JP2002207036A - Method for detecting leucocyte tumor cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for relatively inexpensively detecting tumor cells which can not be detected by a conventional blood analyzing device with a high provability. SOLUTION: (1) The effects of erythrocytes contained in a hematological sample are removed to bring normal leucocytes and leucocyte tumor cells into a state suitable for dyeing. (2) At least one pigment responsive to changes in the state of cells caused by cell stimulation is added, and fluorescent dyeing is performed. (3) An obtained sample is subjected to a flow cytometer to obtain measuring time on each cell and at least one fluorescent intensity for a prescribed time. (4) A cell irritant is added within the prescribed time. (5) A two-dimensional distribution with measurement time and fluorescent intensity as two axes is obtained. (6) Areas made of a prescribed range of a time slot and a prescribed range of fluorescent intensity are set before and after the addition of the cell irritant. (7) The number of cells in each area is counted to obtain the degree of changes in each count value. (8) From the obtained degree of changes, the presence of the leucocyte tumor cells is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring tumor cells by stimulating cells and measuring the difference in response (fluorescence signal) to the stimulus using a flow cytometer (FCM).

[0002]

2. Description of the Related Art In general, diagnosis of tumor formation in blood such as leukemia is performed by a combination of measurement of antigens (surface markers) appearing on the cell surface, identification of genes specifying tumor formation, morphological judgment by microscopic observation, and the like. Will be For example, ATL
To detect neoplastic cells in blood in (adult T-cell leukemia) cases, a method of detecting surface antigens that appear specifically in a disease using a monoclonal antibody labeled with a dye, or a specific causative gene (HTLV gene) ) Is detected.

[0003] FA, a leukemia classification that is currently widely used,
In order to carry out the B classification, morphological examinations using these surface markers and a microscope are indispensable, and genetic tests, which have been actively performed in recent years, are also indispensable depending on diagnostic items. It is known that the types and amounts of antigens appearing on the surface of tumorigenic cells vary depending on the progress of tumorigenicity, and it is necessary to measure with a flow cytometer using an antibody that recognizes this antigen in blood. Leads to the detection of neoplastic cells. Measurement of cell surface markers such as lymphocytes
A method of measuring with a flow cytometer using a monoclonal antibody labeled with a dye is generally used, and 100 or more surface antigens are known.

[0004] Morphological observation with a microscope is an old method that has been used even before automatic measuring instruments became widespread as of today. Commonly used methods for preparing blood smears include double staining with May-Grunwald stain and Giemsa stain, peroxytase staining, Sudan black B staining, and light staining. The tumor cells are identified and classified by observing the size and atypia of the nucleus, the staining state of the cytoplasm, the state of the organelles, etc., by combining these or a plurality of stainings. However, some tumor cells, such as CLL cells, show the same staining properties as normal cells.
In some cases, it cannot be determined as a tumor cell. In such cases, it is necessary to make a diagnosis by comparing it with other test methods (such as measurement of surface markers and genes). In many cases, microscopic morphological features play an auxiliary role in making a diagnosis.

[0005] With the development of molecular biology in recent years, a gene (DNA) test for diagnosing leukemia has been constructed with a system that can be relatively easily performed even in a laboratory, and is routinely performed in some laboratories. ing. Some leukemias, such as ATL, are known to cause tumorigenesis due to specific genes, and detection of such causative genes is essential for making a diagnosis. Furthermore, besides identifying the causative gene, the patterns of genetic diseases that frequently appear in certain leukemias (gene deletion, gene replacement, chromosomal translocation,
Deficiency), and examining them is very important in diagnosing leukemia.

As described above, tumor markers such as leukemia are generally determined by surface marker measurement, morphological observation with a microscope, genetic diagnosis, and the like. It has become. further,
By combining these methods, more detailed classification,
Accurate diagnosis is possible.

[0007]

The above-mentioned examination methods have been clinically established and are currently widely used. However, such examination methods have problems such as high measurement costs and labor costs, and complicated procedures. There is.

[0008] For example, in the measurement of commonly used surface markers, it is difficult to reduce the running cost because the monoclonal antibody used for each measurement is expensive. Further, the measuring equipment used is limited to a flow cytometer capable of optical detection, and the cost of the measuring equipment is very high.

Further, the morphological detection using a microscope is an inspection method which requires labor by an inspection technician having a specific ability and takes a certain amount of time for the measurement, thereby increasing labor costs. Furthermore, the morphological judgment has a part that is left to the subjectivity of the laboratory technician, and in the case of a delicate case in which diagnosis is difficult to make, the diagnosis result may vary depending on the examiner. Similarly, morphological judgment requires the skill of a laboratory technician, and the degree of skill may change the diagnostic result.

In the current field of genetic testing, the part that can be automated is limited, and there is a problem that the testing time is long and the testing cost is high accordingly. For example, detection of the HTLV gene, which is indispensable for the ATL test, requires more than one day and a complicated procedure. The fact that the test requires more than one day requires a re-examination of the patient, and a great psychological burden.

In order to improve the above-mentioned problem, a blood analyzer having an abnormal cell detecting ability has recently been marketed.
(For example, XE-2100: Sysmex Corporation, CD-4000:
Abbott Laboratories) These blood analyzers have the added value of other than blood cell counting, immature cells such as reticulocytes, nucleated red blood cells, immature granulocytes, or atypical cells such as atypical lymphocytes and large lymphocytes. Has the function of detecting Such immature or atypical cells are frequently used in leukemia specimens, and are therefore used as targets for detecting neoplastic cells.

However, these blood analyzers capture the degree of maturity and size of the cells characteristically appearing in the tumorigenic cells, the state of the granules, and the like, and have only made the detection of the morphological characteristics more efficient. It is. Such a blood analyzer has been developed with a view to capturing the morphological characteristics of cells, and cannot detect neoplastic cells that do not have such morphological characteristics. For example, it is very difficult to capture small CLL cells because they are morphologically indistinguishable from mature lymphocytes.

As another method, there is a lymphocyte stimulation test using mitogen stimulation. This test is performed to determine the presence or absence of T cell dysfunction by measuring the degree of lymphocytes that become immature by stimulation. As a result, diseases with T cell deficiency, infection prevention plans,
It is used for follow-up of disease prognosis such as the relationship with the T cell function such as the severity of the underlying disease. This inspection requires complicated operations, but is routinely performed at some facilities. Although this test is useful in knowing many diseases associated with lymphocytes, it does not detect tumorigenesis.

An object of the present invention is to provide a method for detecting tumor cells which is relatively inexpensive and has a high possibility of being undetectable by a conventional blood analyzer.

[0015]

The leukocyte tumor cell detection method of the present invention is characterized by comprising the following steps: (1) eliminating the effects of red blood cells contained in a hematological sample,
Bringing normal leukocytes and leukocyte tumor cells into a state suitable for staining, (2) adding at least one dye that responds to a change in the state of cells caused by cell stimulation, and performing fluorescent staining, (3) then obtained. Subjecting the sample to a flow cytometer to obtain a measurement time for each cell and at least one fluorescence intensity for a predetermined time; (4) adding a cell stimulant within the predetermined time; (5) measuring time; (6) a step of obtaining a two-dimensional distribution with the fluorescence intensity as two axes, (6) setting, before and after the addition of the cell stimulant, an area consisting of a predetermined time period and a predetermined range of the fluorescence intensity, 7) Count the number of cells in each area, determine the degree of change in each count,
(8) a step of detecting the presence of leukocyte tumor cells from the obtained degree of change.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In practicing the present invention, first, a step of bringing leukocytes into a state suitable for staining and removing the influence of red blood cells which is problematic in measurement is performed. The process of removing the effects of red blood cells often refers to white blood cell separation or red blood cell hemolysis. For the measurement of tumor cells, it is necessary to remove the influence of red blood cells as in the measurement of white blood cells using a flow cytometer. As a method for removing the influence of red blood cells, any method can be used as long as it can remove the influence of red blood cells during measurement without damaging white blood cells. As a general method, a method using a leukocyte and mononuclear cell separation solution by a centrifugal specific gravity method, a method using a hemolytic agent that hardly damages leukocytes containing ammonium chloride as a main component, and a method using blood Centrifuge the buffy co
Such methods include a method for directly recovering at, a method using acidic hypotonic hemolysis in which the first solution is returned to a neutral pH of about pH 6 to 8, and a second solution is used as an acidic hypotonic solution.

Next, leukocytes are stained. This staining may be carried out under conditions in which the dye is encapsulated in cells, but usually,
The dye is stained by contacting the dye with the cells at room temperature to about 37 ° C. for several tens of seconds to several tens of minutes.

The following dyes are preferably used for dyeing.

[0019]

Embedded image

5- (and-6) -carboxy SNARF-1-AM (Molecula
r Probes, Inc.) CAS No.:126208-13-7 IUPAC name: Spiro [7H-benzo [c] xanthene- 7,1 '(3H) -i
sobenzofuran] -ar'-carboxylic acid, 3- (acetyloxy)
-10- (dimethylamino) -3'-oxo-, (acetyloxy) methyl es
ter

[0021]

Embedded image

BCECF-AM (Dojindo Laboratories, Inc.) CAS No.:117464-70-7 IUPAC name: Spiro (isobenzofuran-1 (3H), 9 '-(9H) xanthe
ne) -2 ', 7'-dipropanoicacid, 3', 6'-bis (acetyloxy) -5
(or 6)-[[(acetyloxy) methoxy] carbonyl] -3-oxo-,
bis [(acetyloxy) methyl] ester

[0023]

Embedded image

Rho123 (Molecular Probes, Inc.) CAS No.:62669-70-9 IUPAC name: Xanthylium, 3,6-diamino-9- (2- (methoxycarb
onyl) phenyl, chloride

[0025]

Embedded image

Fluo3-AM (Dojindo Laboratories, Inc.) CAS No .: 121714-22-5 IUPAC name: Glycine, N- [4- [6-[(acetyloxy) methox
y]-2,7-dichloro-3-oxo-3H-xanthen-9-yl] -2- [2-
[2- [bis [2-[(acetyloxy) methoxy] -2-oxyethyl] a
mino]-5-methylphenoxy] ethoxy] phenyl]-N- [2-
[(Acetyloxy) methoxy] -2-oxyethyl]-, (acetyloxy)
methyl ester

[0027]

Embedded image

Fura Red AM (Molecular Probes, Inc.) CAS No.:149732-62-7 IUPAC name: Glycine, N- [2-[(acetyloxy) methoxy] -2
-oxoethyl] -N- [5- [2- [2- [bis [2-[(acetyloxy) m
ethoxy] -2-oxoethyl] amino] -5-methylphenoxy] eth
oxy] -2-[(5-oxo-2-thioxo-4-imidazolidinylidene)
methyl] -6-benzofuranyl]-, (acetyloxy) methyl este
r

[0029]

Embedded image

Rhod2-AM (Dojindo Laboratories, Inc.) CAS No.:129787-64-0 IUPAC name: Xanthylium, 9- [4- [bis [2-[(acetyloxy)
methoxy] -2-oxoethyl] amino] -3- [2- [2- [bis [2
-[(Acetyloxy) methoxy]-2-oxoethyl] amino] phenox
y] ethoxy] phenyl]-3,6-bis (dimethylamino)-, brom
ide

[0031]

Embedded image

BTC, AM (Molecular Probes, Inc.) CAS No .: 176767-94-5 IUPAC name: N- [2-[(acetyloxy) methoxy] -2-oxoethy
l] -N- [3- (2-benzothiazolyl) -6- [2- [2- [bis [2-
[(Acetyloxy) methoxy] -2-oxoethyl] amino] -5-met
hylphenoxy] ethoxy] -2-oxo-2H-1-benzopyran-7-yl]
-glycine, (acetyloxy) methyl ester

[0033]

Embedded image

Fura2-AM (Dojindo Laboratories, Inc.) CAS No: 108964-32-5 IUPAC name: 5-Oxazolecarboxylic acid, 2- (6- (bis (2-
((acetyloxy) methoxy) -2-oxoethyl) amino) -5- (2- (2-
(bis (2-((acetyloxy) methoxy) -2-oxoethyl) amino) -5-m
ethylphenoxy) ethoxy) -2-benzofuranyl)-, (acetylox
y) methyl ester

[0035]

Embedded image

Indo 1-AM (Dojindo Laboratories, Inc.) CAS No: 112926-02-0 IUPAC name: 1H-Indole-6-carboxylic acid, 2- [4- [bis
[2-[(acetyloxy) methoxy] -2-oxoethyl] amino] -3-
[2- [2- [bis [2-[(acetyloxy) methoxy] -2-oxoety
l] amino] -5-methylphenoxy] ethoxy] phenyl]-, (a
cetyloxy) methylester

[0037]

Embedded image

Quin 2-AM (Dojindo Research Laboratories, Inc.) CAS No: 83104-85-2 IUPAC name: Glycine, N- [2-[(acetyloxy) methoxy] -2
-oxoethyl] -N- [2-[[8- [bis [2-[(acetyloxy) met
hoxy] -2-oxoethyl] amino] -6-methoxy-2-quinoliny
l] methoxy] -4-methylphenyl]-(acetyloxy) methyl
ester Then, the cell suspension is washed by a method such as centrifugation in order to remove the influence of the dye in the extracellular fluid that is not taken into the cells. However, this washing is a step performed for the purpose of sharply capturing the response to the stimulus shown below, and requires a complicated operation.Therefore, in order to perform the measurement more easily and in a shorter time, this step may be omitted. it can. In the case of the simplified method, a stimulant is added in a state where the dye is suspended in the extracellular fluid, and measurement is performed.

After cell staining, a stimulant is added and the signal is measured. The dyes described above capture changes in intracellular substances and potentials caused by applying cell stimulation.For example, a fluorescent signal is generated according to the concentration of metals such as calcium, magnesium, and potassium in cells. These include those that change, or dyes whose fluorescent signal changes depending on the intracellular pH concentration and the potential of the cell membrane.

The stimulus to the cells may be any method as long as a signal change from the cells can be obtained by the stimulus, and is not limited to a special substance. As an example of stimulation other than chemical substances, electrical stimulation, stimulation by light, and the like are also applicable.

Specific examples of the chemical substance include adenosine 5 'triphosphate (ATP), concanavalin A, porkweed mitogen, phytohemagglutinin, phorbol 12-myristate 13-acetate (PMA), valinomycin, monactin, ionomycin. , A23187, nigericin, monesin, gramicidin A, alamecithin, tri-n
-Butyltin chloride (TBT), inositol triphosphate, anti-CD3 antibody and the like. Where A23187 is
Streptomyces chartreusis is an antibiotic obtained by aerobic cultivation of NRRL 3882, and is an ionophore. As an example of the anti-CD3 antibody, OKT3 (Ortho Diagn
ostic Systems, Inc.).

The amount used varies depending on the substance, but when ATP is used as a stimulant, the concentration may be 0.1 to 50 mM, and more preferably 0.3 to 10 mM. When concanavalin A is used, the concentration may be 0.1 to 10 mg / ml, and more preferably 0.3 to 3 mg / ml. When using PMA, the concentration may be 1 to 100 μg / ml,
More preferably, it is 2 to 50 µg / ml. The concentration when using pork weed mitogen is 10-1000 μg / ml
And more preferably 30 to 300 μg / ml. If ionomycin is used, the concentration should be between 0.1 and 10
μg / ml may be used, and more preferably 1 to 5 μg / ml.

The time from the application of the stimulus to the completion of the measurement is not particularly limited, but is preferably within 30 minutes from the time of adding the stimulant from the viewpoint of shortening the measurement time.
It is desirable to finish within 5 minutes.

In the data analysis, a two-dimensional distribution (cytogram) having measurement time and fluorescence as two axes is obtained and analyzed. Such cytograms have been disclosed in the literature and have been studied using white blood cells of healthy subjects and cell lines that are model cells of tumorigenesis (Journal of Leukocyt
e Biology 49: 369-379 (1991), Cytometry 11: 923-927
(1990)). In the method according to the present invention, a gate is set on the cytogram and the cells contained therein are quantified in order to make the response to the stimulus more clear. This allows
The response to the stimulus can be captured more sharply.

More specifically, a two-dimensional distribution is obtained from the measured fluorescence signals of the individual cells using the measurement time and the fluorescence intensity as two axes, and from the two-dimensional distribution, t0 to t1 before the addition of the cell stimulant is obtained. Time (t0 <t1) and fluorescence intensity f0 to f
An area of 1 (f0 <f1) is set, and the number of cells C0 in that area is determined. In addition, a region from t2 to t3 (t2 <t3) and a fluorescence intensity f0 to f1 after the addition of the cell stimulant is set, and the number of cells C1 in the region is determined.
C0 is calculated to determine the degree of change. Further, if necessary, a region from t4 to t5 (t3 <t4 <t5) and a fluorescence intensity from f0 to f1 may be set, and the number of cells C2 in the region may be obtained to calculate C2 / C0. .

However, in the case of analysis by an automated apparatus, if the measurement start time and measurement end time are set in advance,
Since the measurement can be completed within a certain period of time, it is also possible to obtain a cytogram having two axes of fluorescence and forward scattered light or fluorescence and side scattered light, and to detect the difference between the two cytograms. is there.

Alternatively, a two-dimensional distribution having the scattered light intensity and the fluorescence intensity as two axes is obtained, and a specific leukocyte region is set on the obtained two-dimensional distribution.
A two-dimensional distribution having measurement time and fluorescence intensity as two axes may be obtained, and the obtained two-dimensional distribution may be fractionated at predetermined time intervals to determine an average fluorescence intensity of each fraction.

The inventors measured the response to stimuli using various cells and a dye sensitive to intracellular calcium. As a result, it was found that there was a significant difference between leukocyte cells of healthy subjects and leukocyte tumor cells. I found something. That is, it was found that the response to stimulus was significantly lower for normal monocytes, whereas the response to stimulus was significantly lower for neoplastic cells. Therefore, the presence or absence of leukocyte tumor cells can be detected by capturing the change in the count value or the average fluorescence intensity before and after the addition of the cell stimulant.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

[0050]

Example 1 Step of preparing cells Blood containing anticoagulant (heparin or EDTA), about 20
Add equal volume of PBS to ml and mix well. The cell suspension is carefully layered over the lymphocyte separation solution with a dropper so that the ratio of lymphocyte separation solution (d = 1.077) to cell suspension is 1: 2. Centrifuge at 1800rpm, 4 ℃, 30min. The upper layer of plasma is removed with an aspirator. Carefully collect the lymph layer with a dropper and transfer to another container. Add excess PBS and wash by centrifugation. (1500 rpm, 4 ° C, 10 min., Twice) Loading cells (NaCl 145 mM, KCl 5 mM, CaCl ₂ 1m)
M, MgSO ₄ 0.5 mM, glucose 5 mM, HEPES 10 mM, pH 7.4). Existing flow cytometer (XE-210
0, manufactured by Sysmex Corporation) and the cell concentration was 1 ×
Adjust to 10 ⁶ cells / ml. Next, Pluronic F127 (4% (W / V) in DMSO) (Molecular
Probes, Inc.), 5 μl (final concentration 0.02%), Fluo-3-AM
Add 5 μl of 1 mM stock sol. (In DMSO). Mix well (final concentration: 5 μmol / l) and incubate at 37 ° C for 30 minutes. Load the above solution into the incubated solution
Add 1 ml of ing buf (containing 5% FCS) and incubate at 37 ° C for another 10 minutes. After washing twice with a cell loading buf, 1 ml of a cell suspension (1500 rpm, 10 min) was obtained.

Example 2 Step of stimulating and measuring cells A cell suspension (1 ml) loaded with a dye was applied to a FACSCalibur (Bect
on Dickinson Immunocytometry Systems)
Start measurement. At this time, in addition to forward scattered light, side scattered light, and fluorescence parameter, data of time vs. fluorescence intensity: FL1 is also captured. At 30 seconds after the start of the incorporation, an arbitrary amount of the stimulus is added, and the measurement is resumed immediately after stirring with a voltex mixer. (This operation takes about 5 to 10 seconds) 5 from the start of measurement
Capture minutes of data. The final concentration of the stimulant was 1 mM for ATP stimulation, and 10 μg / ml for PMA stimulation.

Example 3 Step 1 of Analyzing Data An example of measurement of a healthy subject and AML samples and MDS samples according to the above method is shown.

Fluorescence is plotted on the vertical axis and time cytogram is plotted on the horizontal axis (FIGS. 1 to 5) (the horizontal axis of the cytogram is total.
5min.). Next, areas R1, R2, and R3 are set on the cytogram. At this time, R1 is 20 from 5 seconds after the start of measurement.
R2 is a region from 40 seconds to 55 seconds after the start of measurement, R3 is a region from 240 seconds to 255 seconds after, and the fluorescence intensity is a region from 440 channels to 1000 channels, respectively.

Cell number (dot number) in each region of R1, R2 and R3
Is calculated, and the ratio of each region is determined from the total number of captured R1, R2, and R3. Further, a change rate between the respective regions is obtained. The results of ATP stimulation are shown in Table 1, and the results of PMA stimulation are shown in Table 2. Comparison of both data between healthy subjects and AML and MDS patients
In the case of AML and MDS, it was confirmed that the change was significantly small.

[0055]

[Table 1]

[0056]

[Table 2]

Example 4 Step 2 of Analyzing Data According to the method of Examples 1 and 2, peripheral blood samples of healthy subjects and ATL (adult T-cell leukemia) were measured.

First, a cytogram of forward scatter is created on the vertical axis and a side scatter is created on the horizontal axis. Next, clusters of cells such as lymphocytes, monocytes, granulocytes, and tumor cells are gated on the cytogram (FIG. 6). Further, for each of the gated cell clusters, a cytogram of fluorescence is plotted on the vertical axis and time is plotted on the horizontal axis. This cytgram is periodically updated (20
Every second) (FIG. 7), and the average fluorescence intensity of the signal gated to each fraction is determined.

Lastly, the average fluorescence intensity of each cell is plotted with respect to time, and the change in the average fluorescence intensity due to stimulation is quantified. The results are shown in FIGS. In the case of a sample from a healthy subject, a clear response to PMA and ATP stimulation was observed, whereas in the case of an ATL case, almost no response was observed. This method revealed more clearly the change in the fluorescence intensity with respect to the stimulation of the cell population.

[0060]

According to the present invention, a difference in response to a stimulus is evident between a normal cell and a neoplastic cell of a healthy subject, and the neoplastic cell can be detected by using the method of the present invention.

[Brief description of the drawings]

FIG. 1 is a cytogram obtained when a normal sample was stimulated with ATP in Example 3.

FIG. 2 is a cytogram obtained when an AML specimen was stimulated with ATP in Example 3.

FIG. 3 is a cytogram obtained when a normal sample is stimulated with PMA in Example 3.

FIG. 4 is a cytogram obtained when an AML specimen was stimulated with PMA in Example 3.

FIG. 5 is a cytogram obtained when an MDS sample was stimulated with PMA in Example 3.

FIG. 6 is a cytogram of leukocytes obtained when the horizontal axis represents side scattered light intensity and the vertical axis represents forward scattered light intensity in Example 4.

FIG. 7 is a cytogram obtained by taking the measurement time on the horizontal axis and the fluorescence intensity on the vertical axis in Example 4.

FIG. 8 is a diagram showing a change in fluorescence intensity when a healthy subject is stimulated with PMA in Example 4.

FIG. 9 is a diagram showing a change in fluorescence intensity when an ATL sample was stimulated with PMA in Example 4.

FIG. 10 is a diagram showing a change in fluorescence intensity when a healthy subject was stimulated with ATP in Example 4.

FIG. 11 is a diagram showing a change in fluorescence intensity when an ATL sample is stimulated with ATP in Example 4.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) G01N 33/49 G01N 33/49 K (72) Inventor Shinichiro Oguni 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe-shi Sysmex Corporation (72) Inventor Yukio Hamaguchi 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe City Sysmex Corporation (72) Inventor Hitoshi Tsuruta 1-7-1, Sakamoto, Nagasaki-shi, Nagasaki Nagasaki University Hospital Inspection Department (72) Inventor Ken Uehira 1-7-1, Sakamoto, Nagasaki City, Nagasaki Pref. QA19 QQ03 QQ08 QR33 QR48 QR59 QR66 QS36 QS39 QX02

Claims (4)

[Claims] 1. A method comprising the following steps:
Leukocyte tumor cell detection method. (1) removing the effects of red blood cells contained in the hematological sample,
Bringing normal leukocytes and leukocyte tumor cells into a state suitable for staining, (2) adding at least one dye that responds to a change in the state of cells caused by cell stimulation, and performing fluorescent staining, (3) then obtained. Subjecting the sample to a flow cytometer to obtain a measurement time for each cell and at least one fluorescence intensity for a predetermined time; (4) adding a cell stimulant within the predetermined time; (5) measuring time; (6) a step of obtaining a two-dimensional distribution with the fluorescence intensity as two axes, (6) setting, before and after the addition of the cell stimulant, an area consisting of a predetermined time period and a predetermined range of the fluorescence intensity, 7) Count the number of cells in each area, determine the degree of change in each count,
(8) a step of detecting the presence of leukocyte tumor cells from the obtained degree of change. 2. The method for detecting a leukocyte tumor according to claim 1, wherein a dye which produces a difference in fluorescence intensity between normal leukocytes and leukocyte tumor cells by adding a cell stimulant is selected from the following group. Embedded image 5- (and-6) -carboxy SNARF-1-AM BCECF-AM Rho123 Fluo3-AM Fura Red AM Rhod2-AM BTC, AM Fura2-AM Indo 1-AM Quin 2-AM 3. The method for detecting a leukocyte tumor cell according to claim 1, wherein the cell stimulator is selected from the following group. Adenosine 5 'triphosphate, concanavalin A, porkweed mitogen, phytohemagglutinin, phorbol 12-myristate 13-acetate, valinomycin, monactin, ionomycin, A23187, nigericin, monesin,
Gramicidin A, alamesitin, tri-n-butyltin chloride (TBT), inositol triphosphate, anti-CD3 antibody 4. A method comprising the following steps:
Leukocyte tumor cell detection method. (1) removing the effects of red blood cells contained in the hematological sample,
Bringing normal leukocytes and leukocyte tumor cells into a state suitable for staining, (2) adding at least one dye that responds to a change in the state of cells caused by cell stimulation, and performing fluorescent staining, (3) then obtained. Subjecting the sample to a flow cytometer to obtain a measurement time for each cell and at least one scattered light intensity and at least one fluorescence intensity for a predetermined time; (4) adding a cell stimulant within the predetermined time (5) a step of obtaining a two-dimensional distribution having two axes of scattered light intensity and fluorescence intensity; (6) a step of setting a specific leukocyte region on the two-dimensional distribution obtained in (5); (7) A step of obtaining a two-dimensional distribution with the measurement time and the fluorescence intensity as two axes for the leukocyte region set in (6), and a step of fractionating the two-dimensional distribution obtained in (8) and (7) at predetermined time intervals , (7)
(8) a step of determining the average fluorescence intensity of each fraction, and (8) a step of detecting the presence of leukocyte tumor cells from the degree of change in the average fluorescence intensity obtained.