a) What is the duration of exposure of the
device to the patient?
- b) How much hemolysis does the material or
device cause? Does the hemolysis continue
for the entire time the device is exposed
to the patient? Does hemolysis continue after
removal of the device?
- c) What are the relative Risks and Benefits
of other available methods for treating the
condition?
- d) What are the hemolytic properties of these
known treatments? How does the device in
question compare to these other treatments?
- e) How effective is the test device compared
with other forms of treatment? A more effective
device may cause more hemolysis during use but the additional
effectiveness might increase the benefit
to the patient.
C.5 Hemolysis testing - general considerations
C.5.1 Methods
C.5.1.1 Total blood hemoglobin - Classical
methods
Due to many different factors, there are
currently about twenty different assays in
use today for measuring plasma hemoglobin
as an indicator of hemolysis, but no one
method is widely accepted. The concentration
of hemoglobin in plasma is significantly
less than the total blood hemoglobin concentration.
The free plasma hemoglobin concentration
is normally 0 - 10 mg/dL in vivo and whereas
the normal range of total blood hemoglobin
concentration is 11,000 to 18,000mg/dL. Classically,
three analytical methods have been used to
determine total blood hemoglobin (Hb) concentrations
[52].
C.5.1.1.1 Cyanmethemoglobin method
The first classical method, cyanmethemoglobin
detection, is that recommended by the International
Committee for Standardization in Haematology
[53]. The cyanmethemoglobin (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 hemiglobincyanide
which has a broad absorption maxima at 540
nm. For the total hemoglobin concentration,
the spectral interference due to plasma is
minimal and the sample absorbance can be
compared to the HiCN standard solution directly.
However, for the determination of plasma
hemoglobin, unless compensation is made for
endogenous plasma background interference,
the plasma hemoglobin concentration of the
sample will be overestimated. 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.
NOTE - An erythrocyte Iysing agent should
be used for total hemoglobin measurements.
Lysing agents should not be used for cell-free,
plasma hemoglobin assays. Lower than expected
levels may occur if the cells are fixed with
glutaraldehyde or formaldehyde or the hemoglobin
is precipitated.
The broad absorption band of HiCN in this
region enables the use of simple filter type
photometers as well as narrow band spectrophotometers
for detection. Commercially available blood
analyzers have automated quantitations based
on either of the two detection strategies.
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.
Spectrophotometric measurement as haemoglobincynide
is only practical if the amount of Iysis
is > 50mg/l. This method is not recommended.
C.5.1.1.2 Oxyhemoglobin method
The second classical method is more cumbersome
and not widely used today. The oxyhemoglobin
method depends on the formation of HbO2 during
ammonia-hydroxide treatment, and spectrophotometric
detection of this product. No stable reference
preparation is available but this is not
important because all that the method is
required to measure is the percentage of
the total hemoglobin in the original specimen
which is present in the plasma. In any event,
a short-term standard can be prepared from
a fresh blood sample.
C.5.1.1.3 Iron method
The third classical method is based on determining
the hemoglobin iron concentration in solution.
Iron is first separated from Hb, usually
by acid or by aching. It is then titrated
with TiCl3 or complexed with a reagent to
develop color that can be measured photometrically.
This method is too complex for routine work,
and is rarely used.
C.5.1.2 Plasma hemaglobin - Tetramethylbenzidene
method
Currently, there are as many as twenty different
assays for measuring plasma hemoglobin in
use today. The assays can be classified into
two broad categories: those which are direct
optical techniques (i.e. based on quantifying
the oxyhemoglobin absorbance peaks at 415,
541, or 577 nm, directly or through use of
derivative spectrophotometry) and those which
are added chemical techniques (i.e. quantification
of hemoglobin based on a chemical reaction
with reagents such as benzidine-like chromogens
and hydrogen peroxide, of the formation of
cyanmethemoglobin) [54]. A popular method
for determining the concentration of hemoglobin
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 colored product (photometrically detected
at 600 nm) is directly proportional to the
hemoglobin 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
to the hemoglobin cyanide method [52]. 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 -
50 mg/dL) [55], and possible reaction inhibition
(by as much as 40% ) [56] may occur from
calcium chelating anticoagulants (e.g. citrates,
oxalates, EDTA) [55], albumin, [57] or other
non-specific plasma components [56] which
may interfere with H2O2 oxidation. For these
reasons, direct optical methods, such as
those by Harboe [58], Cripps [59], or Taulier
[60] with comparable sensitivity and reproducibility
may be substituted.
C.5.2 Blood and blood component preservation
This section presents the best demonstrated
practices for the preservation of human blood
components by the American Association of
Blood Banks [61] and the Council of Europe
[62].
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 [63], [64], [65],
[66], [67], [68].
Blood clotting is prevented by citrate binding
of calcium. Red blood cells 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
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 must be reduced
by addition of citric acid to the anticoagulant
solution. This provides a suitably high hydrogen
ion concentration at the beginning of red
blood cell 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 overconcentrated.
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 may be used to further
stabilize the cell membrane and prevent hemolysis
[62].
The suitability of containers for the storage
of blood products is evaluated by various
methods that measure the quality of the blood
product [52], [65]. 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 hemoglobin is
measured to assess the viability and quality
of the stored product. The quality of the
stored product may 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.
C.5.3 Protection of employees handling blood
Written procedures are necessary for protecting
employees receiving, handling, and working
with potentially contaminated human blood.
Potentially contaminated materials include
blood and other 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 [69].
C.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 [61].
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
may draw in contaminated air [61].
NOTE - Use of a vacuum tube has the potential
to cause slight hemolysis.
Blood collected in an open system may be
contaminated by exposure to room air and
is not considered sterile. Microbial contamination
is a known cause of hemolysis.
C.5.5 Species selection
Ideally, hemolysis 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 must 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 one hundred percent hemolysis
to obtain total hemoglobin content due to
differences in membrane stability among animal
species. Negative controls should cause minimal
hemolysis so that the activity of the test
material is not masked. Rabbit and human
erythrocytes are reported to have similar
hemolytic properties whereas monkey erythrocytes
are more sensitive and guinea pig erythrocytes
less sensitive [70].
C.5.6 Evaluation of hemolysis - in vitro,
ex vivo, and in vivo exposure to blood or
blood components
Hemolysis may 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 may be made for either of the
following study designs. In the first case,
the test device is compared to reference
control marketed devices with known acceptable
levels of hemolysis. In the second case,
the test subject is evaluated for clinically
significant consequences of hemolysis.
The purpose of in vivo or ex vivo tests is
to characterize the hemolytic potential of
a medical device. The preliminary studies
may be in vitro and may use fresh or outdated human blood or
blood from a nonhuman specie. For medical
devices indicated for ex vivo use, the general practice is to recirculate
blood through the device using conditions
that simulate the intended 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.
C.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 red blood
cells, while other methods describe the preparation
of an extract which is then exposed to red
blood cells. Test selection should be based
upon the device itself and the conditions
in which it will be used. Boundary conditions
to be considered when elevated temperatures
are used are outlined in ISO 10993-12.
C.6 Recommended hemolysis test methods for
medical devices and materials
The following is primarily taken from ASTM
F 756-93), which is a standard that is specific
for testing the hemolytic properties of materials
(mainly due to chemical factors) and is not
sufficient for testing whole medical devices.
C.6.1 In vitro methods
The purpose of in vitro tests is to evaluate
the effect on isolated erythrocytes. Direct
methods determine hemolysis due to physical
interactions of red blood cells with either
the specimen or the extractables from the
test specimen. The dynamic method increases
the physical interaction and, presumably,
the amount of extraction that occurs. Indirect
methods determine hemolysis due to extractables
from the test specimen. Indirect methods
permit separating the extraction phase from
the test system, thereby offering opportunity
to exaggerate extraction conditions and distinguish
physical surface interactions from chemical
effects. [Note: Equipment calibration can
be achieved with a Reference Standard for
hemoglobin analysis [52]].
C.6.2 Preparation of erythrocyte suspension
Blood from one human donor is normally sufficient.
Pooled blood from no more than three animal
donors preferably of the same blood type
is normally sufficient to minimize variation
between donors [70], [71]. A rationale for
the selection of blood donor species should
be provided in the report.
For the purpose of standardization, whole
blood collected in a closed system containing
ACD (acid citrate dextrose) or CPD (citrate
phosphate dextrose) anticoagulant should
be stored no more than 96 hours at 1_C to
6_C.
NOTE - Vigorous mixing of blood should be
avoided to prevent air induced hemolysis.
Blood collected in an open system should
be used immediately. Heparinized whole blood
collected in a closed system should be stored
no more than 2 hours at 1 _C to 6_C in order
to avoid nutrient depletion. After dilution
of whole blood or washed erythrocytes in
crystalloid media, they shall be used within
2 hours because the crystalloid media does
not have adequate nutrients for erythrocyte
preservation. The cell-free plasma hemoglobin
content of whole blood or washed erythrocytes
shall be less than 1.0 mg/mL to avoid artifacts
[61]. [Note: Sterile crystalloid solutions
(saline or phosphate buffered saline) are
not required for the assays because the duration
of the test (less than 4 hours) is insufficient
to allow for significant microbial growth
unless the sample has been collected in an
open system or grossly contaminated during
phlebotomy.]
C.6.3 Method for evaluating material in direct
contact with diluted blood
Dilute whole blood with isotonic saline (0.9%)
to a total hemoglobin concentration of 25.0
+- 2.5 mg/mL.
NOTE - Cooling blood and saline prior to
dilution may reduce inter-assay variability.
This diluted suspension shall be used within
2 hours of its preparation when kept at 1
_C to 6_C or in an ice bath.
Tests on each material and blood sample combination
should be performed in triplicate for each
set of test conditions. Prepare test specimens
of material, as described in ISO 10993-12,
that have been subjected to the same manufacturing
processes and sterilization as the final
product. Prepare six tubes containing strips
or rods of the test specimens (three tubes
for dynamic testing and three tubes for static
testing) and three tubes containing strips
or rods of the Negative Reference Material.
NOTE - Vigorous mixing of blood should be
avoided to prevent air induced hemolysis.
The amount of test specimens and Negative
Reference Material should be 1500 mm2 for
samples > 0.5 mm thickness and 3000 mm2
for samples < 0.5mm thickness., which
is in conformance with the recommendations
of ISO 10993-12.
NOTE- The value of 15.7cm2 in ASTM F 756-93
is based on 50 rods of 10 mm x 1 mm x 3.14.
The change herein is consistent with ISO
10993-12.
Add 5 mL of diluted blood to each tube. Incubate
with the test specimens and Negative Reference
Material. Three test preparations are incubated
for 4 hours at 37 +- 2_C under static conditions
and three are incubated for 1 hour at 37
+- 2_C under dynamic conditions (mixing on
a rocker plate at 30 +- 6 rocks per minute
at a maximum of 45 degrees from the vertical
position).
NOTE - Dynamic mixing conditions should not
cause frothing or protein denaturation which
could contribute to erythrocyte Iysing.
At the end of the contact period, the test
specimens are removed and the diluted blood
samples are centrifuged at 100 to 200 G in
a standard clinical centrifuge for 15 minutes.
Remove and transfer the supernatant fraction
to individual siliconized borosilicate glass
tubes. Recentrifuge the supernatant fraction
at 700 to 800 G in a standard clinical centrifuge
for 5 minutes to remove any remaining erythrocytes
from the supernatant. Remove and transfer
the supernatant into a third borosilicate
tube. Analyze the samples for free hemoglobin
using the method in annex C.5.1.2
C.6.4 Method for indirect contact with diluted
blood
Sufficient saline extracts of the test specimen
and Negative Reference Material are prepared
in order to perform each test in triplicate.
Prepare sufficient replicate tubes containing
4 mL of saline extract and 5 mL of diluted
blood with a hemoglobin concentration of
25 +- 2mg/mL for both static and dynamic
testing of test and control specimens.
NOTE - Vigorous mixing of blood should be
avoided to prevent air induced hemolysis.
Three test preparations are incubated for
4 hours at 37 +- 2_C under static conditions
and three are incubated for 1 hour at 37
+- 2_C under dynamic conditions. The Negative
Reference Material should also undergo static
and dynamic testing.
At the end of the contact period, the test
specimens are removed and the diluted blood
samples are centrifuged at 100 to 200 G in
a standard clinical centrifuge for 15 min.
Remove and transfer the supernatant fraction
to individual siliconized borosilicate glass
tubes. Recentrifuge the supernatant fraction
at 700 to 800 G in a standard clinical centrifuge
for 5 min. to remove any remaining erythrocytes
from the supernatant. Remove and transfer
the supernatant into a third borosilicate
tube. Analyze the samples for free hemoglobin
using the method in annex C.5.1.2
NOTE - It is important that the extracts
be isotonic. Non-isotonic extracts may predispose
cells to hemolysis.
C6.5 Hemolysis tests for blood containers
An in vitro hemolysis assay which uses an
extract of the blood container materials
is recommended in the European Pharmacopeia
[72] and in ISO Standard 3826 (Plastics Collapsible
Containers for Human Blood and Blood Components)
[73]. The extracts are prepared at 110_C
for 30 min.; these are the sterilization
conditions of blood containers with liquid
anticoagulants based on sterility assurance
testing levels). These assays are focused
on the container materials and do not assess
gas permeability or other quality assurance
parameters essential for storage of whole
blood and blood products (see annex C.5.2
for further discussion of this subject).
D.1 International Standards
[1] ISO 5840:1989, Cardiovascular implants
- Cardiac valve prostheses.
[2] ISO 5841 - 1: 1989, Cardiac pacemakers
- Part 1: Implantable pacemakers.
[3] ISO 5841-3:1992, Cardiac pacemakers -
Part 3: Low-profile connectors (IS-1) for
implantable pacemakers.
[4] ISO 7198-1:-1), Cardiovascular implants
- Tubular vascular prostheses - Part 1: Synthetic
vascular prostheses.
[5] ISO 7198-2-1), Cardiovascular implants
- Tubular vascular prostheses - Part 2. Sterile
vascular prostheses of biological origin
- Specification and methods of test.
[6] ISO 7199:-1), Cardiovascular implants
and artificial organs - Blood-gas exchangers.
[7] ISO 10993-12:-1), Biological evaluation
of medical devices - Part 12: Sample preparation
and reference materials.
D.2 National standards
[8] AANSI/AAMI CVP3- 1981, Cardiac valve
prostheses.
[9] ANSI/AAMI VP20- 1986, Vascular graft
prostheses.
[10] ASTM F 756-87, Standard Practice for
assessment of hemolytic properties of materials.
[11] BS 5736-11:1990, Evaluation of medical
devices for biological hazards 3/4 Part 11:
Method of test for hemolysis.
[12] DIN 58 361-4: 1980, Transfusionsbehaltnisse
und Zubehor, Blutbeutel aus Kunststoffen,
Sicherheitstechnische Anforderungen, Prufung,
Uberwachung und Kennzeichnung.
[13] NF 90-300: 1981, Materiel medico-chirurgical
- Oxygenateurs.
D.3 Scientific papers
[14] BOSCH, T., SCHMIDT, B., BLUMENSTEIN,
M. and GURLAND, H.J., Thrombogenicity markers
in clinical and ex vivo assessment of membrane
biocompatibility. Contr Nephrol, 59: 90-98,
1987.
[15] CHENOWETH, D.E., Complement activation
produced by biomaterials. Trans Am Soc Artif
Int Organs 32: 226232, 1986.
[16] BURNS, G.L. PANTALOS, G.M. and OLSEN,
D.B., The calf as a model for thromboembolic
events with the total artificial heart. Trans
Am Soc Artif Int Organs 33: 398-403, 1987.
[17] COOPER, S.L., FABRIZIUS, D.J. and GRASEL,
T.G., Methods of assessment of thrombosis
ex vivo. In: Leonard E.F., Turitto V.T.,
and Vroman L.(Eds): Blood in contact with
natural and artificial surfaces. Ann N.Y.
Acad Sciences 516:572-585, 1987.
[18] Corriveau, D.M. and Fritsma, G.A. (Eds):
Hemostasis and thrombosis in the clinical
laboratory. J.B. Lippincott, Philadelphia,
1988, 443 pp.
[19] DAWIDS, S et al. (Eds): Test procedures
for the blood compatibility of biomaterials.
In preparation, 1992.
[20] DEWANJEE, M.K., Methods of assessment
of thrombosis in vivo, In: Leonard E.F.,
Turitto V.T. and Vroman L. (Eds): Blood in
contact with natural and artificial surfaces.
Ann N.Y. Acad Sciences, 516:541-571, 1987.
[21] DEWANJEE, M.K., KAPADVANJWALA, M. and
SANCHEZ, A., Quantitation of comparative
thrombogenicity of dog, pig, and human platelets
in a hemodialyzer. Am Soc Artif Int Organs
Journal 38: 88-90, 1992.
[22] DIDISHEIM, P., DEWANJEE, M.K., FRISK,
C.S., KAYE, M.P. and FASS, D.N., Animal models
for predicting clinical performance of biomaterials
for cardiovascular use. In: Boretos J.W.
and Eden M. (Eds): Contemporary Biomaterials,
Noyes Publications, Park Ridge, NJ, USA,
pp. 132-179, 1984.
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