U.S. patent application number 10/176272 was filed with the patent office on 2003-11-06 for methods and apparatus for isolating platelets from blood.
Invention is credited to Dorian, Randel, King, Scott R., Storrs, Richard W..
Application Number | 20030205538 10/176272 |
Document ID | / |
Family ID | 29407799 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030205538 |
Kind Code |
A1 |
Dorian, Randel ; et
al. |
November 6, 2003 |
Methods and apparatus for isolating platelets from blood
Abstract
A platelet collection device comprising a centrifugal
spin-separator container with a cavity having a longitudinal inner
surface. A float in the cavity has a base, a platelet collection
surface above the base, an outer surface. The float density is
below the density of erythrocytes and above the density of plasma.
The platelet collection surface has a position on the float which
places it below the level of platelets when the float is suspended
in separated blood. During centrifugation, a layer of platelets or
buffy coat collects closely adjacent the platelet collection
surface. Platelets are then removed from the platelet collection
surface. Movement of a float having a density greater than whole
blood through the sedimenting erythrocytes releases entrapped
platelets, increasing the platelet yield.
Inventors: |
Dorian, Randel; (San Diego,
CA) ; King, Scott R.; (San Francisco, CA) ;
Storrs, Richard W.; (San Francisco, CA) |
Correspondence
Address: |
William B. Walker
W.B. Walker Consultants
805 Morning Sun Drive
Encinitas
CA
92024
US
|
Family ID: |
29407799 |
Appl. No.: |
10/176272 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60377559 |
May 3, 2002 |
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60379951 |
May 10, 2002 |
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60382639 |
May 21, 2002 |
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Current U.S.
Class: |
210/787 ;
210/109; 210/121; 210/512.1; 210/518; 210/789 |
Current CPC
Class: |
B01L 2400/0633 20130101;
A61K 35/00 20130101; B01D 21/26 20130101; B01D 21/0012 20130101;
B01L 3/50215 20130101; G01N 33/49 20130101; Y10T 436/25375
20150115; A61M 2202/0427 20130101; B01D 21/0012 20130101; B01D
21/262 20130101; A61M 1/3693 20130101; B01D 2221/10 20130101; B01L
2200/026 20130101; B01D 21/26 20130101 |
Class at
Publication: |
210/787 ;
210/789; 210/109; 210/121; 210/512.1; 210/518 |
International
Class: |
B01D 021/26 |
Claims
The invention claimed is:
1. A blood platelet separation device comprising a centrifugal
spin-separator container having a cavity with a longitudinal inner
surface; a float positioned within the cavity, the float having a
base and a platelet collection surface above the base, the float
having an outer surface; the float having a density less than the
density of erythrocytes and greater than the density of plasma; and
the platelet collection surface positioned on the float at a level
which places it immediately below the level of platelets after
centrifugation.
2. A blood platelet separation device of claim 1 wherein the cavity
has a cylindrical inner surface and the float has a cylindrical
outer surface.
3. A platelet separation device of claim 2 wherein the distance
between the outer surface of the float and the inner surface of the
tube is less than 0.5 mm.
4. A blood platelet collection device of claim 1 comprising a
flexible inner tube having an inner surface and a float positioned
within the flexible inner tube; the float having an outer surface
in sealing engagement with the inner surface of the flexible tube
in a neutral pressure condition, the sealing engagement preventing
movement of fluid between the outer surface of the float and the
inner surface of the flexible tube in a neutral pressure condition;
the outer surface of the float disengaging from contact with the
inner surface of the flexible tube in an elevated pressure
condition, thus enabling movement of fluid between the outer
surface of the float and the inner surface of the flexible tube in
the elevated pressure condition and movement of the float within
the cylinder; the float having a platelet receptor cavity with a
platelet collection surface positioned immediately below the level
of the platelet layer in separated blood after centrifugation; and
the float having a channel communicating with the platelet receptor
cavity for removing separated platelets after centrifugation.
5. A platelet collection device of claim 4 wherein the float
comprises a proximal segment having a distal surface and a distal
segment having a proximal surface opposed to the distal surface,
the distal surface and the proximal surfaces defining the platelet
receptor cavity.
6. A platelet collection device of claim 5 wherein the outer
container includes a port for introducing blood into the inner tube
at the beginning of a platelet separation process and for removing
platelets from the inner tube at the end of the platelet separation
process.
7. A platelet collection device of claim 6 wherein the port
includes a syringe coupling Luer locking device.
8. A platelet collection device of claim 4 wherein the outer
container has an inner surface for restraining expansion of the
inner tube during centrifugation.
9. A blood platelet separation device of claim 1 wherein the outer
surface of the float is in sliding engagement with the inner
surface of the cavity.
10. A blood platelet separation device of claim 9 wherein the float
comprises a unitary structure with a proximal segment having a
distal surface and a distal segment having a proximal surface
opposed to the distal surface, the distal surface and the proximal
surfaces defining a platelet receptor cavity, the upper surface of
the platelet receptor cavity defining the platelet collection
surface.
11. A platelet collection device of claim 10 wherein the
centrifugal spin-separator is a substantially rigid tube, and the
float comprises a proximal segment having a distal surface, and a
distal segment having a proximal surface opposed to the distal
surface, the distal surface and the proximal surfaces defining the
platelet receptor cavity.
12. A platelet collection device of claim 1 wherein a top surface
of the float constitutes the platelet collection surface, and the
device includes a plunger positioned above the float and
substantially axially concentric with the float and the cavity, the
plunger having a cylindrical outer surface which is spaced from the
inner surface of the cavity; the bottom of the plunger defining a
plasma expressing surface opposed to the platelet collection
surface; and a fluid removal passageway extending through the
plunger to the plasma expressing surface.
13. A platelet collection device of claim 12 having at least one
seal between the outer surface of the plunger and the inner surface
of the cavity, the seal being positioned in sealing engagement with
the outer and inner surfaces.
14. A platelet collection device of claim 11 wherein the top of the
float includes a stop surface positioned above the plasma
collection surface.
15. A platelet collection device of claim 10 wherein the
centrifugal spin-separator is a substantially rigid tube, and the
float has a cylindrical outer surface, the float comprising a
proximal segment having a distal surface, and a distal segment
having a proximal surface opposed to the distal surface, the distal
surface and the proximal surfaces defining the platelet receptor
cavity.
16. A process for separating platelets from whole blood with a
centrifugal spin-separator container having a cavity with a
longitudinal inner surface, a proximal end and a distal end,
including a float positioned within the cavity, the float having a
base and a platelet collection surface above the base, the float
having an outer surface, the distance between the outer surface of
the float and the inner surface of the tube being less than 0.5 mm,
the float having a density less than the density of erythrocytes
and greater than the density of plasma, the platelet collection
surface has a position on the float which places it immediately
below the level of platelets when the float is suspended in fully
separated blood, the process comprising the steps of a) introducing
an amount of whole blood into the cavity, the amount of whole blood
being sufficient to position the level of platelets following
centrifugation at the position of the platelet collection surface,
b) subjecting the centrifugal spin-separator container to
centrifugation forces in the axial direction toward the distal end,
whereby erythrocytes are caused to concentrate at the distal end,
plasma to collect toward the proximal end, and platelets to collect
closely adjacent the platelet collection surface, and c) removing
platelets from the platelet collection surface.
17. A process of claim 16 wherein the float has a density greater
than whole blood, and under the centrifugal forces, the float moves
toward the proximal end through the sedimenting erythrocytes,
releasing entrapped platelets to allow them to collect closely
adjacent the platelet collection surface, whereby the amount of
platelets available for removal is increased.
18. A process of claim 16 wherein the device includes a plunger
positioned above the float and substantially axially concentric
with the float and the cavity, the plunger having a cylindrical
outer surface which is spaced from the inner surface of the cavity;
the bottom of the plunger defining a plasma expressing surface
opposed to the platelet collection surface; and a fluid removal
passageway extending through the plunger through the plasma
expressing surface, wherein the process includes the steps of a)
introducing an amount of whole blood into the cavity, the amount of
whole blood being sufficient to position the level of platelets
following centrifugation at the position of the platelet collection
surface, b) subjecting the centrifugal spin-separator container to
centrifugation forces in the axial direction toward the distal end,
whereby blood cells are caused to concentrate at the distal end,
plasma to collect toward the proximal end, and platelets to collect
on the platelet collection surface, c) advancing the plunger in an
axial direction against the top of the plasma until the plasma
expressing surface is positioned near the platelet collection
surface and spaced apart therefrom, d) extending a platelet
extraction tube through the fluid removal passageway until the end
thereof contacts the platelet layer; and e) removing a platelet
concentrate through the platelet extraction tube.
19. A process of claim 18 wherein the top of the float includes a
stop surface positioned above the plasma collection surface and the
plunger is advanced in an axial direction until the plasma
expressing surface contacts the stop surface.
20. A process for separating platelets from whole blood with a
flexible inner tube having an inner surface, a proximal end, and a
distal end; a float positioned within the flexible inner tube; the
float having an outer surface in sealing engagement with the inner
surface of the flexible tube in a neutral pressure condition, the
sealing engagement preventing movement of fluid between the outer
surface of the float and the inner surface of the flexible tube in
a neutral pressure condition; the float having a platelet receptor
with a platelet collection surface immediately below the level of
the platelets in separated blood after centrifugation; the process
comprising the steps of a) introducing an amount of whole blood
into the inner tube under elevated pressure, the amount of whole
blood being sufficient to position the level of platelets following
centrifugation closely adjacent the level of the platelet support
surface, b) subjecting the tube to centrifugation forces in the
axial direction toward the distal end, causing the outer surface of
the float to disengage from the inner surface of the flexible tube,
enabling movement of fluid between the outer surface of the float
and the inner surface of the flexible tube and movement of the
float within the tube; can causing blood cells to concentrate at
the distal end, plasma to collect at the proximal end, and
platelets to collect at a level closely adjacent the platelet
collection surface; and c) removing platelets from the platelet
collection surface.
21. A process of claim 20 wherein the inner tube is positioned in a
rigid outer tube coaxial therewith, the outer tube having an inner
surface for limiting expansion of the inner tube wall during
centrifugation.
22. A process for separating platelets from whole blood by
centrifugation in a container having a proximal end and a distal
end, including a float positioned within the cavity, the float
having a density less than the density of erythrocytes and greater
than the density of whole blood, the process comprising the steps
of a) introducing whole blood into the cavity, b) subjecting the
container to centrifugation forces in the axial direction toward
the distal end, whereby erythrocytes are caused to concentrate at
the distal end, plasma to collect toward the proximal end, and
platelets to collect in a platelet layer between the erythrocyte
and the plasma collections, wherein during application of the
centrifugal forces, the float moves through the sedimenting
erythrocytes, the movement of the float releasing entrapped
platelets, allowing them to collect in the platelet layer, and c)
removing platelets from the platelet collection surface.
Description
BACKGROUND
[0001] 1. Field
[0002] The present invention concerns apparatuses and methods for
rapid fractionation of blood into erythrocyte, plasma and platelet
fractions. Each fraction may be put to use or returned to the blood
donor. Useful high concentration platelet fractions have platelet
concentrations in excess of two times the concentration in
anti-coagulated whole blood before processing of greater than
2.times.10.sup.6 platelet/.mu.L. The invention has particular value
for rapid preparation of autologous concentrated platelet fractions
to help or speed healing.
[0003] 2. Description of the Prior Art
[0004] Blood may be fractionated and the different fractions of the
blood used for different medical needs. For instance, anemia (low
erythrocyte levels) may be treated with infusions of erythrocytes.
Thrombocytopenia (low thrombocyte (platelet) levels) may be treated
with infusions of platelet concentrate.
[0005] Under the influence of gravity or centrifugal force, blood
spontaneously sediments into three layers. At equilibrium the top,
low-density layer is a straw-colored clear fluid called plasma.
Plasma is a water solution of salts, metabolites, peptides, and
many proteins ranging from small (insulin) to very large
(complement components). Plasma per se has limited use in medicine
but may be further fractionated to yield proteins used, for
instance, to treat hemophilia (factor VIII) or as a hemostatic
agent (fibrinogen).
[0006] The bottom, high-density layer is a deep red viscous fluid
comprising anuclear red blood cells (erythrocytes) specialized for
oxygen transport. The red color is imparted by a high concentration
of chelated iron or heme that is responsible for the erythrocytes
high specific gravity. Packed erythrocytes, matched for blood type,
are useful for treatment of anemia caused by, e.g., bleeding. The
relative volume of whole blood that consists of erythrocytes is
called the hematocrit, and in normal human beings can range from
about 38% to about 54%.
[0007] The intermediate layer is the smallest, appearing as a thin
white band on top the erythrocyte layer and below the plasma, and
is called the buffy coat. The buffy coat itself has two major
components, nucleated leukocytes (white blood cells) and anuclear
smaller bodies called platelets (or thrombocytes). Leukocytes
confer immunity and contribute to debris scavenging. Platelets seal
ruptures in the blood vessels to stop bleeding and deliver growth
and wound healing factors to the wound site.
[0008] Extraction of Platelets
[0009] Extraction of platelets from whole blood has been reviewed
(Pietersz 2000). In transfusion medicine the intention is to
transfuse each patient only with the component that is needed, so
the aim of blood centers is to manufacture blood components as pure
as possible, that is with the least contaminating cells. Platelets
are the most difficult to isolate and purify. Based on data from
Pietersz (2000), even under optimal conditions of centrifugation
(long time at low speed), a significant fraction of platelets
remain trapped within the sedimented erythrocytes.
[0010] Through the years centrifugation methods have been developed
to separate the platelets from red blood cells, white blood cells
and plasma. These methods separate the components both in plastic
bag systems and in apheresis devices, and more recently in
specialized apparatuses. Historically most platelet concentrates
have been harvested from donors and used to treat thrombocytopenia,
i.e., allogenically. More recently the platelet concentrates have
been used to promote wound healing, and the use of autologous
platelet concentrates (sequestration of platelets for treatment of
the platelet donor) has grown.
[0011] The sedimentation of the various blood cells and plasma is
based on the different specific gravity of the cells and the
viscosity of the medium. This may be accelerated by centrifugation
according approximately to the Svedberg equation:
V=(({fraction
(2/9)}).omega..sup.2R(d.sub.cells-d.sub.plasma)r.sup.2)/.eta-
..sub.t
[0012] where
[0013] V=sedimentation velocity,
[0014] .omega.=angular velocity of rotation,
[0015] R=radial distance of the blood cells to the center of the
rotor,
[0016] d=specific gravity,
[0017] r=radius of the blood cells,
[0018] .eta.=viscosity of the medium at a temperature of t.degree.
C.
[0019] Characteristics of blood components are shown in the
table.
1 Diameter Specific gravity Component (.mu.m) (g/ml) Deformability
Adhesion Red cells 5.4 1.100 +++ - Granulocytes 9.6 1.085 + ++
Lymphocytes 7.6 1.070 .+-. .+-. Monocytes 11.2 1.063 + + Platelets
3.2 1.058 + +++ Plasma NA 1.026 NA NA Additive NA 1.007 NA NA
solution
[0020] When sedimented to equilibrium, the component with the
highest specific gravity (density) eventually sediments to the
bottom, and the lightest rises to the top. But the rate at which
the components sediment is governed roughly by the Svedberg
equation; the sedimentation rate is proportional to the square of
the size of the component. In other words, at first larger
components such as white cells sediment much faster than smaller
components such as platelets; but eventually the layering of
components is dominated by density.
[0021] Soft Spin Centrifugation
[0022] When whole blood is centrifuged at a low speed (up to 1,000
g) for a short time (two to four minutes) white cells sediment
faster than red cells and both sediment much faster than platelets
(per Svedberg equation above). At higher speeds the same
distribution is obtained in a shorter time. This produces layers of
blood components that are not cleanly separated and consist of (1)
plasma containing the majority of the suspended platelets and a
minor amount of white cells and red cells, and (2) below that a
thick layer of red cells mixed with the majority of the white cells
and some platelets. The method of harvesting platelet-rich plasma
(PRP) from whole blood is based on this principle. The term
"platelet-rich" is used for this component because most of the
platelets in the whole blood are in the plasma following slow
centrifugation so the concentration of platelets in the plasma has
increased. Centrifugal sedimentation that takes the fractionation
only as far as separation into packed erythrocytes and PRP is
called a "soft spin". "Soft spin" is used herein to describe
centrifugation conditions under which erythrocytes are sedimented
but platelets remain in suspension. "Hard spin" is used herein to
describe centrifugation conditions under which erythrocytes
sediment and platelets sediment in a layer immediately above the
layer of erythrocytes.
[0023] Two Spin Platelet Separation
[0024] Following a soft spin, the PRP can removed to a separate
container from the erythrocyte layer, and in a second
centrifugation step, the PRP may be fractioned into platelet-poor
plasma (PPP) and platelet concentrate (PC). In the second spin the
platelets are usually centrifuged to a pellet to be re-suspended
later in a small amount of plasma.
[0025] In the most common method for PRP preparation, the
centrifugation of whole blood for 2 to 4 min at 1,000 g to 2,500 g
results in PRP containing the majority of the platelets. After the
centrifugation of a unit (450 ml) of whole blood in a 3-bag system
the PRP is transferred to an empty satellite bag and next given a
hard spin to sediment the platelets and yield substantially
cell-free plasma. Most of the platelet poor plasma (PPP) is removed
except for about 50 ml and the pellet of platelets is loosened and
mixed with this supernatant. Optionally one can remove about all
plasma and reconstitute with additive solution. To allow aggregated
platelets to recover the mixture is given a rest of one to two
hours before platelets are again re-suspended and then stored on an
agitator.
[0026] It is believed that centrifugation can damage the platelets
by sedimenting the platelets against a solid, non-physiological
surface. The packing onto such a surface induces partial activation
and may cause physiological damage, producing "distressed"
platelets which partially disintegrate upon resuspension.
[0027] Hard Spin Centrifugation
[0028] If the centrifugation is continued at a low speed the white
cells will sediment on top of the red cells whereas the platelets
will remain suspended in the plasma. Only after extended low speed
centrifugation will the platelets also sediment on top of the red
cells.
[0029] Experiments with a blood processor (deWit, 1975) showed that
centrifugation at a high speed (2,000 g-3,000 g) produces a similar
pattern of cell separation in a shorter time. Initially the cells
separate according to size, i.e., white cells sediment faster than
red cells and platelets remain in the plasma. Soon the red cells
get `packed` on each other squeezing out plasma and white cells.
Because of their lower density, white cells and platelets are
pushed upwards to the interface of red cells and plasma whereas the
platelets in the upper plasma layer will sediment on top of this
interface, provided the centrifugal force is sufficiently high and
sedimentation time is sufficiently long. Plasma, platelets, white
cells and red cells will finally be layered according to their
density. Platelets sedimented atop a layer of red cells are less
activated than those isolated by the "two spin" technique.
[0030] Platelet Yields and Centrifuge Speed
[0031] The so called "buffy coat" consists of the layers of
platelets and white cells (leukocytes) but is usually harvested
along with the lower part of the plasma layer and the upper layer
of the red cell mass. In this application, all references to the
platelet layer are intended to mean the platelet layer if no
leukocytes are present or to the buffy coat layer when leucocytes
are present mixed with the platelets.
[0032] The process and method of this invention can accomplish
platelet isolation and collection with a wide range including both
low and high centrifugation forces. Effective separation does not
require a high g centrifugation; good results have been obtained
with 600 g-1000 g or low speed centrifugation. High speed
centrifugation refers to centrifugal forces greater than 2000 g.
Experiments have shown that long (30 -45 min) centrifugation at a
force of about 700 g gives the most complete separation of whole
blood into components. Such long times are not considered to be
practical and economical for intra-operative autologous
applications. For buffy coat separation one can spin 7 to 10 min at
about 3,000 g to enable separation of whole blood into cell-free
plasma, a buffy coat containing 60 -70% of the white cells and
70-80% of the platelets, and red cells contaminated with
approximately 30% of the white cells and 10-20% of the
platelets.
[0033] Apheresis-single Spin Platelet Separation
[0034] Specialized apparatuses have been invented to perform
apheresis, the separation of platelets from blood while reinfusing
the other components into the donor. This permits donors to give
more platelets than possible with the two-step centrifugation
because loss of erythrocytes limits the volume of blood that blood
donors may give. Typically, a two to three hour apheresis procedure
will produce a platelet product containing 3.times.10.sup.11
platelets, equivalent to 6 or more conventional blood
donations.
[0035] The first demonstration of a single-step method for
preparation of platelet concentrates was reported more than 25
years ago (deWit 1975). In this first attempt complete separation
between the different cellular components could not be achieved, at
least not in one step because of considerable overlap in the
presence of platelets, leukocytes and erythrocytes in the fractions
collected after different centrifugation times and speed. Many
improved apheresis methods and devices have been developed and are
described in cited patents.
[0036] In apheresis methods drawn blood is immediately mixed with
an anticoagulant, centrifuged (Haemonetics, Baxter CS 3000 and
Amicus, Cobe Spectra, Fresenius AS 104, AS 204), and separated into
components according to density. The buffy coat is recognized by
eye or by optical sensors and the platelet-rich layer is directed
to a separate bag. Software of the various manufacturers has been
adjusted to manufacture platelet concentrates without white cell
contamination, some requiring additional filtration after the
platelet harvest, others having special techniques or tools built
into the apheresis systems.
[0037] Leukoreduction
[0038] The PC's resulting from both laboratory two spin processing
and apheresis methods contain donor leukocytes. It was shown the
white cells negatively affect platelet storage and may induce
adverse effects after transfusion due to cytokine formation.
Removal of leukocytes (leukoreduction) from PRP and PC is a major
problem because non-self leukocytes (allogeneic leukocytes) and the
cytokines they produce can cause a violent reaction by the
recipient's leukocytes. In 1999 the FDA Blood Product Advisory
Committee recommended routine leukoreduction of all non-leukocytes
components in the US (Holme 2000). Therefore, much of the prior art
focuses on leukoreduction of platelet concentrates because
non-autologous leukocytes excite deleterious immune reactions.
Since the process of this invention provides a convenient way to
quickly harvest autologous platelets from the patient's blood,
immune reactions are not a risk, and the presence of leukocytes is
of little or no concern.
[0039] Autologous Platelets
[0040] Autologous platelets have been shown to have advantages in
comparison with allogeneic platelets. Concerns about disease
transmission and immunogenic reactions, which are associated with
allogeneic or xenogeneic preparation, are minimized. The fact that
an autologous preparation is prepared at the time of surgery
reduces the risks associated with mislabeling a sample, which might
occur through a laboratory system. The use of autologous platelets
obviates the requirement for time-consuming screening tests.
Platelet activation has less time to develop. Unlike stored
platelets which become partially activated, the activation status
of autologous platelets, when first produced, was found to be
similar to that in the original whole blood (Crawther 2000).
[0041] Platelets may be used as an adjunct for wound healing.
Knighton describes applying autologous platelet releasate to wounds
to enhance healing (Knighton 1986). More recent studies use
platelets themselves. Marx describes platelet preparations that
dramatically accelerate bone healing following dental implant
procedures (Marx 1998). Other researchers make similar claims for
other medical procedures, for instance, treatment of macular holes
(Gehring 1999), improved healing in cosmetic surgery (Man 2001),
and use for hemostasis (Oz 1992).
[0042] In recent years devices originally invented to wash
erythrocytes from shed blood (autotransfusion devices) have been
adapted to permit separation of autologous platelets, usually
intraoperatively. This procedure has the important advantage that
autologous leukocytes cause no reaction from patient leukocytes
because they are self leukocytes, so removal of leukocytes from
PC's is no longer important. For example, sequestration of PRP
reduces allogeneic transfusion in cardiac surgery (Stover 2000).
Autotransfusion devices from a variety of manufacturers (e.g.,
ElectroMedics 500) can be used to make autologous platelet
preparations with high platelet concentrations.
[0043] The autotransfusion equipment used to make autologous
platelet concentrates requires a skilled operator and considerable
time and expense. Most devices require a large prime volume of
blood. The ElectroMedics 500 withdraws 400 to 450 ml of autologous
whole blood through a central venous catheter placed during
surgery. As it withdraws the blood the separator adds citrate
phosphate dextrose (CPD) to achieve anticoagulation. The blood is
then centrifuged into its three basic components. The red blood
cell layer forms at the lowest level, the platelet concentrate
layer in a middle level, and the PPP layer at the top. The cell
separator incrementally separates each layer, from the less dense
to the more dense; therefore it separates PPP first (about 200 ml)
and PC second (about 70 ml), leaving the residual red blood cells
(about 180 ml). Once the PPP is removed, the centrifuge speed is
lowered to 2400 RPM to allow for a precise separation of the PC
from the red blood cells. In fact, the platelets most recently
synthesized, and therefore of the greatest activity, are larger and
mix with the upper 1 mm of red blood cells, so that this layer is
included in the PRP product imparting a red tint.
[0044] Recently devices have been introduced which are specifically
designed to make autologous platelet concentrates intraoperatively;
for example the SmartPReP Autologous Platelet Concentrate System
(Harvest Autologous Hemobiologics, Norwell, Mass.). It requires 90
to 180 cc of blood versus the 500 cc of blood used in most
autotransfusion machines. In addition two other products are near
market introduction, The PlasmaSeal device (PlasmaSeal, San
Francisco, Calif.) and The Platelet Concentrate Collection System
(Implant Innovations, Inc., Palm Beach Gardens, Fla.). While these
devices have somewhat reduced the cost and the time required, a
skilled operator is required for the devices introduced to the
market to date. Therefore, there remains a need for simple and fast
automated methods and devices for making platelet concentrates.
SUMMARY OF THE INVENTION
[0045] The present invention is directed to methods and apparatuses
for simple and fast preparation of autologous platelet concentrates
from whole anti-coagulated blood.
[0046] This discussion includes numerous descriptions of events
within the spinning rotor. Within the frame of reference of the
rotor, the effects of gravity are minimal compared with centrifugal
force. Therefore within the rotor, "top" means the end of the tube
closer to the axis and "bottom" means the end of the tube closer to
the perimeter of the rotor.
[0047] Another aspect of the present invention is that platelets
are not aggregated by pelleting against a surface.
[0048] A further aspect of the invention is the use of a float
having a density less than the density of the erythrocytes and
greater than that of whole blood which rises through the mixture as
the erythrocyte sediment during centrifugation, gently disrupting
the erythrocytes to free trapped platelets, thus greatly increasing
the platelet yield.
[0049] Another aspect of the present invention is that the
apparatuses may be completely automated and require no user
intervention between, first, loading and actuating the device and,
second, retrieving the platelet concentrate.
[0050] Another aspect of the present invention is that different
quantities of blood may be processed by the same apparatus.
[0051] Another aspect of the present invention is that bloods of
different hematocrits and different plasma densities may be
processed by the same apparatus.
[0052] Another aspect of the present invention is that the
concentration of platelets in the product may be varied by
need.
[0053] Another aspect of the present invention is that the
processing includes only a single centrifugation step.
[0054] Another aspect of the present invention is that the
processing is rapid.
[0055] The float collector blood platelet separation device of this
invention comprises a centrifugal spin-separator container having a
separation chamber cavity with a longitudinal inner surface. A
float is positioned within the cavity, the float having a base, a
platelet collection surface above the base, and an outer surface.
The distance between the outer surface of the float and the inner
surface of the cavity can be 0.5 mm, preferably less than 0.2 mm
and optimally less than 0.03 mm. The float has a density less than
the density of erythrocytes and greater than the density of plasma.
The platelet collection surface has a position on the float which
places it immediately below the level of platelets when the float
is suspended in fully separated blood. The cavity can have a
cylindrical inner surface and the float has a complementary
cylindrical outer surface.
[0056] In one embodiment, the device includes a flexible inner
tube, and a float is positioned within the flexible inner tube. The
float has an outer surface in sealing engagement with the inner
surface of the flexible tube in a neutral pressure condition, the
sealing engagement preventing movement of fluid between the outer
surface of the float and the inner surface of the flexible tube in
the neutral pressure condition. The outer surface of the float
disengages from contact with the inner surface of the flexible tube
in an elevated pressure condition, thus enabling movement of fluid
between the outer surface of the float and the inner surface of the
flexible tube in the elevated pressure condition as well as free
movement of the float within the tube. The float has a platelet
receptor cavity positioned to be at the position of platelets in
separated blood after centrifugation. The float has a channel
communicating with the platelet receptor cavity for removing
separated platelets therefrom after centrifugation. In one
configuration, the float comprises a proximal segment having a
distal surface and a distal segment having a proximal surface
opposed to the distal surface, the distal surface and the proximal
surfaces defining the platelet receptor cavity. Preferably, the
outer container includes a port for introducing blood into the
inner tube at the beginning of a platelet separation process and
for removing platelets from the platelet cavity within the inner
tube at the end of the platelet separation process. Optionally, the
port includes a syringe coupling Luer locking device. The outer
container can have an inner surface for restraining expansion of
the inner tube during centrifugation.
[0057] In a still further embodiment, the centrifugal
spin-separator is a substantially rigid tube, and the float
comprises a proximal segment having a distal surface, and a distal
segment having a proximal surface opposed to the distal surface,
the distal surface and the proximal surfaces defining the platelet
receptor cavity. This cavity has a surface which is a platelet
collection surface. The outer surface of the float is preferably in
sliding engagement with the inner surface of the cavity.
[0058] The term "platelet collection surface", as used herein, is
defined to mean a surface which provides support to the platelet or
buffy coat layer. Preferably, the platelet layer is not in direct
contact with the support layer to protect the platelets, and
optimally, the platelets are sedimented on a thin buffer or cushion
layer of erythrocytes resting on the platelet collection
surface.
[0059] In another embodiment, a top surface of the float
constitutes a platelet collection surface. In this form, the device
may include a plunger positioned above the float and substantially
axially concentric with the float and the cavity, the optional
plunger having a cylindrical outer surface which is spaced from a
complementary cylindrical inner surface of the tube. The space can
be so small as to provide an effective liquid seal between the
surfaces, or if the space is larger, at least one seal can be
provided between the outer surface of the plunger and the inner
surface of the cavity, the seal being positioned in sealing
engagement with the outer and inner surfaces. Optionally, the
bottom of the plunger has a plasma expressing surface opposed to
the platelet collection surface; and a fluid removal passageway
extends through the plunger and the plasma expressing surface into
the platelet receptor cavity. Preferably, the top of the float
includes a stop surface extending above the plasma collection
surface.
[0060] The process of this invention for separating platelets from
whole blood with the above devices comprises the steps of first
introducing an amount of whole blood into the cavity, the amount of
whole blood being sufficient, following centrifugation, to elevate
the float above the floor of the separation chamber and position
the platelet collection surface immediately below the level of
platelets. The separation chamber is the cavity within which the
blood is separated into erythrocyte, plasma and platelet (buffy
coat) layers. The centrifugal spin-separator container is subjected
to centrifugation forces in the axial direction toward the distal
end, whereby erythrocytes are caused to concentrate at the distal
end, plasma to collect toward the proximal end, and platelets to
collect on the platelet collection surface. Platelets are then
removed from the platelet collection surface.
[0061] When the device includes a plunger positioned above the
float and substantially axially concentric with the float and the
cavity, process of this invention comprises the steps of
introducing an amount of whole blood into the cavity, the amount of
whole blood being sufficient to position the level of platelets
following centrifugation at the position of the platelet collection
surface. The centrifugal spin-separator container is then subjected
to centrifugation forces in the axial direction toward the distal
end, whereby blood cells are caused to concentrate at the distal
end, plasma to collect toward the proximal end, and platelets to
collect closely adjacent the platelet collection surface. The
plunger is then advanced in an axial direction against the top of
the plasma until the plasma expressing surface is positioned
closely adjacent the platelet collection surface and spaced apart
therefrom. A platelet extraction tube is extended through the fluid
removal passageway until the end thereof contacts the platelet
layer, and a platelet concentrate is removed through the platelet
extraction tube. Optionally, platelet poor plasma can be collected
through the platelet extraction tube into a syringe or other
receptacle while the plunger is being depressed. Platelets can then
be extracted into a separate syringe or other receptacle.
[0062] Optionally, the device can lack a plunger arrangement. In
this embodiment, platelets are removed from the platelet collection
surface suspended in a small volume of plasma retained after first
removing a volume of platelet poor plasma from above the sedimented
platelet layer.
[0063] With embodiments of the device wherein the top of the float
includes a stop surface positioned above the plasma collection
surface, the plunger is advanced in an axial direction until the
plasma expressing surface contacts the stop surface.
[0064] With devices having a float in a flexible tube, the process
comprises the steps of introducing an amount of whole blood into
the inner tube, the amount of whole blood being sufficient,
following centrifugation, to elevate the float above the floor of
the separation chamber and position the platelet collection surface
immediately below the level of platelets. The tube is then
subjected to centrifugation forces in the axial direction toward
the distal end, whereby blood cells are caused to concentrate at
the distal end, plasma to collect at the proximal end, and
platelets to collect at a level closely adjacent the platelet
collection surface. Platelets are then removed from the annular
platelet receptor cavity.
[0065] When the top surface of the float constitutes the platelet
collection surface, the device optionally includes a plunger
positioned above the float and substantially axially concentric
with the float and the cavity. The plunger has a cylindrical outer
surface which is spaced from the inner surface of the cavity; the
bottom of the plunger defining a plasma expressing surface opposed
to a platelet collection surface. A fluid removal passageway
extends through the plunger to the plasma expressing surface. With
this embodiment, the process includes the additional step of moving
the plunger toward the float until the plasma expressing surface is
closely adjacent the platelet layer, and platelets are then removed
through the fluid removal passageway. In this embodiment, plasma is
expressed through the fluid removal passageway as the plunger is
moved toward the float.
BRIEF DESCRIPTION OF DRAWINGS
[0066] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0067] FIG. 1 is a schematic cross-sectional drawing of a
separation device of this invention.
[0068] FIG. 2 shows device of FIG. 1 wherein the plunger of the
syringe is elevated, and the syringe barrel is filled with
blood.
[0069] FIG. 3 shows device of FIG. 1 after the plunger is depressed
to a position forcing the blood into the inner tube.
[0070] FIG. 4 shows device of FIG. 1 after a portion of the blood
has passed between float and the inner tube, filling the bottom of
the inner tube.
[0071] FIG. 5 shows device of FIG. 1 after the blood has separated
into the erythrocyte fraction within which the float rests, the
plasma fraction above the float, and the buffy coat or platelet
layer in the receptor cavity.
[0072] FIG. 6 shows device of FIG. 1 after a fresh syringe has been
connected to the Luer port.
[0073] FIG. 7 shows device of FIG. 1 with the syringe plunger
elevated after drawing platelets from the receptor cavity into the
barrel of the syringe.
[0074] FIG. 8 is an isometric view of an alternative float design
for the separation device of FIG. 1, the bottom of the float having
a hemispherical shape.
[0075] FIG. 9 is a schematic cross-sectional view of a
plunger-float embodiment of the invention.
[0076] FIG. 10 is a schematic cross-sectional view of the
embodiment of FIG. 9 after introduction of anticoagulated blood
into the separation chamber.
[0077] FIG. 11 is a schematic cross-sectional view of the
embodiment of FIG. 9 after centrifugal separation of the blood into
erythrocyte, plasma and platelet layers.
[0078] FIG. 12 is a schematic cross-sectional view of the
embodiment of FIG. 9 after insertion of a syringe needle.
[0079] FIG. 13 is a schematic cross-sectional view of the
embodiment of FIG. 9 after depression of the plunger to a level
which abuts the float stop.
[0080] FIG. 14 is a schematic diagram view of an alternate
embodiment related to the embodiment of FIG. 9 showing the
introduction of blood through a separate fill port of the
embodiment.
[0081] FIG. 15 is a schematic diagram of a still further alternate
embodiment of a plunger-float device of this invention, including a
flexible snorkel tube fixed to the cap.
[0082] FIG. 16 is a schematic cross-sectional view of a
plunger-float embodiment of the invention after centrifugation and
before removal of the platelet layer.
DETAILED DESCRIPTION
[0083] This invention is a blood platelet separation device with
several embodiments. All of the embodiments comprise a centrifugal
spin-separator container having a cavity with a longitudinal inner
surface. A float is positioned within the cavity. The float has a
base and a platelet collection surface above the base. The float
has an outer surface. In general, the distance between the outer
surface of the float and the inner surface of the cavity can be
less than 0.5 mm, preferably less than 0.2 mm and optimally less
than 0.03 mm. For embodiments with a flexible tube, the surfaces
can be in contact. The platelet collection surface has a position
on the float which places it immediately below the level of
platelets when the float is suspended in fully separated blood.
[0084] Patient blood may be obtained by a phlebotomy needle or
central vein cannula or other whole blood collection means. The
blood is immediately mixed with anticoagulant, such as ACD-A or
heparin.
[0085] FIG. 1 is a schematic cross-sectional drawing of a
separation device of this invention. The blood platelet collection
device of this embodiment comprises a flexible inner tube 2 having
an inner surface 4 and a float 6 positioned within the flexible
inner tube. The float has an outer surface 8 in sealing engagement
with the inner surface of the flexible tube when the tube is under
neutral pressure. In this condition, the sealing engagement
prevents movement of fluid between the outer surface of the float
and the inner surface of the flexible tube.
[0086] The outer surface 8 of the float 6 disengages from contact
with the inner surface 4 of the flexible tube 2 when the pressure
in the flexible tube is elevated under centrifugation. This enables
movement of fluid between the outer surface of the float and the
inner surface of flexible tube as well as free movement of the
float within the tube.
[0087] The float has a platelet receptor cavity 10 with a platelet
collection surface 16 in a position to immediately below the level
of platelets in separated blood following centrifugation. The float
6 has a platelet collection channel 11 and a platelet withdrawal
channel 12 communicating with the platelet receptor cavity 10 for
removing separated platelets after centrifugation.
[0088] The float 6 comprises a proximal segment 13 having a distal
surface 14 and a distal segment 15 not having proximal surface 16
opposed to the distal surface 14 . The distal surface 14 and
proximal surface 16 define the platelet receptor cavity 10. The
float 6 has a specific gravity that is less then the specific
gravity of erythrocytes and greater than the specific gravity of
plasma such that at equilibrium the buffy coat platelet layer is
sequestered between the upper and lower members of the float. For
optimum platelet recovery, it is critical that the float rise from
the bottom of the tube as the erythrocytes sediment. This requires
that the float have a density greater than whole blood.
[0089] The platelet collection device of this embodiment includes a
substantially inflexible outer container 18 enclosing the inner
tube 2. The inner surface 22 of the outer container 18 limits
expansion of the inner tube as the pressure in the inner tube 2
increases during centrifugation.
[0090] The outer container includes a port 20 for introducing blood
into the inner tube at the beginning of the platelet separation
process and for removing platelets from the platelet receptor
cavity 10 through channels 11 and 12 at the end of the platelet
separation process. The port can be provided with a Luer lock
device for coupling with a loading syringe and with a platelet
removal syringe.
[0091] Vent channel 17 vents air upward through channel 12 as blood
is introduced into the separation channel 19.
[0092] In this embodiment, the needle or small tube 23 is
preferably fixed to the Luer lock device 20. The tube 23 has an
outer diameter which is smaller than the inner diameter of the
channel 12 to enable it to slide freely in the channel 12 as the
float 6 rises during centrifugation.
[0093] The device of this invention can be used in a simple
operation to produce platelets. It involves the collection of blood
containing an anticoagulant such as heparin, citrate or EDTA in a
syringe; filling the separation tubes with the anti-coagulated
blood from the syringe; centrifugation to separate the blood into
erythrocyte, plasma and platelet buffy coat fractions; and removal
of the platelets buffy coat fraction with another syringe.
[0094] The float can be made of two cones, the bases thereof
optionally concave. The separation chamber cavity preferably has a
concave bottom which mirrors the shape of the lower cone so that
when the buoy is in its initial state, resting at the bottom of the
cavity, there is a small space between the bottom of the lower buoy
and the bottom of the cavity. The flexible tube 2 is preferably an
elastomer sleeve having an inner diameter which is smaller than the
greatest outer diameter of the float so that it holds the float
firmly in place. The outer diameter of the flexible tube 2 is
smaller than the inner diameter of the rigid cylinder 18 so that a
space exists between the inner tube and the rigid cylinder. Small
particles such as smooth spheres, e.g., ball bearings, can be
provided in the space between the two cones to disperse platelets
in the platelet buffy coat layer. The channel 12 terminates
slightly above the base of float 6. A sterile vent 21 allows air to
pass in and out of the device.
[0095] FIGS. 2-7 are sequential schematic cross-sectional drawings
of the device of this invention at the different phases of the
separation process.
[0096] FIG. 2 shows the plunger 24 of the syringe 26 elevated, and
the syringe barrel 28 is filled with blood 30.
[0097] FIG. 3 shows the plunger 24 depressed to a position forcing
the blood 30 into the inner tube 2.
[0098] FIG. 4 shows the position after a portion of the blood 30
passes between float and the inner tube 2 during centrifugation,
filling the bottom 32 of the inner tube.
[0099] FIG. 5 shows the blood components after centrifugation for a
sufficient time to separate the blood components into the
erythrocyte fraction 34 in which the float 6 floats, the plasma
fraction 36 above the float 6, and the buffy coat or platelet layer
38 in the receptor cavity 10. Upon cessation of centrifugation, the
blood remains fractionated into its three components, and the
position of these components remains the same relative to the
float. No longer under pressure produced by centrifugal force, the
elastomer sleeve 2 has shrunk away from rigid cylinder 18 and locks
the buoy in place.
[0100] Surprisingly, with the current invention, a much smaller
fraction of platelets remain associated with the erythrocyte pack,
making higher yields of sequestered platelets possible. The float
rising from the bottom of the device as erythrocytes sediment
fluidizes the erythrocyte pack to release the platelets so they
more readily rise to combine with the buffy coat.
[0101] If resuspension particles are present in the platelet
receptor, the entire device can be shaken or rotated so that the
particles tumble around within the space between the two cones,
disrupting and mixing the buffy coat into a homogeneous suspension.
Alternatively, the platelets can be re-suspended by jetting in and
out of the platelet-containing compartment with the collection
syringe. Alternatively, an air bubble can be trapped within or
introduced into the platelet-containing compartment, and the
platelets can be re-suspended by shaking, inverting or rolling the
device. The suspended buffy coat is then withdrawn though the Lehr
20. The removed volume is displaced by air which enters the device
through vent 21.
[0102] FIG. 6 shows the device with a fresh syringe 40 locked to
the Luer port 20.
[0103] FIG. 7 shows the syringe plunger 42 elevated after drawing
platelets 46 from above the platelet collection surface 16 in the
receptor cavity 10 into the barrel 44 of the syringe. The suspended
platelet layer has been withdrawn through the Luer 20. The removed
volume is replaced by air which enters the device through a sterile
vent 21 and further into the platelet receptor 10. The syringe 40
containing the platelets 46 is then removed for provision of the
platelets to the physician treating a patient (not shown).
[0104] FIG. 8 is an isometric view of the float component 90 with a
platelet receptor 92, a vent channel 93 extending to the interior
of the collection tube 96, and a platelet drainage channel 94
extending from the platelet receptor 92 to the interior of
collection tube 96. This embodiment has a hemi-spherical bottom 98.
The cylinder preferably has a concave bottom which mirrors the
hemi-spherical bottom 98 so that when the buoy is in its initial
state, resting near the bottom of the cylinder, space between the
float and bottom are minimized. The projection 97 extending from
the bottom of the hemispherical bottom 98 insures that a space is
maintained between the bottom of the lower buoy and the bottom of
the cylinder to prevent vacuum sticking of the float to the bottom
of the tube.
[0105] FIG. 9 is a schematic cross-sectional view of a
plunger-float embodiment of the invention. In this embodiment, an
axially concentric float 100 and plunger 102 are contained within
the central cavity 103 of the rigid tube or cylinder 104 with a cap
106. The cap 106 has a vent hole 108 for permitting movement of air
into and out of the tube when blood is added or platelets are
removed. It includes a Luer port 110 for receiving a needle or tube
used for introducing blood into the cylinder and for removing fluid
from the cylinder.
[0106] The float 100 has a bottom surface 112 with a projecting
spacer 113 which rests on the bottom 114 of the tube before
anti-coagulated blood is introduced into the separator. The float
has an upper surface 115 which is positioned to be immediately
below the layer of platelets in separated blood. The upper
structure of the float includes a projection 116, the top edge 118
of which acts as a stop to limit downward movement of the plunger
102 during the process. A platelet collection channel 120 is
positioned in the center of the float. Platelet drainage channels
122 extend from the level of the surface 115 to the interior of the
platelet collection channel 120.
[0107] The float 100 has a density less than separated erythrocytes
and greater than plasma so that it will float on the erythrocyte
layer at a level which places the platelet collection surface 115
immediately below the platelet layer when the blood is separated
into its components. For optimum platelet recovery, it is critical
that the float rise from the bottom of the tube as the erythrocytes
sediment. This requires that the float have a density greater than
whole blood.
[0108] The plunger 102 optionally can have an outer surface 124
which is spaced from the inner surface 126 of the tube 104 or in
sliding engagement therewith. In the illustrated embodiment, seals
128 and 130 which can be O-rings are provided to prevent escape of
liquid between the float and tube surfaces when the plunger 102 is
moved toward the float 100. If the tolerances between the outer
surface 124 and the tube surface 126 are sufficiently small, no
seal is required to prevent escape of liquid between the plunger
and the tube when the plunger is moved toward the float and when
the product is withdrawn.
[0109] The plunger has a bottom surface 132 and a fluid escape or
snorkel tube 134. When the plunger is moved downward toward the
float, the pressure imparted by this bottom surface 132 expresses
liquid below the plunger 102 upward through the snorkel tube 134
into the cavity above the plunger.
[0110] The plunger is provided with a central channel 136 through
which a tube or needle is inserted to remove platelet-rich fluid
from the space between the bottom of the plunger and the top of the
float.
[0111] While this embodiment is illustrated with an outer tube and
a float and plunger with matching outer cylindrical shapes, it will
be readily apparent to a person skilled in the art that the outer
container can have any internal shape which matches the dimensions
of the float and plunger such as a cavity with a square or other
polygonal shape combined with a float and plunger with the
corresponding outer polygonal shape. The cylindrical configuration
is advantageous.
[0112] FIG. 10 is a schematic cross-sectional view of the
embodiment of FIG. 9 after introduction of anti-coagulated blood
138 into the separation chamber 103 through a tube 140 from a
syringe 142.
[0113] FIG. 11 is a schematic cross-sectional view of the
embodiment of FIG. 9 after centrifugal separation of the blood into
erythrocyte 144, plasma 146 and platelet 148 layers. The float 100
has risen to place the platelet collection surface 115 immediately
below the level of the platelets 148.
[0114] FIG. 12 is a schematic cross-sectional view of the
embodiment of FIG. 9 after insertion of a syringe needle 150 of
syringe 143 into the central channel 136 of plunger 102.
[0115] FIG. 13 is a schematic cross-sectional view of the
embodiment of FIG. 9 after depression of the plunger 102 by
pressing the syringe 143 downward, to a level which contacts its
lower surface 132 with the stop tip 118 of the float 100. The
plasma displaced by the plunger 102 has been expressed through the
snorkel tube 134.
[0116] Withdrawal of the piston 150 of the syringe 143 draws a
platelet-rich mixture from the platelet layer through the channels
122 and 120 (FIG. 9) and upward through tube 150 into the syringe
tube 145. The position of the snorkel tube 134 above the liquid
level provides for flow of air to fill the space created by removal
of the platelet suspension.
[0117] FIG. 14 is a variation of the embodiment shown in FIG. 9,
with the addition of an optional port 151. This view shows blood
154 introduced through port 151 from syringe 142, flowing down
channel 136 into the separation chamber 103.
[0118] FIG. 15 is a schematic cross-sectional view of a
plunger-float embodiment of the invention. In this embodiment, an
axially concentric float 160 and plunger 162 are contained within
the separation chamber cavity 163 of rigid tube or cylinder 164
with a cap 166. The float has a platelet collection surface 165
which is positioned to be immediately below the layer of platelets
in separated blood. The cap 166 has a vent hole 168 for permitting
the escape of air from the tube when it blood is added to its
interior. It also has a Luer 170 which receives a needle or tube
for introducing blood into the separation chamber 163 and another
needle or tube for removing fluid containing platelets from closely
adjacent the platelet collection surface 165 following
centrifugation.
[0119] The float 160 has a bottom surface 172 which rests on the
bottom 174 of the tube before anti-coagulated blood is introduced
into the separator. The upper structure of the float includes a
projection 176, the top edge 178 of which acts as a stop to limit
downward movement of the plunger 162 during the process. A platelet
collection channel 180 in the center of the float communicates with
platelet drainage channel 182 extending from the level of the
surface 165.
[0120] The float 160 has a density less than separated erythrocytes
and greater than plasma so that it will float in the erythrocyte
layer at a level which places the platelet collection surface 165
immediately below the platelet layer when the blood is separated
into its components. For optimum platelet recovery, it is critical
that the float rise from the bottom of the tube as the erythrocytes
sediment. This requires that the float have a density greater than
whole blood.
[0121] As the plunger 162 is depressed toward the float 160 after
centrifugation, plasma rises through the flexible snorkel tube 188
into the space 186 above the plunger 162. When platelets are
removed by a tube extending through the central channel 184
(inserted as shown in FIG. 13), air flows through the tube 188 from
its inlet at the top of the tube (above the liquid level) to
replace the liquid being removed.
[0122] The plunger is shown at its highest level to permit
introducing a maximum amount of blood into the separation chamber,
the maximum height being limited by the top 190 of the tube 192
abutting the cap 166. This full extension is permitted by the
flexibility of the snorkel tube 188.
[0123] FIG. 16 is a schematic cross-sectional view of a
plunger-float embodiment of the invention after centrifugation and
before removal of the platelet layer. In this embodiment, a float
200 is contained within the central cavity 202 of the rigid tube or
cylinder 204 with a cap 206. The cap 206 has a vent hole 208 for
permitting movement of air into and out of the tube when blood is
added or platelets are removed. It includes a port 210 for
receiving a needle or tube used for introducing blood into the
cylinder and for removing fluid from the cylinder and a port 212
for receiving a syringe needle (not shown) for collecting platelets
following centrifugation.
[0124] The float 200 has a bottom surface 214 with a projecting
spacer 216 which rests on the bottom of the tube before
anti-coagulated blood is introduced into the separator. The float
rises in the erythrocyte layer during centrifugation. The float 200
has an upper surface 218 which is positioned to be immediately
below the layer of platelets in separated blood. The upper
structure of the float includes a projection 220 which extends
above the platelet or buffy coat layer.
[0125] The float 200 has a density less than separated erythrocytes
and greater than plasma so that it will float in the erythrocyte
layer at a level which places the platelet collection surface 218
immediately below the platelet layer when the blood is separated
into its components. For optimum platelet recovery, it is critical
that the float rise from the bottom of the tube as the erythrocytes
sediment. This requires that the float have a density greater than
whole blood.
[0126] The "Plungerless plunger" device of FIG. 16 is the simplest
and cheapest to manufacture. The user can vary the platelet
concentration factor simply by removing more or less platelet poor
plasma before resuspending the platelets. It requires more user
attention and care to accurately remove the desired amount of
platelet poor plasma. The parasol float system of FIG. 1 and the
plunger-float system of FIG. 9 provide better reproducibility than
the simple float embodiment of FIG. 16.
[0127] This invention is further illustrated by the following
specific, but non-limiting examples.
EXAMPLE 1
Parasol Float Device
[0128] A parasol design platelet concentrator device of the type
depicted in FIG. 1 was constructed. The float was comprised of
polyethylene and polycarbonate in such proportion as to have an
overall density of 1.06 g/ml. The outer diameter of the float was
2.62 cm and its overall length was 4.57 cm. The float together with
two stainless steel balls 0.32 cm in diameter in the platelet
receptor cavity was inserted into the sealed end of a flexible
silicone rubber tube. The flexible tube had an inner diameter of
2.54 cm, a wall thickness of 0.08 cm, and a sealed distal end. The
flexible tube containing the float was housed within a rigid
polycarbonate tube with inner diameter of 2.86 cm and length 11.43
cm. The top of the flexible tube was folded over the top of the
rigid tube and a cap with a 7.62 cm tube 23 (see FIG. 1) was fitted
over the folded top of the flexible tube with tube 23 engaging
channel 12.
[0129] The device was filled with 30 ml of freshly drawn whole
blood anti-coagulated with CPDA-1. The device was centrifuged in an
IEC Centra CL2 centrifuge for 30 minutes at 3000 rpm. Following
centrifugation the tube was swirled vigorously to resuspend the
platelets within the platelet receptor cavity by the agitation
induced by the stainless steel balls. Five cc concentrated
platelets was removed from the platelet receptor cavity through the
platelet extraction tube (23).
[0130] Platelet counts were determined as follows: One half cc of
this sample was diluted with 10 cc of Isoton II isotonic diluent
and centrifuged at 500 g for 1.5 minutes. One half cc of this
diluted sample was diluted in yet another 10 cc of Isoton II and
particles larger than 3 fl counted on a Coulter Z-1 particle
analyzer. This result was compared to the number of particles in a
similarly treated sample of whole blood. These small particles from
treated samples represent the platelets. The sample of concentrated
platelets contained 66% of the platelets present in the introduced
whole blood at a concentration 2.86 times that found in the whole
blood.
[0131] The "Parasol" device shown in FIG. 1 is most difficult and
expensive to manufacture, but is easiest to use. The erythrocyte
concentration is more variable with this product. This results from
different plasma densities, and hematocrit-dependant variability is
present in the amount of displacement of fluid by contraction of
the elastomeric sleeve during deceleration.
EXAMPLE 2
Plunger-float Device with Snorkel
[0132] A platelet concentrator device of the type depicted FIG. 9
was constructed. The float was comprised of polyethylene and
polycarbonate in such proportion as to have an overall density of
1.08 g/ml. The outer diameter of the float was 2.535 cm and its
overall length was 1.2 cm. The float was inserted into a rigid
polycarbonate tube with an inner diameter of 2.540 cm and length
11.43 cm. The bottom of the rigid tube was sealed.
[0133] The device was filled with 25 cc of freshly drawn whole
blood anti-coagulated with CPDA-1. The device was centrifuged in an
IEC CRU 5000 centrifuge for 15 minutes at 1800 rpm. Following
centrifugation the plunger was depressed by inserting a blunt
hypodermic needle connected to a 10 cc syringe through the central
access port until it collided with the stop on the top of the
float. The device was swirled vigorously to resuspend the platelets
within the platelet receptor cavity after withdrawing 0.5 cc
through the hypodermic needle (platelet extraction tube). An
additional 3.5 cc concentrated platelets was removed from the
platelet receptor cavity through the hypodermic needle (platelet
extraction tube).
[0134] One half cc of this sample was diluted with 10 cc of Isoton
II isotonic diluent and centrifuged at 500 g for 1.5 minutes. One
half cc of this diluted sample was diluted in yet another 10 cc of
Isoton II and particles larger than 3 fl counted on a Coulter Z-1
particle analyzer. This result was compared to the number of
particles in a similarly treated sample of whole blood. These small
particles from treated samples represent the platelets. The sample
of concentrated platelets contained 69% of the platelets present in
the introduced whole blood at a concentration 4.30 times that found
in the whole blood.
[0135] The "Plunger" device shown in FIG. 9 has advantage of being
cheap to manufacture and having less variability in percent
erythrocytes in the product. The plunger-float combination provides
a greater concentration factor because the volume between plunger
and float can be smaller and still accommodate the entire range of
plasma densities while keeping the level of the buffy coat within
the gap. Erythrocyte contamination is independent of
hematocrit.
EXAMPLE 3
Plunger-float Device without Snorkel
[0136] A platelet concentrator device of the type depicted in FIG.
9 was constructed, except without the snorkel tube so that the only
fluid communication between the space below the plunger and the
space above the plunger was through a platelet receptor cavity. The
float was comprised of polyethylene and polycarbonate in such
proportion as to have an overall density of 1.08 g/ml. The outer
diameter of the float was 2.535 cm and its overall length was 1.2
cm. The float was inserted into a rigid polycarbonate tube with an
inner diameter of 2.540 cm and length 11.43 cm. The bottom of the
rigid tube was sealed.
[0137] The device was filled with 25 cc of freshly drawn whole
blood anti-coagulated with CPDA-1. The device was centrifuged in an
IEC CRU 5000 centrifuge for 15 minutes at 1800 rpm. Following
centrifugation, the plunger was depressed by inserting a blunt
hypodermic needle connected to a 10 cc syringe through the central
access port and pressing down on the body of the syringe until it
collided with the stop on the top of the float. As the syringe body
was depressed, platelet poor plasma collected in it. The syringe
containing platelet poor plasma was removed and a second syringe
was attached to the needle. The device was swirled vigorously to
resuspend the platelets within the platelet receptor cavity after
withdrawing 0.5 cc through the hypodermic needle (platelet
extraction tube). An additional 3.5 cc concentrated platelets was
removed from the platelet receptor cavity through the hypodermic
needle (platelet extraction tube).
[0138] One half cc of this sample was diluted with 10 cc of Isoton
II isotonic diluent and centrifuged at 500 g for 1.5 minutes. One
half cc of this diluted sample was diluted in yet another 10 cc of
Isoton II and particles larger than 3 fl counted on a Coulter Z-1
particle analyzer. This result was compared to the number of
particles in a similarly treated sample of whole blood. These small
particles from treated samples represent the platelets. The sample
of concentrated platelets contained 74% of the platelets present in
the introduced whole blood at a concentration 4.61 times that found
in the whole blood. Since this concentration is much larger and the
concentration of platements in the erythrocyte layer is much lower
than obtained with simple centrifugation under comparable
conditions without the float, it is clear that the flow of
erythrocyte suspension between the walls of the float and the tube
during centrifugation gently disrupts the erythrocytes and releases
entrapped platelets, allowing them to collect in the platelet or
buffy-coat layer.
[0139] With the "Plunger" device without snorkel used in this
example, the platelet poor plasma is collected in a syringe during
depression of the plunger. This provides all the advantages of
"standard" plunger device plus providing platelet poor plasma in
syringe for anyone who might want to use it, for example, as a
hemostat.
[0140] Various alternative configurations of the device are
possible within the context of the present invention. For example,
the two cones which comprise the buoy can be replaced by funnels or
by cones possessing concavities that communicate between the
various compartments and conduct sedimenting cells between
compartments during sedimentation. Complete fluid isolation of the
various compartments is not essential, provided any openings
between compartments are sufficiently small as to prevent
substantial mixing of the fractions during handling and
resuspension and withdrawal of the buffy coat. Means can be
provided for recovery of platelet depleted plasma and erythrocytes
if desired. The tube and the channel through which blood is
introduced and the buffy coat is withdrawn need not be concentric
or rigid. The elastomeric sleeve can be replaced by a compressible
material, e.g., foam, provided the inner surface which contacts
blood is smooth and does not trap or activate platelets.
* * * * *