JP2635142B2 - Leukocyte classification method and reagent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for classifying and measuring blood cells in the field of clinical examination, and more particularly to a method and a reagent for classifying, identifying, and counting leukocytes in blood.

BACKGROUND ART Leukocytes in peripheral blood of healthy individuals include lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Neutrophils, eosinophils, and basophils are collectively called granulocytes. These have different functions, and can contribute to the diagnosis of disease by counting leukocytes in blood by type.
For example, increased neutrophils are seen in inflammation, myocardial infarction, leukemia, etc., and decreased neutrophils are seen in viral diseases, aplastic anemia, agranulocytosis, etc. Increases in eosinophils are seen in parasitosis, Hodgkin's disease, allergic diseases, and the like. Increased monocytes are seen in remissions of infections, monocytic leukemia, and so on.

The above-mentioned lymphocytes, monocytes, neutrophils, eosinophils, and basophils are so-called normal cells, but in addition to these, abnormalities in peripheral blood in the case of certain blood diseases Cells may appear. For example, blasts may be found in the peripheral blood of patients such as leukemia. Although there are various types of abnormal cells, it is also clinically significant to classify the types and determine the number of cells in each abnormal cell population.

The most commonly used method for classifying and counting leukocytes has been called a blood image test (optic acid method).

In this method, blood is smeared on a slide glass, blood cells are fixed, stained, observed under a microscope, and the morphological characteristics of each leukocyte (white blood cell size, nuclear morphology, cytoplasmic morphology) , The presence or absence of granules) and the degree of staining, determine which blood cell the measurer is, classify and count. At this time, in a general laboratory, 100 to 200 white blood cells are counted, and the percentage (%) of each blood cell in the total number of white blood cells is used as a measured value.

This method involves smearing the blood,
A complicated sample preparation operation such as fixation and staining is required, and classification / counting using a microscope imposes a heavy burden on the observer due to the need to distinguish slight differences in blood cells. It has become. Furthermore, the number of white blood cells to be counted is small, and the blood cells on the smear sample may have an uneven distribution, so that it is difficult even for a skilled measurer to obtain a reproducible measurement value.

For this reason, there is a strong demand for a method capable of automatically classifying and counting leukocytes, and at present, two types of methods have been realized which are roughly divided.

One of the methods is to capture a blood cell image with a video camera or the like and classify the white blood cells by image processing by a computer. Although this method is similar in principle to the conventional calculation method, there are many blood cells that cannot be classified by computer processing, and it has not completely replaced the calculation method. There is also a problem that the device is complicated and large, and the price is high.

Another way to automatically classify and count leukocytes is
This is a method using a flow system. This method uses a sample in which blood cells are suspended in a diluent, flows the sample so that blood cells pass one by one through a thin detector, and analyzes the signal generated by the detector at this time to separate leukocytes. It is to be classified. The method using this flow system is further subdivided into two methods.

The first method is to destroy red blood cells with a lysing agent, flow an electrolyte in which only white blood cells are suspended into the pores, detect a change in impedance of the pores when the blood cells pass through the pores, Leukocytes are classified according to their size.

The second method includes a light source, a flow cell in which cells in a sample flow one by one in a thin channel, a photometric unit that detects light emitted from the cells, and an analyzer that analyzes a detection signal. A flow cysimeter provided is used. In this method, the blood cells are stained, the stained blood cells are irradiated with light, and then the fluorescence and possibly the scattered light emitted from the blood cells are detected together, and the white blood cells are classified according to the intensity of the detection signal. It is.

The second method has practical problems such as a complicated dyeing process being required and a complicated and expensive apparatus including an optical system.

One of the principles underlying the first method is disclosed in Japanese Patent No. 508789 and US Patent No. 3390326. This principle is based on the principle that particles suspended in a fluid medium having a different dielectric constant from the fluid pass through a fluid passage, and a pair of particles provided close to and opposed to each other across the passage in a narrow portion of the passage. This is to detect a change in the electrical impedance between the electrodes caused by the difference in the dielectric constant between the particles and the fluid medium when the particles pass between the electrodes forming the above.

A specific device utilizing this principle is described, for example, in Ichiro Kurokawa, et al .: Experience of Using a Toa Automatic Hematology Counter, Clinical Pathology, Vol. 16, pp. 251-255, 1968.
This device has a 3.5 MHz high-frequency oscillator, applies a high-frequency current to the detection circuit, and detects the change in the electrical impedance between the electrodes of the detection circuit that occurs when blood cells pass through the detection circuit in the suspension. It is to detect.

Measuring leukocytes using this TOA automatic hemocytometer is described, for example, in Ichiro Kurokawa, et al .: Clinical Laboratory, Vol. 11, 148
Pp. 151, 1967, in which saponin is added to a blood cell suspension to lyse erythrocytes, and the amount of residual leukocytes is measured.

There are various types of hemolytic agents other than saponin that lyse erythrocytes. Among them, CTAC (cetyltrimethylammonium chloride) and Tajitalmonionic NPX (Ter
gitolmonionic NPX) shows strong hemolytic power under the conditions used, but also invades the leukocyte protoplasm and almost becomes a naked nucleus. In contrast, saponins remain relatively intact in leukocytes. Therefore, the former two are unsuitable as hemolytic agents for models that detect changes in electrical impedance at high frequencies, such as the TOA automatic hemocytometer.
Kazuo Shintani: Problems in white blood cell count calculation, clinical examination, Vol. 12, 9
00-905, 1963.

By the way, when measuring the number of white blood cells using an automatic blood cell counter, the magnitude of the signal due to the white blood cells is sufficiently large compared to the size of the lysed red blood cell membrane (ghost) and the noise signal. It needs to be discriminated.
In order to confirm this condition, a cumulative particle size distribution curve as shown in FIG. 15 is often used. FIG. 15 shows the signal threshold of the automatic blood cell counter on the horizontal axis, and the number of detection signals above this signal threshold on the vertical axis. In FIG. 15, the part A is a so-called flat part in the cumulative particle size distribution curve, and is measured in a state where the white blood cell signal is sufficiently large compared to the noise signal and the ghost signal to obtain a stable white blood cell count. In this case, it is necessary to adjust the device and the reagent used for the device so that the flat portion of the portion A is extended.

However, when leukocytes were measured using a solution agent saponin in a TOA automatic hemocytometer, it was pointed out that the flat portion was shorter than the DC method described later. For example, Fig. 15 shows Yuji Takamori, et al .: MCC News Nos. 34, 12-2, on the causes of fluctuations in leukocyte particle size distribution and their effects on counts.
Page 1, Toa Special Electric Co., Hyogo, 1969 Dilution of saponin was added after dropping at room temperature for 5 minutes after dilution in Table 1 and the cumulative particle size distribution was measured. Only up to 400.

In this way, the reason why the flat part is short when measuring white blood cells using saponin in the Toa automatic hemocytometer,
PWHelleman, et al .: Scand. J. Haemat, Vol. 6, pp. 160-165, 1969 describes different types of leukocytes (eg, lymphocytes and granulocytes). ) Is attributed to the difference in dielectric properties.

For example, when looking carefully at the cumulative particle size distribution curve in FIG. 15, a second flat portion B can be seen to the right of the first flat portion A. The above fact becomes clearer when the white blood cell count for each threshold level is calculated from the cumulative particle size distribution curve in FIG. 15 and the particle size distribution curve shown in FIG. 16 is drawn. That is, in FIG. 16, to the right of the ghost and noise group of red blood cells indicated by C, there is first a first group of white blood cells indicated by D,
Further to the right is a second population of white blood cells, indicated by E. As described above, since the leukocyte particle size distribution is divided into two groups, it can be said that the flat portion of the cumulative particle size distribution is shortened.

The above shows the fact that the Toa automatic blood cell counter classified and counted two different populations of white blood cells, and the method of detecting the change in electrical impedance at the high frequency described above ( Hereafter, this is referred to as the RF method), which indicates the possibility of further classifying and counting leukocytes into several types and counting them.

However, at that time, the main focus was on the stable and accurate counting of white blood cell counts, so the reason why the flat part of the cumulative particle size distribution curve was short was not pursued in detail, Rather, efforts have been made to adjust the equipment and reagents so that the plateaus are as long as possible and the counts are stable.

On the other hand, the principle underlying the first of the methods using the flow system described above includes, in addition to the above-described RF method,
There are those disclosed in Japanese Patent No. 217947 and U.S. Pat. No. 2,656,508. This principle is based on the principle that a sample placed in a fluid medium with different conductivity from the particles passes through the confined current path, and the change in current caused by the difference in conductivity between the particles and the fluid medium is detected. This is referred to as the DC method below. this
In the DC method, the magnitude of the detected signal is approximately proportional to the volume of the particle.

By combining this DC method and the above-mentioned RF method, information on the volume of the particles is obtained by the DC method, and in addition to the information on the particle size by the RF method, information on the structure of the particles and the properties of the material constituting the particles are obtained. By also obtaining an apparatus for classifying a population of several different types of particles from a system of a mixture of different types of particles in the same suspension, JP 785859 and U.S. Pat.
It is disclosed in 3502974. However, there is no indication in this patent of the possibility of classifying and counting different types of leukocyte populations.

Also, using the above-described RF method, a method of replacing only the contents without destroying blood cells, changing the permittivity of blood cells, and then performing classification and counting, Japanese Patent No. 9368
No. 23 and U.S. Pat. No. 3,838,849. In a second embodiment of this patent, a 1% saponin solution was used.
Addition of 0μ to 100μ whole blood suspended in a phosphate buffered saline solution having a pH of 7.2 to lyse erythrocytes has been shown to show two distinct peaks of erythrocytes, This is in good agreement with the result of the measurement example described above (FIG. 16) using a measuring instrument.

By the way, even when using the RF method and using saponin having a relatively slow action on blood cells, the protoplasm of leukocytes is gradually destroyed, and the signal of leukocytes gradually becomes smaller. If the contents of the blood cells are to be replaced at a normal pH without destroying the blood cells as in Japanese Patent No. 936823, it is necessary to make the concentration of the saponin considerably low and to act on leukocytes slowly. However, in this case, the pretreatment time for measurement is changed to the pretreatment time for normal leukocyte measurement (3 to
5 minutes), it is not practical to use this method in an automatic measuring device that processes a large number of samples in a short time.

On the other hand, a method of changing leukocytes in a relatively short time and classifying leukocytes into three populations based on the difference in the degree of the change has been realized using the DC method.
And U.S. Pat. No. 4,485,175.

In this method, a quaternary ammonium salt described in the above-mentioned Japanese Patent No. 936823 is used as a cell lysing agent. By using it at a low concentration, a volume change is caused in leukocytes. Leukocytes are classified into three populations. However, the results of the embodiment shown in US Pat. No. 4,485,175 are as shown in FIG. 17, in which lymphocytes, monocytes, and granulocytes of three populations of leukocytes are always completely separated and identified. Do not mean.

In addition, the quaternary ammonium salt has a large effect on blood cells even at a low concentration, and causes a hemolysis phenomenon in a short time, so that the contents of blood cells as described in Japanese Patent No. 936823 are reduced. Whether or not it was possible to change only the dielectric constant without exchanging or breaking it was unnecessary because patent 936823 does not show data.

Furthermore, Japanese Patent Publication No. 61-502277 and International Publication Number
WO85 / 05684 states that when classifying and counting leukocytes,
Because quaternary ammonium salts cause too much damage to leukocytes, rather slow-acting saponins are added at high concentrations, and when erythrocytes are lysed, lysis is stopped, followed by flow cytometry or RF and DC. A method for analyzing by a method combining methods is described.

In this method, a fixing agent for stopping the lysis action is required, but after adding it at a predetermined timing, a special treatment such as heating the cells at a high temperature is required. Is not a very effective method.

As described above, of the methods for automatically classifying and counting leukocytes using a flow system, the second method using a flow cytometer has a problem that the apparatus is complicated and expensive. . Further, the first method for detecting the impedance change of the pore portion when the blood cell passes through the pore and classifying the leukocyte according to the magnitude of the detection signal includes a pre-process when the leukocyte is slowly changed. If it takes time, or if a special fixative is added at a predetermined timing or requires heat treatment, or if it strongly changes leukocytes, it will cause excessive damage to leukocytes, and at most There is a problem that only three classifications can be made, and a method that is practically satisfactory has not been obtained. Further, there has not been a technique for classifying and counting abnormal cells such as the blasts described above using the first method.

Further, as the cell lysing agent used in the first method,
Saponin has an unstable hemolytic power, and is too strong for a quaternary ammonium salt as described above.

The method of the present invention does not use saponin or a quaternary ammonium salt as a cell lysing agent, but uses a cell lysing agent having a stable hemolytic ability to lyse red blood cells and damage leukocytes in a short time. By analyzing the DC method and the RF method in combination, the present invention provides dramatic effects, such as five normal cell classifications and abnormal cell classifications, which could not be obtained conventionally, in leukocyte classification.

DISCLOSURE OF THE INVENTION The present invention provides the following leukocyte classification reagent and leukocyte classification method in order to overcome the problems of the above-mentioned conventional method and realize a dramatic effect.

(1) A reagent for classifying leukocytes, which lyses erythrocytes and acts on leukocytes to enable leukocytes to be classified and counted: (a) a general formula R ₁ -R _2- (CH ₂ CH ₂ O) _n- X wherein R ₁ is an alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30; X is SO ₃ Na, COONa, OSO ₃ Na or ONa], a first group of surfactants which are polyoxyethylene-based anionic surfactants; and (b) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30]. A reagent containing at least one surfactant selected from the group consisting of a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant.

(2) A leukocyte classification reagent comprising the following two liquids (a) and (b): (a) a first blood diluent containing a hyperosmotic agent;
A liquid and (b) a second liquid to be added to the blood sample diluted with the first liquid, containing the reagent according to (1) above.

(3) The reagent according to the above (2), wherein the first liquid also contains a surfactant.

(4) The reagent according to the above (1), which comprises a hyperosmotic agent.

(5) A reagent comprising the reagent according to any of the above (1) to (4), further comprising a solubilizing agent for selectively reducing the size of monocytes in leukocytes.

(6) The reagent according to (5), wherein the solubilizing agent is at least one selected from the group consisting of the following five groups.

Solubilizer of the first group (urea-based): urea thiourea 1,1-dimethylurea ethyleneurea methylurethane 1,3-dimethylurea urethane (H ₂ NCOOC ₂ H ₅ ) Solubilizer of the second group: n- Octyl β-D-glucoside CHAPS (3-[(3-chloroamidopropyl) dimethylammonio] -1-propanesulfonate) CHAPSO (3-[(3-chloroamidopropyl) dimethylammonio] -2-hydroxy-1 -Propane sulfonate) MEGA8,9,10 (octanoyl-, nonanoyl- or decanoyl-N-methylglucamide) Sucrose monocaprate N-formylmethylleucylalanine Group 3 solubilizer (guanidines): guanidine thiocyanate Guanylguanidine Guanidine hydrochloride Guanidine rodane salt Guanidine nitrate 1,1,3,3-tetra Laguanidine Guanidine carbonate Guanidine phosphate Guanidine sulfate Solubilizer (bile salt) of the fourth group: Sodium teoxycholate Taurocholate Cholic acid Solubilizer of the fifth group (halogen-substituted acetic acid): Sodium trichloroacetate Sodium tribromoacetate Sodium dichloroacetate Sodium dibromoacetate Sodium monochloroacetate Sodium monobromoacetate (7) Using the reagent according to any of the above (1) to (6), leukocytes are converted into lymphocytes by a particle analysis method such as an RF method and a DC method. A method of classifying into monocytes and granulocytes.

(8) Leukocytes are classified into lymphocytes, monocytes, and granulocytes by the method described in (7) above, and eosinophils that specifically remain in the measurement sample used for the three classifications after a predetermined time. Is counted by only the RF method and the DC method, or the DC method, to classify leukocytes into lymphocytes, monocytes, eosinophils, and granulocytes other than eosinophils.

(9) Leukocytes are classified into lymphocytes, monocytes, and granulocytes by the method described in the above item (7).
Eosinophils specifically left by using the reagent according to any one of the above (6) are subjected to the RF method and the DC method, or the DC method.
By counting only by the method, leukocytes are converted into lymphocytes,
A method of classifying monocytes, eosinophils, and granulocytes other than eosinophils into four types.

(10) Leukocytes are converted into lymphocytes, monocytes, eosinophils, and granules other than eosinophils by the method described in (8) or (9) above.
Classify, on the other hand, by using a basophil measurement reagent that specifically leaves basophils, and counting basophils only by RF method and DC method or DC method, leukocytes to lymphocytes, monocytes, A method of classifying into eosinophils, basophils, and neutrophils.

(11) A method for detecting abnormal cells by a particle analysis method such as an RF method and a DC method using the reagent according to any one of the above (1) to (6).

(12) Using the reagent according to any of the above (1) to (6), leukocytes are classified into lymphocytes, monocytes, and granulocytes by particle analysis methods such as an RF method and a DC method, and A method for detecting abnormal cells.

(13) Solubilizer of the second group: n-octyl β-D-glucoside CHAPS (3-[(3-chloroamidopropyl) dimethylammonio] -1-propanesulfonate) CHAPSO (3-[(3-chloro Amidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate) MEGA 8,9,10 (octanoyl-, nonanoyl- or decanoyl-N-methylglucamide) Sucrose monocaprate N-formylmethylleucylalanine A method for classifying leukocytes using a reagent containing at least one of the following.

By using the leukocyte classification reagent of the present invention, red blood cells in blood are lysed and each leukocyte type is individually damaged. When the thus treated sample is measured by a method combining the RF method and the DC method, white blood cells are classified into three types of lymphocytes, monocytes, and granulocytes due to the difference in the speed of change in the volume of cells depending on the degree of damage to each leukocyte. Separated into two groups. In the present invention, two-dimensional information can be obtained by the RF method and the DC method, and the reagent of the present invention has a characteristic action.
The use of a quaternary ammonium salt as a cell lysing agent and a superior classification of leukocytes can be obtained as compared with the method of U.S. Pat.

Further, using the leukocyte classification reagent of the present invention, the RF method and
When measured by a method that combines the DC method, leukocytes are divided into four populations of lymphocytes, monocytes, eosinophils, and granulocytes other than eosinophils due to the difference in the speed of cell volume change depending on the degree of damage to each leukocyte. Separated. In this case, there are various combinations of the RF method and the DC method.
Leukocytes are classified into lymphocytes, monocytes, and granulocytes by the DC and DC methods, and the eosinophils that remain specifically after a predetermined time are analyzed by RF
Counting by the method and the DC method, or only by the DC method, is one effective method.

Further, as described above, leukocytes are classified into four groups, while basophils are specifically left using a basophil measuring reagent,
The number of basophils is counted by measuring this sample by the RF method and the DC method or by the DC method only. As a result, since the number of eosinophils and the number of basophils are calculated, the number of remaining neutrophils in granulocytes is obtained by subtracting the number of eosinophils and the number of basophils from the number of granulocytes. Desired. As described above, the numbers and ratios of lymphocytes, monocytes, neutrophils, eosinophils, and basophils in leukocytes are determined, and five classifications of leukocytes can be performed.

The method of the present invention does not require the use of an unstable solubilizer saponin as in the method disclosed in JP-T-61-502277, and does not require treatment with a special fixing agent or heat treatment at a high temperature. This is an advantageous method for use in an automatic analyzer.

Next, the characteristic action of the leukocyte classification reagent of the present invention will be described.

The first group of surfactants contained in the reagent of the present invention is an anionic surfactant, and the second group of surfactants is a nonionic surfactant. Both anionic and nonionic surfactants are used in general applications,
It is widely used as a detergent, emulsifier, dispersant, and penetrant in various industrial fields such as fiber, Landry, synthetic fiber, printing ink, metal, photography, food, and medicine.

In contrast, quaternary ammonium salts used as cell lysing agents in the method of US Pat. No. 4,488,175 are cationic surfactants. In general applications, cation surfactants rarely directly apply properties such as wettability, detergency, and surface tension lowering ability based on surface activity.
It is widely used in industrial fields such as textiles, rubber, plastics, pharmaceuticals, civil engineering, ceramics, and petroleum, mainly as disinfectants, disinfectants, waterproofing agents, softeners, antistatic agents, and fixing agents.

In addition, saponin, which has been frequently used as a cell lysing agent, is a natural chemical derived from plants unlike the above-described anionic, nonionic and cationic synthetic surfactants, and depending on the production lot, It is a substance whose chemical structure and chemical properties change.

As described above, since these cell lysing agents have different chemical properties and origins, their actions on leukocytes naturally differ.

Of the above mentioned cell lysing agents, saponins are the slowest acting on cells, and quaternary ammonium salts are the most rapidly damaging cells. On the other hand, it can be said that the strength of the action of the anionic and nonionic surfactants on cells is about the middle between saponin and quaternary ammonium salts.

Table 1 shows the degree of damage to the cells, for example, depending on the size of the lymphocytes. However, the numbers in the table show approximate values.

Table 1 Before lymphocyte damage treatment 170-250 fl After treatment with saponin 100-120 fl After treatment with the surfactant of the present invention 70-80 fl After treatment with a quaternary ammonium salt 50 fl Thus, quaternary ammonium Salts cause too much damage to leukocytes, so for leukocyte classification purposes,
It is not a preferred solubilizer. FIG. 14 shows an example of measuring leukocytes by the RF method and the DC method using a quaternary ammonium salt, but the leukocytes are not separated into a plurality of populations. This is because leukocytes were severely damaged by the quaternary ammonium salt, and most of the cytoplasm was lost, although the cell membrane remained, so the magnitude of the signal intensity detected from each leukocyte type was different between the RF method and the DC method. Are not fractionated on the two-dimensional distribution map due to the disappearance of.

For this reason, it is completely meaningless to use a quaternary ammonium salt as a cell lysing agent in a leukocyte classification method that combines the RF method and the DC method. On the other hand, since saponin acts on leukocytes slowly, there is a problem that the pretreatment time is long as described above, and the hemolytic power is also unstable.

The surfactants of the first and second groups used in the present invention do not damage leukocytes as severely as quaternary ammonium salts, and act more strongly on leukocytes than saponins to classify leukocytes. It damages each leukocyte to a very favorable degree above.

FIG. 9 shows an example in which white blood cells were fractionated by measuring blood before the addition of a cell lysing agent by the RF method and the DC method. Granulocytes a and monocytes b are well separated, but lymphocytes c and erythrocytes i are not separated. This figure is strongly influenced by the simultaneous passage of red blood cells. Therefore,
In this state, leukocytes cannot be classified and counted.

However, even before the addition of the cell lysing agent, it is possible to fractionate leukocytes to some extent by measuring them with the RF method and the DC method if the sample is a sample in which only leukocytes are carefully separated. An example of the fractionation is shown in FIG.

Fractionation is Percoll-Hypaque
Was obtained by separating leukocytes in blood by a specific gravity difference centrifugation method using the method described above. Some red blood cells remain in the sample without being separated. As shown in FIG. 10, it can be seen that granulocytes a, monocytes b, and lymphocytes c can be separated without using a cell lysing agent. However, the operation of separating only white blood cells as described above is very difficult and time-consuming even for a skilled person, and cannot be automated. So practically,
Again, a method of lysing red blood cells and leaving only white blood cells is employed. However, when a cell lysing agent for lysing red blood cells is added, white blood cells are also affected by the lysing agent. Therefore, it is necessary to ensure that granulocytes, monocytes, and lymphocytes are well separated and appear on the two-dimensional distribution map even after erythrocyte lysis. Here, the same cell lysing agent as in Example 2 described below was added to the same blood as that used for the operation of separating only white blood cells, and the white blood cells measured by the RF method and the DC method were separated for 14 to 5 seconds after the addition. FIG. 11 shows such an example. Here, it is shown on the same scale as FIG. It can be seen that the leukocytes have been totally reduced after the addition of the lysing agent. It is also noted that the volume reduction speed of monocytes is particularly large, and in the DC signal intensity, monocytes that were larger than granulocytes before the addition of the solubilizer are smaller than granulocytes after the addition. FIG. 2 based on Example 2 to be described later is shown on a larger scale than FIG. The scales in FIG. 10 and FIG. 2 are different for both the DC and RF methods. That is, since the cells shrink after the addition of the lysing agent, the detection signal also decreases. In FIG. 2, the detection signal is further amplified to make the figure easier to see (the same applies to the following embodiments).

The method of the present invention lyses red blood cells with the reagent of the present invention and damages each white blood cell, and classifies and counts white blood cells based on the difference in the speed of change in cell volume depending on the degree of damage. In addition, since the present invention uses a cell lysing agent, there is no need for the above-described centrifugation.

As described above, it has been described that normal cells of leukocytes can be classified and counted by the action of the reagent of the present invention.Surprisingly, the reagent of the present invention is used to lyse erythrocytes, and the leukocytes are subjected to the DC method and The present inventor has found that when measured by the RF method, various abnormal cells are detected in addition to the normal leukocyte cells, and can be classified and counted. FIG. 18 is an explanatory diagram showing appearance positions of various abnormal cells. The distribution diagram itself in FIG. 18 was obtained when blood of a healthy person was measured using the reagent of the present invention, and each point in the figure is a measurement point obtained by normal cells. When each abnormal cell population exists, it appears near each region surrounded by a solid line in FIG.
Among the abnormal cells, the left shift cell population is in region j, immature granules (IG) are in region k, and blasts are in region l.
Atypical lymphocytes in region m, lymphoblasts in region n,
Nucleated erythrocytes almost appear in the region o, and platelet aggregation cells almost appear in the region p. Neutrophils include lobulated nuclei and rods,
The nucleus is not lobulated but becomes rod-like in younger cells. The increase in immature cells, i.e., rod-shaped nuclei, in leukocytes is called leftward movement. The left migrating cells, immature granulocytes, and blasts are immature, i.e., immature cells of normal leukocytes. It is said that immature cells of leukocytes have a lower specific gravity, and therefore, the signal intensity by the RF method becomes smaller. Therefore, the strongest blast cells in juvenile form appear at the bottom of the figure. In addition, platelet-aggregated cells are those in which platelets are aggregated for some reason and appear in a size comparable to leukocytes, and it is important to identify and remove these cells in order to obtain an accurate leukocyte count.

There is no conventional technique for classifying and counting abnormal cells of leukocytes by the DC method or the RF method or a combination thereof. There is an example in which abnormal cells of leukocytes are detected by the principle of optical measurement (see Japanese Patent Application Laid-Open No. 61-88896). However, according to the principle of optical measurement, the apparatus and procedure become extremely complicated as described above. Further, according to the description in the above publication, all leukocytes other than basophils are naked nuclei. Therefore, in the method described in the above publication, abnormal cells and basophils can be detected, but normal leukocytes other than basophils cannot be classified, and normal leukocytes are classified and counted. This requires a separate measurement channel. For the first time, the method and the reagent of the present invention enable simultaneous classification and counting of normal and abnormal white blood cells.

As a prior art using a reagent containing a polyoxyethylene-based surfactant for the purpose of leukocyte classification, a method disclosed in JP-A-54-22891 and U.S. Pat. -71857 (corresponding European Patent Publication No. 214613).
Each of these methods belongs to the second method among the methods using the flow system described in the section of the prior art of this specification, and is a method using an optical measurement device. Needless to say, the reagents disclosed in the above-mentioned publications cannot be directly applied to the method of the present invention based on the principle of impedance detection because the measurement principle is completely different. Moreover, according to the experiments of the present inventors, some of the polyoxyethylene surfactants disclosed in the above publication are not only unsuitable for the leukocyte classification method of the present invention, but also have an ability to lyse erythrocytes. It turned out to be missing. Rather, a certain type of polyoxyethylene surfactant has been used as a composition for a sheath liquid for a blood cell counter, which is an essential condition for protecting red blood cells (Japanese Patent Laid-Open No. 62-87233). And Japanese Patent Application Nos. 62-261386 and 62-261387.

As understood from the above, there are many polyoxyethylene-based surfactants that do not have the action of dissolving erythrocytes, and in particular, those that are suitable as a composition to be contained in the leukocyte classification reagent in the present invention are: , Are further limited.

The present inventor screened various kinds of anionic or nonionic surfactants including a polyoxyethylene surfactant, and as a result, the surfactants of the first group and the second group according to claim (1) were found. It has been found that it meets the objectives of the present invention.

In the general formula described in claim (1), R ₁
Has a carbon number of 10 to 22 and n (additional mole number) of 8 to 30, the object of the present invention can be achieved. However, in order to better fractionate leukocytes, the carbon atom of R ₁ is required. The number is 12 to 18, and n is preferably 12 to 15.

By the way, when fresh blood immediately after blood collection is measured using the reagent described in claim 1 (hereinafter referred to as a lysis reagent), lymphocytes, monocytes, and granulocytes are well fractionated. Is done. However, several hours after collecting blood, especially abnormal blood,
As an example, if left for 8 hours, a two-dimensional distribution as shown in FIG. 12 can be obtained. A, b, and d in FIG.
The region c indicates the region where granulocytes, monocytes and lymphocytes are present when normal blood is measured immediately after blood collection. The number of cells in the area b of normal monocytes abnormally increases,
The fractionation of lymphocytes c and monocytes b deteriorates, and the number of granules a decreases. This is because, in the case of abnormal blood, white blood cells become weak due to the lapse of time after blood collection and become strongly affected by the added lysing reagent, and some cells in granulocytes, especially neutrophils, are collected. This is because the volume shrank faster than immediately after. In addition, since the lysing reagent acts on the blood suddenly, the lysing reagent does not uniformly act on individual blood cells (the blood cells are not uniformly dispersed in the molten reagent solution).
One factor is that the lytic reagent acts strongly only on some blood cells.

Therefore, the above problem can be solved to some extent only by diluting the blood with a general diluent for uniformly dispersing the blood cells before adding the lysis reagent. However, the present inventor has devised a reagent which can stably classify and count leukocytes even when left for several hours to several tens of hours after blood collection. That is the reagent according to claims (2) to (6).

The reagent described in claim (2) is composed of two liquids. The first liquid is a so-called diluent and contains a hyperosmotic agent. The second liquid is the lysis reagent according to claim (1). First, after diluting blood with a diluting liquid as a first liquid and uniformly dispersing blood cells, a lysing reagent as a second liquid is added to lyse erythrocytes and classify leukocytes.
Counting is possible.

The osmotic pressure of the first liquid is adjusted to 285 mOsm or more by the high osmotic pressure agent contained in the first liquid. A method of using a hypertonic solution (hypertonic solution) as a leukocyte measurement solution in this way is described in the above-mentioned Japanese Patent Application Laid-Open No. 62-71857, but as described above, this publication employs the principle of optical measurement. Therefore, the operation and effect of the hypertonic solution in this publication are completely different from the operation and effect of using the first solution as the hypertonic solution in the present invention. The above publication describes that lymphocytes become serrated blood cells by the action of hypertonic solution on blood cells, thereby improving the discrimination between detection signals of lymphocytes and noise.
It is not clear why the discrimination from noise is improved by becoming a circular sawtooth blood cell because there is no further description in this publication, but there are reasons closely related to the principle of optical measurement. There will be. In the impedance measurement principle of the present invention, the size or internal information of a cell is detected by the DC method or the RF method, and basically affects the strength of the detection signal regardless of how the outer shape of the cell is deformed. Does not reach. The purpose of the present invention in which the first liquid is made to have a high osmotic pressure is to put the blood cells under the high osmotic pressure to make the cell membrane of the blood cells dehydrated and to make the membrane hard, so that the lysis reagent which is the second liquid later The purpose of the present invention is to protect the cell membrane so that the membrane is not subjected to a sudden shock when is added. When blood cells are placed under high osmotic pressure, while the cell membrane is protected in this way, the cell membrane is contracted, and the volume of the blood cell is reduced. In the impedance detection principle, since cell size information is detected as described above, it is difficult to discriminate it from noise when blood cells are excessively reduced in volume. For this reason, the principle of impedance detection has generally avoided keeping blood cells under a high osmotic pressure. However, unexpectedly, even when blood cells are subjected to a high osmotic pressure with the first liquid and then the lysing reagent as the second liquid is applied, it is equivalent to the case where the lysing reagent is applied to the blood from the beginning. Then, it was found that the white blood cell signal and the noise were discriminated. This is probably because the action of the first liquid protected the leukocyte cell membrane. As described above, by using a two-liquid composition and using the first liquid as a high osmotic pressure liquid, the white blood cells are protected from the severe shock caused by the dissolution test as the second liquid, and the discrimination between the white blood cell signal and the noise is prevented. It became possible to secure. This makes it possible to stably classify and count leukocytes without causing a phenomenon in which neutrophils enter the monocyte region in the two-dimensional distribution map even when measuring blood left for several tens of hours. Was. FIG. 13 shows an example in which the same blood as in FIG. 12 was measured for 8 hours with the above-mentioned two-component reagent after being left for 8 hours after blood collection. Granulocytes a, monocytes b, and lymphocytes c are clearly fractionated, and no abnormal increase in the number of cells in the monocyte region is observed. The osmotic pressure of the first liquid is about 285 mOsm
This is required. At lower osmotic pressures, the leukocyte membrane is not adequately protected. As the high osmotic pressure agent, ethylene glycols and alcohols are preferably used.

By the way, as described above, it is ideal to use a two-liquid composition and the first liquid is a high osmotic pressure liquid. On the other hand, the two-liquid composition complicates the analysis procedure or the configuration of the analyzer. Is not preferred. Therefore, a reagent having a one-liquid composition and using the dissolved reagent as a high osmotic pressure liquid was considered (the reagent described in claim (4)). Even with this reagent, the protective action of the blood cell membrane is not as complete as that of the two-component reagent, but even when blood left for several tens of hours is measured, the neutrophils in the two-dimensional distribution map show a monocyte region. Classify leukocytes stably at a practical level without causing invasion
It was confirmed that counting was possible.

Regardless of the two-part composition or the one-part composition, it is more effective if the hyperosmotic liquid contains a fixing agent. This fixative aids the protective action of the hyperosmotic liquid on the leukocyte cell membrane. In the prior art using a reagent containing a fixing agent in leukocyte classification, the above-mentioned JP-A-54-22891 is disclosed.
In addition to JP-A-62-71857 and JP-T-61-502277, there is a method described in U.S. Pat. No. 3,741,875. After the addition of the fixing agent in any of the above prior art,
It is necessary to heat the solution. However, in the present invention, it is not necessary to perform heat treatment even after the addition of the reagent containing the fixing agent. This is because the fixing agent plays an important role in the action of the reaction reagent in any of the above prior arts, so that it is necessary to particularly promote the reaction of the fixing agent by heat treatment, whereas the reagent of the present invention This is because the role of the fixing agent in the above is auxiliary and there is no need to accelerate the reaction by heating. The point that heat treatment is not required is a great advantage in simplifying the analysis method or the analysis device.

Further, in the present invention, the fifth aspect described in claim (6) is provided.
The inclusion of at least one solubilizing agent selected from the group consisting of the two groups is also effective in improving the classification accuracy of leukocytes. The solubilizing agent generally has a function of apparently dissolving a substance which is hardly soluble or insoluble in water at a level higher than its solubility, and is widely applied to the industrial field and the biological field. In the field of medicine, this solubilizing agent is used to convert vitamins and hormones into water-soluble drugs. However, the action of the solubilizer in the present invention is different from the above-mentioned general action. Monocytes in leukocytes have strong reactivity with lysis reagents containing surfactants, and are the cells with the fastest volume reduction speed after the addition of lysis reagents. The present inventor has found that when a solubilizing agent is included in the diluent used, the action of the solubilizing agent promotes the action of the lysing reagent on monocytes, and the monocytes are more rapidly reduced in volume. That is, the action of the solubilizer in the present invention is selective for monocytes. Further, such a solubilizing agent which selectively acts on monocytes is a surfactant which is selected from the above-mentioned first and second groups of surfactants and which is characteristic of the present invention. Is effective not only for reagents containing, but also for all leukocyte classification reagents known from the prior art. Among them, the solubilizer of the second group described in claim (6) is effective.

Incidentally, a surfactant can also be contained in the first liquid (a diluent that has been made into a high osmotic pressure liquid) out of the two-component reagent described above (the reagent described in claim (3)). In this case, the surfactant may be selected from the first and second groups of surfactants described in claim (1), and may be other general surfactants. Is also good. The main action of the surfactant contained in the first liquid is not the lysis action on blood cells, but is close to the action of the above-mentioned solubilizer. That is, since the osmotic pressure of the first liquid is high, the lysis effect of the surfactant is weakened, and among the lysis effects of blood cells caused by the subsequent addition of a lysis reagent, a reserve for promoting the contraction of monocytes is particularly high. It is understood that the treatment is performed on the surfactant in the first liquid. Therefore, it is required that the surfactant in the second liquid has a substantially stronger cell lysis action than the surfactant in the first liquid. Otherwise, the lysis reaction of blood cells will not proceed even after the addition of the second liquid. The above conditions are satisfied, for example, even if the surfactants contained in the first liquid and the second liquid are of the same type and have the same concentration, provided that the first liquid has a higher osmotic pressure.

All the reagents of the present invention contain a buffer for adjusting the solution to a predetermined pH. If the pH is lower than the predetermined pH, the lysis reaction becomes slow, and the leukocytes cannot be classified by the time required for practical use. Conversely, if the pH is higher than the predetermined pH, the lysis reaction is excessively promoted, so that it becomes difficult to perform stable leukocyte classification.

Further, although not characteristic of the present invention, all the reagents of the present invention can contain a general antioxidant and an electric conductivity modifier as required.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are described as follows.

FIG. 1 is a two-dimensional distribution diagram showing the classification of leukocytes in Example 1. The horizontal axis of this two-dimensional distribution diagram represents the relative signal intensity measured by the DC method, and the vertical axis represents the relative signal intensity measured by the RF method. Each point in FIG.
It corresponds to each cell showing the respective DC signal strength and RF signal strength. FIG. 1 shows DC signal strength and RF signal strength, respectively.

FIG. 2 is a two-dimensional distribution diagram showing the classification of leukocytes in Example 2, and the description in the figure is the same as that in FIG.

FIG. 3 is a two-dimensional distribution diagram in which leukocytes are classified in the same manner as in FIG. 2, except that the white blood cells are left for 90 seconds after the addition of the lysing agent.

FIG. 4 is a graph showing classification of leukocytes by only the DC method performed in Example 2 under the same conditions as in FIG.

FIG. 5 is a two-dimensional distribution diagram showing classification of leukocytes performed in Example 4 using a reagent for measuring basophils and a combination of the RF method and the DC method.

FIG. 6 is a graph showing classification of leukocytes performed in Example 4 only by the DC method.

FIG. 7 is a two-dimensional distribution diagram showing classification of leukocytes in Example 5.

FIG. 8 is a two-dimensional distribution diagram showing classification of leukocytes in Example 6.

FIG. 9 is a two-dimensional distribution diagram when a sample obtained by diluting blood without adding a cell lysing agent was measured by the RF method and the DC method.

FIG. 10 is a two-dimensional distribution diagram when a sample obtained by separating only leukocytes without adding a cell lysing agent was measured by the RF method and the DC method.

FIG. 11 is a two-dimensional distribution diagram when the same cell lysing agent as used in Example 2 was added to blood and measured by the RF method and the DC method. The scale of FIG.
It is the same as the scale in Figure 10. The scale in FIG.
The scale of Fig. 11 is enlarged.

FIG. 12 is a two-dimensional distribution diagram when a reagent not containing a hyperosmotic agent was added to blood left for 8 hours after blood collection and measured by the RF method and the DC method.

FIG. 13 is a two-dimensional distribution diagram when a reagent containing a hyperosmotic agent was added to blood left for 8 hours after blood collection and measured by the RF method and the DC method.

FIG. 14 is a two-dimensional distribution diagram showing classification of leukocytes performed by the RF method and the DC method using a quaternary ammonium salt as a cell lysing agent.

FIG. 15 is a cumulative particle size distribution curve for confirming that the magnitude of the white blood cell signal is sufficiently larger than the size of the lysed red blood cell membrane (ghost) or noise signal. In the figure, the horizontal axis represents the signal threshold of the automatic blood cell counter, and the vertical axis represents the number of detection signals equal to or higher than the signal threshold. In FIG. 15, A
And B are flat portions in the cumulative particle size distribution curve.

FIG. 16 is a particle size distribution curve drawn by calculating the number of white blood cells for each threshold level from the cumulative particle size distribution curve of FIG. In the figure, C is a ghost and noise population of red blood cells,
D is the first population of white blood cells, E is the second population of white blood cells.

FIG. 17 shows the DC described in the example of U.S. Pat.
6 is a white blood cell particle size distribution curve obtained by a method.

FIG. 18 is an explanatory diagram showing appearance positions of various abnormal cells on a two-dimensional distribution map.

FIG. 19 is a two-dimensional distribution diagram when blood of an acute lymphocytic leukemia (ALL) patient is measured.

FIG. 20 is a two-dimensional distribution diagram when blood of an adult T-cell leukemia (ATL) patient is measured.

FIGS. 21 and 22 are two-dimensional distribution diagrams when blood of an acute myeloid leukemia (AML) patient is measured.

FIG. 23 is a two-dimensional distribution diagram when leftward movement j and atypical lymphocyte m appear in one sample.

FIG. 24 and FIG. 25 are two-dimensional distribution diagrams when immature granulocyte k appears.

1 to 14 have the following meanings: a: granulocyte b: monocyte c: lymphocyte d: eosinophil e: basophil f: erythrocyte ghost g: eosinophil H: leukocytes other than basophils i: erythrocytes The symbols j to p in FIGS. 18 to 25 have the following meanings: j: left shift k: immature granulocytes (IG) l : Blast m: atypical lymphocyte n: lymphoblast o: nucleated erythrocyte p: platelet aggregation The description of RF and DC in FIGS. 2 to 14, 18 to 25 is the same as that in FIG. BEST MODE FOR CARRYING OUT THE INVENTION Example 1 Sandet EN (trade name of Sanyo Chemical Industries, Ltd., chemical formula C ₁₂ H ₂₅ -O-) which is one of the first group of surfactants as a cell lysing agent An example using (CH ₂ CH ₂ O) ₂ SO ₃ Na) is shown.

Diluent 1 consisting of 5 ml of this cell lysis agent at a concentration of 0.125% and 1 / 60M phosphate buffer and sodium chloride at a concentration of 0.6%
Dilute 60μ of blood with 0ml, pH 7.0, osmotic pressure 10mOsm, liquid temperature
The measurement was started 14 seconds after the addition of the cell lysing agent under the condition of 26 ° C., and the result of measuring leukocytes for 5 seconds is shown in FIG. The horizontal axis of the two-dimensional distribution diagram represents the relative signal intensity measured by the DC method, and the vertical axis of FIG. 1 represents the relative signal intensity measured by the RF method. Each point in FIG.
It corresponds to each cell showing DC signal strength and RF signal strength.

As shown in FIG. 1, leukocytes are lymphocytes, monocytes,
Fractionated into populations presumed to be granulocytes. Since the two-dimensional distribution diagram is drawn by combining the RF method and the DC method in this way, the leukocyte fractionation is better than the conventional leukocyte three-class measurement result using only the DC method shown in FIG. Have been.

Example 2 From a second group of surfactants as cell lysing agents
4.0% EMULMIN 140 [manufactured by Sanyo Chemical Industries, trade name _{(strain), C 18 H 37 -O- (} CH 2 CH 2 O) 14 H to _{56%, C 16 H 33 -O-} (CH 2 CH 2 O) ₁₄ H and _{34%, C 14 H 29 -O-} (CH 2 CH 2 O) a ₁₄ H containing 7%. ] And a concentration of 0.5% Emulgen 420 [trade name of Kao Corporation, C
₁₈ H ₃₇ -O- (CH ₂ CH ₂ O) ₁₃ H], and the other example 1
FIG. 2 shows the results measured under the same conditions as in FIG.

As in Example 1, leukocytes were separated into three populations.
The fact that each population was a lymphocyte, monocyte, or granulocyte was confirmed by measuring a sample from which each leukocyte type was separated, and by a correlation test with a calculation method.

Comparison of FIG. 2 with FIG. 1 shows that the appearance position of monocytes with respect to granulocytes is different. This is because the speed of volume change of each leukocyte varies depending on the type and concentration of the lysing agent. In FIG. 2, since the speed at which monocytes are damaged is particularly large compared to other leukocytes, monocytes larger than granules originally appear at positions smaller than granulocytes (DC signal intensity is smaller). ing.

Further, a blood-containing diluent prepared in the same manner as in Example 1 except that the above-mentioned cell lysing agent was used was left to stand for 90 seconds after the addition of the lysing agent, and the results of measurement for 5 seconds thereafter are shown in FIG. In this way, it was found that only eosinophils remained specifically and were detected separately from other leukocytes. This is explained as being due to the strong eosinophil membrane against this lysing agent. In addition, the results shown in FIG. 4 were obtained even when the measurement was performed only by the DC method under the same conditions, indicating that eosinophils were sufficiently separated from other leukocytes.

Example 3 Assuming that the pH was 10.0 and the other conditions were the same as in Example 2, measurement was performed for 13 to 5 seconds after the addition of the solubilizer. Also in this case, it was shown that only eosinophils were specifically separated as in Example 2.

In the above three examples, the concentration of the cell lysing agent was 0.
125 and 4.5%, but 0.01 to 15%, preferably 0.05
Similar results are obtained in a concentration range of 1010%. pH is
7.0 or 10.0, but 4.0-12.0, preferably 7.0
It may be in the range of 10.0. The osmotic pressure was 120 mOsm, but may be in the range of 50 to 400 mOsm, preferably 100 to 200 mOsm. The liquid temperature was 26 ° C., but it can be measured similarly within a temperature range of 10 to 37 ° C. However, it may be necessary to change the measurement start time after the addition of the solubilizer depending on the temperature.

Further, the composition and concentration of the buffer and the like in the diluent are not limited to the above Examples. Further, in the above embodiment, the cell lysing agent and the diluting solution are different solutions, but both may be integrally prepared from the beginning as one lysing solution.

Example 4 In the above three examples, basophils in granulocytes were not separated. In order to separate and count basophils, for example, the following is performed.

Concentration 1.44% of Emulgen 123-P [the trade name of Kao Corporation, formula _{(C 12 H 25 -O (CH} 2 CH 2 O) 23 H ], concentration 0.435
80 µ of blood was diluted with 10 ml of a basophil measuring reagent containing% o-phthalic acid / potassium, 0.025% hydrochloric acid, and 0.025% nitric acid, pH 3.0, osmotic pressure 60 mOsm, liquid crystal 33 ° C.
The measurement is performed for 13 to 5 seconds after the addition of the solubilizer under the conditions described above.

FIG. 5 shows the measurement results obtained by combining the RF method and the DC method. In addition, FIG. 6 shows the results measured only by the DC method. In any case, only basophils remain specifically, and are distinguished from other leukocytes and measured. This is because all the leukocytes other than basophils were naked nucleated by this lysing agent, leaving only basophils.

In the basophil measurement reagent, the surfactant is not limited to Emulgen 123-P, but may be represented by the general formula R-
O- (CH ₂ CH ₂ O) may be a nonionic surfactant represented by _n H. However, R has 10 to 20 carbon atoms (preferably
12-18) alkyl group, n is 6-100 (preferably 15-4
0). In addition, hydrochloric acid and nitric acid adjust the pH to about 3.0, and at least one may be selected from the group consisting of hydrochloric acid, nitric acid, and acetic acid. The osmotic pressure may be in the range of 10 to 400 (preferably 20 to 100). Liquid temperature is 10-40
The temperature may be in the range of ° C.

As described above, basophils were classified and counted.
Classify and count eosinophils in lymphocytes, monocytes, granulocytes and granulocytes by the method described above, and subtract the number of eosinophils and basophils from the number of granulocytes to obtain the number of neutrophils. By calculation, the numbers and ratios of lymphocytes, monocytes, neutrophils, eosinophils, and basophils in total leukocytes are obtained.

By the above method, it was measured for 105 samples of blood,
Table 2 shows the correlation between the ratios of the five types of leukocytes when this is compared with the visual estimation method when 500 cells are observed per sample.

Table 2 Lymphocytes 0.95 Monocytes 0.43 Neutrophils 0.90 Eosinophils 0.95 Basophils 0.85 Thus, the method of the present embodiment is based on A good correlation was shown, indicating that the method of the present invention sufficiently classified and counted leukocytes into five types. The result of the present embodiment is an epoch-making method that it is possible to classify and discriminate leukocytes into five types for the first time in a method of electrically detecting an impedance change when blood cells pass through a pore. It is something.

Example 5 A composition example of a two-component reagent of the present invention and a measurement example using the reagent are shown.

Reagent composition 1st liquid (diluent) ◎ Buffer Disodium phosphate (12-hydrate) Na ₂ HPO ₄・ 12H ₂ O 9.0 g Monosodium phosphate (anhydrous) NaH ₂ PO ₄ 3.0 g ◎ Antioxidant EDTA- 2K 0.1 g ◎ Electric conductivity regulator NaCl 0.16g ◎ Solubilizer CHAPS 0.4 g ◎ High osmotic pressure agent Ethylene glycol 70.0 g ◎ Fixing agent Formalin 50.0 g ◎ Water (pH 7.2, osmotic pressure 1400mOsm) 1000 ml Second solution (dissolution reagent) ◎ Buffer Disodium phosphate (12-hydrate) Na ₂ HPO ₄・ 12H ₂ O 9.0 g Monosodium phosphate (anhydrous) NaH ₂ PO ₄ 3.0 g ◎ Antioxidant EDTA-2K 0.1 g dl-methionine 1.0 g ◎ fixative formalin 30.0 g ◎ polyoxyethylene based surfactant _{E-212 C 18 H 35 -O-} (CH 2 CH 2 O) 12 -H 0.5 g ◎ water (pH 7.2, osmotic Pressure 920mOsm) 1000 ml 12μ of blood 4 hours after blood collection and first liquid at 26 ℃
After mixing with 2 ml, let stand for 25 seconds and then
FIG. 7 shows the results obtained by adding 1 ml of the liquid to make a 250-fold diluted sample, and measuring from 60 seconds to 6 seconds after the addition of the second liquid. Leukocytes are classified into three types: granulocytes a, monocytes b, and lymphocytes c.

Example 6 Another set of composition examples of the two-component reagent of the present invention and measurement examples using the reagent will be described.

Reagent composition 1st liquid (diluent) ◎ Buffer Disodium phosphate (12-hydrate) Na ₂ HPO ₄・ 12H ₂ O 18.0 g Monosodium phosphate (anhydrous) NaH ₂ PO ₄ 2.3 g ◎ Antioxidant EDTA- 2K 0.1 g dl-methionine 1.0 g Surfactant E-212 C ₁₈ H ₃₅ -O- (CH ₂ CH ₂ O) ₁₂ -H 0.06 g ◎ Water (pH 7.4, osmotic pressure 3000 mOsm) 1000 ml 2nd liquid (dissolution reagent) ◎ Buffer phosphoric acid Disodium (12 hydrate) Na ₂ HPO ₄・ 12H ₂ O 14.0 g Monosodium phosphate (anhydrous) NaH ₂ PO ₄ 1.5 g ◎ Antioxidant EDTA-2K 0.1 g dl-methionine 1.0 g ◎ Electric conductivity regulator sodium chloride NaCl 2.3 g ◎ fixative formalin 100.0 g ◎ polyoxyethylene based surfactant E-212 C ₁₈ H ₃₅ - _{- (CH 2 CH 2 O)} 12 -H 0.06g ◎ water (pH 7.2, osmolality 1380MOsm) first liquid blood 12μ and a liquid temperature of 26 ° C. After 4 hours passed after 1000 ml blood collection
After mixing with 2 ml, let stand for 20 seconds and then
FIG. 8 shows the results obtained by adding 1 ml of the liquid to make a 250-fold diluted sample, and measuring from 40 seconds to 6 seconds after the addition of the second liquid. Leukocytes are classified into three types: granulocytes a, monocytes b, and lymphocytes c.

Leukocyte 3 classification is performed by the method of this Example 6, while
The number of eosinophils was measured by the method of Example 3 and the number of basophils was measured by the method of Example 4. As shown in Table 2, the correlation between the blood of 50 specimens and the calculation method was compared. It is shown in Table 3.

Table 3 Lymphocytes 0.98 Monocytes 0.83 Neutrophils 0.95 Eosinophils 0.95 Basophils 0.81 Comparing the results shown in Table 3 with the results in Table 2, the correlation function of monocytes is significantly improved. You can see that.

In Examples 5 and 6, the pH was 7.2 or 7.4, but the pH may be in the range of 6 to 8.

Example 7 An example in which abnormal leukocyte cells were detected using the reagent described in Example 2 is described below.

FIG. 19 shows an example in which the blood of an acute lymphocytic leukemia (ALL) patient was measured, and lymphoblasts n appeared. Thus, lymphocytic leukemia can be found by detecting the appearance of lymphoblasts.

FIG. 20 shows an example in which the blood of an adult T-cell leukemia (ATL) patient was measured, in which atypical lipocyte m appears. Thus, when the appearance of atypical lymphocytes is detected, a lymphocytic disease is suspected.

FIG. 21 shows an example in which blood of an acute myeloid leukemia (AML) patient was measured, and blast cells 1 appeared. Thus, myelogenous leukemia can be found by detecting the appearance of blasts.

FIG. 22 shows an example in which the blood of an acute myeloid leukemia (AML) patient was similarly measured, and blast cells 1 appeared remarkably.

FIG. 23 shows an example in which leftward movement j and atypical lymphocyte m appear in one sample.

FIG. 24 shows an example in which immature granulocytes k appeared.

FIG. 25 shows a remarkable example in which immature granulocytes k and atypical lymphocytes m appeared.

In order to implement the method of the present invention as a specific analyzer, various configuration examples are conceivable.

The simplest method that can be considered is a first detection unit having a measurement channel of the RF method (hereinafter referred to as an RF channel) and a measurement channel of the DC method (hereinafter referred to as a DC channel), and a second detection unit having only a DC channel. Same as the detection unit 2
A third detection unit having only a DC channel is provided. In the first detection unit, lymphocytes, monocytes, and granulocytes are classified and counted using a leukocyte classification reagent. Counting the number of basophils using a reagent for measuring basophils, leaving only eosinophils using a reagent for classifying leukocytes in the third detection unit, counting the number of eosinophils, The number of balls is obtained by calculation. However, in this method, different measurement samples must be prepared for each of the three detection units, and each requires a different cell lysing agent or diluent and their storage tanks, etc., so that the apparatus becomes complicated and large. There is a disadvantage that.

This can be improved, for example, by using the dissolving agent described in Example 2. That is, a first detection unit having an RF channel and a DC channel and a second detection unit having only a DC channel are provided. In the first detection unit, as described above, lymphocytes, monocytes, and granulocytes The eosinophil count is counted when only the eosinophils specifically remain after a predetermined time has passed, and the second detection unit counts only the basophil count as described above. How to In this method, only two types of cell lysing agents are required, and only two detection units are required, so that the apparatus configuration is much simpler. However, it is necessary to wait for a predetermined time before eosinophils can be counted, which is not preferable for an automatic analyzer that continuously measures multiple samples in a short time.

In order to solve this point, it is preferable to provide a third detection unit having a DC channel as in the first method. Then, after the first detection unit classifies the eosinophils into three, the solution is transferred to the third detection unit. After waiting for a predetermined time, eosinophils are counted. In the meantime, in the first detection unit, a lysing solution for measuring the next sample is prepared, and the measurement of the three classifications proceeds in parallel with the counting of the third detection unit. This makes it possible to continuously measure multiple samples without interruption.

If reducing the processing time per sample is not so important, the third method for measuring eosinophils may be used.
After the counting of basophils in the second detection unit is completed without providing the detection unit, the solution is discharged, and the first detection unit has a DC channel for the lysate that has been measured in three classes. After transferring to the second detection unit and waiting for a predetermined time, eosinophils may be counted.

In addition, when an abnormal cell of white blood cell appears, it is detected by the detection unit for three classifications in the above-described apparatus configuration example.

Although not described here, various other device configuration examples can be easily considered.

INDUSTRIAL APPLICABILITY As described above, the leukocyte classification reagent and the classification method according to the present invention are suitable for classifying leukocytes into 3 to 5 classes and simultaneously classifying and counting normal cells and abnormal cells of leukocytes.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Ikuya Takenaka 6-3-17 Okaidori, Hyogo-ku, Kobe-shi, Hyogo Inside Toa Medical Electronics Co., Ltd. No.3-17, Toa Medical Electronics Co., Ltd. (72) Inventor Hitomi Omi 6-3-17, Okaidori, Hyogo-ku, Kobe-shi, Hyogo Pref. (JP, A) JP-A-60-73356 (JP, A) JP-A-61-502277 (JP, A)

Claims (11)

(57) [Claims] 1. A leukocyte classification reagent comprising the following two liquids (a) and (b): (a) a first blood diluent containing a hyperosmotic agent;
Liquid, and (b) added to the blood sample diluted by the first liquid,
A second liquid comprising a leukocyte classification test that lyses erythrocytes and acts on leukocytes to classify and count leukocytes. (I) General formula: R ₁ -R _2- (CH ₂ CH ₂ O) _n -X (wherein, R ₁ is alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30; X is a first group of surfactants, which are polyoxyethylene-based anionic surfactants represented by: SO ₃ Na, COONa, OSO ₃ Na or ONa), and (ii) ) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30) a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant represented by the following formula: A reagent containing at least one surfactant selected from the group consisting of . 2. The reagent according to claim 1, wherein the first liquid also contains a surfactant. 3. A reagent for classifying leukocytes containing the following components: (a) ethylene glycols as a hyperosmotic agent; and (b) dissolving erythrocytes and acting on leukocytes to classify and count leukocytes. (I) a general formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —X (where R ₁ is an alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms) ; R ₂ is O, Or COO; n is an integer of 8 to 30; X is a first group of surfactants, which are polyoxyethylene-based anionic surfactants represented by: SO ₃ Na, COONa, OSO ₃ Na or ONa), and (ii) ) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30) a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant represented by the following formula: A reagent containing at least one surfactant selected from the group consisting of . 4. The reagent according to any one of claims (1) to (3), further comprising a solubilizer which selectively acts on monocytes in leukocytes to promote their contraction. 5. A leukocyte-classifying reagent comprising the following components: (a) a hyperosmotic agent; (b) a solubilizing agent which selectively acts on monocytes in leukocytes to promote their contraction; c) a leukocyte classification reagent which dissolves red blood cells and acts on leukocytes to classify and count leukocytes; (i) a general formula: R ₁ -R _2- (CH ₂ CH ₂ O) _n -X (Where R ₁ is an alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30; X is a first group of surfactants, which are polyoxyethylene-based anionic surfactants represented by: SO ₃ Na, COONa, OSO ₃ Na or ONa), and (ii) ) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30) a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant represented by the following formula: A reagent containing at least one surfactant selected from the group consisting of . 6. The reagent according to claim 4, wherein the solubilizing agent contained is at least one member selected from the group consisting of the following five groups: Solubilizer (urea-based): urea thiourea 1,1-dimethylurea ethylene urea methylurethane 1,3-dimethylurea urethane (H ₂ NCOOC ₂ H ₅ ) Solubilizer of the second group: n-octyl β-D- Glucoside CHAPS (3-[(3-chloroamidopropyl) dimethylammonio] -1-propanesulfonate) CHAPSO (3-[(3-chloroamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate) MEGA8 , 9, 10 (octanoyl-, nonanoyl- or decanoyl-N-methylglucamide) sucrose monocaprate N-formylmethylleucylalanine Solubilizers of the third group (guanidines): guanidine thioacinate guanylguanidine guanidine hydrochloride guanidine rodane salt guanidine nitrate 1,1,3,3-tetraguanidine guanidine carbonate guanidine phosphate guanidine sulfate Solubilizer (bile salt): Sodium deoxycholate Taurocholate Cholic acid Fifth group solubilizer (halogen-substituted acetic acid): Sodium trichloroacetate Sodium tribromoacetate Sodium dichloroacetate Sodium dibromoacetate Sodium monochloroacetate Monosodium acetate 7. A leukocyte classification reagent which lyses erythrocytes and acts on leukocytes to classify and count leukocytes, wherein (i) a general formula: R ₁ -R _2- (CH ₂ CH ₂ O) _n -X (wherein, R ₁ is alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30; X is a first group of surfactants, which are polyoxyethylene-based anionic surfactants represented by: SO ₃ Na, COONa, OSO ₃ Na or ONa), and (ii) ) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30) a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant represented by the following formula: A reagent containing at least one surfactant selected from the group consisting of A method of classifying leukocytes into lymphocytes, monocytes, and granulocytes by particle analysis methods such as RF method and DC method using 8. The method according to claim 7, wherein the leukocytes are classified into lymphocytes, monocytes, and granulocytes, and the leukocytes are classified into the measurement samples used for the three classifications after a predetermined time. A method of classifying leukocytes into lymphocytes, monocytes, eosinophils, and granulocytes other than eosinophils by counting the remaining eosinophils by the RF method and the DC method, or only the DC method. 9. The method according to claim 7, wherein leukocytes are classified into lymphocytes, monocytes, and granulocytes in the first detection section, while the leukocytes are classified into three types. The eosinophils left specifically by using the reagent described in
A method of classifying leukocytes into lymphocytes, monocytes, eosinophils, and granulocytes other than eosinophils by counting by the RF method and the DC method, or only the DC method in the detection unit. 10. Leukocytes are classified into four types of lymphocytes, monocytes, eosinophils, and granulocytes other than eosinophils by the method according to claim (8) or (9). Basophils using the basophil measurement reagent that specifically leaves
By counting by the DC method or only the DC method, leukocytes are converted to lymphocytes, monocytes, eosinophils, basophils, and neutrophils.
How to classify. 11. A reagent for classifying leukocytes which lyses erythrocytes and acts on leukocytes to classify and count leukocytes, wherein (i) a general formula: R ₁ -R _2- (CH ₂ CH ₂ O) _n -X (wherein, R ₁ is alkyl or alkenyl or alkynyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30; X is a first group of surfactants, which are polyoxyethylene-based anionic surfactants represented by: SO ₃ Na, COONa, OSO ₃ Na or ONa), and (ii) ) General formula: R ₁ —R ₂ — (CH ₂ CH ₂ O) _n —H (where R ₁ is an alkyl or alkenyl or alkyl group having 10 to 22 carbon atoms; R ₂ is O, Or COO; n is an integer of 8 to 30) a second group of surfactants, which is a polyoxyethylene-based nonionic surfactant represented by the following formula: A reagent containing at least one surfactant selected from the group consisting of A method for detecting abnormal cells by particle analysis methods such as RF method and DC method using