JPH0599919A - Leukocyte analyzing method - Google Patents

Abstract

PURPOSE:To realize the classification of five kinds of the normal leukocytes by using acridine orange fluorescence staining solution containing acridine orange fluorescence dyestuff, buffer solution whose pH is higher than physiological pH and physiological isotonic osmotic pressure reagent as analyzing fluorescence staining solution. CONSTITUTION:When leukocyte is stained with acridine orange staining agent, the leukocyte is bonded with neucleuses present in the leukocyte and granules present in cytoplasma. The green or red fluorescence is generated. The neucleus of the cell is mainly constituted of DNA. When the neucleus is bonded with the acridine orange, the green fluorescence indicating the maximum value in the vicinity of 530nm is generated. The granule present in the cytoplasma contains RNA. Therefore, the red fluorescence having the maximum value in the vicinity of 640nm is generated. The osmotic pressure of the acridine orange fluorescence staining solution is set in the vicinity of 290mosm/kg of physiological osmotic pressure. The pH of fluorescence solution is set at a value higher than the normal physiological pH. Thus, the five of the normal leukocytes can be classified. Furthermore, a small amount of erythrocyte hemolysis agent is added into the staining solution. Thus, the surface of the cell cytoplasmic membrane of the leukocyte is changed. In this way, the fluoresence intensity is increased at normal temperature and the staining time is shortened.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a leukocyte analysis method, and more particularly to a leukocyte analysis method for classifying leukocytes among hematology test items in hospitals.

[0002]

2. Description of the Related Art A white blood cell classification method will be described. Normal,
There are five types of normal white blood cells as neutrophils, eosinophils, basophils, monocytes, and lymphocytes as normal white blood cells. Among them, neutrophils, eosinophils and basophils are collectively called granulocytes. These five types of white blood cells are normal white blood cells that normally appear in peripheral blood. Besides these normal white blood cells,
Abnormal blood cells that do not usually appear in peripheral blood include atypical lymphocytes, immature granulocytes, blasts and the like.

Conventionally, blood image examinations such as white blood cell classification have been performed in the form of observing and classifying blood cell morphology by human visual observation under a microscope. Since this method is performed based on a person's judgment by microscopic observation, the classification processing speed is slow and eye fatigue is large. Furthermore, there are problems such as a large statistical error due to the difference in classification and interpretation between the laboratory technicians and the small number of blood cells to be processed.

Therefore, an automatic blood image processing apparatus is proposed. A method used in a conventional typical automatic processing apparatus is a blood cell classification method by a pattern recognition method of a microscope image. In this method, a TV image pickup device is used instead of human eyes, a microscopic image is converted into an electric signal, and image processing / pattern recognition processing is performed by a computer or the like. The processing speed is faster than that of humans, but the number of processed blood cells cannot be increased dramatically. For blood sample preparation, it takes 20 to 30 minutes for pretreatment with May-Giemsa staining, etc.
The overall processing efficiency is not much different from human processing. The statistical error due to the small number of treated blood cells is as large as the case of visual inspection, especially in the examination of a few blood cells. Examples of the minority blood cells include normal blood cells such as monocytes, eosinophils and basophils, and abnormal blood cells such as atypical lymphocytes, immature granulocytes and blasts.

In consideration of the above problems, recently, a method typified by a flow cytometer, that is, a blood cell is irradiated with a laser beam and signals of light scattering, light transmission, and fluorescence intensity from the blood cell are used. A flow system has been proposed in which blood cell classification is performed from these parameters by adding electrical resistance information by a blood cell counter. Regarding these devices, JP-A-49-89390 and JP-A-61-8
8896, Tokushusho No. 61-502277, JP-A-54
There are prior documents such as -14957.

The features of these devices are that the hemocyte suspension is flown into the flow cell and processed using scattered information and fluorescence information from each hemocyte, so the number of hemocytes treated is very large and the statistical error is small. In addition, there are advantages that the pretreatment time is short, the reproducibility is good, and the result is well correlated with visual observation.

[0007]

However, the white blood cell classification method by the above-mentioned flow method has the following problems. In the white blood cell classification method that uses electrical impedance, light scattering parameters, and the like, it is difficult to identify a subtle difference between blood cells because it uses blood cell size information and the overall electrical properties of blood cells. In particular, basophils, eosinophils, and monocytes may be difficult to distinguish from lymphocytes or neutrophils. Moreover, the classification accuracy of these minority blood cells may be worse than the statistically expected accuracy.
Furthermore, the abnormal classification ratio of immature granulocytes, atypical lymphocytes, etc. cannot be known from abnormal spheres. In addition, this method requires a special reagent, and requires devising a method for preparing and analyzing the reagent. Therefore, in the above-mentioned flow method white blood cell classification method, in order to improve the above problems of the flow method, a method is proposed in which blood cells are dyed in advance with a fluorescent dye and white blood cell classification is performed based on the magnitude of fluorescence intensity by laser light. .. Acridine orange is a typical fluorescent dye. In this method, the difference in the amount of fluorescent dye incorporated into cytoplasmic RNA or nuclear DNA depending on the blood cell type is examined from the magnitudes of green and red fluorescence intensities to classify blood cells. According to this method, the difference in the cell membrane permeability of the fluorescent dye also affects, so that the difference in the blood cell types of white blood cells can be clearly known. Regarding this method, a method of using physiological pH and a hypotonic solution and measuring before the dye absorption reaches equilibrium is disclosed in JP-A-50-75473 (Japanese Patent Publication No. 59-8).
No. 53), and a method of using an isotonic solution under physiological conditions as a staining solution is disclosed in JP-A-47-3049. However, in the case of using the above-mentioned isotonic solution, there is a problem that leukocytes can be classified into only three types, granulocytes, monocytes and lymphocytes. On the other hand, when the hypotonic solution is used, the white blood cells are 5 per normal blood cell.
Although it is possible to classify and detect juvenile granulocytes, there were the following problems only with hypotonic staining conditions. (1) White blood cells may be damaged. (2) The red fluorescence intensity is generally weaker than the green fluorescence intensity, and the dye is partially absorbed by red blood cells to emit red fluorescence, which interferes with the fluorescence signal from white blood cells. (3) Since the ratio of white blood cells to red blood cells is 500: 1 and the dilution ratio of the staining solution is small, the simultaneous passage of red blood cells is very large, and the presence of these red blood cells affects the fluorescence signal from the white blood cells. (4) As a result, in particular, it may not be possible to correctly distinguish between monocytes and lymphocytes. In view of the above problems, an object of the present invention is to achieve classification of 5 types of normal leukocytes even with a leukocyte classification method using an isotonic solution, and further to provide abnormal spheres, particularly atypical lymphocytes and immature granules. It is an object of the present invention to provide a leukocyte analysis method which enables classification of spheres and detection of specimens having platelet aggregation.

[0008]

The leukocyte analysis method according to the present invention is a flow-type leukocyte analysis method utilizing laser light, which is (1) acridine orange as an analytical fluorescent staining solution for previously staining leukocytes. Fluorescent dye (2) Buffer solution whose pH is higher than physiological pH (3) Acridine orange fluorescent staining solution containing a reagent consisting of physiological isotonic osmotic pressure is used, and the fluorescence signals of green fluorescence and red fluorescence from white blood cells are detected. The feature is that white blood cells in blood are classified according to size.

As described above, the staining agent used in the leukocyte analysis method of the present invention is acridine orange, which is generally known as a metachromatic fluorescent dye. When leukocytes are stained with this stain, they bind to the nuclei present in leukocytes and the granules present in the cytoplasm to generate green or red fluorescence. The cell nucleus is mainly composed of DNA, and when it binds to acridine orange, it emits green fluorescence having a maximum value around 530 nm. On the other hand, since the granules present in the cytoplasm contain RNA, they emit red fluorescence having a maximum at around 640 nm. As a laser light source, an argon ion laser having an oscillation line near the absorption maximum wavelength of 480 nm of acridine orange is suitable.

As a solution to the classification of the five types of normal white blood cells which is the first problem, the osmotic pressure of the acridine orange fluorescent staining solution is set to a physiological osmotic pressure of about 290 mosm / kg, and the pH of the staining solution is adjusted to physiological. It was found that five types of normal white blood cells can be classified by setting the pH higher than the normal normal pH (8.4 to 8.9).

Furthermore, by adding a small amount of an erythrocyte hemolyzing agent to the above staining solution, the cell membrane surface of leukocytes is changed,
As a result of having the effect of promoting the absorption of the staining solution into blood cells, it became possible to increase the fluorescence intensity and shorten the staining time even at room temperature. As the hemolytic agent, for example, saponin or the like is used, and the addition ratio is 0.025 to 0.035% by weight.

In order to eliminate the influence of the simultaneous passage of a large amount of red blood cells on the white blood cell signal, which is another problem, the red blood cell is destroyed by the action of the red blood cell hemolyzing agent. The impact could be reduced. As a result, the problematic separation of monocytes and lymphocytes is improved, and the classification accuracy and reproducibility of these blood cells are improved. If the concentration is set to sufficiently lyse red blood cells, light scattering information from white blood cells can be used,
The identification accuracy improves as much as the classification parameter increases together with the fluorescence information. However, at higher concentrations, white blood cells will be affected, and the white blood cell classification itself will be affected.

The specific processing of white blood cell classification is as follows. First, fluorescence from blood cells is separated into green and red fluorescence by an optical filter, converted into an electric signal by a photodetector, and then classified by a data processing device. Next, when a two-dimensional cytogram concerning the green and red fluorescence intensity signals obtained from each blood cell is created, a pattern having a characteristic clathra for each blood cell is generated for five types of normal white blood cells. Each white blood cell is classified from this pattern.

Under the conditions of the staining solution according to the present invention, not only five normal white blood cell clusters but also juvenile granulocytes, atypical lymphocytes, etc. form clusters at their own positions. Will also be possible. In addition, a blood sample containing blast cells has its own cytogram, which is very different from the cytogram of normal white blood cells only.Therefore, it is necessary to find a blood sample containing abnormal lymphocytes as well as immature granulocytes and atypical lymphocytes. You can Furthermore, since a cytogram peculiar to platelet aggregation is generated in a blood sample with platelet aggregation, it is possible to detect a sample with platelet aggregation.

[0015]

In the leukocyte analysis method according to the present invention, since the isotonic solution is used in the leukocyte classification method using the above-mentioned staining solution containing acridine orange as a main component, leukocyte destruction under hypotonic conditions that has been conventionally performed. It is possible to perform accurate white blood cell classification without causing Moreover, in the present invention, the normal white blood cells can be classified into 5 categories, whereas the conventional method could only classify into 3 categories under the isotonic condition.

Furthermore, by adding a small amount of an erythrocyte hemolyzing agent to the staining solution of the present invention, the fluorescence intensity is increased and the staining time is shortened. By reducing the influence of erythrocytes, monocytes and lymphocytes are reduced. It became easier to identify.

By using the staining solution of the present invention, not only five normal leukocytes but also abnormal lymphocytes and immature granulocytes can be classified. For blood specimens with platelet aggregation including blast cells, a two-dimensional cytogram different from the 5 types of normal leukocytes is generated, so blood specimens with abnormal spheres including the above-mentioned atypical lymphocytes and immature granulocytes are detected. It is possible to

[0018]

EXAMPLES Examples of the present invention will be specifically described below by classifying them into Examples 1 to 3 according to the contents of the invention. [Example 1] The following table shows a representative example of staining conditions for classifying white blood cells in the present invention. As the buffer solution, a sodium borate-hydrochloric acid buffer solution is used. The osmotic pressure of the dyeing solution is adjusted to 290 mosm / kg, which is a physiological osmotic pressure. Physiologically, the pH of the dyeing solution is set to a value higher than pH by 1 or more, whereby the dyeing solution described in JP-A 47-
Under the physiological osmotic conditions of the analysis method disclosed in No. 3049, only three types of normal leukocytes could be classified,
With this composition, it became possible to classify 5 types of normal white blood cells.

[0019]

[Table 1]

The apparatus configuration for fluorescence measurement is the same as that of an ordinary flow cytometer, and the laser has an oscillation wavelength of 48.
An 8 nm argon ion laser is used. this is,
This is to match the absorption maximum wavelength of acridine orange. As for the fluorescence signal, the green fluorescence and red fluorescence wavelength bands described above are selected by an optical filter and converted into an electrical signal by a photodetector.

In the above-mentioned embodiment, five types of normal white blood cells can be classified essentially without adding a red blood cell hemolyzing agent. However, saponin was used in this example as the red blood cell hemolytic agent. This can improve the effects of scattering, absorption, and fluorescence by the red blood cells described above. The concentration of saponin is 0.025 ~ as shown in Table 1.
Addition in a proportion of 0.035% by weight has a great effect.

In FIG. 1, cytograms of five types of normal white blood cells based on green fluorescence and red fluorescence obtained in this example (FIG. 1 (a)) and changes in fluorescence cytogram depending on the presence or absence of a hemolytic agent (FIG. 1). (B) and (c) are shown. By comparing FIG. 1, it can be seen that the five types of normal white blood cells can be sufficiently classified without adding a hemolytic agent, and that the addition of the hemolytic agent improves the separation of each blood cell.

[Embodiment 2] With the red blood cell hemolyzing agent, the light scattering signal is detected only from white blood cells. The light-scattering signal uses forward scattered light and 90-degree direction side-scattered light. It is known that the forward scattered light corresponds to blood cell size information and the side scattered signal corresponds to cell internal information.
The white blood cell cytogram by the light scattering signal when using the staining solution satisfying the staining conditions of Table 1 was as shown in FIG. In FIG. 2, the horizontal axis represents side scatter and the vertical axis represents forward scatter. There are three clusters. That is, it is a pattern composed of three clusters of lymphocytes L and basophils B, monocytes M, eosinophils E and neutrophils N.

By using the cluster information based on the light scattering signal and the cytogram information based on the fluorescence described above, the classification accuracy of the five classifications of normal white blood cells can be improved. In particular, monocytes form independent clusters in the light-scattering signal, and lymphocytes and basophils overlap, but since the lymphocytes can be correctly identified from the fluorescence cytogram data, the number of basophils can be determined. You can know exactly.

[Embodiment 3] In the fluorescence cytogram of white blood cells shown in the first embodiment, not only the five classifications of normal white blood cells but also the presence of abnormal spheres indicates that these abnormal spheres also exist at a specific position. Was found. Fig. 3 shows typical characteristic patterns of abnormal spheres in the white blood cell fluorescence cytogram.
Shown in. In FIG. 3, (a) is an atypical lymphocyte (AT
L) shows an appearance example, (b) shows an immature granulocyte (IMG) appearance example, and (c) shows a platelet aggregation appearance example.

As shown in FIG. 3 (a), the atypical lymphocytes are
It is distributed in the upper part of normal lymphocytes (the place indicated by the dashed circle), that is, in the position where the green fluorescence is larger. From this fact, the number of atypical lymphocytes can be determined by cutting out the upper part of the lymphocyte position of green fluorescence intensity by data processing.

As shown in FIG. 3 (b), immature granulocytes are distributed on the right side of eosinophils (the place indicated by the ellipse of the broken line) and at the position of large red fluorescence. Therefore, the number of immature granulocytes can be known by considering immature granulocytes as a region having a green fluorescence smaller than that of neutrophils and a red intensity higher than that of eosinophils.

With regard to these atypical lymphocytes or immature granulocytes, it was confirmed that the results of discrimination by the actual microscope and the results of discrimination agree with each other within statistical error.

As shown in FIG. 3 (c), in a blood sample in which platelet aggregation is present, cytograms different from the normal pattern rising from the vicinity of the origin appear to overlap. This aggregation pattern can be detected by processing with a pattern recognition method or the like. Similarly, even in a blood sample in which abnormal blast cells have appeared, a pattern different from that of normal white blood cell clusters usually appears, and an abnormal sample can be detected by a pattern recognition method.

In the above-mentioned Examples, saponin and the like are mainly described as buffers and hemolytic agents shown in Table 1,
It is clear that the present invention includes any buffering agent or hemolytic agent having the same effect in other reagents. In addition, the present invention includes a cell analysis reagent in which reagents according to the leukocyte analysis method according to the present invention are mixed, and cell analysis can be performed using this reagent in a cell analyzer. Therefore, the present invention is not limited to the classification of white blood cells but can be generally applied as a cell analysis method.

[0031]

As is apparent from the above description, the present invention has the following effects. (1) Since the measurement can be performed under physiological isotonic conditions,
Accurate classification of white blood cells with less damage to white blood cells. (2) Since the red blood cell hemolyzing agent is not affected by the interference of absorption, scattering, fluorescence, etc. by red blood cells, the differentiation of various white blood cell clusters is clear and the accuracy of blood cell classification is improved. (3) The staining time can be shortened and each leukocyte can be fractionated in a relatively short time after blood collection. (4) The fluorescence intensity increases. This improves signal detection capability and allows the use of low power lasers. (5) Since the red blood cells are hemolyzed, light scattering information can be used for white blood cell classification. (6) With the above effects, the five normal white blood cell classifications can be classified with high accuracy and reproducibility. (7) It is possible to classify atypical lymphocytes, immature granulocytes, which are abnormal white blood cells, and further, it is possible to detect abnormal blood cell samples including blast cells and platelet aggregation samples.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing the effects of leukocyte fluorescence cytogram and the presence or absence of a hemolytic agent.

FIG. 2 is an explanatory diagram showing an example of a white blood cell cytogram based on a light scattering signal.

FIG. 3 is an explanatory diagram showing an example of a cytogram in the case of a blood sample containing abnormal spheres.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinichi Sakuraba 882 Ichige, Katsuta-shi, Ibaraki Hitachi, Ltd. Naka factory (72) Inventor Norio Kaneko 882, Ichige, Katsuta-shi, Ibaraki Hitachi, Ltd. Naka factory (72) Inventor Nobuyuki Tada No. 882 Ichimo, Katsuta City, Ibaraki Hitachi Measurement Engineering Co., Ltd. (72) Inventor Hiroshi Oki No. 502, Jinmachicho, Tsuchiura City, Ibaraki Prefectural Institute of Mechanical Engineering Co., Ltd. (72) Inventor Isao Yamazaki 502 Jintamachi Machinery Research Institute, Tsuchiura City, Ibaraki Prefecture, inside the Hiritsu Seisakusho Co., Ltd. (72) Inventor Ryo Miyake 502 Jinmachicho, Tsuchiura City, Ibaraki Pref.

Claims (5)

[Claims] 1. A flow-type leukocyte analysis method using laser light, wherein (1) acridine orange fluorescent dye (2) a buffer having a solution pH higher than physiological pH, as an analytical fluorescent staining solution for previously staining leukocytes. Liquid (3) A white blood cell in blood is classified by using acridine orange fluorescent staining solution containing a reagent consisting of physiological isotonic osmotic pressure, and by the magnitude of the fluorescence signal from green fluorescence and red fluorescence from white blood cell. The method of leukocyte analysis. 2. The leukocyte analysis method according to claim 1, wherein the reagent contained in the acridine orange fluorescent staining solution is (1) acridine orange fluorescent dye concentration 0.014.
˜0.018 mM (2) solution pH 8.4 to 8.9 buffer (3) osmotic pressure 290 ± 10 mosm / kg. 3. The leukocyte analysis method according to claim 1, wherein a hemolytic agent is added to the acridine orange fluorescent staining solution. 4. The leukocyte analysis method according to claim 3, wherein saponin is used as the hemolytic agent, and the saponin is added in a proportion of 0.025 to 0.035% by weight. 5. Using the acridine orange fluorescent staining solution for leukocyte analysis according to claim 1, 5 types of normal leukocytes are classified, and atypical lymphocytes and juvenile lymphocytes are classified as abnormal spheres. A leukocyte analysis method characterized by classifying juvenile granulocytes and further detecting a blood sample having platelet aggregation.