U.S. patent application number 10/741396 was filed with the patent office on 2004-07-08 for apparatus and method for biological sample preparation and analysis.
This patent application is currently assigned to Coulter International Corp.. Invention is credited to Burshteyn, Alexander, Joubran, John W., Kuylen, Nazle, Lucas, Frank J..
Application Number | 20040132198 10/741396 |
Document ID | / |
Family ID | 24450629 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040132198 |
Kind Code |
A1 |
Burshteyn, Alexander ; et
al. |
July 8, 2004 |
Apparatus and method for biological sample preparation and
analysis
Abstract
A method for utilizing a filtration device for removing
interferants from a sample containing cells in an automated
apparatus is disclosed. The filtration device includes a
microporous hollow fiber membrane having a plurality of pores sized
to retain cells while allowing smaller diameter interferants to
pass through the membrane. The apparatus also includes a means for
of moving the sample from a sample container to and from the
filtration device. The disclosed method utilizes a vacuum source to
aspirate the sample into a lumen of the hollow fiber membrane so
that the sample is retained in the lumen space until expelled into
an analysis container or transported to an analyzer.
Inventors: |
Burshteyn, Alexander;
(Pembroke Pines, FL) ; Joubran, John W.;
(Escondido, CA) ; Kuylen, Nazle; (Miami, FL)
; Lucas, Frank J.; (Boca Raton, FL) |
Correspondence
Address: |
BECKMAN COULTER, INC.
P.O. BOX 169015
MAIL CODE 32-A02
MIAMI
FL
33116-9015
US
|
Assignee: |
Coulter International Corp.
11800 SW 147 Avenue Mail Code 32-A02
Miami
FL
33196
|
Family ID: |
24450629 |
Appl. No.: |
10/741396 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10741396 |
Dec 19, 2003 |
|
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09611847 |
Jul 7, 2000 |
|
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6692702 |
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Current U.S.
Class: |
436/63 |
Current CPC
Class: |
Y10T 436/25375 20150115;
B01D 63/02 20130101; B01D 61/20 20130101; B01D 61/147 20130101;
Y10T 436/255 20150115; B01D 61/18 20130101; G01N 1/4077
20130101 |
Class at
Publication: |
436/063 |
International
Class: |
G01N 033/48 |
Claims
What is claimed is:
1. An automated method of preparing blood cells for analysis
comprising: a) adding at least one reagent that reacts with blood
cells of a blood cell sample to form a test sample mixture; b)
automatically removing interferants from the test sample mixture to
yield a washed blood cell sample; and c) analyzing the washed blood
cell sample to determine characteristics of the blood cells.
2. The method of claim 1, wherein the reagent comprises at least
one antibody that specifically binds to at least one target antigen
on the surface of the blood cells within the blood cell sample.
3. The method of claim 2, wherein the antibody contains a
fluorescent test label.
4. The method of claim 1, wherein analyzing the washed blood cell
sample is by electrical or optical measurements.
5. The method of claim 1, wherein the reagent comprises a lytic
reagent that lyses erythrocytes in the blood cell sample to be
analyzed.
Description
RELATED U.S. APPLICATION DATA
[0001] Division of co-pending U.S. application Ser. No. 09/611,847
filed Jul. 7, 2000.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of biological
sample preparation and analysis. More particularly, the subject
invention relates to a method and apparatus for enhancing the
sensitivity of blood cell analysis.
BACKGROUND OF THE INVENTION
[0003] Flow cytometry is a well known technique for qualitatively
and quantitatively analyzing a large number of individual cells for
a specific cellular marker in a rapid manner. In a typical
application, a fluorescent molecular probe that selectively binds
to a predetermined cell marker, such as a fluorochrome-conjugated
antibody that specifically binds an intracellular or cell surface
antigen, is added to a cell sample to be analyzed so that the probe
can bind or "stain" the cells within the sample that express the
predetermined cell marker. The sample is then placed in flow
cytometer and illuminated with a light source to enable the
fluorescence associated with each cell in the sample to be
quantified. The magnitude of fluorescence emitted from a particular
cell correlates with the quantity of cell marker on or in that
particular cell. By extrapolating this fluorescence data, the
relative quantity of specific phenotypic markers expressed by cells
in a sample can be rapidly and accurately determined. For an
overview of flow cytometric analysis see, "Flow Cytometry and
Sorting," Myron R. Melamed, Tore Lindmo, and Mortimer L.
Mendelsohn, eds., New York:Wiley-Liss, Inc., (3rd ed., 1995);
Shapiro, H. M., "Practical Flow Cytometry," New York:Wiley-Liss,
Inc., (2nd ed., 1990).
[0004] Sample preparation for flow cytometric analysis is typically
performed in a non-automated fashion, wherein a saturating
concentration of a cell marker-specific probe is added to a cell
sample by manual pipetting, and the mixture is then incubated for a
period of time sufficient to allow the probe to bind the cell
marker of interest. For analyses where red blood cells might cause
interference (e.g., immuno-phenotyping leukocytes), the red blood
cells can be removed from the sample using an agent that
specifically lyses erythrocytes (for example, a hypotonic solution,
ammonium chloride or carboxylic acid). Traditionally, to remove
interfering unbound probe from the cell sample prior to flow
cytometric analysis, the mixture is washed by adding excess buffer
to the mixture, centrifuging the mixture to separate the cells from
the buffer, removing the buffer containing the unbound probe, and
resuspending the cells in fresh buffer. The washing procedure can
be repeated multiple times to further remove any remaining unbound
probe. This non-automated technique is advantageous in that it
results in a relatively clean sample that contains few interferants
(for example, unbound probe or cell debris) which might generate
background noise or interference during the flow cytometric
analysis. For many applications, however, this non-automated
technique is relatively time-consuming, can result in significant
cell loss due to one or more wash steps, and exposes the cells to
the potentially deleterious effects (for example, activation of
enzymatic processes, granule release, cell destruction, high
gravity forces produced by centrifugation, etc).
[0005] While the foregoing technique is acceptable for infrequent
analyses involving a small number of samples, it is less suitable
for protocols involving repeated analyses of a large number of
samples. A more automated procedure is generally preferred when
flow cytometric analysis is employed for clinical diagnostics,
high-throughput screening, or the like. For example, in a typical
clinical assay where leukocytes are immunophenotyped using flow
cytometry, a sample of whole blood is placed into an apparatus that
automatically processes the sample prior to analysis. One such
apparatus is the COULTER.RTM. TQ-Prep.TM. Workstation system
manufactured by Beckman Coulter, Inc. (Miami, Fla.). After adding a
probe to the sample, this apparatus uses computer-controlled
devices to automatically add an agent that lyses erythrocytes in
the sample and a cell fixing agent (for example, paraformaldehyde).
The prepared sample can then be analyzed using a flow cytometer
without further processing. This automated technique is
advantageous in that samples of whole blood can be prepared for
analysis quickly and efficiently.
[0006] A drawback of this lysing technique can be encountered in
applications requiring a high degree of sensitivity. In such
applications, in the absence of a washing step, the automated
technique does not remove interferants, such as unbound probe or
debris from the lysed erythrocytes from the sample. The high
background signal caused by the fluorescence from the unbound
probe, non-specific probe binding, and/or autofluorescence from the
cells and debris can obscure results generated from the
analysis.
[0007] Where a fluorescently-labeled antibody is used to analyze a
cell sample for a marker present in low quantities, the absence of
a washing step can result in high background fluorescence caused by
the unbound antibody present in the sample. Thus, if too many
unbound fluorescent antibody molecules are present in the sample,
the flow cytometer can not distinguish the signal emitted from the
antibody-bound cells from the "noise" generated by the unbound
antibody. That is, the "noise" in the sample overwhelms the
"signal" emanating from the cells of interest. To avoid this, the
signal to noise ratio in the sample can be improved by removing the
interferants by manually washing. An example of manual washing
comprises centrifuging the sample to pellet the cells, decanting
the interferants contained in the supernatant, and resuspending the
cells in fresh buffer. As described above for the non-automated
technique, this manual washing is disadvantageous because it is
time consuming, causes cell damage, and can result in significant
cell loss.
[0008] A need therefore exists for an apparatus and method for
quickly and efficiently removing interferants from a cell sample
prior to analysis. In addition, the apparatus and method should
minimize the risk of exposure to infectious blood because of
operator handling of the blood cell sample. An apparatus that
performs the foregoing method with only negligible cell loss, and
does not expose cells to high gravitational forces or cell packing
caused by centrifugation would be especially advantageous.
SUMMARY OF THE INVENTION
[0009] It has been discovered that filters, such as microporous
hollow fiber membranes, can be utilized in cell sample preparation
devices to quickly and efficiently remove interferants from a cell
sample. More specifically, it has been found that the use of a
hollow fiber membrane having a plurality of pores with a mean
diameter less than the diameter of the cells of interest can be
utilized to remove interferants from a cell sample to improve the
signal-to-noise ratio in a cellular assay. Application of vacuum to
the hollow fiber membrane permits interferants to be removed from a
blood cell sample within a lumen of the filter with little or no
cell damage. As the cells themselves do not pass through pores of
the membrane, compared with conventional continuous filtration
devices, clogging of the filter is less frequent, and cells are
exposed to less deleterious forces. Filters within the invention
can be installed in a cell processing apparatus such that a blood
cell sample can be washed and analyzed automatically.
[0010] Accordingly the invention features an apparatus for
automatically removing interferants from a sample containing cells.
The apparatus includes a vacuum source; a filtration device
comprising an impermeable housing that forms an extramembrane
chamber wherein said chamber contains a filter that selective
retains cells of interest while allowing interferants to pass
through the filter, and wherein said housing contains at least
three port and wherein at least one port is connected by a conduit
to the vacuum source; a conduit from one of said ports in said
housing which is adapted to aspirate a cell sample from a sample
container into the filtration device by said vacuum source; and a
conduit from one of said ports in said housing which fluidly
connects to a buffer reservoir, which provides a means for buffer
to enter into said filtration device to recover the retained cells
through one of said ports.
[0011] In a preferred embodiment, the apparatus for automatically
removing interferants from a sample containing cells includes a
sample container holder adapted for holding a sample container
containing the sample of cells; a filtration device comprising a
filter that selectively retains the cells while allowing the
interferants to pass therethrough; at least one conduit fluidly
connecting the sample container to the filtration device whereby
the sample can move between the sample container and the filtration
device; and a means for recovering the cells from the filtration
device. The filter of the apparatus preferably includes a
microporous hollow fiber membrane having a plurality of pores sized
such that cells are prevented from passing therethrough. For
example, the pores can have a mean diameter of between about 0.1
and 5.0 microns. In preferred versions of the apparatus, the
microporous hollow fiber membrane is fashioned into at least one
tube defining a lumen, the tube having a first port providing a
first opening in the tube, and a second port providing a second
opening in the tube. In this preferred embodiment, the conduit can
be fluidly connected to the at least one lumen via the first port
such that the cell sample can be moved from the cell sample
container through the first port into the at least one lumen. The
second port can be fluidly connected to a buffer reservoir
containing a buffer and also fluidly connected to a detergent
solution reservoir containing a detergent solution. The means for
recovering the cells from the filtration device can include a fluid
pump that can be in fluid communication with a buffer reservoir
suitable for housing a buffer so that the fluid pump can cause the
buffer to flow from the buffer reservoir into the filtration
device. In variations, the fluid pump can also cause the buffer to
flow from the filtration device into the at least one conduit.
[0012] In another aspect of the apparatus of the invention, the
filtration device can also include an impermeable housing that
forms an extramembrane chamber between the impermeable housing and
the microporous hollow fiber membrane. A vacuum source can be
fluidly connected to the extramembrane chamber such that
application of a vacuum from the vacuum source to the extramembrane
chamber causes the sample of cells to be aspirated from the cell
sample container through the at least one conduit into at least one
lumen of the microporous hollow fiber membrane via the first port,
and a portion of the sample of cells to flow through the
microporous hollow fiber membrane into the extramembrane
chamber.
[0013] The apparatus can also include one or more pumps and a
plurality of valves. The pumps provide a hydraulic force for
transporting the buffer from the buffer reservoir into the lumens
of the microporous hollow fiber membrane, and the detergent
solution from the detergent solution reservoir into the lumens of
the microporous hollow fiber membrane; and the plurality of valves
being adapted to open and close such that the vacuum from the
vacuum source can be applied to the extramembrane chamber such that
the buffer, the detergent solution, and portions of the sample of
cells within the lumens of the microporous hollow fiber can be
controllably aspirated from the lumens to the extramembrane
chamber; and the hydraulic force provided from the pumps can be
directed to transport the buffer from the buffer reservoir to the
lumens, the buffer from the buffer reservoir to the cell sample
container, and the detergent solution from the detergent solution
reservoir to the lumens. In another aspect, the apparatus of the
invention can include a computer controller for controlling the
pumps and valves.
[0014] The invention also features a cell analyzing apparatus that
includes both a cell washer for removing interferants from a sample
of cells and a cell analyzer for analyzing the sample of cells. The
cell washer is describe above and the cell analyzer can be any cell
analyzers. Preferably the cell analyzer measures fluorescence, such
as a flow cytometer.
[0015] Also within the invention is an automated method for
removing interferants from a sample containing cells. This method
includes the steps of applying a vacuum force to a blood cell
sample in a first sample container to cause the blood cell sample
to contact a filter; applying a force to the blood cell sample in
contact with the filter, whereby interferants in the blood cell
sample pass through the filter while the cells in the blood cell
sample do not pass through the filter; and recovering the cells
from the filter. In this method, the filter can include a
microporous hollow fiber membrane having a plurality of pores sized
such that the cells are prevented from passing therethrough.
[0016] The invention also features an automated method of analyzing
a phenotypic marker on cells within a sample. This method includes
the steps of adding at least one reagent that reacts with blood
cells to a blood cell sample to form a test sample mixture;
automatically removing interferants from the test sample mixture to
yield a washed blood cell sample; and analyzing the washed blood
cell sample to determine characteristics of the blood cells. The at
least one reagent can be an antibody that specifically binds the
phenotypic marker and the antibody can include a fluorescent test
label. The step of automatically removing interferants from the
test sample mixture can remove greater then 50% of the interferants
from the test sample mixture.
[0017] The method can also further include the step of lysing
erythrocytes in the sample of cells to be analyzed, and/or the step
of quantifying the amount of probe bound to the cells in the test
sample mixture by use of a flow cytometer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic view of an apparatus within the
invention.
[0019] FIG. 2 is a schematic view of a filtration device within the
invention.
[0020] FIG. 3 is a cross-sectional view of the filtration device of
the invention shown with interferants removed from a sample of
cells within a lumen of a hollow fiber membrane of a filtration
device.
[0021] FIGS. 4A-4K are schematic views illustrating the operation
of an apparatus of the invention.
[0022] FIG. 5 is an outline of a method of the invention.
[0023] FIG. 6 is an outline of another method of the invention.
[0024] FIG. 7 is a graph showing data obtained from flow cytometric
analysis of blood cell samples reacted with fluorescent labeled
CD56 monoclonal antibodies. Data are presented as percent debris,
percent CD56.sup.+, and signal-to-noise ratio. Data shown are
averages of ten replicates using one donor.
[0025] FIG. 8 is a graph showing data obtained from flow cytometric
analysis of cell recovery from erythrocyte-lysed and fixed blood
cell samples subject to different washing protocols. Data from
lymphocyte ("Ly.") fractions, monocyte ("Mo.") fractions,
granulocyte ("Gr.") fractions, and a combination of all three
fractions are shown. Fractions were selected based on light
scatter. Data are shown as averages with error bars indicating
standard deviations.
[0026] FIG. 9 is a graph showing data obtained from flow cytometric
analysis of blood cell samples taken from 12 donors and stained for
CD56, erythrocyte-lysed, and fixed. Data are presented as percent
CD56.sup.+, and are shown as averages of two to twelve replicates
per donor. Error bars indicate standard deviation.
[0027] FIG. 10 is a graph showing the amount of cell carryover from
concentrated cell samples washed with a hollow fiber membrane
apparatus. After washing the cell sample and then cleaning the
hollow fiber membrane, blank sample tubes were "washed" using the
same hollow fiber membrane. The number of cells carried over from
the cell sample to the blank sample tube were quantified using flow
cytometry. Data are shown as percent of cells from cell sample
carried over to blank sample. Seventeen samples from one donor were
tested. The percent of carryover cells from the original total
number of events is 0.03% or less. Consequently, FIG. 10 does not
show a bar for the number of cells that were carry overed.
[0028] FIG. 11 is a graph showing data obtained from flow
cytometric analysis of platelet samples stained for CD42b and CD63.
20 ul of anti-CD42b and 20 ul of anti-CD63 fluorescently-labeled
antibodies were incubated with 100 ul of platelet rich plasma
(after gravity sedimentation) for 10 minutes without shaking or
mixing. "Control" samples were not washed; "Sorvall" samples were
washed in a SORVALL.RTM. Cellwasher 2 (E.I. du Pont de Nemours)
using the AUTO mode per the manufacturers instructions; "Quick
Spin" samples were washed according to the Quick Spin protocol
described herein; and "Invention". samples were washed one time
using a hollow fiber membrane apparatus. Data were obtained using a
COULTER.RTM. EPICS.RTM. XL.TM. flow cytometer (Beckman Coulter
Inc., Miami, Fla.) and presented as size (determined based on
forward and orthogonal light scatter), percent CD42b (mean channel
fluorescence), and percent CD63 (mean channel fluorescence). Data
shown are averages of three replicates using one donor.
[0029] FIG. 12 is a graph showing data obtained from flow
cytometric analysis of bone marrow cell samples stained for CD56,
erythrocyte-lysed, and fixed using a TQ-Prep.TM. apparatus (Beckman
Coulter, Inc., Miami, Fla.). "Sorvall" samples were washed in a
SORVALL.RTM. Cellwasher 2 using the AUTO mode per the manufacturers
instructions, and "Invention" samples were washed one time using a
hollow fiber membrane apparatus. Data were obtained using an EPICS
XL flow cytometer and are presented as cell recovery (number of
event in a thirty second run) and signal-to-noise ratio (as
described herein). Data shown are averages of three donors with one
replicate per donor. Error bars represent standard deviation.
[0030] FIG. 13 is a graph showing data obtained from flow
cytometric analysis of blood cell samples stained for hemoglobin.
200 ul of whole blood were cross-linked, permeabilized, and
stabilized using commercially available reagents according to
standard protocols. 20 ul of the prepared permeabilized RBCs were
stained with the following amounts of individual antibodies:
MsIgG1-PE/MsIgG1-FITC-20 ul, PanHb-FITC-10 ul, HbC-FITC-30 ul
(cross reactive with HbAo), HbS-FITC-30 ul, HbF-FITC-30 ul, or
HbA1c-FITC-10 ul; mixed for 20 min; and then washed. "Quick Spin"
samples were washed according to the Quick Spin protocol described
herein; and "Invention" samples were washed one time using a hollow
fiber membrane apparatus. Data were obtained using an EPICS XL flow
cytometer and are presented as signal-to-noise ratios (as described
herein). Data shown are based on one replicate per test
condition.
[0031] FIG. 14 is a graph showing the percent of intact cells
recovered after three wash cycles wherein the wash buffer had
increasing amounts of fetal calf serum.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The below described preferred embodiments illustrate various
adaptations of the invention. Nonetheless, from the description of
these embodiments, other aspects of the invention can be readily
fashioned by making slight adjustments or modifications to the
components and steps discussed below.
[0033] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions will control. In addition, the particular
embodiments discussed below are illustrative only and not intended
to be limiting.
[0034] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs.
[0035] The invention provides an automatic apparatus and automatic
method utilizing a filter to remove interferants from a body fluid
prior to analysis. As used herein the term "automatic" means
performed without direct human intervention. For example, an
automatic apparatus automatically performs a method when a
component of the apparatus, rather than a human operator, performs
one or more steps of the method, even though a human operator might
input instructions into the machine or even perform one of the
steps manually. Similarly, an "automated" method is a method
performed automatically. The term "interferants" means substances
or particles that obscure an analysis. More specifically, the
interferants comprise non-reacted chemical agents; non-reacted
biological agents; and biological particles, such as red blood cell
debris and cellular matter smaller than the cellular matter of
interest. Interferants in a cell sample analyzed fluorescently
typically include unbound fluorescent probe and autofluorescent
cell debris. A particular percentage of interferants is removed
from a sample when either (a) the amount of debris in the sample is
decreased by that percentage or (b) the signal to noise ratio is
improved by that percentage.
[0036] Referring to FIG. 1 of the drawings, a presently preferred
embodiment of a cell wash apparatus 10 includes a sample container
holder 14 and a filtration device 24 mounted to a frame 12. Sample
container holder 14 accommodates a sample container 16 containing a
sample of cells 20 in an arrangement such that an end of a sample
hose 22 can be inserted into the sample of cells 20 which can
contain interferants. Sample hose 22 is fluidly connected to vacuum
source 30 so that actuation of vacuum source 30 supplies a vacuum
force which aspirates the sample of cells 20 from sample container
16 into hose 22. More specifically, there is an absence of air in
filtration device 24 such that when vacuum force 30 is applied, the
cell sample 20 is aspirated form the sample container 16 into the
filtration device 24.
[0037] Vacuum source 30 can take the form of any device that can
provide a vacuum or hydraulic force for moving fluids. For example,
vacuum source 30 can be a fluid pump or an external vacuum line.
Preferably, the vacuum source 30 is a syringe pump, for example a 5
ml syringe pump, that can provide a vacuum to filtration device 24
when its plunger is withdrawn and a forward hydraulic force when
its plunger is depressed.
[0038] Devices that cause a vacuum force rather than a positive
pressure are the preferred form of source 30, because it has been
found that a vacuum is less damaging to cells. More specifically,
the sample of blood cells 20 does not circulate through a pump to
enter into the lumen 66 (not shown). If the cells circulate through
a pump, then cell deformation, aggregation and deterioration occur.
Therefore, the sample of cells 20 enter the lumen 66 by action of a
vacuum force rather than by the action of a force which is applied
to the sample of blood cells 20 which cause the sample of cells 20
to be pushed into the lumen 66.
[0039] Filtration device 24 is attached to frame 12 by a filtration
device fastener 26 and interposed between sample hose 22 and vacuum
source 30 so that application of a vacuum by vacuum source 30
causes aspiration of sample of cells 20 into filtration device 24.
Filtration device 24 can be any device that can remove interferants
such as unbound antibody molecules or cellular debris from sample
of cells 20. In a preferred embodiment, filtration device 24
includes a filter through which interferants can pass. Filters that
can be used include fine mesh screens, flat microfiltration
membranes, spiral wound membrane cartridges, or any other media
that can separate interferants from the cells of interest. In a
more preferred embodiment, the filter is a microporous hollow fiber
membrane that has a plurality of pores sized less than the blood
cells within sample of cells 20 but greater than the
interferants.
[0040] Suitable hollow fiber membranes for use as filtration device
24 can be fashioned by one of skill in the art or can be purchased
from a variety of commercial sources. Hollow fiber membranes useful
in the invention comprise a material which is non reactive with the
cells of interest and can be a hydrophobic or hydrophilic material,
polysulfone, polyestersulfone, nylon, methylacrylates, Peek.TM.
(Upchurch Scientific, Inc.). The filter will have pores sized so
that cells of interest cannot pass therethrough. The pore size will
range from approximately 0.1 microns to about 5 microns in
diameter. Preferably, the pore size will range from approximately
0.1 microns to about 3 microns, which can eliminate platelets as
interferants from the cells of interest. More preferably, the pore
size will range from approximately 0.2 microns to about 2 microns
and most preferably the pore size will range from approximately 0.3
microns to about 1 micron. In the present invention, a pore size of
about 0.65 microns has been successfully used to eliminate
interferants leaving a majority of cellular components for
analysis. One preferred commercially available polysulfone hollow
fiber membrane device having a plurality of pores with a mean
diameter of 0.65 microns is sold as Catalog # CFP-6-D-H22LA by A/G
Technology Corporation (Needham, Mass.). This device is suitable
for removing the majority of interferants from a typical sample of
100 microliter of whole human blood that has been stained with a
fluorescent antibody, erythrocyte-lysed, and diluted to a total
volume of about 4 ml using an isotonic buffer or reagent. Other
devices useful for variations of the invention include CFP-6-D-MB01
(15 cm.sup.2), and CFP-6-D-MM01A (24 cm.sup.2) from A/G Technology
Corporation; and X15E300 04N and X25E201 02N from Spectrum
Laboratories, Inc. (Rancho Dominguez, Calif.).
[0041] A sample hose valve 23 for regulating fluid flow between
sample container 16 and filtration device 24 is positioned on hose
22. Valve 23 can take the form of any device that can control the
flow of fluid through hose 22. Preferably, valve 23 is switchable
between an open position and a closed position. In the open
position, sample 20 can flow between container 16 and filtration
device 24 when a suitable force is applied, such as by vacuum
source 30. In the closed position, the fluid connection is blocked
so that sample 20 cannot flow between container 16 and filtration
device 24. In a preferred variation of the foregoing, valve 23 also
has a partially open position that directs fluid flow from hose 22
to a waste reservoir 39 by another fluid connection.
[0042] Although in the embodiment shown in FIG. 1 the fluid
connection between sample container 16 and filtration device 24 is
provided by sample hose 22 and regulated by valve 23, in an
alternate preferred embodiment, more than one fluid connection can
exist between sample container 16 and filtration device 24. For
example, sample hose 22 can be utilized for transporting sample of
cells 20 from container 16 to filtration device 24 and a return
hose or fluid connector can be provided for returning sample of
cells 20 from device 24 to sample container 16. A fluid flow
regulator analogous to valve 23 can be interposed in the return
hose. In addition, rather than having a fluid connection for
returning sample 20 from device 24 to sample container 16, the
apparatus can feature another pathway for transporting sample 20
from device 24 to a clean sample container, such as an unused test
tube, rather than sample container 16.
[0043] Referring again to FIG. 1, vacuum source valves 32 are
positioned within the fluid connection between vacuum source 30 and
filtration device 24 so that they can control transfer of vacuum
between vacuum source 30 and filtration device 24. Valves 32 are
preferably switchable between an open and a closed position. In the
open position, actuation of vacuum source 30 causes a vacuum force
to be applied to filtration device 24. The vacuum will cause
aspiration of sample 20 from container 16 into device 24. In the
closed position, no force is transmitted between vacuum source 30
and device 24.
[0044] Vacuum source 30 can also be fluidly connected to a waste
reservoir 39 by a waste hose 37. As indicated above, vacuum source
30 is also adapted to provide a forward hydraulic force. This
hydraulic force can be used to move fluid from locations proximal
to vacuum source 30 to waste reservoir 39. For example, when vacuum
source 30 takes the preferred form of a syringe pump, with valves
32 and 23 open, withdrawal of the plunger of the syringe pump
causes a vacuum that aspirates a liquid which contains interferants
and sample of cells 20 to be dispersed into the interior of the
syringe's barrel. Depressing the plunger at this point forcibly
expels the liquid from the syringe. With valves 32 closed, the
liquid is directed through waste hose 37 into waste reservoir 39.
In an alternative variation of the foregoing, rather than using
vacuum source 30, an additional vacuum source, pump, or hydraulic
force transducer can be utilized to move fluid from locations
proximal to vacuum source 30 to waste reservoir 39. This latter
variation is preferred where it is desired to avoid potential cross
contamination between waste reservoir 39 and sample container 16
and their associated fluid connections.
[0045] Filtration device 24 can also be fluidly connected to buffer
reservoir 48 by buffer hose 46. Buffer reservoir 48 is a container
for housing buffer 49 which can be any isotonic solution compatible
with sample of cells 20. Suitable buffers include physiological
saline or phosphate buffered saline (PBS) and Hanks Buffer.
Preferred isotonic solutions for use as buffer 49 include
IsoFlow.TM. buffer, PBS, and IMMUNO-TROL.TM. Final Storage buffer
(all available from Beckman Coulter, Inc., Fullerton, Calif.).
[0046] Interposed between device 24 and reservoir 48, and fluidly
communicating with hose 46 is buffer pump 40. Buffer pump 40
supplies an hydraulic force which moves buffer 49 from reservoir 48
through hose 46, filtration device 24, and sample hose 22 into
sample container 16. Pump 40 can take the form of any device that
can cause a hydraulic force between buffer reservoir 48, device 24,
and sample container 16. For example it can be a vacuum pump,
peristaltic pump, reciprocating pump, or other type of pump known
to those skilled in the art. In preferred embodiments, however, it
is a syringe pump.
[0047] Positioned on hose 46 between reservoir 48 and device 24 is
a buffer valve 42 for controlling flow of buffer between reservoir
48 and device 24. Although it can be any fluid flow regulating
device, valve 42 is preferably a three position stopcock-like valve
that can be placed in either a fill position, a dispense position,
or a closed position. In the fill position, pump 40 is in fluid
connection with buffer reservoir 48 such that it can transmit an
hydraulic force to hose 46 that causes pump 40 to aspirate buffer
49 from buffer reservoir 48 into buffer hose 46 or into the chamber
of the syringe when pump 40 is a syringe pump. In the dispense
position, pump 40 is in fluid communication with device 24 such
that actuation of pump 40, for example depressing the plunger of
the syringe, causes buffer 49 to be transported from pump 40 to
device 24 and, where valve 23 is open, into sample container 16.
Thus, referring to FIG. 1, with valve 42 in the open position,
valves 32 in the closed position and valve 23 in the open position,
actuation of pump 40 can cause buffer 49 to flush sample of blood
cells 20 positioned within filtration device 24 back into sample
container 16. With valve 42 in the closed position, the fluid
connection between reservoir 48, device 24, and container 16 is
blocked.
[0048] Detergent solution reservoir 58 is fluidly connected to
filtration device 24 by detergent solution hose 56. Detergent
solution reservoir 58 is a container for housing a detergent
solution 59 which is suitable for cleaning filtration device 24 and
the fluid connections of apparatus 10. Detergent solution 59 can be
any solution that can remove residual samples, accumulated
deposits, proteins, nucleic acids and the like from the fluid
connections of apparatus 10. For example, detergent solution can be
0.5N NaOH solution, 1N KOH solution, H.sub.3PO.sub.4 solution,
0.05-10% bleach solution, or a similar solution. The detergent
solution can include substances such as Triton X-100 (Rohm and
Haas), Tween 80.RTM. (ICI America), pluronic acids (BASF Corp.),
ethylenediamine tetraacetic acid (EDTA), proteases, nucleases,
azide, and other substances which can clean fluid connections. One
preferred composition for use as detergent solution 59 is the
solution sold under the trade name COULTER CLENZ.RTM. (Beckman
Coulter Inc., Fullerton, Calif.).
[0049] Detergent solution pump 50 supplies a hydraulic force which
moves detergent solution 59 from reservoir 58 through hose 56 into
filtration device 24. Similar to pump 40, pump 50 can take the form
of any device that can cause an hydraulic force between detergent
solution reservoir 58 and device 24. For example it can be a vacuum
pump, peristaltic pump, reciprocating pump or other type of pump
known to those skilled in the art. In preferred embodiments,
however, it is a syringe pump.
[0050] Positioned on hose 56 between detergent solution pump 50 and
buffer hose 56 is a detergent solution valve 52 for controlling
flow of detergent solution 59 between reservoir 58 and device 24.
As with valve 42, although it can be any suitable fluid flow
regulating device, valve 52 is preferably a three position
stopcock-like valve that can be placed in either a fill position, a
dispense position, or a closed position. In the fill position,
detergent solution pump 50 is in fluid connection with detergent
solution reservoir 58 such that it can transmit a hydraulic force
to hose 56 that causes pump 50 to aspirate detergent solution 59
from detergent solution reservoir 58 into detergent hose 56 or into
the chamber of the syringe when pump 50 is a syringe pump. In the
dispense position, pump 50 is in fluid communication with device 24
such that when valve 32 is closed and valve 23 is open, actuation
of pump 50, for example depressing the plunger of the syringe,
causes detergent solution 59 to be transported from pump 50 to
device 24. And when valves 32 are in the open position and valve 23
in the closed position, actuation of pump 50, with or without the
cooperation of vacuum source 30, can cause detergent solution 59 to
wash any fluid or material within filtration device 24 into waste
reservoir 39. With valve 52 in the closed position, the fluid
connection between reservoir 58 and device 24 is blocked.
[0051] In addition to the above-described buffer and detergent
solution devices, other devices can be included within apparatus
10. For example, devices for adding an erythrocyte lysing agent can
be included. Similarly, devices for adding one or more cell marker
probes, such as fluorescently-labeled antigen-specific antibodies,
can be included within apparatus 10. In addition, fluid connections
to one or more cell analyzers, such as hematology and flow
cytometry analyzers, can also be provided. Thus, the invention can
include an apparatus that can automatically process a sample of
whole blood by lysing the red blood cells within the blood cell
sample, adding a cell marker probe to the blood cell sample,
removing the lysed red blood cell debris and unbound cell marker
probe from the blood cell sample, and quantifying the remaining
cells and quantifying specific cell markers using a cell
analyzer.
[0052] In a preferred embodiment, the various components of the
apparatus are controlled by an information processing unit, such as
a computer. That is valves 23, 32, 42, and 52, and vacuum source 30
and pumps 40 and 50 are operatively connected to an information
processing unit (not shown in the drawings) having programmed
therein operating algorithms for switching the valves and actuating
the pumps and vacuum sources. The information processing unit can
be connected to electrical, hydraulic, or mechanical manipulators
such as servos, robotic arms, gears and the like to operate the
pumps, valves, and vacuum source as well as other components of
apparatus 10. For example, in one embodiment, hose 22 can be
attached to a robotic arm that can move hose 22 between sample
container 16 and a different site, for example where another
container is located, according to instructions provided by the
information processing unit.
[0053] Referring now to FIG. 2, a particularly preferred embodiment
of filtration device 24 is shown in further detail. In this
preferred embodiment of the apparatus of the invention, filtration
device 24 includes a hollow fiber membrane 60 fashioned into a tube
having a wall that defines a lumen 66. The filtration device 24
further includes a bottom port 62, which is longitudinal to the
filtration device, so that tubular shaped membrane 60 fluidly
connects sample hose 22 and lumen 66. Fluids, such as sample of
cells 20, can enter lumen 66 from sample hose 22 by port 62.
Filtration device 24 also includes a top port 64, which is
longitudinal to the filtration device, so that tubular shaped
membrane 60 fluidly connects hoses 46 and 56 to lumen 66. Buffer 49
(not shown) can enter lumen 66 from buffer hose 46 by port 64.
Likewise, detergent solution 59 (not shown) can enter lumen 66 from
detergent solution hose 56 by port 64.
[0054] Although the devices in FIGS. 2, 3 and 4 A-K show only one
membrane 60. In another preferred embodiment, device 24 can include
more than 1 membrane 60 which forms more than 1 lumen 66. More
specifically, the filtration device can have 2 membranes each
forming a lumen so that the filtration device contains 2 lumens.
More preferably, the filtration device contains three membranes
which form 3 lumens. Most preferably, the filtration device
contains four membranes which form 4 lumens. It has been found
having more than 1 lumen will increase the processing flow rate. In
addition, having more than 1 lumen will have less fouling and
require less cleaning cycles. However, it is also preferred that
the filtration device contains less than 20 membranes which form
less than 20 lumens, and most preferred that it contains less than
10 membranes which form less than 10 lumens.
[0055] As noted in FIG. 2, the outer surface of filtration device
24 preferably includes a non-reactive impermeable housing 70 which
envelopes hollow fiber membrane 60 and extramembrane chamber 68.
The extramembrane chamber 68 is defined as the space between the
inner wall of housing 70 and the outer wall of tubular membrane 60.
Vacuum and waste port 34, which can be lateral to the filtration
device 24, is an opening that fluidly connects extramembrane
chamber 68 to vacuum source 30 and waste hose 37. Port 34 can thus
project through the wall of impermeable housing 70, such that
application of a vacuum force to port 34, for example from source
30, transfers the vacuum force to extramembrane chamber 68. Vacuum
in chamber 68 causes fluid and interferants 72 to be withdrawn from
lumen 66 across membrane 60 into chamber 68 and out through port
34. After closing valves 32 and applying a forward hydraulic force
from source 30, the withdrawn fluid and interferants 72 can be
transported to waste reservoir 39.
[0056] Device 24 is preferably arranged such that fluid and
interferants can be withdrawn throughout the entire portion of
membrane 60 contained within housing 70. For example, port 34 is
preferably positioned on the device such that a vacuum from vacuum
source 30 is directed approximately perpendicular with respect to
the length of membrane 60. Application of a vacuum in such a
crosswise manner is preferred as compression of cells is reduced
compared to devices that force cells to one end of membrane 60,
which occurs when a pump is used to increase pressure within the
lumen of membrane 60 to expel cells through the pores of the
membrane.
[0057] A preferred mechanism by which filtration device 24
selectively retains the cells of interest while allowing the
interferants to pass through is illustrated in FIG. 3. Sample of
cells 20 is shown in lumen 66 as a mixture comprising cells 74 and
interferants 72, such as unbound probe and cellular debris, which
is dispersed in a liquid medium. Hollow fiber membrane 60 is shown
as having a plurality of pores 65 having a mean diameter of less
than the mean diameter of cells 74 but greater than the diameter of
interferants 72. Interferants 72 can thus pass through pores 65
while the larger diameter cells 74 cannot. Application of a vacuum
to chamber 68, through port 34, causes the liquid in which sample
of cells 20 is dispersed to be withdrawn through pores 65 into
chamber 68 along with interferants 72 contained within the liquid.
Cells 74, being too large to pass through pores 65, are selectively
retained in lumen 66.
[0058] In the embodiment shown in FIG. 3, membrane 60 can be
composed of any suitable material. For example, it can be composed
of a hydrophobic or hydrophilic polymer. In one preferred version
it is composed of microporous polysulfone. Suitable sizes of pores
65 of membrane 60 can be selected by one of skill in the art
depending on the particular characteristics of the cell sample to
be analyzed. For applications where human leukocytes are analyzed,
pores 65 preferably have a mean diameter of between about 0.2 and
2.0 microns, and more preferably have a mean diameter of about 0.3
microns to about 1 micron. The surface area of the membrane 60 can
also be selected by one of skill in the art depending on such
factors as the particular characteristics of the sample to be
analyzed, the sample volume, and the type of membrane used. For
example, for a 100 microliter sample of a whole human blood
processed and then diluted to a total volume of about 4 ml using an
isotonic buffer, 20 cm.sup.2 of a hollow fiber membrane with 0.65
micron diameter pores is sufficient to remove the majority of
interferants in the sample. For a 1 ml sample, preferred lumen
volumes range from about 50 .mu.l to about 2500 .mu.l and
preferably about 200 .mu.l to about 1000 .mu.l, and preferred
extramembrane chamber volumes range from about 100 .mu.l to about
2500 .mu.l and preferably about 500 .mu.l to about 1000 .mu.l.
Other lumen and extramembrane chamber volumes can be preferred
depending on the volume and types of sample. Membrane 60 can also
be treated with non-lytic surfactants such as Pluronic F68 and
Pluronic 25R8 (BASF Corp.) to enhance its reusability without
having a material adverse effect on cell count or cell marker
density on cells in sample 20.
[0059] An overview of a preferred operation of an apparatus of the
invention is shown in FIGS. 4A-4E. In FIG. 4A, apparatus 10 is
shown with sample hose 22 in fluid communication with sample of
cells 20. For example, the sample of cells 20 can be 100 .mu.l of
whole blood having been processed using a lysing reagent, a
stabilizing buffer, and a fixative such as IMMUNOPREP.TM. reagents
(manufactured by Beckman Coulter, Inc., Miami, Fla.). As
illustrated in FIGS. 4B and C, sample 20 is diluted with buffer 49
to facilitate removing a greater percentage of interferants 72. To
transfer a predetermined volume of buffer 49, such as to bring the
total volume of the sample to about 4 ml, from buffer reservoir 48
into sample container 16, apparatus 10 is arranged by a computer
control mechanism (not shown), so that valve 23 is open, and valves
32 and 52 are closed. As shown in FIG. 4B, buffer valve 42 is then
switched to the fill position and buffer pump 40 is activated to
aspirate the predetermined volume of buffer 49. As indicated in
FIG. 4C, valve 42 is then switched to the dispense position and
pump 40 is activated to dispense the aspirated volume of buffer 49
through filtration device 24 into sample container 16 thereby
diluting sample of cells 20.
[0060] As shown in FIG. 4D, sample 20 is then aspirated into
filtration device 24 where interferants are removed from the sample
by having them pass through membrane 60. In this step, apparatus 10
is configured so that valves 42 and 52 are closed, and valves 23
and 32 are open. Vacuum source 30 is then activated to produce a
vacuum to aspirate sample of cells 20 from container 16 into
filtration device 24. While the vacuum is being supplied, the
liquid in sample 20 that contains interferants is passed through
device 24 into vacuum source 30, while cells are retained in device
24, within lumen 66. As shown in FIG. 4E, valves 32 are then closed
and vacuum source 30 is activated to provide a forward hydraulic
force to expel the aspirated liquid through waste hose into waste
reservoir 39.
[0061] As illustrated in FIGS. 4F and G, sample of cells 20 from
which interferants have been removed is then transferred back into
container 16. In this step, apparatus 10 is configured so that
valve 23 is open, and valves 32 and 52 are closed. In FIG. 4F,
buffer valve 42 is then switched to the fill position and buffer
pump 40 is activated to aspirate a predetermined volume of buffer
49, for example 1.25 ml, from buffer reservoir 48. Valve 42 is then
switched to the dispense position and pump 40 is activated to
dispense the aspirated volume of buffer 49 through filtration
device 24 into sample container 16 as illustrated in FIG. 4G.
Movement of buffer 49 through device 24 flushes sample of cells 20
from the device into container 16. In an alternative embodiment
(not shown), an additional fluid connection from device 24 to a
clean sample container rather than sample container 16 can be
provided, such that after the interferants have been removed from
sample of cells 20, the sample can be transported from device 24 to
the clean container.
[0062] The apparatus 10 can be washed as shown in FIGS. 4H-K. The
washing of the apparatus can be after each sample, after a
predetermined number of samples, or upon fouling of the membrane
60. In the washing step, apparatus 10 is set up so that valve 23 is
partially open, and valves 32 and 42 are closed. As shown in FIG.
4H, detergent solution valve 52 is then switched to the fill
position and detergent solution pump 50 is activated to aspirate a
predetermined volume of detergent solution 59, for example 3 ml,
from detergent solution reservoir 58. As indicated in FIG. 41,
valve 52 is then switched to the dispense position and pump 50 is
activated to dispense the aspirated volume of solution 59 through
filtration device 24. Because valve 23 is partially open, solution
59 can flow through hose 100 into waste reservoir 39. To purge any
detergent solution 59 remaining in device 24, as shown in FIG. 4J,
buffer valve 42 is then switched to the fill position and buffer
pump 40 is activated to aspirate a predetermined volume of buffer
49, for example 3 ml, from buffer reservoir 48.
[0063] In FIG. 4K, prior to the buffer dispensing step, valve 23
can be closed and valve 32 can be switched to the open position.
Valve 42 is switched to the dispense position and pump 40 is
activated so that buffer 49 is dispensed and the remaining
detergent solution 59 in the filtration device and buffer 49 are
transferred to waste reservoir 39 by a waste hose. Alternatively,
or in addition, valve 23 is switched to being partially open, and
valve 32 is closed, and valve 42 is switched to the dispense
position and pump 40 is activated to dispense the aspirated volume
of buffer 49 through filtration device 24 and hose 100 into
reservoir 39. The foregoing steps can be repeated so that device 24
is washed with multiple volumes of buffer prior to analysis of the
next sample.
[0064] Referring now to FIG. 5, the invention also includes methods
for removing interferants from a sample of cells. A preferred
method for removing interferants from a sample of cells comprises a
first step 80 of applying a vacuum force to a blood cell sample to
cause the blood cell sample to leave the sample container 16 and
contact a filter. As previously explained, this is accomplished by
a vacuum force, which typically is capable of causing approximately
4 ml of a blood cell sample to be withdrawn from the sample
container and pass through the membrane filter in approximately 7
seconds. As appreciated by one skilled in the art, the amount of
blood cell sample withdrawn from the sample container 16 can be
increased or reduced and the time can also be increase or reduced.
The limitation on the vacuum force is that it will be less than the
amount of force that would cause the cells to aggregate when being
retained in the lumen 66. Preferably, the force will be less than
that which would cause the cells to deform.
[0065] The method includes a second step 82 of applying a force to
the blood cell sample in contact with the filter, whereby
interferants in the blood cell sample pass through the filter while
the cells of interest in the blood cell sample do not pass through
the filter. In a preferred embodiment of the invention, the force
that is applied to the blood cell sample to cause the interferants
to pass through the filter is the same vacuum force which is used
to withdraw the blood cell sample from the sample holder. However,
it is appreciated that the force could be a separate hydraulic
force which after the blood cell sample is withdrawn from the
sample container 16, could be applied to the blood cell sample to
push the blood cell sample into the lumen and through the membrane.
However, it has been found that a vacuum is less damaging to cells.
The limitation on the force is that it will be less than the amount
of force, which would cause the cells to aggregate when being
retained in the lumen 66. Preferably, the force will be less than
that which would cause the cells to deform.
[0066] The method includes a third step 84 of recovering the cells
from the filter. In a preferred embodiment, the cells are recovered
by the apparatus of the invention wherein a volume of buffer is
pumped through the top portion of the lumen causing the cells that
were retained in the lumen to pass through the bottom portion of
the lumen back into the sample container. Alternative, the retained
blood cells can pass through the bottom portion of the lumen into a
new sample container which can be employed to store the recovered
blood cells.
[0067] In a more preferred embodiment of the present method, the
blood cell sample is first diluted with at least one volume of
buffer to each volume of blood cell sample. Even more preferable is
that the blood cell sample be diluted with at least two volumes of
buffer before entering the lumen to remove the interferants. It has
been found that with a one volume dilution of the blood cell sample
that greater than 70% of the interferants are removed from the
blood cell sample, and with a two volume dilution, greater than 80%
of the interferants are removed from the blood cell sample. A three
volume dilution of the blood cell sample is preferred to remove
greater than 90% of the interferants from the blood cell
sample.
[0068] The steps of this method can be accomplished using the
apparatus of the invention which will provide automation of the
steps described above. As defined herein, one cycle of the method
is considered to be one wash cycle of the blood cell sample. More
specifically, one wash cycle of the blood cell sample comprises
applying a vacuum force to a blood cell sample to cause the blood
cell sample contact a filter; applying a force to the blood cell
sample in contact with the filter, whereby interferants in the
blood cell sample pass through the filter while the cells in the
blood cell sample do not pass through the filter; and recoverying
the cells from the lumen. Accordingly, one wash cycle of the blood
cell sample wash cycle of this invention can be performed in less
than 5 minutes. Preferably, one wash cycle of the blood cell sample
is performed in less than 3 minutes, and more preferably less than
1 minute. In an even more preferred embodiment one wash cycle of
the blood cell sample is performed in less than 30 seconds.
Finally, in a most preferred embodiment, one wash cycle of the
blood cell sample is performed in less than 15 seconds.
[0069] It has been found that multiple wash cycles cause the cells
to deteriorate such as shrinkage of the cell membranes and rupture
of the cell membranes. It has been further found that the addition
of a serum substance to the buffer which dilutes the blood cell
sample minimizes the deterioration. As defined herein, serum
substance comprises cholesterol, cholesterol esters, and
cholesterol which has been combined with one or more other
compounds found in serum plasma, and mixtures thereof. Preferably,
such other compounds further comprise lipoproteins and
phospholipids, and mixtures thereof. As appreciated by those
skilled in the art, typically cholesterol will contain
approximately 30% esters. As further appreciated by those skilled
in the art, the lipoprotein will maintain the cholesterol in an
aqueous solution. Preferably, the serum substance is selected from
the group comprising cholesterol, cholesterol esters, lipoprotein
cholesterol, lipoprotein cholesterol esters, cholesterol combined
with phospholipids and mixtures thereof.
[0070] FIG. 14 depicts an increase in the recovery of cellular
events as related to the percent addition of fetal calf serum in a
buffer. In this figure, the blood cell sample was washed 3 times
with a hollow fiber membrane apparatus shown as "Invention" in the
figure. An increase of fetal calf serum indicates that there will
be an increase in the percent of cells recovered after multiple
wash cycles.
[0071] It has also been found that one wash cycle of the blood cell
sample without the addition of a serum substance eliminates the
banana appearance between the lymphocytes and neutrophils
subpopulations in histograms of blood cell samples containing a
high lipid content.
[0072] In an example of the present method, first step 80 is
performed by providing a sample of cells such as a 100 microliters
of whole human blood obtained by venipuncture from a human subject.
If the removal of erythrocytes is desired, the sample can be
diluted in a reagent which lyses red blood cells such as 600
microliters of formic acid, and then further diluted by addition of
a reagent that neutralizes the red blood cell lysing agent such as
265 microliters of a carbonate buffer. Optionally, a fixative such
as 100 .mu.l of a paraformaldehyde solution can also be added to
fix the cell sample. The blood cell sample is diluted to a total
volume of about 4 ml with an isotonic buffer. Suitable reagents for
these steps can be obtained from Beckman Coulter, Inc.
(IMMUNOPREP.TM. reagent system part no. 7546999 or SCATTER PAK.TM.
reagent system). A vacuum force is then applied to the diluted
blood cell sample to cause it to contact a filter. Preferably the
filter is a hollow fiber membrane (e.g., Cat# CFP-6-D-H22LA from
A/G Technology Corporation).
[0073] In second step 82, a force is applied to the blood cells
sample which is in contact with the filter to cause the
interferants in the diluted blood cell sample to pass through the
filter while the cells of interest in the blood cells sample are
retained by the filter. More specifically, the cells of interest in
the diluted blood cell sample do not pass through the filter. When
the hollow fiber membrane is used, the cells of interest will be
retained in the lumen. Preferably, a vacuum force is applied to the
blood cell sample to cause the interferants to pass through the
lumen while the cells of interest are retained in the lumen.
[0074] In a most preferred embodiment, the vacuum force that is
used to cause the interferants to pass through the filter also
aspirates the blood cell sample from the sample container. More
specifically, the filtration device is in fluid communication with
the sample container since it is filled with a buffer. Therefore,
when a sufficient vacuum force is applied to the diluted blood cell
sample in the sample container, the diluted blood cell sample is
aspirated from the sample container into the filtration device and
the interferants pass through the filter. This is accomplished by a
continuous flow of the blood cell sample from the sample container
through the filter. As previously discussed, the apparatus of this
invention can automatically apply the vacuum force necessary to
perform these functions.
[0075] The Third step 84 is recovering the cells from the filter.
This can be accomplished by providing a force, such as a flow of
liquid, to the filter in a direction opposite the direction from
which the blood cell sample contacted the filter in step 80. The
flow of liquid will move the cells of interest away from the
filter. The recovered cells can thereafter be transported by fluid
communication to an analytical instrument. Preferably, the
recovered cells are returned to a test tube that is then
transported to an instrument for analysis.
[0076] Referring now to FIG. 6, methods for analyzing cells for
phenotypic markers are also included in the invention. A preferred
method of analyzing a phenotypic marker on cells within a sample
includes: a first step 90 of adding a probe that binds the
phenotypic market to the sample of cells to be analyzed to form a
test sample mixture; a second step 92 of applying a vacuum force to
a blood cell sample to cause the blood cell sample contact a
filter; a third step 94 of applying a force to the blood cell
sample in contact with the filter, whereby interferants in the
blood cell sample pass through the filter while the cells in the
blood cell sample do not pass through the filter; a fourth step 96
of recovering i the cells from the filter. The method can further
include a fifth step 98 (not ! shown) of quantifying the amount of
probe and differentiating the cell populations.
[0077] Steps 92, 94 and 96 can be performed as described above for
FIG. 5 for Steps 80, 82 and 84 respectively. Step 98 can be
performed by analyzing the test sample from which the interferants
have been removed using flow cytometry or a similar analytical
device.
[0078] For example, in a preferred version of this method, first
step 90, a saturating concentration of a fluorescently-labeled
antigen-specific antibody is added to the blood cell sample to form
the test sample mixture. And fifth step 98 can be performed by
running the processed test sample mixture on a flow cytometer
equipped to quantitatively measure the amount of
fluorescently-labeled antigen-specific antibody associated with
each cell in the processed test sample mixture.
[0079] From the foregoing, it can be appreciated that the apparatus
and methods of the invention facilitate the removal of interferants
from a sample of cells to be analyzed. The invention will be
further described in the following examples, which do not limit the
scope of the invention described in the claims.
EXAMPLE 1
Cell Washing Apparatus
[0080] An apparatus was built with a hollow fiber membrane
cartridge cat# CFP-6-D-H22IA from A/G Technology Corporation. The
apparatus included a carousel-type cell sample holder adapted to
hold several 12.times.75 mm culture tubes. Alternatively, the
apparatus can include other types of tube holders such as a
cassette. The apparatus also included various hoses, valves, and
pumps so that a sample of cells could be aspirated from the tube,
filtered through the hollow fiber membrane cartridge to remove
interferants from the sample, and then returned to the tube. As
described in the detailed description (for example see, discussion
of FIGS. 4A-4K), the apparatus also included various hoses, valves,
and pumps so that waste fluids (for example, filtrate containing
interferants) could be removed to a waste reservoir, and the hollow
fiber membrane could be cleaned for use with additional samples.
The apparatus also included a computerized system for coordinating
the cell sample washing process and the membrane cleaning
procedure. The carousel-type cell sample holder was rotatable and
also controlled by the computerized system such that after
processing a first cell sample, a second tube containing a second
cell sample could be repositioned to allow the second cell sample
to be aspirated from the tube, filtered through the hollow fiber
membrane cartridge to remove interferants from the sample, and then
returned to the second tube. This cycle was repeatable such that
all samples in the carousel could be washed.
EXAMPLE 2
Method of Washing Cells
[0081] Various methods, including a method employing the apparatus
of Example 1, were used for removing interferants from a cell
sample processed according to the general method described below. A
cell population was stained with a fluorescently labeled antibody
according to standard techniques. For example, 100 ul of whole
human blood was obtained by venipuncture from a human subject, and
then 10 ul of a 1 mg/ml solution of an antigen-specific
FITC-labeled antibody was added to the blood sample. Samples were
then incubated for 10 minutes at room temperature, after which
erythrocytes were lysed using Beckman Coulter's IMMUNOPREP research
system and TQ-Prep apparatus according to the manufacturer's
instructions (600 .mu.l of solution A for 8 seconds with mixing;
265 .mu.l of solution B for 10 seconds with mixing; and 100 .mu.l
of solution C for 10 seconds with mixing). Separate aliquots of the
processed blood cells samples were then subjected to one of three
different protocols:
[0082] A. diluted with an isotonic buffer to a total volume of
about 4 ml and then washed 1 time per a "Quick Spin" wash protocol.
The Quick Spin was protocol means centrifuge 400.times.g for 5
minutes using a standard centrifuge, decant supernatant, and
resuspend in 1 ml of an isotonic buffer;
[0083] B. diluted with an isotonic buffer to a total volume of
about 4 ml and then washed 1 time per a "Sorvall" protocol using a
Sorvall.RTM. Cell Washer 2 (auto mode 80 seconds; high speed
2950-3000 rpm; decant 600 rpm) according to the manufacturer's
instructions (washed cells resuspended in final volume of 1 ml
isotonic buffer) ; or
[0084] C. diluted with an isotonic buffer to a total volume of
about 4 ml and then washed 1 time using the apparatus described in
Example 1 (washed cells in final volume of 1 ml isotonic
buffer).
EXAMPLE 3
Analysis of Cell Samples
[0085] Samples of whole human blood were reacted with a fluorescent
labeled monoclonal antibody directed against the cell surface
antigen designated CD56, erythrocyte lysed and fixed according to
Example 2. "TQ-Prep" samples were not washed. "Quick Spin" samples
were washed according to the Quick Spin protocol described in
Example 2. "3 ml predilution" samples were washed one time using a
hollow fiber membrane apparatus according to the protocol described
in Example 2C. "2.times. wash" samples were washed two times
(second wash with a 2 ml predilution) using a hollow fiber membrane
apparatus according to the protocol described in Example 2C. The
processed blood cell samples were then subjected to flow cytometric
analysis using a COULTER EPICS XL flow cytometer according to the
manufacturer's instructions. Results for % debris as determined by
light scatter analysis, % CD56 positive cells, and signal-to-noise
ratio (extrapolated from histograms) are shown in FIG. 7.
[0086] The amount of debris was low for all samples, although more
debris was noted in the samples subjected to two washings with the
hollow fiber membrane apparatus. The increase of debris was caused
by cell degradation because no serum substance was employed in the
diluent. The percent of CD56 cells was about the same whether the
Quick Spin was used or the hollow fiber membrane apparatus was
used. Signal-to-noise ratios were greatly improved over the no wash
control, no matter which washing protocol was used. Washing the
sample two times with the hollow fiber membrane apparatus produced
the best signal-to noise ratio.
[0087] In similar experiments, for unwashed samples the average
percent of debris was 10.3% and the average signal to noise ratio
was 11.4. As defined herein, debris means events falling below
threshold measurement values. In comparison, using the hollow fiber
membrane apparatus, the average percent of debris was 2.5%, which
means that greater than 75% of the original 10.3% of debris was
removed. In addition, the average signal to noise ratio was 23.8,
which means that there was greater than a 200% improvement in the
signal to noise ratio. Using the Quick Spin protocol, the average
percent debris was 2.6% and the average signal to noise ratio was
38.7. In other experiments, when cell samples were washed 2 or 3
times with the hollow fiber membrane apparatus more interferants
were removed and the signal to noise ratio further improved.
EXAMPLE 4
Evaluation of Cell Recovery
[0088] Samples of whole human blood were processed, and washed
according to the protocols described in Example 2, and then
subjected to flow cytometric analysis using an EPICS XL flow
cytometer according to the manufacturer's instructions. As shown in
FIG. 8, results for cell recovery (number of indicated type of
cells recovered from 100 microliter sample of whole blood after
processing) show that little or no cell loss occurs in either the
lymphocyte, monocyte, granulocyte (cell type determined by light
scatter) fractions of the samples. Moreover, cell recovery using
the apparatus of Example I was about equivalent to that obtained
using the Quick Spin protocol. "TQ-Prep" samples (n=5) were not
washed; "Quick Spin" samples (n=5) were washed according to the
Quick Spin protocol described herein. "Auto" samples (n=32) were
washed one time using a hollow fiber membrane apparatus.
EXAMPLE 5
Accuracy
[0089] Samples of whole human blood from several different donors
were stained for CD56, processed, and washed according to the
protocols described in Example 2. "TQ-Prep" samples were not
washed; "Quick Spin" samples were washed according to the Quick
Spin protocol described herein; and "Hollow Fiber" samples were
washed one time using a hollow fiber membrane apparatus. The
samples were then subjected to flow cytometric analysis using an
EPICS XL flow cytometer according to the manufacturer's
instructions. As shown in FIG. 9, the percentage of cells that were
CD56.sup.+ varied from donor to donor but, for any one donor, was
about the same whether the Quick Spin was used or the hollow fiber
membrane apparatus was used.
EXAMPLE 6
Precision
[0090] Thirty-two aliquots of one sample of whole human blood were
stained for CD56, processed, and washed according to the protocols
described in Example 2, and then subjected to flow cytometric
analysis using a COULTER EPICS XL flow cytometer according to the
manufacturer's instructions to determine the percent of CD56+cells
in each aliquot. The average percent of CD56.sup.+ cells among the
aliquots was 17.44% with a standard deviation of 0.74 and a
coefficient of variation of 4.27%. In a similar experiment using 28
aliquots, the average percent of CD56.sup.+ cells among the
aliquots was 15.6% with a standard deviation of 0.6 and a
coefficient of variation of 3.5%.
EXAMPLE 7
Cell Carryover
[0091] Whole blood cell samples were processed as described in
Example 2 and then concentrated to four times normal cell
concentrations. Each sample was then washed using the apparatus of
Example 1 (per the protocol of Example 2C with cleaning of the
hollow fiber membrane after sample washing). The apparatus was then
used to "wash" a blank sample containing only buffer without cells.
The blank sample was analyzed for the presence of cells using a
flow cytometer. As shown in FIG. 10, carryover of cells from test
to test was very low, ranging from 0.00% to 0.03% of cells being
carried over to subsequent analysis.
EXAMPLE 8
Other Applications
[0092] The apparatus and methodology of the invention are also
suitable for other analyses not explicitly described in detail
herein. Such other applications include protein analysis of urine.
In addition, applications which have traditionally utilized
centrifugation as part of their cellular analysis method are
specifically envisioned for use with the disclosed hollow fiber
membrane apparatus and method described herein. For example, many
different cell populations have been analyzed using the apparatus.
Additionally, many different probe types have been used in the
invention. For instance, aside from erythrocyte-depleted whole
blood samples, the hollow fiber membrane apparatus has been
successfully used with cell lines, purified white blood cell
subsets; erythrocytes; platelets; bone marrow cells; and cells in
cerebrospinal, synovial, peritoneal, ascites, pleural, pericardial
fluids and homogenized tissue. The erythrocyte agglutination
techniques commonly practiced in the blood banking field for the
typing of blood and for compatibility testing, which are
traditionally centrifugation dependent, can be readily adapted for
performance using the methodology and apparatus of the invention.
Probes that have been successfully used in the invention include
fluorescently labeled monoclonal antibodies that are specific for
the cell surface antigens such as immunoglobulin, kappa and lambda
factors, CD5, CD7, CD10, CD13, CD19, CD33, CD34, CD38, CD41, CD45,
CD 41, CD42b, CD 61, CD63, CD64, CD71, and CD117; as well as
intracellular antigens such as various types of hemoglobin. Various
other antibody and non-antibody probes such as chemical and
biologic constructs that bind to receptor molecules on the cell
surface, enzymatic substrates which react with cellular enzymes
within the cell, antibody and non-antibody probes which react with
cytoplasmic antigens within the cell, DNA and RNA probes which
react with nucleic acids sequences within the cells and various
intracellular dyes that react with cytoplasmic and nuclear
structures within the cell are expected to be compatible with the
invention. It is thus envisioned that most types of cells and
probes are compatible with the invention, especially if the
selected cell type is larger and the selected probe is smaller than
the pores of the selected hollow fiber membrane.
[0093] For example, referring to FIG. 11, application of the
invention to platelet samples is shown by flow cytometric analysis
of platelet samples stained for CD42b and CD63. Additionally, as
another example, referring to FIG. 12, application of the invention
to bone marrow samples is shown by flow cytometric analysis of bone
marrow cell samples stained for CD56. Cell recovery and
signal-to-noise ratio were comparable between "Invention" which is
the apparatus and method described herein and the Sorvall apparatus
and washing method. Referring now to FIG. 13, application of the
invention for intracellular analysis is shown by flow cytometric
analysis of permeabilized blood cell samples stained for
hemoglobin. Signal-to-noise ratios were comparable between "Hollow
Fiber" which is the apparatus and method described herein and Quick
Spin washing method described in Example 2.
EXAMPLE 9
Cell Washing Apparatus Integrated with a Cell Analyzer
[0094] It is specifically envisioned that the cell washing
apparatus of the invention can be integrated with one or more
conventional cell analyzers thereby obviating a manual step of
transferring a sample of washed cells from the washing device to
the analyzer. For example, the cell washing apparatus described
herein could be integrated with a flow cytometer such as a COULTER
EPICS.RTM. brand flow cytometer by providing robotic means for
transferring a test tube from a cell sample washed using the cell
washing apparatus of the invention such that the tube becomes
positioned so that it can be analyzed in the flow cytometer. As one
example, a conveyor could transport a carousel containing several
washed samples from a position suitable for washing the cells
(e.g., proximal to the cell washing device) to another position
suitable for analyzing the samples (e.g., proximal to the flow
cytometer). Fluid connections and conduits would aspirate washed
cell samples into the flow cytometer for analysis.
[0095] Alternatively, the cell washing apparatus of the invention
can be integrated with one or more hematology instruments. In this
embodiment, the blood cell sample would be washed after lysing the
erythrocytes to remove remaining cellular debris. Still further,
the blood cell sample could be washed prior to any biological or
chemical reaction with the blood cell sample so that interferants
are removed from the blood cell sample.
[0096] While the above specification contains many specifics, these
should not be construed as limitations on the scope of the
invention, but rather as examples of preferred embodiments thereof.
Many other variations are possible. For example, the invention
includes an apparatus for removing interferants from a cell sample
that has only one hydraulic force transducer rather than two pumps
and a vacuum source. The various hoses and valves within this
apparatus can be connected in a manner to cooperate with the sole
hydraulic force transducer, so that the apparatus functions much as
the described preferred embodiments. As another example, a method
of concentrating a cell sample by removing liquid from the sample
using a microporous hollow fiber membrane is included within the
invention. Accordingly, the scope of the invention should be
determined not by the embodiments illustrated, but by the appended
claims and their legal equivalents.
* * * * *