U.S. patent number RE38,131 [Application Number 09/977,768] was granted by the patent office on 2003-06-03 for reagent for measurement of leukocytes and hemoglobin concentration in blood.
This patent grant is currently assigned to Sysmex Corporation. Invention is credited to Yukio Hamaguchi, Yoshiro Ikeuchi, Atsushi Shirakami, Kinya Uchihashi.
United States Patent |
RE38,131 |
Uchihashi , et al. |
June 3, 2003 |
Reagent for measurement of leukocytes and hemoglobin concentration
in blood
Abstract
A reagent for measurement of leukocytes and hemoglobin
concentration in the blood includes a cationic surfactant in an
amount sufficient to lyse erythrocytes and denature hemoglobin, at
least one of the following hemoglobin stabilizers: (a)
sulfosalicylic acid, or its salt, in an amount effective for
promoting the conversion of hemoglobin into methemoglobin, (b) 0.2
to 10.0 g/L of a water-soluble chelating agent having a nitrogen
atom and a carboxyl group, and (c) piperazine, or its salt, in an
amount effective for promoting the conversion of hemoglobin into
methemoglobin, and a buffer for maintaining pH at 4 to 6.
Inventors: |
Uchihashi; Kinya (Kakogawa,
JP), Ikeuchi; Yoshiro (Kobe, JP),
Shirakami; Atsushi (Miki, JP), Hamaguchi; Yukio
(Akashi, JP) |
Assignee: |
Sysmex Corporation (Kobe,
JP)
|
Family
ID: |
14310445 |
Appl.
No.: |
09/977,768 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
057250 |
Apr 8, 1998 |
05968832 |
Oct 19, 1999 |
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Foreign Application Priority Data
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Apr 18, 1997 [JP] |
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9-101808 |
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Current U.S.
Class: |
436/10;
252/408.1; 436/17; 436/174; 436/175; 436/176; 436/18; 436/63;
436/66; 436/8 |
Current CPC
Class: |
G01N
33/5094 (20130101); G01N 33/721 (20130101); Y10T
436/10 (20150115); Y10T 436/107497 (20150115); Y10T
436/2525 (20150115); Y10T 436/108331 (20150115); Y10T
436/101666 (20150115); Y10T 436/25125 (20150115); Y10T
436/25 (20150115) |
Current International
Class: |
G01N
33/50 (20060101); G01N 33/72 (20060101); G01N
031/00 (); G01N 033/72 () |
Field of
Search: |
;436/8,10,17,15,18,63,66,174,175,176 ;252/408.1 ;435/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 444 241 |
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Sep 1991 |
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EP |
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0 794 435 |
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Sep 1997 |
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EP |
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WO 84/04969 |
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Dec 1984 |
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WO |
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Primary Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Pearne & Gordon LLP
Claims
What is claimed is:
1. A reagent for measurement of leukocytes and hemoglobin
concentration in blood, said reagent consisting essentially of: (1)
a cationic surfactant in an amount sufficient to lyse erythrocytes
and denature hemoglobin, (2) at least one hemoglobin stabilizer
selected from the group consisting of the following (a), (b) and
(c): (a) sulfosalicylic acid, or its salt, in an amount effective
for promoting the conversion of hemoglobin into methemoglobin, (b)
0.2 to 10.0 g/L of a water-soluble chelating agent having a
nitrogen atom and a carboxyl group, and (c) piperazine, or its
salt, in an amount effective for promoting the conversion of
hemoglobin into methemoglobin, and (3) a buffer for maintaining pH
at 4 to 6.
2. The reagent as claimed in claim 1, wherein said water-soluble
chelating agent is a salt of EDTA.
3. The reagent as claimed in claim 1, wherein the amount effective
for promoting the conversion of hemoglobin into methemoglobin is
0.2 to 10.0 g/L for sulfosalicylic acid or its salt, and 0.2 to
10.0 g/L for piperazine or its salt.
4. A reagent for measurement of blood leukocyte counts and
hemoglobin concentrations by conversion to methemoglobin, said
reagent containing: (1) a cationic surfactant in an amount
effective to lyse erythrocytes and denature hemoglobin, (2) at
least one or more hemoglobin stabilizers present in a total amount
sufficient to convert the hemoglobin to methemoglobin, said at
least one or more stabilizers being selected from the group
consisting of the following (a), (b) and (c): (a) sulfosalicylic
acid or its salt, (b) 0.2 to 10.0 g/L of a water-soluble chelating
agent having a nitrogen atom and a carboxyl group, and (c)
piperazine or its salt, and (3) a buffer for maintaining pH at 4 to
6. .Iadd.
5. A reagent for measurement of blood leukocyte counts and
hemoglobin concentration in blood, said reagent consisting
essentially of: (1) one or more cationic surfactants in an amount
effective to lyse erythrocytes and denature hemoglobin, (2) 0.2 to
10.0 g/L of a water soluble chelating agent having a nitrogen atom
and a carboxyl group, and (3) a buffer for maintaining pH at 4 to
6..Iaddend..Iadd.
6. A reagent for measurement of blood leukocyte counts and
hemoglobin concentrations by conversion to methemoglobin, said
reagent comprising: (1) one or more cationic surfactants in an
amount effective to lyse erythrocytes and denature hemoglobin, (2)
0.2 to 10.0 g/L of a water soluble chelating agent having a
nitrogen atom and a carboxyl group, said chelating agent being
present in a total amount sufficient to convert the hemoglobin to
methemoglobin, and (3) a buffer for maintaining pH at 4 to
6..Iaddend.
Description
BACKGROUND OF THE INVENTION
This invention relates to a reagent for measurement of leukocytes
and hemoglobin concentration in a blood sample.
Measuring the leukocyte count and hemoglobin concentration in the
blood is very important for the clinical diagnosis of leukemia,
anemia, etc. or for monitoring the clinical course in patients.
Currently, the leukocyte count and hemoglobin concentration can be
measured in a short time by an automatic blood analyzer, and such
an analyzer is in widespread use.
Automatic blood analyzers can be roughly classified into an optical
detection system for detecting light scattering or fluorescence,
and an electric resistance detection system for detecting a change
in impedance occurring when particles pass through an aperture. The
latter system is superior in terms of ease of use. According to the
electric resistance detection system, the measurement of the
leukocyte count is performed by adding a hemolytic agent to a blood
sample to lyse erythrocytes and prepare a sample for leukocyte
measurement with only leukocytes left, and then flowing this sample
through a detector to detect a signal issued.
Normally, leukocytes in the peripheral blood include five types of
cells, lymphocyte, monocyte, neutrophil, eosinophil and basophil.
By formulating the hemolytic agent in a preferred manner, it
becomes possible to classify the leukocytes into 2 or 3
populations, or detect only leukocytes of a particular type. Thus,
leukocyte classification that has necessarily relied on microscopic
observation can be performed automatically in a short time. This
has reduced the burden on laboratory technicians, and has obviated
the need for special technique for laboratory examination.
To measure hemoglobin concentrations, on the other hand, it has
been adopted as the international standard method to convert
hemoglobin into cyanmethemoglobin by the use of Van Kampen reagent
containing a cyanogen compound, and measuring its absorbance at
about 541 nm. For an automatic blood cell analyzer, a method is
known which fragments erythrocytes, and denatures hemoglobin, with
a quaternary ammonium salt having surface activity, obtains a
cyanmethemoglobin-like absorption curve with the use of an alkali
cyanide, and determines hemoglobin as well as leukocytes. However,
harmful cyanogen compounds are contained in the reagents. These
reagents or the samples after measurement need to be subsequently
made atoxic before being discarded.
There is a known reagent which permits leukocyte counting without
using a cyanogen compound for the determination of the hemoglobin
concentration (U.S. Pat. No. 4,185,964). This reagent contains a
water-soluble quaternary ammonium salt having surface activity, and
a small amount of a polycarboxylic acid having up to about 8 carbon
atoms in an amount sufficient to inhibit the lysis of leukocytes.
Hemolytic agents which enable leukocytes to be classified into
three populations are also known. These hemolytic agents contain
quaternary ammonium salts and specific hemoglobin stabilizers
(Japanese Unexamined Patent Publication Nos. 3-137566, 3-252557 and
4-13969).
The above hemolytic agents can denature hemoglobin rapidly, but
have the drawback that the hemoglobin concentration markedly varies
with the liquid temperature of the blood sample. Particularly in
U.S. Pat. No. 4,185,964, the variations are great, since no
stabilizer is used. In Japanese Unexamined Patent Publication Nos.
3-137566, 3-252557 and 4-13969, specific hemoglobin stabilizers are
incorporated, so that the variations are reduced. However, when the
liquid temperature fluctuates, the hemoglobin concentration also
fluctuates. The stabilizing effect of these stabilizers is
insufficient in this case. To obtain stable measurements,
therefore, a unit for keeping the liquid temperature constant is
required. This is one of the factors leading to an increase in the
cost of the apparatus.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a reagent which
does not use a cyanogen compound, can measure the leukocyte count,
and can also measure the hemoglobin concentration stably even when
the liquid temperature of the sample varies.
The reagent for measurement of leukocytes and hemoglobin
concentration according to the present invention contains: (1) a
cationic surfactant in an amount sufficient to lyse erythrocytes
and denature hemoglobin, (2) at least one hemoglobin stabilizer
selected from the group consisting of the following (a), (b) and
(c): (a) sulfosalicylic acid, or its salt, in an amount effective
for promoting the conversion of hemoglobin into methemoglobin, (b)
0.2 to 10.0 g/L of a water-soluble chelating agent having a
nitrogen atom and a carboxyl group, and (c) piperazine, or its
salt, in an amount effective for promoting the conversion of
hemoglobin into methemoglobin, and (3) a buffer for maintaining pH
at 4 to 6.
The use of the reagent for measurement of leukocytes and hemoglobin
concentration according to the present invention enables leukocytes
and hemoglobin concentration to be measured simultaneously or
separately.
The reagent for measurement of leukocytes and hemoglobin
concentration according to the present invention contains a
cationic surfactant in an amount sufficient to lyse erythrocytes
and denature hemoglobin. The cationic surfactant preferably
includes at least one cationic surfactant of the quaternary
ammonium salt type or pyridinium salt type having the structure
indicated below.
Preferred examples are as follows: (a) Quaternary ammonium salts
##STR1## where R.sub.1 represents a C.sub.8 -C.sub.20 alkyl group,
alkenyl group or alkinyl group, R.sub.2, R.sub.3 and R.sub.4
represent, independently of each other, a C.sub.1 -C.sub.8 alkyl
group, alkenyl group or alkinyl group, and X.sup.- represents a
halogen ion. (b) Pyridinium salts ##STR2## where n denotes an
integer of 7 to 19, and X.sup.- represents a halogen ion.
The preferred concentration of the cationic surfactant used in the
reagent of the present invention is 0.1 to 15.0 g/L. The preferred
cationic surfactant is a quaternary ammonium salt, and the
particularly preferable concentration is 0.1 to 4.0 g/L. By
suitably combining the surfactants stated above, leukocytes can be
classified, for example, into lymphocytes and others, or into
lymphocytes, neutrophils and others.
The reagent for measurement of leukocytes and hemoglobin
concentration according to the present invention also contains a
hemoglobin stabilizer in an amount effective for promoting the
conversion of hemoglobin into methemoglobin. The stabilizer is at
least one member selected from the group consisting of (a)
sulfosalicylic acid or its salt, (b) a water-soluble chelating
agent having a nitrogen atom and a carboxyl group, and (c)
piperazine or its salt. Examples of the water-soluble chelating
agent are ethylenediaminetetraacetic acid or its salts,
diaminopropanoltetraacetic acid or its salts,
diaminopropanetetraacetic acid or its salts,
ethylenediaminediacetic acid or its salts, and
ethylenediaminedipropionic acid or its salts. Particularly
preferred are ethylenediaminetetraacetates.
The hemoglobin stabilizer is used in an amount effective for
promoting the conversion of hemoglobin into methemoglobin.
Normally, it is used in a preferred amount in the range of 0.2 to
10.0 g/L. A more preferable concentration differs depending on the
substance used. When sulfosalicylic acid or its salt is used, the
more preferable concentration is 0.2 to 2.0 g/L. When the
water-soluble chelating agent is used, a particularly preferred
concentration is 0.5 to 10 g/L for ethylenediaminetetraacetic acid
salt, 0.5 to 5 g/L for diaminopropanoltetraacetic acid or its salt,
0.5 to 5 g/L for diaminopropanetetraacetic acid or its salt, 0.5 to
5 g/L for ethylenediaminediacetic acid or its salt, or 0.5 to 5 g/L
for ethylenediaminedipropionic acid dihydrochloride. In the case of
piperazine or its salt, it is used preferably in a concentration of
0.5 to 5 g/L. When this stabilizer has a buffer action, it may be
used as part of the buffer.
These stabilizers are presumed to exhibit their stabilizing effect
by binding to the heme of methemoglobin denatured with the
aforementioned cationic surfactant or a combination of the cationic
surfactants.
The buffer is not restricted as long as it can maintain a pH at 4.0
to 6.0. Too low a pH makes leukocytes fragile, thereby adversely
affecting the measurement of the leukocyte count. Too high a pH, on
the other hand, results in poor stability of hemoglobin over time.
Concretely, known buffers can be used, such as Good buffer,
phosphate buffer, succinate buffer, and maleate-TRIS buffer. The
concentration of the buffer is 5 to 50 mM, preferably 15 to 30
mM.
The above-mentioned concentrations of the respective components are
the concentrations used when the blood is directly diluted with the
reagent for measurement of leukocytes and hemoglobin concentration
according to the present invention. Alternatively, the reagent may
be added after the blood is diluted with a diluent (e.g.,
physiological saline solution or Cellpack.TM. (TOA MEDICAL
ELECTRONICS CO., LTD.)). In this case, it suffices to adjust the
concentrations of the respective components so that after addition
of the reagent, these concentrations would fall into the indicated
ranges. The above diluent is preferably one with a pH close to
neutrality (6 to 8) and an osmotic pressure close to isotonicity
(240 to 330 Osm/kg) for maintaining the shape of the blood
cells.
The reagent of the present invention has electric conductivity,
preferably, of 8 to 20 mS/cm, and more preferably, of 10 to 15
mS/cm. The electric conductivity can be adjusted by suitably adding
an electrolyte such as NaCl.
The leukocyte count and the hemoglobin concentration can be
measured in the following manner: A blood sample is prepared using
the reagent for measurement of leukocytes and hemoglobin
concentration according to the present invention. The blood sample
is introduced into an automatic blood cell analyzer. In this
apparatus, a change in impedance occurring during passage of
particles through an aperture is detected to measure the leukocyte
count, and the absorbance of the sample is detected to measure the
hemoglobin concentration. In this case, the leukocyte count and the
hemoglobin concentration can be measured simultaneously or
separately.
Formulation examples of the reagent of the present invention are
offered below.
FORMULATION EXAMPLE 1
Cetyltrimethylammonium chloride 0.3 to 3.0 g/L EDTA-2K 0.5 to 5.0
g/L Phosphate buffer 15 to 30 mM NaCl Proper amount Purified water
1 L
FORMULATION EXAMPLE 2
Lauryltrimethylammonium chloride 0.3 to 10.0 g/L
Stearyltrimethylammonium chloride 0.1 to 1.0 g/L EDTA-2K 0.5 to 5.0
g/L Good buffer 15 to 30 mM NaCl Proper amount Purified water 1
L
FORMULATION EXAMPLE 3
Lauryltrimethylammonium chloride 0.3 to 10.0 g/L
Lauryldimethylaminoacetate betaine 0.5 to 5.0 g/L EDTA-2K 0.5 to
5.0 g/L Succinate buffer 15 to 30 mM NaCl Proper amount Purified
water 1 L
With Formulation Examples 1 to 3, leukocyte and hemoglobin can be
measured simultaneously. By adjusting the type and concentration of
each surfactant, leukocytes can be classified into two or more
populations. The populations of leukocytes refer, for example, to
lymphocytes, monocytes, neutrophils, eosinophils and basophils.
Formulation Example 1 is a combination of the components designed
to classify leukocytes into a single population, while Formulation
Examples 2 and 3 are combinations of the components designed to
classify leukocytes into two or three populations.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the correlation between the hemoglobin
concentration measured by the use of the reagent of the present
invention and the hemoglobin concentration measured by a
conventional method;
FIG. 2 is a graph showing the correlation between the leukocyte
count measured by the use of the reagent of the present invention
and the leukocyte count measured by a conventional method;
FIG. 3 is a graph showing the correlation between the W-SCR
measured by the use of the reagent of Example 2 and the W-SCR
measured by a conventional method;
FIG. 4 is a graph showing the correlation between the W-MCR
measured by the use of the reagent of Example 2 and the W-MCR
measured by a conventional method;
FIG. 5 is a graph showing the correlation between the W-LCR
measured by the use of the reagent of Example 2 and the W-LCR
measured by a conventional method;
FIG. 6 is a graph showing the particle size distribution of
leukocytes measured by the use of the reagent of Example 2; and
FIG. 7 is a graph showing the particle size distribution of
leukocytes measured by the use of the reagent of Example 3.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention will now be described by, but not restricted
to, the following Examples.
EXAMPLE 1
Stability to Changes in Temperature
The reagent of the present invention and reagents as Controls 1 and
2 to be described below were prepared. Each of the reagents was
reacted with a patient's sample at a different temperature, and the
hemoglobin concentration was measured.
Reagent of the Present Invention Lauryltrimethylammonium chloride
3.0 g/L Stearyltrimethylammonium chloride 0.2 g/L EDTA-2K 1.0 g/L
phosphate buffer 20 mM (pH 5.0) NaCl Proper amount (the proper
amount is such an amount that the electric conductivity is about 13
mS/cm) Purified water 1 L Control 1 Lauryltrimethylammonium
chloride 3.0 g/L Stearyltrimethylammonium chloride 0.2 g/L
Phosphate buffer 20 mM NaCl Proper amount (the proper amount is
such an amount that the electric conductivity is about 13 mS/cm)
Purified water 1 L Control 2 Lauryltrimethylammonium chloride 3.0
g/L Stearyltrimethylammonium chloride 0.2 g/L Imidazole 1.0 g/L
Tris-maleate buffer (such an amount that the electric conductivity
is about 13 mS/cm) Purified water 1 L
The blood was diluted 1:500 with each reagent of the above
formulation, and reacted for 30 seconds at 10.degree. C. and
35.degree. C. Then, the absorbances were measured with a
spectrophotometer, and the change rate of the absorbance at
35.degree. C. to the absorbance at 10.degree. C. was calculated.
From this change rate, the amount of changed hemoglobin (converted
value) was determined. The results are shown in Table 1.
TABLE 1 Amount of changed hemoglobin Reagent Change rate (converted
value) Present invention -3.50% -0.53 g/dl Control 1 -15.7% -2.34
g/dl Control 2 -6.1% -0.91 g/dl Change rate = Absorbance at
35.degree. C./absorbance at 10.degree. C.
The reagent as Control 1 had the composition of the reagent of the
present invention from which the stabilizer was removed. This
reagent had a high change rate due to temperature. The reagent of
the present invention contained EDTA-2K as the stabilizer, while
Control 2 contained imidazole as the stabilizer. Both reagents were
lower in change rate than in Control 1 free from the
stabilizer.
A stabilizer other than these substances was usable, but the degree
of the change rate differed according to the type of the stabilizer
used.
EXAMPLE 2
Correlation with a Conventional Method
Composition of reagent Lauryltrimethylammonium chloride 8.7 g/L
Stearyltrimethylammonium chloride 0.8 g/L EDTA-2K 3.0 g/L Phosphate
buffer 60 mM (pH 5.0) NaCl Such an amount that the electric
conductivity is about 13 mS/cm Purified water 1 L
1) Correlation in Hemoglobin Concentration
The hemoglobin concentrations in 113 samples were measured with the
use of the above reagent, Cellpack.TM. (TOA MEDICAL ELECTRONICS
CO., LTD.) as a diluent, and K-4500 (TOA MEDICAL ELECTRONICS CO.,
LTD.). A graph was drawn to show the correlation of the results
with the hemoglobin concentrations measured by a denatured
cyanmethemoglobin method as a control method (an improvement on the
international standard method for use with an automatic blood cell
analyzer; using Stromatolyzer C (TOA MEDICAL ELECTRONICS CO., LTD.)
and making measurement by means of K-4500) (FIG. 1). With this
apparatus, the blood is diluted with the diluent, and then mixed
with the above reagent. Finally, the blood is diluted 1:500, and
the concentrations of the cationic surfactants, EDTA-2K and
phosphate buffer in the reagent drop to 1/3. NaCl is contained in
the diluent as well, and the electric conductivity is almost
unchanged. In the drawing, the X axis represents the conventional
method, while the Y axis represents the method using the reagent of
the present invention. A high correlation was obtained (correlation
coefficient r=0.997, regression line Y=0.951X+0.588), confirming
that hemoglobin was measurable using the reagent of the present
invention.
2) Correlation in Leukocytes
The leukocyte counts were measured in 113 samples with the use of
the above reagent, Cellpack.TM. (TOA MEDICAL ELECTRONICS CO., LTD.)
as a diluent, and K-4500 (TOA MEDICAL ELECTRONICS CO., LTD.). A
graph was drawn for showing the correlation of the results with the
leukocyte counts measured by a control method using Stromatolyzer
3WP.TM. (TOA MEDICAL ELECTRONICS CO., LTD.) and making measurement
by means of K-4500 (FIG. 2). A high correlation was obtained
(correlation coefficient r=0.997, regression line Y=0.986X+1.137),
confirming that leukocytes were measurable as with the conventional
method.
The differential leukocyte counts were also studied in 51 samples,
and as high a correlation as with the conventional method was
obtained. The results are shown in FIGS. 3 to 5. The W-SCR
represents the ratio of small leukocytes after addition of a
hemolytic agent, and this ratio corresponds to the ratio of
lymphocytes. The W-LCR represents the ratio of large leukocytes
after addition of a hemolytic agent, corresponding to the ratio of
neutrophils. The W-MCR represents the ratio of medium-sized
leukocytes after addition of a hemolytic agent, corresponding to
the ratio of other leukocytes. A graph showing the particle size
distribution of the leukocytes is given as FIG. 6.
EXAMPLE 3
Reagent for Classification of Leukocytes into Two Types
Composition of reagent Lauryltrimethylammonium chloride 6.1 g/L
Stearyltrimethylammonium chloride 1.1 g/L EDTA-2K 3.0 g/L Phosphate
buffer 60 mM (pH 5.0) NaCl Such an amount that the electric
conductivity is about 13 mS/cm) Purified water 1 L
Leukocytes were measured with the use of the above reagent,
Cellpack.TM. (TOA MEDICAL ELECTRONICS CO., LTD.) as a diluent, and
K-4500 (TOA MEDICAL ELECTRONICS CO., LTD.). It was confirmed that
this method was able to classify leukocytes into two populations. A
graph showing the particle size distribution of the leukocytes is
given as FIG. 7. In the drawing, the population with a smaller
particle size corresponds to lymphocytes, while the population with
a larger particle size corresponds to other leukocytes.
As noted above, according to the present invention, the leukocyte
count and the hemoglobin concentration can be measured stably even
when the liquid temperature of the blood sample is changed. Thus, a
unit for keeping the liquid temperature of the blood sample
constant is not required, and the cost of an analyzer can be
reduced
Also, the leukocyte count and the hemoglobin concentration can be
measured without the use of a cyanogen compound, so that a special
procedure for liquid waste treatment becomes unnecessary.
* * * * *