U.S. patent application number 17/002089 was filed with the patent office on 2021-05-06 for cellular analysis of body fluids.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to Emily H. Lin, Jiong Wu, Jihping Yang.
Application Number | 20210130866 17/002089 |
Document ID | / |
Family ID | 1000005340796 |
Filed Date | 2021-05-06 |
United States Patent
Application |
20210130866 |
Kind Code |
A1 |
Wu; Jiong ; et al. |
May 6, 2021 |
Cellular Analysis of Body Fluids
Abstract
Herein is provided a simple, reliable and accurate method for
cellular analysis on hematology analyzers. In various aspects, the
methods provide separation and/or differentiation between red blood
cells (RBCs) and white blood cells (WBCs) by utilizing a
fluorescent dye to selectively stain WBCs such that they emit
stronger fluorescence signals. The method provides optimal
detection limits on WBCs and RBCs, thereby allowing analysis of
samples with sparse cellular concentrations. As few as one reagent
may be used to prepare a single dilution for body fluid analysis,
in order to simplify the body fluid analysis. Minimal damage to
WBCs is attained using the lysis-free approach described in aspects
of the disclosure.
Inventors: |
Wu; Jiong; (Pinecrest,
FL) ; Lin; Emily H.; (Cupertino, CA) ; Yang;
Jihping; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
1000005340796 |
Appl. No.: |
17/002089 |
Filed: |
August 25, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15979230 |
May 14, 2018 |
10774359 |
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17002089 |
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13720615 |
Dec 19, 2012 |
9970045 |
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15979230 |
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61580623 |
Dec 27, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5094 20130101;
C12Q 1/04 20130101 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; G01N 33/50 20060101 G01N033/50 |
Claims
1.-21. (canceled)
22. A hematology system comprising: a sample holder for holding a
sample; a mixing receptacle for mixing a staining composition with
at least a portion of the sample; a storage receptacle for storing
the staining composition, wherein the staining composition
comprises a dye that permeates a cell membrane and binds to a
nucleic acid, and wherein the dye is fluorescent when bound to a
nucleic acid; a flow cell; at least one energy source for applying
electromagnetic energy to the flow cell; a detector configured to
detect fluorescence originating from within the flow cell to
differentiate cells with nuclei from cells without nuclei based
only on the presence or absence of the fluorescent dye; and one or
more visible light detectors for detecting scattered visible
light.
23. The hematology system of claim 22, further comprising an
aspirating mechanism for aspirating at least a portion of the
sample from the sample holder and moving it to the mixing
receptacle.
24. The hematology system of claim 22, further comprising an
aspirating mechanism for aspirating the staining composition and
moving it from the storage receptacle to the mixing receptacle.
25. The hematology system of claim 22, wherein the at least one
energy source produces monochromatic light at a wavelength
.lamda.1.
26. The hematology system of claim 25, wherein the dye absorbs
light at wavelength .lamda.1 and emits light at wavelength .lamda.2
when the dye is bound to a nucleic acid.
27. The hematology system of claim 26, wherein the detector detects
light at wavelength .lamda.2.
28. The hematology system of claim 22, further comprising at least
one additional energy source for providing non-monochromatic
visible light.
29. The hematology system of claim 22, wherein the sample is a body
fluid.
30. A method for preparing a hematology system, the method
comprising: providing a mixing receptacle for mixing at least a
portion of a sample to be analyzed with a staining composition;
providing a storage receptacle for storing the staining
composition, wherein the staining composition comprises a dye that
permeates a cell membrane and binds to a nucleic acid, and wherein
the dye is fluorescent when bound to a nucleic acid; providing a
flow cell; establishing a stable core stream to allow the sample to
pass through the flow cell; positioning an energy source for
providing electromagnetic energy to the flow cell; and positioning
a detector for detecting fluorescence emitted from within the flow
cell.
31. A method for preparing a receptacle for use in analyzing a body
fluid, the method comprising adding to the receptacle a composition
comprising a dye that permeates a cell membrane and binds to a
nucleic acid, wherein the dye is fluorescent when bound to a
nucleic acid, and wherein the receptacle is an integrated or
modular part of a hematology system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] Under 35 U.S.C. .sctn. 119(e), this application claims
priority benefit to the filing date of U.S. Provisional Patent
Application Ser. No. 61/580,623, filed on Dec. 27, 2011, the
disclosure of which application is herein incorporated by reference
in its entirety.
BACKGROUND
[0002] A variety of methods are used for cellular analysis,
including visual and/or automated inspection via light or
fluorescent light microscopy. Cellular examinations and analyses of
these types are commonly practiced in order to obtain information
regarding cell lineage, maturational stage, and cell counts in a
sample.
[0003] Flow cytometry is a method for identifying and
distinguishing between different cell types in a non-homogeneous
sample. In the flow cytometer, cells are passed one at a time or
nearly one at a time through a sensing region where each cell is
irradiated by an energy source. Typically, single wavelength light
sources (e.g., lasers, etc.) are used as the energy source and one
or more of a variety of sensors record data based on the
interaction of the cells with the applied energy. Flow cytometry is
commonly used in hematology and has been particularly successful in
the diagnosis of blood cancers. In addition to flow cytometry,
other analytical methods are used in hematology and in
characterizing a population of cells.
[0004] Blood samples tend to have a high concentration of cells.
Analysis of samples with significantly lower concentrations of
cells, whether by flow cytometry or other techniques, is more
difficult and therefore less common. In addition, traditional
hematology analyzers, which are designed to measure whole blood
samples, tend to have limited detection sensitivity for low-end
cell concentrations. In some cases, manual examination of samples
is the only available method for cellular analysis. Improved
methods for analyzing samples with low cell counts are desirable in
the fields of medicine, microbiology, and others.
SUMMARY
[0005] In one aspect, the disclosure provides a method for
analyzing a body fluid containing cells, the method comprising:
staining the body fluid with a fluorescent dye, wherein the
fluorescent dye permeates a cell membrane and binds to a nucleic
acid to form a dye complex within the cell; irradiating the stained
body fluid with energy from an energy source; and measuring a
fluorescence signal emitted by the dye complex in the stained body
fluid.
[0006] In some such aspects, the body fluid comprises less than
about 20 cells/.mu.L.
[0007] In some such aspects, the body fluid comprises more than
about 20 cells/.mu.L.
[0008] In some such aspects, the nucleic acid is selected from a
DNA and RNA.
[0009] In some such aspects, the energy source provides
monochromatic light having a wavelength in the visible spectrum,
and wherein the wavelength of the monochromatic light and the
wavelength of the fluorescence signal are different.
[0010] In some such aspects, unbound fluorescent dye emits less
fluorescent light when irradiated with energy from the energy
source compared with the dye complex.
[0011] In some such aspects, unbound fluorescent dye does not
fluoresce when irradiated with energy from the energy source while
unbound to the nucleic acid, such that cells lacking the dye
complex do not emit a fluorescence signal.
[0012] In some such aspects, the method comprises differentiating
cells with nuclei from cells without nuclei based on the presence
or absence of the fluorescent dye.
[0013] In some such aspects, the measuring involves enumerating and
differentiating RBCs and WBCs.
[0014] In some such aspects, the method does not involve lysing
RBCs prior to the measuring.
[0015] In some such aspects, the body fluid comprises intact WBCs
and RBCs.
[0016] In some such aspects, the measuring is carried out using an
automated hematology analyzer, flow cytometer, or other diagnostic
analyzer for body fluid samples.
[0017] In some such aspects, the measuring comprises flowing the
body fluid through a flow cell in a cytometer.
[0018] In some such aspects, the fluorescent dye is provided in a
composition that further comprises water.
[0019] In another aspect, the disclosure provides a method for
analyzing a fluid, wherein the fluid contains less than about 40
cells/.mu.L, the method comprising: contacting the fluid with a
fluorescent dye, wherein the fluorescent dye permeates a cell
membrane and binds to a nucleic acid to form a dye complex within
the cell; irradiating the fluid with energy from an energy source;
and measuring a fluorescence signal emitted by the dye complex in
the fluid.
[0020] In some such aspects, the fluid contains less than about 20
cells/.mu.L.
[0021] In some such aspects, the fluid contains less than about 5
cells/.mu.L.
[0022] In some such aspects, the fluid is a body fluid.
[0023] In some such aspects, the fluid is a biological fluid.
[0024] In another aspect, the disclosure provides a method for
differentiating cells, the method comprising: contacting the cells
with a solution comprising a fluorescent dye, wherein the
fluorescent dye is water soluble, capable of permeating a cell
membrane, and capable of binding to a nucleic acid; irradiating the
cells with an excitation light from an excitation light source; and
measuring light emissions from the cells and differentiating cells
containing nucleic acids from cells lacking nucleic acids based on
the measuring.
[0025] In another aspect, the disclosure provides a composition for
analyzing a body fluid, the composition comprising water and a
fluorescent dye, wherein the fluorescent dye is water soluble,
capable of permeating a cell membrane, and capable of binding to a
nucleic acid.
[0026] In another aspect, the disclosure provides a hematology
system comprising: a sample holder for holding a sample to be
analyzed; a mixing receptacle for mixing at least a portion of the
sample to be analyzed with a staining composition; a storage
receptacle for storing the staining composition, wherein the
staining composition comprises a dye capable of permeating a cell
membrane and binding to a nucleic acid, and wherein the dye is
fluorescent when bound to a nucleic acid; a flow cell; at least one
energy source for applying electromagnetic energy to the flow cell;
one or more detectors for detecting fluorescence originating from
within the flow cell; and one or more visible light detectors for
detecting scattered visible light.
[0027] In some such aspects, the system comprises an aspirating
mechanism for aspirating at least a portion of the sample to be
analyzed from the sample holder to the mixing receptacle.
[0028] In some such aspects, the system comprises an aspirating
mechanism for aspirating staining composition from the storage
receptacle to the mixing receptacle.
[0029] In some such aspects, the at least one energy source
provides monochromatic light at a wavelength .lamda.1.
[0030] In some such aspects, the dye absorbs light at wavelength
.lamda.1 and emits light at wavelength .lamda.2 when the dye is
bound to a nucleic acid.
[0031] In some such aspects, the detector detects light at
wavelength .lamda.2.
[0032] In some such aspects, the system comprises at least one
additional energy source for providing non-monochromatic visible
light.
[0033] In some such aspects, the sample to be analyzed is a body
fluid.
[0034] In another aspect, the disclosure provides a method for
preparing a hematology system, the method comprising: providing a
mixing receptacle for mixing at least a portion of a sample to be
analyzed with a staining composition; providing a storage
receptacle for storing the staining composition, wherein the
staining composition comprises a dye capable of permeating a cell
membrane and binding to a nucleic acid, and wherein the dye is
fluorescent when bound to a nucleic acid; providing a flow cell;
positioning an energy source for providing electromagnetic energy
to the flow cell; positioning a detector for detecting fluorescence
emitted from within the flow cell.
[0035] In another aspect, the disclosure provides a method for
preparing a receptacle for use in analyzing a body fluid, the
method comprising adding to the receptacle a composition comprising
a dye capable of permeating a cell membrane and binding to a
nucleic acid, wherein the dye is fluorescent when bound to a
nucleic acid, and wherein the receptacle is an integrated or
modular part of a hematology system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 provides a schematic image of an aspect of a body
fluid analysis method described herein. WBCs are separated from
RBCs upon DNA-dye interaction and subsequent emission of
fluorescence. The fluorescence information, as well as other
optical scattering signals, are used in cellular analysis of body
fluid samples. FL1 indicates the fluorescence collected with a
530.+-.20 nm band path filter. ALL and IAS are the light scattering
signals collected at 0 to 1 degrees and 3 to 10 degrees,
respectively.
[0037] FIG. 2A is a scattergram (ALL v. IAS) of cellular analysis
for a body fluid sample. WBC=89/.mu.L (reference=81/.mu.L);
RBCs=2012/.mu.L (reference=1733/.mu.L).
[0038] FIG. 2B is a scattergram (FL1 v. ALL) of cellular analysis
for a body fluid sample. WBCs and RBCs are well separated in
fluorescence (FL1). WBC=89/.mu.L (reference=81/.mu.L);
RBC=2012/.mu.L (reference=1733/.mu.L).
[0039] FIG. 3A is a scattergram (ALL v. IAS) of cellular analysis
of another body fluid sample. WBC=0.95/.mu.L
(reference=0.33/.mu.L); RBC=142/.mu.L (reference=122/.mu.L).
[0040] FIG. 3B is a scattergram (FL1 v. ALL) of cellular analysis
of another body fluid sample. WBCs and RBCs are well separated in
fluorescence (FL1). WBC=0.95/.mu.L (reference=0.33/.mu.L);
RBC=142/.mu.L (reference=122/.mu.L).
[0041] FIG. 4A is an FL1 v. IAS scattergram of the first dilution
of a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0042] FIG. 4B is an FL1 v. IAS scattergram of the second dilution
of a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0043] FIG. 4C is an FL1 v. IAS scattergram of the third dilution
of a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0044] FIG. 4D is an FL1 v. IAS scattergram of the fourth dilution
of a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0045] FIG. 4E is an FL v. IAS scattergram of the fifth dilution of
a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0046] FIG. 4F is an FL v. IAS scattergram of the sixth dilution of
a six level serial dilution of a buffy coat sample. The original
buffy coat sample contains 6.1.times.10.sup.3/.mu.L WBC and
15.times.10.sup.3/.mu.L RBC. The six levels, (A), (B), (C), (D),
(E) and (F), were prepared based upon 1:10, 1:30, 1:100, 1:300,
1:1000 and 1:3000, respectively, dilutions with PBS. All samples
were measured using a dilution on a prototype analyzer.
[0047] FIG. 5 provides correlation graphs of WBC (measured vs.
calculated) among all six levels of diluted buffy coat samples (A)
and three low-end samples only (B). Multiple dots at each level
indicate that multiple runs were performed. The overall
correlations were: Y=1.0239 X-4.9 (R.sup.2=0.9984) for all six
levels and Y=1.0787 X-1.5 (R.sup.2=0.9598) for the three levels
with the lowest cell concentrations.
[0048] FIG. 6 provides correlation graphs of WBC (current method
vs. reference) for 91 body fluid samples. The correlations were
plotted in different ranges: (A) full range (.about.40,000/.mu.L),
(B)<2,000/.mu.L, (C)<200/.mu.L and (D)<50/.mu.L. The
dilution ratio was 1:35 (specimen to labeling reagent).
[0049] FIG. 7 provides correlation graphs of RBC (current method
vs. reference) for 91 body fluid samples. The correlations were
plotted in different ranges: (A) full range (.about.200,000/.mu.L),
(B)<3,000/.mu.L, (C)<200/.mu.L and (D)<50/.mu.L. Samples
with many RBC ghosts were not included in the correlation analysis.
The dilution ratio was 1:35 (specimen to labeling reagent).
[0050] FIG. 8 is a correlation graph of WBC (invented method vs.
reference) for 72 body fluid samples with very low cell
concentrations (WBC<50/.mu.L). The dilution ratio was 1:10
(specimen to labeling reagent).
[0051] FIG. 9 is a correlation graph of RBC (current method vs.
reference) for 38 body fluid samples with very low cell
concentrations (RBC<50/.mu.L). Samples with many RBC ghosts were
not included in correlation analysis. The dilution ratio was 1:10
(specimen to labeling reagent).
DETAILED DISCLOSURE
Definitions
[0052] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
described herein. It is understood that the present disclosure
supersedes any disclosure of an incorporated publication to the
extent there is a contradiction.
[0053] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0054] The term "typically" is used to indicate common practices of
the invention. The term indicates that such disclosure is
exemplary, although (unless otherwise indicated) not necessary, for
the materials and methods of the invention. Thus, the term
"typically" should be interpreted as "typically, although not
necessarily." Similarly, the term "optionally," as in a material or
component that is optionally present, indicates that the invention
includes instances wherein the material or component is present,
and also includes instances wherein the material or component is
not present.
[0055] As used herein, the term "body fluid" refers to fluids
present or obtained from an animal, including fluids such as
cerebrospinal fluid, peritoneal fluid, pericardial fluid, pleural
fluid, synovial fluid, urine, saliva, tears, semen, amniotic fluid,
sputum, and the like, as well as fluids obtained from cysts,
tumors, and the like. Unless otherwise specified, the term "body
fluid" does not include whole blood, although a body fluid may
contain red blood cells (RBCs) and/or white blood cells (WBCs).
[0056] In some aspects, the disclosure provides methods and
materials for analyzing fluids with low cell count. By low cell
count is meant fluids that have less than about 100 cells/.mu.L, or
less than about 80 cells/.mu.L, or less than about 60 cells/.mu.L,
or less than about 40 cells/.mu.L, or less than about 30
cells/.mu.L, or less than about 20 cells/.mu.L, or less than about
10 cells/.mu.L, or less than about 5 cells/.mu.L. In some
embodiments, such low cell count is the result of dilution of a
more concentrated original sample. In other embodiments, such low
cell count is a natural characteristic of the fluid to be analyzed
(i.e., no dilution is necessary to achieve the low cell count).
[0057] The fluids to be analyzed using the methods and materials
described herein are, in some embodiments, selected from a body
fluid. In other embodiments, the fluids to be analyzed are
biological in nature but are not body fluids. In some embodiments,
the fluid to be analyzed is "synthesized," meaning that a
population of cells is added to a fluid, wherein the fluid is
biologically compatible but is not the fluid in which the cells are
normally found. Examples of such fluids include buffered aqueous
solutions and the like.
Materials
[0058] In some embodiments, the methods of interest involve
contacting a population of cells with a labeling composition
suitable to aid in the desired characterization of the cells. In
some embodiments the labeling composition comprises a fluorescent
dye, and optionally further comprises additional components such as
those described herein below. The components of the labeling
composition and the relative concentration of such components may
be varied according to the needs and requirements of the intended
use. The labeling composition, with or without the dye present, may
alternatively be referred to herein as "reagent."
[0059] The methods and materials of interest involve a fluorescent
dye (also referred to herein as a "dye"). The fluorescent dye may
be any suitable dye having the characteristics necessary or
suitable to carry out the methods of interest. For example, in some
embodiments, the dye is water soluble. For example, in some
embodiments, the fluorescent dye has an aqueous solubility of at
least 1 .mu.g/L, or at least 5 .mu.g/L, or at least 10 .mu.g/L, or
at least 20 .mu.g/L, or at least 50 .mu.g/L. In some embodiments,
the dye is stable in aqueous solutions, meaning that the dye does
not significantly degrade on the timescale suitable for the methods
of analysis described herein. For example, the dye is stable in a
buffered aqueous solution (e.g., cell reagent) at ambient
temperatures (e.g., approximately 20-25.degree. C.) for at least 30
min, or at least 1 hr or at least 6 hr, or at least 12 hr, or at
least 24 hours, or at least 2 days, or at least 7 days, or at least
1 month. Also for example, the dye is stable in a buffered aqueous
solution (e.g., cell reagent) under refrigeration (e.g., below
approximately 10.degree. C.) for at least 1 hr, or at least 12 hr,
or at least 24 hours, or at least 2 days, or at least 7 days, or at
least 1 month, or at least 6 months, or at least 12 months.
[0060] In some embodiments, the dye is able to penetrate a cell
membrane, such as the walls of blood cells or the like. In some
embodiments, the dye is further able to penetrate a cell nucleus
once inside a cell.
[0061] In some embodiments, the dye binds to a nucleic acid. In
some embodiments, the dye binds to DNA. In some embodiments, the
dyes of interest bind to DNA but do not bind to RNA.
[0062] In some embodiments, the dyes of interest bind
preferentially to DNA over RNA. In some embodiments, binding of the
dye to a DNA molecule involves hydrogen-bonding or another
non-covalent interaction. In some embodiments, the dye binds to a
single strand of DNA or a single strand of a double-stranded DNA
complex. In some embodiments, the dye binds to both strands of a
double-stranded DNA complex.
[0063] Throughout this specification, a dye bound to DNA is
referred to as a dye complex. In some embodiments, the dye binds to
DNA with a high affinity and a high binding constant. In some
embodiments, and as mentioned herein, the affinity and
concentration of the dye is great enough that the dye is sufficient
to "stain" (post penetration and DNA-binding) at least 250,000
cells/.mu.L.
[0064] In some embodiments, the dye is fluorescent. Thus, the dye
absorbs incident light at one wavelength or one group of
wavelengths around a peak absorption wavelength (also referred to
as a peak excitation wavelength), and emits light at another
wavelength or at a group of wavelengths around a peak emission
wavelength. In some embodiments, the peak absorption wavelength is
different from the peak emission wavelength. Furthermore, in some
embodiments, the peak adsorption wavelength and/or the peak
emission wavelength is/are dependent upon the environment of the
dye. For example, in some embodiments, the peak absorption
wavelength and/or peak emission wavelength for a dye bound to DNA
(i.e., a dye complex) differs from the peak adsorption wavelength
and/or peak emission wavelength for an unbound dye.
[0065] In some embodiments, the peak adsorption wavelength and the
peak emission wavelength for a dye complex differ by at least 10
nm, or at least 15 nm, or at least 20 nm, or at least 25 nm, or at
least 30 nm, or at least 35 nm, or at least 40 nm, or at least 45
nm, or at least 50 nm. In some embodiments, the peak absorption
wavelength for a dye complex is in the range of 400-700 nm. For
example, in some embodiments the peak absorption is in the range of
425-550 nm, or in the range of 450-525 nm, or in the range of
475-500 nm, or in the range of 480-495 nm, or in the range of
485-490 nm. Also for example, in some embodiments the peak
absorption is in the range of 500-700 nm, or in the range of
550-675 nm, or in the range of 600-650 nm, or in the range of
620-640 nm.
[0066] In some embodiments, the peak emission wavelength for a dye
complex is in the range of 425-800 nm, such as in the range of
425-700 nm. For example, in some embodiments, the peak emission is
in the range of 450-650 nm, or in the range of 475-625 nm, or in
the range of 500-550 nm, or in the range of 510-540 nm, or in the
range of 520-530 nm.
[0067] For example, in some embodiments the fluorescent dye is
selected from thiazole orange or
1-methyl-4-[(3-methyl-2-(3H)-benzothiazolylidene)methyl]quinolinium
p-tosylate, thiazole blue,
4-[(3-methyl-2-(3H)-benzothiazolylidene)methyl]-1-[3-(trimethylammonium)p-
ropyl]quinolinium diiodide, 3,3'-dimethyloxacarbocyanine iodide or
3-methyl-2-[3(3-methyl-2(3H)-benzothiazolylidene-1-propenyl]benzoxazolium
iodide, thioflavine T, the stains SYTO.RTM. and TOTO.RTM. (Life
Technologies), ethidium bromide, propidium iodide, acridine orange,
coriphosphine O, auramine O, the stains HOECHST 33258
(2'-(4-hydroxyphenyl)-5-(4
methyl-1-piperizinyl)-2,5'-bi-1H-benzimidazole trihydrochloride
hydrate) and HOECHST 33342.RTM.
(2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperizinyl)-2,5'-bi-1H-benzimidazole
trihydrochloride), 4' 6-diamino-2-phenylindole dihydrochloride
(DAPI), 4',6-(diimidazolin-2-yl)-2-phenylindole dihydrochloride
(DIPI), 7-aminoactinomycin D, actinomycin D, and LDS 751
(2-(4-(4-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzoth
iazolium perchiorate), or a dye sold under the tradename SYBR.RTM.
(Life Technologies, e.g., SYBR Green, SYBR Gold, SYBR 11, etc.), or
the like.
[0068] In some embodiments, in addition to a dye as described
above, a labeling composition of interest further comprises one or
more of the components identified in the following paragraphs. It
will be appreciated that the lists below are representative only,
and that substitutions may be made when desired using chemically
and/or biologically equivalent materials.
[0069] In some embodiments, the labeling composition contains a
surfactant or detergent. Ionic and neutral surfactants can be
employed. For example, zwitterionic surfactants such as steroidal
glucosides (e.g., Big CHAP, Big Doxy CHAP, etc.) and
glucopyranosides, as well as other surfactants known in the art may
be used. Surfactants can be present in the labeling composition in
any suitable amount, such as 0.0001-0.1% (w/v).
[0070] In some embodiments, the labeling composition further
contains a buffer or pH modifier. Examples of buffers or pH
modifiers include, but are not limited to, PBS, MOPS, and HEPES.
Additional examples of buffers or pH modifiers include: acids such
as hydrochloric acid, hydrobromic acid, acetic acid, phosphoric
acid, and the like; bases such as sodium hydroxide, sodium
carbonate, sodium bicarbonate, organic amines (e.g., imidazole,
triethylamine, etc.), and the like; and salts such as sodium
chloride, calcium chloride, and the like. For example, an organic
buffer material such as imidazole may be present in the labeling
composition at a concentration in the range of about 0.001-3%
(w/v). Also for example, an inorganic material such as HCl may be
present in a concentration in the range of about 0.001-5% (w/v).
Similarly, a salt such as NaCl may be present at a concentration in
the range of about 0.001-3% (w/v). As mentioned previously,
equivalents of such materials are known and may be substituted
where appropriate.
[0071] In some embodiments, the methods and materials of interest
further involve additional components as necessary or desired.
Examples of such additional components include colorants,
preservatives, anti-microbial agents, osmolality adjustors, and
co-solvents.
[0072] For example, an anti-microbial agent such as Proclin (e.g.,
Proclin 150, 200, 300, etc.), vancomycin, penicillin, and the like
may be present. The anti-microbial agent may be present at a
concentration in the range of about 0.001-0.5% (w/v).
[0073] In some embodiments, stock solutions of various components
may be made during preparation of the labeling compositions of
interest. For example, stock solutions of the any component (e.g.,
dye compound, etc.) can be made, such as to aid in solubilizing the
components or to accurately measure the component volumes. Stock
solutions can be made using water as solvent, or using an organic
solvent such as dimethyl sulfoxide, isopropanol, ethanol, or
methanol, or using a combination thereof.
[0074] In some embodiments, the methods and materials of interest
involve an aqueous solution of the above-identified components. It
will be appreciated that the concentration of the various
components present in the labeling composition can vary according
to the intended use. The paragraphs above and below provide some
guidelines for concentrations of materials present in the labeling
composition. As indicated herein, in some embodiments the labeling
composition is combined with a fluid to be analyzed (e.g., a body
fluid). Accordingly, although the guidelines provided herein refer
to amounts of various components as present in the labeling
composition, it will be appreciated that such guidelines are also
applicable to the combination of the labeling composition and the
fluid to be analyzed (i.e., in some embodiments, the material that
is actually analyzed in a hematology analyzer as described herein
will contain similar percentages and relative amounts of the
various components).
[0075] In some embodiments, the dye is present in the labeling
composition in a predetermined concentration. It will be
appreciated that the predetermined concentration may vary depending
on the identity of the dye (i.e., the chemical structure, binding
affinity for the nucleic acid target, etc.) as well as the
operating parameters of the analyzer to be used. In some
embodiments, the dye component is present in an amount necessary to
stain at least the number of cells present in the body fluid, such
as 10 cells/.mu.L or greater, or 50 cells/.mu.L or greater. For
example, the dye is present in an amount necessary to stain 100,000
cells/.mu.L or greater, or 200,000 cells/.mu.L or greater, or
250,000 cells/.mu.L or greater. In some embodiments, the amount of
dye compound present is in the range of 0.000001-0.5%, or
0.00001-0.5%, or 0.0001-0.5%, or 0.001-0.1% (w/v).
[0076] In some embodiments, an organic buffering agent is present
in the labeling composition. Examples of such a material include
organic amines, such as triethylamine, trimethylamine, and cyclic
amines such as imidazole, and the like. The organic buffering agent
(along with any other buffers) may be present in an amount
necessary to create and maintain a desired pH in the analyte
composition.
[0077] In some embodiments, the labeling composition containing a
dye of interest is isotonic or substantially isotonic (i.e., within
about 20% of isotonic, or within about 10% of isotonic, or within
about 5% of isotonic). In some embodiments, the labeling
composition containing a dye of interest has neutral pH or
substantially neutral pH (i.e., within a pH range of about 6-8, or
about 6.5-7.5). These characteristics of the labeling composition
aid in reducing damage to cells of any type during sample
preparation and analysis, and therefore result in more accurate
cellular analysis of body fluid samples.
Methods
[0078] In some embodiments, the methods of interest involve
contacting the population of cells with the labeling composition.
In some such embodiments, the contacting involves mixing the
labeling composition with a fluid (e.g., a body fluid) containing
the cells to be analyzed. Thus, in some embodiments the methods of
interest involve diluting a fluid sample containing cells with a
labeling composition, wherein the labeling composition comprises
water and a dye, and optionally comprises other components such as
those provided herein. In some such embodiments, the methods
involve a single dilution step (i.e. adding the labeling
composition to the fluid sample only once) at a predetermined
dilution ratio that provides an optimal concentration of analyte
for fluorescence detection and measurement.
[0079] In some embodiments, because the dyes of interest bind to
DNA, cells that lack DNA are not able to form a dye complex when
exposed to the dye. Cells lacking DNA that have been exposed to the
dye therefore do not exhibit the emission characteristics of the
dye complex when analyzed using the methods described herein (e.g.,
when exposed to a fluorescence excitation energy source) or only
emit minimal or weak fluorescence due to autofluorescence. In
contrast, cells that contain DNA are able to form a dye complex.
Such cells, when stained with a dye of interest, exhibit the
emission characteristics of the dye complex when analyzed using the
methods described herein. In view of this distinction, in some
embodiments the methods of interest provide a means for
differentiating cells containing DNA from cells that lack DNA. In
some embodiments, the methods of interest also provide a means for
differentiating cells containing a nucleus from cells lacking a
nucleus, particularly when the nucleus contains DNA. In some
embodiments, the differentiation is provided based on fluorescence
measurements that either observe fluorescence from a cell
(indicating the presence of DNA and possibly indicating the
presence of a nucleus) or do not observe fluorescence from a cell
(indicating the lack of DNA and possibly indicating the lack of a
nucleus in the cell). Also, non-cellular events (e.g., non-cellular
objects that lack DNA) present in the fluid being analyzed are
differentiated by the lack of characteristic fluorescence
emission.
[0080] For example, when RBCs are exposed to the dye, mature RBCs
(which lack a nucleus and lack DNA) do not form the dye complex.
Therefore, RBCs exposed to the dye do not exhibit the emission
characteristics of the dye complex when analyzed using the methods
described herein. In contrast, WBCs contain a nucleus and DNA, and
therefore form the dye complex when exposed to the dye. In some
embodiments, therefore, the methods of interest allow
differentiation of WBCs from RBCs in a sample containing such
cells. The differentiation is based on the presence or absence of a
fluorescent signal indicative of the formation of the dye
complex.
[0081] In addition to differentiation as described above, the
formation of the dye complex between a dye of interest and DNA also
allows for enumeration of cells containing DNA in a fluid sample
containing cells. For example, the methods of interest include
enumerating WBCs in a fluid sample containing such cells.
Enumeration is based on observation of fluorescence indicative of
the formation of the dye complex in a method suitable for counting
cells. Suitable methods include automated hematology analyzers such
as flow cytometers and other fluorescence-based cell counting
methods.
[0082] As mentioned herein, the methods of interest allow for
enumeration and differentiation of WBCs in a sample containing WBCs
and other objects (e.g., RBCs and non-cellular events). In some
embodiments, the sample is a fluid such as a body fluid, and
contains a very low concentration of cells as described in more
detail herein. In some embodiments, the methods of the present
disclosure further allow for classification of WBCs into 2-part,
3-part or 5-part differentials using multi-angle scattering
technologies. In certain embodiments, RBCs can be further separated
from other non-RBC particles using multi-angle scattering
technologies.
[0083] In some embodiments, the methods of interest do not involve
a lysing agent and do not involve lysing cells prior to recording
data. For example, the methods of interest do not involve adding a
lysing agent to the fluid sample containing cells prior to analysis
of the fluid sample. Thus, in some embodiments, the labeling
composition does not contain a lysing agent. The fluid samples that
are analyzed are not lysates and do not contain cellular contents
that have been released from a cell via lysis. In some embodiments,
the fluid samples to be analyzed contain intact RBCs and such RBCs
are not lysed prior to fluorescence measurements. By "lysing agent"
is meant to include any materials that cause significant cell
lysis, particularly (but not limited to) RBC lysis. Lysing agents
include, but are not limited to, various types of surfactants,
enzymes, antibodies, pH adjusting agents, osmolytes (osmolality
adjusting agents), and the like that are known in the art or later
discovered.
[0084] Because lysing agents are not involved in the preparation of
fluid samples to be analyzed, WBCs present in the fluid are not
damaged as they would or might be were lysing agents employed. This
is particularly advantageous in body fluids containing WBCs,
because the WBCs contained therein are typically more fragile than
WBCs in whole blood.
[0085] As mentioned above, in some embodiments the methods of
interest involve dilution with a predetermined amount of labeling
composition to provide a desired dilution ratio and a desired
analyte concentration. The predetermined amount may be determined
based on detection limits in the apparatus used for analysis of the
fluid sample. In some embodiments, the detection limit is optimized
by adjustments to the sample injection rate (relative to the flow
cell rate), measurement duration, and the like. For example, a
prototype analyzer with 1:10 (blood:reagent) dilution ratio, 4.0
.mu.L per second injection rate, and 60 seconds sample measurement
(data collection), would result in a collection of approximately
240 events for a sample at 10 cells per .mu.L, sufficient for
supporting precise cellular analysis.
[0086] In some embodiments, the methods of interest are suitable
for analyzing any fluid sample with WBCs. As mentioned previously,
such fluid samples include body fluids, and also include other
fluid samples such as prepared aqueous solutions of cells,
plant-based and plant-derived solutions, and like, particularly
where such sample fluids contain low cell counts (e.g., less than
40 cells/.mu.L, or less than 30 cells/.mu.L, etc.).
[0087] In some embodiments, the methods of interest provide a
simple, reliable, and accurate means for enumerating and
differentiating WBCs in a sample fluid containing WBCs and
optionally containing RBCs. Fluorescence detection using automated
hematology analyzers ensures accuracy and reliability, and the use
of single-step dilution provides simplicity in sample preparation.
The sample fluid containing cells is stained with a fluorescent
label contained in a labeling composition, wherein the fluorescent
label permeates cell walls and binds to DNA to create a dye
complex. Stained WBCs emit fluorescent emission signals indicative
of the dye complex, whereas RBCs do not emit such signals or emit
such signals at a significantly lower intensity. The labeling
composition is free of lysing agents and no lysing agents are added
to the sample fluid, thereby protecting the WBCs present. Optimal
detecting limits are determined by adjustment of variables
including sample flow rate, detection frequency, and the like.
[0088] Additional aspects of methods for analyzing WBCs and systems
for performing such methods can be found in co-pending U.S.
Provisional Patent Application Ser. No. 61/482,541, filed May 4,
2011, as well as co-pending U.S. Provisional Patent Application
Ser. No. 61/482,549, filed May 4, 2011, the disclosures of which
are incorporated by reference herein in their entireties.
Output and Advantages
[0089] In some embodiments, analysis of the dyed fluids results in
a variety of information suitable for multi-dimensional data
analysis, graphs, and the like. For example, in some embodiments,
the methods described herein provide an accurate total WBC count.
In some embodiments, the methods provide data as to the relative
proportions of different types of WBCs in a sample (e.g., cell
counts of neutrophils, eosinophils, basophils, lymphocytes, and/or
monocytes, or various combinations thereof). Such data may be used,
for example, as diagnostic information in treating patients.
[0090] As mentioned herein, in some embodiments the methods of
interest involve selectively staining WBCs in a fluid sample by
introducing a dye that selectively binds to DNA and fluoresces when
so bound. Accordingly, analysis of a stained sample fluid includes
recording the stronger fluorescence emissions that originate from
cells containing a dye complex (e.g., WBCs) compared with the weak
or non-existent fluorescence emissions that originate from cells
lacking the dye complex (e.g., RBCs). The fluorescence measurements
can be obtained using, for example, multi-channel optical
scattering analyzers.
[0091] In some embodiments, the materials and methods of interest
allow for cellular analysis of body fluids and other fluids useful,
for example, in medical diagnostics. Cellular analysis of fluid
samples with very low cell counts (e.g., less than 40 per .mu.L, or
less than 10 per .mu.L, etc.) is enabled by the methods herein,
including body fluids having particularly fragile cells. The
simplicity of the methods allows such samples to be analyzed within
1-2 hours after sample draw from a patient, which also aids in
analysis of fragile cells. The lack of cell lysing agents in the
compositions and methods described herein further adds to the
ability to analyze samples containing fragile cells. Damage to
fragile cells in the fluids of interest potentially results in
inaccurate analysis such as inaccurate cell counts and cellular
differentiation.
[0092] The optimal dilution ratios offered by the methods described
herein are a further improvement over traditional hematological
analysis. For example, the dilution ratios (i.e.
blood:diluent=1:300 for RBC dilution) used by traditional
hematology analyzers are not sensitive enough to support measuring
samples with very low cellular concentrations. In contrast, in some
embodiments, the methods herein allow a single dilution to provide
an optimal dilution ratio for optimal fluorescence detection.
[0093] The methods of the present disclosure are simple, e.g., in
some embodiments, only a single reagent and a single dilution are
required. In some embodiments, the dilution ratios can be easily
adjusted and/or optimized. Furthermore, the methods of the present
disclosure are simple to implement, and can be performed, e.g., on
a traditional hematology analyzer, without the need for extensive
adjustments and/or modifications.
[0094] These and other advantages will be apparent to one of skill
in the art based on the disclosure provided herein, including the
examples below.
EXAMPLES
[0095] General Testing Procedure: (A) Modify flow script and
algorithm of a CELL-DYN Sapphire hematology analyzer to allow it to
be used for a body fluid assay. (B) All body fluid (BF) assays are
conducted using (slightly modified) WBC extended counting mode: BF
sample aspiration (.about.120 .mu.L), followed by sample (BF) to
reagent (Sapphire reticulocyte reagent) mixing, ratio at 1 to 35,
followed by mixed sample incubation (40.degree. C..times.25 sec),
followed by incubated sample delivered to flow cell for measurement
(32 sec measurement), followed by data collection, raw data
recorded as .fcs files, followed by use of software such as FCS
Express to analyze the raw data.
[0096] All reference results were measured using standard manual
chamber counting procedures.
[0097] Reagent was formulated using the following components and
concentrations:
TABLE-US-00001 Component Concentration.sup.1 Imidazole 0.3400% 1N
HCl 2.5325% NaCl 0.6800% Proclin 300 0.0315% BIGCHAP 0.0050% SYBR
11 0.0002% DMSO.sup.2 0.0220% Water To 100% .sup.11% = 1 g/100 mL
.sup.2DMSO (dimethyl sulfoxide) was used to make a stock solution
of the dye component.
Example 1
Analysis of Low Cell Count Samples
[0098] A prototype analyzer with 1:35 (blood:reagent) dilution
ratio, 2.3 .mu.L per second injection rate, and 32 seconds sample
measurement (data collection), results in a collection of more than
20 events for a sample at 10 cells per .mu.L.
[0099] FIGS. 2A and 2B show scattergrams of cellular analysis of a
body fluid sample. The cells were enumerated after analysis:
WBC=89/.mu.L (reference=81 .mu.L); RBC=2012/.mu.L
(reference=1733/.mu.L). FIGS. 3A and 3B show scattergrams of
cellular analysis of another body fluid sample. The cells were
enumerated after analysis: WBC=0.95/.mu.L (reference=0.33/.mu.L);
RBC=142/.mu.L (reference=122/.mu.L).
Example 2
Analysis of Very Low Cell Count Samples
[0100] The detection limit was further validated in a study using
diluted buffy coat samples. Six levels of diluted buffy coat
samples were prepared using serial dilutions. FL vs. IAS
scattergrams for the six levels of diluted buffy coat samples are
shown in FIGS. 4A-4F. FIG. 5 shows the correlation of WBC (measured
vs. calculated). Very good correlations were achieved: (A) Y=1.0239
X-4.9 (R.sup.2=0.9984) for all six levels and (B) Y=1.0787 X-1.5
(R.sup.2=0.9598) for the three levels with lowest cell
concentrations.
Example 3
Cellular Analysis of Body Fluid Samples at Dilution Ratio of
1:35
[0101] A comprehensive body fluid study was conducted to evaluate
the methods provided herein. A total of 91 body fluid specimens,
including CSF, plural, peritoneal, and ascites fluids, were
measured and analyzed on a prototype analyzer with 1:35
(blood:reagent) dilution ratio, 2.3 .mu.L per second injection
rate, and 32 seconds sample measurement (data collection).
Reference values of WBC and RBC were achieved by manual chamber
counting.
[0102] Excellent correlations in WBC were achieved between the
methods described above and the reference method (FIG. 6): (A)
Y=1.1305 X-9.8411, R.sup.2=0.997 (Full range, up to approximately
40,000/.mu.L); (B) Y=1.017 X+7.1395, R.sup.2=0.9765
(<2,000/.mu.L); (C) Y=1.0899 X+1.1253, R.sup.2=0.9663
(<200/.mu.L); and (D) Y=1.149 X+1.1461, R.sup.2=0.8459
(<50/.mu.L). Very good correlations in RBC were achieved between
the methods described above and the reference method (FIG. 7): (A)
Y=0.8996 X+403.62, R.sup.2=0.9595 (Full range, up to approximately
200,000/.mu.L); (B) Y=1.0833 X-11.427, R.sup.2=0.9513
(<3,000/.mu.L); (C) Y=0.979 X-1.0747, R.sup.2=0.8284
(<200/.mu.L); and (D) Y=0.9994 X+0.5951, R.sup.2=0.6917
(<50/.mu.L). For RBC analysis, the samples with many RBC ghosts
were not included in the correlation analysis.
Example 4
Cellular Analysis of Body Fluid Samples at Dilution Ratio of
1:10
[0103] Another comprehensive body fluid study was conducted to
evaluate the methods provided herein. A total of 155 body fluid
specimens, including CSF, plural, peritoneal, and ascites fluids,
were measured and analyzed on a prototype analyzer with 1:10
(blood:reagent) dilution ratio, 2.3 .mu.L per second injection
rate, and 32 seconds sample measurement (data collection).
Reference values of WBC and RBC were achieved by manual chamber
counting.
[0104] Excellent correlations in WBC were achieved, even for the
samples with low-end WBC concentrations, between the methods
described above and the reference method (FIG. 8): Y=1.1327
X+1.1937, R.sup.2=0.8195 (WBC<50/.mu.L, 72 samples). Very good
correlations in WBC were achieved, even for the samples with
low-end RBC concentrations, between the methods described above and
the reference method (FIG. 9): Y=0.8244 X+0.6128, R.sup.2=0.7465
(RBC<50/.mu.L, 38 samples). For RBC analysis, the body samples
with many RBC ghosts were not included in the correlation
analysis.
* * * * *