JP4354982B2 - Leukocyte classification and counting method - Google Patents

Description

  The present invention relates to a method for counting and counting leukocytes. More specifically, the present invention relates to a method for classifying and counting leukocytes of a hematological sample using a flow cytometer.

  In recent years, various fully automatic blood cell classification and counting devices applying the principle of a flow cytometer have been provided. However, these devices can classify and count normal white blood cells with high accuracy, but are greatly affected when platelet aggregation occurs.

  On the other hand, the immature leukocytes are kept alive, treated with a hemolytic agent that damages other leukocytes, and then stained with a fluorescent dye that can stain the damaged cells. It has been reported that immature leukocytes and normal leukocytes can be classified and counted simultaneously by measuring (see Patent Document 1).

JP-A-10-206423

However, Patent Document 1 does not describe at all about the separation of leukocytes and platelet aggregation.
In view of the above circumstances, the present invention provides a leukocyte classification and counting method for measuring leukocytes quickly, simply and with high accuracy.

The present invention
(1) treating a hematological sample with a hemolyzing agent for lysing red blood cells;
(2) introducing the treated sample into a flow cytometer and measuring at least scattered light;
(3) A leukocyte classification and counting method comprising a step of classifying platelet aggregation and leukocytes using the difference in forward scattered light width.

  According to the present invention, leukocytes can be measured quickly, simply and with high accuracy.

  The hematological sample referred to in the present embodiment refers to a body fluid sample containing leukocytes, such as a sample collected from peripheral blood, bone marrow puncture fluid, urine and the like.

  The mature leukocytes referred to in the present embodiment refer to mature lymphocytes, monocytes, and granulocytes.

  A leukocyte with an abnormal DNA amount refers to a leukocyte having a DNA amount greater or smaller than a normal DNA amount. However, in the present embodiment, a leukocyte with abnormal DNA amount refers to a leukocyte having a DNA amount larger than a normal DNA amount.

  The immature leukocytes referred to in the present embodiment are immature leukocytes that are usually present in bone marrow and do not appear in peripheral blood. For example, it refers to myeloblast, promyelocyte, myelocyte, posterior myelocyte and the like. Promyelocytes, myelospheres, and retromyelocytes may be collectively referred to as granulocyte immature spheres. Furthermore, cells of differentiation stage before blasts such as myeloid stem cells (CFU-GEMN), neutrophil / macrophage colony forming cells (CFU-GM), eosinophil colony forming cells (CFU-EOS), etc. Leukocyte hematopoietic progenitor cells are also included in the range of immature leukocytes of this embodiment.

  In the present embodiment, platelet aggregation refers to aggregation of two or more platelets.

  The simultaneous passage cell in this embodiment means a state in which two or more cells pass through the detection area of the flow cell almost simultaneously and are counted as one cell.

  In the present embodiment, the scattered light peak is a peak of a signal waveform obtained from scattered light, and the scattered light width is the width of a signal waveform obtained from scattered light.

  In this embodiment, a hematological sample is treated with a hemolytic agent to lyse red blood cells. On the other hand, immature leukocytes are not lysed or damaged by this treatment, and mature leukocytes and leukocytes with abnormal DNA amount are damaged. When a hemolytic agent of a specific composition is allowed to act on cells, the mechanism of action is not clear, but a specific substance passes through the cell membrane by extracting (pulling out) some of the cell membrane lipid components of the specific cell. Open as many pores as possible. This is called damage. As a result, dye molecules can enter and stain in specific cells. Therefore, damaged mature leukocytes and leukocytes with abnormal DNA amount are in a state suitable for staining. On the other hand, immature leukocytes that are not damaged are not stained with a dye because pores that allow the dye to penetrate are not formed. Furthermore, mature leukocytes and leukocytes with abnormal DNA amount differ in the amount of dye binding depending on the amount of DNA contained in the cells. Therefore, when such cells are stained with a dye, the amount of dye dyed differs depending on the amount of DNA, and the fluorescence intensity of the stained cells differs. For example, in the case of a leukocyte with an abnormal DNA amount that is twice the amount of mature leukocytes, the amount of dye that binds is twice that of normal mature leukocytes, and these cells emit fluorescence stronger than mature leukocytes. As a result, a difference in fluorescence intensity may occur between mature leukocytes, leukocytes with abnormal DNA amount and immature leukocytes.

  The hemolytic agent used in the present embodiment preferably comprises a surfactant, a solubilizer, an amino acid, and a buffer.

  Various surfactants can be used, but polyoxyethylene nonionic surfactants are preferred. Specifically, the following formula (II):

（式中、Ｒ^1IIはＣ_9-25のアルキル基、アルケニル基又はアルキニル基；

Ｒ^2IIは、−Ｏ−、

または−ＣＯＯ−；ｎ_IIは１０〜４０である。）を有するものを用いることができる。

^Wherein R ^1II is a C _9-25 alkyl group, alkenyl group or alkynyl group;

R ^2II is —O—,

Or -COO-; n _II is 10-40. ) Can be used.

_Examples of the C _9-25 alkyl group include nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl and the like. Examples of the C _9-25 alkenyl group include dodecenyl, tetradecenyl and the like. _Examples of the C _9-25 alkynyl group include dodecinyl, undecynyl, dodecynyl and the like.

  More specifically, polyoxyethylene (20) lauryl ether, polyoxyethylene (15) oleyl ether, and polyoxyethylene (16) oleyl ether are suitable.

The surfactant can be used in the form of an aqueous solution. For example, the concentration of polyoxyethylene-based nonionic surfactant in water varies depending on the type of surfactant used, but in the above-mentioned polyoxyethylene (20) lauryl ether, 0.1 to 2.0 g / l (preferably 0.5 to 1.5 g / l), 1 to 9 g / l (preferably 3 to 7 g / l) for polyoxyethylene (15) oleyl ether, 5 to 50 g for polyoxyethylene (16) oleyl ether / L (preferably 15 to 35 g / l). If the number of carbons of the hydrophobic group is the same, the polyoxyethylene-based nonionic surfactant has a stronger ability to damage cells as the number of n ^II decreases, and becomes weaker as n ^II increases. Further, if the number of n ^II is the same, the power to damage cells as the number of carbon atoms in the hydrophobic group becomes small becomes stronger. In consideration of this point, the necessary surfactant concentration can be easily determined by experiment using the above values as a guide.

Solubilizers are used to damage and shrink the cell membrane of blood cells. Specifically:
Formula (III):

（式中、Ｒ^1IIIはＣ_10-22のアルキル基；ｎ^IIIは１〜５である。）
のサルコシン誘導体あるいはその塩、
式（ＩＶ）：

( ^Wherein R ^1III is a C _10-22 alkyl group; n ^III is 1 to 5)
Sarcosine derivatives or salts thereof,
Formula (IV):

（式中、Ｒ^1IVは水素原子または水酸基。）
のコール酸誘導体、および
式（Ｖ）：

(In the formula, R ^1IV is a hydrogen atom or a hydroxyl group.)
A cholic acid derivative of formula (V):

（式中、ｎ^Vは５〜７である。）
メチルグルカンアミドから選択される１またはそれ以上を用いることができる。

(Wherein, n ^V is 5-7.)
One or more selected from methyl glucanamide can be used.

_Examples of the C _10-22 alkyl group include decyl, dodecyl, tetradecyl, oleyl and the like.

Specifically, sodium N-lauroyl sarcosinate, sodium lauroylmethyl β-alanine, lauroyl sarcosine, CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate), CHAPSO ([(3 -Colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate),
MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide), MEGA10 (decanoyl-N-methylglucamide) and the like can be preferably used.

  The concentration of the solubilizer is 0.2 to 2.0 g / L for sarcosine acid derivatives or salts thereof, 0.1 to 0.5 g / L for cholic acid derivatives, and 1.0 to 8 for methyl glucanamide. 0.0 g / L is preferred.

  In addition, n-octyl β-glucoside, sucrose monocaprate, N-formylmethylleucylalanine, and the like can be used as the solubilizer, and it is preferably used at a concentration of 0.01 to 50.0 g / L.

  Amino acids are used to immobilize the cytoplasm and cell membrane of immature leukocytes. For example, amino acids constituting the protein can be used, and glutamic acid, valine, and particularly sulfur-containing amino acids such as methionine, cystine and cysteine are preferred, and methionine is most preferred. The amino acid can be used in the range of 1 to 50 g / l. In the case of glutamic acid, 8 to 12 g / l is preferable, and in the case of methionine, 16 to 24 g / l is preferable.

  For the buffer solution, a pH adjuster such as sodium hydroxide is added to Good buffer such as HEPES or phosphate buffer, and if necessary, an osmotic pressure adjustor such as sodium chloride is further added to adjust the pH. It is preferable that the pressure is 5.0 to 9.0 and the osmotic pressure is 150 to 600 mOsm / kg.

The hemolytic agent of this embodiment includes (1) a polyoxyethylene nonionic surfactant; (2) a solubilizer for damaging and reducing the cell membrane of blood cells; (3) amino acids; and (4) a solution. PH 5.0-9.0, buffer solution with osmotic pressure 150-600 mOsm / kg, electric conductivity 6.0-
A hemolytic agent described in JP-A-6-273413 comprising a buffer solution for adjusting to 9.0 mS / cm is preferred.

  The fluorescent dye capable of producing a difference in fluorescence intensity between mature leukocytes, leukocytes with abnormal DNA amount and immature leukocytes of the present embodiment is one that can stain either damaged cells or immature leukocytes. That's fine. A dye that stains damaged cells is preferred. Such a fluorescent dye can stain all cells including blood cells in the sample.

  Examples of the dye for staining damaged cells include a dye having specificity for cell nuclei, particularly DNA, or a dye having specificity for RNA. Several cationic dyes are suitable for this purpose.

  In general, cationic dyes pass through the cell membrane of living cells and stain intracellular components. However, it is well known that certain cationic dyes (eg, ethidium bromide, propidium iodide, etc.) do not pass through living cells and stain only damaged cells.

  Specific examples of the fluorescent dye include ethidium bromide, propidium iodide, ethidium-acridine heterodimer, ethidium azide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide sold by Molecular Probes, Inc. Examples include TOTO-1, TO-PRO-1, TOTO-3, TO-PRO-3, and the like. Furthermore, as a dye suitable for using He—Ne as a light source and a red semiconductor laser, the formula (I):

（式中、Ｒ^1Iは水素原子または低級アルキル基；Ｒ^2I及びＲ^3Iは水素原子、低級アルキル基または低級アルコキシ基；Ｒ^4Iは水素原子、アシル基または低級アルキル基；Ｒ^5Iは水素原子または置換されてもよい低級アルキル基；Ｚは硫黄原子、酸素原子または低級アルキル基で置換された炭素原子；ｎ^Iは１または２；Ｘ^I-はアニオンである。）で示される色素が使用できる。

(Wherein R ^1I is a hydrogen atom or a lower alkyl group; R ^2I and R ^3I are a hydrogen atom, a lower alkyl group or a lower alkoxy group; R ^4I is a hydrogen atom, an acyl group or a lower alkyl group; R ^5I is a hydrogen atom or A lower alkyl group which may be substituted; Z is a carbon atom substituted with a sulfur atom, an oxygen atom or a lower alkyl group; n ^I is 1 or 2; and X ^I- is an anion.) .

The lower alkyl group for R ^1I in the above structural formula means a C _1-6 linear or branched alkyl group, and is a methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl group. Among them, a methyl group and an ethyl group are preferable.

The lower alkyl group in R ^2I and R ^3I is the same as described above, and the lower alkoxy group means a C _1-6 alkoxy group, and examples thereof include methoxy, ethoxy, propoxy and the like, among which methoxy group, ethoxy group Is preferred.

The acyl group in R ^4I is preferably an acyl group derived from an aliphatic carboxylic acid, and examples thereof include acetyl and propionyl. Among them, an acetyl group is preferable. The lower alkyl group is the same as described above.

The lower alkyl group for R ^5I is the same as described above, and the optionally substituted lower alkyl group is a lower alkyl group which may be substituted with 1 to 3 hydroxyl groups, a halogen atom (fluorine, chlorine, bromine or iodine) and the like. An alkyl group means a methyl group or ethyl group substituted with one hydroxyl group.

  The lower alkyl group for Z is the same as described above, and Z is preferably a sulfur atom.

Anions in X ^I− are halogen ions (fluorine, chlorine, bromine or iodine ions), boron halide ions (BF ₄ ⁻ , BCl ₄ ⁻ , BBr ₄ ⁻ etc.), phosphorus compound ions, halogen oxygenate ions, fluorosulfuric acid Examples thereof include ions, methyl sulfate ions, and tetraphenylboron compound ions having a halogen or an alkyl group having a halogen in the aromatic ring as a substituent. Of these, bromine ion or BF ₄ ^- is preferable.

  These dyes can be used alone or in combination of two or more. Specific examples of the above-mentioned dyes include the following dyes, but the present embodiment is not limited thereby.

  A preferred embodiment of the step of treating the hematological sample is to mix the solution containing the hemolytic agent and the fluorescent dye with the hematological sample. Alternatively, the fluorescent dye may be dissolved in a water-soluble organic solvent such as ethylene glycol and mixed with a hemolytic agent at the time of use. In this case, it is preferable because the storage stability of the dye can be improved. The dye concentration can be appropriately determined according to the dye used. For example, in the case of ethidium bromide, 0.01-100 mg / l, preferably 0.1-30 mg / l can be used.

  In the mixing of the hematological sample and the hemolytic agent containing the fluorescent dye, the ratio of the hematological sample to the hemolytic agent containing the fluorescent dye is 1:10 to 1: 1000, the reaction temperature is 20 to 40 ° C., and the reaction time is 5 It can be suitably carried out in seconds to 5 minutes. When the reaction temperature is high, it is preferable to shorten the reaction time. In the present embodiment, the amount of DNA in a sample containing immature leukocytes can be measured by increasing the reaction time in order to stain immature leukocytes. In this case, it is preferable to use a reaction time of, for example, about 10 seconds to 5 minutes.

The measurement sample thus prepared is introduced into a flow cytometer, and the scattered light and fluorescence are measured for each of the stained cells in the sample.
FIG. 18 is a perspective view showing an optical system of a flow cytometer that can be used in this embodiment. In the figure, the beam emitted from the laser 21 irradiates the orifice portion of the sheath flow cell 23 through the collimator lens 22. Forward scattered light emitted from blood cells passing through the orifice part is incident on the photodiode 26 via the condenser lens 24 and the pinhole plate 25.

  On the other hand, for side scattered light and side fluorescence emitted from blood cells passing through the orifice, the side scattered light passes through a condenser lens 27 and a dichroic mirror 28, and is a photomultiplier tube (hereinafter referred to as photomal). The side fluorescence enters the photomultiplier 31 through the condenser lens 27, the dichroic mirror 28, the filter 29, and the pinhole plate 30.

  The forward scattered light signal output from the photodiode 26, the side scattered light signal output from the photomultiplier 29, and the side fluorescent light signal output from the photomultiplier 31 are amplified by amplifiers 32, 33, and 34, respectively. And input to the analysis unit 35.

  Scattered light as used in this embodiment refers to scattered light that can be generally measured with a commercially available flow cytometer, and includes forward low angle scattered light (less than 0 to 5 degrees as an example of the light receiving angle), forward high angle scattered light (light receiving angle). As an example of this, side scattered light etc. are mentioned, Preferably side scattered light is chosen for forward low angle scattered light and further scattered light. Side scattered light reflects internal information such as the nuclear morphology of the cell.

  For fluorescence, a suitable light receiving wavelength is selected depending on the dye to be used. The fluorescent signal reflects the cytochemical properties.

  The light source of the flow cytometer is not particularly limited, and a light source having a wavelength suitable for excitation of the dye is selected. For example, an argon laser, a He—Ne laser, a red semiconductor laser, a blue semiconductor laser, or the like is used. In particular, a semiconductor laser is very inexpensive as compared with a gas laser, and the apparatus cost can be greatly reduced.

  Next, using the difference in the intensity of the scattered light peak and the difference in the scattered light width, the platelet aggregation and simultaneous passing cells and the white blood cells are classified. Specifically, for example, a scattergram is created by taking the forward scattered light width on the X axis and the forward scattered light peak on the Y axis. In the scattergram, for example, as shown in FIG. 1, platelet aggregation and co-passage cells, and leukocytes and ghosts form a population and are distributed. Exclude platelet aggregation and co-passage cells from all cells of this scattergram to classify platelet aggregation and co-passage cells and leukocytes and ghosts. By this operation, platelet aggregation and simultaneously passing cells are prevented from appearing in the region of leukocytes with abnormal DNA amount, and it becomes possible to accurately classify and count leukocytes with abnormal DNA amount.

  Next, using the difference in the intensity of scattered light and the difference in fluorescence of the classified leukocytes, classification and counting are performed on mature leukocytes, leukocytes with abnormal DNA amount, and immature leukocytes. Specifically, for example, taking the fluorescence intensity on the X-axis and the forward scattered light intensity on the Y-axis, a scattergram is created only from a group of blood cell components excluding the above-mentioned platelet aggregation and simultaneously passing cells. In the scattergram, for example, as shown in FIG. 2, each group of mature leukocytes, abnormal DNA amount cells and immature leukocytes and erythrocyte ghosts form a population and are distributed. Then, an area of each group is set using appropriate analysis software, and cells included in the area are classified and counted. In this way, the mature white blood cell count, immature white blood cell count and DNA amount abnormal white blood cell count can be obtained.

  Further, the ratio of mature leukocytes or immature leukocytes to leukocytes with abnormal DNA amount is calculated from the number of leukocytes with abnormal DNA amount and the number of mature leukocytes or immature leukocytes.

  Further, the ratio of immature leukocytes to mature leukocytes is calculated from the mature leukocyte count and immature leukocyte count.

  Further, different types of scattered light are further measured with a flow cytometer, and a scattergram is created using the intensity difference between the scattered light and fluorescence for mature leukocytes. For example, taking the red fluorescence intensity on the X axis and the side scattered light intensity on the Y axis, a scattergram as shown in FIG. 3 is created. In the scattergram, at least three such as lymphocytes, monocytes and granulocytes form a population and are distributed. Then, an area of each group is set using appropriate analysis software, and cells included in the area are classified and counted. As a result, it is possible to classify and count into lymphocytes, monocytes and granulocytes.

  Further, different types of scattered light are further measured with a flow cytometer, and a scattergram is created using the difference in intensity between the scattered light and fluorescence for immature leukocytes. For example, taking the red fluorescence intensity on the X axis and the side scattered light intensity on the Y axis, a scattergram as shown in FIG. 3 is created. In the scattergram, at least two, for example, myeloid blasts and granulocyte immature spheres form a population and are distributed. Then, an area of each group is set using appropriate analysis software, and cells included in the area are classified and counted. As a result, it is possible to classify and count into myeloid blasts and granulocyte immature spheres.

  The above-described step of classifying and counting mature leukocytes and the above-described step of classifying and counting immature leukocytes may be performed as separate steps or simultaneously. At the same time, when a scattergram is created using the intensity difference of the scattered light and the fluorescence intensity difference of the component from which the ghost has been removed, a scattergram as shown in FIG. 3 is obtained. It is preferable because immature leukocytes can be further classified and counted into a plurality of populations.

  The present invention will be described in more detail with reference to the following examples. However, various changes and modifications can be made to the present invention, and therefore the scope of the present invention is not limited by the following examples.

  A reagent comprising an aqueous solution having the following composition was prepared.

(This law)
Polyoxyethylene (16) oleyl ether 24.0 g
N-lauroyl sarcosinate sodium 1.5g
DL-methionine 20.0g
1N-NaOH 0.3g
NaCl 4.0g
Dye of formula (VI) 3.0 mg
HEPES 12.0g
Purified water 1000ml

  1 ml of the above-mentioned reagent was mixed with 33 μl of lymphocytic leukemia patient blood, and after 10 seconds, forward low angle scattered light, side scattered light, and red fluorescence were measured (light source: red semiconductor laser, wavelength: 633 nm). ).

(Standard measurement method of DNA amount)
Citric acid 3Na 100mg
Triton X-100 (Wako Pure Chemical Industries, Ltd.) 0.2g
Propidium iodide (Sigma) 0.2g
RO water 100ml

  1 ml of the above reagent and 100 μl of the above patient's blood were mixed, and after 30 minutes, red fluorescence was measured with a flow cytometer (light source: argon ion laser, wavelength: 488 nm).

Scattergram (Fig. 4) with the forward low angle scattered light width on the X axis, the forward low angle scattered light peak on the Y axis, the red fluorescence intensity on the X axis, and the forward low angle scattered light on the Y axis. A scattergram (FIG. 5) taking the intensity and a scattergram (FIG. 6) taking the side scattered light intensity on the X axis and the red fluorescence intensity on the Y axis are shown.
The blood was stained with May-Grünwald and then visually observed with a microscope. Leukocytes were classified as lymphocytes, monocytes and granulocytes. In addition, the amount of leukocyte DNA was measured by the above-described blood using a standard method for measuring the amount of DNA using a flow cytometer. The obtained results are shown in Table 1 and FIG.

  As a result of the above, it was shown that the method of the present embodiment can measure leukocytes with abnormal DNA amount and immature leukocytes at the same time, quickly and easily with high accuracy comparable to visual observation.

  A reagent comprising an aqueous solution having the following composition was prepared.

(This law)
Polyoxyethylene (16) oleyl ether 24.0 g
N-lauroyl sarcosinate sodium 1.5g
DL-methionine 20.0g
1N-NaOH 0.3g
NaCl 4.0g
Dye of formula (VII) 3.0 mg
HEPES 12.0g
Purified water 1000ml

  1 ml of the above-mentioned reagent and 33 μl of blood from an acute myeloid leukemia (AML) patient were mixed, and after 10 seconds, forward low angle scattered light, side scattered light, and red fluorescence were measured with a flow cytometer (light source: red semiconductor laser, (Wavelength: 633 nm)

(Standard measurement method of DNA amount)
Citric acid 3Na 100mg
Triton X-100 (Wako Pure Chemical Industries, Ltd.) 0.2g
Propidium iodide (Sigma) 0.2g
RO water 100ml

  1 ml of the above reagent and 100 μl of the above patient's blood were mixed, and after 30 minutes, red fluorescence was measured with a flow cytometer (light source: argon ion laser, wavelength: 488 nm).

  Scattergram (Fig. 8) with the forward low angle scattered light width on the X axis, the forward low angle scattered light peak on the Y axis, the red fluorescence intensity on the X axis, and the forward low angle scattered light on the Y axis. A scattergram (FIG. 9) showing the intensity and a scattergram (FIG. 10) showing the side scattered light intensity on the X axis and the red fluorescence intensity on the Y axis are shown.

  The blood was stained with May-Grünwald and then visually observed with a microscope. Leukocytes were classified as lymphocytes, monocytes and granulocytes. The results are shown in Table 2.

  As a result, it was shown that the method of the present embodiment can measure leukocytes with abnormal DNA amount and immature leukocytes simultaneously, quickly, simply and with high accuracy.

  A reagent having the same composition as in Example 1 was used. 1 ml of this method reagent and 33 μl of bone marrow fluid from patients with myelodysplastic syndrome were mixed, and after 7 and 13 seconds, forward low angle scattered light, side scattered light, and red fluorescence were measured with a flow cytometer (light source: red semiconductor) Laser, wavelength: 633 nm).

(Standard measurement method of DNA amount)
1 ml of the standard DNA amount measurement method reagent and 100 μl of the above-mentioned patient's bone marrow fluid were mixed, and after 30 minutes, red fluorescence was measured with a flow cytometer (light source: argon ion laser, wavelength: 488 nm). The scattergram in the case of a reaction time of 7 seconds with the forward low angle scattered light width on the X axis and the forward low angle scattered light peak on the Y axis (FIG. 11), the red fluorescence intensity on the X axis, Scattergram when the reaction time is 7 seconds with the forward low angle scattered light intensity on the axis (FIG. 12), side scattered light intensity on the X axis, and red fluorescence intensity on the Y axis when the reaction time is 7 seconds Scattergram (FIG. 13), scattergram when the reaction time is 13 seconds with the forward low angle scattered light width on the X axis and the forward low angle scattered light peak on the Y axis (FIG. 14), the red fluorescence intensity on the X axis, Scattergram when the reaction time is 13 seconds with the forward low angle scattered light intensity on the Y axis (FIG. 15), when the reaction time is 13 seconds with the side scattered light intensity on the X axis and the red fluorescence intensity on the Y axis The scattergram is shown in FIG.

  After the bone marrow fluid was stained with Meigrunwald, it was visually observed with a microscope. Leukocytes were classified as lymphocytes, monocytes and granulocytes. In addition, the amount of leukocyte DNA was measured using the above blood by a standard method for measuring the amount of DNA using a flow cytometer. The results are shown in Table 3. Table 4 and FIG. 17 show the results of the method according to this embodiment and the standard DNA amount measurement method with a reaction time of 13 seconds.

  From the above, when the reaction time is 7 seconds, mature leukocytes and immature leukocytes can be accurately measured. By setting the reaction time to 13 seconds, leukocytes with abnormal DNA amount contained in immature leukocytes can be detected. .

  As described above, according to this embodiment, leukocytes with abnormal DNA amount can be measured quickly and easily.

It is a conceptual diagram which shows the appearance position of each cell classified and counted by the method of this embodiment. It is a conceptual diagram which shows the appearance position of each cell classified and counted by the method of this embodiment. It is a conceptual diagram which shows the appearance position of each cell classified and counted by the method of this embodiment. FIG. 3 is a scattergram of Example 1 in which a front low angle scattered light width is taken on the X axis and a front low angle scattered light peak is taken on the Y axis. FIG. 3 is a scattergram of Example 1 in which red fluorescent intensity is taken on the X axis and forward low angle scattered light intensity is taken on the Y axis. 3 is a scattergram of Example 1 in which the X-axis represents side scattered light intensity and the Y-axis represents red fluorescence intensity. The results of this method and the standard method for measuring DNA amount are shown. It is a scattergram of Example 2 which took the front low angle scattered light width on the X-axis, and took the front low angle scattered light peak on the Y-axis. It is a scattergram of Example 2 which took the red fluorescence intensity on the X-axis and the front low angle scattered light intensity on the Y-axis. It is a scattergram of Example 2 which took the side scattered light intensity | strength on the X-axis and the red fluorescence intensity on the Y-axis. It is a scattergram in case of reaction time 7 second of Example 3 which took the forward low angle scattered light width on the X-axis and the forward low angle scattered light peak on the Y-axis. FIG. 6 is a scattergram when the reaction time is 7 seconds in Example 3 in which the X-axis represents red fluorescence intensity and the Y-axis represents forward low-angle scattered light intensity. FIG. 5 is a scattergram in a case where the reaction time is 7 seconds in Example 3 with the side scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. It is a scattergram in the case of the reaction time of Example 3 which took the forward low-angle scattered light width on the X-axis and the forward low-angle scattered light peak on the Y-axis and was 13 seconds. It is a scattergram in case of reaction time 13 seconds of Example 3 which took the red fluorescence intensity on the X-axis and the forward low angle scattered light intensity on the Y-axis. It is a scattergram in the case of the reaction time of Example 3 which took the side scattered light intensity | strength on the X-axis and the red fluorescence intensity on the Y-axis. It is a figure which shows the result of the method of this embodiment and the DNA amount standard measuring method of reaction time 13 second. It is a perspective view which shows the optical system of the flow cytometer which can be used by this embodiment.

Claims (7)

(1) a step of treating a hematological sample with a hemolytic agent for lysing red blood cells;
(2) introducing the treated sample into a flow cytometer and measuring at least scattered light;
(3) A method for classifying and counting leukocytes, comprising a step of classifying platelet aggregation and leukocytes using the difference in forward scattered light width.   The leukocyte classification and counting method according to claim 1, wherein in step (3), platelet aggregation and leukocytes are classified using a difference in forward scattered light width and an intensity difference between forward scattered light peaks.   The method according to claim 1, further comprising: (4) classifying and counting mature leukocytes into at least three subpopulations using the difference in intensity of side scattered light for the leukocytes classified in step (3). White blood cell classification and counting method.   The step (1) includes a step of treating a blood sample with a dye, the step (2) further includes a step of measuring the degree of staining, and in the step (4), the side of the leukocytes classified in the step (3) The method of classifying and counting leukocytes according to claim 3, wherein mature leukocytes are classified and counted into at least three sub-populations using the difference in intensity of the scattered light and the degree of staining.   The step (1) includes a step of treating a blood sample with a fluorescent dye, the step (2) further includes a step of measuring fluorescence, and in step (4), the side of the leukocytes classified in step (3) The method of classifying and counting leukocytes according to claim 3, wherein mature leukocytes are classified and counted into at least three sub-populations using the difference in intensity of the scattered light and the intensity of fluorescence.   6. The method for classifying and counting leukocytes according to claim 5, wherein the step (1) includes a step of treating the hematological sample with a hemolytic agent and then treating the sample treated with the hemolytic agent with a fluorescent dye. An orifice that allows the measurement sample prepared in the process of treating the hematological sample with a hemolyzing agent for lysing red blood cells, a light source that irradiates light to the orifice, and scattered light emitted from the orifice to A flow cytometer including a light receiving unit for receiving light;
A leukocyte classification and measurement apparatus comprising: an analysis unit that analyzes a measurement sample by a step of classifying platelet aggregation and leukocytes using a difference in forward scattered light width.

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