U.S. patent application number 11/442631 was filed with the patent office on 2006-12-14 for apparatus and method for separating and concentrating fluids containing multiple components.
Invention is credited to Jennifer E. Woodell-May.
Application Number | 20060278588 11/442631 |
Document ID | / |
Family ID | 46324551 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060278588 |
Kind Code |
A1 |
Woodell-May; Jennifer E. |
December 14, 2006 |
Apparatus and method for separating and concentrating fluids
containing multiple components
Abstract
An apparatus that allows for separating and collecting a
fraction of a sample. The apparatus, when used with a centrifuge,
allows for the creation of at least three fractions in the
apparatus. It also provides for a new method of extracting the
buffy coat phase from a whole blood sample. A buoy system that may
include a first buoy portion and a second buoy member operably
interconnected may be used to form at least three fractions from a
sample during a substantially single centrifugation process.
Therefore, the separation of various fractions may be substantially
quick and efficient.
Inventors: |
Woodell-May; Jennifer E.;
(Warsaw, IN) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 828
BLOOMFIELD HILLS
MI
48303
US
|
Family ID: |
46324551 |
Appl. No.: |
11/442631 |
Filed: |
May 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10932882 |
Sep 2, 2004 |
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11442631 |
May 26, 2006 |
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10445381 |
May 23, 2003 |
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10932882 |
Sep 2, 2004 |
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60383013 |
May 24, 2002 |
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Current U.S.
Class: |
210/787 ; 435/2;
604/82 |
Current CPC
Class: |
B01L 1/52 20190801; B01L
2300/0681 20130101; B01L 2400/0478 20130101; A61K 35/28 20130101;
B01L 3/502 20130101; A61K 35/28 20130101; G01N 33/491 20130101;
B01L 2400/0605 20130101; A61K 35/19 20130101; A61K 2300/00
20130101; A61K 35/14 20130101; B01L 2200/026 20130101; A61K 2300/00
20130101; B01L 3/50215 20130101; A61K 35/17 20130101; B01D 21/262
20130101; B01L 2400/0409 20130101; A61K 35/14 20130101; B01D 21/307
20130101; B01L 3/5021 20130101; B01L 9/54 20130101 |
Class at
Publication: |
210/787 ;
604/082; 435/002 |
International
Class: |
C02F 1/38 20060101
C02F001/38 |
Claims
1. A method for concentrating bone marrow aspirate, the method
comprising: obtaining a volume of bone marrow aspirate from a
mammal; loading the volume of bone marrow aspirate into a separator
comprising two buoys, the separator operable to separate the
aspirate into three or more fractions; centrifuging the separator
creating at least one fraction comprising a concentrated bone
marrow aspirate; and extracting the at least one fraction
comprising a concentrated bone marrow aspirate.
2. The method according to claim 1 wherein the at least one
fraction comprises at least one of the group consisting essentially
of hematopoietic stem cells, stromal stem cells, mesenchymal stem
cells, endothelial progenitor cells, red blood cells, white blood
cells, fibroblasts, reticulacytes, adipose cells, and endothelial
cells.
3. The method according to claim 1 wherein an amount of nucleated
cells in the at least one fraction is greater than or equal to 4
times an amount of nucleated cells found in the volume of bone
marrow aspirate.
4. The method according to claim 1 further comprising applying the
at least one fraction into a patient.
5. A method for concentrating bone marrow aspirate and peripheral
blood, the method comprising: collecting a volume of bone marrow
aspirate and a volume of peripheral blood from a patient; loading
the volume of bone marrow aspirate and the volume of peripheral
blood into a separator comprising two buoys, separator being
operable to separate a combination of the volume of aspirate and
the volume of blood into three or more fractions; centrifuging the
at least one separator operably creating at least one fraction
comprising a concentration of at least one of bone marrow aspirate
and peripheral blood; and withdrawing the at least one fraction
comprising the concentration.
6. The method according to claim 5 wherein the concentration
comprises at least one of the group consisting essentially of
hematopoietic stem cells, stromal stem cells, mesenchymal stem
cells, endothelial progenitor cells, red blood cells, white blood
cells, fibroblasts, reticulacytes, adipose cells, and endothelial
cells.
7. The method according to claim 5 wherein an amount of nucleated
cells in the concentration is greater than or equal to 4 times an
amount of nucleated cells in the combination of the volume of
aspirate and the volume of blood.
8. A method for treating a defect in a mammal using a concentrated
bone marrow aspirate, the method comprising: drawing a volume of
bone marrow aspirate from the mammal; loading the volume of bone
marrow aspirate into a separator comprising two buoys, the
separator being operable to separate the aspirate into three or
more fractions; centrifuging the separator; separating the volume
of bone marrow aspirate into a plurality of fractions including a
fraction comprising a concentrated bone marrow aspirate; extracting
the fraction comprising a concentrated bone marrow aspirate; and
applying the fraction comprising a concentrated bone marrow
aspirate to site of the defect.
9. The method according to claim 8 wherein the fraction comprising
a concentrated bone marrow aspirate is essentially buffy coat
including a plurality of undifferentiated cells.
10. The method according to claim 8 wherein the fraction comprises
at least one of the group consisting of hematopoietic stem cells,
stromal stem cells, mesenchymal stem cells, endothelial progenitor
cells, red blood cells, white blood cells, fibroblasts,
reticulacytes, adipose cells, and endothelial cells.
11. The method according to claim 8 further comprising combining
the fraction with a carrier.
12. The method according to claim 11 wherein the carrier is
selected from the group consisting essentially of collagen,
hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered
solution, fibrin, concentrated fibrin, demineralized bone matrix,
gelatin, porous calcium based ceramics, porous metal, synthetic
fiber matrices, resorbable matrices, autogeneic tissue, allogenic
tissue, xenogeneic tissue, and combinations thereof.
13. The method according to claim 8 further comprising combining an
activating agent with the fraction.
14. The method according to claim 8 further comprising combining a
pharmaceutical agent with the fraction.
15. A method for treating a defect in an animal, the method
comprising: obtaining a volume of bone marrow aspirate and a volume
of whole blood from the animal; loading the volume of bone marrow
aspirate into a first separator comprising two buoys, the separator
being operable to separate the aspirate into three or more
fractions; loading the volume of whole blood into a second
separator comprising two buoys, the separator being operable to
separate the aspirate into three or more fractions; centrifuging
the first separator and the second separators, thereby separating
the volume of bone marrow aspirate into a plurality of fractions
and separating the volume of whole blood into a plurality of
fractions. collecting a fraction of the bone marrow aspirate;
collecting a fraction of the whole blood; and applying at least one
of the fraction of bone marrow aspirate and the fraction of whole
blood to site of the defect.
16. The method according to claim 15 wherein the at least one of
the fraction of bone marrow aspirate and the fraction of whole
blood comprises at least one of the group consisting of
hematopoietic stem cells, stromal stem cells, mesenchymal stem
cells, endothelial progenitor cells, red blood cells, white blood
cells, fibroblasts, reticulacytes, adipose cells, and endothelial
cells.
17. The method according to claim 15 further comprising combining
the at least one of the fraction of bone marrow aspirate and the
fraction of whole blood with a carrier.
18. The method according to claim 17 wherein the carrier is
selected from the group consisting essentially of collagen,
hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered
solution, fibrin, concentrated fibrin, demineralized bone matrix,
gelatin, porous calcium based ceramics, porous metal, synthetic
fiber matrices, resorbable matrices, autogeneic tissue, allogenic
tissue, xenogeneic tissue, and combinations thereof.
19. The method according to claim 17 further comprising adding an
activating agent to the at least one of the fraction of bone marrow
aspirate and the fraction of whole blood
20. The method according to claim 17 further comprising adding a
pharmaceutical agent to the at least one of the fraction of bone
marrow aspirate and the fraction of whole blood.
21. A method for treating a defect in a patient, the method
comprising: drawing a volume of bone marrow aspirate and a volume
of whole blood from the patient; adding a first anticoagulant to
the volume of bone marrow aspirate; adding a second anticoagulant
to the volume of whole blood; loading the volume of bone marrow
aspirate and the volume of whole blood into a separator comprising
two buoys, the separator being operable to separate the volume of
bone marrow aspirate and the volume of whole blood into three or
more fractions; centrifuging the separator, separating the volume
of bone marrow aspirate and the volume of whole blood into a
plurality of fractions. withdrawing a fraction comprising at least
one of the group consisting of hematopoietic stem cells, stromal
stem cells, mesenchymal stem cells, endothelial progenitor cells,
red blood cells, white blood cells, fibroblasts, reticulacytes,
adipose cells, and endothelial cells.; and applying the fraction to
site of the defect.
22. The method according to claim 21 wherein the fraction is
essentially buffy coat including a plurality of undifferentiated
cells.
23. The method according to claim 21 further comprising combining
the fraction with a carrier.
24. The method according to claim 32 wherein the carrier is
selected from the group consisting essentially of collagen,
hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered
solution, fibrin, concentrated fibrin, demineralized bone matrix,
gelatin, porous calcium based ceramics, porous metal, synthetic
fiber matrices, resorbable matrices, autogeneic tissue, allogenic
tissue, xenogeneic tissue, and combinations thereof.
25. The method according to claim 21 further comprising combining
an activating agent with the fraction.
26. The method according to claim 21 further comprising combining a
pharmaceutical agent with the fraction.
27. A method of treating a patient with combination of a
concentrated bone marrow aspirate and buffy coat, the method
comprising: obtaining a volume of a whole blood from the patient;
obtaining a volume of a bone marrow aspirate from the patient;
forming a buffy coat fraction of the whole blood; forming a
concentrated bone marrow aspirate fraction of the bone marrow
aspirate; and applying at least one of the buffy coat fraction or
the concentrated bone marrow aspirate fraction to the patient.
28. The method according to claim 27, wherein the forming a first
fraction of the first whole material and the forming a second
fraction of the second whole material includes: loading the volume
of whole blood and the volume of bone marrow aspirate in a
container; and applying a force to the container to form at least
three fractions.
29. The method according to claim 27, wherein the forming a first
fraction of the first whole material and the forming a second
fraction of the second whole material includes: loading the volume
of whole blood and the volume of bone marrow aspirate in the
container having a separating member, the separating member having
a specific gravity substantially dependent upon at least one of the
at least three fractions; and centrifuging the container to move
the separating member to a selected position relative to the at
least one of the volume of whole blood and the volume of bone
marrow aspirate to substantially physically separate the at least
three fractions.
30. The method according to claim 27 wherein the at least one of
the first fraction or the second fraction comprises at least one of
the group consisting of hematopoietic stem cells, stromal stem
cells, mesenchymal stem cells, endothelial progenitor cells, red
blood cells, white blood cells, fibroblasts, reticulacytes, adipose
cells, and endothelial cells.
31. The method according to claim 27 further comprising combining
the at least one of the first fraction or the second fraction with
a carrier.
32. The method according to claim 31 wherein the carrier is
selected from the group consisting essentially of collagen,
hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered
solution, fibrin, concentrated fibrin, demineralized bone matrix,
gelatin, porous calcium based ceramics, porous metal, synthetic
fiber matrices, resorbable matrices, autogeneic tissue, allogenic
tissue, xenogeneic tissue, and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/932,882, filed on Sep. 02, 2004, entitled
"APPARATUS AND METHOD FOR SEPARATING AND CONCENTRATING FLUIDS
CONTAINING MULTIPLE COMPONENTS", which is a continuation-in-part of
U.S. patent application Ser. No. 10/445,381, filed on May 23, 2003,
entitled "APPARATUS AND METHOD FOR SEPARATING AND CONCENTRATING
FLUIDS CONTAINING MULTIPLE COMPONENTS" that claimed the benefit of
U.S. Provisional Application No. 60/383,013, filed on May 24, 2002.
The disclosures of the above applications are incorporated herein
by reference.
FIELD
[0002] The present teachings relate to a multiple component fluid
and a concentrator/separator and, more particularly, relates to a
container operable with a centrifuge to separate and/or concentrate
various biological components.
BACKGROUND
[0003] Various fluids, such as whole blood or various other
biological fluids, may be separated into their constituent parts,
also referred to as fractions or phases. For example, whole blood
samples may include a plurality of constituents that may be
separated by density in a device such as a centrifuge. The whole
blood sample may be placed in a test tube, or other similar device,
which is then spun in a centrifuge. In the centrifuge the whole
blood is separated into different fractions depending upon the
density of that fraction. The centrifugal force separates the blood
or other sample into different fractions. In addition, various
elements may be added to the test tube to create more than two
fractions. In particular, commonly used gels may be used to divide
the whole blood into a plurality of different fractions which may
include fractions such as platelets, red blood cells, and plasma.
Various other biological fluids may be separated as well. For
example, nucleated cells may be separated and extracted from bone
marrow or adipose tissue sample.
[0004] Many of these systems, however, do not provide a simple or
efficient method to extract any more than one fraction and,
especially, a fraction other than the top fraction. The top
fraction of whole blood is plasma, or other blood constituents
suspended in plasma. Thus, to extract other fractions the plasma
fraction must either be removed for further extracting procedures
or spun again to obtain the constituents suspended in this plasma.
It is difficult to pierce the top fraction without commingling the
sample. Accordingly, obtaining the other fractions is difficult
with commonly known systems.
[0005] Other systems have attempted to alleviate this problem by
providing a float or other device that is disposed within the
sample at the interfaces of the different fractions during the
centrifuge process. Nevertheless, these systems still do not allow
a simple way to remove the different fractions without remixing the
sample fractions. In addition, many of the systems do not allow an
easy and reproducible method to remove the desired sample
fraction.
[0006] Therefore, it is desired to provide a device to allow for
the easy and reproducible removal of a particular fraction which
does not happen to be the top fraction of a sample. It is desired
to remove the required sample without mixing the different
fractions during the extraction process. In addition, it is desired
to provide a device which allows for a consistent extraction which
includes known volumes or concentration of the fraction elements.
Moreover, it is desired to separate and concentrate a selected
fraction with one centrifugation step.
SUMMARY
[0007] The present teachings provide an apparatus that separates
and concentrates a selected fraction or component of a fluid, such
as a biological fluid. For example, undifferentiated cells, such as
mesenchymal stem cells, platelet fraction, buffy coat, or white
blood cell fraction can be separated from bone reaming material,
whole blood, bone marrow aspirate, and other materials. In various
embodiments, the apparatus, when used with a centrifuge, is
generally able to create at least two fractions. The present
teachings also provide for a new method of creating at least three
fractions extracting a third fraction from a sample such as, for
example, a buffy coat fraction.
[0008] In various embodiments, the apparatus includes a container
to be placed in a centrifuge after being filled with a sample and
the container includes a buoy or fraction separator having a
selected density that may be less than one fraction but greater
than a second fraction, that is disposed therein. In various
embodiments, a second buoy may be placed in the container with the
first. The extraction system is connected to the buoy system or to
the collection chamber such that the fraction in the container can
be collected and drawn outside of the chamber. During the
centrifuge processing, the buoy is forced away from a bottom of the
container as the denser fraction collects at the bottom of the
container. The buoy is generally able to physically separate the
denser fraction from another fraction of the sample. In various
embodiments, the fractions can be withdrawn using an extraction
system.
[0009] According to various embodiments, in addition to providing a
first buoy and/or a second buoy, a buoy system may be provided.
Generally, the buoy system may separate the sample into at least
three fractions. The fractions may be separated and extracted from
the container without substantially commingling the various
fractions. Generally, a first buoy and a second buoy operate
together to separate the sample into the various fractions and a
syringe or tube may then be interconnected with a portion of the
buoy system to extract the selected fractions. For example, a first
buoy may be tuned to a density less than the density of a red blood
cell fraction of a whole blood sample, bone marrow aspirate sample,
or combinations thereof, and a second buoy may be tuned to a
density less than the density of a buffy coat fraction.
[0010] According to various embodiments, a method of forming an
enriched scaffold for application relative to an anatomy is
provided. The method may include obtaining a volume of a first
whole material and obtaining a volume of a second whole material. A
first fraction of the first whole material and a second fraction of
the second whole material may be formed. At least one of the first
fraction or the second fraction may be applied to the scaffold and
at least one fraction for application relative to an anatomy is
provided. The method may include obtaining a volume of
heterogeneous whole material and separating the material into the
desired fraction(s). At least one of the fractions may be applied
to the scaffold.
[0011] According to various embodiments, a method of withdrawing a
material directly from a patient and collecting a selected fraction
of the material in a container is provided. The method may include
forming an access to port to the patient. A pressure differential
in a collection container may be formed relative to the patient. A
connection may be made between the patient and the collection
container via the port. The collection container may be filled with
the material and then the material may be separated to form the
selected fraction.
[0012] According to various embodiments, a method for concentrating
bone aspirate can include obtaining a volume of bone marrow
aspirate from a mammal and loading the volume bone marrow aspirate
into a separator, the separator operable to separate the aspirate
into three of more fractions. The method also includes centrifuging
the separator to create a fraction that is a concentrated bone
marrow aspirate and extracting and removing the fraction from the
separator.
[0013] According to various embodiments, a method for concentrating
bone marrow aspirate and blood includes collecting bone marrow
aspirate and blood from a patient then loading the bone marrow
aspirate and blood into a separator that can separate the aspirate
and the blood into three or more fractions. The method includes
centrifuging the separator containing the bone marrow aspirate and
the blood creating a fraction that has a concentrated bone marrow
aspirate and a concentrated blood. In various embodiments, such a
concentration may be referred to as a buffy coat. The method also
include withdrawing the fraction comprising the concentrate or
buffy coat.
[0014] According to various embodiments, a method for treating a
defect in a mammal using a concentrated bone marrow aspirate
includes drawing bone marrow aspirate from the mammal and loading
the bone marrow aspirate into a separator that can separate the
bone marrow aspirate into three or more fractions. The method
includes centrifuging the separator separating the bone marrow
aspirate into fractions and one fraction is concentrated bone
marrow aspirate. The method also can include the concentrated bone
marrow aspirate and applying the bone marrow aspirate to a site of
a defect in the mammal.
[0015] According to various embodiments, a method for treating a
defect in a patient includes drawing bone marrow aspirate and whole
blood from the patient then adding anticoagulants to the bone
marrow aspirate and the blood. The method includes the loading of
the bone marrow aspirate and the blood into a separator that can
separate the bone marrow aspirate and blood into three or more
fractions. The method also includes centrifuging the separator then
withdrawing a fraction comprising at least one of the group
consisting of hematopoietic stem cells, stromal stem cells,
mesenchymal stem cells, endothelial progenitor cells, red blood
cells, white blood cells, fibroblasts, reticulacytes, adipose
cells, and endothelial cells, then applying the fraction to the
site of the defect in the patient.
[0016] According to various embodiments, a method of treating a
patient with a combination of concentrated bone marrow aspirate and
buffy coat is provided. The method includes obtaining blood and
bone marrow aspirate from the patient, forming a buffy coat
fraction of the whole blood and forming a concentrated bone marrow
aspirate fraction, and applying at least one of the buffy coat or
concentrated bone marrow aspirates to the patient.
[0017] Further areas of applicability of the present teachings will
become apparent from the detailed description provided hereinafter.
It should be understood that the detailed description and specific
examples, while indicating various embodiment of the teachings, are
intended for purposes of illustration only and are not intended to
limit the scope of the teachings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present teachings will become more fully understood from
the detailed description and the accompanying drawings,
wherein:
[0019] FIG. 1 is a plan view of a separator including a depth gage
affixed to a plunger in a tube according to various embodiments of
the present teachings;
[0020] FIG. 2 is a cross-section view taken along line 2-2 of FIG.
1;
[0021] FIG. 3 is an exploded of the separator apparatus according
to various embodiments;
[0022] FIG. 4 is a kit including the separator according to an
embodiment of the present teachings;
[0023] FIG. 5A is a plan view of the separator, according to
various embodiments, being filled;
[0024] FIG. 5B is a plan view of a blood sample in the separator
after the centrifuge process;
[0025] FIG. 5C is a plan view of the plunger plunged into the tube
with the depth gage to further separate the blood sample;
[0026] FIG. 5D is a plan view of the buffy coat and the plasma
fractions being extracted from the separator according to various
embodiments;
[0027] FIG. 6A is a side plan view of a buoy system according to
various embodiments;
[0028] FIG. 6B is a cross-sectional view of the buoy system of FIG.
6a;
[0029] FIG. 7A is a plan view of a separator, according to various
embodiments, being filled;
[0030] FIG. 7B is a plan view of a separator, according to various
embodiments, after a centrifugation process;
[0031] FIG. 7C is a plan view of a separator system, according to
various embodiments, being used to extract a selected fraction
after the centrifugation process;
[0032] FIG. 7D is a plan view of a second fraction being extracted
from the separator according to various embodiments;
[0033] FIG. 8 is a schematic view illustrating an assisted blood
withdrawal device according to various embodiments; and
[0034] FIG. 9 is a block diagram illustrating a method for applying
selected fractions of a fluid.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS
[0035] The following description of various embodiments is merely
exemplary in nature and is in no way intended to limit the
teachings, its application, or uses. Although the following
description exemplary refers to a bone reaming material, whole
blood and/or bone marrow aspirate separation, it will be understood
that the present teachings may be used to separate and concentrate
any appropriate material. It will be further understood that many
multi-component materials containing particles may be separated.
The components or fractions are generally intermingled in the whole
sample but may be separated with a centrifuge device that causes
increased local gravity or gravitational forces.
[0036] With reference to FIGS. 1-3, according to various
embodiments, a separator 10, also referred to as a concentrator, is
illustrated. The separator 10 generally includes a tube or
container 12 that is adapted to hold a fluid sample, such as an
anti-coagulated whole blood sample, for further processing. It will
be understood that the tube 12 may hold other solutions including
constituents of more than one density, such as bone marrow or a
mixture of whole blood and bone marrow. The tube 12 includes a top
or open end 12a, which is closeable, and a bottom or closed end
12b. The bottom 12b may also be selectively closeable.
[0037] Disposed within the tube 12 is a first piston or buoy 14
that is able to move along a central axis A of the tube 12. The
buoy 14 is generally nearer the bottom end 12b of the tube 12
rather than the open end 12a. The buoy 14 is able to move along a
central axis A of tube 12. Also disposed within the tube 12 is a
second piston or plunger 16. The plunger 16 is also able to move
within the tube 12 generally between a position closer to the open
end 12a to a position closer to the closed end 12b of the tube 12.
A cap 18 substantially mates with the open end 12a of the tube 12
to close the tube 12 save for ports formed in the cap 18. Extending
from the cap 18 is a plasma valve or port 20 that communicates with
an area, described further herein, within the tube 12 defined
between the plunger 16 and the cap 18. It will be understood that
the plasma port 20 is merely exemplary in nature and simply allows
for removal of a selected fraction of a sample such as, for
example, plasma from whole blood.
[0038] The cap 18 also includes a depth gage port 19. Extending
from the plunger 16 and through the depth gage port 19 is a first
plunger port 22. A depth guide or gage 24 includes a female
connector 26 adapted to connect with the first plunger port 22. The
depth gage 24 also includes a depth gage housing or cannula 28. The
depth gage housing 28 defines a depth gage bore 30. Incorporated in
the depth gage housing 28 and extending distal from the end mating
with the plunger 16 is a neck 32. The neck 32 includes external
neck threads 34. The external neck threads 34 are adapted to engage
appropriate internal threads of a mating member.
[0039] The mating member may include a compression nut 36 that
mates with the external neck threads 34 to lock a depth gage rod 38
in a predetermined position. A split bushing 39 is also provided to
substantially seal the depth gage housing 28 when the depth gage
rod 38 is locked in place. The depth gage rod 38 extends through
the depth gage housing 28 and terminates at a rod handle 40. The
rod handle 40 may be a form easily manipulated by a human operator.
The depth gage rod 38 extends coaxially with axis A of the tube 12.
The depth gage rod 38 extends through the plunger 16 a
predetermined distance and may be locked at that distance with the
compression nut 36.
[0040] Although the tube 12 is described herein as a cylinder, it
will be understood that other shapes may be used, such as polygons.
The internal portions, such as the cap 18, buoy 14, and plunger 16,
would also include this alternate shape. Preferably the tube 12 is
formed of a thermal plastic material which is flexible under the
forces required to separate blood. The tube 12 may be made of a
material that includes the properties of both lipid and alcohol
resistance. These properties helps increase the separation speed
and decrease the amount of material which may cling to the tube
wall 42. For example, Cyrolite MED2.RTM. produced by Cyro
Industries of Rockaway, N.J., may be used to produce the tube
12.
[0041] The tube 12 has a tube wall 42 with a thickness of between
about 0.01 millimeters and about 30.0 millimeters, although the
tube wall 42 may be any appropriate thickness. The thickness of the
tube wall 42 allows the tube wall 42 to flex during the centrifuge
process yet be rigid enough for further processing of a blood
sample disposed in the tube 12. The tube 12 is closed at the bottom
end 12b with a tube bottom 44 formed of the same material as the
tube wall 42 and is formed integrally therewith. Generally, the
tube bottom 44 has a thickness which is substantially rigid under
the forces required to separate the sample such that it does not
flex.
[0042] The buoy 14 includes an upper or collection face 46 that
defines an inverse cone or concave surface. Generally, the cone has
an angle of between about 0.5.degree. to about 45.degree., and may
be about 0.5.degree. to about 90.degree. from a vertical axis,
wherein the apex of the cone is within the buoy 14. The collection
face 46 forms a depression in the buoy 14 which collects and
concentrates material during the separation process. Additionally,
the buoy 14 has a bottom face 48 that defines an inverse cone,
dome, or covered surface. The buoy bottom face 48 includes an apex
50 that engages the tube bottom 44 before a buoy edge 52 engages
the tube bottom 44. The buoy 14 includes a material that is a
substantially rigid such that the buoy edges 52 never meet the tube
bottom 44. Therefore, there is a gap or free space 54 formed
between the buoy edges 52 and the tube bottom 44 along the
perimeter of the buoy 14.
[0043] The separator 10 is generally provided to separate a
multi-component fluid that generally includes various components or
constituents of varying densities that are commingled or mixed
together. The separator 10 includes the buoy 14 that is of a
selected density depending upon a selected constituent of the
multi-constituent liquid. Although the buoy 14 may be tuned or of
any selected density, the following example relates to separation
of whole blood to various components. Therefore, the buoy 14 will
be discussed to include a selected density relative to whole blood
separation. It will be understood, however, that the buoy 14 may be
of any appropriate density depending upon the multi-component fluid
being separated including, for example, the concentrating of bone
marrow aspirate.
[0044] The buoy 14 may be formed of any appropriate material that
may have a selected density. For example, when the separator 10 is
to separate blood, the buoy 14 generally has a density which is
greater than that of red blood cells in a whole blood sample, but
less than the plasma or non-red blood cell fraction of a whole
blood sample. For blood, the density of the buoy 14 may be between
about 1.00 g/cc to about 1.12 g/cc or between about 1.02 g/cc and
about 1.09 g/cc.
[0045] To achieve the selected density, the buoy 14 may be formed
as a composite or multi-piece construction, including a plurality
of materials. Particularly, a first or outside portion 56 defines
the upper or collection face 46 and the buoy edges 52 and is formed
of the same material as the tube 12. The outside portion 56 defines
a cup or void into which a plug or insert 58 is placed. The insert
58 has a mass such that the density of the entire buoy 14 is within
the selected range, for example, the range described above.
Generally, a high density polyethylene may be used, but the
material and size of the insert 58 may be altered to produce the
desired density of the buoy 14. Alternatively, the buoy 14 may be
formed of a single suitable material that has a density in the
selected range. Nevertheless, the buoy 14 formed unitarily or of a
single material would still include the other portions described in
conjunction with the buoy 14.
[0046] The outside portion 56 of the buoy 14 also defines the
outside circumference of the buoy 14. The outside circumference of
the buoy 14 is very close to the internal circumference of the tube
12. Due to the operation of the buoy 14, however, described further
herein, there is a slight gap between the outside of the buoy 14
and the inside of the tube 12. Generally, this gap is between about
1 and about 10 thousandths of an inch around the entire
circumference of the buoy 14. Generally, it is desired that the
distance between the outside circumference of the buoy 14 and the
inside circumference of the tube 12 is great enough to allow a
selected material or component to pass. For example, in whole blood
the distance is selected so that red blood cells may pass through
the gap without being lysed, damaged, or activated.
[0047] The plunger 16 includes a plunger front or collection face
60 and a plunger wall 62 that extends from the plunger front face
60. The plunger wall 62 extends relatively perpendicular to the
plunger front face 60 and substantially parallel to the tube wall
42. Extending from the center of the plunger 16 is a sample
collection projection 64. Extending from the top of the sample
collection projection 64 is the first plunger port 22. The sample
collection projection 64 includes a plunger sample collection bore
68 defined therethrough. The plunger sample collection bore 68
terminates at a sample collection aperture 70 that is substantially
in the center of the plunger front face 60. The plunger front face
60 also defines an inverse cone where the sample collection
aperture 70 is the apex of the cone. The plunger front face 60
defines a cone with an angle substantially similar or complimentary
to the collection face 46 of the buoy 14. In this way, the plunger
front face 60 may mate substantially completely with the collection
face 46 for reasons described more fully herein.
[0048] The plunger 16 also includes a back face 72. Extending from
the plunger front face 60 to the back face 72 is a bore 74. A check
valve 76 is operably connected to the bore 74. The check valve 76
allows a liquid to move from the plunger front face 60 to the back
face 72 while not allowing the liquid to move from the back face 72
to the plunger front face 60. Therefore, the check valve 76 is
substantially a one-way valve which allows a material to move in
only one direction. The check valve 76 may also operate
automatically allowing flow in only one predetermined direction.
Alternatively, the check valve 76 may be operated manually and
include a portion extending from the check valve 76 requiring
manipulation to stop or start a flow through the check valve
76.
[0049] The plunger 16 may be made out of any appropriate material
which does not interfere with the separation of the fractions of
the fluid, such as whole blood. The plunger 16, however, is made of
a material that is flexible or at least partially deformable. A
flexible material allows the plunger 16 to have an external
circumference defined by the plunger walls 62 that is substantially
equal to the internal circumference of the tube 12. Because of the
deformability of the plunger 16, however, the plunger 16 is still
able to move within the tube 12. The plunger 16 is able to move
through the tube 12 and also substantially wipe the interior of the
tube wall 42. This creates, generally, a moveable seal within the
tube 12. Thus, substantially no material escapes the action of the
separator 10 when the plunger 16 is plunged into the tube 12. This
also helps concentrate the portion of the sample desired to be
collected, described more fully herein.
[0050] The cap 18 provides a structure to substantially close the
tube 12. The cap 18 particularly includes a plate 78 that has an
external circumference substantially equal to the external
circumference of the tube 12. Extending from the plate 78 and into
the tube 12 is a flange 80. The external circumference of the
flange 80 is substantially equal to the internal circumference of
the tube 12. In this way, the cap 18 substantially closes the tube
12. It will be understood the cap 18 may be in any form so long as
the cap 18 substantially closes and/or seals the tube 12 when
installed.
[0051] Formed through the center of the plate 78 is the depth gage
port 19. The depth gage port 19 is also adapted to receive the
sample collection projection 64. The first plunger port 22 extends
above the plate 78 through the depth gage port 19. The
circumference of the depth gage port 19 is substantially equal to
the external circumference of the sample collection projection 64
such that a liquid seal is formed. The plate 78 defines a sample
face 84 that includes an interior side of the cap 18. The area
between the sample face 84 of the cap 18 and the back face 72 of
the plunger 16 define a plasma collection area 86. Although the
plasma collection area 86 is exemplary called the plasma collection
area, it will be understood that the plasma collection area 86 may
also collect any appropriate fraction of the sample that is
positioned within a separator 10. The plasma collection area 86 is
merely an exemplary name and an example of what material may be
collected in the area of the separator 10. As discussed herein, the
separator 10 may used to separate whole blood into various
fractions, therefore, the plasma collection area 86 is used to
collect plasma. The plasma collection area 86 also allows a space
for the check valve 76 to be installed.
[0052] A second bore 88 is formed in the plate 78. Extending
through the second bore 88 is the plasma collection valve 20. In
liquid communication with the plasma collection valve 20 is a
plasma collection tube 92. The plasma collection tube 92 has a
length such that the plasma collection tube 92 is able to extend
from the plasma collection valve 20 to substantially the tube
bottom 44. The plasma collection tube 92, however, is flexible
enough such that it may be folded or compressed to fit within the
plasma collection area 86 when the plunger 16 is substantially near
the open end 12a of the tube 12. The plasma collection tube 92 may
also be connected to a hose barb 93 that includes a plasma
collection bore 93a. The plasma collection bore 93a is
substantially level with the plunger back face 72. Alternatively,
the plasma collection bore 93a may be positioned below the plunger
back face 72 but in fluid communication with the plasma collection
tube 92.
[0053] The outboard side of the plasma collection valve 20 may
include external threads 94 to mate with internal threads of a
plasma valve cap 96. Therefore, the plasma collection valve 20 may
be selectively opened and closed via the plasma valve cap 96. It
will be understood, however, that other appropriate means may be
used to open and close the plasma collection valve 20 such as a
clip or a plug. It will be understood that the plasma collection
valve 20, plasma collection tube 92, plasma collection bore 23a may
be used to collect any appropriate material or fraction from the
separator 10.
[0054] Also formed in the plate 78 is a vent bore 98. The vent bore
98 allows air to flow into the plasma collection area 86 as the
plunger 16 is being plunged into the tube 12. The vent bore 98 may
include a filter 100 such that liquid cannot escape from the tube
12. The filter 100 allows air to enter or escape from the plasma
collection area 86 while maintaining the liquid seal of the tube 12
produced by the cap 18.
[0055] Selectively attachable to the first plunger port 22 is the
depth gage 24. The female connector 26 interconnects the depth gage
housing 28 to the first plunger port 22. Internal threads in the
female connector 26 mate with an external thread 102 formed on the
first plunger port 22. It will be understood, however, that other
engagement mechanisms between the depth gage 24 and the plunger 16
may be used. For example, a snap connection rather than a threaded
connection between the two may be used.
[0056] The depth gage housing 28 is formed to be substantially
rigid. Suitable materials, when sized properly, include
polycarbonate and CYRO MED2.RTM.. The material preferably is both
rigid and does not substantially react with the sample. It is rigid
enough to provide a mechanism to plunge the plunger 16 into the
tube 12. In addition, the external circumference of the depth gage
housing 28 is substantially equal to the circumference of the depth
gage port 19 in the plate 78. Therefore, as the plunger 16 is being
plunged into the tube 12 with the depth gage 24, no liquid material
is allowed to escape around the depth gage housing 28 and through
depth gage port 19.
[0057] Formed within the depth gage housing 28 is the bore 30 which
receives the depth gage rod 38. The depth gage rod 38 extends
through the plunger sample collection bore 68 of the sample
collection projection 64 and protrudes through the sample
collection aperture 70 a predetermined length. The depth gage rod
38 extends through the sample collection aperture 70 a length such
that when an end 104 of the depth gage rod 38 meets the buoy 14,
the volume defined by the collection face 46 and the plunger front
face 60 is between about 5% and about 30% of the total volume of
the sample that the tube 12 holds. The projection of the depth gage
rod 38 allows for an easily reproducible collection amount and
concentration over several trials.
[0058] The compression nut 36 locks the depth gage rod 38 in the
predetermined position. Nevertheless, once the plunger 16 has been
plunged to the desired depth in the tube 12, the compression nut 36
may be loosened so that the depth gage rod 38 may be removed from
the plunger 16 and the depth gage housing 28 without moving the
plunger 16. A syringe or other appropriate device may then be
affixed to the external neck threads 34 of the depth gage 24 to
extract the fraction or phase that is between the plunger front
face 60 and the collection face 46. As described further herein,
the fraction or phase that is left between the plunger front face
60 and the collection face 46 may be the buffy coat of a whole
blood sample. Nevertheless, it will be understood that the fraction
between the plunger front face 60 and the collection face 46 may be
any appropriate fraction of the sample that is disposed in the
separator 10.
[0059] The separator 10 may be provided alone or in a kit 200, as
illustrated in FIG. 4. The kit 200 may be placed in a tray 202
which is covered to provide a clean or sterile environment for the
contents of the kit 200. The kit 200 may include at least a first
separator 10 and a second separator 10'. A first depth gage 24 and
a second depth gage 24' are also provided, one for each separator
10, 10'. The kit 200 also generally includes a first syringe 204,
including a needle, to draw a biological sample, such as blood from
a patient. The first syringe 204 may also be used to place the
sample in the first separator 10. After centrifuging the sample a
second device or syringe 210 may be used to extract a first
fraction of the sample, while a third device or syringe 212 may be
used to extract a second fraction of the sample. Also, a tourniquet
214 and other medical supplies, such as gauze 216 and tape 218, may
be provided to assist the practitioner. It will be understood the
elements of the kit 200 are merely exemplary and other appropriate
items or elements may be included.
[0060] With reference to FIGS. 5A-5D, a method using the separator
10 is illustrated according to various embodiments. The following
example relates specifically to the taking and separating of a
sample of whole blood from a patient. Nevertheless, it will be
understood that another appropriate biological material may be
separated and concentrated using the separator 10. For example,
bone marrow may be separated and concentrated using the separator
10. The various fractions of the bone marrow are similar to the
fractions of whole blood. Generally, the bone marrow includes a
fraction that includes substantially dense material and a second
phase that is less dense and has other components suspended therein
such as, for example, nucleated cells. The bone marrow sample may
be positioned in the separator 10, similarly to the whole blood as
described herein, and separated in a substantially similar manner
as the whole blood. The separator 10 can then be used to remove
nucleated cells from the bone marrow sample (which may be referred
to as buffy coat), whereas the separator 10, as described herein,
is used to remove the buffy coat from the whole blood which
includes platelets and other appropriate materials (which may be
referred to as platelet rich plasma (PRP)).
[0061] A mixture of whole blood and bone marrow may be positioned
in the separator 10 for separation and concentration. Similar
methods and steps will be used to separate the mixture of whole
blood and bone marrow with a main difference being the material
that is separated. It will also be understood that various
centrifuge times or forces may be altered depending upon the exact
material that is being separated with the separator 10. It will
also be understood that the separation of whole blood, bone marrow,
or a mixture of whole blood and bone marrow are merely exemplary of
the materials that may be separated using the separator 10.
[0062] According to various embodiments, and with reference to
FIGS. 5A-5D and to a whole blood sample, a sample of whole blood
taken from a patient is placed in the tube 12 with an anticoagulant
using the first syringe 204 or other appropriate delivery method.
In particular, the first syringe 204 may be connected to the first
plunger port 22. After which the blood sample is provided to the
tube 12 via the sample collection bore 68 and sample collection
aperture 70. A cap 220 is then placed over the first plunger port
22 to substantially seal the tube 12.
[0063] After the whole blood sample is delivered to the tube 12,
the separator 10 is placed in a centrifuge. The second separator
10', substantially identical to the first, is placed opposite the
first separator 10 including the sample in a centrifuge. The second
separator 10' may also include a second sample or may include a
blank, such as water, so that the centrifuge is balanced. The
second separator 10' balances the centrifuge by both weight and
dynamics.
[0064] The separator 10 is then spun in the centrifuge in a range
between about 1,000 and about 8,000 RPM. This produces a force
between about 65 and about 4500 times greater than the force of
normal gravity, as generally calculated in the art, on the
separator 10 and the blood sample placed in the separator 10. At
this force, the more dense material in a whole blood sample is
forced toward the bottom end 12b of the tube 12. The dense
material, such as red blood cells or a red blood cell fraction 222,
collects on the tube bottom 44. Because the buoy 14 has a density
that is less than the red blood cell fraction 222, it is forced in
a direction toward the top end 12a of the tube 12 in the
centrifuge. Nevertheless, because the buoy 14 is denser than a
plasma fraction 224, the buoy 14 does not reach the top end 12a of
the tube 12.
[0065] The forces also affect the tube wall 42. The forces compress
the tube 12 linearly along axis A thereby bowing or flexing the
tube wall 42. As the tube wall 42 compresses it increases the
diameter of the tube 12 making it easier for the buoy 14 to move in
the direction of the top 12a of the tube 12. In addition, the
bottom face 48, defining an inverse cone, helps the initial
movement of the buoy 14. Because the buoy 14 is not substantially
flat along its bottom, it does not form a vacuum interaction with
the tube bottom 44. Therefore, the initial movement of the buoy 14
away from the tube bottom 44 is quicker than if the bottom of the
buoy 14 was flat.
[0066] During the centrifuge process, the red bloods cells of the
red blood cell fraction 222 force the buoy 14 in the direction of
the top end 12a of the tube 12 because the buoy 14 is less dense
than the red blood cell fraction 222. Although the whole blood
sample, including the red blood cells, is loaded above the buoy 14,
the red blood cells are able to move between the buoy 14 and the
tube wall 42 because the circumference of the buoy 14 is less than
the internal circumference of the tube 12. During the centrifuge
process, the buoy 14 stops at an interface of a plasma fraction 224
and the red blood cell fraction 222 because of the selected or
tuned density of the buoy 14.
[0067] With particular reference to FIG. 5B, the centrifuge process
has been completed and the buoy 14 has moved to the interface of
the red blood cell fraction 222 and plasma fraction 224. After the
centrifuge has slowed or stopped, and before or after the tube 12
has been removed from the centrifuge, the tube wall 42 decompresses
which helps support the buoy 14 at the interface position. It is
also understood that applying an external pressure to the tube 12
via fingers or another apparatus may help stabilize the buoy 14
during the plunging procedure described herein.
[0068] On or near collection face 46 is a middle fraction 226,
including a small, yet concentrated, amount of red blood cells,
white blood cells, platelets, and a substantial portion of a buffy
coat of the blood sample. Although the plasma is also present near
the collection face 46 at this point, the solid portions of the
buffy coat are more compressed against the collection face 46. The
position of the buoy 14 also helps in this matter. Because the buoy
14 is a single body it defines the interface of the plasma fraction
224 and the red blood cell fraction 222. Also the density of the
buoy 14 assures that it has not passed into the plasma fraction
224. Therefore, the fractions remain separated after the centrifuge
process. In addition because the buoy 14 is tuned to the density of
the red blood cell fraction 222, it is not affected by variations
in the density of the plasma fraction 224 and the buoy's 14
position is always at the interface of the red blood cell fraction
222 and the plasma fraction 224.
[0069] With particular reference to FIG. 5C, the depth gage 24 is
affixed to the first plunger port 22 of the sample collection
projection 64. After connecting the depth gage 24 to the first
plunger port 22, the plunger 16 is plunged into the tube 12 by
pushing on the depth gage 24. As this is performed the plasma
fraction 224, formed and separated above the buoy 14, is able to
flow through the check valve 76 into the plasma collection area 86.
This displacement of the plasma fraction 224 allows the plunger 16
to be plunged into the tube 12 containing the blood sample.
[0070] The plunger 16 is plunged into the tube 12 until the point
where the end 104 of the depth gage rod 38 reaches the buoy 14. The
volume left in the collection face 46 is the middle fraction 226
and is determined by the depth gage 24. It may be adjusted by
selectively determining the amount that the depth gage rod 38
extends below the plunger front face 60. By adjusting the depth
gage 24, the concentration of the middle fraction 226 can be
adjusted depending upon the desires of the operator.
[0071] The plasma fraction 224 is held in the plasma collection
area 86 for later withdrawal. Therefore, the use of the plunger 16
and the buoy 14 creates three distinct fractions that may be
removed from the tube 12 after only one spin procedure. The
fractions include the red blood cell fraction 222, held between the
buoy 14 and the tube bottom 44. The middle or buffy coat fraction
226 is held between the plunger 16 and the buoy 14. Finally, the
plasma fraction 224 is collected in the plasma collection area 86.
In various embodiments, the middle fraction 226 may be a platelet
rich plasma (PRP) fraction and the plasma fraction 224 may be a
platelet poor plasma (PPP) fraction.
[0072] The middle fraction 226 may be extracted from the tube 12
first, without commingling the other fractions, through the sample
collection bore 68. With particular reference to FIG. 5D, the depth
gage rod 38 may be removed from the depth gage housing 28. This
creates a sample collection cannula which includes the depth gage
bore 30, the sample collection bore 68, and the sample collection
aperture 70. After the depth gage rod 38 has been removed, the
second syringe 210 may be affixed to the depth gage housing 28 via
the external neck threads 34. The second syringe 210 may be
substantially similar to the first syringe 204.
[0073] Before attempting to withdraw the middle fraction 226 the
separator 10 may be agitated to re-suspend of the platelets and
concentrated red blood cells in a portion of the plasma remaining
in the collection face 46. This allows for easier and more complete
removal of the middle fraction 226 because it is suspended rather
than compressed against the collection face 46. A vacuum is then
created in the second syringe 210 by pulling back the plunger 16 to
draw the middle fraction 226 into the second syringe 210.
[0074] As the middle fraction 226 is drawn into the second syringe
210 the plunger 16 moves toward the buoy 14. This action is allowed
because of the vent bore 98 formed in the cap 18. Atmospheric air
is transferred to the plasma collection area 86 through the vent
bore 98 to allow the middle fraction 226 to be removed. This also
allows the movement of the plunger 16 toward the buoy 14. This
action also allows the plunger 16 to "wipe" the collection face 46.
As the plunger front face 60 mates with the collection area 46 the
middle fraction 226 is pushed into the sample collection aperture
70. This ensures that substantially the entire middle fraction 226
collected in the collection area 46 is removed into the second
syringe 210. It can also increase the repeatability of the
collection volumes. In addition, because the second syringe 210
does not protrude out the sample collection aperture 70, it does
not interfere with the collection of the middle fraction 226. Once
the plunger front face 60 has mated with the collection face 46
there is substantially no volume between the plunger 16 and the
buoy 14.
[0075] Once the middle fraction 226 is extracted the second syringe
210 is removed from the first plunger port 22. Also the extraction
of the middle fraction 226 leaves the plasma fraction 224 and the
red blood cell fractions 222 separated in the tube 12. At this
point, a third syringe 212 may be affixed to the plasma collection
valve 20. The third syringe 212 is connected to the external
threads 94 of the plasma collection valve 20 to ensure a liquid
tight connection. It will be understood, however, that another
connection mechanism such as a snap or compression engagement may
be used to connect the third syringe 212 to the plasma collection
valve 20.
[0076] A vacuum is then created in the third syringe 212 to draw
the plasma fraction 224 from the plasma collection area 86 through
the plasma collection tube 92. As discussed above, the plasma
collection tube 92 is connected to the hose barb 93. Therefore, the
plasma flows through the plasma collection bore 93a through the
hose barb 93, and then through the plasma collection tube 92. It
will be understood that the plasma collection tube 92 may
alternatively simply rest on the plunger back face 72 to collect
the plasma fraction 224. In this way, the plasma fraction 224 may
be removed from the blood separator 10 without commingling it with
the red blood cell fraction 222. After the plasma fraction 224 is
removed, the separator 10 may be dismantled to remove the red blood
cell fraction 222. Alternatively, the separator 10 may be discarded
in an appropriate manner while retaining the red blood cell
fraction 222.
[0077] The separator 10 allows for the collection of three of a
whole blood sample's fractions with only one centrifugation spin.
The interaction of the buoy 14 and the plunger 16 allows a
collection of at least 40% of the available buffy coat in the whole
blood sample after a centrifuge processing time of about 5 minutes
to about 15 minutes. The complimentary geometry of the plunger
front face 60 and the collection face 46 help increase the
collection efficiency. Although only the cone geometry is discussed
herein, it will be understood that various other geometries may be
used with similar results.
[0078] The plunger front face 60 being flexible also helps ensure a
complete mating with the collection face 46. This, in turn, helps
ensure that substantially the entire volume between the two is
evacuated. The process first begins with the suction withdrawal of
the middle fraction 226 via the second syringe 210, but is
completed with a fluid force action of the middle fraction 226 as
the plunger front face 60 mates with the collection face 46. As the
plunger front face 60 mates with the collection face 46, the fluid
force assists in removal of the selected fraction.
[0079] The plunger 16 also substantially wipes the tube wall 42.
Because the plunger 16 is formed of a flexible material it forms a
seal with the tube wall 42 which is movable. Therefore,
substantially no liquid is able to move between the plunger wall 62
and the tube wall 42. Material is substantially only able to go
past the plunger front face 60 via the check valve 76.
[0080] The complimentary geometry also helps decrease the
collection time of the middle fraction 226. Therefore, entire time
to prepare and remove the middle fraction 226 is generally about 5
to about 40 minutes. This efficiency is also assisted by the fact
that the separator 10 allows for the removal of the middle fraction
226 without first removing the plasma fraction 224, which includes
the buffy coat, and re-spinning the plasma fraction 224. Rather,
one spin in the separator 10 with the whole blood sample allows for
the separation of the buffy coat for easy extraction through the
plunger 16.
[0081] As discussed above, the separator 10 may be used to separate
any appropriate multi-component material. For example, a bone
marrow sample may be placed in the separator 10 to be centrifuged
and separated using the separator 10. The bone marrow sample may
include several fractions or components that are similar to whole
blood fractions or may differ therefrom. Therefore, the buoy 14 may
be altered to include a selected density that is dependent upon a
density of a selected fraction of the bone marrow. The bone marrow
may include a selected fraction that has a different density than
another fraction and the buoy 14 may be designed to move to an
interface between the two fractions to allow for a physical
separation thereof. Similar to the whole blood fraction, the
plunger 16 may then be moved to near a collection face 46 of the
buoy 14. The fraction that is then defined by the collection face
46 and the plunger 16 may be withdrawn, as described for the
removal of the buffy coat from the whole blood sample. For example,
the middle fraction 226 in the bone marrow sample may include a
fraction of undifferentiated or stem cells. In various embodiments,
the middle fraction 226 in a bone marrow sample may include
hematopoietic, stem cells, stromal stem cells, mesenchymal stem
cells, endothelial progenitor cells, red blood cells, white blood
cells, fibroblasts, reticulacytes, adipose cells, or endothelial
cells. In various embodiments, the middle fraction 226 is
concentrated bone marrow aspirate.
[0082] It will also be understood that mixtures of various fluids
may be separated in the separator 10. For example, a mixture of
whole blood and bone marrow may be positioned in the separator 10
at a single time. The buoy 14 may be tuned to move to an interface
that will allow for easy removal of both the buffy coat, from the
whole blood sample, and the undifferentiated cells, from the bone
marrow sample. Nevertheless, it will be understood that the
separator 10 may be used within any appropriate biological material
or other material having multiple fractions or components therein.
Simply, the buoy 14 may be tuned to the appropriate density and the
plunger 16 may be used to cooperate with the buoy 14 to remove a
selected fraction.
[0083] According to various embodiments and with reference to FIGS.
6A and 6B, a buoy system 300 is illustrated. The buoy system 300
generally includes a first buoy or fraction separator member 302
and a second buoy member or fraction separator 304. The first buoy
302 and the second buoy 304 may be operably interconnected with a
buoy system cylinder or member 306. The buoy system 300 may be
placed in a tube, such as the tube 12. The tube 12 may be formed of
any appropriate material, such as the Cryolite Med.RTM.2 as
discussed above. Nevertheless, the buoy system 300 may be designed
to fit in the tube 12 or may be formed to fit in any appropriate
member that may be disposed within a selected centrifuging device.
It will be understood that the following discussion relating to
buoy system 300 to be substantially matched to the size of the tube
12 is merely exemplary. As the buoy 14 may be sized to fit in any
appropriate tube, the buoy system 300 may also be sized to fit in
any appropriate tube. It will be further understood that the tube
12 may be any appropriate shape. The tube 12 need not only be
cylindrical but may also be or include conical portions, polygonal
portions, or any other appropriate shapes.
[0084] The first buoy 302 of the buoy system 300 may be generally
similar in geometry to the buoy 14. It will be understood that the
first buoy member 302 may be formed in the appropriate manner
including shape or size to achieve selected results. Nevertheless,
the first buoy member 302 generally includes an exterior diameter
that may be slightly smaller than the interior diameter of the tube
12. Therefore, the first buoy member 302 may be able to move within
the tube 12 during the centrifugal process. Also, as discussed
above, the tube 12 may flex slightly during the centrifuging
process, thus allowing the first buoy member 302 to include an
exterior diameter substantially equivalent to the interior diameter
of the tube 12. As discussed further herein, during the
centrifugation process, a portion of the fraction of a sample may
pass between the exterior wall of the first buoy member 302 and the
tube 12.
[0085] The first buoy member 302 may generally include a density
that is substantially equivalent to a first or selected fraction of
the sample. If the sample to be separated includes whole blood and
is desired to separate the red blood cells from the other portions
of the sample, the first buoy member 302 may have a selected
density that may be about 1.00 grams per cc (g/cc) to about 1.10
g/cc. It will be understood that the density of the first buoy
member 302 may be any appropriate density, depending upon the
fraction to be separated, and this range of densities is merely
exemplary for separating red blood cells from a whole blood
sample.
[0086] In addition, the first buoy member 302 includes a collection
face or area 308 at a proximal or upper portion of the first buoy
member 302. The collection face 308 generally defines a concave
area of the first buoy member 302 and may have a selected angle of
concavity. The buoy assembly 300 defines a central axis D. The
collection face 308 defines a surface E that is formed at an angle
.gamma. to the central axis D of the buoy system 300. The angle
.gamma. may be any appropriate angle and may be about 0.5.degree.
to about 90.degree.. The angle .gamma. may, however, be between
about 45.degree. and 89.5.degree.. Nevertheless, it will be
understood that the angle .gamma. may be any appropriate angle to
assist in collection of a selected fraction or portion of the
sample by the first buoy member 302.
[0087] A bottom or lower surface 310 of the first buoy member 302
may define a bottom face. The bottom face 310 may also be formed at
an angle D relative to the central axis D. The bottom surface 310
defines a surface or plane F that may be formed at an angle .DELTA.
relative to the central axis D of the buoy system 300. The angle
.DELTA. may be any appropriate angle and may be about 90.degree. to
about 160.degree.. For example, the angle .DELTA. may be about
15.degree.. Similarly to the buoy bottom face 48, the bottom
surface 310 defines an apex 312 that may first engage the bottom
12d of the tube 12, such that most or the majority of the bottom
surface 310 does not engage the tube 12. As illustrated further
herein, the apex 312 allows for a free space or gap to be formed
between the bottom face 310 of the first buoy member 302 and the
bottom 12b of the tube 12.
[0088] The second buoy member 304 may include an outer diameter
substantially equivalent to the outer diameter of the first buoy
member 302. Therefore, the second buoy 304 may move with the first
buoy 302, particularly if the second buoy 304 is interconnected
with the first buoy 302 with the buoy central cylinder 306.
Nevertheless, the second buoy member 304 may be allowed to move
substantially freely within the tube 12 during the centrifuging
process.
[0089] The second buoy member 304 also includes an upper or
superior surface 314 that defines a plane G that is formed at an
angle relative to the central axis D of the buoy system 300. The
angle .epsilon. of the plane G relative to the central axis D of
the buoy system 300 may be any appropriate angle. For example, the
angle .epsilon. may be about 90.degree. to about 150.degree..
Generally, the angle .epsilon. may assist in allowing a selected
fraction or a portion of the sample to pass over the top surface
314 and past the second buoy member 304 during the centrifuging
process.
[0090] The second buoy member 304 also define a bottom or inferior
surface 316 that also defines a plane H that may be formed at an
angle K relative to the central axis D of the buoy system 300. The
angle K may be any appropriate angle, such as about 90.degree. to
about 150.degree.. Nevertheless, the angle K may be substantially
complimentary to the angle .gamma. of the collection face 308 of
the first buoy member 302. For example, if the angle .gamma. is
about 80.degree., the angle K may be about 100.degree., such that
substantially 180.degree. or a straight line is formed when the
first buoy member 302 engages the second buoy member 304. This may
be for any appropriate reason, such as extraction of a fraction
that may be disposed near the collection face 308 of the first buoy
member 302. Nevertheless, the angle K may be any appropriate angle
as the angle .gamma..
[0091] The second buoy member 304 may be formed to include any
appropriate density. For example, the second buoy member 304 may
include a density that is less than the plasma fraction of a whole
blood sample. It will be understood that the second buoy member 304
may include any appropriate density and a density that is less than
the plasma fraction of a whole blood sample is merely exemplary.
Nevertheless, if a whole blood sample is desired to be separated
and the plasma sample is to be substantially separated from another
fraction, the second buoy member 304 may include a density that is
less than the plasma fraction of the whole blood sample. Therefore,
the density of the second buoy member 304 may be about 0.01 g/cc to
about 1.03 g/cc. As described herein, if the second buoy member 304
includes a density less than the plasma fraction of a whole blood
sample and the first buoy member 302 includes a density greater
than that of the red blood cells, the buoy system 300 may be
substantially positioned near an interface between the red blood
cell fraction and the plasma fraction of a whole blood sample.
Therefore, as discussed above, and further described herein, the
platelet or buffy coat fraction of the whole blood sample may be
substantially collected near or in the collection face 308 of the
buoy system 300.
[0092] The buoy post 306 may operably interconnect the first buoy
member 302 and the second buoy member 304. The buoy post 306 may be
any appropriate connection member. The buoy post need not be a
single cylindrical portion. For example the buoy post 306 may
include one or more members interconnecting the first buoy member
302 and the second buoy member 304, such as around a perimeter
thereof. In addition, the buoy post 306 may include any appropriate
shape or geometry.
[0093] The buoy system post 306 may be rigidly affixed to the first
buoy member 302 and the second buoy member 304, such that the first
buoy member 302 may not move relative to the second buoy member 304
and vice versa. Alternatively, the buoy post 306 may be slide ably
connected to either or both the first buoy member 302 and the
second buoy member 304. According to various embodiments, the buoy
post 306 is generally fixedly connected to the first buoy member
302 and slide ably interconnected to the second buoy member 304.
The buoy post 306 may include a catch portion or lip 320 that is
able to engage a portion of the second buoy member 304, such that a
range of travel of the second buoy member 304, relative to the
first buoy member 302 is limited. Nevertheless, the range of travel
of the second buoy member 304 toward the first buoy member 302 may
be substantially unlimited until the second buoy member 304 engages
the first buoy member 302.
[0094] In various embodiments, the buoy post 306 may also define a
central cannula or bore 322. The post bore 322 may include a
connection portion 324 substantially defined near an upper or a
proximal end of the buoy post 306. This may allow for
interconnection of various components with the buoy post 306, such
that various components may be moved through the bore 322 from an
exterior location. The buoy post 306 may also define a port or
cannula 326 that connects the post cannula 322 with the collection
face 308. Therefore, a substance may travel through the post
cannula 322 and through the port 326. Various substances may then
be provided to or removed from the collection face 308 of the first
buoy member 302.
[0095] In various embodiments, the buoy system 300 may be used to
separate a selected multi component sample, such as a whole blood
sample. With continuing reference to FIGS. 6A and 6B, and reference
to FIGS. 7A-7D, a method of using the buoy system 300, according to
various embodiments, is illustrated and described. With reference
to FIGS. 7A-7D, like reference numerals are used to indicate like
portions of the tube 12 and the associated mechanisms described in
FIGS. 1-3. Therefore, it will be understood that the buoy system
300 may be used with the tube 12 or any other appropriate tube or
container system or apparatus. Nevertheless, for simplicity, the
description of a method of use of the buoy system 300 will be
described in conjunction with the tube 12.
[0096] The tube 12 may include the cap 18 that further defines a
plasma valve or port 20. Extending through the cap 18 and
interconnecting with a first flexible tube or member 92, the plasma
port 20 may be used to extract a selected fraction of the sample
that is positioned above the second buoy member 304. The tube 12
may also define a second port 21, which may also be referred to as
a platelet rich plasma (PRP) port. As discussed herein, a second
flexible member, such as a flexible tube 21a, may interconnect the
PRP port 21 and a connection portion 324 of a buoy cylinder 306. As
illustrated above, the first tube 92 may also be interconnected
with a selected portion of the system, such as the top surface 314
of the second buoy member 304. As illustrated above, a valve may be
positioned and is operably interconnect the tube 92 with the upper
surface 314 of the second buoy member 304. Nevertheless, such a
valve is not necessary and it may be provided merely for
convenience.
[0097] Other portions of the blood separator system 20,
particularly those portions of the tube 12 and the cap 18 that have
various valves connected therewith may be included in the tube 12
and used with the buoy system 300. Nevertheless, once the buoy
system 300 is interconnected, it may be positioned in the interior
of the tube 12 and the syringe 204 used to place a sample into the
tube 12. The sample may be expressed from the syringe 204 into the
interior of the tube 12 and the sample may be any appropriate
sample, such as a whole blood sample. Nevertheless, it will be
understood, such as discussed above, various other samples may be
used, such as bone marrow samples, a mixture of bone marrow and
whole blood or non-biological fluids or materials. It will be
understood that two buoys 302 and 304 may generally be near one
another when the sample is positioned in the tube 12, but are
illustrated apart for clarity of the present discussion.
[0098] Also, the sample may be placed in the tube 12 according to
various embodiments. As described above, an anticoagulant or other
components may be mixed with the whole blood sample, if a whole
blood sample is used, before the whole blood sample is positioned
within the tube 12. As is apparent to one skilled in the art, an
anticoagulant or other components may be mixed with a bone marrow
aspirate sample or a sample of bone marrow aspirate and whole
blood, if such samples are used before such sample is positioned
within the tube 12. The syringe 204 is connected with the plunger
port 22 extending from the cap 18, although a plunger may not be
used in various embodiments.
[0099] After the sample is positioned within the tube 12, as
described above, a cap may be positioned over the port 22, such
that the sample is not allowed to escape from the tube 12. After
the sample is placed in the tube 12 and the cap placed on the port
22, the tube 12 including the sample and the buoy system 300 may be
centrifuged.
[0100] With reference to FIG. 7B, after a centrifugation of the
tube 12, including the buoy system 300, substantially three
fractions of the sample may be formed. A first fraction 330 may be
positioned between the bottom face 310 and the bottom of the tube
44. A middle fraction 332 may be positioned between the collection
face 308 and the bottom surface 316 of the second buoy 304. In
addition, a third fraction 334 may be positioned between the upper
surface 314 and the cap 18 of the tube 12. Generally, the first
fraction 330, the middle fraction 332, and the third fraction 334
are substantially physically separated with the buoy system 300.
During the centrifugation process, the tube 12 may flex slightly to
allow for ease of movement of the buoy system 300 through the tube
12 and the sample. Nevertheless, the buoy system 300, during the
centrifugation process, substantially creates the three fractions
330, 332, and 334 without the operation of an operator. Therefore,
the formation of at least three fractions may be substantially
simultaneous and automatic using the buoy system 300.
[0101] The buoy system 300 substantially separates the fractions
330, 332, and 334, such that they may be easily removed from the
tube 12. For example, with reference to FIG. 7C, a syringe or other
instrument 340 may be used to extract the middle fraction 332 by
interconnecting a cannula or bored tube 342 with the connection
portion 324 of the buoy cylinder 306. By drawing the plunger 344
into the extraction syringe 340, a vacuum or upward force is
produced within the extraction syringe 340. This force draws the
middle fraction 332 through the ports 326 of the buoy post 306 and
through the buoy cannula 322. Therefore, the middle fraction 332
may be extracted from the tube 12 without substantially commingling
the middle fraction 332 with either the first fraction 330 or the
third fraction 334. The middle fraction 332 is drawn in the
direction of arrow M through the cannula 322 and into the
extraction syringe 340.
[0102] It will be understood that the second tube 21a may also be
used. The extraction syringe 340 may be interconnected with the PRP
port 21 that is interconnected with the connection portion 324 of
the buoy cylinder 306. As discussed herein, the buoy cylinder
allows access to the middle fraction 332 (platelet rich portion)
between the buoy portions. Thus, it will be understood, that access
may be obtained and the middle fraction 332 (platelet rich portion
or buffy coat of the sample), between the two buoys, may be
extracted in a plurality of ways. The illustrations and method
described herein is merely exemplary. For example, if bone marrow
aspirate is used as the sample, the PRP port 21 would allow for
extraction of undifferentiated nucleated cells. In various
embodiments, the PRP port 21 allows for extraction of the buffy
coat.
[0103] Alternatively, if the post 306 is not provided other
portions may be provided to gain access to the middle fraction 332.
For example, if a plurality of members are provided around the
perimeter of the first buoy 302 and the second buoy 304 a valve
portion, such as a puncture-able valve, may be provided in the
second buoy 304 to be punctured with an object. In this way an
extraction needle may puncture the valve to gain access to the
middle fraction 332. Regardless, it will be understood that the
buoy system 300 may be able to form a plurality of fractions, such
as the three fractions 330, 332, and 334 and at least the middle
fraction 332 may be extracted without substantially commingling the
various fractions.
[0104] During the extraction of the middle fraction 332 through the
cannula 322, the second buoy member 304 may move in the direction
of arrow M toward the first buoy member 302. As described above,
the collection face 308 of the first buoy member may include an
angle .gamma. that is substantially complementary to the bottom
face 316 of the second buoy member 304. Therefore, if the second
buoy member 304 is allowed to move along the buoy cylinder 306, the
bottom face 316 of the second buoy member 304 may be able to
substantially mate with the collection face 308 of the first buoy
member 302. Alternatively, if the second buoy member 304 is not
allowed to move, the second buoy member may be provided with a vent
port or valve, such that the extraction of the middle fraction 332
from the collection face 308 may not be hindered by the buildup of
undesirable forces. Nevertheless, if the second buoy member 304 may
move, the interaction of the bottom face 316 of the second buoy
member 304 may assist in substantially removing the entire middle
fraction 332 from the tube 12. As described above, the bottom face
60 of the plunger 16 may also serve a similar purpose when engaging
the collection face 46 of the buoy 14.
[0105] With reference to FIG. 7D, once the middle fraction 332 has
been extracted from the tube 12, the second buoy member 304 may
substantially mate with a portion of the first buoy member 302. As
discussed above, the second buoy member 304 may substantially only
mate with the first buoy member 302 if the second buoy member 304
is able to substantially move relative to the first buoy member
302. Therefore, it will be understood that the second buoy member
304 need not necessarily mate with the first buoy member 302 and is
merely exemplary of an operation of various embodiments.
Nevertheless, once the middle fraction 332 has been extracted from
the tube 12, the port 20 may be used in conjunction with a selected
instrument, such as a plasma extraction syringe 212 to remove the
plasma or the third fraction 334 from the tube 12 using the
extraction tube 92 interconnected with the port 20.
[0106] As described above, the tube 92 allows for extraction of the
third fraction 334 from the tube 12 without commingling the third
fraction 334 with the remaining first fraction 330 in the tube 12.
Therefore, similar to the separator and extraction system 10, three
fractions may be substantially formed within the tube 12 with the
buoy system 300 and may be extracted without substantially
commingling the various fractions. Once the third fraction 334 is
extracted from the tube 12, the buoy system 300 may be removed from
the tube 12, such that the first fraction 330 may be removed from
the tube 12. Alternatively, the first fraction 330 may be discarded
with the tube 12 and the buoy system 300 as a disposable system.
Alternatively, the system may be substantially reusable, such that
it can be sterilized and may be sterilized for various uses.
[0107] The description of the method of use of the buoy system 300
is exemplary of a method of using a system according to various
other embodiments. It will be understood, however, that various
specifics may be used from various embodiments to allow for the
extraction of selected fractions. For example, the centrifugation
process may be substantially a single step centrifugation process.
The buoy system 300, according to various embodiments, may allow
for the formation of three fractions during a single centrifugation
process. This centrifugation process may occur at any appropriate
speed, such as about 1000 RPM to about 8000 RPM. This speed may
produce a selected gravity that may be approximately 4500 times
greater than the normal force of gravity. Nevertheless, these
specifics are not necessary to the operation of the buoy system 300
according to various embodiments. The buoy system 300, according to
various embodiments, may be used to extract a plurality of
fractions of a sample after only a single centrifuging process and
without substantially commingling the various fractions of the
sample.
[0108] With reference to FIG. 8, the blood collection and
separation system that includes the tube 12, according to various
embodiments, may be filled with a multi-component fluid or
solution, such as blood from a patient, is illustrated. The tube 12
may include any appropriate separation system, such as the
separation system 300. Nevertheless, in addition to filling the
tube 12 with a fluid from the syringe 204 any appropriate method
may be used to fill the tube 12. For example, when a solution,
including a plurality of components, is placed into the tube 12 it
may be collected directly from a source.
[0109] For example, a patient 350 may be provided. The patient 350
may be provided for a selected procedure, such as generally an
operative procedure or other procedure that requires an intravenous
connection 352, such as a butterfly needle, to be provided in the
patient 350. The intravenous connection 352 generally provides a
tube 354 extending therefrom. The tube 354 may be used to withdraw
fluids from the patient 350 or provide materials to the patient
350, such as medicines or other selected components. Nevertheless,
the intravenous connection 352 is generally provided for various
procedures and may be used to fill the tube 12.
[0110] The tube 354 may interconnect with the plunger port 22 or
any appropriate portion of the tube 12. The port 22 may be used to
connect with the tube 354 in a similar manner as it would connect
with the syringe 204, if the syringe 204 was provided.
Nevertheless, it will be understood that the tube 354 may be
provided directly to the tube 12 from the patient 350. This may
reduce the number of steps required to fill the tube 12 and reduce
possible cross-contamination from the patient 350 with the various
components. Moreover, making a connection directly with the patient
350 may make the withdrawal and collection of blood from the
patient 350 more efficient.
[0111] Once the tube 354 is interconnected with the tube 12 the
pressure differential between the patient 350, such as the
intravenous pressure of the blood, may be used to fill the tube 12
to a selected volume. In addition, a vacuum system 356 may be
provided The vacuum system 356 may include a vacuum inducing
portion or member 358, such as a resilient bulb. The vacuum
inducing member 358 may be interconnected with the tube 12 through
a selected connecting portion 360.
[0112] The vacuum connecting portion 360 may interconnect with an
orifice 362. The orifice 362 may be interconnected or extend from
the cap 18 or provided in any appropriate portion with the tube 12.
Nevertheless, a first one-way valve 364 may be provided along the
connection portion 360 or near the orifice 362. The first one-way
valve 364 provides that a flow of a fluid, such as a gas, may pass
in a first direction but not in a second. A second one-way valve
366 may also be provided downstream from the first one-way valve
364. In this way, a vacuum may be created with the vacuum inducing
member 358, such that air is drawn out of the tube 12 and removed
through the second one-way valve 366 in the direction of arrow V.
Due to the first and second one-way valves 364, 366 the air is
generally withdrawn from the tube 12 without substantially allowing
the air to flow back into the tube 12. Thus, a vacuum can be
created within the tube 12 to assist with removing a selected
volume of fluid, such as blood, from the patient 350.
[0113] Because the tube 12 may be filled substantially directly
from the patient 350, the collection of the fluid, such as blood,
may be provided substantially efficiently to the tube 12. Although
any appropriate mechanism may be used to assist in withdrawing the
blood from the patient 350 the vacuum system 356 may be provided
including the vacuum inducing member 358. Any appropriate vacuum
creating device may be used, such as a mechanical pump or the like.
Nevertheless, the tube 12 may be filled for use during a selected
procedure.
[0114] As discussed above, the tube 12 may be used to separate a
selected portion of the blood obtained from the patient 350
substantially intraoperatively. Therefore, the collection or
separation of the various components may be substantially
autologous and substantially intraoperatively. Moreover, obtaining
the fluid directly from the patient 350 may increase the efficiency
of the procedure and the efficiency of the intraoperative or the
operative procedure.
[0115] With reference to FIG. 9, the separator 10 may be used to
separate any appropriate material. The material may be separated
for any purpose, such as a surgical procedure. For example, a
selected fraction of a bone marrow aspirate or a bone marrow
portion may be produced with the separator 10 according to various
embodiments. The selected fraction of the bone marrow aspirate may
include various components, such as undifferentiated cells. The
various undifferentiated cells may be positioned in a selected
scaffold or relative to a selected portion of a patient for
providing a volume of the undifferentiated cells to the patient. As
known to those skilled in the art, a selected portion of the
patient may include bone, cartilage, connective tissue, or any
other tissue. Also as known by those skilled in the art, a selected
portion of the patient may include a defect in bone, cartilage,
connective tissue and/or any other tissue. It will be understood
that the method described according to FIG. 9 is merely exemplary
of various embodiments that may be used to provide a selected
fraction of a bone marrow aspirate or other material to a patient
or selected position. The selected portion may be placed on the
scaffold in any appropriate manner, such as by spraying, dipping,
infiltrating, or any appropriate method.
[0116] A method of selecting or creating a selected fraction of a
bone marrow aspirate in a selected scaffold according to a method
400 is illustrated in FIG. 9. Generally, the method 400 may start
in block 402 in obtaining a bone marrow aspirate volume. The bone
marrow aspirate (BMA) may be obtained in any selected or generally
known manner. For example, a selected region of bone, such as a
portion near an operative procedure, may be used to obtain the bone
marrow aspirate. Generally, an accessing device, such as a syringe
and needle, may be used to access an intramedullary area of a
selected bone. The BMA may then be withdrawn into the syringe for
various procedures. Once a selected volume of the BMA is obtained
in block 402, the BMA may be positioned in the separator 10
according to various embodiments in block 404. The BMA may be
positioned in any appropriate separator, such as those described
above including the separator 10. Once the BMA is positioned in the
separator 10, a selected fraction of the BMA may be separated from
the BMA in block 406.
[0117] BMA is a complex tissue comprised of cellular components
(that contribute to bone growth) including red and white blood
cells, their precursors and a connective tissue network termed the
stroma. Bone marrow stromal cells or mesenchymal stem cells have
the potential to differentiate into a variety of identifiable cell
types including osteoblasts, fibroblasts, endothelial cells,
reticulocytes, adipocytes, myoblasts and marrow stroma. The
selected fraction of the BMA may include undifferentiated cells or
any appropriate portion of the BMA. The fractionation or separation
of various fractions of the BMA may allow for a volume of BMA to be
taken from a single location and the separation or concentration of
the selected portion may be performed in the separator 10.
Generally, obtaining a small volume of the selected portion from a
plurality of locations may be used to obtain an appropriate volume
of BMA or selected fraction of the BMA. Nevertheless, the separator
10 may allow for separating a selected volume from a single
location from which the BMA is obtained. This may reduce the time
of a procedure and increase the efficiency of obtaining the
selected fraction of the BMA.
[0118] In addition to obtaining a volume of the BMA in block 402, a
volume of whole blood may be obtained in block 408. The volume of
blood obtained in block 408, according to any appropriate
procedure, including those described above, may then be positioned
in the separator 10, in block 410. The whole blood may be
positioned in any appropriate separator, such as those described
above or a separator to separate a selected fraction of the whole
blood. As described above, the whole blood may be separated into an
appropriate fraction, such as a fraction including a platelet
portion or buffy coat. The whole blood may be separated into
selected fractions in block 412. It will be understood that the BMA
and the whole blood volume may be obtained substantially
simultaneously or consecutively in block 402 and 408. In various
embodiments, concentrated BMA, such as the middle fraction 332, 226
comprising nucleated cells in separator 10 has a concentration of
nucleated cells that is at least 4 times the concentrate of
nucleated cells in BMA. Similarly, the selected fractions of the
BMA obtained in block 406 and whole blood obtained in block 412 may
also be performed substantially sequentially or simultaneously. For
example, the separator 10 including the volume of the BMA may be
positioned in a separating device, such as a centrifuge,
substantially opposite, so as to balance, the separator 10
including the volume of the whole blood. Therefore, a single
separation, such as centrifuge procedure may be used to separate
both the BMA and the whole blood into selected fractions. This
again may increase the efficiency of the procedure to provide both
a selected fraction of the BMA and a selected fraction of the whole
blood substantially simultaneously.
[0119] The selected fractions of the BMA and the whole blood,
provided in block 406 and 412 may be harvested in block 414. The
selected fractions of the BMA and the whole blood, may be harvested
in block 414 for appropriate purposes, such as those described
herein. The separator 10 may be used to obtain the selected
fractions of the BMA and the whole blood, through various
procedures, such as those described above.
[0120] A plurality of separators 10 may be used to obtain a larger
quantity of the selection fractions and such quality of selected
fractions may be pooled together. In various embodiments, the BMA
aspirate can be concentrated alone or in combination with whole
blood. In various embodiments, whole blood may be added to the
separator 10, and the resulting buffy coat fraction (middle
fraction 332, 226) may not only contain the at least 4 times
greater concentration of nucleated cells from bone marrow, and may
include at least 5 times greater concentration of white blood cells
from the whole blood and at least 8 times greater concentration in
platelets from the whole blood. In addition, circulating stem cells
from whole blood may be concentrated with the mature white blood
cells in the buffy coat.
[0121] After harvesting the selected fractions of the BMA and the
whole blood in block 414, the selected fraction of the BMA may be
positioned on an appropriate scaffold in block 416. The scaffold in
block 416 may be any appropriate scaffold, such as synthetic bone
substitutes or allergenic tissue. Examples of a scaffold include,
but are not limited to, bone, cartilage, bone substrates, ceramics,
biopolymers, collagens, metal, and the like. The scaffolds may be
used for appropriate procedures, such as hard or soft tissue
grafting, including uses in non-union or chronic wounds. The
undifferentiated cells of the BMA may allow for a substantial
source of cells for use during a substantially natural healing
after an operative procedure, for example, the natural healing of a
patient may use the supplied undifferentiated cells. In such a
natural healing of a patient, the undifferentiated cells may be
applied to a wound, a defect, a graft site, a bone, cartilage,
connective tissue. and the like. Therefore, the scaffold may be
positioned in a selected portion of the anatomy and the cells may
be allowed to grow and differentiate into selected portions in the
implanted position.
[0122] It is well known to those skilled in the art that bone
marrow contains hematopoietic and mesenchymal stems cells that are
precursors for most of the cells found within the body. In general,
the hematopoietic cells, along with angiogeneic growth factors,
such as those found in platelets, will promote angiogenesis.
Angiogenesis is a necessary stage in wound healing of most tissues
and treatment of ischemia. The mesenchymal stem cells are the cells
responsible for the formation of bone, tendon, ligament, articular
cartilage, muscle, fat, intervertebral discs, meniscus, skin, and
any other structural tissue found in the body. In various
embodiments, a concentration and delivery of these precursor cells,
with or without platelet concentrate, will improve therapeutic uses
over the native bone marrow.
[0123] In addition to positioning the selected fractioning of the
BMA and the scaffold in block 416, the platelets of the whole blood
may be positioned on or near the scaffold of block 418. The
platelets of the whole blood fraction positioned in the scaffold of
block 418 may assist the undifferentiated cells and the anatomy
into which the scaffold is positioned to allow for a substantially
efficient and complete healing. The platelet rich fraction of the
whole blood sample may include various healing and growth factors
that may assist in providing an efficient and proper healing in the
anatomy. Therefore, the undifferentiated cells of the BMA, or other
selected fraction obtained from the separation of the BMA, and the
selected fraction of the whole blood, obtained from the separator,
may be used with the scaffold to provide a substantially efficient
implant.
[0124] In some embodiments, harvest block 414 may be combined with
a growth factors which may include any of the well-known growth
factors such as Platelet-Derived Growth Factor (PDGF), Transforming
Growth Factor Beta (TGF-.beta.), Insulin-Like Growth Factor (IGF),
Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF),
Vascular Endothelial Growth Factor (VEGF), Bone Morphogenetic
Proteins (BMPs), and vectors for gene therapy. In various
embodiments, harvest block 414 may be combined with cellular
solutions, suspensions, and materials including osteoblasts,
osteoprogenitor cells, chondroblasts, stem cells, or fibroblasts
may also be used, as may solutions or suspensions containing other
therapeutic agents such as antibiotics, analgesics, pharmaceutical
agents, antithrombinolytics, or chemotherapeutic agents. In various
embodiments, the buffy coat, platelet fraction, and/or
undifferentiated cell fraction (the middle fraction 332, 226) may
be combined with an activator such as thrombin solutions and the
like. In various embodiments the middle fraction 332, 226 may be
used alone for cartilage repair. In various embodiments, the
platelet rich fraction may be used to fill a cartilage defect with
a fibrin matrix, or it may be mixed with other cell sources such as
autologous chondrocytes, synovial cells, bone marrow cells, or to
mix with the blood clot formed during microfracture. In addition,
the separator 10, or any appropriate separator, such as that
described above, may allow for a substantially quick and efficient
separation of the BMA and the whole blood into an appropriate
fraction for use in the procedure. Other examples include pooling
whole blood and/or BMA from different sites of the anatomy or from
different sources.
[0125] In various embodiments, the concentrated bone marrow cells
can also be included with a carrier to aide in delivery and to help
maintain the cells' location after implantation. Examples of
carriers can include fibrin, concentrated fibrin, demineralized
bone matrix, gelatin, collagen, porous calcium based ceramics,
porous metal, synthetic fiber matrices, or resorbable matrices. In
addition, carriers can be made from other autogeneic, allogenic,
and xenogeneic tissues.
[0126] After the selected portion of the BMA and the whole blood
are positioned on the scaffold in blocks 416 and 418 the scaffold
may be implanted in block 420. As described above, the scaffold may
be implanted in any appropriate position in the block 420 for
various procedures. It will be understood that the scaffold may be
implanted for any appropriate procedure and may allow for
positioning the selected portion of the BMA, such as
undifferentiated cells, and the selected portion of the whole
blood, such as platelets, relative to a selected portion of the
anatomy. The scaffold may allow for a bone ingrowth, such as
allowed with the undifferentiated cells, to assist in healing of a
selected portion of the anatomy. In various embodiments,
concentrated bone marrow aspirate can be used in articular
cartilage repair. The middle fraction 332, 226 can be added to a
focal defect or to an osteoarthritic defect. The defects can be in
any joint that contains articular cartilage. In various
embodiments, the middle fraction 332, 226 can promote the formation
of repair tissue. In addition, these cells can be added to a
microfracture technique in order to increase the mesenchymal cells
present in the defect and increase the amount and quality of repair
tissue that forms. In various embodiments, the middle fraction 332,
226 can be delivered in one of the carriers listed above. In
various embodiments, for delivery to cartilage, the middle fraction
332, 226 in an autologous fibrin or concentrated fibrin is
activated with an activating solution so that it forms a 3-D gel in
situ and holds the middle fraction 332, 226 within the cartilage
defect. In various embodiments, the concentrated bone marrow can
also be used in meniscus repair. The concentrated bone marrow can
be used to fill a tear with the meniscus, or in can be used to soak
a graft used to replace the meniscus after a full or partial
meniscectomy. In various embodiments, concentrated bone marrow
aspirate used in combination with platelet rich plasma can also be
used for repair of meniscus. In various embodiments, concentrated
bone marrow aspirate can be used to repair bone. In various
embodiments, the middle fraction 332, 226 can used alone, or mixed
with an appropriate carrier such as demineralized bone matrix,
calcium based ceramics, fibrin, or concentrated fibrin. The defects
in bone could be found in long bones, cranium, sternum, and spine.
One specific placement of concentrated bone marrow aspirate would
be to deliver the undifferentiated cells to a freeze dried
demineralized bone product (such as the Bonus DBM product from
Biomet Biologics) under vacuum. In various embodiments, the
concentrated bone marrow can infiltrate the graft, and the plasma
in the bone marrow may hydrate the bone matrix and create an
injectable carrier. The undifferentiated cells may have
differentiating growth factors included within the demineralized
bone to stimulate cartilage and bone formation. In various
embodiments, the concentrated bone marrow can be delivered to the
patient with a growth factor that will induce proliferation,
chemotaxis, and/or morphogeneis. Examples of growth factors that
could be used include PDGF, TGF-b, IGF, VEGF, EGF, CTGF, FGF, and
any of the BMPs.
[0127] The description of the invention is merely exemplary in
nature and, thus, variations that do not depart from the gist of
the invention are intended to be within the scope of the invention.
Such variations are not to be regarded as a departure from the
spirit and scope of the invention.
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