U.S. patent number 3,858,795 [Application Number 05/408,940] was granted by the patent office on 1975-01-07 for method for washing blood cells.
This patent grant is currently assigned to International Equipment Company. Invention is credited to John E. Joyce.
United States Patent |
3,858,795 |
Joyce |
January 7, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
METHOD FOR WASHING BLOOD CELLS
Abstract
Method for washing blood cells by forcing blood cells to the
bottom of a stationary liquid in a container, where the liquid has
a greater density at the bottom of the container than at the
top.
Inventors: |
Joyce; John E. (South Weymouth,
MA) |
Assignee: |
International Equipment Company
(Needham Heights, MA)
|
Family
ID: |
26987498 |
Appl.
No.: |
05/408,940 |
Filed: |
October 23, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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330880 |
Feb 8, 1973 |
3813618 |
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Current U.S.
Class: |
494/37;
494/17 |
Current CPC
Class: |
A61M
1/3692 (20140204); A61M 1/3695 (20140204); G01N
1/28 (20130101); A61M 1/3693 (20130101) |
Current International
Class: |
A61M
1/36 (20060101); G01N 1/28 (20060101); B04b
005/02 () |
Field of
Search: |
;233/1D,14R,14A,15,26,27
;210/83,84,516,DIG.23 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Cancer, Vol. 12, pages 591, 592, May-June 1959. .
Mary C. Morgan, Jean J. Szafir, Separation of Platelets from Whole
Blood by Silicone Liquids, Blood 18:89-94, 1961..
|
Primary Examiner: Krizmanich; George H.
Attorney, Agent or Firm: Kenway & Jenney
Parent Case Text
This is a continuation, division, of application Serial No. 330,880
filed February 8, 1973 now U.S. Pat. No. 3,813,618.
Claims
I claim:
1. A method for washing blood cells comprising the steps of loading
a vessel with a liquid which is less dense than blood cells in such
a manner that the liquid increases in density from the top of the
vessel to the bottom thereof, thereafter forcing blood cells to be
washed from the top of the vessel toward the bottom of the vessel
through the stationary liquid to strip the blood cells and thereby
produce washed blood cells.
2. The method as set forth in claim 1 wherein said blood cells are
forced through said liquid by a centrifugation.
3. A method for washing blood cells comprising the step of
producing a density gradient within a liquid contained by a vessel
by introducing two liquids of different densities both of which are
less dense than blood cells into separate compartments of a
generator, initially allowing the liquids to flow from said
compartments to a mixing zone, varying continuously the amount of
liquid flowing from one compartment relative to the amount of
liquid flowing from a second compartment and delivering said
liquids from said mixing zone into a vessel and thereafter forcing
blood cells to be washed from the top of the vessel containing a
liquid to the bottom thereof.
4. The method as set forth in claim 3 wherein said blood cells are
forced through said liquid by a centrifugation.
5. A method for washing blood cells comprising the steps of:
introducing two liquids of different densities both of which are
less dense than blood cells into separate compartments of a
generator, said generator being located in the upper portion of a
vessel having a configuration which enables it to be placed in a
centrifuge, each of said compartments having an opening for the
flow of liquid into the lower portion of said vessel, said
compartments being separated by a partition which causes the amount
of liquid flowing from one compartment to continuously vary
relative to the amount of liquid flowing from the other
compartment, said liquids being introduced into said compartments
so that a liquid gradient which increases in density is produced in
the lower portion of the vessel;
loading a sample of blood to be washed into the lower portion of
said vessel;
centrifuging the vessel to cause the liquid in the compartments to
flow into the lower portion of the vessel and produce a liquid
density gradient which increases in density from the top of the
vessel to the bottom thereof with the sample of the blood floating
on the liquid in the lower portion; and,
continuing centrifuging to force the blood cell through the
stationary liquid gradient to strip the blood cells and thereby
produce washed blood cells.
6. The method as set forth in claim 5 including the step of
detaching the lower portion of the vessel away from the upper
portion to yield readily accessible washed blood cells in the lower
portion of the vessel.
7. The method as set forth in claim 6 wherein the lower portion is
detached from the upper portion by cutting the vessel with a pair
of scissors.
8. The method as set forth in claim 6 wherein the lower portion is
sealed to provide a container for storing the washed blood
cells.
9. The method as set forth in claim 5 wherein the lower portion of
the vessel is loaded into liquid which is denser than the blood
cells to provide a dense cushion which prevents the blood cells
from reaching the bottom of the lower portion of the vessel when
centrifuged.
Description
BACKGROUND OF THE INVENTION
The method and apparatus disclosed in the following specification
is especially useful for washing red blood cells with two different
liquids so that the red blood cells can be analyzed.
Before a patient requiring a blood transfusion can safely receive
blood from a donor, the donor's blood must be tested in order to
ascertain that it is compatible with the blood of the patient. In
order to test blood for such compatibility, the blood cells must be
washed to strip the blood cells of the plasma or serum that
surrounds them. Such washing is normally accomplished by repeatedly
diluting the blood cells with a saline solution. Such dilution is
repeated until a sample of the blood cells is sufficiently free of
plasma or serum such that an effective test with Coomb's serum or
other reagent may be performed. Such dilutions often require as
many as four washing and decanting steps prior to the addition of
Coomb's serum or cross-matching reagents.
SUMMARY OF THE INVENTION
The present invention provides a means for stripping plasma or
serum from red blood cells by passing the red blood cells through a
liquid density gradient. Using the generator of the present
invention, a test tube may be efficiently filled with two liquids
having different densities in a configuration such that the
concentration of a first, or lower density, liquid is greatest
adjacent the top of the test tube, while the concentration of a
second, or higher density, liquid is at a maximum at the bottom of
the test tube.
When red blood cells are floated on a liquid in a test tube
containing such a density gradient and then centrifuged, red blood
cells are forced to pass downwardly into the test tube through a
density gradient which strips all the plasma from the red blood
cells before the red blood cells reach the bottom of the test tube.
When the test tube is removed from the centrifuge and decanted, the
blood cells are retained in the bottom of the test tube and are
ready for testing.
Accordingly, it is an object of the present invention to provide a
novel apparatus for producing a known and controllable liquid
concentration gradient.
Another object of the present invention is to provide an apparatus
which will enable the attainment of a predictable volume gradient
in a container from two different liquids.
A further object of the invention is to provide a new method for
producing a liquid concentration gradient from two different
liquids.
Still another object of the present invention is to provide an
apparatus for producing a liquid concentration gradient which will
enable the washing of red blood cells in a single centrifuging
step.
Yet another object of the present invention is to provide a new
method for washing red blood cells.
Another object of the present invention is to provide a new method
for washing red blood cells whereby the plasma surrounding the red
blood cells is stripped therefrom as the red blood cells are forced
through a liquid having a density gradient.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a diagram showing the operation of the liquid
concentration gradient generator of the present invention;
FIG. 2 is a cross-sectional view of a generator of the type shown
diagrammatically in FIG. 1;
FIG. 3 is a diagram showing a suitable orientation for a test tube
in relationship to a delivery conduit when the gradient generator
of FIG. 2 delivers the most dense liquid initially;
FIG. 4 is a perspective view of an alternate embodiment of a
gradient generator in which changeable inserts are used to vary the
proportion of the gradient which results;
FIG. 5 is a cross-sectional view showing an alternate embodiment of
the generator of the present invention; and
FIGS. 6-9 are shematic views showing the utilization of the
generator set forth in FIG. 5.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The operation of concentration gradient generator 10 of the present
invention is shown diagrammatically in FIG. 1 and includes
compartment 12 and compartment 14 which are formed by a barrier 13.
At the bottom of generator 10 are liquid outlets 15 and 16 which
allow passage or flow of liquid from compartments 12 and 14 into
conduits 18 and 20 respectively. Outlets 15 and 16 comprise equal
size apertures in generator 10. Conduits 18 and 20 are joined at a
junction 22 to form single delivery conduit 24. A suitable valve 17
is provided in order to stop the flow of liquid through delivery
conduit 24 during the filling cycle.
To produce a gradient with generator 10, both compartments 12 and
14 are filled with liquids. Since the purpose of generator 10 is to
produce a known and controllable concentration gradient in a vessel
such as test tube 26, the liquid in compartment 12 differs in some
way from the liquid in compartment 14.
For purposes of washing blood cells, in accordance with the present
invention, the liquids in the two compartments differ from each
other by being solutions of different concentrations or two sugar
solutions of different concentrations. For the reasons set forth
below, when generating liquid density gradients, it is desirable to
fill compartment 12 with the higher density liquid and compartment
14 with the lower density liquid.
Although the invention is primarily directed to forming liquid
density gradients, it is to be understood, however, that the
gradient generator of the present invention has a utility for
producing many kinds of liquid gradients, for example, a gradient
generator in accordance with the present invention can be
advantageously utilized to produce refractive index gradients,
ionic strength gradients, viscosity gradients, pH gradients, and
optical density gradients.
In order to facilitate understanding the operation of generator 10,
in FIG. 1, the liquid in compartment 12 is represented
diagrammatically by a plurality of squares, whereas the liquid in
compartment 14 is represented diagrammatically by a plurality of
circles. It is to be understood, however, that such symbols have
absolutely no relationship to the properties of the liquids which
are intended to be placed in compartments 12 and 14. The use of
circles and squares is simply a device to aid in understanding how
concentration or volume gradients are produced by the apparatus of
the present invention.
Prior to the allowance of any flow through outlets 15 and 16 of the
gradient generator 10, compartment 12 is filled with an appropriate
liquid by introduction of that liquid through the top of
compartment 12 as is indicated by arrow 28. Compartment 14 is
similarly filled by introducing a different liquid in compartment
14 through the top as is indicated by arrow 30.
For best operation of generator 10, compartments 12 and 14 are
initially filled to an equal level. Compartments 12 and 14 will
remain essentially equal. However, for liquids with widely
different densities, the levels will have heights approximately
inversely proportional to the densities. Thus, prior to operation
of generator 10, the level of liquid in both compartments 12 and 14
is equal as is indicated at level 32. With delivery conduit 24 in
an open or flow condition, for any given interval of time,
dt.sub.i, the level of liquid in both compartments 12 or 14 will
remain equal. Thus, for a first unit interval of time, dt.sub.i,
during which liquid flows through outlets 15 and 16, as shown by
arrows 34, the level of the upper surface of the liquids in both
compartments 12 and 14 will drop to a lower but equal level as is
shown by level 36.
The time that it takes for the level to drop from level 32 to level
36, due to flow of liquid through outlets 15 and 16, is shown by
the bracket dt.sub.i in FIG. 1. During the initial time interval,
dt.sub.i, the amount and relative proportion of liquid which flows
through outlets 15 and 16 is shown diagrammatically in test tube 26
by the bracket dt.sub.i on test tube 26. As is shown in FIG. 1, as
the liquid level in compartments 12 and 14 drops from level 32 to
level 36, the amount of liquid which will be delivered into test
tube 26 from compartment 14 is that amount represented
schematically by the circles between levels 32 and 36, whereas the
amount of liquid from compartment 12 that will be delivered into
test tube 26 is represented by the squares between levels 32 and
36.
Thus, as can be seen from FIG. 1, initially a large volume of low
density liquid from compartment 14 is delivered into test tube 26
along with a relatively smaller volume of high density liquid from
compartment 12.
The volume of high density liquid from compartment 12 that is
delivered through delivery conduit 24, increases as the level of
liquid in the compartments 12 and 14 drops, while on the other
hand, the volume of liquid which is delivered from compartment 14
decreases as the level in compartment 14 drops. This relationship
continues until both compartments 12 and 14 are empty.
As is shown in FIG. 1, the volume relationship during the final
time interval (dt.sub.f), is reversed from the volume relationship
during the initial time interval (dt.sub.i). That is, as the level
of liquid in compartments 12 and 14 drop from level 38 to bottom
40, the volume proportion of the liquids delivered into test tube
26 is equal but opposite to that proportion delivered during the
initial time interval. The foregoing is based on the fact that the
actual intervals of time represented by dt.sub.i and dt.sub.f are
equal and that barrier 13 is set to provide such a linear gradient.
Of course, the invention is not intended to be limited to
embodiments in which barrier 13 provides such a gradient. As is
also apparent, the volumes of the compartments may be unequal, but
the invention is more easily understood when the volumes of
compartments 12 and 14 are equal.
It should also be noted that because a large amount of low density
fluid is delivered during the initial interval, that liquid will be
displaced by the heavier liquids which follow. For this reason, the
liquids delivered during the initial period, dt.sub.i, are shown on
the top of test tube 26.
Although it is possible to reverse the relationship of the liquids
within compartments 12 and 14 so that high density liquid is in
compartment 14 and low density liquid is in compartment 12 and
thereby provide for the delivery of more dense fluid followed by
less dense fluid, such an arrangement is not as desirable for blood
washing applications as the arrangement shown in FIG. 1. The reason
for this fact is that if the more dense fluid is delivered first,
the less dense fluid which follows perculates through the more
dense fluid. Such perculation results in a certain amount of mixing
within test tube 26. Of course, as is apparent, any mixing of a
liquid gradient is undesirable when the purpose of the
concentration gradient is to provide stabilized sedimentation for
the transport of suspended matter from one liquid to another by
gravitational or centrifugal forces.
An acceptable arrangement for delivering high density fluid first,
results with the delivery conduit 24 terminated at the upper end of
the test tube, as is shown in FIG. 3. One undesirable aspect of the
embodiment shown in FIG. 3 is that some splattering occurs when the
end of the delivery conduit 24 is spaced a great distance from the
bottom of the test tube. Thus, the lower density liquid, which
follows the higher density liquid, can splatter and result in some
mixing within test tube 26. At this point, it should be noted,
however, that a small degree of mixing is not always a significant
problem. Furthermore, as set forth below, in connection with the
discussion of the embodiment of the invention shown in FIG. 3, with
wettable liquids and with the test tube orientation shown in FIG.
3, it is possible to deliver the less dense liquid first without
any appreciable mixing. For blood test applications, however, the
preferred embodiment is to deliver the lower density liquid first
as shown in FIG. 1.
A more detailed view of the generator shown diagrammatically in
FIG. 1 appears in FIG. 2, where generator 10 comprises a vessel 42
which is adapted to contain a quantity of a liquid. Generator 10 is
inclusive of four walls 44 and a bottom 46. Since FIG. 2 has a
front portion broken away, the front wall does not appear in the
view. Vessel 42 is divided into two equal compartments 12 and 14 by
a barrier 13 running diagonally within vessel 42. When conduits 18
and 20 are open, fluids contained in compartments 12 and 14 will
merge at junction 22 and flow into mixer 48. Mixer 48 may be any
conventional in-line laboratory mixer. In fact, acceptable
gradients result without a mixer in the delivery system.
Because outlets 15 and 16 comprise equal size apertures in
communication with conduits of equal diameters, as the liquid level
in compartments 12 and 14 drops, larger amounts of liquid from
compartment 14 is mixed with decreasing amounts of liquid from
compartment 12.
Should the level of liquid in vessel 42 reach a point that is lower
than the highest point of the conduits, a siphon that is
established causes the liquids to flow under the influence of
gravity, provided no significant pressure drop is created in the
mixer 48. If such a pressure drop is created, however, a suitable
pump 49 may be employed after the mixer 48 to draw the liquids
through the conduits. Thus, either a pump or a gravity feed may be
used to deliver the liquids in conduit 24 to a container. Also, as
is shown in FIG. 2, conduit 24 may be branched to enable liquid
gradients to be formed simultaneously in test tubes 26.
Since one important application of gradient generator 10 is to make
density gradients, as for example, solutions of different
concentrations, design precautions are employed to prevent transfer
of the less dense fluid in one compartment into the other
compartment and domination of flow by viscosity factors rather than
by gravitational or body force factors. At this point, it should be
noted that the size of outlets 15 and 16, as well as the inside
diameter of conduits 18 and 20 are selected so that the resistance
to flow of liquids through the outlets and conduits are controlled
by the relative pressure in the chambers, with the viscosity of the
two liquids are appreciably affecting the flow. At this point, it
should be noted that aperture sizes from each compartment may
differ since the viscosities of the two liquids may dictate certain
desirable balanced flow resistances between each chamber and the
mixing zone. For example, a 20 percent sucrose solution is about
twice as viscous as water. If the more viscous fluid (20 percent
sucrose) were to flow through a conduit whose diameter is larger
than the diameter of the conduit of the water by a factor of
.sqroot.2, then the resistance to flow per unit length per unit
flow rate would be equal. Of course, in some embodiments of the
invention such a feature might be desirable. Furthermore, since the
flow rates are always varying, it is desirable to locate the
junctions of the conduits near the entry to the compartments (as is
shown in FIG. 1) so that the pressure drop or flow resistance is
governed by a single conduit.
As is set forth above, the system shown in FIG. 2 produces the
least dense end of the gradient initially. Thus, liquid emerging
from conduit 24 at the bottom of test tubes 26 floats on top of the
more dense phases which follow. A suitable variation for the
delivery of liquids through conduit 24 is possible when the
solutions in compartments 12 and 14 are reversed so that the denser
end of the gradient is delivered into test tube 26 initially. When
the denser end of the gradient is delivered initially, it is
advantageous to position test tube 26 at an angle relative to
delivery conduit 24 and orient test tube 26 and delivery conduit 24
relative to each other so that the end of delivery conduit 24 is in
the vicinity of the upper end of test tube 26. The arrangement
shown in FIG. 3 allows wettable liquids flowing through conduit 24
to slide down the side of the test tube 26 without disturbing the
gradient formed therein and without splattering.
Another embodiment of the liquid concentration-volume gradient
generator, in accordance with the present invention, is shown in
FIG. 4. In FIG. 4, generator 50 is comprised of two symmetrical
rectangular compartments 52 and 54. Like generator 10 of FIG. 2,
generator 50 is inclusive of liquid containing vessel 56 having
four walls 58 and a bottom. A barrier wall 60 divides vessel 56
into compartments 52 and 54. Equal size apertures at the bottom of
compartments 52 and 54 allow the passage of liquid into conduits 62
and 64. Conduits 62 and 64 have equal diameters and are also joined
at a junction 66 to a delivery conduit 68. Both compartments 52 and
54 are open at the top. As is also apparent, for some gradient
configurations, compartments 52 and 54 may have unequal volumes.
Furthermore, as stated above, the apertures may be unequal.
With the device, as shown in FIG. 4, variations of the gradient
that can be produced is accomplished with various inserts 70, which
are shaped to yield the desired gradient.
In all embodiments of the invention, the concentration of the
liquids emerging from the delivery conduit will depend upon the
volumetric portion of each liquid leaving their respective
compartment. These volumetric portions depend on the liquid surface
area exposed on each liquid in its respective compartment which is
determined by the combined effect of the barrier separating the
compartments and the depth of each liquid in each compartment. The
depth of the liquid is a function of the density of the liquid in
the compartment and the gravitational force acting on the liquid.
For practical consideration, with most liquids, both of these
factors are constant. Therefore, for compartments with equal size
apertures, the volumetric portioning of the liquids flowing from
the compartments is solely a function of the specific positioning
of the barrier separating the compartments. For example, in FIG. 2,
barrier 13 is shown as a straight angled partition which generates
an approximately linear gradient whose shape is approximately
equivalent to the rate of change of volume with depth in the
compartments.
Another embodiment of a generator, in accordance with the present
invention, is shown in FIG. 5. In FIG. 5, the gradient generator 72
is formed in the upper part of a vessel 74 which has a test
tube-like configuation enabling vessel 74 to be placed in a
centrifuge. Vessel 74 is both a gradient generator and a test tube
in which the blood cells to be washed are centrifuged. The upper
portion of vessel 74 which forms gradient generator 72 contains two
chambers 76 and 78 which are formed by a barrier 77. With the
embodiment of the present invention represented by vessel 74 of
FIG. 5, it is advantageous to pre-load chambers 76 and 78 with the
liquids from which a gradient is to be produced. It should be
immediately apparent that chambers 76 and 78 can advantageously be
constructed to conform to chambers 12 and 14 of the embodiment
described in connection with FIG. 2. Thus, like the embodiment as
represented by FIG. 2, the preferred manner of pre-loading
generator 72 is to place the less dense liquid in chamber 78, with
the more dense liquid being placed in chamber 76. Chambers 76 and
78 each contain a capillary opening 80 and 82 communicating with a
capillary passageway 86. The diameters of opening 80 and 82 and the
inside diameter of passageway 86 are preferably of a value between
the range of 0.5 mm to 2.0 mm. Capillary passageway 86 terminates
at the bottom portion of vessel 74 which forms a test tube 88 for
blood to be tested. Capillary opening 80 and 82 are designed so
that under a normal gravitational field, no liquid flows through
these openings. However, under an increased gravitational field,
such as the field produced by a centrifuge, liquid in chambers 76
and 78 would flow through openings 80 and 82. Alternatively,
openings 80 and 82 can be plugged with a non-wetting material. When
vessel 74, having such a plug, is centrifuged, the additional
gravitational field created by the centrifuge breaks the plug
causing liquid to flow through the openings.
Test tube portion 88 and generator portion 72 of vessel 74 are
joined together so that generator 72 can be broken away from test
tube portion 88.
The test tube portion 88 has an opening 90 formed therein for
loading a sample of blood cells to be washed. Once vessel 74 is
loaded, placed in a centrifuge and centrifuged, the liquids in
chambers 76 and 78 flow into test tube portion 88. The sample of
blood to be tested, which is injected into test tube 88, initially
floats on the concentration gradient delivered into test tube
portion 88.
Upon further centrifugation, the blood sample will be forced down
through the liquid in test tube portion 88 and thereby washed.
After completion of such washing and the removal of vessel 74 from
the centrifuge, vessel 74 is broken to yield a test tube containing
washed blood cells which are readily accessible for further
testing. In one embodiment of the invention, vessel 74 is formed of
a compliant moldable thermoplastic material such as polyvinyl
chloride. The advantage of such a material is that it is easily cut
by a pair of scissors. Thus, a vessel formed of this material can
be cut, after the cells are washed, above the liquid in the vessel
to yield a detached test tube portion 88. Thereafter, the liquid
above the washed cells can be poured off. To store washed blood
cells, the polyvinyl chloride walls of test tube portion 88 are
pinched above the washed blood cells. Pinching seals the test tube
portion 88 making storage of the blood cells in test tube portion
88 convenient.
FIGS. 6-9 are schematic diagrams showing the utilization of the
embodiment of the invention set forth in FIG. 5.
As is shown in FIG. 6, a sample of blood cells to be washed 92 is
injected into test tube portion 88 of vessel 74.
As is shown in FIG. 7, the gradient liquid materials are loaded
into chambers 76 and 78 with high density fluid being loaded into
chamber 76 and low density fluid being loaded into chamber 78. It
is also advantageous to include the total required amount of
Coomb's serum as two equal aliquots in chambers 76 and 78, although
this step is optional.
As is apparent, the actual order of steps set forth in FIGS. 6 and
7 may be reversed, for example, it is advantageous to pre-package
vessel 74 with fluids in compartments 76 and 78 and add the blood
sample in the laboratory. With such pre-loading, the only liquid
that is added to vessel 74 in the laboratory is the blood sample
itself.
With generator 72 and test tube portion 88 loaded, vessel 74 is
placed in a centrifuge and centrifuged as is shown in FIG. 8.
During the initial phases of centrifuging, a density gradient 94 is
generated in test tube portion 88 with the sample of blood cells to
be washed 92 floating on density gradient 94. The centrifugally
induced forces are strong enough to allow liquids to flow from
compartments 76 and 78 to form the gradient in test tube portion
88. After continued centrifugation, red blood cells are forced
through gradient 94 to the bottom of test tube portion 88 as is
shown in FIG. 9.
Thereafter, vessel 74 is broken in two to yield test tube portion
88 which contains washed red blood cells.
If desired, excess supernatant is poured off and the washed red
blood cells are examined.
By utilization of the liquid gradient generator of the present
invention, more efficient washing of blood cells is possible.
Examples illustrating the technique for washing blood cells with
the generator of the present invention appear below.
EXAMPLE 1
When utilizing the embodiment of the generator represented by FIG.
2 of the drawing, the generator is loaded with liquids of varying
density. Into compartment 12 is placed a 12 percent by weight
solution of ficoll in normal saline, with compartment 14 being
filled to an equal level with normal saline. The specific gravity
of the ficoll solution is 1.07, and the specific gravity of normal
saline is 1.03. The holding capacity of generator 10 is calculated
and designed so that when both compartments 12 and 14 are filled to
the top, test tubes 26 will be able to receive and contain all of
the liquid held in the generator compartments. Thus, each test tube
receives an aliquot portion of the total gradient produced by the
generator. After test tubes 26 are filled, a sample of blood to be
tested is placed on top of the liquid in the test tube. For a 10 ml
test tube containing about 8 mls of the liquid gradient, 1 ml of
blood to be tested is placed in the test tube.
The test tube containing the sample of blood to be washed is then
placed in a centrifuge and centrifuged at a speed of 3,000 rpms for
1 minute, or 1,000 rpms for 10 minutes. The test tube is removed
from the centrifuge and the liquid above the washed blood cells is
decanted. The washed blood cells on the bottom of the test tube are
then tested with Coomb's serum or another antibody preparation. Of
course, the result of the Coomb's serum test is controlled by the
type of blood tested.
EXAMPLE 2
When utilizing the embodiment of the invention represented by FIG.
5 of the drawing, blood cells are washed in the following manner.
Chambers 76 and 78 are pre-loaded with liquids from which the
gradient is to be formed. Chamber 76 is loaded with 2.5 mls of a 12
percent by weight solution of ficoll in normal saline, and chamber
78 is loaded with 2.5 mls of normal saline. A 0.2 mls sample of
blood is then injected through opening 90 of vessel 74. Vessel 74
is then placed in a centrifuge and centrifuged at a speed of 2,000
rpms for one half a minute which forces the liquids in chambers 76
and 78 into test tube portion 88. To accomplish the foreging flow,
capillary openings 80 and 82 are 0.5 mm in diameter. Thereafter,
the vessel 74 is centrifuged at a speed of 3,000 rpms for 1 minute
which forces the blood cells through the liquids in test tube
portion 88. The vessel 74 is then removed from the centrifuge and
broken in half to yield test tube portion 88. The liquid in test
tube portion 88 is then decanted to yield the blood cells which are
ready for further examination.
In the foregoing examples, the dense phase of the gradient was
formed from a 12 percent solution of ficoll in normal saline. In
connection with the term "ficoll," ficoll is a synthetic high
polymer made by the copolymerization of sucrose and
epichlorohydrin. The molecules have a branced structure and have a
high content of hydroxyl groups leading to a very good solubility
in aqueous media. No ionized groups have been found in ficoll. The
average molecular weight of ficoll is 400,000 .+-. 100,000. It has
an intrinsic viscosity of around 0.17 dl/g. Ficoll can be purchased
from Pharmacia Fine Chemicals, Inc., 800 Centennial Avenue,
Piscataway, New Jersey 08854.
It should be noted that a gradient for washing blood cells can be
formed from a number of materials. The important consideration is
that the most dense portion of the gradient be less dense than the
blood cells. In this regard, the specific gravity of blood cells is
about 1.20. In accordance with the present invention, the gradient
through which the blood is to be washed should have a specific
density varying between about 1.03 to 1.15, although excellent
results are possible with a gradient having a specific gravity
variation of 1.03 to 1.09. When a ficoll solution is utilized for
the dense phase of the gradient, it is advantageous to use normal
saline which has a specific gravity of 1.03 as the less dense phase
of the gradient.
Blood cells can also be treated in accordance with the present
invention preparatory to freezing. In such freezing preparation,
washed cells are "glycerized" after being removed from their serum
or plasma. This step can be integral with the wash procedure in
accordance with the present invention either by:
1. including the glycerine or desired reagent (Coomb's serum) at
the end of the gradient as a separate zone denser than the
gradient's heavy end, but lighter than the blood cells; or
2. as a constant concentration in the gradient itself similar to
saline. The gradient generated will be of uniform reagent
concentration but of varying concentration gradient solute.
It is also desirable to have a dense cushion on the bottom of the
test tube to prevent the blood cells from reaching the bottom of
the test tube. In this regard, a liquid on the bottom of the test
tube having a specific gravity of 1.25 can be effectively utilized
for such a cushion. A dense cushion is particularly desirable when
the washed blood cells are to be frozen. When ficoll in normal
saline is employed, the solution can include ficoll in a range of
about 12 - 20 percent by weight.
In addition to ficoll, dextran may also be used to form gradients.
In accordance with the present invention, a 12 - 20 percent by
weight solution of dextran in normal saline can be employed to form
density gradients. Dextran is an anhydroglucose polymer, produced
by numerous strains of Leuconostoc and closely related bacteria in
sucrose-containing solutions. Most of the glucosidic linkages are
.alpha.--D--1.fwdarw.6, but to a lesser extent, 1.fwdarw.3-- and
1.fwdarw.4--linkages also appear. The presence of these
non-1.fwdarw.6--linkages is evidence of the branching of the
chains. The ratio of 1.fwdarw.6 to non-1.fwdarw.6-linkages can vary
within wide limits (3-14) for dextran from different sources, with
consequent differences in chemical and physical properties.
Pharmacia dextran, produced by Leuconostoc mesenteroides, strain B
512 has 5 - 10 percent of non-1.fwdarw.6-linkages.
Native dextran is soluble in water, resulting in very viscous
solutions. Its molecular weight distribution is extremely wide,
covering a range of molecular weights from hundreds of millions
down to oligosaccharides. Dextran can also be purchased from
Pharmacia Fine Chemicals, Inc., 800 Centennial Avenue, Piscataway,
New Jersey 08854.
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
present embodiments are therefore to be considered in all respects
as illustrative and not restrictive, the scope of the invention
being indicated by the appended claims rather than by the foregoing
description, and all changes which come within the meaning and
range of equivalency of the claims are therefore intended to be
embraced therein.
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