Note: Descriptions are shown in the official language in which they were submitted.
CA 02617465 2008-01-28
TRIPLE SPIN. DOUBLE POOL AND REVOLUMIZATION PROCESS FOR
CONCENTRATING PLATELETS AND DERIVATIVE PLATELET
CONCENTRATE
1. FIELD OF THE INVENTION:
[0001] The present invention relates to improved methods of manufacture of
highly
concentrated platelet formulations, for use in wound healing and bone
regeneration, and
in particular dental healing and bone regeneration.
2. BACKGROUND OF THE INVENTION:
[0002] Platelets or thrombocytes are blood cells that play a critical role in
healing and the
generation of new tissue. Platelets promote the formation of blood clots that
act as a
physical barrier at a site of rupture of a blood vessel, thereby preventing
infection and
avoiding further blood loss. Additionally, platelets have the capacity to
stimulate
angiogenesis (formation of new blood vessels) and increase collagen formation,
cell
division and cell growth.
[0003] Enriched platelet concentrates are known to possess advantages in
healing and
formation of new tissue. The use of platelet rich plasma (PRP) in surgical
sites has been
shown to rapidly produce both hard and soft tissue regeneration and repair.
Whitman et
al. 1997. Platelet gel: An autologous alternative to fibrin glue with
applications in oral and
maxillofacial surgery. J. Maxillofac Surg. 55:1294-9. In one example, a linear
relationship
was demonstrated between release of growth factor PDGF-AB and the number of
platelets, where the highest concentration of platelets used was 1.6
million/pl or
approximately 8 times baseline. Marx, Robert E. and Garg, Arun K. 2005. Dental
and
Craniofacial Application of Platelet-Rich Plasma. Quintessence Publishing Co.
Inc. 31-
47, 53-75.
[0004] Consequently, it is desirable to produce platelet concentrates, such as
PRP,
having elevated platelet concentrations. PRP is commonly produced by
centrifuging
whole blood to separate a supernatant of platelet-containing plasma from the
remainder
of the blood components; centrifuging the supernatant to separate platelet
poor plasma
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(PPP) from a precipitate or pellet of highly concentrated platelets; and re-
suspending the
pellet in a small amount of plasma or other approved solution to form a
concentrated
PRP or other platelet concentrate To date, automated centrifuges and other
centrifugation techniques have been commonly used to carry out the extraction
and
concentration of platelets, but such techniques have been limited in their
capacity to
concentrate platelets, and have produced, at best, concentrates having
platelet
concentrations of approximately 8 times baseline, with peak platelet
concentrations of
about 4400 x 103/pl. G. Weibrich et al. Curasan PRP kit vs. PCCS PRP System.
Clin.
Oral Impl. Res. 13: 2002/437 - 443.
[0005] There is a requirement in the art of dental and other wound healing and
bone
regeneration for improved platelet concentrate formulations to further
accelerate wound
healing and bone regeneration.
3. SUMMARY OF THE INVENTION
[0006] The present invention involves highly concentrated platelet
formulations, including
greater than 8 times baseline, preferably approximately 23 times to 45 times
baseline,
and optimally up to approximately 60 times baseline (where baseline is defined
as the
initial concentration of platelets in a sample of whole blood). Platelet
concentrate
solutions of approximately 23 times baseline have been shown by the applicant
in
particular to yield markedly superior results to the current art in dental
wound healing
and bone regeneration. The attainment of such highly concentrated platelet
samples
has not been achievable by conventional techniques.
[0007] In the manufacture of the above highly concentrated platelet
formulations, the
present invention employs a novel series of centrifugation steps in
combination with the
strategic pooling and revolumization of platelet rich substrates to produce a
highly
concentrated platelet concentrate (hcPC) or highly concentrated platelet rich
plasma
(hcPRP) having a platelet concentration greater than 8 times baseline, and
commonly in
the range of approximately 23 times baseline to 45 times baseline, and up to
approximately 60 times baseline.
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[0008] Another aspect of the invention is the concentration of platelets by
means of a
novel serial centrifugation, strategic pooling and revolumization process,
whereby
platelet rich samples isolated from one centrifuge step (whether in a
supernatant or in a
resuspension) are strategically pooled, and redistributed (or "revolumized")
such that
the volume of the extracted platelet rich sample from a prior centrifuge
container is
increased within its subsequent centrifuge container prior to a subsequent
centrifugation
step.
[009] For clinical practicality, such as for use in a dental office, a first
centrifugation (first
spin) of whole blood followed by a first collection, strategic pooling and
revolumization
(first pooling and revolumization) of platelet rich supernatant; thereafter a
second
centrifugation (second spin) followed by a second collection, strategic
pooling and
revolumization (second pooling and revolumization) of platelet rich
precipitate in
suspension; and thereafter a third centrifugation (triple spin) followed by a
decantation
of platelet poor supernatant overlying a highly concentrated platelet rich
precipitate,
yields a highly concentrated grouping of platelets. The further step of re-
suspending the
platelet rich precipitate in a small remainder of the overlying platelet poor
supernatant
gives rise to hcPRP having a platelet concentration of greater than 8 times
baseline, and
commonly approximately 23 to 45 times baseline, and up to approximately 60
times
baseline. In conversion to absolute concentrations, platelet concentrations of
hcPRP of
greater than 4800 x 103/NI - 10000 x 10' /NI, and optimally up to
approximately 15000 x
103/pl are achievable by the current inventive process, which is of greater
concentration
as compared to what has been achievable in the prior art.
[0010] In accordance with the present invention, the essence of the disclosed
process
for concentrating platelets can be summarized in the following general steps:
a. performing a first centrifugation on an anticoagulated sample of blood;
b. extracting and pooling a first platelet rich sample;
c. performing a second centrifugation on said first platelet rich sample,
whereby
the average volume of said first platelet rich sample placed in each
subsequent centrifuge container is greater than the average extracted volume
from each previously utilized centrifuge container;
d. withdrawing and pooling a second platelet rich sample;
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e. performing a third centrifugation on said second platelet rich sample,
whereby
the average volume of said second platelet rich sample placed in each
subsequent centrifuge container is greater than the average previous
extracted volume from each previously utilized centrifuge container; and
f. decanting at least part of a supernatant overlying a third highly
concentrated
platelet rich sample.
[0011] The final step of suspending the third highly concentrated platelet
rich sample,
which is embodied as a pellet, with preferably a small portion of the
overlying
supernatant atop of the third highly concentrated platelet rich sample forms
hcPRP at
concentrations of greater than 8 times baseline, commonly in the range of
approximately
23 to 45 times baseline, and up to approximately 60 times baseline.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 is a side elevation view of a number of first, second and
third containers
following centrifugation, and depicts the transfer of platelet rich plasma
from the first
containers through to the third container.
5. DETAILED DESCRIPTION AND PREFERRED EMBODIMENT
[0013] The present invention relates to a novel, highly concentrated platelet
formulation
and method of manufacturing the same, in view of using such formulation in the
promotion or acceleration of wound healing and bone regeneration, preferably
in the
field of dental surgery.
[0014] The following details the preferred method according to the inventor.
It will be
understood that some of the steps may be practiced in a different order from
which they
are set out below. The order in which the steps appear in this specification
is not
necessarily the order in which they must be performed but indicates the
preferred order.
[0015] In accordance with the preferred embodiment of the present invention,
there is
provided a platelet concentrate having a novel platelet concentration of
greater than 8
times baseline, and commonly in the range of approximately 23 times baseline
to 45
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times baseline, and up to approximately 60 times baseline, as manufactured by
the
following process:
a. placing a sample of whole blood into a first plurality of containers;
b. adding an anticoagulant to at least part of said sample at any point prior
to
initiation of step c;
c. performing a first centrifugation of said sample within said first
plurality of
containers;
d. decanting a first supernatant of platelet rich plasma from each of said
first
plurality of containers, following said first centrifugation;
e. performing a first pooling of said first supernatant;
f. placing said first supernatant once pooled into a second plurality of
containers;
g. performing a second centrifugation on said first supernatant within said
second plurality of containers, such as to form a second supernatant and a
first precipitate within each of said second plurality of containers;
h. decanting at least a portion of said second supernatant from each of said
second plurality of containers;
i. forming a resuspension of said first precipitate;
j. performing a second pooling of said resuspension into at least one
container;
k. performing a third centrifugation on said resuspension within said at least
one
container, such as to form a third supernatant, and a second precipitate; and
1. decanting at least a portion of said third supernatant;
wherein the average volume of said first supernatant within each of said first
plurality of containers is less than the average volume of said first
supernatant within each of said second plurality of containers, and wherein
the average volume of said re-suspension within each of said second plurality
of containers is less than the average volume of said re-suspension within
said at least one container; and
whereby said second precipitate comprises a highly concentrated platelet
concentrate (hcPC).
CA 02617465 2008-01-28
[0016] The creation of hcPRP of concentrations of greater than 8 times
baseline,
commonly in the range of approximately 23 times baseline to 45 times baseline
and up
to approximately 60 times baseline is accomplished by a final step of
preferably
suspending the second precipitate into a small amount of the third overlying
supernatant
or another suitable solution approved by regulatory authorities. hcPRP
concentrations of
greater than 4800 x 103/pl - 10000 x 103 /pl, and up to approximately 15000 x
103/pl,
assuming a typical initial baseline concentration of approximately 220 x
103/pl, are
readily achievable by the disclosed process. Optionally, if a large enough
sample of
whole blood is initially collected, a more concentrated sample of hcPRP can be
obtained
wherein the final pellet of hcPC is mixed to form a suspension with a portion
of the first
supernatant of platelet rich plasma, which may be saved from step (d) above.
[0017] In a particular example of the above method with prescribed volumes,
spin
speeds and centrifugation durations included, the process preferably consists
of the
following steps:
(i) collecting approximately 60 - 120 ml of whole blood treated with an
anticoagulant and dividing it in approximately equal amounts between 4 and
and most preferably 6 centrifuge-adapted first containers;
(ii) centrifuging each first container at a speed consistent with
approximately 100
to 140 g, and most preferably 120g, for approximately 8 to 12 minutes, most
preferably 10 minutes to separate from the blood a first supernatant;
(iii) decanting the first supernatant from each first container into a number
of
second, centrifuge-adapted containers that is less, and preferably half the
number of the first containers (preferably 3), so that each second container
has approximately an equal volume of first supernatant;
(iv) centrifuging each second container at a speed consistent with
approximately
700 to 1100 g, most preferably 900 g for approximately 8 to 12 minutes, most
preferably 10 minutes to separate the first supernatant into a second
supernatant and a first platelet pellet;
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(v) decanting the second supernatant from each second container such that
approximately 1.0 - 2.0, preferably approximately 1.5 ml of the second
supernatant remains therein;
(vi) re-suspending in each second container the first platelet pellet in the
second
supernatant to form a first platelet rich plasma;
(vii) transferring the first platelet rich plasma from each second container
to a
number of third, centrifuge-adapted containers (preferably 1);
(viii) centrifuging the third container at a speed consistent with
approximately 200
to 600g, most preferably 400g for approximately 8 to 12 minutes and
preferably for 10 minutes to separate the first platelet rich plasma into a
third
supernatant and a second platelet pellet;
(ix) decanting the third supernatant from the third container such that up to
approximately 1.0 - 3ml, preferably 2 ml of the third supernatant and the
second platelet pellet remain; and
(x) suspending the second platelet pellet in an amount of approved solution,
preferably approximately 1.0 - 3 ml, preferably 2 ml of the remaining third
supernatant, to form the second, most highly concentrated platelet rich
plasma (hcPRP).
[0018] The invention provides a platelet concentrate or platelet rich plasma
that has a
baseline platelet concentration greater than 8 times baseline, commonly
approximately
23 to 45 times baseline, and up to approximately 60 times baseline, which is a
concentration higher than that found in platelet concentrates or platelet rich
plasma of
the prior art.
[0019] Platelet concentrate, hcPC or hcPRP of a desired concentration may
thereby be
produced by obtaining a prescribed volume of anticoagulant-treated whole blood
and, in
accordance with the select method, centrifuging the blood and derivative
platelet rich
concentrate at the prescribed speed and duration and pooling and re-adjusting
(or
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increasing) the volume of the derivative platelet rich concentrate within
subsequent
centrifuge tubes (or containers) on each re-centrifugation step.
[0020] The steps of manufacture of hcPC or hcPRP as disclosed to the present
invention
preferably occurs in a dental or other equivalent medical office, wherein the
initial whole
blood sample is collected from the patient ("autologously") at the time or
just prior to
dental surgery in which the patient will require superior dental wound
healing. The
product hcPRP is particularly useful in the promotion or acceleration of
osteogenesis, a
vital component of post dental surgical healing, and more particularly in
implant surgery.
[0021] The hcPRP of the present invention is osteoconductive, osteoinductive,
osteogenic and likely chondrogenic. New bone is formed from the hcPRP in
individuals
when the hcPRP, preferably having a platelet concentration of approximately 23
times
baseline, is placed at a surgical site, preferably the sinus. Insertion or
grafting of bone
material is thereby avoided.
[0022] In one example, whole blood is obtained having a typical baseline
platelet
concentration of approximately 213 x 103/pl. Commencing with a typical blood
volume of
60 ml, re-suspending the second platelet pellet in approximately 2 ml of third
supernatant provides a platelet rich plasma with a platelet concentration of
greater than
4800 x 103/NI, which represents an approximately twenty-three-fold increase in
platelet
concentration.
[0023] Figure 1 illustrates centrifugation of first (12), second (14) and
third (16)
containers and pooling of platelet rich plasma from the first through to the
third
containers in accordance with the invention. The preferred centrifuge is an
FDA
approved manual centrifuge. In the preferred embodiment, the centrifuge
accepts at
least 6 containers at a time. The preferred first, second and third containers
are
Vacutainers or similar containers designed for centrifugation.
[0024] In an example of use of the present invention, a number of first
containers of
equal size are selected, preferably 6 as depicted in Figure 1. A volume of
whole blood,
preferably 60 ml, is distributed between the first containers such that each
first container
holds an approximately equal volume of blood. The blood must be treated prior
to
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centrifugation with an anticoagulant, preferably sodium citrate, to reduce
aggregation of
the platelets. The anticoagulant may be added to the blood before or after the
blood has
been transferred to the first containers.
[0025] All the first containers are then simultaneously centrifuged at a
prescribed speed
and duration, preferably at approximately 120g for approximately 10 min. This
separates
the heavier components of the blood, such as red blood cells, from the
platelet rich
plasma, which forms an upper layer or first supernatant that is distinct from
the heavier
components in the sediment layer. At this stage, the platelets remain
suspended in the
plasma of the first supernatant.
[0026] Following completion of the first centrifugation, the first supernatant
in each first
container is decanted or removed from the sediment layer and pooled into a
number of
second containers (of preferably equal size to the first), that is half the
number of the first
containers, preferably three (3). Decanting may be carried out with the aid of
a pipette or
other suitable instrument. Following decanting, each second container holds an
approximately equal portion of the total volume of the first supernatant
derived from the
first containers.
[0027] All the second containers are simultaneously centrifuged at a
prescribed speed
and duration, preferably at approximately 900g for approximately 10 min. In
the
preferred embodiment, a counterweight is used to balance the three (3) second
containers prior to commencing centrifugation. The second centrifugation
causes
formation in each second container of a first platelet pellet and a second
supernatant of
platelet-poor plasma.
[0028] Following completion of the second centrifugation, the second
supernatant in
each second container is decanted by a pipette or other suitable means, such
that a
reduced volume of second supernatant remains. In the preferred embodiment,
approximately 1.5 ml of the second supernatant remains in each second
container
following decanting. The first platelet pellet in each second container in
then re-
suspended in the second supernatant remaining therein, thereby forming a first
platelet
rich plasma. Re-suspension is preferably carried out by aspiration and
ejection repeated
three times. The first platelet rich plasma in each second container is then
pooled into a
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single third container, which for ease of convenience is of equal size to the
other
containers. In the preferred embodiment, this results in approximately 4.5 ml
of first
platelet rich plasma in the third container.
[0029] The third container is then centrifuged at a prescribed speed and
duration,
preferably at approximately 400g for approximately 10 min. In the preferred
embodiment,
a counterweight is used to balance the third container prior to commencing
centrifugation. The third centrifugation causes formation in the third
container of a
second platelet pellet and a third supernatant of platelet-poor plasma.
[0030] Following completion of the third centrifugation, the third supernatant
in the third
container is decanted by a pipette or other suitable means, such that a
reduced volume
of third supernatant remains. In the preferred embodiment, approximately 2 ml
of the
third supernatant remains following decanting.
[0031 ] The second platelet pellet is next suspended in an appropriate
approved solution
of a desired volume to result in a platelet concentrate or platelet rich
plasma with a
desired platelet concentration of approximately 23 times baseline to 45 times
baseline,
and up to approximately 60 times baseline. Preferably, the second platelet
pellet is re-
suspended by aspiration and ejection repeated three times in approximately 2ml
of the
third supernatant remaining in the third container, so as to form hcPRP of
greater than 8
times baseline, commonly in the range of approximately 23 times baseline to 45
times
baseline, and up to approximately 60 times baseline. The resulting hcPC or
hcPRP is
then used for any purposes as are known in the art.
[0032] Preferably, the anticoagulant within the hcPRP is reversed, and
thrombin
(preferably autologously derived or optionally derived from an animal donor)
is utilized to
activate the hcPRP, which is thereafter ready for dental surgical application.
The
activated hcPRP is then preferably applied to a carrier, such as a re-
absorbable collagen
sponge, which can applied and retained on a surgical wound site.
[0033] The present invention also relates to kits comprising materials and
instructions
necessary to practice the present methods. Preferred kits include ones
comprising
instructions for practicing a method in accordance with the present invention,
along with
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a blood collection kit, a centrifuge, or both. A manual centrifuge is
preferred, optimally
with 6 rotor positions. Preferably, the centrifuge has variable speed. A set
of re-
absorbable collagen sponges may be included. Preferably, the above mentioned
instructions take the form of a hcPRP instruction manual.
[0034] While the embodiment disclosed comprises a preferred method of
manufacture of
platelet concentrate performed at the time of a dental surgery, within or
directly
proximate to a dentist's office, and with an autologously obtained blood
sample taken
from a patient, alternatively, it is also possible to concentrate non-
autologously (or
"homologously") obtained donor platelets from a remote location by the
disclosed
methods of the present invention. In this latter alternative embodiment, a
dentist or other
surgeon would purchase donor platelet concentrate and have it shipped from the
remote
location, which may, for example, comprise an independent blood collection and
processing unit. In this alternative method, a patient in need of platelet
concentrate
would have their blood typed, and the appropriate donor hcPC, hcPRP, or other
highly
concentrated platelet concentrate formulation would be obtained and stored
prior to a
planned dental procedure.
[0035] With a larger input sample of whole blood available, as described
earlier, it is also
possible to further concentrate product hcPRP by utilizing saved PRP from a
previous
centrifugation step, as opposed to utilizing PPP derived from the final
centrifugation step
(or other approved non-platelet rich solution), for the final step of re-
suspending the
hcPC pellet to form a useful suspension.
[0036] Furthermore, while the preferred embodiment to the present invention
discloses
a method of manufacturing platelet concentrate performed manually by a medical
practitioner or lab assistant in conjunction with a centrifuge and blood
collection kit
(including the manual steps of extracting supernatant, pooling and
revolumizing, and
suspending supernatant or other approved solution to form a suspension),
alternatively
the disclosed methods could be fully automated by a stand alone centrifuge
system,
which enables the aforementioned triple spin, double pool, and revolumization
process
(to form hcPC), and optionally the final re-suspension process (to form hcPRP,
or other
highly concentrated platelet solution), without substantial need of further
intervention of a
human medical practitioner or equivalent thereof.
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[0037] In this alternative embodiment of automated centrifugation, a
centrifuge would be
adapted to receive an anti-coagulated blood sample from an operator, and then
perform
independently any of the aforementioned steps, enabling formation of hcPC and
optionally hcPRP (or other equivalent platelet rich solution), after which,
the desired
platelet rich sample product would be collected by an operator.
[0038] While specific embodiments of the invention have been described and
illustrated,
such embodiments should be considered illustrative of the invention only, and
not as
limiting the invention as construed in accordance with the accompanying
claims.
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