U.S. patent application number 10/226800 was filed with the patent office on 2003-12-25 for method for measurement of nucleated red blood cells.
Invention is credited to Britton, Ted W., Li, Jing, Li, Yi.
Application Number | 20030235917 10/226800 |
Document ID | / |
Family ID | 26861630 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235917 |
Kind Code |
A1 |
Li, Yi ; et al. |
December 25, 2003 |
METHOD FOR MEASUREMENT OF NUCLEATED RED BLOOD CELLS
Abstract
A method for differentiating and enumerating nucleated red blood
cells in a blood sample is described. The method includes the steps
of lysing red blood cells of a blood sample with a lytic reagent,
measuring nucleated blood cells by DC impedance measurement in a
non-focused flow aperture, differentiating nucleated red blood
cells from other cell types, and reporting nucleated red blood
cells in the blood sample. The method further includes subtracting
nucleated red blood cells and other interference materials from the
count of remaining blood cells, and reporting a corrected white
blood cell count of the blood sample. Additionally, the method
further includes measuring spectrophotometric absorbance of the
sample mixture at a predetermined wavelength of a hemoglobin
chromogen formed upon lysing the blood sample, and reporting
hemoglobin concentration of the blood sample.
Inventors: |
Li, Yi; (Miami, FL) ;
Li, Jing; (Miami, FL) ; Britton, Ted W.;
(Sunrise, FL) |
Correspondence
Address: |
BECKMAN COULTER, INC.
P.O. BOX 169015
MAIL CODE 32-A02
MIAMI
FL
33116-9015
US
|
Family ID: |
26861630 |
Appl. No.: |
10/226800 |
Filed: |
August 23, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10226800 |
Aug 23, 2002 |
|
|
|
10165699 |
Jun 7, 2002 |
|
|
|
10165699 |
Jun 7, 2002 |
|
|
|
09917533 |
Jul 27, 2001 |
|
|
|
6410330 |
|
|
|
|
Current U.S.
Class: |
436/10 ; 422/73;
422/82.05; 422/82.09; 436/164; 436/17; 436/63; 436/66; 436/8 |
Current CPC
Class: |
G01N 2015/008 20130101;
G01N 2015/1062 20130101; G01N 33/80 20130101; G01N 2015/0069
20130101; Y10T 436/107497 20150115; G01N 15/14 20130101; Y10T
436/10 20150115; G01N 2015/0073 20130101; Y10T 436/101666 20150115;
G01N 15/12 20130101 |
Class at
Publication: |
436/10 ; 436/8;
436/63; 436/66; 436/164; 436/17; 422/73; 422/82.05; 422/82.09 |
International
Class: |
G01N 031/00; G01N
033/48 |
Claims
What is claimed is:
1. A method of differentiating nucleated red blood cells from other
cell types in a blood sample comprising steps of: (a) mixing a
blood sample with a lytic reagent to lyse red blood cells, and to
form a blood sample mixture, (b) measuring said blood sample
mixture by a DC impedance measurement, and obtaining a blood cell
distribution of said blood sample mixture, (c) differentiating
nucleated red blood cells from other cell types from obtained blood
cell distribution, and (d) reporting nucleated red blood cells in
said blood sample.
2. The method of claim 1 wherein said reporting nucleated red blood
cells comprises reporting a presence of nucleated red blood cells
in said blood sample.
3. The method of claim 1 wherein said reporting nucleated red blood
cells comprises reporting numbers of nucleated red blood cells per
one hundred white blood cells.
4. The method of claim 1 wherein said measuring said blood sample
mixture by a DC impedance measurement further comprises counting
white blood cells in said blood sample mixture.
5. The method of claim 4 wherein said reporting nucleated red blood
cells comprises reporting numbers of nucleated red blood cells in
per unit volume of said blood sample.
6. The method of claim 1 wherein said measuring said blood sample
mixture by a DC impedance measurement is performed using a
non-focused flow aperture.
7. The method of claim 6 wherein said non-focused flow aperture has
an aperture aspect ratio of length versus width of 0.7 and
greater.
8. The method of claim 7 wherein said non-focused flow aperture has
an aperture aspect ratio of length versus width of 1.0 and
greater.
9. The method of claim 8 wherein said non-focused flow aperture has
an aperture aspect ratio of length versus width of about 1.2.
10. The method of claim 1 wherein said mixing a blood sample with a
lytic reagent comprises diluting said blood sample with a blood
diluent to form a diluted blood sample, and mixing said diluted
blood sample with said lytic reagent.
11. The method of claim 1 wherein said mixing a blood sample with a
lytic reagent comprises mixing said blood sample with a lytic
reagent containing a salt to simultaneously dilute and lyse said
blood sample.
12. The method of claim 1 further comprising measuring
spectrophotometric absorbance of said blood sample mixture at a
predetermined wavelength of a hemoglobin chromogen formed upon
lysing said blood sample, and reporting hemoglobin concentration of
said blood sample.
13. The method of claim 12 wherein said absorbance of said blood
sample mixture is measured between about 510 nm and about 560
nm.
14. A method of correcting white blood cell count comprising the
steps of: (a) mixing a blood sample with a lytic reagent to lyse
red blood cells, and to form a blood sample mixture, (b) measuring
said blood sample mixture by a DC impedance measurement to obtain a
blood cell distribution and a count of remaining blood cells, (c)
differentiating nucleated red blood cells and other interference
materials from white blood cells from obtained blood cell
distribution, (d) subtracting nucleated red blood cells and other
interference materials from said count of remaining blood cells,
and (e) reporting a corrected white blood cell count in said blood
sample.
15. The method of claim 14 further comprising reporting the
presence of nucleated red blood cells in said blood sample.
16. The method of claim 14 further comprising reporting numbers of
nucleated red blood cells in said blood sample.
17. The method of claim 14 wherein said measuring nucleated blood
cells is performed using a non-focused flow aperture.
18. The method of claim 17 wherein said non-focused flow aperture
has an aperture aspect ratio of length versus width of 0.7 and
greater.
19. The method of claim 18 wherein said non-focused flow aperture
has an aperture aspect ratio of length versus width of 1.0 and
greater.
20. A method of concurrently differentiating nucleated red blood
cells, enumerating white blood cells, and measuring hemoglobin
concentration of a blood sample comprising steps of: (a) mixing a
blood sample with a lytic reagent to lyse red blood cells, and to
form a blood sample mixture, (b) measuring said blood sample
mixture by a DC impedance measurement to obtain a blood cell
distribution and a count of remaining blood cells, (c)
differentiating nucleated red blood cells and other interference
materials from white blood cells from obtained blood cell
distribution, (d) subtracting nucleated red blood cells and other
interference materials from said count of remaining blood cells,
(e) measuring spectrophotometric absorbance of said blood sample
mixture at a predetermined wavelength of a hemoglobin chromogen
formed upon lysing said blood sample, (f) reporting said nucleated
red blood cells in said blood sample, (g) reporting numbers of
white blood cells in said blood sample, and (h) reporting a
hemoglobin concentration of said blood sample.
21. The method of claim 20 wherein said measuring nucleated blood
cells is performed using a non-focused flow aperture.
22. The method of claim 21 wherein said non-focused flow aperture
has an aperture aspect ratio of length versus width of 0.7 and
greater.
23. The method of claim 22 wherein said non-focused flow aperture
has an aperture aspect ratio of length versus width of 1.0 and
greater.
24. The method of claim 20 wherein said mixing a blood sample with
a lytic reagent comprises diluting said blood sample with a blood
diluent to form a diluted blood sample, and mixing said diluted
blood sample with said lytic reagent.
25. The method of claim 20 wherein said mixing a blood sample with
a lytic reagent comprises mixing said blood sample with a lytic
reagent containing salts to simultaneously dilute and lyse said
blood sample.
26. The method of claim 20 wherein said absorbance of said blood
sample mixture is measured between about 510 nm and about 560
nm.
27. A method of analyzing a nucleated blood cells in a blood sample
comprising the steps of: (a) mixing a blood sample with a lytic
reagent to lyse red blood cells, and to form a blood sample
mixture, (b) measuring said blood sample mixture by a DC impedance
measurement to obtain a blood cell distribution, (c)
differentiating nucleated blood cells using said blood cell
distribution obtained in step (b) to obtain an improved separation
among cell types using a non-focused flow aperture having an
aperture aspect ratio of length versus width of about 1.2 and
greater, and (d) reporting subpopulations of nucleated blood cells
in said blood sample.
28. The method of claim 27 wherein the subpopulations of nucleated
blood cells comprise nucleated red blood cells and white blood
cells.
29. The method of claim 27 wherein the subpopulations of nucleated
blood cells comprise subpopulations of white blood cells.
30. The method of claim 27 wherein said mixing a blood sample with
a lytic reagent comprises diluting said blood sample with a blood
diluent to form a diluted blood sample, and mixing said diluted
blood sample with said lytic reagent.
31. The method of claim 27 wherein said mixing a blood sample with
a lytic reagent comprises mixing said blood sample with a lytic
reagent containing a salt to simultaneously dilute and lyse said
blood sample.
32. The method of claim 27 further comprising measuring
spectrophotometric absorbance of said blood sample mixture at a
predetermined wavelength of a hemoglobin chromogen formed upon
lysing said blood sample, and reporting hemoglobin concentration of
said blood sample.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for determination
of nucleated red blood cells in a blood sample. More specifically
the method differentiates nucleated red blood cells from other cell
types using a direct current impedance measurement in a non-focused
flow aperture, and enumerates nucleated red blood cells in a blood
sample.
BACKGROUND OF THE INVENTION
[0002] Normal peripheral blood contains mature red blood cells
which are free of nucleus. Nucleated red blood cells (NRBCs), or
erythroblasts, are immature red blood cells. They normally occur in
the bone marrow but not in peripheral blood. However, in certain
diseases such as anemia and leukemia, NRBCs also occur in
peripheral blood. Therefore, it is of clinical importance to
measure NRBCs. Traditionally, differentiation and enumeration of
NRBC are performed manually. The process involves the smearing of a
blood sample on a microscope slide and staining the slide, followed
by manual visual analysis of the individual slide. The NRBC
concentration is reported as numbers of NRBC per 100 white blood
cells. Usually, 200 white blood cells and the numbers of NRBC
present in the same region on a blood smear are counted and the
numbers are divided by 2 to express the NRBC concentration as the
numbers of NRBC/100 WBC. This approach is extremely time-consuming
as well as being subjective to the interpretation of the individual
analyzing the slide.
[0003] In recent years, several fluorescence flow cytometry methods
have been developed for differentiating NRBCs. These methods
utilizes specific nuclear staining technique to distinguish NRBCs
from other cell types because it is difficult to differentiate
NRBCs based on their electronic or optical properties.
[0004] U.S. Pat. No. 5,298,426 (to Inami et al.) discloses a
fluorescence method for differentiating NRBCs. The method utilizes
a two-step staining using a first fluid and a second fluid. Inami
et al. teaches that the first fluid contains an
erythroblast-staining dye that diffuses into nucleated red blood
cells to specifically stain their nuclei, and then separating a
group of NRBCs from other cell groups on a two-dimensional plot
whereby the results of NRBC differentiation are computed.
[0005] U.S. Pat. No. 5,559,037 (to Kim et al.) discloses a method
for flow cytometric analysis of NRBCs and leukocytes. The method
comprises lysis of red blood cells and NRBC cytoplasm from a whole
blood sample to expose the NRBC nuclei to a vital nuclear stain and
minimizing the permeation of the vital nuclear stain into the
leukocytes and analyzing the sample by measuring fluorescence and
two angles of light scatter. This method features a triple
triggering method which blocks the signals from debris (fluorescent
and non-fluorescent) and identifies the signals which fall below
the ALL trigger but above the fluorescence trigger (FL3) as NRBCs.
ALL is the axial loss of light or the light scatter signals
detected at 0.degree. from the incident light. Therefore,
pre-gating signals in more than one dimension are required in this
method for identification of NRBC population. In addition, the
method requires heating of the reagent to 42.degree. C. in order to
obtain the NRBC and leukocyte differentiations.
[0006] U.S. Pat. No. 5,648,225 (to Kim et al) discloses a method of
using a multipurpose lysing reagent for subclassification of
nucleated blood cells. The method comprises the steps of lysing a
blood sample with the multipurpose lysing reagent which contains a
nuclear stain, incubating the sample mixture at an elevated
temperature, and determining the nucleated blood cells including
NRBCs with an automated electro-optical hematology
instrumentation.
[0007] U.S. Pat. No. 5,879,900 (to Kim et al) discloses a method of
differentiating NRBCs, damaged white blood cells (WBC), WBC and a
WBC differential in a blood sample by flow cytometry. The method
includes lysing a blood sample; staining NRBCs and any damaged
white blood cells with a vital nuclear stain; analyzing the sample
mixture by measuring at least one fluorescence, and at least one
light scatter signals in a range from 0.degree. to 10 and 30 to
10.degree.; constructing a three-dimensional plot from the
fluorescence and light scatter signals; and differentiating and
enumerating WBC, NRBC, damaged WBC and a WBC subclass
differential.
[0008] EP 1 004 880 A2 discloses reagents and a method for
discrimination and counting of nucleated red blood cells. The
method includes the steps of lysing red blood cells, staining white
blood cells and NRBCs, assaying the sample by measuring at least
one scattered light parameter, and at least one fluorescence
parameter.
[0009] U.S. Pat. No. 5,874,310 (to Li et al) discloses a method for
differentiation of nucleated red blood cells. The method includes
lysing mature red blood cells and analyzing the sample in a flow
cell by light scatter measurement to differentiate NRBCs from other
cell types. The light scatter measurement is performed by using two
low angle light scatter signals of less than 10.degree.. The method
further includes a concurrent differentiation of white blood cells
using electronic and optical analysis, wherein the electronic
analysis is a DC impedance measurement.
[0010] U.S. Pat. No. 5,917,584 (to Li et al) discloses a method for
differentiation of nucleated red blood cells. The method includes
lysing mature red blood cells in a blood sample; analyzing the
sample in a flow cell by two angles of light scatter measurement to
differentiate NRBCs from other cell types, wherein the second light
scatter signal is a medium angle or a right-angle light scatter
signal.
[0011] The above described methods enable differentiation and
enumeration of NRBCs and leukocytes by fluorescence flow cytometry
and light scatter measurements. However, fluorescence and light
scatter measurements are complex and expensive detection
methods.
[0012] Many current non-fluorescence automated hematology
analyzers, such as Abbott Cell-Dyn.RTM. 3500, COULTER.RTM.
Gen*S.TM., Bayer Advia*120.RTM., and Sysmex.TM. NE-9000 are only
able to provide NRBC flagging for the possible presence of NRBCs in
an analyzed blood sample when the instruments sense an increased
amount of signals near blood cell debris area of an obtained cell
distribution histogram. However, such techniques frequently
generate false positive flagging because many other blood
abnormalities can cause increased signals at the same area, such as
platelet clumps and sickle cells, as well as red cell debris from
insufficiently lysed blood samples. In these methods NRBCs are not
distinctly identified. Instead, only a common NRBC sample
distribution pattern in a histogram or a dotplot is recognized by
the instrument which can be confused with a similar pattern
generated by above-mentioned other causes.
[0013] Furthermore, a well known problem with NRBC containing
samples is erroneous white blood cell count (WBC) reported by
hematology analyzers on these samples. Since the nuclear volumes of
NRBC are close to those of white blood cells, and they are commonly
counted as white blood cells on hematology analyzers which measure
the sizes of blood cells, resulting an elevation of WBC. Therefore,
correction of NRBC contribution to the WBC reported from hematology
analyzer is required for samples containing NRBC. Current practice
in the clinical laboratory is to subtract the numbers of NRBC
obtained by manual count from the WBC count reported by the
hematology analyzers. This is time consuming and error prone.
[0014] On the other hand, measurement of hemoglobin (Hgb)
concentration of blood samples is an integral part of blood
analysis, which is important for disease diagnosis and for
monitoring responses to medical treatment. It is desirable to be
able to accomplish multiple diagnostic analyses such as enumerating
nucleated blood cells and measuring hemoglobin concentration of a
blood sample using the same reagent and concurrent
measurements.
[0015] Among the many well known methods for hemoglobin
determination, the cyanmethemoglobin method has been recommended as
a standard by the International Committee for Standardization in
Hematology. However, the presence of cyanide in the reagent waste
has caused enormous environmental concern. In last ten years, a
tremendous effort has been given to develop automated hemoglobin
analysis methods without utilizing cyanide.
[0016] U.S. Pat. No. 5,242,832 (to Sakata) discloses a method using
a cyanide-free lysing reagent for counting white blood cells and
measuring the hemoglobin concentration in blood samples.
PCT/US95/02897 (to Kim) discloses a cyanide-free method and reagent
for determining hemoglobin in a whole blood sample. No capability
of counting leukocytes, nor differentiating nucleated red blood
cells is taught by Kim. U.S. Pat. No. 5,763,280 and 5,882,934 (to
Li et al) disclose cyanide-free reagents for measuring hemoglobin
in a blood sample, counting leukocytes, and differentiating
leukocyte subpopulations. However, none of the above described
hemoglobin measurement methods enables differentiation of nucleated
red blood cells from other cell types.
[0017] Based on foregoing, there exists a need for a simple and
less costly analysis method for differentiating and enumerating
nucleated red blood cells. Furthermore, it is desirable to have a
multifunctional test method for enumeration of nucleated blood
cells, differentiation of nucleated red blood cells from other cell
types, and measurement of hemoglobin concentration in one
concurrent test.
SUMMARY OF THE INVENTION
[0018] In one embodiment, the present invention relates to a method
of differentiating nucleated red blood cells from other cell types
in a blood sample. The method comprises steps of mixing a blood
sample with a lytic reagent to lyse red blood cells, and to form a
blood sample mixture; measuring the blood sample mixture by a DC
impedance measurement in a non-focused flow aperture, and obtaining
a blood cell distribution of the blood sample mixture;
differentiating nucleated red blood cells from other cell types;
and reporting nucleated red blood cells in the blood sample. The
non-focused flow aperture has an aperture aspect ratio of 0.7 and
greater. Reporting nucleated red blood cells includes reporting the
presence of nucleated red blood cells in the blood sample, and
reporting numbers of nucleated red blood cells per one hundred of
white blood cells in the blood sample, or numbers of nucleated red
blood cells in an unit volume of the blood sample.
[0019] In a further embodiment, the present invention relates to a
method of correcting white blood cell count. The method comprises
the steps of mixing a blood sample with a lytic reagent to lyse red
blood cells, and to form a blood sample mixture; measuring the
blood sample mixture by a DC impedance measurement to obtain a
blood cell distribution and a count of remaining blood cells;
differentiating nucleated red blood cells and other interference
materials from white blood cells; subtracting nucleated red blood
cells and other interference materials from the count of remaining
blood cells; and reporting a corrected white blood cell count in
the blood sample. The method further comprises reporting nucleated
red blood cells in the blood sample.
[0020] In another embodiment, the present invention relates to a
method of concurrently differentiating nucleated red blood cells,
enumerating white blood cells, and measuring hemoglobin
concentration of a blood sample. The method comprises steps of
mixing a blood sample with a lytic reagent to lyse red blood cells,
and to form a blood sample mixture; measuring the blood sample
mixture by a DC impedance measurement to obtain a blood cell
distribution and a count of remaining blood cells; differentiating
nucleated red blood cells and other interference materials from
white blood cells; subtracting nucleated red blood cells and other
interference materials from the count of remaining blood cells;
measuring spectrophotometric absorbance of the blood sample mixture
at a predetermined wavelength of a hemoglobin chromogen formed upon
lysing the blood sample; reporting the nucleated red blood cells in
the blood sample; reporting numbers of white blood cells in the
blood sample; and reporting a hemoglobin concentration of the blood
sample.
BRIEF DESCRIPTION OF DRAWINGS
[0021] FIG. 1A, 1B and 1C are the DC histograms of a normal blood
sample and two clinical abnormal blood samples containing nucleated
red blood cells, respectively. The samples were processed according
to the procedure described in Example 1 and analyzed on an
experimental hematology analyzer using non-focused flow apertures
having a length of 120.mu. and a width of 100.mu..
[0022] FIG. 2A and 2B show histograms of a normal blood sample and
a clinical sample containing nucleated red blood cells. The samples
were processed according to the procedure described in Example 2
and analyzed on an experimental hematology analyzer using a
non-focused flow aperture having a length of 70.mu. and a width of
100.mu..
[0023] FIG. 3A and 3B show histograms of a clinical sample
containing nucleated red blood cells processed according to the
procedure described in Example 1 and analyzed on two experimental
hematology analyzers using non-focused flow apertures having a
length of 120.mu. and a width of 100.mu., and a length of 85.mu.
and a width of 70.mu., respectively.
[0024] FIG. 4A shows a histogram of a whole blood sample obtained
according to the procedure described in Example 4 using the lytic
reagent composition of Example 4. FIG. 4B shows a spectrum of the
same sample processed with the lytic reagent composition of Example
4 according to the procedure described in Example 4.
[0025] FIG. 5A shows the correlation of NRBC concentration obtained
by the method of the present invention described in Example 5 to
the manual reference results. FIG. 5B shows the correlation of the
hemoglobin concentration obtained using the method of the present
invention described in Example 5 to that obtained on COULTER Gen*S.
FIG. 5C shows the correlation of corrected WBC obtained using the
method of the present invention described in Example 5 to the
corrected WBC obtained by correcting the count obtained on COULTER
COUNTER.RTM. ZBI with manual NRBC count.
[0026] FIG. 6 shows a histogram of the present invention for
differentiating nucleated red blood cells from white blood cells
and further differentiating subpopulations of white blood cells
such as lymphoid and myeloid subpopulations.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The first embodiment of the present invention is directed to
a method for differential analysis of nucleated red blood cells.
More specifically the method enables differentiation of nucleated
red blood cells from other cell types in a blood sample by a direct
current impedance measurement with a non-focused flow aperture.
[0028] A method of differentiating nucleated red blood cells from
other cell types in a blood sample comprising steps of: mixing a
blood sample with a lytic reagent to lyse red blood cells, and to
form a blood sample mixture; measuring said blood sample mixture by
a DC impedance measurement in a non-focused flow aperture, and
obtaining a blood cell distribution of said blood sample mixture;
and differentiating nucleated red blood cells (NRBCs) from other
cell types.
[0029] To lyse a blood sample, the blood sample can be diluted
first by a blood diluent, then mixed with a sufficient amount of a
lytic reagent to lyse red blood cells. For the purpose of the
present invention, the blood diluent contains a sufficient amount
of salt or salts for impedance measurement of the sample mixture.
Suitable examples of salts are alkaline metal salts.
[0030] A blood diluent is commonly used on a hematology analyzer to
dilute a blood sample for measuring red blood cells, where the
blood diluent is adjusted to isotonic by salts for maintaining the
blood cell volumes. It is convenient to use commercially available
isotonic blood diluents for the purpose of the present invention,
although isotonicity is not required for differential analysis of
NRBCs.
[0031] A lytic reagent suitable to use with a blood diluent for the
present invention comprises an aqueous solution of:
[0032] (a) a quaternary ammonium salt or salts, represented by
following molecular structure: 1
[0033] wherein R.sub.1 is an alkyl, alkenyl or alkynyl group having
12 to 16 carbon atoms; R.sub.2, R.sub.3 and R.sub.4 are alkyl
groups having 1 to 4 carbon atoms and X.sup.- is chloride or
bromide anion;
[0034] (b) an ethoxylated alkyl phenol, wherein the alkyl group has
6 to 12 carbon atoms, and the number of ethylene oxide is in a
range from about 10 to about 50; and
[0035] (c) an ethoxylated alcohol represented by following
molecular structure:
R.sub.1--R.sub.2--(CH.sub.2CH.sub.2O).sub.n--H
[0036] wherein R.sub.1 is an alkyl, alkenyl or alkynyl group having
10 to 22 carbon atoms, R.sub.2 is --O--, and n is between 20 and
35.
[0037] Suitable examples of quaternary ammonium salts are
tetradecylammonium bromide, hexadecyltrimethylammonium bromide and
dodecyltrimethylammonium chloride. Suitable examples of ethoxylated
alcohol are Plurofac A38 prill surfactant, from BASF Corp., New
Jersey, and Hetoxol STA-30, from Heterene, Inc., New Jersey.
Suitable examples of ethoxylated phenol is Igepal SS-837, from
Rhne-Poulenc, New Jersey, and Chemax NP-30, from Chemax Inc., South
Carolina.
[0038] Alternatively, a lytic reagent further containing a
sufficient amount of a salt or salts for impedance measurement can
be used for lysing a blood sample without a separate blood diluent.
Suitable examples of salts are alkaline metal salts, such as
sulfates, chlorides, phosphates, and citrates.
[0039] The differential analysis of NRBCs is performed in a
non-focused flow aperture using DC impedance measurement. When a
particle, such as a blood cell, passes through the aperture, an
electrical signal can be measured due to conductivity or impedance
change. The pulse shape, height and width, is directly related to
the size of a particle, and can be converted to the size of the
particles measured. When two or more particles of different sizes
are measured, the histogram obtained from the measurement can
represent size distribution of the particles.
[0040] The detection methods used for blood cell counting and
sizing by a blood analyzer equipped with a DC impedance measurement
device are generally described in U.S. Pat. No. 2,656,508 (to
Coulter), and U.S. Pat. No. 3,810,011 (to Coulter, et al), which
are hereby incorporated by reference in its entirety.
[0041] It is found that the aperture aspect ratio, defined as a
ratio of the aperture length versus the aperture width, affects the
separation of different sizes of blood cells, in particular the
NRBC population from other nucleated blood cells. With the method
of the present invention, separation of the NRBC population from
the other cell types can be achieved by using an aperture aspect
ratio of 0.7 and greater.
[0042] It has been understood that aperture aspect ratio affects
the flow profile of a flow passing through the aperture, which in
turn, affects trajectory of particles in the flow. In general, with
a fixed aperture width, the rate of a flow at the center of the
flow increases with increasing the length of the aperture.
Therefore, with an increase of the aperture aspect ratio, a flow
rate gradient, from the sides of the flow which interface with the
wall of aperture toward center of the flow, increases. In the
presence of such a flow rate gradient, particles suspended in a
flow passing through the aperture tend to move to the center of the
flow. Therefore, under such a condition, particles have a similar
behavior to the particles passing through a focused flow aperture.
A focused flow aperture can be used in the present invention for
measuring nucleated blood cells, particularly for differentiating
blood cells having similar sizes. However, the cost of a focused
flow aperture is much higher than a non-focused flow aperture.
[0043] On the other hand, it is known that at a cross section of an
aperture, an imposed electrical field has a different strength
along the cross section. Consequently, the particles passing
through a non-focused flow aperture can generate various pulse
shapes because each particle may experience a different electrical
field depending on its position along the cross section of the
aperture. These pulse distortions cause distortion of the particle
size distribution in the measured histogram. Historically, pulse
editing has been broadly used in the art to edit out seriously
distorted pulses, and improve particle size differentiation to a
certain degree. It is understood that with increase of the aperture
aspect ratio, electrical field gradient along the cross section,
from the center to the side wall of the aperture, decreases.
Consequently, electrical pulses generated from the particles not
passing the center of the aperture have less distortions because of
the presence of a more homogeneous electrical field along the cross
section.
[0044] Therefore, with an increase of aperture aspect ratio the two
effects, control of particle trajectory in the non-focused flow
aperture and reduction of electrical field gradient along the cross
section of the aperture, improve ability of differentiation of
different sizes of particles.
[0045] It has been found using a non-focused flow aperture with an
aperture aspect ratio of 0.7 and greater, the NRBC population can
be differentiated from closely sized other nucleated blood cells,
particularly lymphocytes. Preferably, an aperture aspect ratio of
1.0 and greater is used. More preferably, an aperture aspect ratio
about 1.2 is used.
[0046] To further increase the aperture aspect ratio, the
separation of NRBC from other cell types can be further improved.
However, when the aperture aspect ratio is 1.5 or above, the
throughput of the sample mixture passing through the aperture for
measurement reduces significantly, and it can render the
measurement incompatible to the throughput requirement of a
hematology instrument. Therefore, it should be understood that an
aperture aspect ratio about 1.2 is selected based on a balance
between the population separation and throughput of the measurement
for a practical reason. Theoretically, an aperture aspect ratio
above 1.2 can be used for separating the NRBCs from other cell
types.
[0047] FIG. 1A, 1B and 1C show DC histograms of blood samples
processed according to the method of the present invention,
following the procedure described in Example 1, and analyzed on an
experimental hematology analyzer using non-focused flow apertures
having a length of 120.mu. and a width of 100.mu.. The aperture
aspect ratio is 1.2. FIG. 1A is a histogram of a normal blood
sample. As shown, a normal blood sample, after lysing the red blood
cells with a lytic reagent, exhibits a bi-module distribution of
the nucleated blood cells. In this case, all nucleated blood cells
are from the white blood cells. The major cell population in the
left peak is lymphocytes. On the left side of this peak, the area
of the histogram is clean.
[0048] FIG. 1B and 1C are histograms of two clinical abnormal blood
samples containing nucleated red blood cells. The blood samples
shown in FIG. 1B and 1C contain 230 NRBC/100 WBC and 9 NRBC/100
WBC, respectively, determined by the manual reference method. The
NRBC population shows as an additional peak on the left side of
white blood cells.
[0049] FIG. 2A and 2B show histograms of a normal blood sample and
a clinical sample containing nucleated red blood cells processed
according the procedure described in Example 2, and analyzed using
a non-focused flow aperture having a length of 70.mu. and a width
of 100.mu.. The aperture aspect ratio is 0.7. This experimental
hematology analyzer has a different signal amplification scale from
the analyzer used in Example 1. As shown, the NRBC population
appears on the left of white blood cells.
[0050] FIG. 3A and 3B show histograms of a clinical sample
containing nucleated red blood cells processed following the
procedure described in Example 1, and analyzed on two experimental
hematology analyzers, as described in Example 3. The histogram
shown in FIG. 3A was obtained using non-focused flow apertures
having a length of 120.mu. and a width of 100.mu.. The histogram
shown in FIG. 3B was obtained using non-focused flow apertures
having a length of 85.mu. and a width of 70.mu.. Although the two
types of apertures are substantially different in length and width,
they both have the same aperture aspect ratio of 1.2. Apparently,
the two histograms show a similar population distribution. In both
cases, differentiation of NRBC population from other nucleated
blood cells is achieved.
[0051] Using the method of the present invention, since NRBC
population is differentiated from other cell types, particularly
from the white blood cells, the presence of NRBC population in a
sample analyzed can be identified and reported. To report the
presence of clinically abnormal populations in a blood sample is
often called flagging on hematology analyzers, which is an
important feature for assisting clinical diagnosis.
[0052] Furthermore, using the method of the present invention the
NRBC population can be enumerated. When the threshold of a DC
detector is set below the size of NRBCs as shown in the figures,
the NRBC population can be enumerated together with white blood
cells. After differentiating the NRBC population from other cell
types based on the population distribution of an obtained DC
histogram, NRBC concentration of an analyzed sample can be
calculated. The NRBC concentration can be reported as the numbers
of NRBC per hundred of white blood cells (NRBC/100 WBC), which is
the same unit of the manual reference. Alternatively, the numbers
of NRBC can also be reported as an absolute number per unit volume
of a blood sample by multiplying the ratio with WBC count of the
blood sample.
[0053] In a further embodiment of the present invention, the method
can further comprise correction of white blood cell counts.
Historically, when white blood cells are counted using a direct
current impedance method, the NRBCs are counted, or partially
counted with white blood cells because they are not differentiated
from other nucleated blood cells. The interference caused by NRBCs
can result in elevated and erroneous white blood cell counts. With
the method of the present invention, upon differentiating the
NRBCs, the contribution of this population to the white blood cell
count can be subtracted from the total count of nucleated blood
cells.
[0054] Additionally, it is known that some blood samples are more
difficult to lyse than normal blood samples. In some cases, if
blood cell membranes do not dissolve sufficiently during sample
preparation to the sizes below the DC detection threshold, cell
debris can also interfere the white blood cell count. Usually, cell
debris has relatively small volumes, and it appears on the left of
NRBCs on the histogram.
[0055] However, with the method of the present invention the
interference materials can be better separated from the white blood
cells because of improved separations among different cell types,
and can be subsequently removed from the total count of the
remaining blood cells in the sample mixture. Therefore, a corrected
white blood cell count can be reported by the method.
[0056] The term of interference materials has a broad meaning
herein, which includes any particulate materials that are not white
blood cells, but measured in the sample mixture by the DC impedance
measurement during the measurement of white blood cells.
[0057] In an additional embodiment, the present invention also
relates to a method of concurrently differentiating nucleated red
blood cells, and measuring hemoglobin concentration of a blood
sample. The method comprises the steps of (a) mixing a blood sample
with a lytic reagent to lyse red blood cells, and to form a blood
sample mixture, (b) measuring the blood sample mixture by a DC
impedance measurement in a non-focused flow aperture, and obtaining
a blood cell distribution of the blood sample mixture, (c)
differentiating nucleated red blood cells from other cell types,
(d) measuring spectrophotometric absorbance of the blood sample
mixture at a predetermined wavelength of a hemoglobin chromogen
formed upon lysing the blood sample, (e) reporting the presence of
nucleated red blood cells, and (f) reporting hemoglobin
concentration of the blood sample. As described previously, the
method can further enumerate NRBCs and report numbers of NRBCs per
hundred white blood cells.
[0058] Furthermore, the method can further include steps of
obtaining a count of remaining blood cells by the DC impedance
method, subtracting nucleated red blood cells from the count of
remaining blood cells, and reporting numbers of white blood cells
in the blood sample.
[0059] The method of measuring nucleated blood cells by a DC
impedance measurement in a non-focused flow aperture, and
differentiating nucleated red blood cells from other cell types has
been described above. To concurrently measure hemoglobin
concentration of the blood sample, the lysing reagent comprises a
hemoglobin ligand which forms a stable hemoglobin chromogen upon
lysing the red blood cells. Suitable ligands include tetrazole and
derivatives, imidazole and its derivatives, alkaline salts of
benzoic acid and derivatives, and quinaldic acid. A description of
these ligands can be found in U.S. Pat. No. 5,763,280 (to Li et
al), and which is hereby incorporated by reference in its entirety.
Suitable examples of imidazole derivatives are methylimidazole, and
ethylimidazole. Alternatively, the hemoglobin ligands can be added
in a blood diluent if the diluent is used to dilute the blood
sample as described previously. Moreover, a lytic reagent
containing a salt or salts, which enables impedance measurement
without using a separate blood diluent, can also include the
hemoglobin ligands for the hemoglobin measurement. Example 4
illustrates such an example of the method that enables
differentiation of NRBCs and measuring hemoglobin concentration of
a blood sample using a single lytic reagent without a separate
blood diluent.
[0060] The total hemoglobin concentration of the blood sample can
be determined by measuring spectrophotometric absorption of the
blood sample mixture at a predetermined wavelength. The wavelengths
are different for different hemoglobin chromogens. With the ligands
described above, the absorptions can be measured between about 510
nm and about 560 nm.
[0061] The utility of the method of the present invention in
analyzing clinical samples is demonstrated in Example 5. Totally 95
normal and 74 clinical whole blood samples containing NRBCs were
analyzed on an experimental hematology analyzer with the instrument
configuration detailed in Example 1, using the lytic reagent
composition of Example 5 and Isoton.RTM. III as diluent. The same
samples were also analyzed by COULTER.RTM. GEN*S hematology
analyzer, and COULTER COUNTER.RTM. ZBI as references. The numbers
of NRBC per 100 WBC were obtained from a 500 cell manual count
following NCCLS standard method, and used as the reference.
[0062] The DC histograms obtained on the experimental hematology
analyzer were analyzed by an experimental algorithm to
differentiate the NRBCs from the white blood cells, and to report
the numbers of NRBC per 100 WBC. Then the NRBCs were subtracted
from the total nucleated cells counted to obtain a correct WBC
count.
[0063] FIG. 5A shows the results of NRBC enumeration, which
demonstrates a good linear correlation between the results obtained
using the method of the present invention and the manual
reference.
[0064] FIG. 5B shows the correlation between hemoglobin
concentration obtained by the method described in the example and
that obtained on COULTER.RTM. GEN*S. The results demonstrate an
excellent linear correlation for hemoglobin measurement.
[0065] FIG. 5C shows the correlation of the corrected WBC count
between the reference and the results obtained by the method of the
present invention. The reference results were obtained by
subtracting the manual NRBC results from the WBC count obtained on
the COULTER COUNTER.RTM. ZBI. As shown, the results obtained by the
method of the present invention correlate excellently to the
reference.
[0066] In a further aspect, the present invention is directed to an
apparatus for differentiating nucleated blood cells in a blood
sample. The apparatus comprises (a) means for mixing a blood sample
with a lytic reagent system to lyse red blood cells and to form a
blood cell sample mixture; (b) means for measuring the blood sample
mixture by a DC impedance measurement in a non-focused aperture,
and obtaining a blood cell distribution of the blood sample
mixture; and (c) means for differentiating nucleated red blood
cells from other cell types from obtained blood cell distribution.
The non-focused aperture has preferably an aperture aspect ratio of
1.0 and greater.
[0067] The following examples are illustrative of the invention and
are in no way to be interpreted as limiting the scope of the
invention, as defined in the claims. It will be understood that
various other ingredients and proportions may be employed, in
accordance with the proceeding disclosure.
EXAMPLE 1
[0068] A reagent of the following composition was used for lysing a
blood sample and for analyzing the nucleated blood cells.
1 tetradecyltrimethylammonium bromide 25.0 g Igepal SS-837 (from
Rhne-Poulenc) 15.0 g Plurofac A38 prill surfactant (from BASF
Corp.) 4.0 g distilled water adjusted to 1 liter pH 5.0
[0069] 28 .mu.l of a whole blood sample was aspirated by an
experimental hematology analyzer, diluted with 6 ml of Isoton.RTM.
III (Beckman Coulter, Inc., Miami, Fla.), then mixed with 1 ml of
above lytic reagent composition to lyse red blood cells. The sample
mixture was drawn through a set of three non-focused flow apertures
(arranged in parallel) by a constant vacuum. The apertures had a
length of 120.mu. and a width of 100.mu.. The nucleated blood cells
were counted by a DC impedance measurement, and a histogram of the
blood cells, after pulse editing, was also produced (averaged from
the measurements of three apertures).
[0070] FIG. 1A shows a histogram of a fresh normal blood obtained
following the above procedure, which shows a bi-module distribution
of the white blood cells. FIG. 1B and 1C show two clinical samples
measured following the above procedure. The clinical samples
contain 230 NRBC/100 WBC, and 9 NRBC/100 WBC, respectively. As
seen, a distinct population of NRBC appears on the left side of the
white blood cells. The NRBC population was differentiated from the
white blood cells, and the ratio between the NRBCs and white blood
cells (.times.100) was reported as the numbers of NRBC/100 WBC.
Alternatively, the NRBC can also be reported as absolute count in
the blood sample by incorporating the total count of white blood
cells.
EXAMPLE 2
[0071] FIG. 2A and 2B show two histograms of a normal blood sample
and a clinical sample containing NRBCs, respectively, processed
according the procedure described in Example 1, and measured on an
experimental hematology analyzer which had a single non-focused
flow aperture. The aperture had a length of 70.mu. and a width of
100.mu.. The aperture aspect ratio is 0.7. This experimental
hematology analyzer has a different signal amplification scale from
the analyzer used in Example 1. As shown, the NRBC population
appears on the left of white blood cells.
EXAMPLE 3
[0072] FIG. 3A and 3B show two histograms of a clinical sample
containing NRBCs processed following the procedure described in
Example 1, and measured on two experimental hematology analyzers
which used same reagents, and instrument configurations except that
one had non-focused flow apertures having a length of 120.mu. and a
width of 100.mu., and the other had non-focused flow apertures
having a length of 85.mu. and a width of 70.mu.. The histogram
shown in FIG. 3A was obtained using the non-focused flow aperture
having a length of 120.mu. and a width of 100.mu.. The histogram
shown in FIG. 3B was obtained using the non-focused flow apertures
having a length of 85.mu. and a width of 70.mu.. Although the two
types of apertures are substantially different in length and width,
they have the same aspect ratio of 1.2. Apparently, the two
histograms showed a similar population distribution. In both cases,
differentiation of NRBC population from other nucleated cells was
achieved.
EXAMPLE 4
[0073]
2 A reagent of the following composition was prepared.
tetradecyltrimethylammonium bromide 3.48 g Igepal SS-837 (from
Rhne-Poulenc) 2.09 g Plurofac A38 prill surfactant (from BASF
Corp.) 0.56 g tetrazole 0.28 g Na.sub.2SO.sub.4 7.94 g NaCl 3.46 g
Na.sub.2EDTA 0.09 g ADA 1.21 g antimicrobials 0.98 g BHT
(predissolved in ethanol) 0.01 g distilled water adjusted to 1
liter pH 5.8 Osmolality 312 mOsm
[0074] A clinical whole blood sample containing 31 NRBC/100 WBC
(determined by manual reference method) was analyzed on the
experimental hematology analyzer described in Example 1 with the
same instrument configuration, but using the above lytic reagent
composition as both the lysing reagent and the diluent. FIG. 4A
shows the obtained histogram, which illustrates differentiation of
NRBCs from white blood cells.
[0075] Additionally, 11.6 .mu.l of the same sample was diluted and
mixed by 2903 .mu.l of the above lytic reagent composition. The
photometric absorption spectrum of the sample was measured
immediately on a Beckman DU 7500 spectrophotometer. FIG. 4B is the
obtained spectrum. As shown, the hemoglobin concentration of the
sample can be measured between about 510 nm to about 560 nm.
EXAMPLE 5
[0076]
3 A reagent of the following composition was prepared.
tetradecyltrimethylammonium bromide 25.0 g Igepal SS-837 (from
Rhne-Poulenc) 15.0 g Plurofac A38 prill surfactant (from BASF
Corp.) 4.0 g tetrazole 2.0 g BHT (predissolved in ethanol) 0.04 g
distilled water adjusted to 1 liter pH 2.9
[0077] 95 normal and 74 clinical whole blood samples containing
NRBCs were analyzed on the experimental hematology analyzer
described in Example 1 except that the aperture had a length of 85
and a width of 70.mu.. The analyzer uses the above lytic reagent
and Isoton.RTM. III as diluent. The absorption of a sample mixture
was also measured at 525 nm on the analyzer immediately after
counting of the nucleated blood cells. Hemoglobin concentration of
the sample was reported by the analyzer. Then the same samples were
also analyzed by COULTER.RTM. GEN*S hematology analyzer, and
COULTER COUNTER.RTM. ZBI. The COULTER.RTM. GEN*S was operated under
its standard configuration according to manufacturer's manual,
using Lyse S.RTM. III diff (Beckman Coulter, Inc. Miami, Fla.) as
lysing reagent and Isoton.RTM. III as diluent. The white blood
cells of the blood samples were counted on COULTER COUNTER.RTM. ZBI
following NCCLS reference procedure for WBC count. The threshold on
COULTER COUNTER.RTM. ZBI was set at 7.5 to ensure nucleated red
blood cells being counted. A 500 cell manual count was obtained by
three medical technologists for all clinical samples as a reference
for nucleated red blood cells, and reported as numbers of nucleated
red blood cells observed per 100 WBC counted (no. NRBC/100
WBC).
[0078] The DC histogram obtained was analyzed by an experimental
algorithm to differentiate the NRBCs from the white blood cells,
and to report the numbers of NRBC per 100 WBC. Then the NRBCs were
subtracted from the total nucleated blood cells counted to obtain a
correct WBC count.
[0079] FIG. 5A shows the results of NRBC enumeration obtained
following the above process versus manual reference. The
correlation coefficients, slopes and intercepts of the regression
lines showed a good linear correlation for NRBC.
[0080] FIG. 5B shows the correlation between hemoglobin
concentration obtained on the experimental hematology analyzer and
that obtained on COULTER.RTM. GEN*S. The result demonstrates an
excellent linear correlation for hemoglobin concentration.
[0081] FIG. 5C shows the correlation between the corrected WBC
count obtained on the experimental hematology analyzer using
procedure described above, and obtained by subtracting the manual
NRBC results from the WBC count obtained on COULTER COUNTER.RTM.
ZBI. The results illustrate an excellent correlation between the
method of the present invention and the reference method.
[0082] Example 6 further demonstrates the present invention for
differentiating nucleated red blood cells from white blood cells
and further differentiating subpopulations of white blood cells
such as lymphoid and myeloid subpopulations.
EXAMPLE 6
[0083] A reagent of the following composition was used for lysing a
blood sample and for analyzing the nucleated blood cells.
4 tetradecyltrimethylammonium bromide 35.0 g Igepal SS-837 (from
Rhne-Poulenc) 12.8 g Sodium chloride 9.0 g Potassium cyanide 0.25 g
distilled water adjusted to 1 liter pH 9.3
[0084] 28 .mu.l of a whole blood sample was aspirated by an
experimental hematology analyzer, diluted with 6 ml of Isotono.RTM.
3E (Beckman Coulter, Inc., Miami, Fla.), then mixed with 1 ml of
above lytic reagent composition to lyse red blood cells. The sample
mixture was drawn through a set of three non-focused flow apertures
(arranged in parallel) by a constant vacuum. The apertures had a
length of 100.mu. and a width of 83.mu.. The nucleated blood cells
were counted by a DC impedance measurement, and a histogram of the
blood cells, after pulse editing, was also produced (averaged from
the measurements of three apertures).
[0085] FIG. 6 shows a histogram of a clinical blood sample
containing 4 NRBC/100 WBC obtained following the above procedure.
As shown, three nucleated blood cell types are differentiated one
from another. More specifically, the NRBC peak appears on the left
side of the white blood cells. The white blood cells further
differentiate into two subpopulations, lymphoid (lymphocytes)
population and myeloid (neutrophils, monocytes, eosinophils, and
basophils) population. This example illustrates utility of the
method of the present invention for differentiating nucleated red
blood cells from the white blood cells, and for differentiating
subpopulations of white blood cells.
* * * * *