U.S. patent application number 10/795710 was filed with the patent office on 2004-09-02 for method for counting leukocytes and leukocyte counter.
This patent application is currently assigned to ARKRAY, INC.. Invention is credited to Okubo, Akio, Yamada, Shigeki.
Application Number | 20040171088 10/795710 |
Document ID | / |
Family ID | 13233619 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171088 |
Kind Code |
A1 |
Okubo, Akio ; et
al. |
September 2, 2004 |
Method for counting leukocytes and leukocyte counter
Abstract
A method for counting leukocytes which comprises liberating
elastase from granulocytes contained in a specimen, adding an
anti-granulocyte elastase antibody to the thus liberated elastase,
measuring the antibody bonded to the elastase to thereby determine
the concentration of the elastase, and then calculating therefrom
the number of leukocytes contained in the specimen with the use of
the ratio of the leukocyte count to the elastase concentration.
This method makes it possible to conveniently and less expensively
count leukocytes at a high accuracy comparable to the one
established by using a conventional automatic blood cell counter,
as the figure shows.
Inventors: |
Okubo, Akio; (Kyoto, JP)
; Yamada, Shigeki; (Kyoto, JP) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
ARKRAY, INC.
Kyoto
JP
|
Family ID: |
13233619 |
Appl. No.: |
10/795710 |
Filed: |
March 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10795710 |
Mar 8, 2004 |
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09623582 |
Aug 31, 2000 |
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6743591 |
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09623582 |
Aug 31, 2000 |
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PCT/JP99/01202 |
Mar 11, 1999 |
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Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 2333/966 20130101 |
Class at
Publication: |
435/007.21 |
International
Class: |
G01N 033/567 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 13, 1998 |
JP |
10/63589 |
Claims
1. A method for counting leukocytes comprising: liberating elastase
from neutrophils, eosionphils and basophils (hereafter, these three
blood cells are referred to as "granulocytes") in leukocytes
contained in a specimen; adding an antigranulocyte elastase
antibody to the thus liberated elastase; measuring the
antigranulocyte elastase antibody bonded to the elastase to thereby
determine the concentration of the elastase; and calculating the
number of leukocytes contained in the specimen from the thus
determined concentration of elastase by using a known ratio of the
leukocyte count, the granulocyte count or the neutrophil count to
the concentration of elastase.
2. The method according to claim 1, comprising: bonding the same
protease inhibitor as a protease inhibitor contained in the
specimen to the liberated elastase, and then adding an
antigranulocyte elastase antibody to the elastase.
3. The method according to claim 2, wherein the specimen is whole
blood and the protease inhibitor is .alpha.1-antitrypsin.
4. The method according to claim 2 or 3, wherein the protease
inhibitor is added in the range from 5 to 10 times weight as the
granulocyte elastase.
5. The method according to any one of claims 1 to 3, comprising
causing an antigen-antibody reaction between an antibody particle
in which the antigranulocyte elastase antibody is bonded to a latex
particle and the elastase liberated from granulocytes, and
determining the degree of agglutination of the latex particle to
thereby measure the antigranulocyte elastase antibody bonded to the
elastase.
6. The method according to claim 5, wherein the antibody particle
in which the antigranulocyte elastase antibody is bonded to the
latex particle is added at the rate of 0.1 to 5 mg with respect to
1 ml of the specimen.
7. The method according to any one of claims 1 to 3, comprising
bonding the elastase liberated from granulocytes to the
antigranulocyte elastase antibody bonded to a solid phase; adding a
labeled antigranulocyte elastase antibody therein in this state;
and measuring the labeled antigranulocyte elastase antibody bonded
to the elastase.
8. The method according to claim 7, wherein a label of the labeled
antigranulocyte elastase antibody is at least one label selected
from the group consisting of an enzyme label, a radioactive label
and a fluorescent label.
9. The method according to claim 7, wherein the label of the
labeled antigranulocyte elastase antibody is an enzyme and a
detectable product is produced by a reaction between the enzyme and
its substrate.
10. The method according to claim 9, wherein the enzyme is
peroxidase and its substrate is orthophenylene diamine and hydrogen
peroxide.
11. The method according to any one of claims 1 to 3, wherein the
known ratio is a statistical ratio of the leukocyte count or the
granulocyte count to the elastase concentration.
12. The method according to any one of claims 1 to 3, wherein the
known ratio is the elastase concentration with respect to one
granulocyte cell.
13. The method according claim 12, wherein the elastase
concentration with respect to one granulocyte cell is in the range
from 1 to 5 ng/ml.
14. The method according to claim 12, wherein the number of whole
leukocytes is calculated from the number of granulocytes by using a
known ratio of a granulocyte count to the number of whole
leukocytes.
15. A leukocyte counter used for performing the method for counting
leukocytes according to claim 1, comprising a means for calculating
the number of leukocytes in a specimen from the concentration of
elastase liberated from granulocytes in the specimen by using a
known ratio of a leukocyte count, a granulocyte count or a
neutrophil count to the concentration of elastase.
16. The leukocyte counter according to claim 15, comprising a means
for liberating elastase from granulocytes in the specimen, a means
for adding an antigranulocyte elastase antibody to the thus
liberated elastase, a means for measuring the antigranulocyte
elastase antibody bonded to the elastase to thereby determine the
concentration of elastase; and a means for calculating the number
of leukocytes contained in the specimen from the determined
concentration of elastase by using a known ratio of a leukocyte
count, a granulocyte count or a neutrophil count to the
concentration of elastase.
17. A reagent kit used for the method for counting leukocytes
according to claim 1, including an antigranulocyte elastase
antibody.
18. A reagent kit used for the method for counting leukocytes
according to claim 5, including an antibody particle in which an
antigranulocyte elastase antibody is bonded to a latex
particle.
19. A reagent kit used for the method for counting leukocytes
according to claim 7, including a labeled antigranulocyte elastase
antibody and an antigranulocyte elastase antibody bonded to a solid
phase.
20. A reagent kit according to any one of claims 17 to 19,
including the same protease inhibitor as the protease inhibitor
contained in a specimen to be determined.
21. The reagent kit according to claim 20, wherein the specimen to
be determined is whole blood and the protease inhibitor is
.alpha.1-antitrypsin.
22. The reagent kit according to any one of claims 17 to 19,
further including a lysing material.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for counting
leukocytes contained in a specimen and to a leukocyte counter used
therefor.
BACKGROUND ART
[0002] In a blood test, blood cell counting is frequently carried
out and its clinical significance is great. For example, an
erythrocyte count permits examining the presence and the degree of
anemia and further permits clinical diagnosis of various cases
caused by oxygen deficiency in the tissue. On the other hand, a
leukocyte plays an important role in a defense function of fighting
bacteria or virus entering the body, namely, an immunological
function. In particular, granulocytes or leukocytes have a function
of liberating protease such as elastase, etc. by the stimulation of
bacteria or foreign proteins entering the body, and a function of
allowing elastase etc., to respond the bacteria or foreign
proteins. Since the leukocyte count is increased or decreased in
the case of many diseases, it is important to count leukocytes in a
screening for diseases. In such diseases as require an early
determination or cure, the leukocyte counting is a particularly
important examination item. Furthermore, since the leukocyte count
fluctuates in accordance with ages of subjects, daytime or
night-time, seasons, and other factors, the leukocyte count is
therefore desired to be determined in routine medical treatments
appropriately and accurately.
[0003] At present, a method for counting blood cells is crudely
classified into two types, i.e., a visual counting method and an
automatic counting method. In the visual counting method, blood
cells are counted by microscopic examination on a calculating
board. The visual counting method includes a technique for counting
blood cells without any treatment and a technique for counting
blood cells after staining nuclei of blood cells with dye. In the
automatic counting method, blood is diluted to a certain amount and
then allowed to pass through a thin flow passage so as to detect
and count blood cells by using electrical resistance or scattered
light. In this method, a specific blood cell counter is generally
used. In these counting methods, blood cells themselves are counted
actually either by human eyes or by using a counter. However, these
methods have the below mentioned problems.
DISCLOSURE OF INVENTION
[0004] In the above-mentioned visual counting method, an error in
counting value occurs because of mistake about the kind of cells
caused by erythrocytes being deficient in hemolysis or the
hemagglutination of blood cells, in homogeneous broadening of
leukocytes on the calculating board, or the skill of laboratory
technicians. Furthermore, in the visible counting method, a
nucleated cell other than a leukocyte, for example, an erythroblast
cell, etc. is counted as a leukocyte. Therefore, when the nucleated
cell other than a leukocyte is present in peripheral blood, it is
necessary to analyze the ratio of leukocytes to the other cells by
using a blood smear preparation and to correct the value counted.
Moreover, the visual counting method is a manual method, and
therefore it takes a long time to count cells.
[0005] In the automatic counting method, the exact counted value
may not be obtained because of the appearance of erythroblast
cells, deposition of fibrin, platelet agglutination, deficiency in
hemolysis of erythrocytes, and the like. Moreover, some measurement
devices permit classifying the counted results count by a grain
size distribution measurement function or a pattern recognition.
Such devices make it possible to detect diseases easily. However,
such devices are expensive and large. Not only such special devices
but also general devices used for counting blood cells in the
automatic counting method are large and expensive. Furthermore, the
maintenance is complicated, for example, cleaning of a liquid flow
passage, etc. is complex. Therefore, such devices are effective for
such institutes as laboratories in large-scaled hospitals or
examination centers, etc., which deal with a large number of
specimens. However, for medium size hospitals or practitioners,
etc. which deal with a small number of specimens, the use and the
maintenance of the large-size device is a heavy burden.
[0006] It is therefore an object of the present invention to
provide a method for counting leukocytes capable of counting
leukocytes accurately and conveniently by using a small-size and
inexpensive device and a leukocyte counter used therefor.
[0007] In order to achieve the above-mentioned object, a method for
counting leukocytes of the present invention includes: liberating
elastase from neutrophils, eosionphils and basophils (hereafter,
these three blood cells are referred to as "granulocytes") in
leukocytes contained in a specimen; adding an antigranulocyte
elastase antibody to the thus liberated elastase; measuring the
antigranulocyte elastase antibody bonded to the elastase to thereby
determine the concentration of the elastase; and calculating the
number of leukocytes contained in the specimen from the thus
determined concentration of elastase by using a known ratio of the
leukocyte count, the granulocyte count or the neutrophil count to
the concentration of elastase.
[0008] Thus, the counting method of the present invention is a
method for indirectly calculating the number of leukocytes by
measuring the concentration of elastase contained in the
granulocyte instead of actually counting leukocytes.
[0009] There are five classes of leukocytes, i.e., neutrophils,
eosinophils, basophils, monocytes and lymphocytes. Three cells,
i.e. neutrophils, eosinophils, basophils in all are called
granulocytes. Moreover, the leukocyte count and the percentage of
each class of leukocyte (a differential leukocyte count) in blood
are not different between sexes and are substantially constant,
although the percentage of each class of leukocyte is somewhat
different between children and aged people. For example, in adult
blood, the leukocyte count is about 6700 cells/.mu.l and the
average percentage of each class of leukocyte is; neutrophil 55.3%,
eosinophil 3.5%, basophil 0.5%, monocyte 5.0% and lymphocyte 36.6%.
Furthermore, the granulocyte is the largest in number among
leukocytes and contains a large amount of elastase inside.
Granulocyte elastase is liberated when the granulocyte is exposed
to a stimulation or a damage by phagocytes or inflammation, thus
responding to lesion. In blood, the above-mentioned elastase is
present in its deactivated state by bonding to a protease inhibitor
such as .alpha.1-antitrypsin, or the like. The average retention
time in blood is about 10 hours. Once the elastase is excreted into
the tissue or urine, it does not return to the circulating blood
again. Furthermore, the elastase concentration in the plasma is
generally about one-several hundredth of the elastase concentration
in granulocyte and is substantially negligible. Furthermore, the
granulocyte elastase is different from a elastase derived from the
pancreas and an antibody for the granulocyte elastase does not
cross-react with the elastase derived from the pancreas. The
present invention uses such principles. More specifically,
granulocytes are lysed so as to liberate the elastase from the
granulocyte, and then the concentration of the liberated elastase
is measured by an immunological technique. Consequently, even if
the specimen is blood, the concentration can be measured exactly.
From the measurement value, the number of leukocytes in the
specimen can be calculated based on a known ratio of the leukocyte
count, etc. to the elastase concentration. Thus, since the
leukocytes are counted by using the concentration of elastase in
granulocytes, the counting method is not affected by a nucleated
cell other than a leukocyte, for example, an erythroblast cell,
etc.; the deposition of fibrin; platelet agglutination; and
deficiency in hemolysis of erythrocytes. Furthermore, the counter
used for the method can be miniaturized, the cost for counting can
be reduced and furthermore the counted value is not affected by the
skill of laboratory technician because the immunological techniques
is used.
[0010] In the present invention, the leukocytes denote both
individual leukocytes, i.e. neutrophils, eosinophils, basophils,
monocytes and lymphocytes, and whole leukocytes. Therefore, in the
present invention, the number of individual blood cells
constituting leukocytes, such as granulocyte including neutrophil,
eosinophil and basophil, etc. may be counted, and the number of
whole leukocytes may be counted.
[0011] It is preferable that the method of the present invention
includes bonding the same protease inhibitor as a protease
inhibitor contained in the specimen to the liberated elastase, and
then adding an antigranulocyte elastase antibody into the specimen.
With such a preferable embodiment, when a protease inhibitor is
contained in the specimen such as blood etc., the effect by the
protease inhibitor can be eliminated. Therefore, it is preferable
that when the specimen is whole blood, the protease inhibitor is
.alpha.1-antitrypsin, because .alpha.1-antitrypsin is the largest
in amount (about 90%) among the protease inhibitors in blood.
[0012] In the counting method of the present invention, it is
preferable that a latex agglutination immunoassay be employed as
the immunological technique. More specifically, it is preferable
that the method of the present invention includes causing an
antigen-antibody reaction between an antibody particle in which the
antigranulocyte elastase antibody is bonded to a latex particle and
the elastase liberated from granulocytes, and determining the
degree of agglutination of the latex particle to thereby measure
the antigranulocyte elastase antibody bonded to the elastase. The
latex agglutination immunoassay is preferred because it is a
homogeneous immunoassay in which an antigen-antibody reaction is
fast, and a B/F separation is not required, and therefore, it
includes only a few steps so as to be carried out conveniently and
at low cost. In addition, the method of the present invention
includes bonding the elastase liberated from granulocytes to the
antigranulocyte elastase antibody bonded to a solid phase; adding a
labeled antigranulocyte elastase antibody therein in this state;
and measuring the labeled antigranulocyte elastase antibody bonded
to the elastase. Depending upon the labels, the immunological
technique may be an enzyme immunoassay, a radioimmunoassay, a
fluorescence immunoassay, and the like.
[0013] In the counting method of the present invention, as the
known ratios, for example, the following first and second ratios
are preferably used.
[0014] The first ratio is a statistical ratio of the leukocyte
count or the granulocyte count to the elastase concentration. This
statistical ratio includes, for example, a regression linear
expression, etc. obtained by separately measured values of the
elastase concentration and the number of leukocytes, etc. in the
specimen such as blood. The regression linear expression includes a
regression linear expression between the elastase concentration and
the granulocyte count, a regression linear expression between the
elastase concentration and the number of whole leukocytes, and the
like.
[0015] The second ratio is the elastase concentration with respect
to one granulocyte cell. From this ratio, the number of
granulocytes contained in the specimen can be calculated. The
elastase concentration with respect to one granulocyte cell is
generally 1 to 5 ng/ml, preferably 2 to 2.5 ng/ml. Therefore, the
number of whole leukocytes in the specimen may be calculated from
the number of granulocytes by using a known ratio of a granulocyte
count to the number of whole leukocytes.
[0016] Moreover, as the known ratio of the leukocyte count to the
elastase concentration of the present invention, the conventionally
known ratio may be used, or a ratio that was calculated in advance
in accordance with the kinds of specimen may be used. Furthermore,
the counting of leukocytes may be carried out by either the
above-mentioned visual method or the automatic counting method.
Also, the method for measuring the elastase concentration is not
particularly limited, however, it is preferable to use the
immunological techniques employed in the present invention.
[0017] Next, the leukocyte counter of the present invention is used
for performing the method for counting leukocytes according to the
present invention and the method includes a means for calculating
the number of leukocytes in a specimen from the concentration of
elastase liberated from granulocytes in the specimen by using a
known ratio of a leukocyte count, a granulocyte count or a
neutrophil count to a concentration of elastase.
[0018] It is preferable that the above-mentioned leukocyte counter
includes a means for liberating elastase from granulocytes in the
specimen, a means for adding an antigranulocyte elastase antibody
to the thus liberated elastase, a means for measuring the
antigranulocyte elastase antibody bonded to the elastase to thereby
determine the concentration of elastase; and a means for
calculating the number of leukocytes contained in the specimen from
the determined concentration of elastase by using a known ratio of
a leukocyte count, a granulocyte count or a neutrophil count to the
concentration of elastase.
[0019] Next, the reagent kit of the present invention is used for
performing the method for counting leukocytes according to the
present invention and the kit includes an antigranulocyte elastase
antibody.
[0020] The reagent kit of the present invention used for the method
for counting leukocytes employing the latex agglutination
immunoassay of the present invention includes an antibody particle
in which an antigranulocyte elastase antibody is bonded to a latex
particle.
[0021] The reagent kit used for the counting method of the present
invention using a labeled antibody has an antigranulocyte elastase
antibody bonded to a labeled antigranulocyte elastase antibody and
to a solid phase. It is preferable that the labeled antigranulocyte
elastase antibody is an enzyme-labeled antigranulocyte elastase
antibody.
[0022] It is preferable that the reagent kit of the present
invention includes the same protease inhibitor as the protease
inhibitor contained in a specimen to be determined. Furthermore,
when the subject specimen is whole blood, the protease inhibitor is
preferably an .alpha.1-antitrypsin. Furthermore, the reagent kit of
the present invention includes a lysing substance.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a graph showing the relationship between the
leukocyte count determined by the counting method of the present
invention and the leukocyte count determined by using an automatic
blood cell counter in one embodiment of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] In the counting method of the present invention, first,
granulocytes are lysed. The lysing method is not particularly
limited and the conventionally known methods can be employed.
Examples of such methods include a physical method using ultrasonic
waves, etc., a method using the difference in the osmotic pressure,
a chemical method using a surface active agent, etc., and the like.
Above all, from the viewpoint of the operability etc., a method of
immersing a specimen in an aqueous solution of a surface active
agent is preferred. Examples of the surface active agent include
dodecyltrimethylammonium bromide, dodecyltrimethylammonium
chloride, saponin, lecithin, cholic acid, sodium dodecyl sulfate,
3-[(cholic amide propyl) dimetyl ammonio]-2-hydroxy-1-pr-
opanesulfonic acid, polyoxyethylene octylphenyl ether,
polyoxyethylene sorbitol ester, and the like. The concentration of
the active surface agent is not particularly limited and it is
generally 0.001 to 5 weight %, preferably 0.05 to 1 weight %.
[0025] Next, the elastase liberated from granulocyte is measured by
an immunological technique.
[0026] Moreover, as mentioned above, it is preferable that when the
specimen is whole blood, prior to this measurement, a protease
inhibitor is added to the specimen in order to form a free-state
elastase liberated from the granulocytes into a complex of the
protease and the elastase. It is because the elastase liberated
from the granulocytes is in a free state and exists as a complex
that is bonded to the protease inhibitor in the plasma and because
a subject to be measured is preferably a complex. Examples of the
protease inhibitor include, in addition to .alpha.1-antitrypsin,
.alpha.2-macroglobulin, and the like. Furthermore, the adding rate
of the protease inhibitor is appropriately decided depending upon
the concentration of the specimen, and the like, however, it is
generally 5 to 10 times, preferably 8 to 12 times of weight with
respect to the weight as granulocyte elastase.
[0027] As the immunological technique, as mentioned above, there
are, for example, the latex agglutination immunoassay, the enzyme
immunoassay, a radioimmunoassay, the fluorescent immunoassay, and
the like.
[0028] The antigranulocyte elastase antibody used in the present
invention can be prepared by separating from the blood serum of a
specifically immunized animal by using granulocyte elastase by a
usual method. Furthermore, it can also be prepared by the usual
hybridoma method. The animal used for preparing antibody includes,
for example, sheep, rabbit, etc. However, the present invention is
not limited to them.
[0029] The latex agglutination method uses an antigranulocyte
elastase antibody bonded to latex particles. This method determines
the elastase concentration by measuring the degree of the
agglutination without performing B/F separation, because the latex
particles are agglutinated, and thus a B/F separation is not
required. In the determination of the agglutination degree, a
spectrophotometer is generally used and the transmitted light is
determined as turbidity. As the agglutination degree is increased,
scattered light is increased to thereby reduce the transmitted
light and the turbidity is enhanced. Moreover, the measurement
wavelength is generally 400 to 1500 nm, preferably 500 to 800 nm.
Furthermore, the adding rate of the antigranulocyte elastase
antibody bonded to latex particles is appropriately decided in
accordance with the kinds of specimens or the concentration, etc.
When the specimen is whole blood, the adding rate is generally 0.1
to 5 mg/ml, preferably 1 to 3 mg/ml. Furthermore, the agglutination
degree may be determined by using nephelometry. Furthermore, the
agglutination degree may be determined visually. In this case, the
use of the commercially available reagent for the visual
determination permits the highly accurate determination.
[0030] The antigranulocyte elastase antibody bonded to latex
particles can be prepared by usual methods. For example, antibody
may be physically adsorbed or covalently-bonded to latex particles
at the rate of 200 ng/cm.sup.2. The latex particles have an average
particle size of 0.1 to 0.2 .mu.m and include polyethylene that is
graft-copolymerized to polystyrene. Moreover, as the
antigranulocyte elastase antibody bonded to latex particles,
commercially available products may be used.
[0031] As mentioned above, the enzyme immunoassay includes: first,
bonding elastase liberated from granulocyte to an antigranulocyte
elastase antibody that is immobilized to a solid phase; further
adding the antigranulocyte elastase antibody labeled with an enzyme
in this state; separating the unbound antibody by means for
washing, etc.; and thereby measuring the enzyme-labeled
antigranulocyte elastase antibody bonded to the elastase by the
reaction between the enzyme and the substrate.
[0032] As the combination of the enzyme and the substrate, the
combination capable of producing detectable products by a reaction
between the enzyme and the substrate is preferred. Examples of
combinations include, a combination of peroxidase, hydrogen
peroxide and orthophenylene diamine, and a combination of alkaline
phosphatase and 4-nitrophenyl phosphate. In a case where the
combination of peroxidase, hydrogen peroxide and orthophenylene
diamine is used, orthophenylene diamine develops color by the
enzymatic reaction. Thus, the color is assayed at the wavelength of
492 nm.
[0033] Furthermore, the antibody immobilized to the solid phase can
be prepared by usual methods. For example, the antibody can be
immobilized to the solid phase by an acid treatment, a heat
treatment, and the like. Furthermore, the enzyme-labeled antibody
can be prepared by usual methods. For these antibodies, commercial
products may be used.
[0034] Furthermore, the rate of the immobilized antibody and the
addition rate of the enzyme-labeled antibody can be decided
appropriately in accordance with the kinds of specimens, the
concentration, and the like. Furthermore, examples of the solid
phase include a bead surface, tube surface, and the like.
[0035] The radioimmunoassay uses an antigranulocyte elastase
antibody labeled with a radioactive substance instead of the
enzyme. The radioimmunoassay is carried out by the same way as the
enzyme immunoassay except that the radioactive substances are
assayed. An example of the radioactive substance includes
126I--Na.
[0036] The fluorescent immunoassay uses an antigranulocyte elastase
antibody labeled with a fluorescent substance instead of the
enzyme. The fluorescent immunoassay is carried out by the same way
as the enzyme immunoassay except that the fluorescent substances
are assayed. An example of the fluorescent substance includes,
rhodamine, fluorescein, coumarin, and the like.
[0037] Next, from the concentration of the elastase obtained by the
above-mentioned immunological techniques, the number of leukocytes
in the specimen is calculated by using a known ratio of the
leukocyte count to the concentration of granulocyte elastase. As
the known ratio, for example, the statistic ratio or the
concentration of elastase to one granulocyte cell can be used.
Furthermore, it is preferable that the ratio is decided
appropriately in accordance with the kinds of specimens. For
example, when the specimen is whole blood of adult, whole blood of
child or whole blood of aged person, respective ratios may be
used.
[0038] Next, it is preferable that the leukocyte counter of the
present invention includes a means for liberating elastase from
granulocyte in the specimen, a means for adding an antigranulocyte
elastase antibody to the thus liberated elastase, a means for
measuring the antigranulocyte elastase antibody bonded to the
elastase to thereby determine the concentration of elastase; and a
means for calculating the number of leukocytes contained in the
specimen from the determined concentration of elastase by using a
known ratio of a leukocyte count, a granulocyte count or a
neutrophil count to the concentration of elastase. With this
counter, the number of leukocytes can be calculated fully
automatically.
[0039] The means for liberating elastase and means for adding the
antibody include elements such as a pump for divided injection etc.
of the reagent, a valve, a nozzle, a stirrer, a container, a
mechanism for adjusting temperature, etc. Furthermore, the means
for measuring the elastase concentration includes, for example, an
optical measurement system, etc. in addition to the above-mentioned
elements. Furthermore, the calculating means includes, for example,
a calculating mechanism programmed so that the number of
granulocytes or the number of whole leukocytes in the specimen can
be calculated from the elastase concentration and the
above-mentioned known ratio, and a mechanism (display, printer,
etc.) for displaying the calculated results.
[0040] In this way, the counter for performing the counting method
of the present invention also can be made to be the same size as A4
size paper. The counter of the present invention is extremely small
and inexpensive as compared with that of the conventional automatic
counting method.
[0041] The counter for counting leukocytes of the present invention
has at least a means for calculating the number of leukocytes from
the concentration of elastase liberated from granulocytes in the
specimen by using the known ratio of a leukocyte count, a
granulocyte count or a neutrophil count and the elastase
concentration. Other means are not always necessary. For example,
the reagent or antibody may be added by hand. Furthermore, the
elastase concentration may be measured by using the optical
measuring system or may be measured visually.
[0042] Next, as the reagent kit of the present invention, the
reagents exemplified in the counting methods can be used.
[0043] Hereinafter, the present invention will be described by way
of examples and comparative examples.
EXAMPLE 1
[0044] Specimen was prepared by diluting adult whole blood with
phosphate buffered saline (pH 7.4) into various concentrations (5
types). Each of these specimens was added to 0.1 weight % aqueous
solution of dodecyltrimethylammonium chloride so as to lyse
leukocyte. Each of them was diluted with 0.01 weight % of
polyoxyethylene sorbitol ester containing .alpha.1-antitrypsin so
as to prepare a plurality of test liquids. The elastase
concentration of 100 .mu.l of each test liquid was measured by the
above-mentioned technique (the enzyme immunoassay) by using an
immobilized antibody (antihuman granulocyte elastase mouse
monoclonal antibody) and an enzyme-labeled antibody
(peroxidase-labeled antihuman granulocyte elastase sheep polyclonal
antibody).
[0045] More specifically, first, 100 .mu.l of each test liquid was
bonded to the immobilized antibody in a reaction solution and
washed thereof. Then, the enzyme-labeled antibody was added and
washed thereof. Finally, hydrogen peroxide and orthphenylene
diamine were added to the reaction solution. Color developed by the
enzyme reaction was determined by the absorbance at the wavelength
of 492 nm and the elastase concentration was calculated by using a
calibration curve that had been made in advance.
[0046] On the other hand, a regression linear expression between
the number of whole leukocytes and the elastase concentration was
formed with respect to another adult whole blood. The number of
whole leukocytes was counted by using an automatic blood cell
counter (K-4500 produced by TOA MEDICAL ELECTRONICS CO., LTD.).
Furthermore, the concentration of granulocyte elastase was measured
by the same way as the above-mentioned enzyme immunoassay. This
regression linear expression (1) is shown below. The number of
whole leukocytes in each specimen was calculated from the
concentration of elastase by using this regression linear
expression (1). Table 1 shows the results.
Y=2132.9897X-245.6519 (1)
[0047] X: concentration of granulocyte elastase (.mu.g/ml)
[0048] Y: number of whole leukocytes (cell/.mu.l)
[0049] Furthermore, as control, the number of leukocytes in each
specimen was counted by using the automatic blood cell counter
(K-4500 produced by TOA MEDICAL ELECTRONICS CO., LTD.). The results
are also shown in Table 1.
EXAMPLE 2
[0050] The number of whole leukocytes was calculated by using the
elastase concentration with respect to one neutrophil cell, i.e.,
0.9084 ng/ml and the ratio of the neutrophil count to the number of
the whole leukocytes, i.e., 55.3%. The determination of the
elastase concentration with respect to one neutrophil cell was
calculated from only neutrophils counted by the usual method (the
visual method) and the elastase concentration measured by the same
technique as Example 1 (the enzyme immunoassay) except that a
regression linear expression in Example 1 was not used. Table 2
shows the
COMPARATIVE EXAMPLE
[0051] Five kinds of test liquid were prepared the same way as in
Example 1. 10 .mu.l of each test liquid was added to 250 .mu.l of
Good's buffer solution (HEPES, pH7.5) and incubated at 37.degree.
C. for 3 minutes. To this test liquid, 4 mg/ml of aqueous solution
of AAPV (methoxysuccinyl-Ala-Ala-Pro-Val-nitroanilide) was added,
stirred and incubated at 37.degree. C. for 6 minutes. Thereafter,
the absorbance of the reaction solution was measured at the
wavelength of 405 nm and the elastase activity was determined.,
Furthermore, as a control, similar to Example 1, the number of
whole leukocytes was counted by using the automatic blood cell
counter (K-4500 produced by TOA MEDICAL ELECTRONICS CO., LTD.).
Table 3 shows the results.
1TABLE 1 Example 1 Number of control Elastase Calculated number
leukocytes concentration of leukocytes (cell/.mu.l) (.mu.g/ml)
(cell/.mu.l) 1000 0.36 522 2100 1.51 2975 4200 2.02 4063 6300 2.99
6132 8400 4.01 8308
[0052]
2TABLE 2 Example 2 Number of control Elastase Calculated number
leukocytes concentration of leukocytes (cell/.mu.l) (.mu.g/ml)
(cell/.mu.l) 1000 0.36 717 2100 1.51 3006 4200 2.02 4021 6300 2.99
5952 8400 4.01 7983
[0053]
3TABLE 3 Comparative Example Number of control leukocytes Elastase
activity (cell/.mu.l) (U/ml) 730 4.3 1090 24.8 1540 7.8 1970 52.8
2670 33.4
[0054] As shown in Tables 1 and 2, in Examples 1 and 2, the number
of whole leukocytes calculated from the elastase concentration and
the number of whole leukocytes counted by using the automatic blood
cell counter were highly correlated (correlation coefficient
r=0.986). On the other hand, in the comparative example, the
correlation between the elastase concentration (the enzyme
activity) and the number of whole leukocyte counted by using the
automatic blood cell counter was low (correlation coefficient
r=0.6268). This is thought to be because in the comparative
example, the activity of elastase was inhibited by the protease
inhibitor (.alpha.1-antitrypsin, etc.) in the specimen with the
result that the elastase concentration was measured by the enzyme
immunoassay.
EXAMPLE 3
[0055] Five kinds of specimens were prepared the same way as in
Example 1. The elastase concentration of each of these 5 specimens
was measured by the below mentioned latex agglutination
immunoassay. More specifically, first, each of the above-mentioned
specimens was added to 0.1 weight % of saponin aqueous solution so
as to lyse leukocyte. This was diluted in the same way as in
Example 1 to prepare test liquid. 10 .mu.l of this test liquid was
added to 250 .mu.l of phosphate buffered saline (pH7.4), then 50
.mu.l of latex suspension (concentration: 2.0 mg/ml), which had
been sensitized by antihuman granulocyte elastase antibody, was
added to the test liquid, and incubated at 37.degree. C. for 15
minutes. The latex suspension was prepared by physically adsorbing
the above-mentioned antibody to the latex particles, which have an
average particle size of 0.1 to 0.2 .mu.m and in which polyethylene
was graft-copolymerized to polystyrene at the rate of about 200
ng/cm.sup.2. Then, the absorbance (turbidity) of this buffer
solution was determined at the wavelength of 660 nm by using a
spectrophotometer and the elastase concentration was measured. From
the numeral value, the number of leukocytes in the specimen was
calculated in the same way as in Example 1. Furthermore, as a
control, the number of leukocytes in each specimen was counted by
using the automatic blood cell counter (K-4500, TOA MEDICAL
ELECTRONICS CO., LTE.). Table 4 shows the results.
4TABLE 4 Example 3 Number of control Elastase Calculated number
Dilution level of leukocytes concentration of leukocytes specimen
(cell/.mu.l) (.mu.g/ml) (cell/.mu.l) 1/16 1100 0.40 1284 1/8 1600
1.70 1996 1/4 3300 3.04 2766 1/2 6600 9.53 6498 1 13200 21.32
13277
[0056] As shown in Table 4, in Example 3, the number of leukocytes
calculated from the elastase concentration and the number of whole
leukocytes counted by using the automatic blood cell analyzer were
highly correlated (correlation coefficient r=0.998).
EXAMPLE 4
[0057] The number of leukocytes in the specimen was calculated in
the same way as in Example 3 by using 50 cases of adult whole blood
(n=50) as the specimens. Furthermore, as controls, the number of
leukocytes of each of the above-mentioned specimens (n=50) was
counted by using the automatic blood cell counter (K-4500 produced
by TOA MEDICAL ELECTRONICS CO., LTD.). FIG. 1 shows the results.
FIG. 1 is a graph showing the relationship between the number of
leukocytes counted by the counting method according to the present
invention and the number of leukocytes counted by using the
automatic blood cell counter concerned in each specification
(n=50).
[0058] As shown in FIG. 1, in each specimen (n=50), the number of
leukocytes calculated from the elastase concentration and the
number of whole leukocytes counted by using the automated analyzer
were highly correlated (correlation coefficient r=0.973).
INDUSTRIAL APPLICABILITY
[0059] As mentioned above, according to the method for counting
leukocytes of the present invention, the number of leukocytes in a
specimen was calculated from the concentration of elastase
contained in granulocytes, which was measured by the immunological
technique. With this method, leukocytes can be counted without
being affected by nucleated cells other than leukocytes, for
example, erythroblast cells, etc., deposition of fibrin, platelet
agglutination, and deficiency in hemolysis of erythrocytes.
Furthermore, the counter can be miniaturized and the counting cost
can be reduced according to the method of the present invention.
Furthermore, since this method uses the immunological technique, it
is not influenced by the skill of laboratory technicians.
* * * * *