JPH11326323A - Method for classifying and counting erythroblast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of measuring erythroblasts accurately, easily, and inexpensively even on a hematological sample deteriorated with age. SOLUTION: (1) A fluorescence-labeled antibody which specifically combines with leucocytes is added to a hematological sample to subject leucocytes to fluorochrome. (2) The permeability of nuclei acid fluorochromes, which normally do not permeate through cell membranes, through cell membranes is accelerated only with respect to erythroblasts (3) to dye the nuclei of erythroblasts. (4) Next, the obtained sample is supplied for a flow cytometer to measure at least two fluorescence signals of an individual cell. (5) Then erythroblasts are classified and counted from the difference in the fluorescence strength. The fluorescence-labeled compound of the fluorescence-labeled antibody in the process (1) is preferably at least one type of compound selected from a group of phycoerythrin, fluorescein isothiocyanate, allophycocyanin. Texas red, CY5, peridinin chlorophyll complex, and their phycoerythrin combination.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for classifying and counting erythroblasts, and more particularly to a method for classifying and counting erythroblasts using flow cytometry.

[0002]

2. Description of the Related Art In the field of clinical examination, classification and counting of erythroblasts can provide extremely useful information for diagnosing disease and observing the course of the disease. It is advantageous because it can. In other words, erythroblasts, also called nucleated erythrocytes, are usually present in the bone marrow and are not present in peripheral blood except for newborns. It indicates a possible disease such as leukemia, hemolytic anemia, iron deficiency anemia, pernicious anemia, and other non-hematological / oncological disorders. Therefore, performing classification and counting of erythroblasts is very useful for diagnosis of these diseases and observation of the progress of these diseases.

Conventionally, in order to classify and count erythroblasts, it has been common practice to prepare a smear of blood, perform appropriate staining, and then perform classification and counting while observing with a microscope. However, such a method requires complicated pre-processing for observation and requires considerable skill of an observation technician to obtain accurate results.

On the other hand, in recent years, various fully automatic leukocyte classification / counting apparatuses utilizing the principle of a flow cytometer have been provided, and methods for analyzing blood components using such apparatuses have been proposed.

For example, Japanese Patent Application Laid-Open No. 4-268453 discloses an acid hypotonic treatment, staining with a fluorescent dye for staining nuclei of erythroblasts, and detecting scattered light and fluorescence with a flow cytometer. A method for classifying and counting erythroblasts is described.

Further, Japanese Patent Application Laid-Open No. Hei 5-34251 discloses that
A method for measuring erythroblasts by acid hypotonic treatment, staining with four kinds of dyes including Astrazone Yellow 3G and neutral red, which are fluorescent dyes, measuring red fluorescence and green fluorescence with a flow cytometer. Is described.

Further, Japanese Patent Publication No. Hei 8-507147 discloses a specific amount of a non-quaternary ammonium salt, an aliphatic aldehyde, a non-phosphate buffer, a reagent having a specific pH and a specific osmotic pressure, A method of measuring nucleated red blood cells by measuring forward scattered light or fluorescence-side scattered light with a flow cytometer using a nuclear dye such as ethidium homodimer is described.

Further, US Pat. No. 5,559,037 discloses that
The cell membrane of erythrocytes and erythroblasts is lysed, the leukocytes are not stained, but stained with a biological nuclear dye that can stain erythroblasts. Is described. However, in these methods, the change over time of the blood sample after blood collection,
Not only erythroblasts, but also cell membranes of leukocytes are easily damaged, so that when erythroblasts are stained, some of the leukocytes are stained with a dye.For example, erythroblasts are detected by scattered light and fluorescence. Further, there is a problem that the appearance position of leukocytes overlaps, and erythroblasts cannot be measured accurately. In particular, when lymphoid cells are damaged, it is more difficult to clearly distinguish damaged lymphocytes from erythroblasts, and it is not possible to accurately grasp the appearance of erythroblasts. There's a problem.

In recent years, in order to reduce medical expenses and increase the efficiency of medical institutions, blood samples collected from patients at each medical institution have been collected at a certain specialized institution, and intensive examinations have been conducted there. In such cases, it is not unusual to require one day or more from blood collection to measurement.

[0010] Furthermore, in some lymphoblast-appearing specimens or specimens in which the cell membrane of leukocyte cells is susceptible to damage by a hemolytic agent due to chemotherapy, etc., erythroblasts can be accurately obtained even if they have not changed over time. Is difficult to classify.

Japanese Patent Publication No. 8-1434 discloses that after addition of thiazole orange to a sample, two types of fluorescently labeled antibodies, anti-CD45 and anti-CD71, are added to the sample, and at least three types of fluorescent antibodies are obtained using a flow cytometer. A method is described for detecting signals in a channel and at least two types of light scattering channels to identify nucleated red blood cells and the like. According to this method, nucleated red blood cells can be measured by combining a specific antibody and a dye. However, in this method, since two types of antibodies and one type of fluorescent dye are used, there is a problem in that the measurement reagent becomes very expensive. Therefore, it is required to analyze erythroblasts at low cost.

Further, JP-A-2-73157 discloses that two kinds of fluorescent nucleic acid dyes and a fluorescent-labeled monoclonal antibody are used, and a flow cytometer is used for at least three kinds of fluorescent channels and at least two kinds of light scattering channels. Methods for detecting signals and analyzing various cells, including nucleated red blood cells, are described. However, in this method, in order to distinguish erythroblasts from leukocytes, the cells are stained with a fluorescent-labeled monoclonal antibody and side scattered light is measured.However, a method for distinguishing platelets, debris from erythroblasts has been proposed. Since it is not described, erythroid cells cannot be accurately counted.

Also, Japanese Patent No. 2620810 discloses that
Lyse erythrocytes, add fluorescently labeled monoclonal antibody,
A method is described in which a fixing agent is added, a nucleic acid dye that preferentially binds to DNA is added, and fluorescence and scattered light are detected by a flow cytometer. However, according to this method, first, in order to perform a process of lysing red blood cells in the sample, a centrifugal washing operation must be performed immediately thereafter.
Absolute counting becomes difficult. In addition, since this operation is complicated, there is a problem that a remarkable difference occurs in the measurement result depending on the skill of the measurement technician.

As described above, erythroblasts can be accurately, easily, and inexpensively measured in hematological samples that have passed a long time after blood collection, and erythroblasts are classified and counted according to their maturity. There is a need for a method that can do that.

[0015]

According to the present invention, according to the present invention, (i) a fluorescently-labeled antibody that specifically binds to leukocytes is added to a hematological sample, and leukocytes are fluorescently stained; It has a fluorescence spectrum that can be distinguished from the fluorescence of the antibody, and enhances the cell membrane permeability of the nucleic acid fluorescent dye that does not normally pass through the cell membrane only in erythroblasts. (Iv) Then, the obtained sample is subjected to a flow cytometer to measure at least two fluorescence signals of individual cells, and (v) erythroblasts comprising classification and counting of erythroblasts from these fluorescence intensity differences. A sphere classification and counting method is provided.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the hematological sample used in step (i) includes peripheral blood, bone marrow fluid, lymph tissue,
A body fluid sample containing leukocytes and erythroblasts, such as a sample collected by urine or apheresis, is meant.

The antibody in the fluorescently labeled antibody that specifically binds to leukocytes includes an anti-CD45 antibody and the like, and commercially available antibodies can be used.

Phycoerythrin (phycoerythrin) is used as a fluorescent-labeled compound for converting the above antibody into a fluorescent-labeled antibody.
n), fluorescein isothiocyanate (fluorescein
isothiocyanate (FITC), allophycocyanin (al
lophycocyanin), Texas Red, CY
5. Peridinin chlorophyll complex (peridini
n chlorophyll complex) and their phycoerythrin conjugates. These fluorescent labeling compounds preferably have a different fluorescence spectrum from the nucleic acid fluorescent dye described below. Of these, phycoerythrin and fluorescein isothiocyanate are preferred.

The mixing ratio between the hematological sample and the fluorescently labeled antibody can be appropriately adjusted depending on the condition of the hematological sample to be used, the type of the fluorescently labeled antibody, and the like.
A capacity ratio of about 2: 1. At this time, the reaction temperature and the reaction time can be appropriately adjusted, and for example, it is preferably about 15 to 30 minutes at room temperature and about 30 to 45 minutes in an ice bath.

In the step (ii) of the present invention, the cell membrane permeability of the nucleic acid fluorescent dye which does not normally permeate the cell membrane is enhanced only in erythroblasts.

Examples of the nucleic acid fluorescent dye include propidium iodide, N-methyl-4- (1-pyrene) vinyl-propidium iodide, ethidium bromide, TOTO-1, TOTO-3, YOYO-1, and YOYO-3. , BOBO-1, BOBO-
3, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2), POPO-1, POPO-3, BO-P
RO-1, YO-PRO-1, TO-PRO-1 and the like. Above all,
Propidium iodide is preferred. As described above, these nucleic acid fluorescent dyes preferably have a different fluorescence spectrum from the fluorescently labeled compound of the fluorescently labeled antibody that specifically binds to leukocytes in step (ii). The final concentration of the nucleic acid fluorescent dye is about 0.003 to 200 mg / L,
Preferably it is about 0.03-70 mg / L, more preferably 0.3-35 mg / L. Here, the final concentration is
Hematology sample to be subjected to the flow cytometer, the concentration in the mixture of the fluorescently labeled antibody and the nucleic acid fluorescent dye, or even when using other reagents as described below, in the mixture to be subjected to the flow cytometer Means concentration.

As a method for enhancing the cell membrane permeability of the above-mentioned nucleic acid fluorescent dye only in erythroblasts, for example, the obtained hematological sample (containing at least erythroblasts and leukocytes) is used.
First, a low osmotic pressure first solution comprising a buffer for keeping the pH in an acidic range is added and mixed, and the hematological sample and the first solution are neutralized, and the solution pH is adjusted to a pH suitable for staining. And a method of adding and mixing a second liquid consisting of a buffering agent for adjusting the osmotic pressure and an osmotic pressure adjusting agent for adjusting the osmotic pressure to maintain the form of leukocytes.

The first liquid in the step has an acidic pH range, for example, 2.0 to 2.0, in order to effectively lyse erythrocytes.
It is maintained at about 5.0, more preferably about 2.5 to 4.0, and still more preferably about 3.0 to 3.5. If the pH is too low, not only erythrocytes but also leukocytes, erythroblasts and fluorescently labeled antibodies that specifically bind to leukocytes cause excessive damage, while if the pH is too high, erythrocytes are fragmented. This is not preferred, because the action of oxidizing is clearly prevented.

Examples of the buffer for maintaining the above pH include a buffer having an acid dissociation constant pKa of about 3.0 ± 2.0. Specific examples include malic acid, succinic acid, citric acid, phosphoric acid, Good's buffer and the like. The concentration of the buffer is not particularly limited as long as it is necessary to maintain the first solution at a pH of about 2.0 to 5.0, and for example, 5 to 50 mM / L.

The osmotic pressure of this buffer must be low, for example, 100 mOsm / kg
· H ₂ O of about or less osmotic pressure and the like, more preferably 10~60mOsm / kg · H ₂ O about. The type of the osmotic pressure adjusting agent used for adjusting the osmotic pressure to such an osmotic pressure is not particularly limited, and examples thereof include alkali metal salts and saccharides. Specifically, sodium chloride, sucrose and the like can be used at a concentration of about 0.1 g / L to 2.0 g / L. However, when the above-mentioned osmotic pressure is compensated only by the above-mentioned buffer, the osmotic pressure adjusting agent may not be used.

The reaction time between the hematological sample and the first liquid requires a sufficient time to complete the hemolysis of red blood cells, for example, about 5 seconds to 120 seconds, preferably about 10 seconds to 60 seconds, More preferably, it is about 20 to 40 seconds.
The mixing ratio between the hematological sample and the first liquid is not particularly limited, but considering the measurement with a flow cytometer, it is 1: 5.
A capacity ratio of about 1: 200 is preferable.

The second solution in the process neutralizes the mixture of the hematological sample and the first solution, and adjusts the solution pH to a value suitable for staining.
H and a osmotic pressure adjusting agent for adjusting the osmotic pressure to maintain the form of leukocytes.

The pH of the second liquid suitable for neutralizing the acid in the first liquid and dyeing is, for example, about 5.0 to 11.0, and preferably about 7.5 to 10.0. . The type of buffer used to maintain the pH is not particularly limited, but a buffer having a pKa around 9.0 ± 2.0 is preferable. Specific examples include phosphoric acid, HEPES, and tricine. The concentration of the buffer was adjusted so that the second solution had a pH of 5.0 to 1.
The concentration is not particularly limited as long as it is necessary to maintain the concentration at about 1.0, and usually about 5 to 100 mM / L.

The range of the osmotic pressure suitable for maintaining the form of leukocytes is, for example, 300 to 1000 mOsm / kg · H.
_{About 2} O, more preferably 400 to 600 mOsm / k
g · H ₂ O. The type of the osmotic pressure adjusting agent used for adjusting the osmotic pressure to such an osmotic pressure is not particularly limited, and examples thereof include alkali metal salts and saccharides. Specifically, sodium chloride, sucrose, etc. is 10.0 g / L to 2 g
It can be used at a concentration of about 0.0 g / L.

The mixing ratio of the first liquid and the second liquid depends on the pH and amount of the first liquid used previously, the concentration of the osmotic pressure adjusting agent of the first liquid, the pH of the second liquid, and the permeation of the second liquid. It can be appropriately adjusted depending on the concentration of the pressure adjusting agent and the like. For example, the pH of the first liquid is 3.
0, the osmotic pressure is about 16 mOsm / kg · H ₂ O, the pH of the second liquid is 7.5, and the osmotic pressure is 400 mOsm / kg · H ₂ O
In the case of about O, the mixing ratio of the first liquid and the second liquid is 1: 1 to 1
About 1: 5 is preferable.

In the step (ii) of the present invention, the osmotic pressure after mixing the first and second liquids is 100 to 500 mOsm in order to maintain the leukocyte form of the hematological sample.
/ Kg · H ₂ O, more preferably in the range of 200 to 400 mOsm / kg · H ₂ O. When the osmotic pressure after mixing the first liquid and the second liquid is out of this range, it is preferable that the second liquid further contains an osmotic pressure compensating agent. The kind of the osmotic pressure compensating agent is not particularly limited, but usually, a substance such as an alkali metal or a saccharide for keeping biological cells at a physiological osmotic pressure is preferable.

In the step (iii) of the present invention, erythroblast nuclei are stained. Staining the erythroblast nucleus is a step of staining the hematological sample treated in the above step with a nucleic acid fluorescent dye. Specifically, there is a method in which a nucleic acid fluorescent dye is previously added to the first liquid or the second liquid, and the first liquid or the second liquid containing the nucleic acid fluorescent dye is mixed with a hematological sample. .
Alternatively, a solution containing a nucleic acid fluorescent dye may be prepared, and this solution may be added. In particular, it is preferable that the nucleic acid fluorescent dye is added to the first liquid in advance. The time required for staining the erythroblast nuclei at this time is about 1 to 120 minutes after mixing the hematological sample and all the reagents, preferably about 3 to 30 minutes, more preferably 5 to 10 minutes. About a minute.

The flow cytometer used in step (iv) of the present invention is not particularly limited, and a commercially available one can be used. With such a flow cytometer, at least 2
Measure two fluorescence signals. The fluorescence signal at this time differs depending on the labeling compound of the fluorescently labeled antibody to be used, the type of the nucleic acid fluorescent dye, for example, a combination of red fluorescence and green fluorescence,
A combination of red fluorescence and orange fluorescence, a combination of orange fluorescence and green fluorescence, and the like can be given. Among them, a combination of red fluorescence and green fluorescence is preferable.

In the step (v) of the present invention, the erythroblasts are obtained based on the difference between the intensity of at least two fluorescent signals described above.
Classification can be counted. For example, when two fluorescent signals are measured, two axes are defined as fluorescence based on a fluorescently labeled antibody that specifically binds to leukocytes and fluorescence based on a nucleic acid fluorescent dye to obtain a two-dimensional distribution (for example, a scattergram). Is preferred. The distribution area of leukocytes and erythroblasts is set from this two-dimensional distribution, the number of cells in each area is counted, and the number of cells of erythroblasts is divided by the number of cells of leukocytes. The ratio can be determined.

In addition, the leukocyte membrane is excessively damaged due to the effects of drug administration and the like, and the fluorescence based on a fluorescently labeled antibody (green fluorescence in the example) that specifically binds to leukocytes and the fluorescence based on a nucleic acid fluorescent dye (in the example). When the erythroblast discrimination is not clear on the two-dimensional distribution having two axes of (red fluorescence), in step (iv), (a) simultaneously measuring the scattered light signal, the scattered light signal (for example, (A side scattered light, forward scattered light, preferably side scattered light) and a fluorescent signal based on a fluorescently labeled antibody are respectively obtained as a two-dimensional distribution, and a leukocyte population is identified from the distribution (FIG. 6). (B) Identify the corresponding leukocyte population distribution region on the two-dimensional distribution with the nucleic acid fluorescent dye and the fluorescence based on the fluorescently labeled antibody as two axes (FIG. 7), and (c) and (b). The boundary (A) between the leukocyte population and the erythroid population on the three-dimensional distribution By setting, erythroblasts can be discriminated more accurately.

Further, in the step (ii), when the concentration of the nucleic acid fluorescent dye is in the range of 0.003 mg / L to 10 mg / L, at least two different maturation levels of red are different due to the difference in fluorescence intensity based on the nucleic acid fluorescent dye. It can be classified as blast. The concentration of the nucleic acid fluorescent dye is more preferably in the range of 0.03 mg / L to 3 mg / L.

In this case, in step (v), erythroblasts having different maturity levels can be classified and counted from the difference in fluorescence intensity based on the nucleic acid fluorescent dye. In other words, by setting the distribution area of erythroblasts from the two-dimensional distribution, and further setting the area of erythroblasts at each maturation stage within the area of erythroblasts by the difference in fluorescence intensity based on the nucleic acid fluorescent dye, Can be counted. Furthermore, by dividing the number of erythroid cells at each maturation stage by the total number of erythroblasts,
The ratio of erythroblasts at each maturity stage to total erythroblasts can also be determined.

To classify erythroblasts according to the stage of maturation,
For example, proerythroblasts, basophil erythroblasts, polychromatic erythroblasts,
It refers to classification and counting of normal erythroblasts and the like into at least two erythroblast groups.

[0039]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the method for classifying and counting erythroblasts of the present invention will be specifically described below. Example 1 First, a reagent having the following composition was prepared. Fluorescent labeled antibodies · FITC-labeled anti-CD45 antibody first solution (pH 3.0, _{osmolality: 16mOsm / kg · H 2 O} ) · buffers Citric acid monohydrate 2.10 g / L disodium phosphate 0.56 g / L ・ Nucleic acid fluorescent dye Propidium iodide 100 mg / L ・ Purified water second liquid (pH 7.5, osmotic pressure: 420 mOsm / kg ・ H ₂ O) ・ Buffer monosodium phosphate dihydrate 0.95 g / L Phosphorus Disodium acid 6.24 g / L ・ Osmotic pressure adjusting agent Sodium chloride 10.2 g / L ・ Purified water

First, 50 μL of a patient's blood was added to peripheral blood treated with an anticoagulant to prepare a hematological sample. In addition,
The peripheral blood and the patient's blood used at this time had passed 8 hours after collection. FITC-labeled anti-CD45 antibody 1
0 μL is added to the obtained hematology sample, and
Incubated for minutes.

Thereafter, 500 μL of the first solution was added, and the mixture was incubated at room temperature for about 30 seconds.
Add 00 μL, incubate at room temperature for about 5 minutes,
The individual cells contained in the obtained hematological sample were analyzed using a flow cytometer equipped with a 488 nm argon ion laser as a light source at 530 nm (green), 6 nm.
Fluorescence at a wavelength of 50 nm (red) was measured.

FIG. 1 shows a scattergram depicting the distribution of individual cells using the green fluorescence intensity and the red fluorescence intensity as coordinate axes. In FIG. 1, four populations of white blood cells, red fluorescently stained white blood cells, erythroblasts, and ghosts were observed. In the analysis, as shown in FIG. 2, white blood cells and red fluorescently stained white blood cells were windowed (W
Surrounded by 1), the number of leukocytes alone was counted, and the total number of leukocytes was counted.

Next, all erythroblasts were placed in a window (W
Surrounded by 2), the number of erythroblasts alone was counted, and the total number of erythroblasts was counted. The ratio of erythroblasts to leukocytes was determined by dividing the total erythroblast count by the total leukocytes determined above.

Separately from Example 1, erythroblasts were classified and counted using the same hematological sample as in Example 1 by a conventional method (May-Grunwald-Giemsa staining, 1000 counts). FIG. 3 shows a correlation diagram between the number of erythroblasts measured by the flow cytometer of the present example and the number of erythroblasts measured by the method.

From FIG. 3, the correlation coefficient R was 0.991, and it was confirmed that the method of Example 1 had extremely high accuracy in the classification and counting of erythroid cells.

Example 2 In the same manner as in Example 1, peripheral blood (8 hours, 24 hours and 48 hours after blood collection, blood was stored at room temperature) and 2
Leukocytes and erythroblasts were measured using the blood of human patients. 4 (a) to 4 (c) and 5 (a) to
(C) shows each.

The ratio of total erythroblasts to total leukocytes was determined for each sample by the same method as in Example 1. Table 1 shows the results.

[Table 1]

FIGS. 4A to 4C and FIGS. 5A to 5C.
As is clear from Table 1 and Table 1, it was confirmed that almost the same measurement results were obtained regardless of the lapse of time according to the method of this example.

Example 3 First, a reagent having the following composition was prepared. Fluorescent labeled antibodies · FITC-labeled anti-CD45 antibody first solution (pH 3.0, _{osmolality: 16mOsm / kg · H 2 O} ) · buffers Citric acid monohydrate 2.10 g / L disodium phosphate 0.56 g / L ・ Nucleic acid fluorescent dye Propidium iodide 1 mg / L ・ Purified water second liquid (pH 7.5, osmotic pressure: 420 mOsm / kg ・ H ₂ O) ・ Buffer monosodium phosphate dihydrate 0.95 g / L Phosphorus Disodium acid 6.24 g / L ・ Osmotic pressure adjusting agent Sodium chloride 10.2 g / L ・ Purified water

First, FITC-labeled anti-CD45 antibody 10 μm
To L, 50 μL of a patient's blood in which erythroblasts appeared in peripheral blood treated with an anticoagulant was added, and the mixture was incubated at room temperature for about 15 minutes. Thereafter, 500 μL of the first solution was added, and the mixture was incubated at room temperature for about 30 seconds.
After incubating at room temperature for about 5 minutes, the individual cells contained in the obtained hematological sample were analyzed at 530 nm using a flow cytometer equipped with a 488 nm argon ion laser as a light source.
(Green) and 650 nm (red) fluorescence were measured.

FIG. 8 shows a scattergram depicting the distribution of individual cells using the green fluorescence intensity and the red fluorescence intensity as coordinate axes. In FIG. 8, six populations of leukocytes, red fluorescent stained leukocytes, mature erythroblasts, immature erythroblasts 1, immature erythroblasts 2, and ghosts were observed. In the analysis, as shown in FIG. 9, white blood cells and red fluorescently stained white blood cells were surrounded by a window (W1), the number of white blood cells alone was counted, and the total number of white blood cells was counted.
Next, all erythroblasts were surrounded by a window (W2), the number of erythroblasts alone was counted, and the total number of erythroblasts was counted.

Further, the erythroblasts in the window (W2) are set as stages I, II and III, respectively.
3), surrounded by a window (W4) and a window (W5), and the respective numbers were counted. By dividing the obtained number of erythroblasts in each window by the total number of erythroblasts,
The ratio of erythroblasts at each stage to total erythroblasts was determined.

In addition, apart from Example 3, a hematological sample similar to that of Example 3 was used (Maygrunwald-
(Giemsa staining) and classified into pro-erythroblasts, basophil erythroblasts, polychromatic erythroblasts and erythroblasts of normal erythroblasts.

Table 2 shows the results measured by the flow cytometer of Example 3 and the results measured by the method. In addition,
In Table 2, Stages I, II and III correspond to immature erythroblast 2, immature erythroblast 1 and mature erythroblast, respectively, in FIG.

[Table 2]

Table 2 shows that the results of the present method and the method of the present invention are almost the same, confirming that the method of Example 3 has extremely high accuracy in classification and counting of erythroblasts.

Example 4 In the same manner as in Example 3, erythroblasts were measured using peripheral blood (8 hours after blood collection, blood was stored at room temperature) and blood of 24 blood disease patients. Separately from Example 4, erythroblasts were measured using the same hematological sample as in Example 4 by a manual method (May-Grunwald-Giemsa staining).

FIGS. 10 and 11 show correlation diagrams between the number of erythroblasts measured by the flow cytometer of the present embodiment and the numbers of erythroblasts at stage II and stage III measured by the manual method. From FIGS. 10 and 11, it was confirmed that the method of Example 4 had extremely high accuracy in classification and counting at each stage of erythroblasts.

[Brief description of the drawings]

FIG. 1 is a scattergram measured by the erythroblast classification and counting method of the present invention.

FIG. 2 is a schematic diagram of FIG.

FIG. 3 is a diagram showing a correlation between the number of erythroblasts measured by the method and the method of the present invention.

FIG. 4 is a scattergram showing a change over time in erythroblast measurement by the erythroblast classification and counting method of the present invention.

FIG. 5 is a scattergram of another sample showing a change over time in erythroblast measurement by the erythroblast classification and counting method of the present invention.

FIG. 6 is a scattergram when a scattered light signal is combined with the erythroblast classification and counting method of the present invention.

FIG. 7 is a scattergram when Gho + NRBC in FIG. 6 is further distinguished from erythroblasts and ghosts.

FIG. 8 is a scattergram showing the distribution of individual cells using green fluorescence intensity and red fluorescence intensity as coordinate axes according to the erythroblast classification and counting method of the present invention.

FIG. 9 is a schematic diagram of FIG. 1;

FIG. 10 is a diagram showing a correlation between the number of stage II erythroblasts measured by the method and the method of the present invention.

FIG. 11 is a diagram showing a correlation between the number of stage III erythroblasts measured by the method and the method of the present invention.

[Explanation of symbols]

 WBC whole leukocyte NRBC erythroblast leu leukocyte Gho ghost Ly lymphocyte Mo monocyte Gran granulocyte

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tomoyuki Tsuji 1-5-1 Wakihama Kaigandori, Chuo-ku, Kobe City Inside Sysmex Corporation (72) Inventor Takashi Sakata 1-5-1, Wakihama-kaigandori, Chuo-ku, Kobe City No. 1 Sysmex Corporation (72) Inventor Yukio Hamaguchi 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe City Sysmex Corporation

Claims (12)

[Claims] (1) adding a fluorescently labeled antibody that specifically binds to leukocytes to a hematological sample to fluorescently stain leukocytes; and (ii) obtaining a fluorescence spectrum that can be distinguished from the fluorescence of the fluorescently labeled antibodies. (Iii) fluorescently stain nuclei of erythroblasts with a nucleic acid fluorescent dye, and (iv) then obtain the obtained sample. Is subjected to a flow cytometer to measure at least two fluorescence signals of individual cells, and (v) classification and counting of erythroblasts from the difference in fluorescence intensity. 2. The fluorescently labeled antibody that specifically binds to leukocytes in step (i) is a fluorescently labeled antibody that recognizes an antigen expressed on the surface of leukocytes and binds to the antigen.
The erythroblast classification and counting method described in the above. 3. The method of claim 3, wherein the fluorescently labeled compound of the fluorescently labeled antibody in step (i) is phycoerythrin, fluorescein isothiocyanate, allophycocyanin, Texas Red, CY5,
The erythroblast classification and counting method according to any one of claims 1 to 4, wherein the method is at least one compound selected from the group consisting of a peridinin chlorophyll complex and a phycoerythrin conjugate thereof. 4. In the step (ii), the step of enhancing the cell membrane permeability of the nucleic acid fluorescent dye which does not normally permeate the cell membrane only by erythroblasts is performed by adding an acidic pH to the hematological sample obtained in the step (i). A low osmotic pressure first solution comprising a buffer for maintaining the blood solution is added and mixed to neutralize the first solution containing the hematological sample, and the solution p
2. The method according to claim 1, comprising adding and mixing a second liquid comprising a buffer for adjusting H to a pH suitable for staining and an osmotic pressure adjusting agent for adjusting the osmotic pressure to maintain the form of leukocytes. Erythroblast classification and counting method. 5. The method for classifying and counting erythroblasts according to claim 1, wherein the step (iii) of staining the nuclei of erythroblasts is a step of mixing with a nucleic acid fluorescent dye. 6. The nucleic acid fluorescent dye of the step (ii) comprising propidium iodide, N-methyl-4- (1-pyrene) vinyl-propidium iodide, ethidium bromide,
TOTO-1, TOTO-3, YOYO-1, YOYO-3, BOBO-1, BOBO-3, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2), POPO-1, POPO- 3, BO-PRO-1,
The erythroblast classification and counting method according to any one of claims 1 to 5, wherein the method is at least one kind of dye selected from the group consisting of YO-PRO-1 and TO-PRO-1. 7. A two-dimensional distribution is obtained from the measured fluorescence signals of individual cells, with the fluorescence based on a fluorescently labeled antibody that specifically binds to leukocytes and the fluorescence based on a nucleic acid fluorescent dye as two axes, respectively. Item 7. The method for classifying and counting erythroid cells according to any one of Items 1 to 6. 8. The method for classifying and counting erythroblasts according to claim 1, wherein a distribution area of erythroblasts is set from the two-dimensional distribution, and the number of cells in the area is counted. 9. The erythroblasts for leukocytes are obtained by setting distribution areas of leukocytes and erythroblasts from the two-dimensional distribution, counting the number of cells in each area, and dividing the number of erythroblasts by the number of leukocytes. The method for classifying and counting erythroblasts according to any one of claims 1 to 7, wherein the ratio is determined. 10. The nucleic acid fluorescent dye is used in an amount of 0.003 mg / L or less.
The erythroblast according to claim 5, comprising staining the erythroblast according to the maturity using the concentration range of 10 mg / L, and classifying the erythroblast into at least two regions corresponding to the maturity of the erythroblast. Classification and counting method. (1) A two-dimensional distribution is obtained from the measured fluorescence signals of the individual cells, using fluorescence based on a fluorescently labeled antibody that specifically binds to leukocytes and fluorescence based on a nucleic acid fluorescent dye as two axes, respectively. (2) In the two-dimensional distribution, erythroblasts are separated by at least 2 due to the difference in fluorescence intensity based on the nucleic acid fluorescent dye.
The erythroblast classification and counting method according to claim 10, wherein erythroblasts having different maturity levels are classified by setting regions to be classified into groups and counting the number of cells in each region. 12. In the two-dimensional distribution, the number of cells in each region of all erythroblasts and at least two erythroblasts of different maturity is counted, and the number of cells of two erythroblasts of different maturity is counted. The erythroblast classification and counting method according to claim 11, wherein each ratio of erythroblasts having different maturity levels to all erythroblasts is obtained by dividing by the number of blasts, respectively.