U.S. patent application number 14/599641 was filed with the patent office on 2021-03-18 for multilayered blood product.
The applicant listed for this patent is Reapplix ApS. Invention is credited to Niels Erik HOLM, Rasmus LUNDQUIST.
Application Number | 20210077533 14/599641 |
Document ID | / |
Family ID | 1000005430064 |
Filed Date | 2021-03-18 |
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United States Patent
Application |
20210077533 |
Kind Code |
A9 |
LUNDQUIST; Rasmus ; et
al. |
March 18, 2021 |
MULTILAYERED BLOOD PRODUCT
Abstract
A blood product (10), a method for preparing the blood product,
a blood product obtainable by the method and a blood product
preparing container means. The blood product comprises components
from whole blood, especially fibrin, thrombocytes and leukocytes.
The blood product (10) comprises a first layer (21), a second layer
(22) and a third layer (23). The second layer (22) is adjacent to
the first layer (21) and the third layer (23). The first layer (21)
defines a first outer surface (24) of the blood product (10) and
the third layer (23) defining a second outer surface (25) of the
blood product (10). The first layer (21) comprises a majority of
fibrin, the second layer (22) comprises a majority of thrombocytes
and the third layer (23) comprises a majority of leukocytes.
Inventors: |
LUNDQUIST; Rasmus;
(Bronshoj, DK) ; HOLM; Niels Erik; (Birkerod,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Reapplix ApS |
Birkerod |
|
DK |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150132739 A1 |
May 14, 2015 |
|
|
Family ID: |
1000005430064 |
Appl. No.: |
14/599641 |
Filed: |
January 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13058954 |
Feb 14, 2011 |
8980301 |
|
|
PCT/DK2009/050209 |
Aug 24, 2009 |
|
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14599641 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0644 20130101;
A61K 35/19 20130101; A61K 35/15 20130101; A61K 35/33 20130101; C12N
2501/905 20130101; A61K 38/36 20130101; C12N 5/0642 20130101 |
International
Class: |
A61K 35/19 20060101
A61K035/19; A61K 35/33 20060101 A61K035/33; A61K 38/36 20060101
A61K038/36; A61K 35/15 20060101 A61K035/15; C12N 5/078 20060101
C12N005/078; C12N 5/0787 20060101 C12N005/0787 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2008 |
DK |
PCT/DK2008/000299 |
Claims
1. A method for preparing a blood product (10) from a volume of
whole blood (5), the method comprising the following steps: a)
placing the volume of whole blood (5) in a container means (1), the
container means (1) comprising a first material defining an inner
surface (3) in which the whole blood (5) is in contact with, b)
activating coagulation of the whole blood (5), c) separating the
whole blood (5) into erythrocytes (11), serum (9) and blood product
(10) by a centrifugal force (6) acting on the whole blood (5)
placed in the container means (1), whereby the whole blood (10)
separates into layers comprising erythrocytes (11), blood product
(10) and serum (9) due to the differences in densities between the
erythrocytes (11), blood product (10) and serum (9), the blood
product (10) comprising fibrin, leukocytes and thrombocytes, the
applied centrifugal force (6) being at least 1000 times greater
than the gravity force, e.g. g, acting on the whole blood, the
applied centrifugal force (6) being of such a value and time that
the components in the blood are separated into a 3 layers blood
product (10), and d) removing the blood product (10) from the
container means (1).
2. The method according to claim 11, wherein the centrifugal force
is applied for at least 30 seconds.
3. The method according to claim 11, wherein the coagulation in
step b) is activated by the first material defining the inner
surface (3).
4. The method according to claim 11, wherein the coagulation in
step b) is activated by exposing the whole blood (5) to an object
(7), wherein the object (7) is a glass bead.
5. The method according to claim 11, wherein the first material of
the inner surface (3) of the container means (1) is chosen from a
group consisting of polypropylene, polyethylene, polycarbonate,
polyamide, acrylonitrile butadiene styrene, styrene, modified
styrene, polyurethane and other polymer materials.
6. The method according to claim 11, wherein the inner surface (3)
of the container means (1) is surface treated, by coating to lower
friction between the blood product (10) and the inner surface (3)
of the first material.
7. The method according to claim 11, wherein the centrifugal force
(6) is greater than an adhesive force acting between the inner
surface (3) and the blood product (10).
8. The method according to claim 11, wherein the blood product (10)
adhering to the inner surface (3) is detached from the inner
surface (3), at least once, during step c).
9. The method according to claim 11, wherein the method further
comprises a compacting step, where the blood product (10) is
compacted by a compacting means (8), wherein the compacting means
(8) is a filter, placed in the container means (1).
10. The method according to claim 11, wherein the method further
comprises an isolation step, where the erythrocytes (11) are
isolated from the blood product (10) during step c).
11. The method according to claim 11, wherein the method further
comprises a washing step where the blood product (10) is washed, so
substantially all erythrocytes (11) and/or serum (9) attached to
the blood product (10) are detached.
12. The method according to claim 11, wherein step b) precedes step
a).
13. The method according to claim 12, wherein step b) occurs at
least 1 minute before step a).
14. The method according to claim 11, wherein step b) occurs
concurrently with step c).
15. The method according to claim 11, wherein a first substance
chosen from a group consisting of fibroblasts, keratinocyte cells
and hyaluronic acid is added to the whole blood.
16. The method according to claim 9, wherein the compacting means
(8), has a first fixed position and a second position.
17. The method according to claim 16, wherein the compacting means
(8), is fixed in the first fixed position by a deformation in the
container means (1) wall.
18. The method according to 11, wherein plasma comprising fibrin
and a buffy coat comprising leukocytes and thrombocytes are used
instead of whole blood (5).
Description
[0001] This is a Divisional Application of U.S. patent application
Ser. No. 13/058,954, filed Feb. 14, 2011, an application filed as a
national stage under 371 of Application No. PCT/DK2009/050209 filed
Aug. 24, 2009, the content of each of which is hereby incorporated
by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to a multilayered blood
product, a method for preparing the blood product, a blood product
obtainable by the method and a blood product preparing container
means.
BACKGROUND ART
[0003] The human coagulation system is able to stop bleeding and
initiate healing. The function of the system is well known and
extensively investigated. However, the importance of coagulation
products in the initiation of healing has only been recognized
recently.
[0004] Blood products, such as fibrin sealants and platelets
concentrates, are produced by isolating the platelet rich plasma
(PRP) from anti-coagulated whole blood. The presence of platelets
and plasma partly imitates the natural human coagulation system
upon thrombin activation. This leads to a platelet containing
autologous concentrate of growth promoting factors in a fibrin
matrix. Such a composition can be used for covering wound surfaces
and is claimed to initiate healing.
[0005] "Platelet-rich fibrin (PRF): A second-generation platelet
concentrate. Part I: Technological concepts and evolution, Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 101:E37-44" by
David M. Dohan et al describes how to prepare a platelet rich solid
fibrin network from whole blood without adding any additives or
reagents. The PRF protocol is: A blood sample is taken without
anticoagulant in 10-mL glass tubes or glass coated plastic which
are immediately centrifuged at approximately 400 g for 10 minutes.
The absence of anticoagulant implies the activation in a few
minutes of most platelets of the blood sample in contact with the
glass tube walls and the release of the coagulation cascades.
Fibrinogen is initially concentrated in the top part of the tube,
before the circulating thrombin transforms it into fibrin. A fibrin
clot is then obtained in the middle of the tube, extending from the
upper part of the red corpuscles at the bottom of the tube to the
acellular plasma at the top. Platelets are trapped massively in the
fibrin meshes. The success of this technique entirely depends on
the speed of blood collection and transfer to the centrifuge.
Indeed, without anticoagulant, the blood samples start to coagulate
almost immediately upon contact with the tube glass, and it takes a
minimum of a few minutes of centrifugation to concentrate
fibrinogen in the middle and upper part of the tube. Quick handling
is the only way to obtain a clinically usable PRF clot. If the
duration required to collect blood and launch centrifugation is
overly long, failure will occur: The fibrin will polymerize in a
diffuse way in the tube and only a small blood clot without
consistency will be obtained. In conclusion, the PRF protocol makes
it possible to collect a fibrin clot charged with serum and
platelets. By removing the clot from the tube, manually cutting of
the red cells part, and manually driving out the fluids trapped in
the fibrin matrix (serum), practitioners will obtain autologous
fibrin membranes.
[0006] However this fibrin network includes a red thrombus
containing a substantial part of red blood cells, which have to be
manually cut off. Furthermore the components of the produced fibrin
network, such as fibrin, leukocytes and thrombocytes, are arbitrary
distributed and enmeshed within the product. The recovery of
leukocytes are not described and at the low g force used, the
recovery of leukocytes is low as some will be located in the red
cell part. The enmeshment of cells within the fibrin leads to
absent or slow release of these cells and thereby inhibits the
contact-dependent anti-microbicidal potential of the included
leucocytes.
[0007] "Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;
101:E37-44 and 2006; 101:E45-50" by Dohan et al describes a network
that does not represent a platelet concentrate in a shape and
structure, which is directly applicable for covering wound
surfaces. To obtain a shape and form/rigidity useable for covering
wound surfaces and prevent red blood cell inclusion, the known
platelet rich solid fibrin network will have to be reshaped
manually and compressed. Furthermore, the method comprises several
steps and cannot be prepared in one closed system and is therefore
not convenient for clinical use.
[0008] "Cell separation in the buffy coat. Biorheology. 1988;
25(4):663-73" by Sutton et al describes how anti-coagulated full
blood will separate into several layers upon centrifugation or
passive sedimentation; Red blood cells, leukocytes and platelets
(=buffycoat) and plasma. Further, by using centrifugation force of
10000 g for 10 minutes and using a float of density of 1.053, the
buffy coat can be fixed by Glutaraldehyde, and removed for
investigation; however, this cannot be used clinically due to the
toxicity of the Glutaraldehyde. Several methods for extracting the
buffycoat from anti-coagulated blood exists including the use of
density defined substances (ie. Lymphoprep). In addition to the
need for anti-coagulated blood the extracted cells will be
suspended--and mixed (disorganized)--in the plasma that inevitably
will be included.
[0009] EP 1637145A describes a method of filtration of cells from a
suspension (eg. blood cells including platelets and leukocytes)
through a sheet like porous membrane, leaving the cells in the
membrane as described. The sheet porous material can be prepared
from fibrin. However, no layered structure is obtained, the cell
are trapped in depth in the porous material and the use of
allogeneic fibrin raises the risk of cross infection from other
humans. Furthermore, the method comprises several steps and cannot
be prepared in one closed system and is therefore not convenient
for clinical use.
[0010] Known methods are limited in their use, especially clinical
use. The addition of anticoagulants prior to cell separation lead
to products not completely autologous, furthermore the release of
substances promoting wound healing (e.g. growth factors) requires
mixing with other non-autologous substances (e.g. thrombin,
Ca.sub.2.sup.+ etc.) leading to homogenous final products without
the desired distribution of cells.
[0011] Known methods excluding anti-coagulation lead to a
disorganized distribution of cells, the cells are locked inside the
product, effectively limiting the release and potential of these
cells. Furthermore these methods need manual handling outside a
closed system to obtain a product physically suitable for clinical
use, an inadequately defined handling that leads to a variable
outcome with a lower than optimal cell yield. Furthermore, the
manual handling will require labor time (cost) and prolong the
preparation time.
[0012] Methods describing a well defined layered structure depend
on anti-coagulation and addition of toxic components, not suitable
for clinical use, for the fixation and self-sustainability of the
structure obtained.
DISCLOSURE OF INVENTION
[0013] The object of the invention is to provide a new and improved
blood product which overcomes or ameliorates at least one of the
disadvantages of the prior art or which provides a useful
alternative. The object of the invention is furthermore to provide
a new and improved method for obtaining the blood product which
overcomes or ameliorates at least one of the disadvantages of the
prior art or which provides a useful alternative.
[0014] The object of the invention is obtained by a blood product
comprising components from whole blood, especially fibrin,
thrombocytes and leukocytes, the blood product comprising a first
layer, a second layer and a third layer, the second layer being
adjacent to the first layer and the third layer, the first layer
defining a first outer surface of the blood product and the third
layer defining a second outer surface of the blood product, the
first layer comprising a majority of fibrin, the second layer
comprising a majority of thrombocytes and the third layer
comprising a majority of leukocytes. Hereby, a blood product with a
multilayered structure is provided, each layer provides different
functionality due to different composition of each of the layers.
The blood product is self-supporting, compact and solid, and the
blood product has a structure, such that the blood product is
directly applicable for the intended use. By majority is meant,
that a component, such as fibrin, thrombocytes or leukocytes,
comprises at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, 99% or even 100%, or any interval that can be defined
from combinations of these mentioned percentages, volume and/or
mass wise of the respective layer and/or volume- and/or mass wise
of the blood product. The first, the second and the third layer are
each continuous and/or substantially parallel to each other, e.g.
forming a stratified and/or multilayered blood product.
[0015] The blood product preferably consists of three layers, e.g.
a first layer comprising a majority of fibrin, a second layer
comprising a majority of thombocytes and a third layer comprising a
majority of leukocytes. The blood product has preferably a maximum
width/thickness-ratio of 1, 2, 3, 4 or 5, however the ratio may be
up to 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or even up to 100,
where the width is measured along the layers of the blood product
and the thickness is measured perpendicular to the layers of the
blood product.
[0016] In another aspect of the invention, the blood product solely
consists of components from whole blood. Hereby, a blood product is
provided that solely consists of components from whole blood,
meaning that no additives are added to the whole blood and/or the
blood product, and that the blood product is directly derivable
from whole blood, fractions of whole blood or combinations of
fractions of whole blood.
[0017] In another aspect of the invention, the blood product is
autologous.
[0018] In another aspect of the invention, the blood product is
flexible. Hereby, a blood product is provided that can withstand
applied stress during normal use without rupturing. Due to the
flexibility of the blood product, the blood product conforms most
continuous contours whereto the blood product is applied.
[0019] In another aspect of the invention, the blood product
further comprises a first substance chosen from a group comprising
fibroblasts, keratinocyte cells and hyaluronic acid. Hereby, a
blood product is provided which includes additional cells known to
be important for skin regeneration and thereby further improves the
healing potential of chronic wounds, especially wounds in areas
with low or unviable adjacent tissue. Hyaluronic acid, a known
component of skin, has the potential to increase the water binding
capacity of the blood product as well as increase the potential for
incorporation/infiltration of the blood product in areas of tissue
loss.
[0020] The invention also relates to, a blood product for
therapeutic use and/or use of a blood product according to the
aforementioned for therapeutic use.
[0021] The invention also relates to use of a blood product for
manufacturing of a medicament for therapeutic use.
[0022] The invention also relates to a blood product for treatment
of a wound and/or use of a blood product according to the
aforementioned for treatment of a wound.
[0023] The invention also relates to use of a blood product for
manufacturing of a medicament for treatment of a wound. Hereby, a
blood product is provided which is particular suitable for
manufacturing of a medicament for treatment of a wound. By applying
the second outer surface defined by the third layer against the
wound, the wound is kept and/or maintained substantially sterile,
e.g. free of infection, as the third layer comprises a majority of
leukocytes, which are the first active cells and thus controls
infection and attracts other cells including macrophages, while the
second layer comprises a majority of thrombocytes which comprises
growth promoting factors that stimulates the fibroblast cells,
while the first layer of the blood product comprises a majority of
fibrin, and thus the first outer surface of the blood product
provides an effective protection against contamination from the
surroundings; the first layer furthermore comprises growth
promoting factors, which is released over time. By applying the
second outer surface defined by the third layer against the wound,
the wound is kept and/or maintained substantially free of
infection, as the third layer comprises a majority of leukocytes
which are easily released from the product. Leukocytes are cells of
the immune system defending the body against infection and foreign
bodies, thus they control infection and further attracts other
cells including macrophages. The second layer comprises a majority
of thrombocytes which comprises growth promoting factors that
stimulates the cells. As the leukocytes quickly will be released
from the product, the second layer will face the wound surface for
optimal delivery of growth promoting substances to the wound. The
first layer of the blood product comprises a majority of fibrin,
and thus the first outer surface of the blood product provides an
effective protection against contamination from the surroundings;
the first layer furthermore comprises growth promoting factors,
which is released over time.
[0024] The invention also relates to a blood product for autologous
use and/or use of a blood product according to any of claims 1-7
for autologous use.
[0025] The invention also relates to use of a blood product for
manufacturing of an autologous medicament.
[0026] The invention also relates to a blood product for surgical
use, e.g. to seal of an area, to prevent post surgical adherence
and/or use in anastomosis procedures and/or use of a blood product
according to the aforementioned for surgical use.
[0027] The invention also relates to use of a blood product for
manufacturing of a medicament for surgical use.
[0028] In another aspect of the invention, the blood product is for
anastomosis. Use of a blood product obtainable by a method
according to the aforementioned for anastomosis.
[0029] In another aspect of the invention, the blood product is
used for manufacturing of a medicament for anastomosis.
[0030] The invention relates also to a method for preparing a blood
product from a volume of whole blood, the method comprising the
following steps: a) placing the volume of whole blood in a
container means, the container means comprising a first material
defining an inner surface in which the whole blood is in contact
with, b) activating coagulation of the whole blood, c) separating
the whole blood into erythrocytes, serum and blood product by a
centrifugal force acting on the whole blood placed in the container
means, whereby the whole blood separates into layers comprising
erythrocytes, blood product and serum due to the differences in
densities between the erythrocytes, blood product and serum, the
blood product comprising fibrin, leukocytes and thrombocytes, the
applied centrifugal force being at least 1000 times greater than
the gravity force, e.g. g, acting on the whole blood, the
centrifugal force varying inversely with the time of the
centrifugation, and d) removing the blood product from the
container means. Hereby, a method is provided whereby a blood
product is derivable from whole blood. The method can be processed
in one cycle in a closed system, since there is no need for an
isolation of the erythrocytes; however the method can also be
performed using an isolation of the erythrocytes.
[0031] The yield of the method for extracting fibrin from the whole
blood is at least above 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 97%, 98%, 99% or even 100%, while the yield of the method
for extracting leukocytes from the whole blood is at least above
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or
even 100%, while the yield of the method for extracting
thrombocytes from the whole blood is at least above 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or even 100%. The
obtainable blood product has a volume less than 30%, 20%, 15%, 10%
or even less than 5% of the volume of the whole blood. Hereby, a
method is provided whereby a blood product is obtainable by e.g.
centrifugation giving rise to a centrifugal force. The applied
centrifugal force is at least 1000, 2000, 3000, 4000, 5000, 6000,
7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000,
17000, 18000, 19000 or even 20000 times greater than the gravity
force, e.g. g, acting on the whole blood, or the applied
centrifugal force is within any interval that can be defined from
combinations of the mentioned numbers. The centrifugal force is
applied for at least 30 seconds, 40 seconds, 50 seconds, 60
seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes,
12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17
minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22
minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27
minutes, 28 minutes, 29 minutes or 30 minutes, or the centrifugal
force is applied within any interval that can be defined from
combinations of the mentioned numbers.
[0032] In one aspect of the invention, the yield of the method for
extracting fibrin from the whole blood is at least above 60%.
[0033] In one aspect of the invention, the yield of the method for
extracting leukocytes from the whole blood is at least above
50%.
[0034] In one aspect of the invention, the yield of the method for
extracting thrombocytes from the whole blood is at least above
60%.
[0035] In one aspect of the invention, the centrifugal force is
applied for at least 30 seconds.
[0036] In one aspect of the invention, the blood product solely
consists of components from whole blood. Hereby, a method is
provided whereby a blood product is derivable from whole blood,
thus solely consisting of components from whole blood. Thus the
method is performed without adding any additives to the whole blood
and/or the blood product.
[0037] In another aspect of the invention, the coagulation in step
b) is activated by the first material defining the inner surface.
Hereby, a method is provided in which the coagulation is initiated
when the whole blood is brought in contact with the inner surface,
thus it can be avoided to add any object or the like to the whole
blood to initiate coagulation.
[0038] In another aspect of the invention, the coagulation in step
b) is activated by exposing the whole blood to an object, such as a
glass bead. Hereby, a method is provided in which the coagulation
is initiated when the whole blood is brought in contact with the
object added to the whole blood. Hereby, the coagulation can be
initiated at a chosen point of time, which is optimal for the
method.
[0039] In another aspect of the invention, the first material of
the inner surface of the container means is chosen from a group
comprising of polypropylene, polyethylene, polycarbonate,
polyamide, acrylonitrile butadiene styrene, styrene, modified
styrene, polyurethane and other polymer materials. The polymers in
the mentioned group can furthermore be glass-filled. Hereby, a
method is provided where a container means with an inner surface of
a first material can be typical test tubes or the like made from
all kinds of polymers, metal or glass. The material can also be
chosen so the material property provides a minimal adhesive
force/friction between the blood product and the inner surface.
Polyamide and polyurethane are preferred as these materials
initiates coagulation within a preferred level of activation which
is higher than the level obtained using other polymers.
[0040] In another aspect of the invention, the inner surface of the
container means is surface treated, e.g. coated, in order to lower
friction between the blood product and the inner surface of the
first material. Hereby, a method is provided where a container
means with an inner surface of a first material can be typical test
tubes or the like made from all kinds of polymers, metal or glass.
The inner surface can be surface treated and/or coated to obtain a
minimal adhesive force/friction between the blood product and the
inner surface.
[0041] In another aspect of the invention, the centrifugal force is
greater than an adhesive force acting between the inner surface and
the blood product. Hereby, a method is provided where the
centrifugal force is dominant as compared to the adhesive force,
which secures a well defined layered structure of the blood
product. The centrifugal force can be at least 10, 100, 1000, 5000,
10000, 20000, 50000, 100000, 1000000 or even 10000000 times greater
than the adhesive force, or the centrifugal force is within any
interval that can be defined from combinations of the mentioned
numbers. In another aspect of the invention, the centrifugal force
and centrifugation time is of such strength that the adhesive force
acting between the inner surface and the fibrin is broken/released
and thereby allowing the fibrin layer to be compacted/compressed.
Hereby, a method is provided where the centrifugal force is
dominant as compared to the adhesive force, which secures a well
defined layered structure of the blood product. The centrifugal
force needed to release the adhesion to the wall will depend on the
fibrin density. The fibrin density will depend on several factors
including coagulation activation, fibrin concentration, time, etc.
The centrifugation force can be at least 10, 100, 1000, 5000,
10000, 20000, 50000, or even 100000, g, or the centrifugal force is
within any interval that can be defined from combinations of the
mentioned numbers.
[0042] In another aspect of the invention, the blood product
adhering to the inner surface is detached from the inner surface,
at least once, during step c). Hereby, a method is provided where
the possible adhesion of the blood product to the inner surface can
be dealt with by separating the blood product at least once during
step c). The separation can be done by mechanical means such as by
cutting or the like. The compacting of the fibrin can then be
performed at lower g-force as the g-force does not need to release
the fibrin from the wall.
[0043] In another aspect of the invention, the method further
comprises a compacting step, where the blood product is compacted
by a compacting means, such as a filter placed in the container
means. Hereby, a method is provided where the blood product can be
compacted by e.g. a filter. The filter can be placed fixed or
movable in the container means and the filter can be used to
isolate the blood product.
[0044] In another aspect of the invention, the method further
comprises an isolation step, where the erythrocytes are isolated
from the blood product during step c). Hereby, a method is provided
where the erythrocytes are isolated from the serum and the blood
product during the method.
[0045] In another aspect of the invention, the method further
comprises a washing step where the blood product is washed, so
substantially all erythrocytes and/or serum attached to the blood
product are detached. Hereby, a method is provided so the blood
product is substantially clean from other components, that of the
blood product itself. The serum formed during the centrifugation
may be used as a washing fluid.
[0046] In another aspect of the invention, the container means is a
tube comprising an open end, closable by a detachable lid, and a
closed end. Hereby, a method is provided where a standard test tube
or the like can be used to perform the method.
[0047] In another aspect of the invention, step b) precedes step
a). Hereby, the coagulation can be activated before the whole blood
is placed in the container means, thus the container means does not
need to comprise any coagulation activator and/or the whole blood
does not need to comprise a coagulation activator when the whole
blood is placed in the container means. The coagulation can as an
example, be activated during blood drawing by placing glass beads
in the blood drawing tubing, or choosing a tubing material that
will activate blood. Thus it can be avoided to add any object or
the like to the whole blood to initiate coagulation.
[0048] In another aspect of the invention, step b) occurs at least
1 minutes before step a). Hereby a method is provided where very
fast handling is not necessary. Step b) can occur at least 30
seconds, 40 seconds, 50 seconds, 60 seconds, 1 minute, 2 minutes, 3
minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9
minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14
minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes
or even 20 minutes before step a). The level of activation of
coagulation in step b) can also be controlled by the method. This
allows the time between step b) and a) to be prolonged as the cell
separation in step c) has to occur before fibrin levels is
sufficient to inhibit cell separation.
[0049] In another aspect of the invention, step b) occurs
concurrently with step c). Hereby, the coagulation can be activated
at an optimum point of time for the method during step c). The
coagulation can be activated/initiated by a coagulation activator
integrated into the container means, and/or by using a container
material that will activate the blood. The coagulator material can
be an object, such as glass beads, added to the whole blood in the
container means.
[0050] In another aspect of the invention, a first substance chosen
from a group comprising fibroblasts, keratinocyte cells and
hyaluronic acid is added to the whole blood. Hereby, a blood
product is obtainable which includes additional cells known to be
important for skin regeneration and thereby further improves the
healing of chronic wounds, especially wounds in areas with low or
unviable adjacent tissue. Hyaluronic acid, a known component of
skin, has the potential to increase the water binding capacity of
the blood product as well as increase the potential for
incorporation/infiltration of the blood product in areas of tissue
loss.
[0051] In another aspect of the invention, the compacting means,
such as a filter, has a first fixed position and a second position.
The filter is fixed in a position in the part of the container
means containing the erythrocytes during the first part of the
centrifugation where the lecucytes and thrombocytes has been
separated, while the fibrin in the serum has not been compacted.
Provided that the density of the filter is less than that of serum,
a release of the filter will cause the filter to be transferred to
the top of the tube and thereby collecting the leucocyte and
thrombocyte layer and compacting the fibrin layer.
[0052] In another aspect of the invention, the compacting means,
such as a filter, is fixed in the first fixed position by a
deformation in the container means wall. The filter is fixed by
deforming the tube wall. The filter is released by removing the
deformation of the wall.
[0053] In another aspect of the invention, plasma comprising fibrin
and a buffy coat comprising leukocytes and thrombocytes are used
instead of whole blood. Hereby, a method is provided where whole
blood excluding erythrocytes can be used, thus the blood product is
directly derivable from whole blood, fractions of whole blood or
combinations of fractions of whole blood.
[0054] In another aspect of the invention, the method is performed
within 15 minutes. The method is performed within at least 1
minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7
minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes,
13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18
minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23
minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28
minutes, 29 minutes or at least 30 minutes.
[0055] In another aspect of the invention, the blood product
obtained by the method is the blood product according to any of
claims 1-8.
[0056] The invention also relates to a blood product obtainable by
a method according to any of claims 17-41 comprising components
from whole blood, especially fibrin, thrombocytes and leukocytes,
the blood product comprising a first layer, a second layer and a
third layer, the second layer being adjacent to the first layer and
the third layer, the first layer defining a first outer surface of
the blood product and the third layer defining a second outer
surface of the blood product, the first layer comprising a majority
of fibrin, the second layer comprising a majority of thrombocytes
and the third layer comprising a majority of leukocytes. Hereby, a
blood product is obtainable with a multilayered structure; where
each of the layers provides different functionality due to each
layers different composition. The blood product is self-supporting,
compact and solid, and the blood product has a structure, such that
the blood product is directly applicable for the intended use. By
majority is meant, that a component, such as fibrin, thrombocytes
or leukocytes, comprises at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99% or even 100%, or any interval that can
be defined from combinations of these mentioned percentages,
volume- and/or mass wise of the respective layer or volume- and/or
mass wise of the blood product. The first, the second and the third
layer are each continuous and/or substantially parallel to each
other, e.g. forming a stratified and/or multilayered blood product.
The blood product preferably consists of three layers, e.g. a first
layer comprising a majority of fibrin, a second layer comprising a
majority of thombocytes and a third layer comprising a majority of
leukocytes. The blood product has preferably a maximum
width/thickness-ratio of 1, 2, 3, 4 or 5, however the ratio may be
up to 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or even up to 100,
where the width is measured along the layers of the blood product
and the thickness is measured perpendicular to the layers of the
blood product.
[0057] In another aspect of the invention, a majority of the fibrin
comprised in the blood product is comprised in the first layer.
Hereby, a blood product is obtainable where a majority of the
fibrin comprised in the entire blood product is comprised in the
first layer.
[0058] In another aspect of the invention, a majority of the
thrombocytes comprised in the blood product is comprised in the
second layer. Hereby, a blood product is obtainable where a
majority of the thrombocytes comprised in the entire blood product
is comprised in the second layer.
[0059] In another aspect of the invention, a majority of the
leukocytes comprised in the blood product is comprised in the third
layer. Hereby, a blood product is obtainable where a majority of
the leukocytes comprised in the entire blood product is comprised
in the third layer.
[0060] In another aspect of the invention, the blood product solely
consists of components from whole blood. Hereby, a blood product is
obtainable that solely consists of components from whole blood,
meaning that no additives are added to the whole blood and/or the
blood product, and the blood product is directly derivable from
whole blood, fractions of whole blood or combinations of fractions
of whole blood.
[0061] In another aspect of the invention, the blood product is
autologous.
[0062] In another aspect of the invention, the blood product is
flexible. Hereby, a blood product is obtainable that can withstand
applied stress during normal use without rupturing. Due to the
flexibility of the blood product, the blood product conforms most
continuous contours whereto the blood product is applied.
[0063] In another aspect of the invention, the blood product
further comprises a first substance chosen from a group comprising
fibroblasts, keratinocyte cells and hyaluronic acid. Hereby, a
blood product includes additional cells known to be important for
skin regeneration and thereby further improves the healing of
chronic wounds, especially wounds in areas with low or unviable
adjacent tissue. Hyaluronic acid, a known component of skin, has
the potential to increase the water binding capacity of the blood
product as well as increase the potential for
incorporation/infiltration of the blood product in areas of tissue
loss.
[0064] In another aspect of the invention, the blood product is for
therapeutic use. Use of a blood product obtainable by a method
according to the aforementioned for therapeutic use.
[0065] In another aspect of the invention, the blood product is
used for manufacturing of a medicament for therapeutic use.
[0066] In another aspect of the invention, the blood product is for
treatment of a wound. Use of a blood product obtainable by a method
according to the aforementioned for treatment of a wound.
[0067] In another aspect of the invention, the blood product is
used for manufacturing of a medicament for treatment of a wound.
Hereby, a blood product is obtainable which is particular suitable
for manufacturing of a medicament for treatment of a wound. By
applying the second outer surface defined by the third layer
against the wound, the wound is kept and/or maintained
substantially sterile, e.g. free of infection, as the third layer
comprises a majority of leukocytes, which are the first active
cells and thus controts infection and attracts other cells
including macrophages, while the second layer comprises a majority
of thrombocytes which comprises growth promoting factors that
stimulates wound healing and/or granulation, while the first layer
of the blood product comprises a majority of fibrin, and thus the
first outer surface of the blood product provides an effective
protection against contamination from the surroundings; the first
layer furthermore comprises growth promoting factors, which is
released over time. By applying the second outer surface defined by
the third layer against the wound, the wound is kept and/or
maintained substantially free of infection, as the third layer
comprises a majority of leukocytes which are easily released from
the product. Leukocytes are cells of the immune system defending
the body against infection and foreign bodies, thus they control
infection and further attracts other cells including macrophages.
The second layer comprises a majority of thrombocytes which
comprises growth promoting factors that stimulates the cells. As
the leukocytes quickly will be released from the product, the
second layer will face the wound surface for optimal delivery of
growth promoting substances to the wound. The first layer of the
blood product comprises a majority of fibrin, and thus the first
outer surface of the blood product provides an effective protection
against contamination from the surroundings; the first layer
furthermore comprises growth promoting factors, which is released
over time.
[0068] In another aspect of the invention, the blood product is
used for autologous use. Use of a blood product obtainable by a
method according to the aforementioned for autologous use.
[0069] In another aspect of the invention, the blood product is
used for manufacturing of an autologous medicament.
[0070] The invention also relates to a blood product for surgical
use, e.g. to seal of an area, to prevent post surgical adherence
and/or use in anastomosis procedures and/or use of a blood product
according to the aforementioned for surgical use.
[0071] In another aspect of the invention, the blood product is for
anastomosis. Use of a blood product obtainable by a method
according to the aforementioned for anastomosis.
[0072] In another aspect of the invention, the blood product is
used for manufacturing of a medicament for anastomosis.
[0073] The invention also relates to a blood product preparing
container means for preparing a blood product according to any of
claims 1-8, wherein the blood product preparing container means
comprises polyamide and/or polyurethane. Hereby, a blood product
preparing container means is provided which activates the
coagulation of whole blood, or more specific provides a complement
activation, and at the same time comprises a polymer material,
which is preferred over glass containers, due to the polymers
fragility and/or costs, and other polymere containers due to their
coagulation-inactive properties. The fact that polyamide and/or
polyurethane activates the coagulation of whole blood is surprising
and provides a better alternative to the typical known glass
container means and coagulation-inactive polymere container means,
as the blood product preparing container means combines the
advantages of the known glass container means and polymere
container means.
[0074] The invention also relates to use of a blood product
preparing container means according to claim 58 for manufacturing
of a blood product according to any of claims 1-8.
[0075] The invention also relates to a blood product preparing
container means, wherein the blood product preparing container
means comprises polyamide and/or polyurethane. Hereby, a blood
product preparing container means is provided which activates the
coagulation of whole blood, or more specific provides a complement
activation, and at the same time comprises a polymer material,
which is preferred over glass containers, due to the polymers
fragility and/or costs, and other polymere containers due to their
coagulation-inactive properties. The fact that polyamide and/or
polyurethane activates the coagulation of whole blood is surprising
and provides a better alternative to the typical known glass
container means and coagulation-inactive polymere container means,
as the blood product preparing container means combines the
advantages of the known glass container means and polymere
container means.
[0076] The invention also relates to use of a blood product
preparing container means according to claim 60 for manufacturing
of a blood product.
BRIEF DESCRIPTION OF THE DRAWING
[0077] The invention is explained in detail below with reference to
the drawing, in which
[0078] FIG. 1 shows a cross sectional view of a first embodiment of
the container means according to the invention,
[0079] FIG. 2 shows a cross sectional view of a second embodiment
of the container means according to the invention,
[0080] FIG. 3 shows a cross sectional view of a third embodiment of
the container means according to the invention,
[0081] FIG. 4 shows a cross sectional view of a fourth embodiment
of the container means according to the invention,
[0082] FIG. 5 shows a cross sectional view of the first embodiment
of the container means according to the invention,
[0083] FIG. 6 shows a cross sectional view of the second embodiment
of the container means according to the invention,
[0084] FIG. 7 shows a cross sectional view of the third embodiment
of the container means according to the invention,
[0085] FIG. 8 shows a cross sectional view of the fourth embodiment
of the container means according to the invention,
[0086] FIG. 9 illustrates a cross sectional view of an embodiment
of the blood product according to the invention, and
[0087] FIG. 10 shows a cross sectional view of an embodiment of the
blood product according to the invention.
DETAILED DESCRIPTION
[0088] FIGS. 1 to 4 illustrates respectively a first, second, third
and fourth embodiment of a container means 1. The container means 1
in the four embodiments comprises a cavity having an inner surface
3. The cavity being defined by a wall 4, a closed end 12 and an
open end closeable by a lid 2. The container means 1 defines a
volume of whole blood 5 and is closed to the surroundings via the
lid 2 mounted on the open end of the container means 1. The
container means 1 can be made of any material, and the material can
be chosen, so that the material of the container means 1 due to
contact with the whole blood 5 via the inner surface 3 activates
the coagulation cascade of the whole blood 5, which is the case for
the first embodiment in FIG. 1 and the third embodiment in FIG. 3.
Alternatively, the material of the container means 1 can be chosen
so that the material is inactive in relation to the whole bloods 5
coagulation cascade, which is the case for the second embodiment in
FIG. 2 and the fourth embodiment in FIG. 3, where an object 7, made
of a material that activates the bloods 5 coagulation cascade,
instead is added to the whole blood 5, such that the object 7 is
used to activate the whole bloods 5 coagulation cascade. The inner
surface 3 of the container means 1 in the four embodiments can be
coated and/or surface treated in order to lower the friction
between the inner surface 3 and any components of the blood 5.
Furthermore the material of the container means 1 can be chosen, so
that a low friction between the inner surface 3 and any component
of the blood 5 is obtained, e.g. by choosing a material with a low
protein binding capacity. In the third and fourth embodiment shown
in FIGS. 3 and 4 a compaction means 8, such as a filter, is placed
in the container means 1, whereby the blood product 10 is compacted
against the compaction means.
[0089] The compacting means 8 can be locked in the closed end 12 of
the container means 1, where the erythrocytes are located, in the
initial part of the process. At a later point in time the
compacting means 8 can be released and provided that the density of
the compacting means 8 is lower that the plasma, the compacting
means will be forced to the top of the plasma. By removing the lid
2, the blood product 10 can be removed from the container means 1.
The compaction means 8, or part of the compacting means, can be
used to support the blood product 10 during transport from the
container means 1.
[0090] The whole blood 5 in all four embodiments are subjected to a
centrifugal force 6 acting downwards as illustrated, however the
centrifugal force 6 is not limited to act in the shown direction.
The centrifugal force 6 functions as a separation means, since the
components of the whole blood 5 have different densities and thus
will respond differently to the centrifugal force 6.
[0091] FIGS. 1 to 4 illustrates the four embodiments of the
container means 1 at a point of time where the centrifugal force 6
just been applied, hence no separation of the whole blood 5 is
visible, while FIGS. 5 to 8 respectively shows the first, second,
third and fourth embodiment of the container means 1 at a point of
time where the centrifugal force 6 has been acting sufficiently
long and with sufficiently magnitude, hence the whole blood 5 has
separated into serum 9, with the fibrin distributed through this
serum part, platelets and leucocytes layer 10 and erythrocytes 11.
At a later point in time the compacting means 8 can be released and
provided that the density of the compacting means 8 is lower than
that of the serum, the compacting means will be forced to the top
of the serum and thereby releasing the fibrin from the wall and
compacting the fibrin. By removing the lid 2, the blood product 10
can be removed from the container means 1. The compaction means 8,
or part of the compacting means, can be used to support the blood
product 10 during transport from the container means 1.
[0092] FIG. 9 illustrates a schematic cross sectional view of the
blood product 10 comprising a first layer 21, a second layer 22 and
a third layer 23, where the second layer is adjacent to the first
layer 21 and the third layer 23. The first layer 21 defines a first
outer surface 24 of the blood product 10 and the third layer 23
defines a second outer surface of the blood product 10. The first
layer 21 comprises a majority of fibrin, while the second layer 22
comprises a majority of thrombocytes and the third layer 23
comprises a majority of leukocytes. Thus the blood product 10 has a
well defined multilayer structure with each layer having different
compositions and therefore having different functionalities.
[0093] FIG. 10 shows a cross sectional view of a blood product
obtained experimentally by using a test tube made of polypropylene
as container means.
[0094] The invention has been described with reference to a
preferred embodiment. However, the scope of the invention is not
limited to the illustrated embodiment, and alterations,
combinations and modifications can be carried out without deviating
from the scope of the invention.
EXAMPLES
Example 1
[0095] 1. Take a plastic container (eg. a 2 ml microcentrifuge
tube) and add a coagulation trigger, eg. 4 glass beads (diameter 2
mm) [0096] 2. Draw blood into the plastic container. [0097] 3. Mix
the blood and coagulation activator end-over-end for 2 min. [0098]
4. Spin the container at 16200 g for 20 min. [0099] 5. After this
the blood product will be formed in a layer between the red blood
cells and the serum. [0100] 6. Remove the blood product using
forceps.
[0101] In this method the needed spin time and spin speed will
depend on the power of activation. E.g. if the coagulation
activation is high, the cells must be separated before they are
trapped in the fibrin network, this will require a high spin speed.
Due to the high spin speed, spin time can be low.
Example 2
[0102] 1. Take a standard 50 ml centrifugation tube container and
ad a coagulation trigger, eg. glass beads. [0103] 2. Draw blood
into the centrifugation tube. [0104] 3. Mix the blood in the tube
for 1 min. [0105] 4. Spin the tube at 3000 g for 20 min [0106] 5.
Open the lid and loosen the fibrin (formed in the serum in the top
part) from the wall (using a thin plastic stick or needle) [0107]
6. Spin the sample another 5 min at 3000 g. [0108] 7. After this
the blood product will be formed in a layer between the red cell
and serum. [0109] 8. Remove the blood product using forceps
Example 3
[0109] [0110] 1. Take a 20 ml container (inner diameter: 26 mm)
prepared out of polyamide or polyurethane [0111] 2. Ad a lid and
create a vacuum inside the container [0112] 3. Insert a needle in
the patient [0113] 4. Connect the needle to the container without
losing the vacuum [0114] 5. Draw blood into the plastic container
by the help of the vacuum. [0115] 6. Spin the tube at 15000 g for
12 min [0116] 7. After this the blood product will be formed in a
layer between the red cell and serum. [0117] 8. Remove lid and
remove the blood product with for forceps
Example 4
[0117] [0118] 1. Take a 20 ml container (inner diameter: 26 mm)
prepared out of polyamide or polyurethane [0119] 2. Place a disk in
the bottom of the container. The density of the disk shall be less
than 1, preferable as low as possible. [0120] 3. Fixate the disk in
the bottom of the container, eg compressing the wall of the
container. [0121] 4. Ad a lid and create a vacuum inside the
container [0122] 5. Insert needle in the patient [0123] 6. Connect
the needle to the container without losing the vacuum [0124] 7.
Draw blood into a plastic container by the help of the vacuum.
[0125] 8. Spin the tube at 3000 g for 8 min. At this stage the
leucocytes will be on top of the red blood cells and the fibrin
will be polymerized through the upper serum layer as the g force is
low and not able to compress it on top of the platelets. [0126] 9.
Release the disk in the bottom [0127] 10. Spin the tube at 3000 g
for 2 min. This will cause the disk to move to the top and thereby
collecting the leukocytes and platelets and compress the fibrin
layer into one sheet. [0128] 11. Remove lid and remove the blood
product, that is placed in top of the contain
Example 5
[0128] [0129] 1. Take a 20 ml container (inner diameter: 26 mm)
prepared out of polyamide or polyurethane [0130] 2. Place a disk in
the bottom of the container. The density of the disk shall be less
than 1, preferable as low as possible. [0131] 3. Fixate the disk in
the bottom of the container, eg by compressing the wall of the
container. [0132] 4. Create a vacuum inside the container and ad a
lid [0133] 5. Insert a needle in the patient [0134] 6. Connect the
needle to the container without losing the vacuum [0135] 7. Draw
blood into a plastic container by the help of the vacuum. [0136] 8.
Spin the tube at 3700 g for 3 min. At this stage the leucocytes
will be on top of the red cells and the fibrin will be polymerized
through the upper serum layer as the g force is low and not able to
compress it on top of the platelets. [0137] 9. Wait for 8 minutes.
[0138] 10. Release the disk in the bottom [0139] 11. Spin the tube
at 3000 g for 2 min. This will cause the disk to move to the top
and thereby collecting the leukocytes and platelets and compress
the fibrin layer into one sheet. [0140] 12. Remove lid and remove
the blood product, that is placed in top of the container
[0141] As it appear from the above examples, the combination of
coagulation activation, spin speed (g), spin time, rest time
between spin can be varied within some limits.
[0142] These are illustrated on below tables:
TABLE-US-00001 Coagulation Coagulation Spin 1. Spin time after or
activation speed time during 1 spin 2. Spin Low Low Long Long Yes,
high Low Low Long Long Yes, can be low if fi- brin adhesion to con-
tainer wall is mechan- ical released from wall or adheasion to the
wall is low. Low High Short Long Yes, high High High Long Short No
High High Short Short Yes, high
[0143] If processing with a disk:
TABLE-US-00002 Coagulation Coagulation Spin 1. Spin time after or
2. Spin were the activation speed time during 1 spin disk is
released Low Low Long Long Low Low High Short Long Low High High
Long Short Low High High Short Short Low
Example 6
Optimization of Relative Centrifugation Force and Time
[0144] 1. Full blood was drawn into 6 ml EDTA tubes (Vacutainer,
BD.RTM.). [0145] 2. 20 ml container (inner diameter: 26 mm)
prepared out of polyamide or polyurethane were filled with 18 ml
and spun at different RCFs for different times [0146] 3. Samples
(.about.300 .mu.l) were taken by a syringe at the bottom (approx 5
mm above the bottom) and top (approx 5 mm below the surface) after
the above spin times--afterwards the spin was continued to obtain
the next sample. Bottom samples were diluted 1:1 with D-PBS. [0147]
4. Samples were analyzed by automated cell counting (XE 2100,
Sysmex Corporation) and cell numbers in the original sample
calculated. Results are given in millions of cells per ml.
[0148] Millions of platelets per milliliter in the upper (top) and
lower (bottom) part of blood centrifuged at the given relative
centrifugation force (g) as a function of time (min):
TABLE-US-00003 Minutes 0 1 3 5 7 11 2000 g top 178 480 224 113 59
13 2000 g bottom 178 7 5 3 2 4 3000 g top 178 378 103 38 14 0 3000
g bottom 178 3 2 5 3 1 3700 g top 178 364 97 27 8 0 3700 g bottom
178 2 11 2 1 0
[0149] Millions of Leucocytes per milliliter in the upper (top) and
lower (bottom) part of blood centrifuged at the given relative
centrifugation force (xg) as a function of time (min):
TABLE-US-00004 Minutes 0 1 3 5 7 11 2000 g top 7.43 0.28 0 0 0 0
2000 g bottom 7.43 0.89 0.02 0.13 0.26 0.12 3000 g top 7.43 0.19 0
0 0.01 0 3000 g bottom 7.43 0.07 0.04 0.11 0.02 0.02 3700 xg top
7.43 0.05 0.03 0 0 0 3700 xg bottom 7.43 0.03 0.59 0.07 0.02
0.01
Example 7
Growth Factor Release as a Response to Chronic Wound Fluid
[0150] 1. The blood product was generated by the method given in
example 1. [0151] 2. The blood products was cut in half and placed
in 100 .mu.l PBS1% BSA or 100 .mu.l chronic wound fluid (collected
over 24 hrs from a venous leg ulcer) and incubated at 37 degrees
celcius. [0152] 3. After given time points (1, 2, 3.5, 7, 14, 22
and 29 hours) the samples were spun at 16000 g for 10 min, and the
supernatant transferred to a new tube, added 1/10 th (81 .mu.l
sample+9 .mu.l PI) Protease Inhibitor (Complete.RTM., Roche) and
frozen at -80 degree celcius. [0153] 4. Platelet derived growth
factor--AB levels in were determined by using an ELISA kit
(DuoSet.RTM. ELISA cat. No. DY222, R&D systems) as described by
the manufacturer. PDGF-AB concentrations in the original samples
were calculated.
[0154] Platelet derived growth factor AB (PDGF-AB) release from
blood product (ng/ml blood product) as a function of time
(hrs).
TABLE-US-00005 Time (hours) 1 2 4 7 22 29 Chronic Wound fluid
(control) 0 0 0 0 0 0 Blood product in PBS 153 172 160 174 206 233
Blood product in chronic 426 490 602 497 234 275 wound fluid
Example 8
[0155] 1. The blood product was generated by the method given in
example 1. [0156] 2. Two blood products were incubated in 1 ml DMEM
(PAA, Germany) at 37 C and 5% CO2 for 48 hrs. [0157] 3. In parallel
2 blood products were incubated in 1 ml DMEM (PAA, Germany)
including lipopolysaccharide ((10 ng/ml) (LPS derived from
Escherichia coli; L2654; Sigma-Aldrich) at 37 C and 5% CO2 for 48
hrs. [0158] 4. After 48 hrs the media were transferred to
microcentrifuge tubes and spun 20 min at 16200 xg. The supernatants
were frozen at -80 C until analysis. [0159] 5. Proteome profiler
Arrays ARY007 and ARY005 (both R&D systems) were performed as
described by R&D systems, except that buffer 4 (ARY007) were
used in both kits. 420 ul supernatant were diluted in 500 ul buffer
4 and 580 ul buffer 5 as described by R&D systems. [0160] 6.
Reactivity with arrays were detected using chemiluminescent HRP
substrate (Immobilon Western.TM., Millipore, US). [0161] 7. Light
emission were captured using a Fluorchem 3000 system (Alpha
Innotech, US). Mean pixel density were extracted by the Flourchem
software (Alpha Innotech, US).
TABLE-US-00006 [0161] Blood product + Blood product
Lipopolysaccharide Substance detected (Mean pixel density) (Mean
pixel density) CXCL8 (IL-8) 2605 404 CXCL10 (IP-10) 305.5 48.5
MMP-8 518 214 IL-1ra 1825.5 1117.5 IL-16 171 105 MMP-9 2663.5
1729.5 Angiopoietin-1 676.5 537.5 PDGF-AB/PDGF-BB 851.5 704.5
PDGF-AA 1357 1159.5 CXCL16 300 256.5 TIMP-1 2507.5 2170.5
Endostatin 187 175.5 Angiogenin 689 650.5 MIF 1989 1880.5 IGFBP-2
1942 1883.5 IGFBP-3 444 468 EGF 612.5 670.5 IGFBP-1 291.5 401
sICAM-1 635.5 894.5 PAI-1 1940 2897.5 CCL5 (RANTES) 4104 6151.5
CD26 458.5 828.5 VEGF 246.5 891.5 CXCL1 (GRO-alpha) 336 2236 CCL4
(MIP-1-beta) 7 117.5 CCL2 (MCP-1) 6.5 334 G-CSF 7.5 429.5 IL-1Beta
7 600 IL-6 8 2094.5
* * * * *