U.S. patent number 3,852,194 [Application Number 05/314,270] was granted by the patent office on 1974-12-03 for apparatus and method for fluid collection and partitioning.
This patent grant is currently assigned to Corning Glass Works. Invention is credited to Anthony R. Zine, Jr..
United States Patent |
3,852,194 |
Zine, Jr. |
December 3, 1974 |
APPARATUS AND METHOD FOR FLUID COLLECTION AND PARTITIONING
Abstract
Partitioning assemblies and partitioning or seal members,
utilized with containers (adapted to serve as fluid specimen
collection or fluid-retaining tubes) for effecting partitioning of
two differing-density fluid phases of a centrifugally separated
fluid specimen, at a position not lower than the fluid phase
interface, wherein the partitioning members include a separating
amount of a gel-like material. This gel-like material, by having a
specific gravity intermediate those of the separated fluid phases,
is adapted to move within the container in response to centrifugal
force, only to the vicinity of the fluid phase interface. The
gel-like material thereupon is further adapted to make a
transversely continuous semi-rigid contact seal with an annular
portion of the container inner surface to thereby effect a seal
that partitions the fluid phases. The gel-like material may also be
used in combination with a spool member having a
container-contacting outer surface and a central axial orifice,
with the gel-like material making a transversely-continuous contact
seal within the spool central axial orifice. Three-phase
partitioning may also be accomplished by using first and second
gel-like materials having specific gravities intermediate those of
the first-second and second-third differing-density phases,
respectively. The partitioning or seal members may also be utilized
in closed system (evacuated) fluid collection tubes or may be hand
inserted into opened (atmospheric pressure) tubes after specimen
collection. Also set forth is a method for effecting partitioning
of centrifugally separated fluid phases within a container.
Inventors: |
Zine, Jr.; Anthony R. (Corning,
NY) |
Assignee: |
Corning Glass Works (Corning,
NY)
|
Family
ID: |
23219282 |
Appl.
No.: |
05/314,270 |
Filed: |
December 11, 1972 |
Current U.S.
Class: |
210/789; 436/17;
494/38; 494/16; 494/81 |
Current CPC
Class: |
G01N
33/491 (20130101); Y10T 436/107497 (20150115) |
Current International
Class: |
G01N
33/49 (20060101); B01d 021/26 () |
Field of
Search: |
;23/258.5 ;106/287SB
;210/65,83,84,512,DIG.23 ;233/1A,1R,26 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Hart; Charles N.
Assistant Examiner: Mukai; Robert G.
Attorney, Agent or Firm: Turner; Burton R. Patty, Jr.;
Clarence R.
Claims
I claim:
1. A fluid collection and partitioning assembly for collecting a
specimen of blood within a sealed fluid collection chamber,
centrifically separating the heavier and lighter fluid phases of
said blood specimen, and physically and chemically partitioning the
separated phases, comprising:
a. a container having an open end and a closed end;
b. gel-like means initially positioned within said container
adjacent said closed end for forming a transversely continuous
contact seal with an annular surface portion within said container
at a subsequently formed interface between said heavier and lighter
phases;
c. closure means for vacuum-sealing said open end of said container
and for defining a closed fluid collection chamber containing said
gel-like means therewithin, said closure means being pierceable by
a needle for supplying blood to said closed fluid collection
chamber which is adapted to draw the blood specimen
therewithin;
d. said gel-like means being a thixotropic material and including a
mixture of a fluid which is generally inert to body fluids and a
powdered inorganic filler; and
e. said gel-like means having a specific gravity intermediate those
of said lighter and heavier phases and being of such a thixotropic
composition such that during the centrifugation of said blood
specimen into its component phases, said gel-like material is
flowable from its initial position adjacent said closed end toward
said sealed open end and effects a semi-rigid seal at the interface
of said separated fluid phases which physically and chemically
partitions said phases within said container.
2. A method of partitioning a heavier phase from a lighter phase of
a centrifugally separated fluid specimen within a container which
comprises:
a. providing a container having a closed end and an open end;
b. initially positioning thixotropic gel-like means having a
specific gravity intermediate those of said lighter and heavier
fluid phases within said container in spaced relation from said
open end;
c. evacuating and sealing said container to provide a closed fluid
collection chamber therewithin;
d. supplying a fluid specimen to said closed chamber;
e. subjecting said specimen and gel-like means to a centrifugal
force to separate said fluid specimen into a heavier phase and a
lighter phase and simultaneously move said gel-like means toward
the interface of said phases; and
f. establishing a continuous semi-rigid gel-like seal across the
interior of said container between said heavier phase and said
lighter phase within said container.
3. A method of collecting a multiphase fluid specimen, separating
said specimen into at least two differing-density phases, and
partitioning said phases comprising:
a. providing an open-ended container with thixotropic gel-like
material having a specific gravity intermediate those of the two
phases of a fluid specimen to be collected and separated;
b. vacuum-sealing the open end of said container, containing said
gel-like material, with a needle-pierceable closure;
c. drawing a specimen through said closure;
d. applying centrifugal force to said specimen and gel-like
material and simultaneously forcibly moving the phases of said
specimen and said gel-like material toward relative positions
within said container corresponding to their respective specific
gravities;
e. terminating said centrifugal force after said specimen has
separated into differing-density phases and a substantial portion
of said gel-like material has reached a position intermediate said
phases, and
f. at such position, utilizing said gel-like material to partition
said separated differing-density phases.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an apparatus and method for the
collection and partitioning of at least two phases of a multi-phase
fluid within a container. More specifically, it pertains to the
collection of whole blood and, after the separation thereof, the
partitioning of blood serum or blood plasma from the blood cells.
If desired further fractionating and partitioning of, for example,
the blood serum may be accomplished.
2. Prior Art
In the standard evacuated blood sampling tubes, such as the system
illustrated in U.S. Pat. No. 2,460,641 to Kleiner, a glass tube has
one permanently closed end and the other end is closed by a rubber
stopper having a pair of opposite top and bottom axial recesses
separated by an intermediate diaphragm. A cup-like holder having a
double ended hollow needle, with one end terminating axially within
the holder and the other end terminating axially outside the
holder, is used to receive the stoppered end of the glass tube,
with the inner needle end being adapted to extend through the
stopper diaphragm into the evacuated tube. The outer needle end is
injected into the patient's vein and then, by forward thrust on the
tube, the puncturing of the stopper diaphragm is completed to
withdraw the blood. When the desired quantity of blood has been
collected in the tube, the filled tube is removed from the cup-like
holder thereby obtaining a stopper-sealed collection tube housing a
blood sample.
Blood or another fluid collected in the previously-described
collection device is then generally taken to the laboratory for
processing. The contents may be utilized as whole blood or
separated into a lighter phase (serum or plasma) and a heavier
phase (cells). If, for example, it is desired to obtain blood serum
(after an initial time period during which the filled tube assembly
is allowed to stand) the filled tube assembly is placed into a
centrifuge which completes separation into two blood phases.
Disposed at the bottom of the tube will be a heavy phase or high
density portion of the fluid consisting of packed red blood cells,
while disposed at the upper part of the tube will be the lighter
phase or low density portion of the fluid which is blood serum. The
separated serum is then analyzed, generally after first being
removed from the tube assembly by decanting and/or siphoning.
It is well known that once the blood phases are separated, if the
lighter phase is not removed from the tube within a short time,
interaction will occur between the separated phases and inaccurate
test results will be obtained. In addition, even if the lighter
phase is presently removed from the container there are the hazards
of contamination of the sample and of possible mismarking of the
removed sample. Furthermore, there are also hazards to the
laboratory personnel who may be exposed to disease-carrying blood
samples containing, for example, hepatitic serums.
Coleman, in U.S. Pat. No. 3,508,653, made an advance over the blood
sampling tube of Kleiner by introducing and attaching a resilient
piston directly beneath the tube closure or stopper, with the
piston being adapted to be punctured during the initial filling of
the sampling tube. After initial centrifugation, in order to obtain
the desired blood phase separation, and in response to further
centrifugal force, the piston is designed to move downwardly
through the light blood phase, with the piston being adapted to
permit upward flow of the light phase therearound, i.e., between
the container inner wall surface and the outer peripheral surface
of the piston. The piston, which has a wiper portion that makes an
initial sealing contact with the container inner surface, loses
this sealing contact during its downward movement (to permit the
flow of fluid therearound) and thereafter is designed to make a
final sealing contact with the container inner surface at a
position not lower than a position intermediate the separated
phases by stopping the downward movement by terminating the applied
force. In addition, the piston, which is initially detachably
secured to the stopper, requires passageway means and a vent
opening therewithin to facilitate the passage of gases to permit
descent of the piston but resist the passage of fluids
therethrough.
While the Coleman device provides a unitary sealing member between
the blood cells and the plasma or serum, it does have several
shortcomings. The piston and stopper must be held in intimate
contact with each other, otherwise blood which flows into any space
between them during the tube filling operation will remain above
the piston, and the blood cells will contaminate the lighter phase.
Once these blood cells find their way above the piston wiper, they
cannot be separated, since no mechanism or method has been provided
to permit them to move below the piston.
In addition, there are no positive means incorporated into the
Coleman device to prevent blood cells from moving upward past the
piston wiper. Actual observations in the laboratory confirm that in
spite of the general downward movement of the heavy phase, due to
the influence of centrifugal force, some blood cells do indeed
become caught up in the fast-moving light phase stream and are
carried past the piston wiper into the upper chamber of the tube.
As noted, once the cells find their way above the piston wiper,
there is no way to return them to the lower portion of the
tube.
Since the introduction of the blood sample into the tube may also
permit some air to enter the tube upon withdrawal from the patient
and since some gases are evolved from the blood sample, they must
be vented from below the piston to eliminate the retarding effect
they will have on the downwardly moving piston through a buoyancy
effect. While Coleman speaks of incorporating a vent opening into
the piston design, actual experience has shown that the vent
cannnot readily be incorporated into the design at manufacture but
is preferably made by the technician during the blood drawing
operation, thereby putting the burden of creating a satisfactory
vent upon the skill of the operator. The needle puncture in the
piston diaphragm (for the filling of the tube) serves as a vent for
air and gases during piston descent. An improperly punctured
diaphragm vent may either refuse to operate at all or may rupture
and blow out when the piston impacts the fluid surface during
centrifugation and thus completely loses its ability to act as a
seal between the light and heavy blood phases during piston
descent. In either instance, unfortunately the separation step
becomes aborted.
Lawhead, in U.S. Pat. application Ser. No. 228,573 filed Feb. 23,
1972, made an advance over the method and apparatus of Coleman by
introducing spools or partitioning assemblies for use with rigid
tubular containers for effecting either the physical or complete
physical and chemical partitioning of two centrifugally separated
fluid phases. These spools have a central axial orifice, a
resilient, annular, container-contacting wiper portion and an
integral annular skirt portion. By having specific gravities
intermediate those of the separated fluid phases, the spools are
adapted to move downwardly in the tubular containers, in response
to centrifugal force, only to the vicinity of the fluid phase
interface, with fluid flow occurring freely only through the spool
central axial orifice. Partitioning of the separated phases is
effected by the combination of the spool in conjunction with either
a natural plug of the heavy phase fluid or a float member having a
similar specific gravity.
While the Lawhead device produces excellent sealing between the
separated phases, it does require different diameter parts for
different diameter tubes, which of course is an economic
disadvantage in a low unit cost system.
Weichselbaum, in U.S. Pat. No. 3,464,890, sets forth a method of
separating plasma from whole blood which comprises bringing into
contact with the blood a separating amount of inert particulate
material, e.g., polystyrene beads having a coating of
anti-coagulant and having a specific gravity intermediate that of
plasma and blood. This loose material is placed into the blood
sample prior to phase separation and upon separation these
particles tend to establish a barrier between the plasma and cells.
This system, however, will not tolerate any subsequent jarring or
unusual motion since this will tend to destroy the barrier.
Furthermore, this system will not tolerate shipping and cannot be
utilized for mailing to testing laboratories.
Adler, in U.S. Pat. No. 3,647,070, sets forth a method and
apparatus for a barrier at the interface between plasma and packed
cells in centrifuged blood samples, which barrier means are adapted
to sink through the plasma layer, and upon being wetted and
expanded by the plasma, expanded into firm contact with each other
and the walls of the container to form a barrier. While this system
appears to be quite workable, it is limited to post centrifugation
insertion of the barrier means which is a definite disadvantage
from the cost, time and contamination standpoint.
The use of silicones for centrifuge fractionating of blood samples
is well known and is set forth in articles by Seal, S. H. in
Cancer. 1959 12:590-595; McCrea, L. E. in J. of Urol. 1961.
85:1006-1010; as well as Morgan M. C. and Szafir j. J. in Blood.
1961. 18:89-94. These articles basically describe the use of
silicone fluids (blended to specific gravities intermediate those
of the two phases sought to be separated) with blood samples, with
the silicone fluid, upon centrifugation, forming a fluid barrier
between the desired two phases. However, since the barrier is only
a fluid barrier the desired phase cannot be removed by decanting
and even in pipetting there is a problem of possible contamination
of the removed phase with silicones. Furthermore, these liquid
barriers will neither tolerate any subsequent jarring nor are they
adaptable to shipping.
SUMMARY OF THE INVENTION
The instant invention, both in terms of apparatus and method,
responds to each of the previously-described prior art shortcomings
in a manner so as to completely eliminate any further concern
regarding such problems.
The several embodiments of the partitioning assemblies and
partitioning or seal members of this invention are utilized with
containers that are adapted to serve as fluid collection or fluid
retaining tubes.
The partitioning or seal members include a predetermined or
separating amount of a gel-like material, preferably hydrophobic,
substantially thixotropic and generally inert to the separated
fluid phases that are to be partitioned. This gellike material,
such as a mixture of a silicone fluid and hydrophobic silicon
dioxide powder, which has a specific gravity intermediate those of
the fluid phases, is positioned within the container either before
or after fluid collection. Due to its specific gravity, the
gel-like material is adapted to move within the container in
response to centrifugation, with the gel-like material being
adapted to stop moving when it reaches the vicinity of the fluid
phase interface. The gel-like material thereupon is further adapted
to make a transversely-continuous, semi-rigid, contact seal with an
annular portion of the container inner surface, thereby effecting a
seal that physically and chemically partitions the fluid
phases.
While the gel-like material may be used by itself to form a
semi-rigid partitioning or seal member, it may also be used in
combination with a spool member having a container-contacting outer
surface portion and a central axial orifice. The spool member,
which is preferably initially positioned below the container
stopper or closure, by having a specific gravity that is
intermediate those of the separated fluid phases, is adapted to
move downwardly within the container in response to centrifugal
force. The fluid phases flow freely only through the spool central
axial orifice, with the spool being adapted to stop moving
downwardly when it reaches the vicinity of the fluid phase
interface. The gel-like material, which in this combination is
preferably initially located adjacent to the bottom of the
container, by reason of its specific gravity, moves upwardly within
the container and is adapted to make a transversely continuous
semi-rigid contact seal with at least an annular surface portion of
the spool central axial orifice.
The partitioning or seal members of this invention may also be
utilized to partition at least three differing density phases of a
separated multi-phase fluid specimen at positions substantially at
the interfaces of these fluid phases. This three-phase partitioning
may be accomplished by using first and second gel-like material
having specific gravities intermediate those of the first-second
and second-third differing density phases, respectively. These
gel-like materials are adapted to make separate
transversely-continuous, semi-rigid, contact seals with different
annular portions of the container inner surface thereby effecting
seals that partition the three separated phases.
The partitioning assemblies and partitioning or seal members of
this invention may be utilized in several different operational
sequences. One operational sequence applies specifically to a fluid
collection and partitioning assembly that is intended to remain
closed (vacuum sealed) from the time of manufacture through
sampling, preparation and centrifugation of its contents until the
lighter phase is removed after centrifugation.
In another operational sequence, the partitioning or seal member is
hand-inserted or dispensed into an opened collection tube (i.e., at
atmospheric pressure) after sample collection, prior to
centrifugation.
In the "closed system" concept sequence, the gel-like material may
be positioned anywhere within the collection tube, while in the
"hand-insertion" concept sequence, the gel-like material is
preferably dispensed into the tube either as a floating capsule or
positioned on the side of the tube below the tube closure.
When three or more phase partitionings are desired, both "closed
system" and "hand insertion" concept sequences, as well as
combinations thereof, may be employed, with one or more
centrifugation steps being required.
One method of establishing the partitioning of heavier phase from
the lighter phase of a centrifugally separated fluid specimen
within a container involves providing the container with a
predetermined amount of a gel-like material having a specific
gravity intermediate those of the separated phases. Moving the
gel-like material within the container through at least one of the
fluid phases (in response to centrifugal force) establishes a flow
of at least one of the fluid phases within the container. A
transversely-continuous semi-rigid contact seal is established with
an annular portion of the container inner surface when the gel-like
material reaches a position in the vicinity of the fluid phase
interface thereby partitioning the lighter and heavier fluid
phases. Thereafter the applied force is terminated.
Other advantages and features of the instant invention will be
understood from the following description in conjunction with the
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates one of the fluid collection and partitioning
assemblies of this invention, ready for use, with the partitioning
or seal member in the form of a gel-like material being initially
position adjacent to the normally closed end of the tubular
container.
FIG. 2 is the assembly of FIG. 1 after the introduction of a
homogenized fluid sample thereinto.
FIG. 3 illustrates the assembly of FIG. 2 shortly after the start
of centrifugation, which begins to separate the homogenized sample
into at least two differing-density fluid phases, with the gel-like
material beginning to move away from its initial position.
FIG. 4 illustrates that in the assembly of FIG. 3, as
centrifugation continues, the gel-like material is approaching the
interface between the two differing-density fluid phases.
FIG. 5 illustrates the assembly of FIG. 4 upon the completion of
centrifugation, with the gel-like material being located at the
interface between the differing-density fluid phases and making a
transversely continuous contact partition or seal to thereby
physically and chemically partition the two separated phases.
FIG. 6 illustrates another embodiment of the fluid collection and
partitioning assemblies of this invention, having a spool poised
beneath the closure member of the container and having a
predetermined amount of gel-like material positioned adjacent to
the naturally closed end of the container, with the differing
density fluid being disposed therebetween.
FIG. 7 illustrates the assembly of FIG. 6 upon the completion of
centrifugation, with the spool and gel-like material being located
at the interface between the differing density fluid phases and
coacting to make a transversely-continuous contact seal to thereby
partition these phases.
FIG. 8 is a sectional view, partially broken away, of one of the
fluid collection and partitioning assemblies of this invention
wherein the gel-like material is dispensed into the fluid
collection assembly after the fluid collection is completed.
FIG. 9 is a sectional view, partially broken away, of another
embodiment of the fluid collection and partitioning assemblies of
this invention, having separately-positioned first and second
gel-like materials of differing densities and at least a
three-phase fluid specimen disposed therebetween.
FIG. 10 illustrates an assembly, such as that of FIG. 9, upon the
completion of centrifugation, with the first and second gel-like
materials being located in the form of transversely-continuous
partitioning members or contact seals at the interfaces between the
first-second and the second-third differing density phases,
respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now to the drawings in detail, FIGS. 1-5 illustrate one
of the fluid collection and partitioning assemblies or container
assemblies of this invention both in terms of the various
components in correct relationship to each other as well as the
operational sequence of the various parts thereof.
FIGS. 1-5 depict a fluid collection and partitioning assembly, more
specifically, a blood collection and partitioning assembly or
container assembly 11 consisting of a container or collection tube
12; a predetermined amount of a gel-like material 30; and a stopper
or closure 20; all of which will now be described in more
detail.
Collection tube 12, which is preferably made of glass, plastic or
other material, and which is preferably also transparent, has a
normally closed bottom end 14 and an open upper end 16 for
receiving a self-sealing stopper or closure 20 formed of medical
grade butyl rubber or other suitable material. Closure 20 may be of
the shape and material described herein or it may be of other
suitable known types. Stopper 20 as shown, is shaped so as to have
a flanged end 22 which abuts and overlies annular end face 18 of
collection tube open end 16. Stopper 20 is further provided with a
diaphragm or septum 24 which forms a transversely-continuous seal
with an annular surface portion of tube inner wall surface 13.
Stopper 20, together with collection tube 12, defines a sealed,
closed fluid receiving chamber 26, which in the arrangement shown
in FIG. 1 is adapted, (after previously having been evacuated) to
maintain a negative pressure (vacuum) of about 24 inches Hg for an
extended period of time. Thus, stopper 20, serves as a sealing
closure to preserve the interior vacuum and provides a septum 24
through which the sampling needle (not shown) can reach chamber 26
without destroying its integrity. No invention is claimed for
either the previously described collection tube 12 or stopper 20,
per se.
Again, as shown in FIG. 1, a predetermined amount of a gel-like
material 30 preferably is initially positioned adjacent to closed
end 14 of tube 12. This predetermined or separating amount (such as
about 1 ml) of gel-like material 30 preferably is hydrophobic,
thixotropic and generally inert to body fluids. One example of such
a gel-like material is a mixture of a silicone fluid and very fine
hydrophobic silicon dioxide powder. Hydrophobic silicon dioxide
(SiO.sub.2) may be defined as silicon dioxide that is treated so as
to repel water, with one example of a hydrophobic silicon dioxide
powder being Silanox.sup.TM 101 (manufactured by the Cabot
Corporation of Boston, Massachusetts and described in Cabot
brochure SGEN-1) hydrophobic fumed silicon dioxide, which is a
fumed silicon dioxide having trimethylsilyl groups bonded to the
surface thereof. Another example of a hydrophobic silicon dioxide
powder is AEROSIL R972 (sold by DEGUSSA INC. Pigments Div., New
York, N.Y. and described in Technical Bulletin 31), wherein the
silicon dioxide is rendered hydrophobic by reacting the silanol
groups on the surface with dimethyl dichlorsilane.
Silicon fluid may be defined as a polysiloxane liquid such as for
example DOW Corning 360 Medical Fluid (a dimethyl polysiloxane
liquid manufactured by the DOW CORNING Corporation of Midland,
Michigan and described in DOW CORNING Bulletins CPO-1072, March,
1972 and CPO-158-1, March 1972). Other examples of silicone fluids
are DOW CORNING 200 and 510 (a methylphenyl polysiloxane)
fluids.
The following specific example of a gel-like material is given in
illustration of the present invention and is not intended to be
limiting on the invention.
One hundred grams of DOW CORNING 360 Medical Fluid (350
centistokes; specific gravity about 0.97) were mixed with 15.7
grams of Silanox.sup.TM 101 silane-modified silicon dioxide
(specific gravity about 2.2) to produce 115.7 grams of a gel-like
material having a specific gravity of about 1.05.
Table 1 illustrates, among others, a number of mixtures of gel-like
materials that may be utilized in this invention:
Sample Sili- Viscosity Grams of Grams of Grams of Resulting No.
cone (centistokes) Silicone S-101 R-972 S.G.
__________________________________________________________________________
2 DC-360 100 100 3.0 16.0 1.059 8 DC-510 100 100 11.0 -- 1.047 10
DC-510 100 100 10.0 2.0 1.053 11 DC-510 100 100 9.5 2.5 1.053 12
DC-510 100 100 9.0 3.0 1.053 13 DC-360 100 100 2.0 15.0 1.050 14
DC-360 100 100 4.0 13.0 1.050 24 DC-360 350 100 1.0 15.0 1.051
__________________________________________________________________________
Table I DC-360 is DOW CORNING 360 Medical Fluid. DC-510 is DOW
CORNING 510 Fluid. S-101 is Silanox.sup. TM 101 hydrophobic
SiO.sub.2. R-972 is DEGUSSA R 972 hydrophobic SiO.sub.2.
It should be noted that the specific gravity (S.G.) of whole blood
is 1.05-1.06 while the S.G. of the light phase (blood serum) is
1.02-1.03 and the S.G. of the heavy phase (blood cells) is
1.08-1.09. Therefore the specific gravity of gel-like material 30
has to be below that of the heavy phase and above that of the light
phase, i.e., generally in the range from about 1.035 to about 1.06,
with the preferred range being 1.04-1.055.
With reference to one of the operational sequences of this
invention, FIG. 1 illustrates fluid collection and partitioning
assembly 11, ready for use, with stopper 20 together with
collection tube 12 defining a sealed, closed evacuated fluid
receiving chamber 26. Contained within chamber 26 is gel-like
material 30 which is positioned adjacent to normally closed end 14
of tube 12.
The FIG. 2 assembly depicts the FIG. 1 assembly with the addition
of a multi-phase fluid sample 34, such as whole blood. After a
correct venipuncture has been made on the patient, the inner or
butt end of the needle (not shown) is pushed through stopper
diaphragm portion 24, thereby permitting the vacuum within the
assembly to draw blood freely into tube 12.
FIG. 3 illustrates the assembly of FIG. 2 shortly after the start
of centrifugation which begins to separate homogeneous fluid sample
34 into a lighter phase 38 and a heavier phase 42. The interface 44
between lighter and heavier phases 38 and 42, respectively, is
shown, for the sake of clarity, in the form of a dash on either
side of tube 12. During centrifugation, heavier phase 42, because
of its higher specific gravity (relative to lighter phase 38)
starts to move toward tube bottom 14, leaving less dense lighter
phase 38 thereabove. In contrast, gel-like material, by virtue of
its lower specific gravity (1.035-1.06) and its location on tube
bottom 14 starts to move upward, toward lighter phase 38. It must
be remembered that since gel-like material 30 is very resilient, it
does not move all at once, but rather, under the influence of
centrifugal force, it becomes gradually elongated and starts to
pull away from its initial position.
The FIG. 4 assembly shows the FIG. 3 assembly, as centrifugation
continues, with gel-like material 30 still in an elongated form,
but now fully removed from its initial position, with the upper end
of gel-like material 30 being located in the vicinity of fluid
phase interface 44. It should be noted that a thin layer 32 of
gel-like material remains at its initial position, i.e., at tube
bottom 14.
It should be noted that due to the resilience of the gel-like
material 30, movement of fluid can occur in either direction, i.e.,
red blood cells, fibrin or other heavy-phase bodies can usually
move downwardly through gel-like material 30 under the persuasion
of centrifugal force. At the same time, any lighter phase fluid
remaining below gel-like material 30, again under the persuasion of
centrifugal force, can usually move upwardly through material 30.
The operation of the instant invention is such that it does not
differentiate between gases and liquids and permits both to flow
through material 30 without prejudice. The flow, either of gases or
liquids, is neither restricted nor otherwise influenced in any way
by the gel-like material 30. Each phase is free to seek its own
flow path and its ultimate position within tube 12 is influenced
solely by the persuasion of centrifugal force.
FIG. 5 illustrates the assembly of FIG. 4 upon the completion of
centrifugation, i.e., all the parts are now in final position. Upon
the completion of centrifugation the maverick lighter components or
cells of heavier phase 42 (previously in or above material 30),
still having a specific gravity greater than that of material 30,
have now eased into or through material 30, with material 30
resting at a density level equivalent to its own specific gravity.
As shown in FIG. 5 member 30 has now consolidated so as to make a
transversely-continuous semi-rigid contact seal or partitioning
member 48 with an annular surface portion of tube inner surface 13.
If the homogenized test fluid 34 is whole blood, then the heavier
phase 42 is now blood cells and the lighter phase may be either
blood serum or blood plasma, depending upon whether or not the
whole blood sample was coagulated or not coagulated,
respectively.
It should be understood that the thickness or axial dimension of
the transversely-continuous contact seal made by partitioning or
seal member 48 is, among other things, of course also dependent
upon the amount of gel-like material that is initially introduced
into tube 12. In addition, the seal need not be of uniform shape or
thickness across its transverse dimension as long as it has at
least one transversely-continuous portion. Uniformity of the seal
is influenced by such factors as the viscosity of the gel-like
material, the amount of material present, the speed and type
(horizontal or anglehead centrifuge) of centrifugation (and
resulting g-force) as well as the centrifugation time.
It should be noted that gel-like material 30, which makes up
transversely-continuous semi-rigid seal member 48, is substantially
thixotropic, i.e., at rest it acts substantially like a material in
a thixotropic state. It is not intended that this definition of the
gel-like material, which also may be described as semi-solid,
semi-rigid, substantially non-flowable, or resistant to flow at
rest, be a limitation on the invention herein described, since the
behavior of the material is, at this time, not yet completely
subject to a full exacting explanation. It should suffice to say
that gel-like material 30 appears to have a very high viscosity, is
thermoplastic in nature, will act substantially as a fluid during
centrifugation and will again set up to a gel when allowed to
stand.
In the form of seal-member 48, gel-like material 30 is
substantially rigid and allows decanting of the lighter fluid phase
from the tube or container 12 without disrupting its seal with the
tube inner surface. In addition, the partitioned sample will
readily tolerate subsequent jarring and is entirely adaptable to
shipping (such as to a remote laboratory for example).
While the previously-described examples of gel-like materials 30
are mixtures of silicone fluids and silicone dioxide powders it
must be understood that these mixtures are not to be considered as
limiting this invention. Any gel-like material is useful in the
context of this invention if it meets the following basic
requirements:
1. Specific gravity (or density) intermediate between those of the
two fluid phases sought to be separated.
2. Non-interaction with the fluid phases sought to be
separated.
3. Substantially non-flowable (semi-rigid) at rest.
In the previously-described examples, the silicon fluid may be
thought of as a liquid or base material (an oil) and the silicon
dioxide powder as a solid (a filler), with the latter serving both
to adjust the specific gravity of the former to the desired value
and to gel the oil, i.e., to convert it into a semi-rigid gel-like
material or grease (with the terms gel-like and grease being used
synonymously). Thus, as long as they meet the previously noted
three basic requirements, almost any liquid and filler combination
may be utilized, with examples of oils including esters of
polyacids (such as dioctylsebacate, dibutylphthalate and
tributylphosphate) and mineral oils (hydrocarbons). Examples of
fillers include titania, zirconia, asbestos, wood flour and finely
divided organic polymers (such as polyethylene, polypropylene,
fluorocarbons and polyesters, etc.) In addition, depending on the
specific gravity of the base material, the fillers may be used to
either increase or decrease the specific gravity of the former.
Furthermore, again as long as the three basic requirements are met,
the gel-like material may be made up of but a single component
(such as a silicone) material or may be mixtures of one or more
base materials and one or more fillers.
Up to this point the only operational sequence described has been
one wherein gel-like material 30 is initially positioned adjacent
to closed end 14 of tube 12, as shown in FIGS. 1-5. However, other
initial placements of material 30 are entirely possible, i.e.,
material 30 may be placed anywhere within fluid receiving chamber
26. For example, as shown in FIG. 9, a predetermined amount of
gel-like material 30a may be positioned on a portion of tube inner
surface 13 below stopper 20. When used in the sequence shown in
FIGS. 1-5, in lieu of material 30, upon centrifugation, gel-like
material 30a (which is substantially similar to material 30), by
virtue of its specific gravity (1.035-1.06) will move downwardly
through lighter phase 38 (specific gravity 1.02-1.03) and
eventually rest at the density level equivalent to its own specific
gravity. The end result, as shown in FIG. 5, will be substantially
the same regardless of whether material 30 moves up from tube
bottom 14 or material 30a moves down from the vicinity of stopper
20.
The operational sequences described up to now have been limited to
a fluid collection and partitioning assembly 11 consisting of
collection tube 12, stopper 20 and contact seal or partitioning
member 48, wherein tube 12 together with stopper 20 defines a
sealed, closed, evacuated fluid receiving chamber 26. This
operational sequence, as shown in FIGS. 1-5, applies specifically
to a fluid collection and partitioning assembly that is intended to
remain closed from the time of manufacture, through sampling,
preparation and centrifugation of its contents until the lighter
phase is to be removed after centrifugation. Of necessity, the
gel-like material must be placed into the tube (prior to the
evacuation thereof) at the factory. This sequence will hereinafter
be referred to as the "closed system" concept to differentiate it
from a "hand or user insertion" concept.
In an operational sequence utilizing the "hand-insertion" concept,
a predetermined amount of gel-like material such as for example 30a
in FIG. 9 or 30b (also substantially similar to material 30) in
FIG. 8 is dispensed into an opened collection tube after sample
collection, preferably either after coagulation has been completed
or after partial phase separation has been effected (upon
completion of coagulation). The gel-like material can be inserted
into an opened collection tube even before coagulation has been
completed, however, since blood cells exhibit a tendency to harden
on the walls of the opened tube it is preferable to delay the
opening of the collection tube until coagulation has been completed
therein.
With reference to the operational sequence utilizing the
"hand-insertion" concept, FIGS. 2 and 3, sans material 30, may be
utilized to illustrate a well-known evacuated blood collection tube
assembly comprised of collection tube 12 and stopper 20. Once blood
sample 34 has been introduced into this assembly and preferably
either after coagulation (FIG. 2) or after partial phase separation
(FIG. 3), stopper 20 is removed and gel-like material 30a (FIG. 9),
or 30b (FIG. 8) is dispensed into tube 12. Thereafter, stopper 20,
in accordance with good medical practice, preferably is placed back
on tube 12 and centrifugation can begin (FIG. 2, sans material 30)
or be continued (FIG. 3, sans material 30). Hereinafter, the
operational sequence proceeds in a manner and with a result
identical to that already described with reference to the "closed
system" concept. Substantially similar results are obtained
regardless of whether the gel-like material is dispensed directly
into the fluid sample, as is material 30b in FIG. 8, or positioned
on an inner surface portion of the tube, as is material 30a in FIG.
9.
FIGS. 6 and 7 disclose another embodiment of the fluid collection
and partitioning assemblies of this invention wherein a
predetermined amount of gel-like material 30 coacts with a spool 52
to effect complete physical and chemical partitioning of two
differing-density fluid phases. The assembly shown in FIG. 6, i.e.,
tube 12, stopper 22, gel-like material 30, fluid 34 and spool 52,
can be the result of at least two different concept sequences,
namely: (1) a "closed system" concept wherein spool 52 and gel-like
material 30, are both located in a sealed, closed, fluid receiving
chamber 26 as shown in FIG. 1, into which fluid sample 34 has
thereafter been introduced, or (2) a "hand-insertion" concept
wherein spool 52 is introduced into a collection tube 12 (upon
stopper removal) after fluid sample 34 has been collected (as shown
in FIG. 2).
Spool 52, which has an annular, generally cylindrically-shaped main
body portion 54 having a diameter less than the inside diameter of
collection tube 12, also has an upper, outwardly-tapering, annular,
resilient, wiper or outer surface portion 56 having a maximum outer
free diameter greater than that of portion 54, with portion 56
being adapted to sealingly contact tube inner wall surface 13.
Spool 54 also has a lower skirt portion 58 and a central axial
orifice 62. Spool 52 may be of the type disclosed in co-pending
U.S. Pat. application Ser. No. 228,573 filed Feb. 23, 1972 (which
is a continuation in part of application Ser. No. 178,274 filed
Sept. 7, 1971) and is also assigned to the assignee of this
invention.
With reference to the operational sequence of the FIG. 6 and 7
embodiments, FIG. 6 shows the fluid collection and partitioning
assembly immediately prior to centrifugation, while FIG. 7 depicts
the assembly upon the completion of centrifugation. During
centrifugation, gel-like material 30 behaves in the manner already
described with reference to FIGS. 3 and 4 except that material 30
coacts with spool 52 to make a transversely-continuous contact seal
to separate phases 38 and 42. Spool 52, which is preferably made of
a resilient material such as medical grade rubber, preferably has a
specific gravity intermediate those of the fluid phases to be
separated (in the case of human blood the intermediate
S.G.=1.035-1.06). At the start of centrifugation, spool 52, because
of its specific gravity, starts to move downward, away from the
vicinity of stopper 20, toward lighter phase 38, which in turn
flows upwardly through spool central axial orifice 62. It should be
noted that all fluid flow takes place through orifice 62 and no
fluid is permitted, nor can it possibly take place, between the
outer surface of spool 52 and tube inner surface 13. Furthermore,
fluid flow can occur through orifice 62 in either direction,
depending upon the initial position of spool 52 relative to the
various density components of the fluid which are to be separated.
The operation of spool 52 is such that it does not differentiate
between gases or liquids and each phase is free to seek its own
flow path and its ultimate position within tube 12 is influenced
solely by the persuasion of centrifugal force. Upon the completion
of centrifugation (FIG. 7) the skirt portion 58 of spool 52 has
entered heavier phase 42 and gel-like material 30, again as a
result of the applied centrifugal force, has started to enter
lighter phase 38 by extending at least partially through spool
central axial orifice 62. Gel-like material 30 is adapted to make a
transversely-continuous contact seal member 64 with at least an
annular portion of orifice 62. Thus spool 52 together with gel-like
material 30 forms a transversely-continuous partitioning assembly
66 with an annular surface portion of tube inner surface 13.
Basically, spool 52 acts as a constriction within tube 12 since
sealing of the differing density fluids from one another at tube
inner surface 13 has been continuous (by reason of spool wiper 56)
since spool 52 began its descent through the fluid and the
separated fluid phases have never been in contact with each other
in this area. Final sealing is accomplished within spool central
axial orifice 62 due to the action of gel-like material 30, and is
purposefully designed to occur at or just above the fluid phase
interface 44 to ensure the absence of any heavy phase components
within the lighter phase sample. The exact positioning of gel-like
material 30 with reference to spool skirt portion 58 and orifice 62
depends upon the amount and viscosity of material 30 as well as the
centrifugal force applied.
Up to this point the embodiments described have been limited to the
partitioning of two differing-density fluid phases of a
centrifugally separated fluid specimen at the interface of the
fluid phases. Often it may be desirable to partition at least three
differing-density phases of centrifugally separated multi-phase
fluid specimen at positions substantially at the interfaces of the
differing-density fluid phases. This goal can be accomplished by
utilizing n-1 differing-density gel-like materials to partition n
number of differing-density fluid phases.
FIG. 9, which is a partially broken away sectional view of another
embodiment of the fluid collection and partitioning assemblies of
this invention, shows a first gel-like material 30, having a first
specific gravity, adjacent to tube bottom 14 and a second gel-like
material 30a, having a second specific gravity, attached to tube
inner wall 13 in an area below stopper 20. An at least three-phase
fluid 68 having first or heaviest density 70, second or
intermediate density 72, and third or lightest density 74, fluid
components is also contained within tube 12. Gel-like material 20
has a specific gravity intermediate those of first and second fluid
phase components 70, 72, respectively, while material 30a has a
specific gravity intermediate those of second and third fluid phase
components 72, 74 respectively.
FIG. 10 which illustrates an assembly, such as that of FIG. 9, upon
the completion of centrifugation, with first and second gel-like
materials 30, 30a, being located in the form of
transversely-continuous semi-solid partitioning members or contact
seals 48, 48a, between first-second (70-72) and second-third
(72-74) differing density fluid phases, respectively. As already
previously described, gel-like material 30 moves upward away from
tube bottom 14 and material 30a moves downward away from the area
below stopper 20, under the influence of centrifugal force until
they reach the fluid gradient levels, i.e., the interfaces closest
to their own specific gravity.
The end result, i.e., the partitioning of fluid phases 70, 72 and
74 shown in FIG. 10 may be achieved in a number of different
operational sequences. In the "closed system" concept technique,
both materials 30 and 30a are contained (at tube bottom 14 and in
the area below stopper 20, respectively) within a closed, evacuated
fluid receiving chamber, into which fluid sample 68 is thereafter
introduced (see FIG. 9). A subsequent single centrifugation step
produces the partitioning shown in FIG. 10, with both materials 30
and 30a leaving thin layers of gel-like materials 32 and 32a
respectively at their initial positioning areas.
In the "hand insertion" concept technique, after fluid sample 68
has been collected, a first gel-like material, having a first
specific gravity intermediate those of heaviest and intermediate
fluid phases 70, 72, respectively, is dispensed into collection
tube 12, either in the shape of material 30b (FIG. 8) or material
30a (FIG. 9). Thereafter, the assembly is centrifuged a first time
and this first gel-like material forms transversely-continuous
partitioning or seal member 48 between phases 70 and 72. Then, a
second gel-like material, having a second specific gravity
intermediate those of the intermediate and lightest fluid phases,
72 and 74, respectively, is dispensed into collection tube 12,
again either in the shape of material 30b (FIG. 8) or material 30a
(FIG. 9). After a second centrifugation, the second gel-like
material forms transversely-continuous partitioning or seal member
48a between phases 72 and 74.
Several combination "closed system and hand-insertion" concept
techniques are also possible. In one such combination, a first
gel-like material, having a specific gravity intermediate those of
heaviest and intermediate phases 70, 72, respectively, is contained
within a closed, evacuated fluid receiving chamber (FIG. 1). After
the introduction of fluid 68 a second gel-like material, having a
specific gravity intermediate those of the intermediate and
lightest phases 72, 74, respectively, is dispensed into collection
tube 12 (either in the shape of material 30b or material 30a, as
shown in FIGS. 8 and 9, respectively). A subsequent single
centrifugation step produces the partitioning shown in FIG. 10. In
a variation of this combination technique, the assembly is
centrifuged a first time after the introduction of fluid 68 so as
to produce a first transversely-continuous partitioning or seal
member 48 (such as that shown in FIG. 5), between phases 70 and 72.
Thereafter, the second gel-like material is dispensed into
collection tube 12 (in either of the shapes as previously noted)
and a second centrifugation step then forms a second
transversely-continuous member 48a between phases 72 and 74.
It should be noted that with any multi-phase fluid, having three or
more phases, an initial separating of the heaviest phase from the
remaining phases can be accomplished by means of either of the two
phase separation techniques herein discussed, e.g. by means of
partitioning or seal member 48 (FIG. 5) or partitioning assembly 66
(FIG. 7), using either the "closed system" or "hand-insertion"
concept techniques. Further separations of the remaining phases can
thereafter be accomplished by successively dispensing in gel-like
materials of decreasing specific gravities and successively
centrifuging the collection tube assembly. For example, a whole
blood sample may be initially be separated into blood cells and
blood serum and thereafter, while remaining in the same container,
the blood serum may be further fractionated into separate
components. Whole human blood has a given, generally quite uniform,
specific gravity between 1.05 and 1.06. Centrifuged blood however
has many layers or constituents of varying specific gravities, from
the heaviest at the bottom to the lightest at the top, with the
greatest visible demarcation occurring at the serum/red cell
interface.
The principles of this invention may be utilized in partitioning
assemblies for fluids other than human blood, e.g., any fluid
separable into at least two differing density phases may be
separated using a gel-like material (or a gel-like material and
spool combination) having a specific gravity intermediate those of
the phases sought to be separated. Any gel or gel-like material
that is hydrophobic, substantially thixotropic, and generally inert
to the fluids to be separated, may be utilized.
While the invention has been described in connection with possible
forms or embodiments thereof, it is to be understood that the
present disclosure is illustrative rather than restrictive and that
further changes or modifications may be resorted to without
departing from the spirit of invention or scope of the claims which
follow.
* * * * *