U.S. patent application number 10/442306 was filed with the patent office on 2003-10-30 for apparatus for obtaining increased particle concentration for optical examination.
Invention is credited to Berndt, Klaus W., Frank, Robert S., Livingston, Dwight, Scrivens, Brian G..
Application Number | 20030202908 10/442306 |
Document ID | / |
Family ID | 27613737 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030202908 |
Kind Code |
A1 |
Scrivens, Brian G. ; et
al. |
October 30, 2003 |
Apparatus for obtaining increased particle concentration for
optical examination
Abstract
The present invention relates to a method and apparatus for
analyzing a blood or other biological fluid sample in a quiescent
state, whereby particulate constituents of biological samples that
contain sparse populations of interesting cellular species can be
enumerated and inspected using an optical scanning instrument.
Specifically, this invention relates to a method and apparatus for
obtaining increased cellular or particulate concentrations within
the use of said optical scanning method.
Inventors: |
Scrivens, Brian G.; (Colora,
MD) ; Livingston, Dwight; (Fallston, MD) ;
Frank, Robert S.; (Ellicott City, MD) ; Berndt, Klaus
W.; (Timonium, MD) |
Correspondence
Address: |
BECTON, DICKINSON AND COMPANY
1 BECTON DRIVE
FRANKLIN LAKES
NJ
07417-1880
US
|
Family ID: |
27613737 |
Appl. No.: |
10/442306 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10442306 |
May 20, 2003 |
|
|
|
09670744 |
Sep 27, 2000 |
|
|
|
6599480 |
|
|
|
|
Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01L 2300/04 20130101;
B01L 2300/0887 20130101; G01N 1/40 20130101; B01L 2300/0681
20130101; G01N 1/405 20130101; G01N 15/0272 20130101; B01L 3/502753
20130101; B01L 2400/0406 20130101; Y10T 436/25375 20150115 |
Class at
Publication: |
422/101 |
International
Class: |
G01N 001/18 |
Claims
What is claimed is:
1. An apparatus for obtaining a liquid sample having an increased
cellular or particulate concentration for optical examination,
comprising: a separation chamber; a wall surrounding said
separation chamber; a sample entrance; venting channels; a
separation wall in said separation chamber dividing said chamber
into two compartments, said separation wall having at least one
separation channel; and a flow path in said separation chamber.
2. The apparatus according to claim 1 wherein said liquid sample is
blood.
3. The apparatus according to claim 1 wherein said apparatus
further comprises a lid portion.
4. The apparatus according to claim 3 wherein said lid portion has
at least one opening for sample delivery.
5. The apparatus according to claim 1 wherein said separation wall
has at least one first separation channel and at least one second
separation channel.
6. The apparatus according to claim 5 wherein said separation
channels are of different sizes.
7. The apparatus according to claim 6 wherein said first separation
channel is about 3 to 10 .mu.m deep by 5 to 50 .mu.m wide.
8. The apparatus according to claim 6 wherein said second
separation channel is about 0.5 to 1.5 .mu.m deep by 50 to 1000
.mu.m wide.
9. The apparatus according to claim 1 wherein said apparatus
further comprises a plurality of notches in said separation
chamber.
10. The apparatus according to claim 1 wherein said apparatus
further comprises a plurality of notches in said first compartment
of said separation chamber.
11. The apparatus according to claim 1 wherein said apparatus
further comprises a plurality of notches in said second compartment
of said separation chamber.
12. The apparatus according to claim 1 wherein said apparatus
further comprises a plurality of notches in said first compartment
and second compartment of said separation chamber.
13. The apparatus according to claim 1 wherein said separation wall
has a plurality of separation channels of the same size.
14. The apparatus according to claim 1 wherein said separation wall
has a plurality of separation channels of different sizes.
Description
[0001] This is a divisional of U.S. Ser. No. 09/670,744, filed Sep.
27, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to a method and apparatus for
analyzing a blood or other biological fluid sample in a quiescent
state, whereby particulate constituents of biological samples that
contain sparse populations of cellular species of interest can be
enumerated and inspected using an optical scanning instrument.
Specifically, this invention relates to a method and apparatus for
obtaining an increased cellular or particulate concentration within
the use of said optical scanning method.
BACKGROUND OF THE INVENTION
[0003] The formation of appropriate cellular or particulate layers
for later optical examination is important to many fields. One of
these fields is hematology, where several methods and devices have
been described for obtaining clinically useful cell
concentrations.
[0004] A method and apparatus for analyzing a blood or other
biologic fluid sample in a quiescent state without the need for
separate fluid streams passing through the blood sample during the
analysis is described in U.S. Ser. Nos. 09/248,135 and 09/249,721.
Although this method simplifies the analysis procedure and yields
the full compliment of CBC parameters it also possesses several
disadvantages. One disadvantage of the apparatus is that the
concentration of cells in the examination layer is not controlled.
This can lead to difficulties in optically examining cell volume
and morphology. Another disadvantage of the aforementioned
apparatus is that the field of cells may be too sparse in
clinically relevant samples to complete scanning in a timely
manner.
[0005] U.S. Pat. Nos. 5,627,041 and 5,912,134 describe an apparatus
and method for cytometric measurement of cell populations using
fluorescent markers. However, a disadvantage of this method is
that, if the sample under test is blood, it requires addition of
diluent in such quantities that white blood cells (WBCs) with
depressed counts are not very numerous in the sample and may
require extremely long examination times to locate them. Moreover,
if the cell counts within the undiluted blood are low, clinically
relevant cell populations present in the sample may not be detected
in the diluted sample. For example, patients who undergo
chemotherapy regimens may have depressed white cell counts in the
range of 1000 cells/.mu.L and less. Cytometric examinations are
typically searching for a sub-population of these cells, further
reducing the likelihood of locating them.
[0006] U.S. Pat. No. 4,790,640 discloses a wedge shaped device for
trapping rigid particles, such as sickle cells in blood. However, a
disadvantage of the device is that the selection of cell sizes is
accomplished by thickness of the chamber alone, which can exhibit
substantial manufacturing variation over the examination area,
causing a corresponding loss of ability to separate by size.
[0007] Consequently, it would be desirable to have a method and
apparatus for obtaining the desired cellular concentration in a
blood or other biologic sample which can mitigate the effect of a
separate dilution step and addition of diluting fluids.
SUMMARY OF THE INVENTION
[0008] It is an objective of the present invention to provide
channels in a separation wall inside a separation chamber, said
channels having appropriate size and dimensions to allow at least
one undesired particle species to pass while excluding larger
particles from passing through, thereby arriving at a predetermined
increased volume fraction of the desired particles.
[0009] It is another objective of the present invention to
incorporate at least two separating channels in a separation wall
in a separation chamber, the channels having channel sizes selected
to allow at least one cell species and the substantially liquid
component of the sample to pass through them, arriving at a desired
concentration of larger cell types in the first compartment in
front of the. separation wall. A further embodiment of the present
invention is to have a plurality of separating channels in the
separating wall having one channel size selected to allow at least
one cell species and the substantially liquid component of the
sample to pass through them.
[0010] It is a further objective of the present invention to
regulate the volume fraction of cellular or particle components of
a specimen by means of an array of separating channels which effect
their selection by means of size exclusion during flow between two
adjoining compartments.
[0011] It is another objective of the present invention to increase
the concentration of larger particles for cytometric examination of
sparse populations by allowing only smaller particles and
substantially liquid components to pass through the channels into a
subsequent chamber.
[0012] It is a still further objective of the present invention to
create an accurate spacing between two opposing containment walls
to allow for the optimal formation of desired regions where all
particles of interest lie in the same focal plane, allowing an
accurate determination of the chamber thickness without relying on
extraneous equipment and manipulations for height calibration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is an isometric view of the particle separation
chamber with the lid removed showing the separation channels and
adjacent cell examination areas.
[0014] FIG. 2 is an isometric view of the bottom portion of the
separation chamber showing the disposition of the separation
channels.
[0015] FIG. 3 shows a detailed view of another embodiment of the
separation channels.
[0016] FIG. 4 shows an isometric view of another embodiment of the
separation channels.
[0017] FIG. 5 is a schematic view of a particle separation chamber
with increased analytical sensitivity.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to a method and apparatus for
obtaining an increased cellular or particulate concentration in a
substantially dilute sample. The apparatus includes a sample
chamber which has opposing sample containment walls, at least one
of which is transparent. The sample chamber is separated into
adjoining compartments which are in fluid communication by means of
a multiplicity of channels aligned substantially parallel to each
other and which traverse a separating wall between the two
compartments. Filling of the chamber results in a substantially
quiescent sample ready for further examination.
[0019] In a preferred embodiment, a chamber is used to manipulate
blood components and one type of channel in the array is of
sufficient size and dimensions to allow red blood cells (RBCs) to
pass while excluding larger white blood cells (WBCs). The
dimensions of the channel to create the desired size exclusion are
nominally, in a preferred embodiment, 3 to 10 .mu.m deep.times.5 to
50 .mu.m wide. Dimensions within this range have been selected to
effect the desired volume flow rate of RBCs, or particle-containing
sample. In a modification of the invention, another type of channel
in the array is of sufficient size and dimensions to exclude RBCs
and WBCs from passing while allowing the liquid component of the
specimen to pass freely. The dimensions of the channel to create
the desired size exclusion are nominally, in a preferred
embodiment, 0.5 to 1.5 .mu.m deep.times.50 to 1000 .mu.m wide.
Dimensions within this range are selected to effect the desired
volume flow rate of the liquid-only portion of the sample.
[0020] As the blood sample or particle containing specimen flows
from the first compartment and through the array of channels into
the subsequent compartment, the relative volume fraction of WBCs,
RBCs, or other particles is increased in the first compartment. The
two streams, one containing substantially undiluted blood or
particles, and the other, a liquid-only fraction, recombine in the
subsequent compartment to form a liquid sample having a reduced
volume fraction of cells or particles in the subsequent
compartment. A further benefit of the invention is to provide a
thickness within the chamber during manufacture, which is more
accurate than would otherwise be obtained since the wall
surrounding the interior of the chamber acts to hold the two
opposing sample containment walls, that function as optical windows
apart at a fixed and accurate distance.
[0021] FIG. 1 is merely exemplary and is not intended to limit the
present invention in any way. A separation chamber in accordance
with the present invention can have notches in one or more
compartments of the separation chamber, or no notches at all.
[0022] In the embodiment of the present invention demonstrated in
FIG. 1, the separation chamber is surrounded by a wall (1) which is
used to carry an optically. transparent lid (not shown) allowing
for optical observation and to contain the liquid sample. The
separation chamber is divided into two compartments by a separation
wall (2) which has separation channels on top of it. Details of
different embodiments for separation wall (2) are shown in FIGS. 2,
3, and 4. Returning to FIG. 1, fluid fills the first compartment
through a sample entrance (3), which is surrounded by an extension
(11) of wall (1), and moves into the first compartment (4) of the
chamber. Upon proceeding, the liquid encounters a first notch (5)
displaced laterally across the flow path. The notch creates a
momentary barrier to progression of the advancing fluid meniscus
until the meniscus has contacted the notch across the whole width
of the chamber. Eventually the fluid wets into the notch, allowing
the fluid to advance to the next notch (6) where the meniscus is
again evened out. The fluid advance continues in this manner until
it reaches the separation wall (2). The fluid continues to advance
through the separation wall and separation channels to the second
compartment (7) until finally stopping at its far end (8). Adequate
venting of air while the chamber fills with liquid is provided by a
series of venting-channels (13) on top of wall (1). To allow for a
free passage of air out of channels (13), walls (1) and (11) are
surrounded by a moat (12). During the filling process, the
particles of interest, are prevented from passing through the
separation channels that traverse separation wall (2), and as a
consequence, concentrate in the first compartment (4). After the
fluid fills the subsequent first and the second compartment it
becomes quiescent, allowing optical examination.
[0023] FIG. 2 shows a separation wall having two different channels
of different sizes (9) and (10). The channel labeled (9) is sized
to allow only particles smaller than a certain size to pass and the
channel labeled (10) is sized to allow only liquid to pass. It has
been found through experimentation that these channels can be sized
appropriately to exclude cell or particle sizes of interest.
[0024] FIG. 3 shows a separation wall (16) with a plurality of one
type of separating channel (17) disposed laterally across its full
width.
[0025] FIG. 4 shows another embodiment of the separating channels,
with a plurality of a first channel (14) which allows cellular or
particulate species to pass through it, while allowing a liquid
portion devoid of this species to pass through a plurality of a
second channel (15) disposed in an array alongside the first
channel (14).
[0026] In another embodiment of the present invention, which is
depicted in FIG. 5, increased analytical sensitivity can be
attained by enlarging the volume of the second compartment,
allowing the sample portion in the first compartment to accumulate
larger cells over an increased volume.
[0027] In the embodiment of FIG. 5, the separation chamber is
surrounded by a wall (21) which is again used to carry an optically
transparent lid (not shown) allowing for optical observation and to
contain the liquid sample. The separation chamber is divided into
two compartments (22) and (23) by a separation wall (24) which has
the separation channels on top of it. Separation wall (24) is
U-shaped in order to make it as long as possible. In operation,
fluid fills the first compartment (22) through a sample entrance
(25), which is surrounded by an extension (27) of wall (21), and
moves into the first compartment (22) of the chamber. Upon
proceeding, the liquid encounters a first notch (25) displaced
laterally across the flow path. The notch creates a momentary
barrier to progression of the advancing fluid meniscus until the
meniscus has contacted the notch across the whole width of the
chamber. Eventually the fluid wets into the notch, allowing the
fluid to advance to the next notch (26) where the meniscus is again
evened out. The fluid advance continues in this manner until it
reaches separation wall (24). The fluid continues to advance
through separation wall (24) and the separation channels to the
second compartment (23) which is surrounded by wall extension (28).
Wall (21) and wall extension (28) are equipped with air-venting
channels (29) and (30), respectively. To allow for a free passage
of air out of channels (29) and (30), walls (21), (27), and (28)
are surrounded by a moat (31) in base plate (32). The lid is
resting on walls (21), (27), and (28).
[0028] During the filling process, the particles of interest are
prevented from passing through the separation channels that
traverse separation wall (24), and as a consequence, concentrate in
the first compartment in a U-shaped area (33) in front of
separation wall (24). As mentioned above, compartment (23) behind
separation wall (24) has an increased volume, which, in connection
with the elongated U-shaped separation wall (24) allows one to
process a larger sample volume, as compared to the embodiments
shown in FIGS. 1, 2, 3, and 4. In other words, the embodiment of
FIG. 5 is suitable to achieve reasonable particle concentrations
for analysis in front of separation wall (24), even if the particle
concentration in the incoming sample is rather low.
* * * * *