FIELD OF THE INVENTION
[0001] The present invention is in the field of biomedical technology. In particular, the
present invention relates to the dynamic evaluation of the hemocompatibility of materials.
BACKGROUND OF THE INVENTION
[0002] The evaluation of blood interaction properties, in particular of hemocompatibility,
of materials employed in the fabrication of biomedical devices such as implants, stents,
drug delivery systems and cardiopulmonary bypasses requires the use of blood incubation
systems which simulate physiological flow conditions as realistically as possible.
In the design and development of such biomedical devices it is of uppermost importance
to predict any possible interactions and complications, which might occur once the
device is in contact with blood, like thrombosis, coagulation, protein absorption,
or hemolysis complement system activation.
[0003] Regarding hemocompatibility, the conditions under which such blood interaction properties
tests must be carried out are regulated by the DIN EN ISO 10993-4 norm, which recommends
the use of dynamic evaluations, which reproduce better the real physiological flow
conditions inside the body. However, the norm does not describe a particular test
bench with which the tests should be carried out. Several kinds of testing systems
are available. Most of the currently available systems for dynamic evaluation of hemocompatibility
have serious limitations, especially regarding restricted ability to change the tested
materials and poor access to the test surface of the same. Further problems include
the unwanted influencing of the results of the experiment due to damage of blood cells
during the test.
[0004] In view of the necessity of the reproducibility of the experiments and the comparability
between different test samples, the use of simple geometries for the sample materials
whose blood interaction properties are to be tested is preferable.
[0005] Given the limitations of the currently available testing systems, there is room for
technical improvements in the dynamic evaluation of blood interaction properties.
A testing system overcoming the deficiencies of most current systems could furthermore
pave the way to standardised testing, like for example for dynamic hemocompatibility
testing in the DIN norm.
[0006] US 2011/0045993 discloses a microfluidic device for testing the interaction between a fluid sample
and a test material comprising a sample inlet, a sample outlet and a flow cavity extending
between the sample inlet and the sample outlet for flow of a fluid sample through
the flow cavity. The flow cavity comprises a test area to which at least one test
material can be attached. When the fluid sample is made to flow through the flow cavity,
a shear force between the test area and the fluid sample flowing over the test area
is substantially constant.
[0007] US 2014/0287451 describes a flow chamber having an inlet port, an exit port and a flow channel extending
between the inlet port and the exit port. The fluid channel is configured to hold
a sample so that a fluid flowing from the inlet port to the exit port through the
flow chamber contacts a side of the sample. The test chamber further has an optically
transparent viewing window adjacent to the flow channel so that the sample can be
observed in real-time while it is exposed to the fluid.
SUMMARY OF THE INVENTION
[0008] The problem underlying the invention is to provide means and a method for dynamically
testing blood interaction properties of a sample material with reduced damage of blood
cells during the test and the possibility to easily change the sample material. This
problem is solved by an apparatus according to claim 1 and a method according to claim
10.
[0009] The apparatus of the invention comprises at least one test chamber for receiving
a blood sample. Herein, the term "blood sample" is understood to have a broad meaning
and covers any liquid substance comprising at least parts of human or animal blood.
[0010] The test chamber comprises at least two reservoirs, a fluid channel extending between
the at least two reservoirs and a receptacle for receiving the material sample. The
receptacle might for example be any geometric configuration allowing for the arrangement
of the material sample in the test chamber, like but not restricted to, an indent
or a depression. The receptacle is arranged with respect to the fluid channel such
that when the blood sample is received in the test chamber and the material sample
is received in the receptacle, the blood sample flowing in the fluid channel may flow
in contact with the material sample. Note that the term "test chamber" has been chosen
to indicate that the reservoirs and the fluid channel form a hollow structure for
accommodating the blood sample, but shall not imply any further structural limitations.
In particular, it does not imply that the reservoirs and the fluid channel are necessarily
rigidly connected. Instead, the reservoirs and the fluid channel could for example
be connected by flexible tubes or the like.
[0011] The apparatus further comprises a driving arrangement configured for providing a
reciprocating movement of at least a part of the at least one test chamber such as
to alternatingly raise one of the reservoirs with respect to the other, thereby causing
the blood sample in the fluid channel to flow back and forth from one reservoir to
the other. Herein, the driving arrangement can be any type of mechanical, magnetic,
electric or electronic system or any combination thereof suitable for providing the
reciprocating movement of the test chambers or parts thereof.
[0012] According to the invention, the reciprocating movement of the test chamber provided
by the driving arrangement alternatingly raises one of the reservoirs with respect
to the other such that gravity causes the blood sample in the fluid channel to flow
in contact with the material sample alternatingly from the momentarily higher reservoir
to the momentarily lower reservoir. This way the blood sample interacts dynamically
with the material sample, thereby simulating the physiological conditions inside true
blood vessels. This allows for the dynamic evaluation of blood interaction properties
without the use of a pump to drive the blood sample flow, which significantly reduces
the damage to blood cells in the course of the test of blood interaction properties
and hence the influence of the testing system itself over the results of the test.
As will be shown in the examples below, this advantageous feature is confirmed by
experimental results showing that the invention induces upon the blood sample a much
lower release of free hemoglobin in plasma than standard testing setups. Hereby a
major deficiency of most known experimental setups for dynamic hemocompatibility testing,
which are based in the use of pumps for driving the blood flow, is overcome.
[0013] Additionally, by not using a pump the necessity of removing the air from the test
chambers prior to incubation, which could induce a waste of blood, is eliminated.
This introduces a clear advantage with respect to known test systems for dynamic hemocompatibility
testing based on the use of tubes, like the standard "Chandler loop".
[0014] Both reservoirs typically have the same form and volume. The reservoirs might have
a cylindrical form, although other geometries are possible. The reservoirs may be
connected to the fluid channel through at least one opening. In addition, the reservoirs
may be open at their upper end so as to allow the fluid to flow in the fluid channel
freely under the influence of gravity without any restriction due to a pressure gradient
in the test chamber.
[0015] Preferably, the amount of the blood sample in the test chamber is chosen such that
the volume of the blood sample is bigger than the added volume of the fluid channel
and of one of the reservoirs and smaller than the added volume of the fluid channel
and of both reservoirs. This way, at any stage of the reciprocating movement of the
test chamber, the fluid channel is completely filled with the blood sample, while
the excess volume of the blood sample not fitting in the fluid channel partially fills
the reservoirs. When both reservoirs have the same volume and one of the reservoirs
is raised with respect to the other, a larger amount of the blood sample accumulates
in the reservoir momentarily having the lower position. When both reservoirs are arranged
equally high, both reservoirs are partially filled with the same amount of the blood
sample. The volume of the reservoirs is preferably big enough so as to guarantee that
no blood sample flows out of the test chamber during the operation of the apparatus.
Other arrangements regarding the volume of the blood sample, the fluid channel and
the reservoirs are possible, so as to enable different flowing configurations.
[0016] According to a preferred embodiment of the invention, the driving arrangement comprises
a rocking platform or a rocker that provides a reciprocating tilting movement of the
at least one test chamber. The at least one test chamber is then placed or arranged
on the platform of the rocking platform. The adjustment of the frequency and maximum
angle of the reciprocating tilting movement of the test chamber driven by the rocking
platform allows for an easy and direct control of the parameters determining the flow
conditions of the blood sample in the test chamber, such as the flow rate or the wall
shear rate.
[0017] The reciprocating tilting movement of the test chamber driven by the driving arrangement
is typically a tilting of the test chamber with respect to a horizontal rest position,
wherein the tilting movement takes place in a vertical plane. This vertical plane
preferably contains the direction corresponding to the longitudinal direction of the
test chamber. Preferably, the longitudinal direction of the test chamber and the longitudinal
direction of the fluid channel coincide, such that the instant axis of rotation of
the tilting movement of the test chamber is perpendicular to the longitudinal direction
of the fluid channel. As a consequence of that, as will be evident to the skilled
person, a fluid flow inside the fluid channel only occurs along the longitudinal direction
of the fluid channel. However, other configurations might be used.
[0018] In a preferred embodiment of the invention, the at least one test chamber comprises
a first part and a second part, which are releasably secured to one another, for example
by means of screws or other means for pressing the parts together. In addition, means
may be provided for fixing the at least one test chamber to the driving arrangement.
By releasing either of the parts, access to the receptacle is obtained, which allows
the material sample to be easily placed, exchanged or removed. This facilitates the
investigation of a broader range of sample materials while keeping the assembly of
the apparatus parts simple, thereby minimising the risk of operating errors and reducing
operation time. Furthermore, the simple structure of the test chamber renders its
fabrication technically easier and hence less costly and its operation more accessible
and intuitive. In addition, easy access to the inner space of the test chamber makes
it possible to analyse the properties of the surface of the same before its contact
with the blood sample as well as the changes caused by the interaction with the blood
sample after interaction.
[0019] According to a preferred embodiment of the invention, the receptacle is configured
for receiving a planar material sample. The planar geometry of the receptacle allows
for an easy operation of the apparatus when it comes to placing, exchanging or removing
the material sample and reduces the time needed for preparation or manipulation of
the apparatus. It further provides easy and direct access to the test surface of the
material sample, both before and after it has interacted with the blood sample in
the apparatus and hence facilitates the investigation of said test surface. Herein,
"test surface" refers to the surface of the material sample facing the inside of the
fluid channel. This enables for example the detailed characterization of the chemical
composition, the structure, the coating or the roughness of the test surface of the
material sample before its contact with the blood sample. It further allows investigation
of the blood components adhered to the test surface after the material sample and
the blood sample have interacted in the apparatus.
[0020] In a preferred embodiment of invention, the planar material sample to be received
in the receptacle has the form of a stripe. Herein a stripe is understood as having
a generally rectangular shape with one of its dimensions significantly longer than
the other one, for example at least three times longer. This allows for the use of
planar material samples with standardized geometry, dimensions and structure, thereby
offering a better control over the universe of possible test objects for analysis
and comparison. In addition, such a stripe-like shape contributes to the reduction
of the influence of the apparatus upon the results of testing by allowing for a very
reproducible washing procedure of the material samples. Washing steps are commonly
employed in the course of testing and in previously known testing systems they are
typically performed by pipetting a wash solution on the sample, which may lead to
uncontrolled detachment of adhered cells from the surface of the material sample due
to strong shear stress and hence to uncontrolled changes in the cell concentrations
on the surface of the material sample.
[0021] According to a preferred embodiment of the invention, the at least one test chamber
comprises a top confining part and a bottom confining part, wherein the top confining
part and the bottom confining part can be tightly and releasably secured to one another
and the receptacle is arranged within the bottom confining part, such that when the
material sample is received in the receptacle and the top confining part and the bottom
confining part are tightly secured to one another, a cavity between the top confining
part and the material sample is formed that acts as the fluid channel. The top confining
part of the test chamber is preferably made of silicone rubber, although other material
compositions are possible. The bottom confining part may be formed by the second part
of the at least one test chamber. According to this embodiment, when the blood sample
is introduced into the test chamber and made to flow in the fluid channel, the blood
sample flows between top confining part of the test chamber and the material sample.
This configuration reduces the contact of the blood sample with surfaces not to be
tested, which for example suppresses unwanted blood activation and uncontrolled interactions.
This preferred embodiment further allows for configurations in which, provided a suitable
amount of the blood sample is filled into the test chamber, the material sample that
constitutes the bottom part of the fluid channel is entirely covered with the blood
sample and hence is not in contact with air during the blood interaction properties
test, thereby eliminating possible uncontrolled effects that could be caused by the
interaction with air.
[0022] In a preferred embodiment of the invention, the apparatus further comprises a transition
region between the fluid channel and each of the reservoirs, said transition region
comprising rounded edges to prevent or at least reduce the formation of eddies in
the blood sample flow in said transition region. The prevention or reduction of the
formation of eddies in the blood sample contributes to avoid unwanted blood activation
like clotting, a better control of the testing conditions and the flow parameters
and allows for testing the material sample under conditions that better reproduce
physiological flow conditions.
[0023] According to a preferred embodiment of the invention, the driving arrangement is
adapted for simultaneously providing the reciprocating tilting movement of at least
two, preferably of at least four and most preferably of at least six test chambers.
This makes it possible to test several material samples simultaneously thereby reducing
the time required for testing different material samples. In the case that the driving
arrangement is a rocking platform, at least some of the test chambers might be stapled
on top of each other on the platform of the rocking platform and optionally secured
thereto. This does not only increase the throughput of the test, but may also increase
its reproducibility and significance by making it possible to obtain identically obtained
blood samples and material samples simultaneously subjected to evaluation under the
same testing conditions, thereby reducing the need of any waiting time that may influence
the test results in an uncontrolled manner or render the repetition of experiments
necessary.
[0024] Regarding the volume of the blood sample, a compromise should be found between volumes
of blood sample sufficient for performing the analysis and avoiding a waste of blood,
which is in general a limited resource for experimental purposes. Accordingly, in
a preferred embodiment of the invention, the fluid channel has a volume between 2.5
cm
3 and 20 cm
3, preferably between 3.0 cm
3 and 12 cm
3, and most preferably between 4.5 cm
3 and 8 cm
3.
[0025] In a preferred embodiment, the fluid channel has a rectangular cross-section. This
geometry of the fluid channel makes it possible to approximately resemble physiological
flow conditions inside blood vessels while preserving the ability to easily change
the material sample and the direct access to the receptacle, the material sample and
the test surface of the latter.
[0026] According to a preferred embodiment of the invention, the cross-section of the fluid
channel has a width to height ratio between 1 and 40, preferably between 10 and 30
and most preferably between 15 and 25. Preferably, the fluid channel has a cross-section
between 10 mm
2 and 30 mm
2. This has the advantage of maximising the contact surface of the material sample
with respect to a given cross-section of the fluid flow.
[0027] A further aspect of the invention relates to a method for evaluating blood interaction
properties of a material sample comprising the steps of:
- a) providing a material sample and inserting the material sample into the receptacle
of an apparatus for testing blood interaction properties of a material sample according
to any of the embodiments described above;
- b) filling a blood sample into the at least one test chamber;
- c) operating the driving arrangement for providing a reciprocating movement of the
at least one test chamber such as to alternatingly raise one of the reservoirs with
respect to the other, thereby causing the blood sample in the fluid channel to flow
back and forth from one reservoir to the other in direct contact with the material
sample; and
- d) performing an analysis of one or both of the material sample and the blood sample
after the material sample and the blood sample have interacted in the apparatus.
[0028] In a preferred embodiment of the invention, the step of providing a material sample
comprises preheating the material sample to a temperature between 35°C and 42°C, preferably
between 36°C and 38°C, so as to reproduce physiological conditions.
[0029] In another preferred embodiment of the invention, the method may further comprise,
prior to step b), a step for preparing the blood sample. This may for example comprise
mixing blood with an anticoagulant or preparing a blood sample with a predefined cell
concentration so as to minimise the donor-dependent factor influencing the result
of the evaluation. This temperature is preferably maintained during the blood interaction
properties evaluation procedure.
[0030] According to a preferred embodiment of the invention, the method may further comprise,
prior to step b), a step of measuring of properties of the blood sample before interaction
with the material sample in the apparatus. In particular this may comprise measuring
a cell concentration of the blood sample.
[0031] Step c) of operating the driving arrangement may comprise configuring it to provide
the reciprocating movement of the at least one test chamber according to predefined
parameters, like for example reciprocating frequency, maximum height of the reservoirs
or operation time. Said parameters might be constant or variable in time. Herein,
the reciprocating frequency refers to the rate at which each of the reservoirs alternatingly
occupies the highest attainable position with respect to a horizontal rest position
and may be measured in revolutions per minute (rpm) according to the number of times
one of the reservoirs successively occupies the highest position in a period of time
of one minute. The maximum height refers to the maximum height with respect to a horizontal
rest position.
[0032] In the case that the driving arrangement is a rocking platform, the parameters determining
the flow conditions of the blood sample in the fluid channel might be the reciprocating
frequency and the maximum tilt angle. Herein the maximum tilt angle refers to the
maximum tilt angle of the at least one test chamber with respect to the horizontal
rest position, that is the angle between the longitudinal axis of the test chamber
and the horizontal rest position when one of the reservoirs has achieved the highest
attainable position.
[0033] The choice of the reciprocating frequency and the maximum height of the reservoirs
or the maximum tilt angle should be such that the resulting flowing conditions of
the blood sample in the fluid channel at least approximately resemble physiological
conditions. The concrete correlation between reciprocating frequency and maximum height
of the reservoirs or maximum tilt angle on the one side and flowing conditions of
the blood sample on the other side can be calculated from the corresponding fluid
mechanics equations, as will be shown in the examples below. According to this requirement,
in a preferred embodiment of the invention, step c) of operating the driving arrangement
may comprise configuring said driving arrangement to provide the reciprocating tilting
movement of the at least one test chamber with a reciprocating frequency between 2
and 50 rpm and a maximum tilt angle between 1 and 20 degrees or a maximum height of
the reservoirs that corresponds to such maximum tilt angle.
[0034] According to a preferred embodiment of the invention, step c) of operating the driving
arrangement may comprise configuring said driving arrangement to operate for a period
of time between 1 and 240 minutes, preferably between 60 and 90 minutes. This has
been experimentally determined to be long enough for the interaction of the blood
sample with the material sample to have measureable consequences and hence to ensure
good simulation of the in-vivo application of the material sample.
[0035] According to various embodiments of the invention, step d) of performing an analysis
of one or both of the material sample and the blood sample after the material sample
and the blood sample have interacted in the apparatus may comprise any kind of analysis
aiming at characterizing the blood interaction properties of the tested material.
In particular, said step may comprise one or more of
- a platelet adhesion test to measure the density of platelets adhered to the material
sample during incubation;
- a scanning electron microscopy analysis of the material samples to recognize platelets
adhered to the material sample during incubation;
- a platelet deprivation test of the blood sample to measure changes in the platelet
concentration of the blood sample during incubation.
- a hemolysis test of the blood sample to measure the hemolysis caused during incubation;
Specific details of the different kinds of analysis will be given below in the examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036]
- Fig. 1
- shows a schematic representation of an apparatus according to an embodiment of the
invention comprising a test chamber and a driving arrangement.
- Fig. 2
- is an exploded view of the components of the test chamber of Fig. 1.
- Fig. 3
- shows in detail the test chamber of Fig. 1 and 2.
- a) is a sectional view of the test chamber of Fig. 1 and 2.
- b) shows a perspective view of the test chamber of Fig. 1 and 2.
- Fig. 4
- shows in detail the top confining part of the test chamber of Fig. 2 and 3.
- a) is a base view of the top confining part of Fig. 2 and 3.
- b) is a perspective bottom view of the top confining part of Fig. 2 and 3.
Fig. 5 displays the shear rates experienced by two different test fluids during incubation
in the apparatus of Fig. 1 depending on the reciprocating frequency.
Fig. 6 displays the platelet deprivation in whole blood after 60 minutes incubation
in the apparatus of Fig. 1 at a reciprocating frequency of 30 rpm of four different
test materials.
Fig. 7 displays the platelet adhesion on the surfaces of four different test materials
of a platelet suspension after 60 minutes incubation in the apparatus of Fig. 1 at
a reciprocating frequency of 15 rpm.
Fig. 8 displays a comparison of the platelet adhesion on the surfaces of four different
test materials after 60 minutes incubation in the apparatus of Fig. 1 under static
conditions and dynamic flow conditions with a tilting frequency of 15 rpm.
Fig. 9 displays the measured quantity of free hemoglobin in blood plasma and the index
of hemolysis experienced by whole blood after 30, 60 and 90 minutes incubation in
the apparatus of Fig. 1 with tilting frequencies of 10 rpm and 30 rpm and the corresponding
values of a statically incubated blood sample right after blood collection and after
90 minutes incubation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] For the purposes of promoting an understanding of the principles of the invention,
reference will now be made to a preferred embodiment illustrated in the drawings,
and specific language will be used to describe the same. It will nevertheless be understood
that no limitation of the scope of the invention is thereby intended, such alterations
and further modifications in the illustrated apparatus and such further applications
of the principles of the invention as illustrated therein being contemplated as would
normally occur now or in the future to one skilled in the art to which the invention
relates.
[0038] Figure 1 shows a schematic representation of an apparatus 10 for testing blood interaction
properties of a material sample according to a preferred embodiment of the invention.
The apparatus 10 comprises a test chamber 20 for receiving a blood sample and a driving
arrangement 30. The test chamber 20 comprises two reservoirs 21, a fluid channel 22
extending between the two reservoirs 21, and a receptacle 23 (see Fig. 2 and 3) for
receiving a material sample, not shown in Fig. 1. The receptacle 23 is arranged with
respect to the fluid channel 22 in such a way that when the blood sample is received
in the test chamber 20 and the receptacle 23 receives the material sample, the blood
sample flowing in the fluid channel 22 may flow in contact with the material sample.
The driving arrangement 30 is configured for providing a reciprocating movement of
the test chamber 20 such as to alternatingly raise one of the reservoirs 21 with respect
to the other, so that gravity causes the blood sample in the fluid channel 22 to flow
in contact with the material sample in the receptacle 23 alternatingly from the momentarily
higher reservoir to the momentarily lower reservoir.
[0039] The driving arrangement 30 can generally be any type of mechanical, magnetic, electric
or electronic system or any combination thereof suitable for providing the reciprocating
movement of the test chamber 20. In Fig. 1, the driving arrangement 30 is symbolically
represented by a triangle, which stands for any suitable arrangement, which in this
particular case, comprises a platform 31 on which the test chamber 20 is placed and
provides a reciprocating tilting movement of the test chamber 20. In particular, the
driving arrangement 30 might be a rocking platform or rocker comprising a platform
on which several test chambers can be placed for being simultaneously driven.
[0040] Both reservoirs 21 have the same cylindrical shape and identical volume. The reservoirs
21 are connected to the fluid channel 22 through at least one opening 210. The transition
region between the fluid channel 22 and each of the reservoirs 21 around the opening
210 comprises rounded edges, not shown in Fig. 1 (see Fig. 4), to prevent or at least
to reduce the formation of eddies in a blood sample flowing in said transition region.
In addition, the reservoirs 21 have a second opening 212 to establish atmospheric
pressure in the reservoirs 21, thereby allowing the fluid in the fluid channel 22
to flow freely under the influence of gravity.
[0041] In the embodiment shown, the amount of the blood sample in the test chamber 20 is
chosen such that the volume of the blood sample is bigger than the added volume of
the fluid channel 22 and of one of the reservoirs 21 and smaller than the added volume
of the fluid channel 22 and of both reservoirs 21. This way, at any stage of the reciprocating
tilting movement of the test chamber 20, the fluid channel 22 is completely filled
with the blood sample, while the excess volume of the blood sample not fitting in
the fluid channel partially fills the reservoirs 21. When the tilt angle of the test
chamber 20 is different from 0, like in the top and the bottom situation displayed
in Fig. 1, a bigger amount of the blood sample accumulates in the reservoir momentarily
having the lower position. When the tilt angle of the test chamber 20 is zero, as
is the case in the middle situation displayed in Fig. 1, the test chamber 20 momentarily
is arranged horizontally and both reservoirs 21 are partially filled with the same
amount of the blood sample. In the present embodiment, the fluid channel 22 has a
rectangular cross-section with a width to height ratio of 14 and a volume of 4,5 cm
3.
[0042] Figure 2 shows an exploded view of the components of the test chamber 20 of Fig.
1. The test chamber 20 comprises a first part 201, a top confining part 220 of the
fluid channel 22 and a second part 202 confining the fluid channel 22. The second
part 202 comprises the receptacle 23 for receiving a material sample 40. Between the
first part 201 and the second part 202 of the test chamber 20, the top confining part
220 of the fluid channel 22 is arranged, such that when the first part 201 and the
second part 202 are secured to one another by means of screws 203, the top confining
part 220 of the fluid channel 22 is clamped into the receptacle 23. The base of the
receptacle 23 in the second part 202 then constitutes a bottom confining part of the
fluid channel 22, and a rectangular cavity 221, not seen in the exploded view of Fig.
2 is formed between the top confining part 220 of the fluid channel 22 and the second
part 202, which constitutes the fluid channel 22. In particular, when a material sample
40 is received in the receptacle 23, a fluid flowing in the rectangular cavity 221
flows in contact with said material sample 40. As displayed in Fig. 2, the material
sample 40 received in the receptacle 23 has the form of a stripe. The material sample
40 can be tightly secured to the top confining part 220 of the fluid channel 22 by
securing together the first part 201 and the second part 202 of the test chamber 20,
within which the top confining part 220 of the fluid channel 22 is arranged. The material
sample 40 then entirely covers the receptacle 23, so that it forms the bottom of the
fluid channel 22. The top confining part 220 of the fluid channel 22 is made of silicone
rubber.
[0043] Figure 3 shows a cross-sectioned and a perspective view of the test chamber 20 of
Fig. 1 and 2. Here, the screws 203 are holding together the first part 201 and the
second part 202 of the test chamber 20. The top confining part 220 is arranged within
the first part 201 and the second part 202. The material sample 40 is received in
the receptacle 23 arranged within the second part 202. Further Fig. 3 depicts a rectangular
cavity 221, which is formed between the top confining part 220 and the material sample
40 through which the blood sample may flow, and which constitutes the fluid channel
22.
[0044] Figure 4 displays in detail the top confining part 220 of the test chamber 20 of
Fig. 2 and 3. The top confining part 220 comprises the reservoirs 21 and an elongated
recess 222 along the longitudinal direction of the top confining part 220 extending
between the reservoirs 21. When the top confining part 220 is arranged between the
material sample 40 and the first part 201 in the way shown in Fig. 2 and 3, and the
first part 201 is secured to the second part 202, the second part 202 acts as a bottom
confining part and a rectangular cavity 221 is formed between the material sample
40 and the top confining part 220, which forms the fluid channel 22. Each of the reservoirs
21 is connected to the recess 222 through at least one opening 210. The transition
region between the recess 222 and each of the reservoirs 21 around the opening 210
comprises rounded edges, which help preventing the formation of eddies in the blood
sample when the rectangular cavity 221 acts as the fluid channel 22.
EXAMPLES
[0045] In order to test the suitability of an apparatus and methods according to embodiments
of the invention for testing blood interaction properties of a material sample and
to compare their performance to that of other testing systems known from the prior
art, hemocompatibility of four common polymeric materials was investigated using such
apparatus and methods.
[0046] A preliminary assessment of the flowing conditions of the blood samples in the fluid
channel based on an experimental survey of the corresponding flow parameters was followed
by the preparation of the blood samples and material samples. These were disposed
in the apparatus and subsequently subjected to the methods according to various embodiments
of the invention.
[0047] The obtained results confirm the suitability of the apparatus and methods according
to an embodiment of the invention for dynamically testing blood interaction properties
and highlight the improvements over previously known testing systems.
Apparatus
[0048] An apparatus 10 as shown in Fig. 1 to 3 was used. The driving arrangement 30 was
a rocking platform (Duomax 1030, Heidolph). The fluid channel 22 of the test chamber
20 had a rectangular cross-section and a top confining part 220 of silicone rubber.
The dimensions of the fluid channel 22 were: width = 14 mm, length = 218 mm, height
= 1 mm or 2 mm. The reservoirs 21 had cylindrical shape and the dimensions were: diameter
= 17 mm and height = 20 mm. The receptacle 23 of the apparatus had a rectangular stripe-shape
and the reservoirs 21 had rounded edges at the passage from the fluid channel 22 to
the reservoirs 21 avoiding the formation of eddies in the blood sample. The longitudinal
axis of the test chamber 20 was aligned with the rocking platform such that the tilting
movement be in the direction of said longitudinal axis. Up to 8 test chambers 20 were
simultaneously loaded onto the rocking platform.
Flow parameters
Test fluids
[0049] Substitute liquids were used in the preliminary assessment of the flowing conditions
so as not to waste real blood for this purpose. A mixture of 35% glycerine (glycerol
anhydrous, Applichem GmbH, Germany) and 65% saline solution was used to simulate whole
blood, and distilled water was used to simulate platelet suspension. These fluids
were chosen because the viscosity and density are similar to those of the corresponding
true biological fluids. Their kinematic viscosity was measured with a capillary viscometer
(Ubbelohde AVS 310, Schott-Gerate GmbH, Germany) and the density was measured with
a density meter (DensityMeter DMA 4100M, Anton Paar GmbH, Austria).
Assessment of the fluid conditions in the channel
[0050] The glycerine solution and the distilled water were respectively pipetted into a
2-mm-high and a 1-mm-high fluid channel. Two different kinds of fluid channel were
used due to the different viscosities of whole blood and the platelet suspension.
The test chambers were placed on the rocking platform and this was configured to operate
with a maximum tilt angle of 5°. By adding coloured fluid in one of the reservoirs,
it was possible to visualise the velocity profile of the fluids for tilting frequencies
varying between 2 and 50 rpm. The velocity of the fluid in the centre-line of the
fluid channel (
v0) was recorded over time. The flow rate in the fluid channel
Q was calculated using

where
b stands for the width,
h for the height of the fluid channel and
v is the mean velocity of the fluid over the cross section of the fluid channel. The
mean velocity can be calculated from the velocity in the centre-line using

where
K1 is a correction factor that adapts the velocity profile to the case of a rectangular
section that was calculated using the formula of
Bruss used in Theoretical Microfluidics. Oxford University Press. 2009:37-70. K2 is another correction factor that adapts the amplitude of the periodic flow depending
on the reciprocating frequency and is a function of the Womersley number.
K2 was calculated using the formula of
Loudon and Tordesillas shown in The use of the dimensionless Womersley number to characterise
the unsteady nature of internal flow. J. theor. Biol. 1998;191:63-78. Assuming the ideal case of a Poiseuille flow between two plates, the constants
K1 and
K2 were taken to be 1. This assumption was found to be justified for tilting frequencies
over 7.5 rpm. The wall shear rate γ in the fluid channel was calculated by means of
the Poiseuille law:

Experimental flow parameters
[0051] The main flow velocity of the test fluids over a tilting period corresponded to a
sinus curve and the maximum amplitude
vmax obtained during a tilt movement was found to depend on the reciprocating frequency.
The highest values of
vmax were 11.3 cm/s at 30 rpm for the glycerine solution in the 2-mm-high fluid channel
and 6.3 cm/s at 10 rpm for distilled water in the 1-mm-high fluid channel. For higher
tilting velocities, the inertia of the fluid limited a higher flow velocity. The measured
density and viscosity of the test fluids were comparable to those of whole blood and
platelet suspension so that the flow characteristics in the fluid channel should be
approximately equivalent. The maximum Reynolds number was 120, which guarantees laminar
flow conditions for all configurations and tilting velocities. The Wormersley number
α was smaller than 1 for all configurations at 30 rpm, which guaranteed quasi-steady
flow conditions for tilting frequencies up to 30 rpm. The obtained wall shear rate
γ for whole blood and the platelet suspension depending on the reciprocating frequency
is represented in figure 5. In both cases the shear rate increased with the reciprocating
frequency up to a maximum value of 339 s
-1 for whole blood and 380 s
-1 for the platelet suspension. In view of the values of the shear rates reached in
the fluid channel of the tested apparatus, the flow conditions in the fluid channel
corresponded to those characteristic of veins and arteries. By adjusting the reciprocating
frequency depending on the geometry of the fluid channel, it was possible to conduct
the experiment under varying well-defined flow conditions resembling those in true
blood vessels. This shows that the apparatus according to an embodiment of the invention
is suitable for hemocompatibility testing in flow conditions which are comparable
to the physiological flow in blood vessels
Sample preparation
Blood sample
[0052] Different tests were carried out using true blood samples formed by a platelet suspension
or by whole blood. Human blood was obtained from healthy and voluntary donors and
mixed with an anticoagulant, this being citrate (Vacuatte® ACD-A tubes, 9 ml, Greiner
Bio-One International GmbH, Germany) in the tests with platelet suspensions and heparin
(Vacuette® Lithium Heparin tube 9 ml, Greiner Bio-One International GmbH, Germany)
in the tests with whole blood. Immediately after the blood withdrawal, the cell concentration
was measured with a hematology analyser (KX-21N, Sysmex Europe GmbH, Germany). For
the preparation of the platelet suspensions, the freshly collected blood was centrifuged
at 1750 g for 6 minutes. The resulting overlying supernatant and buffy coat concentrating
most of the white blood cells and platelets were separated and underwent a second
centrifugation at 2250 g for 9 minutes. Then the supernatant was removed and the resulting
concentrated platelet suspension was adjusted to a concentration of 600×10
3 platelets/µl (PLT/µl) using phosphate buffered saline.
Material samples
[0053] Planar material samples having the form of a stripe of low-density polyethylene (PE,
type DOWLEX™ 2107GC, Dow Chemical Company, USA), polypropylene (PP, type PCGH10, Sabic,
Saudi Arabia), polyoxymethylene (POM, type HOSTAFORM® MT24U01, Celanese Corporation,
USA) and silicone (type SILPURAN® UR 9030/60, Wacker Chemie AG, Germany) were used.
The material samples of those four polymers were cleaned with 70%-2-propanol and vacuum
dried for a minimum of 12 hours. The surface of the material samples was characterised
before conducting the hemocompatibility tests in order to compare the results with
values from the literature. The contact angle with water was measured to examine the
wetting properties of the material samples with respect to aqueous liquids (Device
OCA 20, DataPhysiy Instruments GmbH, Germany). For each material, ten measurements
were conducted immediately after wetting with 2 µl drops. Topology and roughness of
the surfaces of the material samples were assessed with a confocal microscope (µSurf,
NanoFocus Ag, Germany).
Analysis methods
Platelet deprivation test
[0054] The material samples were introduced into the receptacle of the test chambers with
2-mm-high fluid channels, which were then preheated to 37°C. The test chambers were
filled with 8 ml of whole blood and then placed on the rocking platform for 60 minutes
to operate according to the flow parameters indicated above at a constant temperature
of 37°C. After that, the incubated blood samples were removed from the test chambers
and the cell concentration was immediately measured. Three measurements were made
for each material sample.
[0055] A reduction of the number of platelets in the blood after incubation is linked to
thrombogenic events such as the adhesion of platelets on the surface of the material
samples and the formation of platelet aggregates. In view of the blood donor dependent
variability of platelet number and function, each value was normalised with the mean
value of the platelet count for all materials after testing.
Platelet adhesion test
[0056] The same procedure was conducted with test chambers having 1-mm-high fluid channels
filled with 4.5 ml of platelet suspension. During the 60 minutes incubation step,
blood platelets could adhere to the surface of the material samples. After incubation,
the platelet suspensions were removed from the test chambers and 5 ml of phosphate
buffered saline heated to 37°C was pipetted into one reservoir, made to flow through
the fluid channel and removed from the other reservoir. This guaranteed that all samples
underwent exactly the same wash procedure and only the adhered platelets remained
on the surface of the material samples. Then the material samples were removed from
the test chambers and three fragments (diameter 12 mm) of each stripe were stamped
and placed in a microtiter plate with 300 µl lysis buffer solution (Sodium Citrate
Tribasic Dihydrate 0.1 M, p-Nitrophenyl-Phosphat 5 mM, Triton X-100 1%, pH 5.4) for
1 hour at 37°C. The chemicals for the lysis buffer were all purchased from Sigma-Aldrich
Co. LLC, USA. The purpose of the lysis buffer is that of dissolving the adhered platelets
and releasing the enzyme ACP (Acid Phosphatase) which reacts with p-Nitrophenyl-Phosphat
resulting in a colour change. The colour change is proportional to the number of thrombocytes
dissolved in the lysis buffer and may be measured by means of UV spectroscopy so as
to quantify the amount of platelets adhered to the surfaces of the material samples.
After incubation, the reaction was stopped with 210 µl sodium hydroxide (NaOH 2 mol/L,
Merck KGaA, Germany), and 170 µl of the solution was pipetted in a 96-wells microtiter
plate. The photometric absorbance of 170 µl of the solution was measured at 405 nm
with an ELISA reader (Multiscan FC, Type 357, Thermo Fisher Scientific AG, USA). In
order to convert the absorbance values into the number of adhered platelets per square
millimetre (PLT/mm
2), a standard curve was applied which was generated using a dilution series of platelet
suspensions.
Hemolysis tests
[0057] The damage caused to red blood cells during testing was assessed with the quantification
of the release of free hemoglobin in plasma of the blood samples consisting in whole
blood induced by the flow in the apparatus. Fluid channels according to preferred
embodiments of the invention and silicone material samples were cleaned and put together
in clamping devices. Fresh human blood was withdrawn from a voluntary donor, collected
in heparinised tubes (Vacuette® Lithium Heparin tube 9 ml, Greiner Bio-One International
GmbH, Germany) and pooled. Seven test chambers having a 2-mm-high fluid channel were
used. 8 ml blood was pipetted into each test chamber. Three test chambers were placed
on a rocking platform configured to operate with a maximum tilt angle of 5° at a reciprocating
frequency of 10 rpm. Three test chambers were placed on a separate rocking platform
configured to operate with a maximum tilt angle of 5° and a reciprocating frequency
of 30 rpm. One test chamber was left under static conditions. The temperature was
kept at 37°C and after 30 minutes, 60 minutes and 90 minutes, blood was collected
from one of the dynamically incubated chambers. Blood from the statically incubated
chamber was collected after 90 minutes. In order to quantify the hemolysis that had
been induced during incubation, the amount of free haemoglobin in the obtained plasma
of the blood samples was measured. For this purpose, the collected blood samples were
centrifuged at 1000 g for 10 minutes. Plasma was drawn off and recentrifuged at 1000
g for another 10 minutes. The supernatant was withdrawn and its optical absorption
was recorded with a UV/VIS spectrophotometer (Nanodrop™ 1000, thermo Scientific™,
USA). Hemoglobin has three absorbance peaks at 451 nm, 541 mm and 577 nm. Because
of the small amounts of hemoglobin in plasma, the quantification method described
by
Harboe in "A method for determination of hemoglobin in plasma by near-ultraviolet
spectrophotometry. Scandinav. J. Clin. & Lab. Investigation 1959;11:66-70" based on the analysis of the highest peak at 450 nm was chosen. The index of hemolysis
(IH) was defined as the ratio of free hemoglobin in plasma (PHb) to the total hemoglobin
in the whole blood (THb) depending on the haematocrit (HCT) of the blood:

Results
Hemocompatibility testing
[0058] The analysis of hematocrit measurements indicated a reduction of the number of platelets
in the blood samples during incubation in the apparatus according to an embodiment
of the invention. This can be due to platelet adhesion on the surface of the material
sample and to activation or aggregation of platelets in the flowing blood samples.
Figure 6 shows the reduction in the number of platelets after contact with the four
different kinds of material sample. The only significant difference in the platelet
deprivation was found between PP and silicone, were silicone was the material with
the least depraved platelets and PP the one which caused the highest platelet deprivation,
37% more platelet reduction than on silicone. The results showed significant differences
for most of the test materials.
[0059] As shown in Fig. 7, the amount of detected platelets per mm
2 surface area (PLT/ mm
2) of material sample varied between 17,000 PLT/ mm
2 for silicone and 4000 PLT/ mm
2 for POM. PP and PE have intermediate amounts of adhered platelets on their surface
with respectively 6000 PLT/ mm
2 and 12000 PLT/ mm
2. The most platelets were found on the silicone material sample and they exhibited
an advanced stage of adhesion with spreading of pseudopodia and formation of platelet
aggregates. No aggregates were found on the material samples of PE, PP and POM, and
the cells were observed to be isolated and dispersed on the surface. More platelets
were found on PE than on PP and in both cases they were strongly activated and spread
numerous pseudopodia. A smaller amount of platelets was found on POM and they were
less activated than on other materials.
[0060] Figure 8 shows the difference in the platelet adhesion when the same cell suspension
is simultaneously incubated under static (0 rpm) or dynamic (15 rpm) flow conditions.
Very few platelets were detected after incubation under static flow conditions and
variations between the different test materials were minimal, from 1660 PLT/ mm
2 for silicone to 2020 PLT/ mm
2 for PP. However, after incubation the amount of adhered platelets increased greatly
and pronounced differences between the different test materials could be measured,
from 2800 PLT/ mm
2 for POM to 21,700 PLT/ mm
2 for silicone. This confirms that the detection of platelets was absolutely caused
by the flow-induced adhesion of the platelets on the surface of the material samples
and excludes the possibility that the detected platelets came from residual blood
which may not have been removed by the washing procedure.
[0061] Notably, greater variations of the number of adhered platelets could be detected
under dynamic flow conditions. Shear-induced platelet activation is a well-documented
phenomenon that occurs both in in-vitro systems and in in-vivo biological systems.
In view of this, hemocompatibility testing of materials should be conducted under
dynamic flow conditions for the best characterization of the platelet/material interactions.
The apparatus according to various embodiments of the invention can be used for the
measurement of platelet adhesion and platelet depravation with very good accuracy.
Hemolysis induced by the apparatus
[0062] The levels of plasma hemoglobin detected after incubation of the blood samples in
the test chambers and the corresponding index of hemolysis are shown in Fig. 9. Directly
after blood collection, the amount of free hemoglobin in plasma was 0.2 mg/dl and
after 90 minutes static incubation in the apparatus, the amount of plasma hemoglobin
was 0.6 mg/dl. The difference between the two values was presumably due to the blood
damage by pipetting. The amount of plasma hemoglobin for the dynamically incubated
blood samples ranged between 0.5 and 1.5 mg/dl, which is still in the lower range
of free hemoglobin in blood plasma for healthy subjects (0 mg/dl to 10 mg/dl). Hemoglobin
release in plasma was slightly elevated by high shear rates (340 s
-1 at 30 rpm, arterial flow conditions) with respect to low shear rates (135 s
-1 at 10 rpm, venous flow conditions). A time dependence of the hemoglobin release could
also be observed and the maximum plasma hemoglobin (1.42 mg/dl) was reached after
90 minutes at 30 rpm. The index of hemolysis (IH) was calculated as indicated above
using the values HCT = 40.4% and THb = 14.2 g/dl. The IH reached a maximum value of
0.006%.
1. An apparatus (10) for testing blood interaction properties of a material sample (40),
said apparatus comprising:
at least one test chamber (20) for receiving a blood sample comprising: at least two
reservoirs (21); a fluid channel (22) extending between the at least two reservoirs
(21); and a receptacle (23) for receiving the material sample (40), wherein the receptacle
(23) is arranged with respect to the fluid channel (22) such that when the blood sample
is received in the test chamber (20) and the material sample (40) is received in the
receptacle (23), the blood sample flowing in the fluid channel (22) may flow in contact
with the material sample (40); and
a driving arrangement (30);
wherein the driving arrangement (30) is configured for providing a reciprocating movement
of the at least one test chamber (20) or parts thereof such as to alternatingly raise
one of the reservoirs (21) with respect to the other, thereby causing the blood sample
in the fluid channel (22) to flow back and forth from one reservoir to the other.
2. The apparatus (10) according to claim 1, wherein the driving arrangement (30) comprises
a rocking platform or a rocker and the reciprocating movement is a reciprocating tilting
movement.
3. The apparatus (10) according to any of the preceding claims, wherein the at least
one test chamber (20) comprises a first part (201) and a second part (202) releasably
secured to one another, wherein release of the first part (201) or the second part
(202) provides access to the receptacle (23) for placement or removal of the material
sample (40).
4. The apparatus (10) according to any of the preceding claims, wherein the receptacle
(23) is configured for receiving a planar material sample (40), wherein the planar
material sample (40) preferably has the form of a stripe.
5. The apparatus (10) according to any of the preceding claims, wherein the at least
one test chamber (20) comprises a top confining part (220) and a bottom confining
part, wherein the top confining part (220) and the bottom confining part can be tightly
and releasably secured to one another and the receptacle (23) is arranged within the
bottom confining part, such that when the material sample (40) is received in the
receptacle (23) and the top confining part and the bottom confining part are tightly
secured to one another, a cavity (221) between the top confining part (220) and the
material sample (40) is formed that acts as the fluid channel (22), wherein the top
confining part (220) is preferably made of silicone rubber.
6. The apparatus (10) according to any of the preceding claims, further comprising a
transition region between the fluid channel (22) and each of the reservoirs (21),
said transition region comprising rounded edges to prevent or at least reduce the
formation of eddies in a blood sample flow in said transition region.
7. The apparatus (10) according to any of the preceding claims, wherein the driving arrangement
(30) is adapted for simultaneously providing said reciprocating movement of at least
two, preferably of at least four and most preferably of at least six test chambers
(20).
8. The apparatus (10) according to any of the preceding claims, wherein the fluid channel
(22) has a volume between 2.5 cm3 and 20 cm3, preferably between 3.0 cm3 and 12 cm3 and most preferably between 4.5 cm3 and 8 cm3.
9. The apparatus (10) according to any of the preceding claims, wherein the fluid channel
(22) has a rectangular cross-section, and/or wherein the fluid channel (22) has a
cross section between 10 mm2 and 30 mm2, and/or wherein the cross-section of the fluid channel (22) has a width to height
ratio between 1 and 40, preferably between 10 and 30 and most preferably between 15
and 25.
10. A method for evaluating blood interaction properties of a material sample (40) comprising
the steps of:
providing a material sample (40) and inserting the material sample (40) into the receptacle
(23) of an apparatus (10) for testing blood interaction properties of a material sample
(40) according to any of claims 1 to 9; and
filling a blood sample into the at least one test chamber (20);
operating the driving arrangement (30) for providing a reciprocating movement of the
at least one test chamber (20) such as to alternatingly raise one of the reservoirs
(21) with respect to the other, thereby causing the blood sample in the fluid channel
(22) to flow back and forth from one reservoir to the other in direct contact with
the material sample (40); and
performing an analysis of one or both of the material sample (40) and the blood sample
after the material sample (40) and the blood sample have interacted in the apparatus
(10).
11. The method of claim 10, wherein the step of providing a material sample (40) comprises
preheating the material sample (40) to a temperature between 35°C and 42°C, preferably
between 36°C and 38°C.
12. The method of any of claims 10 to 11, further comprising prior to step b) a step of
preparing the blood sample by mixing blood with an anticoagulant and/or a step of
measuring a cell concentration of the blood sample.
13. The method of any of claims 10 to 12, wherein the step of operating the driving arrangement
(30) comprises configuring said driving arrangement (30) to provide the reciprocating
movement of the at least one test chamber (20) with a reciprocating frequency between
2 and 50 rpm and a maximum tilt angle between 1 and 20 degrees or a maximum height
of the reservoirs (21) that corresponds to such maximum tilt angle, and/or wherein
the step of operating the driving arrangement (30) comprises configuring said driving
arrangement (30) to operate for a period of time between 1 and 240 minutes, preferably
between 60 and 90 minutes.
14. The method of any of claims 10 to 13, wherein the step of performing an analysis of
the material sample (40) comprises a platelet adhesion test and/or a scanning electron
microscopy analysis.
15. The method of any of claims 10 to 14, wherein the step of performing an analysis of
the blood sample comprises a platelet deprivation test and/or a hemolysis test.