Patent Application
20140051071
The present invention relates to a blood analyzer and a blood analysis method for optically measuring a blood specimen and classifying hemocytes contained in the blood specimen, and a computer program product for enabling a computer to analyze blood.
Five types of leukocyte consisting of lymphocytes, monocytes, basophils, eosinophils, and neutrophils are present in normal peripheral blood, and many blood cell counting apparatuses have the function of classifying leukocytes contained in a blood specimen into the five types. On the other hand, cells that are not present in normal peripheral blood appear in peripheral blood affected with diseases such as viral infectious diseases and hematopoietic organ tumors. Abnormal leukocytes that appear in peripheral blood include abnormal mononuclear leukocytes and, for example, “abnormal lymphocytes”, which are neoplastic mature lymphocytes, appear as abnormal mononuclear leukocytes in peripheral blood affected with diseases such as chronic lymphatic leukemia and malignant lymphoma. On the other hand, “blasts (myeloblasts and lymphoblasts)”, which are immature lymphocytes, appear as abnormal mononuclear leukocytes in peripheral blood affected with acute leukemia. Moreover, “atypical lymphocytes”, which are lymphocytes activated by stimulation of antigen, appear as abnormal mononuclear leukocytes in peripheral blood affected with diseases such as viral infectious disease and drug allergy. Distinguishably detecting abnormal lymphocytes from atypical lymphocytes and blasts in peripheral blood is very useful in screening or diagnosis of diseases such as chronic lymphatic leukemia in which abnormal lymphocytes appear in peripheral blood.
US 2009/0023129A1 and US 2010/0151509A1 disclose distinguishably detecting abnormal leukocytes including abnormal lymphocytes from normal leukocytes using reagents for classifying leukocytes into five or four categories. Specifically, these documents disclose that a hemolyzing agent containing a cationic surfactant and a nonionic surfactant, and a stain solution containing a fluorescent dye for staining nucleic acid are used as the reagents for classifying leukocytes to develop differences of fluorescence intensity and scattered light intensity between abnormal leukocytes and normal leukocytes, and to distinguishably detect abnormal leukocytes from normal leukocytes based on the differences.
Japanese Unexamined Patent Application Publication No. JP 2007-263894A discloses distinguishably detecting myeloblasts from mature leukocytes and immature granulocytes using predetermined reagents (see FIGS. 1, 2, and 5). Specifically, this document discloses that a hemolyzing agent containing a nonionic surfactant and a solubilizing agent, and a stain solution containing a fluorescent dye for staining nucleic acid are used as the above-described reagents to develop differences of fluorescence intensity and scattered light intensity between myeloblasts, mature leukocytes and immature granulocytes, and to distinguishably detect myeloblasts from mature leukocytes and immature granulocytes based on the differences.
Japanese Unexamined Patent Application Publication No. JP 2010-237147A discloses distinguishably detecting lymphoblasts and myeloblasts from mature leukocytes and immature granulocytes using predetermined reagents (see FIGS. 13A and 13B). Specifically, this document discloses that a hemolyzing agent containing a nonionic surfactant and a solubilizing agent, and a stain solution containing a fluorescent dye for staining nucleic acid are used as the above-described reagents to distinguishably detect lymphoblasts and myeloblasts from mature leukocytes and immature granulocytes.
However, in the method disclosed in US 2009/0023129A1 described above, abnormal lymphocytes and blasts appear in the same area in a scattergram of side fluorescence intensity and side scattered light intensity, and therefore cannot be distinguished from each other (see FIG. 2). Furthermore, US 2009/0023129A1 describes nothing about an appearance of atypical lymphocytes.
US 2010/0151509A1 shows the area in which abnormal lymphocytes appear in a scattergram of side fluorescence intensity and side scattered light intensity (see FIG. 4 and FIG. 7), but describes nothing about an appearance of blasts and atypical lymphocytes. Furthermore, since the reagents described in US 2010/0151509A1 have similar characteristics to the reagents described in US 2009/0023129A1 and, in addition, the area in which abnormal lymphocytes appear in a scattergram shown in US 2010/0151509A1 is in the same position as the area in which abnormal lymphocytes and blasts appear in a scattergram shown in US 2009/0023129A1, it is assumed that, even in US 2010/0151509A1, blasts also appear in the area described above in which abnormal lymphocytes appear, and blasts and abnormal lymphocytes cannot be distinguished from each other.
Furthermore, neither JP 2007-263894A nor JP 2010-237147A describes distinguishably detecting abnormal lymphocytes from other hemocytes.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
A first aspect of the present invention is a blood analyzer comprising: a sample preparation portion for preparing a measurement sample by mixing a hemolyzing agent not substantially containing a cationic surfactant, but containing a nonionic surfactant, a blood specimen, and a fluorescent dye for staining nucleic acid; a light source for irradiating the measurement sample prepared by the sample preparation portion with light; a light-receiving portion for receiving fluorescence and scattered light that are produced by cells in the measurement sample when the light source irradiates the measurement sample with light to output a fluorescence signal relating to the received fluorescence and a scattered light signal relating to the received scattered light; a cell analysis portion for detecting cells showing fluorescence intensity and scattered light intensity in a predetermined range as abnormal lymphocytes based on the fluorescence signal and the scattered light signal output by the light-receiving portion; and an output portion for performing output based on a result of a detection made by the cell analysis portion.
A second aspect of the present invention is a blood analysis method comprising steps of: preparing a measurement sample by mixing a hemolyzing agent not substantially containing a cationic surfactant, but containing a nonionic surfactant, a blood specimen, and a fluorescent dye for staining nucleic acid; irradiating the measurement sample prepared with light; receiving fluorescence and scattered light that are produced by cells in the measurement sample when the measurement sample is irradiated with light to obtain a fluorescence signal relating to the received fluorescence and a scattered light signal relating to the received scattered light; detecting cells showing fluorescence intensity and scattered light intensity in a predetermined range as abnormal lymphocytes based on the fluorescence signal and the scattered light signal; and performing output based on a result of a detection.
A third aspect of the present invention is a computer program product comprising: a computer readable medium, and software instructions, on the computer readable medium, for enabling a computer to perform predetermined operations comprising: receiving a fluorescence signal and a scattered light signal relating to fluorescence and scattered light that are produced by cells in a measurement sample prepared by mixing a hemolyzing agent not substantially containing a cationic surfactant, but containing a nonionic surfactant, a blood specimen, and a fluorescence dye for staining nucleic acid when the measurement sample is irradiated with light; detecting cells showing fluorescence intensity and scattered light intensity in a predetermined range as abnormal lymphocytes based on the fluorescence signal and the scattered light signal; and outputting an information based on a result of a detection.
Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.
Configuration of Blood Analyzer
A blood specimen that is peripheral blood collected from a patient is housed in a specimen container (blood collecting tube). A plurality of specimen containers are held in a sample rack, and the sample rack is carried by the specimen carrying unit 4, and thereby the blood specimen is supplied to the measurement unit 2.
The configuration of the measurement unit will be described.
As shown in
The sample preparation portion 22 includes a mixing chamber MC. The suction tube 211 suctions a predetermined amount of a whole blood specimen from the specimen container T using the syringe pump. The specimen thus suctioned is transferred to the position of the mixing chamber MC, and a predetermined amount of the whole blood specimen is dispensed to the mixing chamber MC using the syringe pump. The sample preparation portion 22 also includes a heater H for heating the mixing chamber MC.
The sample preparation portion 22 is connected via a tube with a reagent container 221a for housing a first reagent, a reagent container 221b for housing a second reagent, and a reagent container 223 for housing a sheath fluid (diluting fluid). The sample preparation portion 22 is also connected with a compressor, and the respective reagents can be drawn from the corresponding reagent containers 221a, 221b, and 223 with the pressure generated by the compressor.
The first reagent is a hemolyzing agent for distinguishably detecting abnormal lymphocytes, atypical lymphocytes and blasts. The hemolyzing agent that contains a nonionic surfactant and substantially no cationic surfactant can be used as the first reagent. Use of the hemolyzing agent allows erythrocytes to be hemolyzed and the cell membranes of normal leukocytes and abnormal mononuclear leukocytes (atypical lymphocytes, abnormal lymphocytes, and blasts) to be damaged. Accordingly, normal leukocytes and abnormal mononuclear leukocytes are more likely to be stained with a fluorescent dye that will be described below.
An “abnormal lymphocyte” refers to a neoplastic mature lymphocyte. The abnormal lymphocytes appear in peripheral blood of patients who have a disease such as chronic lymphatic leukemia and malignant lymphoma. Furthermore, an “atypical lymphocyte” refers to a lymphocyte that is activated by stimulation of antigen and changes its form in response to the stimulation. The atypical lymphocytes appear in peripheral blood of patients who have a disease such as viral infectious disease and drug allergy.
A “blast” refers to an immature lymphocyte such as a myeloblast and a lymphoblast. The myeloblasts appear in peripheral blood of patients who have acute myelocytic leukemia, and the lymphoblasts appear in peripheral blood of patients who have acute lymphocytic leukemia.
Here, it is preferable that the nonionic surfactant is a polyoxyethylene-based nonionic surfactant. Specific examples of polyoxyethylene-based nonionic surfactants include those represented by structural formula (I) below:
R1—R2—(CH2CH2O)n-H (I)
where R1 is an alkyl, alkenyl, or alkynyl group having 9 to 25 carbon atoms; R2 is —O—, —COO—, or:
and n is an integer of 10 to 40.
Specific examples of surfactants represented by structural formula (I) above include polyoxyethylene (15) oleyl ether, polyoxyethylene (15) cetyl ether, polyoxyethylene (16) oleyl ether, polyoxyethylene (20) oleyl ether, polyoxyethylene (20) lauryl ether, polyoxyethylene (20) stearyl ether, and polyoxyethylene (20) cetyl ether, with polyoxyethylene (20) oleyl ether being preferable. The first reagent may contain one or two or more surfactants.
The concentration of surfactant contained in the first reagent can be suitably selected according to the kind of surfactant, the osmotic pressure of the hemolyzing agent, and like factors. For example, when the surfactant is polyoxyethylene oleyl ether, the concentration of surfactant contained in the first reagent is 0.5 to 50.0 g/L and preferably 1.0 to 20.0 g/L.
The first reagent may contain in addition to the nonionic surfactant a solubilizing agent to sufficiently shrink the hemolyzed erythrocytes so that the hemolyzed erythrocytes form a ghost population that does not adversely affect measurement. The solubilizing agent is used to assist an action for hemolyzing the erythrocytes due to the nonionic surfactants. Anionic surfactants can be used as solubilizing agents that may be contained in the first reagent, and examples thereof include sarcosine derivatives, cholic acid derivatives, methylglucanamide, n-octyl-β-glucoside, sucrose monocaprate, and N-formylmethylleucylalanine, with sarcosine derivatives being particularly preferable. The first reagent may contain one or two or more solubilizing agents.
Examples of sarcosine derivatives include compounds represented by structural formula (II) below or salts thereof
where R1 is a C10-22 alkyl group and n is 1 to 5. Specific examples of sarcosine derivatives include sodium N-lauroylsarcosinate, sodium lauroyl methyl β-alanine, and lauroyl sarcosine, with sodium N-lauroylsarcosinate being particularly preferable.
Examples of cholic acid derivatives include compounds represented by structural formula (III) below or salts thereof
where R1 is a hydrogen atom or a hydroxyl group. Specific examples of cholic acid derivatives include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and CHAPSO ([(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate).
Examples of methylglucanamides include compounds represented by structural formula (IV) below:
where n is 5 to 7. Specific examples of methylglucanamides include MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide), and MEGA10 (decanoyl-N-methylglucamide).
The concentration of solubilizing agent contained in the first reagent may be suitably selected according to the kind of solubilizing agent used. For example, when a sarcosine derivative is used as a solubilizing agent, the concentration of solubilizing agent contained in the first reagent is 0.05 to 3.0 g/L and preferably 0.1 to 1.0 g/L. When a cholic acid derivative is used, the concentration of solubilizing agent contained in the first reagent is 0.1 to 10.0 g/L and preferably 0.2 to 2.0 g/L. When a methylglucanamide is used, the concentration of solubilizing agent contained in the first reagent is 1.0 to 8.0 g/L and preferably 2.0 to 6.0 g/L. When n-octyl β-glucoside, sucrose monocaprate or N-formylmethylleucylalanine is used, the concentration of solubilizing agent contained in the first reagent is 0.01 to 50.0 g/L and preferably 0.05 to 30.0 g/L.
The pH of the first reagent is preferably 5.0 to 9.0, more preferably 6.5 to 7.5, and even more preferably 6.8 to 7.3. The pH of the first reagent can be adjusted with a buffer or a pH adjustor. Examples of buffers include Good's buffers such as HEPES, MOPS (3-morpholinopropanesulfonic acid) and MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), and phosphate buffers. Examples of pH adjustors include sodium hydroxide and hydrochloric acid.
The osmotic pressure of the first reagent can be suitably determined according to the kind of surfactant described above and the concentration thereof in the first reagent. A specific example of the osmotic pressure of the first reagent may be 10 to 600 mOsm/kg. The osmotic pressure of the first reagent may be adjusted by adding sugar, amino acid, sodium chloride, or the like to the first reagent. Specific examples of sugars include monosaccharides, polysaccharides, and sugar alcohols. Glucose and fructose are preferable as monosaccharides. Arabinose is preferable as a polysaccharide. Xylitol, sorbitol, mannitol, and ribitol are preferable as sugar alcohols. A sugar to be added to the first reagent is preferably a sugar alcohol and particularly preferably xylitol. When xylitol is added to the first reagent, the concentration of xylitol in the first reagent is preferably 1.0 to 75.0 g/L and particularly preferably 20.0 to 50.0 g/L. Specific examples of amino acids include valine, proline, glycine, and alanine, with glycine and alanine being particularly preferable. When glycine is added to the first reagent, the concentration of glycine in the first reagent is preferably 1.0 to 50.0 g/L and particularly preferably 10.0 to 30.0 g/L.
The electric conductivity of the first reagent is preferably 0.01 to 3 mS/cm and particularly preferably 0.1 to 2 mS/cm. In addition, a chelating agent, a preservative, or the like may be added to the first reagent. Examples of chelating agents include EDTA-2K and EDTA-3Na. Examples of preservatives include Proxel GXL (manufactured by Avecia) and Material TKM-A (API Corporation).
Due to the use of the first reagent, normal leukocytes and abnormal mononuclear leukocytes are more likely to be stained with a fluorescent dye that will be described below, and in addition, abnormal mononuclear leukocytes develop a difference in staining degree, size or other features of abnormal lymphocytes, atypical lymphocytes, and blasts. It is therefore possible based on the fluorescence signal (fluorescence intensity) and the scattered light signal (scattered light intensity) derived from hemocytes to distinguishably detect abnormal lymphocytes, atypical lymphocytes, and blasts in abnormal mononuclear leukocytes.
The second reagent is a stain solution for fluorescently staining nucleated cells in a blood sample. A fluorescent dye capable of staining nucleic acid is contained in the second reagent. There is no particular limitation on the fluorescent dye as long as it is capable of fluorescently staining nucleic acid. Such a dye barely stains erythrocytes that do not have nucleic acid, but stains nucleated hemocytes such as abnormal lymphocytes having nucleic acid. The fluorescent dye capable of staining nucleic acid can be suitably selected according to the light irradiated from a light source. Examples of fluorescent dyes capable of staining nucleic acid include propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylenebis[[3-[[4-[[(3-methylbenzothiazol-3-ium)-2-yl]methylene]-1,4-dihydroquinoline]-1-yl]propyl]dimethylaminium].tetraiodide (TOTO-1), 4-[(3-methylbenzothiazol-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)propyl]quinolinium diiodide (TO-PRO-1), N,N,N′,N′-tetramethyl-N,N′-bis[3-[4-[3-[(3-methylbenzothiazol-3-ium)-2-yl]-2-propenylidene]-1,4-dihydroquinolin-1-yl]propyl]-1,3-propandiaminium tetraiodide (TOTO-3), 2-[3-[[1-[3-(trimethylaminio)propyl]-1,4-dihydroquinolin]-4-ylidene]-1-propenyl]-3-methylbenzothiazol-3-ium diiodide (TO-PRO-3), and fluorescent dyes represented by structural formulas (V) to (XVIII) below.
where R1 and R2 each are a lower alkyl group; n is 1 or 2; X− is an anion; and Z is a sulfur atom, an oxygen atom, or a carbon atom substituted with a lower alkyl group.
In structural formula (V), the lower alkyl group is a linear or branched alkyl group having 1 to 6 carbon atoms. Specific examples of lower alkyl groups include a methyl group, an ethyl group, a propyl group, a butyl group, an iso-butyl group, a sec-butyl group, a tert-butyl group, a pentyl group, and a hexyl group, with a methyl group and an ethyl group being preferable. Z is preferably a sulfur atom. Examples of anions represented by X− include halogen ions (fluorine, chlorine, bromine, and iodine ions), boron halide ions (BF4−, BCl4−, BBr4−, and the like), phosphorus compound ions, halogen oxoacid ions, fluorosulfuric acid ions, methylsulfuric acid ions, and ions of tetraphenylboron compounds having a haloaromatic ring or an alkyl group having a halogen as a substituent, with an iodine ion being preferable.
Among the fluorescent dyes represented by structural formula (V), a particularly preferable fluorescent dye capable of staining nucleic acid is NK-321 represented by structural formula (VI) below:
where R1 and R2 each are a lower alkyl group; n is 1 or 2; and X− is an anion.
The lower alkyl group and the anion X− in structural formula (VII) are the same as those in structural formula (V).
Among the fluorescent dyes represented by structural formula (VII), a particularly preferable fluorescent dye capable of staining nucleic acid is represented by structural formula (VIII) below:
where R1 is a hydrogen atom or a lower alkyl group; R2 and R3 each are a hydrogen atom, a lower alkyl group, or a lower alkoxy group; R4 is a hydrogen atom, an acyl group, or a lower alkyl group; R5 is a hydrogen atom or a lower alkyl group that may be substituted; Z is a sulfur atom, an oxygen atom, or a carbon atom substituted with a lower alkyl group; n is 1 or 2; and X− is an anion.
The lower alkyl group and the anion represented by X− in structural formula (IX) are the same as those in structural formula (V). The lower alkoxy group refers to an alkoxy group having 1 to 6 carbon atoms. Specific examples of lower alkoxy groups include a methoxy group, an ethoxy group, and a propoxy group, with a methoxy group and an ethoxy group being particularly preferable. The acyl group is preferably an acyl group derived from an aliphatic carboxylic acid. Specific examples of acyl groups include an acetyl group and a propionyl group, with an acetyl group being particularly preferable. Examples of substituents of the lower alkyl group that may be substituted include a hydroxyl group and halogen atoms (fluorine, chlorine, bromine, and iodine). The lower alkyl group that may be substituted may be substituted by 1 to 3 substituents. It is particularly preferable that the lower alkyl group that may be substituted is a lower alkyl group substituted with one hydroxyl group. Z is preferably a sulfur atom and X− is preferably a bromine ion or BF4−.
Among the fluorescent dyes represented by structural formula (IX), particularly preferable fluorescent dyes capable of staining nucleic acid are represented by any of the three structural formulas (X) to (XII) below:
where X1 and X2 are independently Cl or I.
Among the above-described fluorescent dyes capable of staining nucleic acid, a particularly preferable fluorescent dye contained in the second reagent is NK-321 represented by the structural formula (XIX) below:
The concentration of above-described fluorescent dye capable of staining nucleic acid in the second reagent is preferably 10 to 500 mg/L and particularly preferably 30 to 100 mg/L. The second reagent may contain one or two or more fluorescent dyes capable of staining nucleic acid.
The detection portion 23 includes an optical detector D that can conduct a specimen measurement by flow cytometry. In the specimen measurement using the optical detector D, a measurement sample obtained by mixing the blood specimen, the first reagent, and the second reagent is supplied to the optical detector D, and optical information (fluorescence intensity, forward scattered light intensity, and side scattered light intensity) is detected in hemocytes in the measurement sample by the optical detector D at this time. The optical information obtained by the specimen measurement is supplied to the information processing unit 3, thereby determining whether any abnormal lymphocyte is present in the blood specimen and whether any blast is present therein.
The sheath flows system 232 is configured to cause the measurement samples to flow through the flow cell 231 in such a state where the measurement samples are each enclosed in the sheath fluid. The beam spot formation system 233 is configured to allow light irradiated from a semiconductor laser 237 to be irradiated onto the flow cell 231 through a collimator lens 238 and a condenser lens 239. The beam spot formation system 233 also includes a beam stopper 240.
The forward scattered light receiving system 234 is configured to focus forward scattered light with a forward focusing lens 241, and receive the light that has passed through a pinhole 242 with a photodiode (forward scattered light receiving portion) 243.
The side scattered light receiving system 235 is configured to focus side scattered light with a side focusing lens 244, reflect a portion of the light at a dichroic mirror 245, and receive the reflected light with a photodiode (side scattered light receiving portion) 246.
Light scattering is a phenomenon that occurs when light changes the direction of its movement due to the presence of particles such as hemocytes in the movement direction as impediments. Information relating to the size and the material of the particles can be obtained by detecting such scattered light. In particular, information relating to the size of the particles (hemocytes) can be obtained from forward scattered light. Meanwhile, information about the interior of the particles can be obtained from side scattered light. When hemocyte particles are irradiated with laser light, the intensity of side scattered light is dependent on the complexity of the cell interior (the shape, size, and density of the nucleus, and the granular amount). Therefore, the intensity of these scattered light beams can be utilized for classification of leukocytes, detection of blasts, and the like.
The fluorescence receiving system 236 is configured to allow the light that has transmitted through the dichroic mirror 245 to further transmit through a spectral filter 247, and receive the transmitted light with an avalanche photodiode (fluorescence receiving portion) 248.
When a hemocyte that has been stained by a fluorescent substance is irradiated with light, the hemocyte emits light having a wavelength longer than the wavelength of the irradiated light. The intensity of fluorescence is increased if the hemocyte has been stained well, and information relating to the staining degree of the hemocyte can be obtained by measuring the fluorescence intensity. Accordingly, the difference in fluorescence intensity can be utilized for detection of leukocytes, detection of abnormal lymphocytes, detection of blasts, and the like.
Referring back to
The specimen container carrying portion 25 also includes a specimen container setting portion 25b having a hole into which the specimen container T can be inserted. After completion of agitation, the specimen container T gripped by the hand portion 25a described above is moved such that the gripped specimen container T is inserted into the hole of the specimen container setting portion 25b. Then, the gripping members are moved away from each other, thereby releasing the specimen container T from the hand portion 25a and setting the specimen container T in the specimen container setting portion 25b. The specimen container setting portion 25b can be moved horizontally in Y1 and Y2 directions in
A bar code reading portion 26 is provided inside the measurement unit 2. The specimen container setting portion 25b can be moved to a bar code reading position 26a in the vicinity of the bar code reading portion 26 and to the suction position 21a where the specimen is suctioned by the specimen suction portion 21. When the specimen container setting portion 25b is moved to the bar code reading position 26a, the bar code of the specimen is read by the bar code reading portion 26. When the specimen container setting portion 25b is moved to the suction position, the specimen is suctioned by the specimen suction portion 21 from the specimen container T that has been set.
Next, the configuration of the information processing unit 5 will be described. The information processing unit 5 is configured by a computer.
The CPU 51a can execute a computer program loaded into the RAM 51c. The computer 5a functions as the information processing unit 5 by the CPU 51a executing a computer program 54a for blood analysis and control of the measurement unit 2 and the specimen carrying unit 4 as will be described later.
Various computer programs, including, for example, an operating system and application programs, for being executed by the CPU 51a, and the data used for execution of such computer programs are installed in the hard disk 51d. The computer program 54a for enabling the CPU to execute processing described later is also installed in the hard disk 51d. The computer program 54a is an event-driven computer program.
The readout device 51e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read out the computer programs or data recorded in a portable recording medium 54. The computer program 54a for enabling the computer to function as the information processing unit 5 is stored in the portable recording medium 54. The computer 5a can read out the computer program 54a from the portable recording medium 54, and install the computer program 54a in the hard disk 51d.
For example, a multitasking operating system such as Windows (registered trademark) manufactured and sold by Microsoft Corporation, US is installed in the hard disk 51d. The following description is given assuming that the computer program 54a according to this embodiment runs on that operating system.
The input/output interface 51f is configured, for example, by a serial interface such as USB, IEEE1394, or RS-232C, a parallel interface such as SCSI, IDE, or IEEE1284, and an analog interface made up of a D/A converter, an A/D converter, and the like. An input portion 53 made up of a keyboard and a mouse is connected to the input/output interface 51f, and the user can input data into the computer 5a using the input portion 53. Furthermore, the input/output interface 51f is connected to the measurement unit 2 and the specimen carrying unit 4. This enables the information processing unit 5 to control each of the measurement unit 2 and the specimen carrying unit 4.
The communication interface 51g is an Ethernet (registered trademark) interface. The communication interface 51g is connected to a host computer 6 via a LAN (see
Hereinafter, the operation of the blood analyzer 1 according to this embodiment will be described. The blood analyzer 1 can perform a specimen measurement using the optical detector D. The specimen measurement steps include a measurement step in which a measurement sample is measured and a data processing step in which the measurement data obtained in the measurement step is subjected to analysis processing.
First, the sample rack L holding the specimen container T is placed on the specimen carrying unit 4 by the operator. The sample rack L is carried by the specimen carrying unit 4, and the specimen container T housing a specimen to be measured is positioned in the specimen supply position 43a. Next, the specimen container T is gripped by the hand portion 25a of the measurement unit 2, and the specimen container T is taken out from the sample rack L. The hand portion 25a then causes oscillating movement, and thereby the specimen inside the specimen container T is agitated. Next, the specimen container T is inserted into the specimen container setting portion 25b, and the specimen container setting portion 25b is moved in the Y direction. After the bar code of the specimen is read by the bar code reading portion 26, the specimen container T reaches the suction position. Then, a measurement step described below is performed.
Measurement Step The measurement step will be described first. In the measurement step, the blood analyzer 1 mixes a whole blood specimen (17.0 μL), a first reagent (1000 μL), and a second reagent (20 μL) to prepare a measurement sample, and measures the measurement sample by flow cytometry using the optical detector D.
In this embodiment, the following reagents are used as the first reagent.
The above-listed ingredients were mixed, and NaOH was further added to adjust the pH to 7.3. The osmotic pressure of the first reagent was 37 mOsm/Kg, and the electric conductivity thereof was 0.745 mS/cm.
NK-321 (50 mg/L) dissolved in ethylene glycol was used as the second reagent.
Next, the CPU 51a controls the measurement unit 2 to supply, to the mixing chamber MC, the first reagent (1000 μL) from the reagent container 221a, the second reagent (20 μL) from the reagent container 221b, and the whole blood specimen (17.0 μL) from the suction tube 211 (step S102). In this step S102, the specimen supplied to the mixing chamber MC is a portion of the whole blood specimen suctioned by the suction tube 211 in the step S101 described above.
Next, the CPU 51a waits 18.5 seconds and determines whether 18.5 seconds have elapsed since the supply of the first reagent, the second reagent and the whole blood specimen to the mixing chamber MC (step S103). Here, the mixing chamber MC has been heated to 34.0° C. by the heater. Thus, the mixed solution of the first reagent, the second reagent and the blood specimen is heated at 34.0° C. for 18.5 seconds to prepare the measurement sample.
Then, optical measurement is conducted on the measurement sample with the optical detector D (step S104). Specifically, in the processing of step S104, the measurement sample and the sheath fluid are simultaneously supplied to the flow cell 231 of the optical detector D. At that time, forward scattered light is received by the photodiode 243, side scattered light is received by the photodiode 246, and fluorescence is received by the avalanche photodiode 248. In this optical measurement, a red semiconductor laser having an excitation wavelength of 633 nm is used as a light source to detect fluorescence (red fluorescence) having a wavelength of 650 nm or more as a fluorescence signal. Output signals (analog signals) output from these various light-receiving elements of the optical detector D are converted into digital signals by an A/D converter (not shown), and then converted into measurement data that is digital data through predetermined signal processing. The measurement data is transmitted to the information processing unit 5. In this signal processing, a forward scattered light signal (forward scattered light intensity), a side scattered light signal (side scattered light intensity), and a fluorescence signal (fluorescence intensity) are obtained as feature parameters contained in the measurement data. This completes the measurement step. As will be described later, the CPU 51a of the information processing unit 5 performs predetermined analysis processing on the measurement data to detect abnormal lymphocytes or blasts and generate analysis result data containing this detection result, and stores the analysis result data in the hard disk 51d.
Next, the data processing step will be described.
In the step S202, the CPU 51a detects a cell group of abnormal lymphocytes (hereinafter, referred to as an “abnormal lymphocyte group”) using the measurement data, and counts the number of hemocytes CN1 contained in the detected abnormal lymphocyte group (step S202).
Processing of step S202 will be described with reference to a schematic diagram.
In this embodiment, the range of forward scattered light intensity and fluorescence intensity indicated by the dashed line in
Next, the CPU 51a determines whether CN1 is greater than a predetermined threshold T1 (step S203). The threshold T1 is a reference value for determining whether any abnormal lymphocyte is present in a blood specimen. In step S203, if CN1 is greater than the threshold T1, it is determined that abnormal lymphocytes are present in the blood specimen. If CN1 is less than or equal to the threshold T1, it is determined that no abnormal lymphocytes are present in the blood specimen.
If CN1>T1 in step S203 (YES in step S203), the CPU 51a sets an abnormal lymphocyte flag provided in the RAM 51c to “1” and sets a blast flag to “0” (step S204). Here, the abnormal lymphocyte flag is a flag indicating the presence or absence of abnormal lymphocytes in a blood specimen. The abnormal lymphocyte flag indicates the presence of abnormal lymphocytes if it is set to “1”, and indicates the absence of abnormal lymphocytes if it is set to “0”. The blast flag is a flag indicating the presence or absence of blasts in the blood specimen. The blast flag indicates the presence of blasts if it is set to “1”, and indicates the absence of blasts if it is set to “0”. Then, the processing executed by the CPU 51a moves to step S209.
On the other hand, if CN1≦T1 in step S203 (NO in step S203), the CPU 51a detects a cell group of blasts (hereinafter, referred to as a “blast group”) using the measurement data, and counts the number of hemocytes CN2 contained in the detected blast group (step S205).
The processing of step S205 will be described in detail.
Next, the CPU 51a determines whether CN2 is greater than a predetermined threshold T2 (step S206). The threshold T2 is a reference value for determining whether any blast is present in a blood specimen. In step S206, if CN2 is greater than the threshold T2, it is determined that blasts are present in the blood specimen. If CN2 is less than or equal to the threshold T2, it is determined that no blasts are present in the blood specimen.
If CN2>T2 (YES in step S206), the CPU 51a sets the blast flag provided in the RAM 51c to “1” and sets the abnormal lymphocyte flag to “0” (step S207). Then, the processing executed by the CPU 51a moves to step S208.
On the other hand, if CN2≦T2 (NO in step S206), the CPU 51a sets each of the abnormal lymphocyte flag and the blast flag provided in the RAM 51c to “0” (step S208). Then, the processing executed by the CPU 51a moves to step S209.
In the step S209, the CPU 51a stores the thus obtained analysis result (including the abnormal lymphocyte flag and blast flag) in the hard disk 51d. Next, the CPU 51a causes the image display portion 52 to display an analysis result screen showing the analysis result stored in the hard disk 51d (step S210), and ends the data processing.
Next, examples of scattergrams obtained when measuring a specific blood specimen are shown, and the analysis of the measurement data performed by the blood analyzer 1 according to this embodiment will be described.
As shown in
When the blood specimen A was checked by hand method (visual check using a microscope), the ratio of the number of myeloblasts to the number of total leukocytes was 15%, and the ratio of the number of each of lymphoblasts, abnormal lymphocytes and atypical lymphocytes to the number of total leukocytes was 0%. Therefore, it can be seen that detection of blasts in the blood specimen A by the blood analyzer 1 is appropriate.
As shown in
When the blood specimen B was checked by hand method, the ratio of the number of lymphoblasts to the number of total leukocytes was 23.5%, and the ratio of the number of each of myeloblasts, abnormal lymphocytes and atypical lymphocytes to the number of total leukocytes was 0%. Therefore, it can be seen that detection of blasts in the blood specimen B by the blood analyzer 1 is appropriate.
As shown in
When the blood specimen C was checked by hand method, the ratio of the number of abnormal lymphocytes to the number of total leukocytes was 9%, and the ratio of the number of each of myeloblasts, lymphoblasts and atypical lymphocytes to the number of total leukocytes was 0%. Therefore, it can be seen that detection of abnormal lymphocytes in the blood specimen C by the blood analyzer 1 is appropriate.
As shown in
When the blood specimen D was checked by hand method, the ratio of the number of atypical lymphocytes to the number of total leukocytes was 7%, and the ratio of the number of each of myeloblasts, lymphoblasts and abnormal lymphocytes to the number of total leukocytes was 0%. Therefore, it can be seen that no detection of both blasts and abnormal lymphocytes in the blood specimen D by the blood analyzer 1 is appropriate.
As described above, when a blood specimen containing abnormal lymphocytes, but not containing blasts and atypical lymphocytes is measured by the blood analyzer 1, abnormal lymphocytes are detected and blasts are not detected. Similarly, when a blood specimen containing blasts, but not containing abnormal lymphocytes and atypical lymphocytes is measured by the blood analyzer 1, blasts are detected and abnormal lymphocytes are not detected. Furthermore, when a blood specimen containing atypical lymphocytes, but not containing abnormal lymphocytes and blasts is measured by the blood analyzer 1, neither abnormal lymphocytes nor blasts are detected. That is, with the blood analyzer 1 according to the embodiment, it is possible to detect abnormal lymphocytes and blasts separately.
Abnormal lymphocytes, blasts, and atypical lymphocytes do not appear in normal peripheral blood. As shown in
When the blood specimen E was checked by hand method, the ratio of the number of each of myeloblasts, lymphoblasts, abnormal lymphocytes, and atypical lymphocytes to the number of total leukocytes was 0%. Therefore, it can be seen that detection of neither blasts nor abnormal lymphocytes in the blood specimen E by the blood analyzer 1 is appropriate.
With the configuration described above, the blood analyzer 1 can detect abnormal lymphocytes and blasts separately by measuring, with the optical detector D, the measurement sample prepared by mixing the blood specimen, the first reagent containing a hemolyzing agent, and the second reagent containing a fluorescent dye for staining nucleic acid.
Hereinafter, a blood analyzer according to this embodiment will be described.
First, configuration of a blood analyzer according to this embodiment will be described.
The configuration of the measurement unit will be described.
The sample preparation portion 220 is connected via a tube with a reagent container 221a for housing a first reagent, a reagent container 221b for housing a second reagent, a reagent container 222a for housing a third reagent, a reagent container 222b for housing a fourth reagent, and a reagent container 223 for housing a sheath fluid (diluting fluid). The sample preparation portion 220 is also connected with a compressor, and the respective reagents can be drawn from the corresponding reagent containers 221a, 221b, 222a, 222b, and 223 with the pressure generated by the compressor.
The third reagent is a hemolyzing agent for measuring nucleated erythrocytes (NRBCs). Examples of this hemolyzing agent for measuring NRBCs include a Stromatolyser NR Lyse manufactured by Sysmex Corporation. The fourth reagent is a stain solution containing a fluorescent dye for measuring NRBCs. Examples of this stain solution for measuring NRBCs include a Stromatolyser NR Dye manufactured by Sysmex Corporation.
Note that the first reagent, the second reagent and the sheath fluid are the same as those in Embodiment 1, and therefore the description thereof is omitted.
Furthermore, since configurations of other elements of the blood analyzer 200 according to this embodiment are the same as those of the blood analyzer 1 according to Embodiment 1, the same elements are denoted by the same reference numerals, and the descriptions thereof are omitted.
Next, the specimen measuring operation of the blood analyzer 200 according to this embodiment will be described.
The blood analyzer 200 according to this embodiment can perform an abnormal lymphocyte/blast measurement and an NRBC measurement using the optical detector D. The measurement steps include a first measurement step in which a measurement sample for abnormal lymphocyte/blast is measured, a second measurement step in which a measurement sample for NRBC is measured, and a data processing step in which the measurement data obtained in the first measurement step and the second measurement step are subjected to analysis processing.
First, the sample rack L holding the specimen container T is placed on the specimen carrying unit 4 by the operator. The sample rack L is carried by the specimen carrying unit 4, and the specimen container T housing a specimen to be measured is positioned in the specimen supply position 43a. Next, the specimen container T is gripped by the hand portion 25a of the measurement unit 202, and the specimen container T is taken out from the sample rack L. The hand portion 25a then makes an oscillating movement, and thereby the specimen inside the specimen container T is agitated. Next, the specimen container T is inserted into the specimen container setting portion 25b, and the specimen container setting portion 25b is moved in the Y2 direction. After the bar code of the specimen is read by the bar code reading portion 26, the specimen container T reaches the suction position. Then, the first measurement step and the second measurement step are performed.
In the first measurement step, a measurement sample for abnormal lymphocytes/blasts is prepared by supplying a predetermined amount of the first reagent, a predetermined amount of the second reagent, and a predetermined amount of the whole blood specimen to the first mixing chamber MC1, and optical measurement is conducted on the measurement sample for abnormal lymphocytes/blasts with the optical detector D. Since this first measurement step is the same as the measurement step according to Embodiment 1, the detailed description thereof is omitted. In the first measurement step, a first measurement data which is a digital data containing feature parameters of a forward scattered light signal (forward scattered light intensity), a side scattered light signal (side scattered light intensity), and a fluorescence signal (fluorescence intensity) is generated, and the first measurement data is transmitted to the information processing unit 5.
Next, the second measurement step will be described. The second measurement step is performed in such a manner that it partially overlaps in time with the first measurement step. In the second measurement step, the blood analyzer 200 mixes the whole blood specimen (17.0 μL), the third reagent (1.0 mL), and the fourth reagent (0.030 mL) to prepare an NRBC measurement sample, and measures the NRBC measurement sample by flow cytometry using the optical detector D.
Next, the CPU 51a waits 7.0 seconds and determines whether 7.0 seconds have elapsed since the supply of the third reagent, the fourth reagent and the whole blood specimen to the second mixing chamber MC2 (step S302). Here, the second mixing chamber MC2 has been heated to 41.0° C. by the heater. Thus, the mixed solution of the third reagent, the fourth reagent and the blood specimen is heated at 41.0° C. for 7.0 seconds to prepare an NRBC measurement sample.
Then, optical measurement is conducted on the NRBC measurement sample with the optical detector D (step S303). Specifically, in the processing of step S303, the NRBC measurement sample and the sheath fluid are simultaneously supplied to the flow cell 231 of the optical detector D. At that time, forward scattered light is received by the photodiode 243, and side scattered light is received by the photodiode 246, and fluorescence is received by the avalanche photodiode 248. Output signals (analog signals) output from these various light-receiving elements of the optical detector D are converted into digital signals as in the first measurement step (measurement step according to Embodiment 1) described above, and then converted into second measurement data that is digital data through predetermined signal processing. The second measurement data is transmitted to the information processing unit 5. In this signal processing, a forward scattered light signal (forward scattered light intensity), a side scattered light signal (side scattered light intensity), and a fluorescence signal (fluorescence intensity) are obtained as feature parameters contained in the second measurement data. This completes the second measurement step. As will be described later, the CPU 51a of the information processing unit 5 performs predetermined analysis processing on the second measurement data, thereby generating analysis result data containing the numeric data of NRBCs, and stores the analysis result data in the hard disk 51d.
Next, the data processing step will be described.
In step S402, the CPU 51a detects a cell group of abnormal lymphocytes or NRBCs (hereinafter, referred to as an “abnormal lymphocyte/NRBC group”) using the first measurement data, and counts the number of hemocytes CN21 contained in the detected abnormal lymphocyte/NRBC group (step S402).
In this embodiment, the detection area A1 in
Next, the CPU 51a determines whether CN21 is greater than a predetermined threshold T21 (step S403). The threshold T21 is a reference value for determining whether any abnormal lymphocyte or any NRBCs is present in a blood specimen. In step S403, if CN21 is greater than the threshold T21, it is determined that abnormal lymphocytes or NRBCs are present in the blood specimen. If CN21 is less than or equal to the threshold T21, it is determined that neither abnormal lymphocytes nor NRBCs are present in the blood specimen.
If CN21>T21 in step S403 (YES in step S403), the CPU 51a classifies the NRBC group and other hemocyte groups using the second measurement data and counts the number of nucleated erythrocytes CN22 (step S404). The processing will be described in detail.
Next, the CPU 51a determines whether CN22 is greater than a predetermined threshold T22 (step S405). The threshold T22 is a reference value for determining whether any NRBC is present in a blood specimen. In step S405, if CN22 is greater than the threshold T22, it is determined that NRBCs are present in the blood specimen. If CN22 is less than or equal to the threshold T22, it is determined that no NRBCs are present in the blood specimen.
If CN22>T22 in step S405 (YES in step S405), the CPU 51a sets the NRBC flag provided in the RAM 51c to “1”, and sets each of the abnormal lymphocyte flag and the blast flag to “0” (step S406). Here, the NRBC flag is a flag indicating the presence or absence of NRBCs in a blood specimen. The NRBC flag indicates the presence of NRBCs if it is set to “1”, and indicates the absence of NRBCs if it is set to “0”. Note that the abnormal lymphocyte flag and blast flag are the same as in Embodiment 1, and therefore the descriptions thereof are omitted. Then, the processing executed by the CPU 51a moves to step S412.
On the other hand, if CN22≦T22 in step S405 (NO in step S405), it is determined that no NRBCs are present in the blood specimen. That is, in step S402, it is established that a hemocyte group detected in the detection area A1 is an abnormal lymphocyte group. Accordingly, in this case, the CPU 51a sets the abnormal lymphocyte flag provided in the RAM 51c to “1”, and sets each of the NRBC flag and the blast flag to “0” (step S407). Then, the processing executed by the CPU 51a moves to step S412.
If CN21≦T21 in step S403 (NO in step S403), the CPU 51a detects a blast group using the first measurement data, and counts the number of hemocyte CN23 contained in the detected blasts group (step S408). Note that the processing of step S408 is the same as that of step S205 described in Embodiment 1, and therefore the description thereof is omitted.
Next, the CPU 51a determines whether CN23 is greater than a predetermined threshold T23 (step S409). The threshold T23 is a reference value for determining whether any blast is present in a blood specimen. In step S409, if CN23 is greater than the threshold T23, it is determined that blasts are present in the blood specimen. If CN23 is less than or equal to the threshold T23, it is determined that no blasts are present in the blood specimen.
If CN23>T23 (YES in step S409), the CPU 51a sets the blast flag provided in the RAM 51c to “1”, and sets each of the abnormal lymphocyte flag and the NRBC flag to “0” (step S410). Then, the processing executed by the CPU 51a moves to step S412.
On the other hand, if CN23≦T23 (NO in step S409), the CPU 51a sets each of the abnormal lymphocyte flag, the blast flag and the NRBC flag provided in the RAM 51c to “0” (step S411). Then, the processing executed by the CPU 51a moves to step S412.
In step S412, the CPU 51a stores the thus obtained analysis result (including the abnormal lymphocyte flag, the blast flag and the NRBC flag) in the hard disk 51d. Next, the CPU 51a causes the image display portion 52 to display an analysis result screen showing the analysis result stored in the hard disk 51d (step S413), and ends the processing.
Next, examples of scattergrams obtained when measuring a specific blood specimen are shown, and the analysis of the measurement data performed by the blood analyzer 200 according to this embodiment will be described.
As shown in
As shown in
As described above, when a blood specimen containing abnormal lymphocytes and containing none of blasts, NRBCs and atypical lymphocytes is measured by the blood analyzer 200, abnormal lymphocytes are detected, and blasts and NRBCs are not detected. Similarly, when a blood specimen containing NRBCs and containing none of abnormal lymphocytes, blasts and atypical lymphocytes is measured by the blood analyzer 200, NRBCs are detected, and neither abnormal lymphocytes nor blasts are detected. Similarly, when a blood specimen containing blasts and containing none of abnormal lymphocytes, NRBCs and atypical lymphocytes is measured by the blood analyzer 200, blasts are detected, and neither abnormal lymphocytes nor NRBCs are detected. Furthermore, when a blood specimen containing atypical lymphocytes and containing none of abnormal lymphocytes, blasts and NRBCs is measured, neither abnormal lymphocytes, blasts nor NRBCs are detected. That is, with the blood analyzer 200 according to the embodiment, it is possible to detect abnormal lymphocytes, blasts and NRBCs separately.
Although not shown, when the NRBC flag is set to “1” and each of the abnormal lymphocyte flag and the blast flag is set to “0”, “NRBC present” is indicated in the flag indication field FLG on the analysis result screen shown in
With the configuration described above, the blood analyzer 200 can detect abnormal lymphocytes, blasts and NRBCs separately by measuring, with the optical detector D, an abnormal lymphocyte/blast measurement sample prepared by mixing the blood specimen, the first reagent containing a hemolyzing agent, and the second reagent containing a fluorescent dye for staining nucleic acid, and measuring, with the optical detector D, an NRBC measurement sample prepared by mixing the blood specimen, the third reagent, and the fourth reagent. Furthermore, the blood analyzer 200 can suppress misdetections of NRBCs as abnormal lymphocytes.
Note that the reaction temperature and the reaction time during mixing of the blood specimen, the first reagent, and the second reagent in the sample preparation portion 22 may be suitably set according to the state of damage or staining of the hemocytes contained in the blood specimen, without any particular limitation. Specifically, the reaction time and the reaction temperature may be adjusted such that the reaction time is short when the reaction temperature is high and the reaction time is long when the reaction temperature is low. More specifically, it is preferable that the blood specimen and the reagents are mixed at a temperature of 20° C. to 45° C. for 3 to 40 seconds.
Although the above-described embodiment has addressed a configuration in which the first reagent containing a hemolyzing agent and the second reagent containing a fluorescent dye that can stain nucleic acid are used to perform the measurement step, the present invention is not limited thereto. It is possible to adopt a configuration in which the measurement sample is prepared by mixing the blood specimen with a single reagent containing a hemolyzing agent and a nucleic acid staining dye, and abnormal lymphocytes and blasts are detected using the measurement sample. In this case, the concentrations of the surfactant and the fluorescent dye are adjusted to the above-described concentrations when the reagents have been mixed.
Although the above-described embodiment has addressed a configuration in which the presence or absence of abnormal lymphocytes in the blood specimen is determined based on whether the number of hemocytes CN1 that appeared within the detection area A1 is greater than the threshold T1 for abnormal lymphocytes with respect to forward scattered light intensity and fluorescence intensity in the measurement data, the present invention is not limited thereto. It is also possible to adopt a configuration in which the ratio of the number of hemocytes that appeared within the detection area A1 for abnormal lymphocytes to the total number of leukocytes is obtained, and whether abnormal lymphocytes are present is determined by determining whether the obtained ratio is greater than a predetermined reference value. Likewise, for detection of blasts as well, it is also possible to adopt a configuration in which the ratio of the number of hemocytes that appeared within the detection area A2 for blasts to the total number of leukocytes is obtained, and whether any blast is present is determined by determining whether the obtained ratio is greater than a predetermined reference value.
Although the above-described embodiment has addressed a configuration in which the measurement sample is optically measured by flow cytometry to obtain an optical signal including fluorescence intensity, forward scattered light intensity, and side scattered light intensity, and the fluorescence intensity and the scattered light intensity are used to detect a cell group not containing blasts and atypical lymphocyte, but containing abnormal lymphocytes in a blood specimen, the present invention is not limited thereto. It is also possible to adopt a configuration in which scattered light information other than forward scattered light intensity and side scattered light intensity, such as wide angle forward scattered light intensity, is obtained along with fluorescence intensity, and a cell group not containing blasts and atypical lymphocytes, but containing abnormal lymphocytes is detected in the predetermined range of the scattered light information and the fluorescence intensity.
Although the above-described embodiment has addressed a configuration in which the control of the measurement unit 2 and the processing of the measurement data are performed by the CPU 51a executing the above-described computer program 54a, the present invention is not limited thereto. It is also possible to adopt a configuration in which the control of the measurement unit 2 and the processing of measurement data are performed by dedicated hardware, such as FPGA or ASIC, that can perform the same processing as that performed by the computer program 54a.
Although the above-described embodiment has addressed a configuration in which a single computer 5a executes all the processing of the computer program 54a, the present invention is not limited thereto. It is also possible to adopt a distributed system in which the same processing as that of the above-described computer program 54a is executed by a plurality of devices (computers) in a distributed manner.
In Embodiment 2 described above, although the CPU 51a determines that a hemocyte group that appeared in the detection area A1 is an NRBC group if CN22>T22 in S405 of the data processing step, the CPU 51a may further perform steps for determining an appearance of blasts (S408 and S409) if CN22>T22 in S405. Both NRBCs and blasts are blood cells which are normally present in bone marrow. Therefore, both NRBCs and blasts may appear in peripheral blood due to bone marrow diseases.
In Embodiment 2 described above, although the CPU 51a determines whether NRBCs are present, and whether abnormal lymphocytes are present, in the data processing step, the CPU 51a may perform the step for determining whether NRBCs are present independently of the step for determining whether abnormal lymphocytes are present.
The same value as the threshold T21 that is used to determine whether abnormal lymphocytes are present, or the different value from the T21 may be used as the threshold T22 that is used to determine whether NRBCs are present.
The present inventors conducted a performance evaluation experiment of a blood analysis method performed with the above-described blood analyzer 1.
In this experiment, a plurality of blood specimens was examined with a microscope by hand method, and the numbers of lymphoblasts, abnormal lymphocytes and atypical lymphocytes were counted. If the ratio of the number of lymphoblasts to the number of total leukocytes was not less than 2%, the blood specimen was determined to be “positive” for lymphoblasts. If the ratio of the number of abnormal lymphocytes to the number of total leukocytes was not less than 10%, the blood specimen was determined to be “positive” for abnormal lymphocytes. If the ratio of the number of atypical lymphocytes to the number of total leukocytes was not less than 2%, the blood specimen was determined to be “positive” for atypical lymphocytes. Furthermore, the blood specimen in which no appearances of lymphoblasts, abnormal lymphocytes and atypical lymphocytes were observed was determined to be a “negative specimen”.
In this experiment, the specimen that was determined to be positive for lymphoblasts by the above-described microscopic examination and was not determined to be positive for abnormal lymphocytes and atypical lymphocytes (hereinafter, referred to as a “lymphoblast positive specimen”) was measured by flow cytometry by following the procedure below. Likewise, in this experiment, the specimen that was determined to be positive for abnormal lymphocytes by the microscopic examination and was not determined to be positive for lymphoblasts and atypical lymphocytes (hereinafter, referred to as an “abnormal lymphocyte positive specimen”) was measured by flow cytometry. The specimen that was determined to be positive for atypical lymphocytes by the microscopic examination and was not determined to be positive for lymphoblasts and abnormal lymphocytes (hereinafter, referred to as an “atypical lymphocyte positive specimen”) was also measured by following the procedure below. Furthermore, the above-described “negative specimen” was measured by flow cytometry.
The following reagents were used for the measurement by flow cytometry.
Hemolyzing Agent
MOPS: 2.09 g/L
polyoxyethylene (20) oleyl ether: 1.25 g/L
sodium N-lauroylsarcosinate: 0.268 g/L
EDTA-2K: 0.5 g/L
The above-listed ingredients were mixed, and NaOH was further added to adjust the pH to 7.3. The osmotic pressure of the first reagent was 37 mOsm/Kg, and the electric conductivity thereof was 0.745 mS/cm.
Staining Reagent
NK-321: 50 mg/L
NK-321 (50 mg/L) dissolved in ethylene glycol was used as the second reagent.
A measurement sample was prepared by mixing a whole blood specimen (17.0 μL), a hemolyzing agent (1000 μL), and a staining reagent (20 μL), and measured by flow cytometry using the optical detector. A red semiconductor laser having an excitation wavelength of 633 nm was used as a light source to detect fluorescence (red fluorescence) having a wavelength of 650 nm or more as a fluorescence signal.
In the scattergram of forward scattered light intensity and fluorescence intensity obtained by performing the measurement on each specimen, the number of hemocytes that appeared in the area corresponding to the detection area A1 shown in
In the scattergram of forward scattered light intensity and side scattered light intensity obtained by measuring the specimens, the number of hemocytes LC1# that appeared in the area corresponding to the detection area A2 shown in
Experimental results on the lymphoblast positive specimen are shown in Table 1. Note that values in Table 1 show the number of specimens (similarly in Tables 2 to 4).
As a result of measuring all of 11 lymphoblast positive specimens by flow cytometry, no specimen was determined to be positive for abnormal lymphocytes. That is to say, the number of specimens in which abnormal lymphocytes were detected by flow cytometry measurement is 0, and the number of specimens in which no abnormal lymphocytes were detected was 11, among the lymphoblast positive specimens. As a result of flow cytometry measurement, the number of specimens that was determined to be positive for blasts was 9, and the number of specimens that was determined to be negative for blasts was 2, among the 11 lymphoblast positive specimens. That is to say, blasts were detected in 81.8% of the lymphoblast positive specimens by flow cytometry measurement. This shows that lymphoblasts contained in a lymphoblast positive specimen do not substantially appear in the detection area A1 for detecting abnormal lymphocytes, and appear in the detection area A2 for detecting blasts. That is, with the blood analysis method according to the embodiment, it is possible to accurately detect blasts in a blood specimen containing lymphoblasts, and not containing abnormal lymphocytes and atypical lymphocytes without misdetection of abnormal lymphocytes.
Experimental results on the abnormal lymphocyte positive specimen are shown in Table 2.
As a result of flow cytometry measurement, the number of specimens that was determined to be positive for abnormal lymphocytes was 8, and the number of specimens that was determined to be negative for abnormal lymphocytes was 4, among the 12 abnormal lymphocyte positive specimens. That is to say, abnormal lymphocytes were detected in 66.7% of the abnormal lymphocyte positive specimens by flow cytometry measurement. As a result of measuring all of the 12 abnormal lymphocyte positive specimens by flow cytometry, no specimen was determined to be positive for blasts. That is to say, the number of specimens in which blasts were detected by flow cytometry measurement is 0, and the number of specimens in which no blasts were detected was 12, among the abnormal lymphocyte positive specimens. This shows that abnormal lymphocytes contained in an abnormal lymphocyte positive specimen appear in the detection area A1 for detecting abnormal lymphocytes, and do not substantially appear in the detection area A2 for detecting blasts. That is, with the blood analysis method according to the embodiment, it is possible to accurately detect abnormal lymphocytes in a blood specimen containing abnormal lymphocytes, and not containing lymphoblasts and atypical lymphocytes without misdetection of blasts.
Experimental results on the atypical lymphocyte positive specimen are shown in Table 3.
As a result of flow cytometry measurement, the number of specimens that was determined to be positive for abnormal lymphocytes was 2, and the number of specimens that was determined to be negative for abnormal lymphocytes was 32, among the 34 atypical lymphocyte positive specimens. That is to say, abnormal lymphocytes were not detected in almost all of the atypical lymphocyte positive specimens by flow cytometry measurement. As a result of measuring all of 34 atypical lymphocyte positive specimens by flow cytometry, no specimen was determined to be positive for blasts. That is to say, the number of specimens in which blasts were detected by flow cytometry measurement is 0, and the number of specimens in which no blasts were detected was 34, among the atypical lymphocyte positive specimens. This shows that atypical lymphocytes contained in an atypical lymphocyte positive specimen do not substantially appear in the detection area A1 for detecting abnormal lymphocytes and in the detection area A2 for detecting blasts. That is, with the blood analysis method according to the embodiment, blasts and abnormal lymphocytes are rarely misdetected in a blood specimen containing atypical lymphocytes, and not containing lymphoblasts and abnormal lymphocytes.
Experimental results on the negative specimen are shown in Table 4.
As a result of flow cytometry measurement, the number of specimens that was determined to be positive for abnormal lymphocytes was 11, and the number of specimens that was determined to be negative for abnormal lymphocytes was 1168, among the 1179 negative specimens. That is to say, abnormal lymphocytes were not detected in almost all of the negative specimens by flow cytometry measurement (where abnormal lymphocytes were detected in 0.9% of all of the negative specimens). As a result of flow cytometry measurement, the number of specimens that was determined to be positive for blasts was 4, and the number of specimens that was determined to be negative for blasts was 1175, among the 1179 negative specimens. That is to say, blasts were not detected in almost all of the negative specimens by flow cytometry measurement (where blasts were detected in 0.3% of all of the negative specimens). This shows that atypical lymphocytes contained in an atypical lymphocyte positive specimen do not substantially appear in the detection area A1 for detecting abnormal lymphocytes and in the detection area A2 for detecting blasts. That is, with the blood analysis method according to the embodiment, blasts and abnormal lymphocytes are rarely misdetected in a blood specimen containing none of atypical lymphocytes, lymphoblasts and abnormal lymphocytes.
A blood analyzer, a blood analysis method and a computer program of the present invention are useful as a blood analyzer and a blood analysis method for optically measuring a blood specimen and detecting a cell group contained in the blood specimen, and a computer program for enabling a computer to analyze blood.
The foregoing detailed description and accompanying drawings have been provided by way of explanation and illustration, and are not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be obvious to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-100810 | Apr 2011 | JP | national |
This application is a continuation of PCT/JP2012/058589 filed on Mar. 30, 2012, which claims priority to the Japanese Application No. 2011-100810 filed on Apr. 28, 2011. The entire contents of these applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2012/058589 | Mar 2012 | US |
| Child | 14063788 | US |
