U.S. patent application number 16/653489 was filed with the patent office on 2020-05-07 for nucleated red blood cell analysis system and method.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to Michael R. Buhl, Marilou Coleman, Emily H. Lin, Giacomo Vacca, Jiong Wu.
Application Number | 20200141858 16/653489 |
Document ID | / |
Family ID | 47090460 |
Filed Date | 2020-05-07 |
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United States Patent
Application |
20200141858 |
Kind Code |
A1 |
Wu; Jiong ; et al. |
May 7, 2020 |
Nucleated Red Blood Cell Analysis System and Method
Abstract
Systems and methods for analyzing blood samples, and more
specifically for performing a nucleated red blood cell (nRBC)
analysis. The systems and methods screen a blood sample by means of
fluorescence staining and a fluorescence triggering strategy, to
identify nuclei-containing particles within the blood sample. As
such, interference from unlysed red blood cells (RBCs) and
fragments of lysed RBCs is substantially eliminated. The systems
and methods also enable development of relatively milder
reagent(s), suitable for assays of samples containing fragile white
blood cells (WBCs). In one embodiment, the systems and methods
include: (a) staining a blood sample with an exclusive cell
membrane permeable fluorescent dye; (b) using a fluorescence
trigger to screen the blood sample for nuclei-containing particles;
and (c) using measurements of light scatter and fluorescence
emission to distinguish nRBCs from WBCs.
Inventors: |
Wu; Jiong; (Los Gatos,
CA) ; Coleman; Marilou; (Newark, CA) ; Lin;
Emily H.; (Cupertino, CA) ; Buhl; Michael R.;
(San Ramon, CA) ; Vacca; Giacomo; (San Jose,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
47090460 |
Appl. No.: |
16/653489 |
Filed: |
October 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15704974 |
Sep 14, 2017 |
10481072 |
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16653489 |
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14808720 |
Jul 24, 2015 |
9778163 |
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15704974 |
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13456738 |
Apr 26, 2012 |
9103759 |
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14808720 |
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61482545 |
May 4, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2015/008 20130101;
G01N 2015/1477 20130101; G01N 2015/1488 20130101; G01N 2015/1402
20130101; G01N 21/6428 20130101; G01N 2015/0069 20130101; G01N
15/1459 20130101; G01N 15/1434 20130101; G01N 33/80 20130101; G01N
2015/1006 20130101; G01N 33/49 20130101; G01N 15/147 20130101; G01N
2021/6439 20130101 |
International
Class: |
G01N 15/14 20060101
G01N015/14; G01N 33/80 20060101 G01N033/80; G01N 21/64 20060101
G01N021/64; G01N 33/49 20060101 G01N033/49 |
Claims
1-18. (canceled)
19. A method of performing a nucleated red blood cell (nRBC)
analysis with an automated hematology analyzer, the method
comprising: (a) staining a sample of whole blood with a cell
membrane permeable, nucleic acid binding fluorescent dye; (b)
exciting the sample from step (a) with an excitation source as the
sample traverses a flow cell in the hematology analyzer; (c)
collecting a plurality of light scatter signals and a fluorescence
emission signal from the excited sample; (d) prior to performing an
nRBC differential analysis, excluding nuclei-free particles and
retaining nuclei-containing particles using only a fluorescence
trigger configured in the hematology analyzer and that is limited
to fluorescence emission signals and is set to a fluorescence
magnitude that is greater than fluorescence emission signals from
RBCs, including RBC fragments, and is less than fluorescence
emission signals from white blood cells (WBCs) and nRBCs; and (e)
performing the nRBC analysis on the nuclei-containing particles
collected in step (d).
20. (canceled)
21. The method of claim 19, wherein the excitation source has a
wavelength of from about 350 nm to about 700 nm.
22. The method of claim 19, wherein the fluorescence emission
signal is collected at a wavelength of from about 360 nm to about
750 nm, by a band-pass filter or a long-pass filter.
23. The method of claim 19, wherein the plurality of light scatter
signals include: (1) axial light loss, (2) intermediate angle
scatter, and (3) 90.degree. side scatter.
24. The method of claim 19, wherein the plurality of light scatter
signals include: (1) axial light loss, (2) intermediate angle
scatter, (3) 90.degree. polarized side scatter, and (4) 90.degree.
depolarized side scatter.
25. The method according to claim 19, wherein performing the nRBC
analysis on the nuclei-containing particles comprises
distinguishing a plurality of WBCs and a plurality of nRBCs from
one another based on the magnitude of the fluorescence emission
signal.
26. The method according to claim 19, wherein performing the nRBC
analysis comprises distinguishing a plurality of WBCs and a
plurality of nRBCs from one another based on the plurality of light
scatter signals.
27. The method according to claim 19, wherein performing the nRBC
analysis on the nuclei-containing particles comprises
distinguishing a plurality of WBCs and a plurality of nRBCs from
one another based on both the magnitude of the fluorescence
emission signal and the plurality of light scatter signals.
28. The method according to claim 24, wherein the axial light loss
signals are measured at 0.degree. scatter.
29. The method according to claim 24, wherein the intermediate
angle scatter signals are measured at about 3.degree. up to about
15.degree. scatter.
30. The method according to claim 24, wherein the polarized side
scatter signals are measured at about 90.degree. scatter.
31. The method according to claim 24, wherein the depolarized side
scatter signals are measured at about 90.degree. scatter.
32. The method according to claim 19, further comprising incubating
the blood sample of step (a) for an incubation period of time.
33. The method according to claim 32, wherein the incubation period
of time is less than 25 seconds.
34. The method according to claim 32, wherein the incubation period
of time is less than 17 seconds.
35. The method according to claim 32, wherein the incubation period
of time is less than 9 seconds.
36. The method according to claim 32, wherein the blood sample is
incubated at a temperature ranging from 30.degree. C. to 50.degree.
C.
37. The method according to claim 32, wherein the blood sample is
incubated at a temperature of about 40.degree. C.
38. The method according to claim 19, wherein a single fluorescent
dye is used to identify, quantify, and analyze nRBCs and a
plurality of WBC subpopulations at once.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application No. 61/482,545,
titled, Method For Analyzing Nucleated Red Blood Cells, and filed
on May 4, 2011, the entire disclosure of which is incorporated by
reference herein.
[0002] This application is also related to application Ser. No.
______, filed on Apr. 26, 2012, titled "WHITE BLOOD CELL ANALYSIS
SYSTEM AND METHOD," with Atty Dkt No: ADDV-016 (11040USO1); and
application Ser. No ______, filed on Apr. 26, 2012, titled
"BASOPHIL ANALYSIS SYSTEM AND METHOD," with Atty Dkt No: ADDV-018
(11042USO1), the entire disclosures of which are herein
incorporated by reference in their entirety.
BACKGROUND
[0003] This invention relates to hematology systems and methods.
More specifically, this invention relates to systems and methods
for analyzing blood samples to identify, classify, and/or quantify
nucleated red blood cells (nRBCs) in a sample of blood.
[0004] Nucleated red blood cells are often present in the fetus and
in the peripheral blood of newborns. However, nRBCs are considered
to be abnormal for adults. The presence of nRBCs in an adult's
peripheral blood stream is usually an indication of serious marrow
stress. Studies have shown that the appearance of nRBCs in the
blood stream is highly correlated with severe disease stages and/or
poor prognosis for critically ill patients. Therefore, accurate
identification and quantification of nRBCs has become increasingly
important for clinical diagnostics.
[0005] Because nRBCs share numerous similarities with white blood
cells (WBCs), the concentration of nRBCs in a blood sample is
typically reported as a percentage of total WBCs in the blood
sample (i.e., % nRBC=nRBCs/WBCs.times.100%). Traditional approaches
to analyze nRBCs include: (1) separating nRBCs from WBCs by size;
(2) differentiating nRBCs from WBCs by means of light scattering;
or (3) analyzing nRBCs by means of fluorescence emission detection
after lysis and staining with a cell membrane impermeable
fluorescent dye(s).
[0006] Each of the above-listed techniques has shown weaknesses in
clinical practices. For example, it is difficult to completely
eliminate fragments of lysed red blood cells (RBCs) in rapid
hematology measurements. Because fragments of RBCs and the nuclei
of nRBCs may be similar in size and light scattering
characteristics, analysis based on size and/or light scattering is
sometimes misleading. Meanwhile, analysis based on fluorescence
emission may be adversely affected by: (1) "under-lysing" of the
sample such that the cell membrane impermeable dye cannot reach the
nuclei of the nRBCs; (2) "over-lysing" of the sample such that
nuclei of the WBCs are stained and interfere with the nRBC count;
(3) the existence of fragile lymphocytes, such that WBCs are
unexpectedly hyper-sensitive to a lysing reagent (giving false
positives); and/or (4) the existence of lyse-resistant nRBCs, such
that the nRBCs are unexpectedly insensitive to a lysing reagent
(giving false negatives). In practice, over-lysing or under-lysing
is common on account of the variation in membrane rigidity of blood
cells among samples of blood. As such, dependence on known light
scatter and/or fluorescence emission detection techniques may
result in an inaccurate and unreliable analysis for nRBCs, thereby
preventing correct diagnoses and treatment for critically ill
patients.
BRIEF SUMMARY
[0007] Provided herein are systems and methods for analyzing blood
samples, and more specifically for performing a nRBC analysis. The
systems and methods screen a blood sample by means of fluorescence
staining with a cell membrane permeable fluorescent dye. A
fluorescence triggering strategy is then used to identify,
distinguish, and separate nuclei-containing particles (e.g., nRBCs
and WBC) from non-nuclei-containing particles (e.g., RBCs and/or
RBC fragments) within the blood sample. As such, interference from
unlysed RBCs and fragments of lysed RBCs can be substantially
eliminated prior to subsequent analysis. For example, in one
embodiment, the systems and methods include: (a) staining a blood
sample with an exclusive cell membrane permeable fluorescent dye;
(b) using a fluorescence trigger to screen the blood sample for
nuclei-containing particles; and then (c) using measurements of
light scatter and fluorescence emission to distinguish nRBCs from
WBCs. The systems and methods enable development of relatively
milder reagent(s), suitable for assays of samples containing
fragile WBCs.
BRIEF DESCRIPTION OF THE FIGURES
[0008] The accompanying drawings, which are incorporated herein,
form part of the specification. Together with this written
description, the drawings further serve to explain the principles
of, and to enable a person skilled in the relevant art(s), to make
and use the systems and methods presented. In the drawings, like
reference numbers indicate identical or functionally similar
elements.
[0009] FIGS. 1A-1D show histograms of a sample of whole blood,
showing WBCs, nRBCs, and residues of RBCs following lysis.
[0010] FIG. 1A is a histogram showing measurements of axial light
loss (ALL).
[0011] FIG. 1B is a histogram showing measurements of intermediate
angle scatter (IAS).
[0012] FIG. 1C is a histogram showing measurements of 90.degree.
polarized side scatter (PSS).
[0013] FIG. 1D is a histogram showing measurements of fluorescence
(FL1).
[0014] FIG. 2 is a schematic diagram illustrating a hematology
instrument.
[0015] FIGS. 3A-3F are cytograms illustrating an analysis of a
sample of blood containing nRBCs at a % NRBC of 75%.
[0016] FIG. 3A is a cytogram depicting axial light loss vs.
intermediate angle scatter.
[0017] FIG. 3B is a cytogram depicting 90.degree. polarized side
scatter vs. axial light loss.
[0018] FIG. 3C is a cytogram depicting 90.degree. polarized side
scatter vs. intermediate angle scatter.
[0019] FIG. 3D is a cytogram depicting FL1 vs. axial light
loss.
[0020] FIG. 3E is a cytogram depicting FL1 vs. intermediate angle
scatter.
[0021] FIG. 3F is a cytogram depicting FL1 vs. 90.degree. polarized
side scatter.
[0022] FIGS. 4A-4F are cytograms illustrating an analysis of a
sample of blood containing nRBCs as a % NRBC of 1.0%.
[0023] FIG. 4A is a cytogram depicting axial light loss vs.
intermediate angle scatter.
[0024] FIG. 4B is a cytogram depicting 90.degree. polarized side
scatter vs. axial light loss.
[0025] FIG. 4C is a cytogram depicting 90.degree. polarized side
scatter vs. intermediate angle scatter.
[0026] FIG. 4D is a cytogram depicting FL1 vs. axial light
loss.
[0027] FIG. 4E is a cytogram depicting FL1 vs. intermediate angle
scatter.
[0028] FIG. 4F is a cytogram depicting FL1 vs. 90.degree. polarized
side scatter.
[0029] FIG. 5 is a plot illustrating correlation of % NRBC as
determined by manual gating vs. reference results, wherein the
reference results were obtained by a microscope review.
[0030] FIG. 6 is a plot illustrating correlation of % NRBC as
determined by manual gating vs. reference results, wherein the
reference results were obtained by a "CELL-DYN" Sapphire.TM.
hematology analyzer.
[0031] FIG. 7 is a plot illustrating correlation of % NRBC, as
determined by auto-clustering vs. reference results, wherein the
reference results were obtained by a microscope review.
[0032] FIG. 8 is a plot illustrating correlation of % NRBC, as
determined by auto-clustering vs. reference results, wherein the
reference results were obtained by a "CELL-DYN" Sapphire.TM.
hematology analyzer.
DETAILED DESCRIPTION
[0033] Provided herein are systems and methods for analyzing blood
samples, and more specifically for performing an nRBC analysis to
identify, classify, and count nRBCs in a blood sample. In general,
the systems and methods disclosed screen nuclei-containing events
vs. non-nuclei-containing events by means of fluorescence staining
and a fluorescence triggering strategy. As such, interference from
unlysed red blood cells (RBCs), such as lysis-resistant red blood
cells (rstRBCs), and RBC fragments is substantially eliminated
prior to subsequent analysis. In other words, the systems and
methods described herein utilize at least one fluorescent dye and a
fluorescence triggering system to screen events containing nuclei,
to thereby accurately and reliably identify and quantify WBCs and
nRBCs. A combination light scattering information and fluorescence
information is then used to further separate nRBCs from WBCs. The
systems and methods disclosed thereby ensure accurate counting and
differentiation of nRBCs, WBCs, and WBC sub-populations. The
systems and methods also enable development of relatively milder
WBC reagent(s), suitable for assays of samples containing fragile
lymphocytes (or other fragile WBCs), including aged samples.
[0034] In one embodiment, for example, the systems and methods
disclosed herein include: (a) staining a blood sample with an
exclusive cell membrane permeable fluorescent dye; (b) using a
fluorescence trigger to screen the blood sample for
nuclei-containing particles; and (c) using measurements of light
scatter and fluorescence emission to distinguish nRBCs from WBCs.
Systems and methods in accordance with the present invention show
great advantages over traditional methods because the interference
from fragments of RBCs is substantially eliminated, and the results
of the assay become much less sensitive to the lysing strength. In
other words, traditional nRBC analysis techniques are highly
sensitive to lysing strength. If the strength of the lysing agent
is too weak, the fragments of RBCs or unlysed RBCs were also
collected in the analysis of WBCs. These fragments of RBCs or
unlysed RBCs overlapped nRBCs in many dimensions, resulting in
difficulty in analyzing nRBCs. On the other hand, if the strength
of the lysing agent was too strong (in order to better deal with
fragments of RBCs), a certain percentage of lymphocytes could be
damaged and could be recognized as nRBCs. Therefore, the strength
of the lysing agent is a problem in the analysis of WBCs. A key
feature of the methods described herein is the reduction of
interference from RBC fragments or unlysed RBCs in the analysis of
WBCs and nRBCs. Accordingly, variations in the strength of the
lysing agent can be better tolerated. Even if the lysing agent is
weaker than usual, the unlysed RBCs do not interfere with the assay
for nRBCs.
[0035] In the systems and methods described herein, quantification
and identification of nRBCs and WBCs can be obtained simultaneously
in a single assay. Alternatively, in the systems and methods
described herein, quantification and identification of nRBCs can be
carried out without analysis of WBCs.
(1) Use of Fluorescent Dye(s).
[0036] WBCs and nRBCs contain a relatively high concentration of
DNA in their nuclei. Mature RBCs, however, do not contain DNA.
Therefore, a fluorescent dye is selected to differentiate two
classes of blood cells; namely, the blood cells containing nucleic
acids and the blood cells not containing nucleic acids. The purpose
of the dye is to penetrate into live cells easily, bind DNA with
high affinity, and emit strong fluorescence with adequate Stokes
shift when the dye is excited by an appropriate source of light.
The peak absorption of the dye in the visible band substantially
matches the wavelength of the source of light (within 50 nm of the
wavelength of the source of light, more preferably, within 25 nm of
the wavelength of the source of light), in order to be properly
excite the dye and achieve optimal results.
[0037] The fluorescent dye selected is preferably: 1) capable of
binding nucleic acids, 2) capable of penetrating cell membranes of
WBCs and nRBCs, 3) excitable at a selected wavelength when
subjected to a source of light, 4) emits fluorescence upon
excitation by the source of light, and 5) is biostable and soluble
in a liquid. The dye may be selected from group consisting of:
acridine orange, SYBR 11, SYBR Green series dye, hexidium iodide,
SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO RNA
Select, SYTO 24, SYTO 25 and any equivalents thereof. The dye is
used to "activate" WBCs and nRBCs, and "screen out" unlysed RBCs
and fragments of RBCs based on a fluorescence trigger configured in
the hematology analyzer. The dye is typically present at a
concentration of from about 0.1 ng/mL to about 0.1 mg/mL. While
various dyes are available, the dye selected is generally paired
with the excitation source of the hematology analyzer such that a
single exclusive dye is used to stain and excite fluorescence
emission in nRBCs and all WBC sub-populations intended to be
identified, quantified, and/or analyzed. As such, a single (i.e.,
exclusive) dye can be used to identify, quantify, and analyze nRBCs
and all the WBC subpopulations at once.
[0038] In one embodiment, a fluorescent dye is provided in a
reagent, with combinations of 1) at least one surfactant, 2) at
least one buffer, 3) at least one salt, and/or 4) at least
antimicrobial agent, in sufficient quantities for carrying out
staining and activating up to 1,000.times.10.sup.3 cells per
microliter. The at least one surfactant, such as "TRITON" X-100 or
saponin, is used to destroy the membranes of RBC, and reduce the
sizes of fragments of RBCs. The at least one surfactant is
typically present at a concentration of from about 0.001% to about
5%. The at least one antimicrobial agent, such as those from
"TRIADINE" or "PROCLIN" families, is used to prevent the
contamination of the reagent from microbes. The concentration of
the at least one antimicrobial agent is sufficient to preserve the
reagent for the shelf life required. The at least one buffer, such
as phosphate buffered saline (PBS) or
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), is used
to adjust the pH of reaction mixture for controlling lysis of RBCs
and preserving WBCs. The at least one buffer is typically present
at a concentration of from about 0.01% to about 3%. The pH
typically ranges from about 3 to about 12. The at least one salt,
such as NaCl or Na.sub.2SO.sub.4, is used to adjust the osmolality
to increase the effect of lysing and/or optimize WBC preservation.
The at least one salt may be present at a concentration of from
about 0.01% to about 3%. In certain cases, the at least one buffer
can serve as the at least one salt, or the at least one salt can
serve as the at least one buffer. In general, lower osmolality, or
hypotonicity, is used to accelerate the lysis of RBCs. The
osmolality typically ranges from about 20 to about 250 mOsm.
[0039] Lysis of RBCs can be made to occur at a temperature above
room temperature (e.g., between about 30.degree. C. to about
50.degree. C., such as about 40.degree. C.) over a relatively short
period of time (e.g., less than about 25 seconds, less than about
17 seconds, or even less than about 9 seconds), following mixing of
the sample of blood and the reagent at a ratio of about one part by
volume sample to about 35 parts by volume reagent. The data for
analysis is collected with a plurality of optical channels and at
least one fluorescence channel.
[0040] FIGS. 1A-1D show histograms of a sample of whole blood,
showing the separation of WBCs, nRBCs, and residues of RBCs
following lysis, based on optical and fluorescence measurements.
More specifically, FIG. 1A is a histogram showing separation of
particles based on measurements of axial light loss (ALL). FIG. 1B
is a histogram showing separation of particles based on
measurements of intermediate angle scatter (IAS). FIG. 1C is a
histogram showing separation of particles based on measurements of
90.degree. polarized side scatter (PSS). FIG. 1D is a histogram
showing separation of particles based on measurements of
fluorescence (FL1). In the histograms, the horizontal axis
indicates the value of the detection channel (or the names of the
channels, i.e., ALL, IAS, PSS, or FL1). The vertical axis indicates
counts of components of the sample of blood. In the histograms, the
lines 100 indicate residues of RBCs, lines 200 indicate WBCs, and
lines 300 indicate nRBCs. As shown by comparing FIG. 1D to FIGS.
1A-1C, fluorescence information, rather than optical measurements,
shows much better separation between the nuclei-containing
particles (e.g., WBCs and nRBCs) and non-nuclei-containing
particles (residues of RBCs). As used herein, "residues of RBCs" is
synonymous with "fragments of RBCs."
(2) Use of a Fluorescence Trigger.
[0041] Blood cells emit different magnitudes of fluorescence
signals upon excitation of the fluorescent dye by a source of
light. The differences in magnitude of fluorescence signals arise
from the quantity of nucleic acids, namely DNA, inside the cells.
The greater the quantity of DNA, the greater the likelihood of
higher fluorescence signals. Also, efficacy of penetration of cell
membranes, size of the dye, binding kinetics between the dye and
DNA, affinity between the dye and DNA, and other factors, affect
the fluorescence signals. Mature RBCs emit minimal fluorescence
signals because there is no DNA within mature RBCs. nRBCs emit very
strong fluorescence signals, because not only is DNA inside nuclei
of nRBCs, but also the staining is easier because membranes of
nRBCs are destroyed during the lysis procedure. Unlysed RBCs or RBC
fragments do not emit fluorescence, although they may emit very
weak auto-fluorescence. As shown with reference to FIG. 1D, the
cells that emit much stronger fluorescence signals are the cells
having nuclei, namely, all WBCs and nRBCs (when present).
[0042] As such, the systems and methods presented herein use a
fluorescence trigger for collecting and analyzing WBCs and nRBC.
For example, a fluorescence trigger, usually set between signals
from RBCs and signals from WBCs and nRBCs, can be used to collect
signals from WBCs and nRBCs for subsequent analysis.
(3) Use of a Plurality of Optical Channels and at Least One
Fluorescence Channel for Analysis.
[0043] As used herein, the expression "fluorescence information"
means data collected from a fluorescence channel of a hematology
analyzer. As used herein, the expression "fluorescence channel"
means a detection device, such as a photomultiplier tube, set at an
appropriate wavelength band for measuring the quantity of
fluorescence emitted from a sample.
[0044] In one embodiment, the blood sample analysis is conducted by
means of Multiple Angle Polarized Scattering Separation technology
(MAPSS), with enhancement from fluorescence information. At least
one photodiode, or at least one photomultiplier tube, or both at
least one photodiode and at least one photomultiplier tube, are
needed to detect light scattered by each blood cell passing through
a flow cell. Two or more photodiodes are used for measuring ALL
signals, which measure about 0.degree. scatter, and IAS signals,
which measure low angle (e.g., about 3.degree. to about 15.degree.)
scatter. Two or more photomultiplier tubes are used for detecting
90.degree. polarized side scatter (PSS) signals and 90.degree.
depolarized side scatter (DSS) signals. Additional photomultiplier
tubes are needed for FL1 measurements within appropriate wavelength
range(s), depending on the choice of wavelength of the source of
light. Each event captured on the system thus exhibits a plurality
of dimensions of information, such as ALL, IAS (one or more
channels), PSS, DSS, and fluorescence (one or more channels). The
information from these detection channels is used for further
analysis of blood cells.
[0045] FIG. 2 is a schematic diagram illustrating the illumination
and detection optics of an apparatus suitable for hematology
analysis (including flow cytometry). Referring now to FIG. 2, an
apparatus 10 comprises a source of light 12, a front mirror 14 and
a rear mirror 16 for beam bending, a beam expander module 18
containing a first cylindrical lens 20 and a second cylindrical
lens 22, a focusing lens 24, a fine beam adjuster 26, a flow cell
28, a forward scatter lens 30, a bulls-eye detector 32, a first
photomultiplier tube 34, a second photomultiplier tube 36, and a
third photomultiplier tube 38. The bulls-eye detector 32 has an
inner detector 32a for 0.degree. light scatter and an outer
detector 32b for 7.degree. light scatter.
[0046] In the discussion that follows, the source of light is
preferably a laser. However, other sources of light can be used,
such as, for example, lamps (e.g., mercury, xenon). The source of
light 12 can be a vertically polarized air-cooled Coherent Cube
laser, commercially available from Coherent, Inc., Santa Clara,
Calif. Lasers having wavelengths ranging from 350 nm to 700 nm can
be used. Operating conditions for the laser are substantially
similar to those of lasers currently used with "CELL-DYN" automated
hematology analyzers.
[0047] Additional details relating to the flow cell, the lenses,
the focusing lens, the fine-beam adjust mechanism and the laser
focusing lens can be found in U.S. Pat. No. 5,631,165, incorporated
herein by reference, particularly at column 41, line 32 through
column 43, line 11. The forward optical path system shown in FIG. 2
includes a spherical plano-convex lens 30 and a two-element
photo-diode detector 32 located in the back focal plane of the
lens. In this configuration, each point within the two-element
photodiode detector 32 maps to a specific collection angle of light
from cells moving through the flow cell 28. The detector 32 can be
a bulls-eye detector capable of detecting axial light loss (ALL)
and intermediate angle forward scatter (IAS). U.S. Pat. No.
5,631,165 describes various alternatives to this detector at column
43, lines 12-52.
[0048] The first photomultiplier tube 34 (PMT1) measures
depolarized side scatter (DSS). The second photomultiplier tube 36
(PMT2) measures polarized side scatter (PSS), and the third
photomultiplier tube 38 (PMT3) measures fluorescence emission from
440 nm to 680 nm, depending upon the fluorescent dye selected and
the source of light employed. The photomultiplier tube collects
fluorescent signals in a broad range of wavelengths in order to
increase the strength of the signal. Side-scatter and fluorescent
emissions are directed to these photomultiplier tubes by dichroic
beam splitters 40 and 42, which transmit and reflect efficiently at
the required wavelengths to enable efficient detection. U.S. Pat.
No. 5,631,165 describes various additional details relating to the
photomultiplier tubes at column 43, line 53 though column 44, line
4.
[0049] Sensitivity is enhanced at photomultiplier tubes 34, 36, and
38, when measuring fluorescence, by using an immersion collection
system. The immersion collection system is one that optically
couples the first lens 30 to the flow cell 28 by means of a
refractive index matching layer, enabling collection of light over
a wide angle. U.S. Pat. No. 5,631,165 describes various additional
details of this optical system at column 44, lines 5-31.
[0050] The condenser 44 is an optical lens system with aberration
correction sufficient for diffraction limited imaging used in high
resolution microscopy. U.S. Pat. No. 5,631,165 describes various
additional details of this optical system at column 44, lines
32-60.
[0051] The functions of other components shown in FIG. 2, i.e., a
slit 46, a field lens 48, and a second slit 50, are described in
U.S. Pat. No. 5,631,165, at column 44, line 63 through column 45,
line 26. Optical filters 52 or 56 and a polarizer 52 or 56, which
are inserted into the light paths of the photomultiplier tubes to
change the wavelength or the polarization or both the wavelength
and the polarization of the detected light, are also described in
U.S. Pat. No. 5,631,165, at column 44, line 63 through column 45,
line 26. Optical filters that are suitable for use herein include
band-pass filters and long-pass filters.
[0052] The photomultiplier tubes 34, 36, and 38 detect either
side-scatter (light scattered in a cone whose axis is approximately
perpendicular to the incident laser beam) or fluorescence (light
emitted from the cells at a different wavelength from that of the
incident laser beam).
[0053] While select portions of U.S. Pat. No. 5,631,165 are
referenced above, U.S. Pat. No. 5,631,165 is incorporated herein by
reference in its entirety.
[0054] The optical and fluorescence information collected may then
be used to distinguish (or differentiate) nRBCs from WBCs (and WBC
sub-populations). For example, two-dimensional cytograms (e.g.,
cytograms showing PSS vs. ALL; ALL vs. IAS; and/or FL1 vs.
ALL/IAS/PSS) can be used to identify and distinguish particles.
[0055] FIGS. 3A-3F are cytograms illustrating an analysis of a
sample of blood containing nRBCs at a % NRBC of 75%. More
specifically, FIG. 3A is a cytogram depicting axial light loss vs.
intermediate angle scatter. FIG. 3B is a cytogram depicting
90.degree. polarized side scatter vs. axial light loss. FIG. 3C is
a cytogram depicting 90.degree. polarized side scatter vs.
intermediate angle scatter. FIG. 3D is a cytogram depicting FL1 vs.
axial light loss. FIG. 3E is a cytogram depicting FL1 vs.
intermediate angle scatter. FIG. 3F is a cytogram depicting FL1 vs.
90.degree. polarized side scatter. The % NRBC values obtained from
the cytograms were 80.7 (as determined by manual gating using PSS
v. ALL) and 76.3 (as determined by auto-clustering analysis carried
out with MATLAB software).
[0056] FIGS. 4A-4F are cytograms illustrating an analysis of a
sample of blood containing nRBCs as a % NRBC of 1.0%. More
specifically, FIG. 4A is a cytogram depicting axial light loss vs.
intermediate angle scatter. FIG. 4B is a cytogram depicting
90.degree. polarized side scatter vs. axial light loss. FIG. 4C is
a cytogram depicting 90.degree. polarized side scatter vs.
intermediate angle scatter. FIG. 4D is a cytogram depicting FL1 vs.
axial light loss. FIG. 4E is a cytogram depicting FL1 vs.
intermediate angle scatter. FIG. 4F is a cytogram depicting FL1 vs.
90.degree. polarized side scatter. The % NRBC values obtained from
the cytograms were 1.2 (as determined by manual gating using PSS v.
ALL) and 1.2 (as determined by auto-clustering analysis carried out
with MATLAB software).
[0057] In a study using a total of 136 samples containing nRBCs,
both manual microscope reviews and "CELL-DYN" Sapphire.TM.
hematology analyzer results were used as reference for
quantifications of nRBCs. Acridine orange, at a concentration of 3
.mu.g/mL, was included in the a reagent. The sample of blood and
the reagent were mixed at a ratio of one part by volume sample to
35 parts by volume of reagent. The mixture was incubated for a
period of 25 seconds at a temperature of 40.degree. C. A sample
measurement duration of 9 seconds was applied, with FL1 used as the
sole trigger. Measurements of ALL, IAS, PSS, and FL1 were collected
for each sample. The data was analyzed using both manual gating
(PSS vs. ALL, or PSS vs. IAS) and auto-clustering analysis using
MATLAB software. The correlations of % nRBC between the results
from the method of the present invention, and reference results
were reported as shown in FIGS. 5-8.
[0058] FIG. 5, for example, compares results (obtained by manual
gating) against reference results, wherein the reference results
were obtained by a microscope review. As shown, the systems and
methods of the present invention produced results in accordance
with the slope and R.sup.2 formula of: Y=1.0404X;
(R.sup.2=0.968).
[0059] FIG. 6 compares results (obtained by manual gating) against
reference results, wherein the reference results were obtained by a
"CELL-DYN" Sapphire.TM. hematology analyzer. As shown, the systems
and methods of the present invention produced results in accordance
with the slope and R.sup.2 formula of: Y=1.1004X;
(R.sup.2=0.956).
[0060] FIG. 7 compares results (obtained by auto-clustering)
against reference results, wherein the reference results were
obtained by a microscope review. As shown, the systems and methods
of the present invention produced results in accordance with the
slope and R.sup.2 formula of: Y=1.0215X; (R.sup.2=0.963).
[0061] FIG. 8 compares results (obtained by auto-clustering)
against reference results, wherein the reference results were
obtained by a "CELL-DYN" Sapphire.TM. hematology analyzer. As
shown, the systems and methods of the present invention produced
results in accordance with the slope and R.sup.2 formula of:
Y=1.0800X (R.sup.2=0.950)
[0062] Bhattacharyya distances (BD) between nRBCs and lymphocytes
were also calculated based upon the mean positions of the clusters
and distribution coefficients of variation. The cluster separation
is considered to be "good" if BD exceeds 3 and is considered to be
"acceptable" if BD is greater than 2 and less than or equal to 3.
The average BD value for the tests were 4.64.+-.1.89, ranging from
2.10 to 11.95, for the 136 samples. Good separations (in which BD
exceeds 3) between nRBCs and lymphocytes were observed in more than
85% (116/136) of samples containing nRBCs.
[0063] In contrast to the difficulty of balancing "over-lysing" and
"under-lysing" for traditional methods of measuring nRBCs, the
methods described herein preserve WBCs and provide the best
separation between nRBCs and lymphocytes (the WBC sub-population
closest to nRBCs). Further, interference from fragments of RBCs is
substantially eliminated.
Additional Embodiments
[0064] In another embodiment, there is provided a hematology
analyzer for conducting a nucleated red blood cell (nRBC) analysis
on a blood sample that has been dyed with a fluorescent dye,
wherein the fluorescent dye is cell membrane permeable and nucleic
acid binding. The analyzer comprises an excitation source
positioned to excite particles within the blood sample. The
analyzer also comprises a plurality of detectors including: (1) an
axial light loss detector positioned to measure axial light loss
from the excited blood sample, (2) an intermediate angle scatter
detector positioned to measure intermediate angle scatter from the
excited blood sample, (3) a side scatter detector positioned to
measure 90.degree. side scatter from the excited blood sample, and
(4) a fluorescence detector positioned to measure fluorescence
emitted from the excited blood sample. The analyzer also comprises
a processor configured to: (a) receive the measurements of (1)
axial light loss, (2) intermediate angle scatter, (3) 90.degree.
side scatter, and (4) fluorescence from the plurality of detectors,
and (b) perform a nRBC differential analysis of the blood sample,
based on all four measurements, for particles that emit
fluorescence above a fluorescence threshold. The side scatter
detector may be a polarized side scatter detector positioned to
measure 90.degree. polarized side scatter from the excited blood
sample. The hematology analyzer may further comprise a depolarized
side scatter detector positioned to measure 90.degree. depolarized
side scatter from the excited blood sample. The processor may be
further configured to pre-screen the received measurements to
remove from consideration any particles that do not meet the
fluorescence threshold. The axial light loss detector can measure
axial light loss at 0.degree. scatter. The intermediate angle
scatter detector can measure light angle scatter at about 3.degree.
to about 15.degree.. The plurality of detectors can include one or
more photomultiplier tubes. The excitation source may be a laser
configured to emit light at a wavelength corresponding to the
fluorescent dye. The fluorescent dye can be selected to correspond
with the excitation source.
[0065] The hematology analyzer may further comprise an incubation
subsystem for diluting the blood sample with a reagent. The reagent
can include the fluorescent dye and a lysing agent. The reagent may
alternatively comprise (a) at least one surfactant, (b) at least
one buffer or at least one salt, (c) at least one antimicrobial
agent, and (d) the fluorescent dye. The incubation subsystem may be
configured to incubate the blood sample with the reagent for a
period of time of less than about 25 seconds, less than about 17
seconds, and/or less than about 9 seconds. The incubation subsystem
may be configured to incubate the blood sample with the reagent at
a temperature ranging from about 30.degree. C. to about 50.degree.
C., such as a temperature of about 40.degree. C.
[0066] In another embodiment, there is provided a method of
performing a nucleated red blood cell nRBC analysis with an
automated hematology analyzer. The method comprises the steps of:
(a) diluting a sample of whole blood with a reagent, wherein the
reagent includes a red blood cells (RBC) lysing agent and a cell
membrane permeable, nucleic acid binding fluorescent dye; (b)
incubating the diluted blood sample of step (a) for an incubation
period of less than about 25 seconds, at a temperature ranging from
about 30.degree. C. to about 50.degree. C.; (c) delivering the
incubated sample from step (b) to a flow cell in the hematology
analyzer; (d) exciting the incubated sample from step (c) with an
excitation source as the incubated sample traverses the flow cell;
(e) collecting a plurality of light scatter signals and a
fluorescence emission signal from the excited sample; and (f)
performing a nRBC analysis based on all the signals collected in
step (e), while removing from consideration any particles within
the diluted blood sample that do not meet a fluorescence threshold
based on the fluorescence emission signal.
[0067] The reagent may include: (a) at least one surfactant, (b) at
least one buffer or at least one salt, (c) at least one
antimicrobial agent, and (d) at least one fluorescent dye. The
excitation source may have a wavelength of from about 350 nm to
about 700 nm. The fluorescence emission may be collected at a
wavelength of from about 360 nm to about 750 nm, by a band-pass
filter or a long-pass filter. The plurality of light scatter
signals may include: (1) axial light loss, (2) intermediate angle
scatter, and (3) 90.degree. side scatter. The plurality of light
scatter signals may include: (1) axial light loss, (2) intermediate
angle scatter, (3) 90.degree. polarized side scatter, and (4)
90.degree. depolarized side scatter.
[0068] In yet another embodiment, there are provided systems and
methods for counting nRBCs by means of an automated hematology
analyzer. The systems and method include means for and the steps
of: (a) diluting a whole blood sample containing nucleated red
blood cells with at least one white blood cell reagent; (b)
incubating the diluted sample of step (a) for a sufficient period
of time within a selected temperature range to lyse red blood
cells, allow nuclei of nucleated red blood cells to be exposed to
the at least one white blood cell reagent, and allow at least one
fluorescent dye to stain nuclei of nucleated red blood cells, and
preserve white blood cells; (c) delivering the incubated sample of
step (b) to a flow cell as a stream; (d) exciting the incubated
sample with a source of light as the incubated sample traverses the
flow cell; (e) collecting a plurality of optical scatter signals
and at least one fluorescence emission signal simultaneously; and
(f) differentiating and quantifying nucleated red blood cells by
means of the optical information and fluorescence information
collected in step (e). The systems and methods further include: (1)
the use of at least one fluorescent dye to bind and stain nucleic
acids in WBCs and the nuclei of nRBCs in a given sample of blood
during the procedure for lysing RBCs, and to induce fluorescence
emissions upon being excited by photons from a given source of
light, such as a laser beam at an appropriate wavelength; (2) the
use of a fluorescent trigger to separate and collect events that
emit strong fluorescence (i.e., WBCs and nRBCs); and (3) the use of
a plurality of optical channels and at least one fluorescent
channel for collecting and analyzing data in order to identify
nRBCs, and to separate nRBCs from WBCs. The systems and method
described herein allows simultaneous analysis of WBCs and nRBCs. No
additional reagent, preparation of samples, or analytical procedure
is needed. Therefore, the method is efficient, cost-effective, and
practical for modern diagnostic use.
[0069] In one embodiment, the invention is directed towards one or
more computer systems capable of carrying out the functionality
described herein. For example, any of the method/analysis steps
discussed herein may be implemented in a computer system having one
or more processors, a data communication infrastructure (e.g., a
communications bus, cross-over bar, or network), a display
interface, and/or a storage or memory unit. The storage or memory
unit may include computer-readable storage medium with instructions
(e.g., control logic or software) that, when executed, cause the
processor(s) to perform one or more of the functions described
herein. The terms "computer-readable storage medium," "computer
program medium," and "computer usable medium" are used to generally
refer to media such as a removable storage drive, removable storage
units, data transmitted via a communications interface, and/or a
hard disk installed in a hard disk drive. Such computer program
products provide computer software, instructions, and/or data to a
computer system, which also serve to transform the computer system
from a general purpose computer into a special purpose computer
programmed to perform the particular functions described herein.
Where appropriate, the processor, associated components, and
equivalent systems and sub-systems thus serve as examples of "means
for" performing select operations and functions. Such "means for"
performing select operations and functions also serve to transform
a general purpose computer into a special purpose computer
programmed to perform said select operations and functions.
CONCLUSION
[0070] The foregoing description of the invention has been
presented for purposes of illustration and description. It is not
intended to be exhaustive or to limit the invention to the precise
form disclosed. Other modifications and variations may be possible
in light of the above teachings. The embodiments were chosen and
described in order to best explain the principles of the invention
and its practical application, and to thereby enable others skilled
in the art to best utilize the invention in various embodiments and
various modifications as are suited to the particular use
contemplated. It is intended that the appended claims be construed
to include other alternative embodiments of the invention;
including equivalent structures, components, methods, and
means.
[0071] The above Detailed Description refers to the accompanying
drawings that illustrate one or more exemplary embodiments. Other
embodiments are possible. Modifications may be made to the
embodiment described without departing from the spirit and scope of
the present invention. Therefore, the Detailed Description is not
meant to be limiting. Further, the Summary and Abstract sections
may set forth one or more, but not all exemplary embodiments of the
present invention as contemplated by the inventor(s), and thus, are
not intended to limit the present invention and the appended claims
in any way.
* * * * *