Patent Application
20100248247
The present invention relates to a blood analyzer for optically measuring a blood sample and classifying a cell group contained in the blood sample into plural populations. Further, the present invention relates to a method for determining the existence and nonexistence of lymphoblasts in a blood sample.
In the blood plasma of peripheral blood, there are floating red blood cells, platelets, and white blood cells. Blood examination inspecting these cells can provide a variety of clinical information. For this reason, there have been examined a large number of samples. The blood examination is carried out using a hematocytometer. The hematocytometer provides automatic measurements of red blood cell counts, platelet counts, white blood cell counts, hemoglobin concentrations, and the like in the blood.
There are five types of white blood cells in normal peripheral blood, i.e., lymphocytes, monocytes, basophils, eosinophils, and neutrophils. Many hematocytometers have a function of classifying white blood cells in a blood sample into five types. Meanwhile, in diseases such as hematological malignancies, there is an appearance of cell types which are not present in the normal blood. For example, in acute lymphocytic leukemia (ALL), there is an appearance of a large number of lymphoblasts in the peripheral blood. Accordingly, the detection of lymphoblasts in the peripheral blood is very useful in the diagnosis of acute lymphocytic leukemia.
U.S. Pat. No. 6,004,816 discloses a method for classification of white blood cells, including the steps of:
1) mixing a blood sample with a hemolytic agent which lyses red blood cells in the blood sample to such a degree as not to impede measurement, thereby bringing normal or abnormal blood cells to a state suitable for staining;
2) mixing the sample prepared in step 1) with a dye which is represented by a certain structural formula, and specifically binds to cellular RNA to increase in fluorescence intensity, thereby fluorescent-staining nucleated cells in the blood sample;
3) measuring an assay sample prepared in step 2) with a flow cytometer to measure scattered light and fluorescence; and 4) classifying normal white blood cells into at least 5 populations, and counting them, by the use of the intensities of the scattered light and the fluorescence measured in step 3).
This patent document 1 discloses definite separation, classification and counting of atypical lymphocytes from normal white blood cells.
JP-A-2007-263958 discloses a method for classification of blood cells, including classifying a differentiation and maturing stage of myelocytic cells and B lymphoid cells, using an antibody against a certain cellular marker. JP-A-2007-263958 discloses the classification of lymphoblasts, by the use of side-scattered light and a fluorescence-labeled CD45 antibody.
US 2005202400 discloses a method for classifying and counting white blood cells, which includes:
(1) a step of staining cells, with a dye which has specificity to cell nuclei, particularly DNA, or a dye which has specificity to RNA;
(2) a step of introducing the thus prepared sample into a flow cytometer;
(3) a step of measuring scattered light and fluorescence for the respective stained cells in the sample, and classifying white blood cells and coincidence cells/platelet clumps utilizing a difference in the intensity of a scattered light peak and a difference in the scattered light width; and
(4) a step of classifying and counting mature white blood cells, white blood cells with an abnormal DNA amount and immature white blood cells, utilizing a difference in the scattered light intensity and a difference in the fluorescence intensity of classified components.
The method for classifying white blood cells disclosed in U.S. Pat. No. 6,004,816 can classify atypical lymphocytes from normal white blood cells and count them. However, the detection of lymphoblasts cannot be accomplished with this method.
The method for classification of blood cells disclosed in JP-A-2007-263958 requires the use of expensive fluorescence-labeled antibodies in measurements. As a consequence, there is a problem associated with increased measurement costs.
The method for classifying and counting white blood cells disclosed in US 2005202400 enables the classification and counting of white blood cells with an abnormal DNA amount, including lymphoblasts. However, cell types other than lymphoblasts are also included within the white blood cells with an abnormal DNA amount. Therefore, it is impossible to correctly detect whether or not lymphoblasts are present in the sample of interest.
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 blood sample supply section for supplying a blood sample;
a sample preparation section for preparing an assay sample by mixing the blood sample supplied from the blood sample supply section with a nucleic acid-staining fluorescent dye;
a light source for irradiating the assay sample;
an optical detecting section for receiving fluorescence emitted from the irradiated assay sample; and
a controller for performing operations comprising:
detecting a cell group comprising lymphoblasts, contained in the assay sample, on the basis of the fluorescence received by the optical detecting section, and
outputting an information on an appearance of the lymphoblasts in the blood sample, on the basis of the detection results.
A second aspect of the present invention is a method for determining the existence and nonexistence of lymphoblasts in a blood sample, comprising steps of:
preparing an assay sample by mixing a blood sample and a nucleic acid-staining fluorescent dye;
irradiating the assay sample;
measuring fluorescence emitted from the irradiated assay sample;
detecting a cell group comprising lymphoblasts, contained in the assay sample, on the basis of the measured fluorescence; and
The preferred embodiments of the present invention will be described hereinafter with reference to the drawings.
In this embodiment, there is provided a blood analyzer for detecting lymphoblasts in a blood sample, including mixing the blood sample with a nucleic acid-staining fluorescent dye to prepare an assay sample, and measuring the assay sample by an optical flow cytometer.
[Configuration of Blood Analyzer]
<Configuration of Measurement Unit>
Next, the configuration of the measurement unit will be described.
As shown in
The sample preparation section 22 is provided with a first mixing chamber MC1 and a second mixing chamber MC2. The aspiration tube 211 aspirates a given amount of a complete blood sample from the sample container T by a syringe pump (not shown). Then, the aspirated sample is transferred to positions of the first mixing chamber MC1 and the second mixing chamber MC2. Then, a given amount of the complete blood sample is dispensed and supplied to each of the chambers MC1 and MC2, by the syringe pump. In addition, the sample preparation section 22 is provided with a heater H for warming the first mixing chamber MC1 and the second mixing chamber MC2.
The sample preparation section 22 is connected via a tube to a reagent container 221 for accommodating a first reagent, a reagent container 222a for accommodating a second reagent, a reagent container 222b for accommodating a third reagent, and a reagent container 223 for accommodating a sheath fluid (diluent). Further, the sample preparation section 22 is connected to a compressor (not shown). The respective reagents can be aliquoted from the reagent containers 221, 222a, 222b, and 223, in response to a pressure generated by the compressor.
The first reagent is a reagent for detecting a blood cell group composed of lymphoblasts and nucleated red blood cells (hereinafter, referred to as “lymphoblast/nucleated red blood cell group”). The first reagent contains a hemolytic agent and a fluorescent dye. As the hemolytic agent contained in the first reagent, a known hemolytic agent may be employed which is used for measuring white blood cells. The use of the hemolytic agent results in damage to cell membranes of red blood cells and mature white blood cells, which contributes to shrinkage of the damaged blood cells. More specifically, the hemolytic agent contains a surfactant which damages cell membranes of red blood cells and mature white blood cells, and a solubilizing agent which reduces the size of damaged blood cells.
As the surfactant contained in the hemolytic agent, a nonionic surfactant is preferred. As the nonionic surfactant, a polyoxyethylene-based nonionic surfactant is preferred. As a specific polyoxyethylene-based nonionic surfactant, exemplified is a surfactant having the following structural formula (I):
R1—R2—(CH2CH2O)n—H (I)
wherein
R1 is a C9-C25 alkyl group, alkenyl group or alkynyl group,
R2 is —O—, —COO— or
and
n is an integer of 10 to 40.
Specific examples of the surfactant represented by the structural formula (I) 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, polyoxyethylene(20) cetyl ether, and the like. In particular, polyoxyethylene (20) oleyl ether is preferred. Further, the hemolytic agent may contain one or more surfactants.
A concentration of the surfactant in the first reagent can be appropriately selected depending on the type of surfactants or the osmotic pressure of the hemolytic agent. For example, when the surfactant is polyoxyethylene oleyl ether, a concentration of the surfactant in the first reagent is in the range of 0.5 to 50.0 g/L, and preferably 1.0 to 20.0 g/L.
Examples of the solubilizing agent contained in the hemolytic agent include a sarcosine derivative, a cholic acid derivative, methylglucanamide, n-octyl β-glucoside, sucrose monocaprate, N-formylmethylleucylalanine and the like. Particularly preferred is the sarcosine derivative. In addition, the hemolytic agent may contain one or more solubilizing agents.
Examples of the sarcosine derivative may include a compound represented by the following structural formula (II):
wherein R1 is a C10-C22 alkyl group, and n is 1 to 5; and a salt thereof.
Specific examples of the sarcosine derivative may include sodium N-lauroylsarcosinate, sodium lauroyl methyl β-alanine, lauroylsarcosine, and the like. In particular, sodium N-lauroylsarcosinate is preferred.
Examples of the cholic acid derivative may include a compound represented by the following structural formula (III):
wherein R1 is a hydrogen atom or a hydroxyl group; and a salt thereof.
Specific examples of the cholic acid derivative may include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), and the like.
Examples of the methylglucanamide may include a compound represented by the following structural formula (IV):
wherein n is 5 to 7.
Specific examples of the methylglucanamide may include MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide), MEGA10 (decanoyl-N-methylglucamide), and the like.
A concentration of the solubilizing agent in the first reagent can be appropriately selected depending on the type of solubilizing agents to be used. For example, when a sarcosine derivative is used as the solubilizing agent, a concentration of the solubilizing agent in the first reagent is in the range of 0.05 to 3.0 g/L, and preferably 0.1 to 1.0 g/L. When a cholic acid derivative is used as the solubilizing agent, a concentration of the solubilizing agent in the first reagent is in the range of 0.1 to 10.0 g/L, and preferably 0.2 to 2.0 g/L. When a methylglucanamide is used as the solubilizing agent, a concentration of the solubilizing agent in the first reagent is in the range of 1.0 to 8.0 g/L, and preferably 2.0 to 6.0 g/L. When n-octyl β-glucoside, sucrose monocaprate, and N-formylmethylleucylalanine are used as solubilizing agents, a concentration of the solubilizing agent in the first reagent is in the range of 0.01 to 50.0 g/L, and preferably 0.05 to 30.0 g/L.
There is no particular limit to the fluorescent dye which is contained in the first reagent and is capable of staining a nucleic acid, as long as it is capable of fluorescent-staining the nucleic acid. The use of such a dye can stain nucleated blood cells, such as lymphoblasts having nucleic acid and nucleated red blood cells, while poorly staining red blood cells having no nucleic acid. Further, the nucleic acid-staining fluorescent dye can be appropriately selected by light irradiated from a light source. Specific examples of the nucleic acid-staining fluorescent dye may include propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylene bis[[3-[[4-[[(3-methylbenzothiazol-3-ium)-2-yl]methylene]-1,4-dihydroquinolin]-1-yl]propyl]dimethylaminium]•tetraiodide (TOTO-1), 4-[(3-methylbenzothiazol-2(3H)-ylidene)methyl]-1-[3-(trimethylamino)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-propanediaminium•tetraiodide (TOTO-3), and 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 the following structural formulae (V) to (XIII).
<Structural Formula (V)>
wherein R1 and R2 are each 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 by a lower alkyl group.
The lower alkyl group of the structural formula (V) means a C1-C6 straight or branched alkyl group. Specific example of the lower alkyl group may include a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, and the like. In particular, methyl and ethyl groups are preferred. The Z is preferably a sulfur atom. The anion with regard to X− includes halogen ions (fluorine, chlorine, bromine and iodine ions), boron halide ions (BF4−, BCl4−, BBr4−, etc.), phosphorus compound ions, halooxy-acid ions, fluorosulfate ions, methyl sulfate ions, and tetraphenyl boron compound ions which have a halogen or halogeno-alkyl group as a substituent, in an aromatic ring. Particularly, iodine ions are preferred.
Among the compounds represented by the structural formula (V), a particularly preferable nucleic acid-staining fluorescent dye is NK-321 represented by the following structural formula.
<Structural Formula (VI)>
wherein R1 and R2 are each a lower alkyl group; n is 1 or 2; and X− is an anion.
The lower alkyl group and the anion X− in the structural formula (II) are as defined in the structural formula (I).
Among the compounds represented by the structural formula (VI), a particularly preferable nucleic acid-staining fluorescent dye is one represented by the following structural formula.
<Structural Formula (VII)>
wherein R1 is a hydrogen atom or a lower alkyl group; R2 and R3 are each 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 which may be substituted; Z is a sulfur atom, an oxygen atom, or a carbon atom which is substituted by a lower alkyl group; n is 1 or 2; and X− is an anion.
The lower alkyl group and the anion X− in the structural formula (VII) are as defined in the structural formula (V). The lower alkoxy group represents a C1-C6 alkoxy group. Specific examples of the lower alkoxy group may include methoxy, ethoxy, propoxy groups, and the like. In particular, methoxy and ethoxy groups are preferred. The acyl group is preferably an acyl group derived from an aliphatic carboxylic acid. Specific examples of the acyl group may include an acetyl group, a propionyl group, and the like. In particular, an acetyl group is preferred. Examples of the substituent of the lower alkyl group which may be substituted may include a hydroxyl group, and a halogen atom (fluorine, chlorine, bromine or iodine). The lower alkyl group which may be substituted may be substituted by 1 to 3 substituents. In particular, the lower alkyl group which may be substituted is preferably a lower alkyl group substituted by one hydroxyl group. Z is preferably a sulfur atom, and X− is preferably a bromine ion or BF4−.
Among the compounds represented by the structural formula (VII), particularly preferable nucleic acid-staining fluorescent dyes are represented by the following three structural formulae.
<Structural Formula (VIII)>
wherein X1 and X2 are independently Cl or I.
<Structural Formula (IX)>
<Structural Formula (XI)> (NK-1049)
<Structural Formula (XII)> (NK-98)
<Structural Formula (XIII)> (NK-141)
Among the above-exemplified nucleic acid-staining fluorescent dyes, a particularly preferable fluorescent dye contained in the first reagent is NK-321 represented by the following structural formula.
A concentration of the nucleic acid-staining fluorescent dye in the first reagent may be in the range of 10 to 500 mg/L. Particularly preferred is in the range of 30 to 100 mg/L. Further, the first reagent may contain one or more nucleic acid-staining fluorescent dyes.
A pH of the first reagent may be in the range of 5.0 to 9.0. Preferred is a pH of 6.5 to 7.5. Particularly preferred is a pH of 6.8 to 7.3. The pH of the first reagent may be adjusted by a buffer or a pH-adjusting agent. Examples of the buffer may include a Good buffer such as HEPES, 3-morpholinopropanesulfonic acid (MOPS) or 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), a phosphate buffer, and the like. Examples of the pH-adjusting agent may include sodium hydroxide, hydrochloric acid, and the like.
An osmotic pressure of the first reagent can be appropriately set depending on the type of the above-mentioned surfactants or the concentration thereof in the first reagent. A specific osmotic pressure of the first reagent may be in the range of 10 to 600 mOsm/kg. Further, an osmotic pressure of the first reagent may be adjusted by adding sugars, amino acids, sodium chloride or the like to the first reagent. Specific examples of the sugars may include monosaccharide, polysaccharide, sugar alcohol and the like. For the monosaccharide, glucose or fructose is preferred. For the polysaccharide, arabinose is preferred. For the sugar alcohol, xylitol, sorbitol, mannitol, or ribitol is preferred. As the sugar which is added to the first reagent, preferred is a sugar alcohol, particularly xylitol. When xylitol is added to the first reagent, a concentration of xylitol in the first reagent is preferably in the range of 1.0 to 75.0 g/L, and particularly preferably 20.0 to 50.0 g/L. Specific examples of the amino acid may include valine, proline, glycine, alanine, and the like. In particular, preferred is glycine or alanine. When glycine is added to the first reagent, a concentration of glycine in the first reagent is preferably in the range of 1.0 to 50.0 g/L, and particularly preferably 10.0 to 30.0 g/L.
An electric conductivity of the first reagent is preferably in the range of 0.01 to 3 mS/cm. Particularly preferred is in the range of 0.1 to 2 mS/cm. Further, a chelating agent, a preservative or the like may be added to the first reagent. As the chelating agent, exemplified is EDTA-2K, EDTA-3Na, or the like. As the preservative, exemplified is Proxel GXL (manufactured by Avecia), material TKM-A (manufactured by API Corporation), or the like.
The second reagent is a hemolytic agent for the measurement of nucleated red blood cells (NRBC). Examples of the hemolytic agent for the measurement of NRBC may include Stromatolyser NR hemolytic reagent (manufactured by Sysmex Corporation). The third reagent is a staining solution for the measurement of NRBC. Examples of the staining solution for the measurement of NRBC may include Stromatolyser NR dye solution (manufactured by Sysmex Corporation). The fourth reagent is a sheath fluid which is supplied to a sheath flow cell which will be illustrated hereinafter. The sheath fluid is also used as a diluent. For example, the sheath fluid may be Cellpack (II) (manufactured by Sysmex Corporation).
The detecting section 23 includes an optical detector D which is capable of performing WBC measurement (white blood cell counting) and DIFF measurement (white blood cell classification). The optical detector D is configured such that the detection of WBC (mature white blood cells), NRBC (nucleated red blood cells), and lymphoblasts (L-Blast) can be performed by a flow cytometry method using semiconductor lasers. By using the detecting section 23, it is possible to achieve five classifications of white blood cells (WBC) into neutrophils (NEUT), lymphocytes (LYMPH), eosinophils (EO), basophils (BASO) and monocytes (MONO). When it is desired to measure a lymphoblast/nucleated red blood cell group, an assay sample (L-Blast assay sample), which is a mixture of a blood sample and a first reagent, is supplied to the optical detector D. When it is desired to measure NRBC, an assay sample (NRBC assay sample), which is a mixture of a blood sample, a second reagent and a third reagent, is supplied to the optical detector D.
The sheath flow system 232 is configured such that the assay sample flows in the flow cell 231 in a state of being surrounded by a sheath fluid. The beam spot-forming system 233 is configured such that the light irradiated from a semiconductor laser 237 passes through a collimator lens 238 and a condenser lens 239 so as to irradiate the flow cell 231. In addition, the beam spot-forming system 233 is provided with a beam stopper 240.
The forward-scattered light receiving system 234 is configured such that the forward-scattered light is condensed by a forward-condensing lens 241, and the light passing through a pin hole 242 is received by a photodiode (forward-scattered light receiving section) 243.
The side-scattered light receiving system 235 is configured such that the side-scattered light is condensed by a side-condensing lens 244, and a part of the light is reflected on a dichroic mirror 245 so as to be received by a photodiode (side-scattered light receiving section) 246.
Light scattering is a phenomenon occurring such that particles such as blood cells act as an obstacle to light in the advancing direction thereof, and the light is changed in the advancing direction by the particles. By detecting the scattered light, information on the size or material of the particle can be obtained. In particular, the information on the size of the particle (blood cell) can be obtained from the forward-scattered light. In addition, the information on the content of the particle can be obtained from the side-scattered light. When a laser light is irradiated to the blood cell particle, the side-scattered light intensity depends on the complexity of the inside of the cell (shape, size, density, or granulated amount of nucleus). Therefore, these scattered light intensities can be used in the measurement of a lymphoblast/nucleated red blood cell group, the measurement of nucleated red blood cells, the classification of white blood cells, and the like.
The side-fluorescent light receiving system 236 is configured such that the light that passed through the dichroic mirror 245 further passes through a spectral filter 247 and is received by an avalanche photodiode (fluorescence receiving section) 248.
When light is irradiated to blood cells stained with a fluorescent material, light is generated of which the wavelength is longer than that of the irradiated light. If staining is sufficiently performed, the fluorescence intensity becomes stronger. By measuring the fluorescence intensity, the information on the staining degree of the blood cell can be obtained. Therefore, differences in the (side) fluorescence intensity can be used in the measurement of a lymphoblast/nucleated red blood cell group, the measurement of nucleated red blood cells, the classification of white blood cells, and the like.
Returning to
In addition, the sample container transport section 25 is provided with a sample container setting section 25b which includes a hole section through which the sample container T can be inserted. The sample container T grasped by the above-mentioned hand section 25a moves after the stirring is completed. Then, the grasped sample container T is inserted into the hole section of the sample container setting section 25b. Thereafter, the grasping members are away from each other, so that the sample container T is released from the hand section 25a, whereby the sample container T is set in the sample container setting section 25b. The sample container setting section 25b can horizontally move in the Y1 and Y2 directions in the drawing by a driving force of a stepping motor (not shown).
In the measurement unit 2, a bar-code reading section 26 is provided. The sample container setting section 25b can move to a bar-code reading position 26a near the bar-code reading section 26 and an aspirating position 21a carried out by the sample aspiration section 21. When the sample container setting section 25b moves to the bar-code reading position 26a, the set sample container T is horizontally rotated by a rotation mechanism (not shown) and the sample bar-code is read by the bar-code reading section 26. Accordingly, even when the bar-code label BL1 of the sample container T is positioned on the opposite side with respect to the bar-code reading section 26, the bar-code label BL1 can face the bar-code reading section 26 by rotating the sample container T, whereby the bar-code reading section 26 can definitely read the sample bar-code. In addition, when the sample container setting section 25b is moved to the aspirating position, the sample is aspirated from the set sample container T by the sample aspiration section 21.
<Configuration of Sample Transport Unit>
Next, the configuration of the sample transport unit 4 will be described. As shown in
The before-analysis rack holding section 41 has a quadrangular shape in plane view, and the width thereof is slightly larger than the width of the sample rack L. The before-analysis rack holding section 41 is formed to be lower by one stage than the surrounding surface. On an upper face of the before-analysis rack holding section 41, the before-analysis sample racks L are disposed. In addition, the rack sending sections 41b are provided in both faces of the before-analysis rack holding section 41 so as to be protruded inward. The rack sending sections 41b protrude so as to contact the sample rack L. In this state, the rack sending sections are moved backward (a direction so as to be closer to the rack transport section 43) and thus the sample rack L is moved backward. The rack sending sections 41b are configured to be driven by a stepping motor (not shown) which is provided below the before-analysis rack holding section 41.
As shown in
In addition, as shown in
As shown in
As shown in
The after-analysis rack holding section 42 has a quadrangular shape in plane view, and the width thereof is slightly larger than the width of the sample rack L. The after-analysis rack holding section 42 is formed to be lower by one stage than the surrounding surface. On an upper face of the after-analysis rack holding section 42, the analyzed sample racks L are held. The after-analysis rack holding section 42 is connected to the above-mentioned rack transport section 43. And, as described above, the sample rack L is transported from the rack transport section 43 by the rack delivery section 46.
According to the configuration as described above, the sample transport unit 4 moves the sample rack L disposed on the before-analysis rack holding section 41 to the rack transport section 43, and is further transported by the rack transport section 43, whereby the sample can be supplied to the measurement unit 2. In addition, the sample rack L accommodating the samples which are completely aspirated is moved to the after-analysis rack delivery position 461 by the rack transport section 43, and is delivered to the after-analysis rack holding section 42 by the rack delivery section 46. When the plural sample racks L are disposed on the before-analysis rack holding section 41, the sample racks L accommodating the samples which are completely analyzed are sequentially delivered to the after-analysis rack holding section 42 by the rack delivery section 46. These plural sample racks L are then stored in the after-analysis rack holding section 42.
<Configuration of Information Processing Unit>
Next, the configuration of the information processing unit 5 will be described. The information processing unit 5 is composed of a computer.
The CPU 51a can execute a computer program loaded to the RAM 51c. The CPU 51a executes a computer program 54a for analyzing blood and for controlling the measurement unit 2 and the sample transport unit 4, which will be described later, so that the computer 5a functions as the information processing unit 5.
The ROM 51b is composed of a mask ROM, a PROM, an EPROM, an EEPROM or the like. The computer program executed by the CPU 51a, the data used for the computer program, and the like are recorded in the ROM 51b.
The RAM 51c is composed of a SRAM, a DRAM or the like. The RAM 51c is used to read the computer program 54a recorded in the hard disk 51d. In addition, the RAM 51c is used as an operating area of the CPU 51a when the CPU 51a executes a computer program.
In the hard disk 51d, various computer programs for execution by the CPU 51a, such as an operating system and an application program, and data which is used to execute the computer programs, are installed. The computer program 54a to be described later is also installed in the hard disk 51d. In addition, the computer program 54a is an event-driven computer program.
The reading device 51e is composed of a flexible disk drive, a CD-ROM drive, a DVD-ROM drive or the like. The reading device 51e can read the computer program or data recorded in a portable recording medium 54. In the portable recording medium 54, the computer program 54a for prompting the computer to function as the information processing unit 5 is stored. The computer 5a can read the computer program 54a from the portable recording medium 54 and install the computer program 54a in the hard disk 51d.
The computer program 54a is provided by the portable recording medium 54 and can also be provided from an external device, which is connected to the computer 5a by an electric communication line (which may be wired or wireless) to communicate therewith, through the electric communication line. For example, when the computer program 54a is stored in a hard disk of a server computer on the internet, the computer 5a can access the server computer to download the computer program and install the computer program in the hard disk 51d.
Furthermore, in the hard disk 51d, for example, a multitasking operating system such as Windows (registered trademark), which is made and distributed by Microsoft corporation in U.S.A, is installed. In the following description, the computer program 54a according to this embodiment operates on the above operating system.
The I/O interface 51f is composed of, for example, a serial interface such as USB, IEEE1394 or RS-232C, a parallel interface such as SCSI, IDE or IEEE1284, and an analog interface including a D/A converter and an A/D converter. The input section 53 composed of a keyboard and a mouse is connected to the I/O interface 51f. The user can use the input section 53 so as to input data to the computer 5a. In addition, the I/O interface 51f is connected to the measurement unit 2 and the sample transport unit 4. Therefore, the information processing unit 5 can control the measurement unit 2 and the sample transport unit 4, respectively.
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
The image output interface 51h is connected to the image display section 52 composed of an LCD or a CRT so as to output a picture signal corresponding to the image data provided from the CPU 51a to the image display section 52. The image display section 52 displays an image (screen) in accordance with an input picture signal.
[Measurement Operation of Blood Analyzer 1]
Hereinafter, an operation of the blood analyzer 1 according to this embodiment will be described.
<Sample Measurement Operation>
First, the sample measurement operation of the blood analyzer 1 according to this embodiment will be described. The blood analyzer 1 can perform the measurement of a lymphoblast/nucleated red blood cell group, and the measurement of NRBC (nucleated red blood cells), using an optical detector D. The measurement process is composed of a first measurement process for measuring an L-Blast assay sample, a second measurement process for measuring an NRBC assay sample, and a data processing process for analyzing and processing the measurement data obtained by the first measurement process and the second measurement process.
First, an operator places the sample rack L holding the sample containers T on the before-analysis rack holding section 41. The rack sending sections 41b contact the sample rack L placed on the before-analysis rack holding section 41, and are moved backward and then transported to the rack transport section 43. Thereafter, the sample rack L is transported by the rack transport section 43, and the sample container T where a sample to be measured is accommodated is positioned at the sample supply position 43a. Next, the sample container T is grasped by the hand section 25a of the measurement unit 2, and the sample container T is taken out from the sample rack L. The hand section 25a is then oscillated, so that the sample in the sample container T is stirred. Next, the sample container T is inserted into the sample container setting section 25b. Next, the sample container setting section 25b moves in the Y direction, the sample bar-code is read by the bar-code reading section 26, and then the sample container T arrives at the aspirating position. Thereafter, the following first measurement process and second measurement process are performed.
First Measurement Process
First, the first measurement process will be described. The blood analyzer 1 prepares, in the first measurement process, an L-Blast assay sample by mixing a complete blood sample (19.0 μL) and a first reagent (1.02 mL). Then, the L-Blast assay sample is measured by an optical detector D in accordance with a flow cytometry method.
Here, the first reagent was used which is composed of the following components.
Further, the following three samples were used as complete blood samples.
In Table 1, “O” represents that there are target blood cells (lymphoblasts or nucleated red blood cells), and “X” represents that there are no target blood cells.
Next, the CPU 51a controls the measurement unit 2, whereby the first reagent (1.02 mL) from the reagent container 221 and the complete blood sample (19.0 μL) from the aspiration tube 211 are respectively supplied to the first mixing chamber MC1 (Step S102). The CPU 51a determines whether or not 18.5 seconds have passed after a supply of the first reagent and the complete blood sample to the first mixing chamber MC1 (Step S103), and waits for 18.5 seconds. Here, the first mixing chamber MC1 is warmed to 35.0° C. by a heater. Accordingly, a mixture of the first reagent and the blood sample is warmed to 35.0° C. for 18.5 seconds, and therefore the L-Blast assay sample is prepared.
Then, the L-Blast assay sample is subjected to optical measurement using an optical detector D (Step S104). Specifically, in processing of Step S104, the L-Blast assay sample and a sheath fluid are simultaneously supplied to a 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 then the avalanche photodiode 248. Output signals (analog signals) being output by the respective light-receiving elements of the optical detector D are converted into digital signals by an A/D converter (not shown). And, a given signal processing is performed by a signal processing circuit (not shown), such that the digital signals are converted into digital data, i.e. first measurement data. The converted first 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 side fluorescence signal (side fluorescence intensity) can be obtained as feature parameters contained in the first measurement data. In this way, the first measurement process is completed. In addition, as will be illustrated hereinafter, the CPU 51a of the information processing unit 5 performs a given analysis processing on the first measurement data. Accordingly, the analysis result data is generated including numerical data such as NEUT, LYMPH, EO, BASO, MONO and WBC, and the analysis result data is recorded in the hard disk 51d.
Second Measurement Process
Next, the second measurement process will be described. The second measurement process is temporally overlapped with a part of the first measurement process. The blood analyzer 1, in the second measurement process, prepares an NRBC assay sample by mixing a complete blood sample (17.0 μL) with a second reagent (1.0 mL) and a third reagent (0.030 mL). The NRBC assay sample is measured in the optical detector D by a flow cytometry method. As the second reagent, the above-mentioned Stromatolyser NR hemolytic reagent was used. As the third reagent, the above-mentioned Stromatolyser NR dye solution was used.
Next, the CPU 51a determines whether or not 7.0 seconds have passed after a supply of the second reagent, the third reagent and the complete blood sample to the second mixing chamber MC2 (Step S202), and waits for 7.0 seconds. Here, the second mixing chamber MC2 is warmed to 41.0° C. by a heater. Accordingly, a mixture of the second reagent, the third reagent and the blood sample is warmed to 41.0° C. for 7.0 seconds, and therefore the NRBC assay sample is prepared.
Then, the NRBC assay sample is subjected to optical measurement using an optical detector D (Step S203). Specifically, in processing of Step S203, the NRBC assay sample and the sheath fluid are simultaneously supplied to a 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 then the avalanche photodiode 248. Output signals (analog signals) being output by the respective light-receiving elements of the optical detector D are converted into digital signals, in the same manner as in the first measurement process. A given signal processing is then performed such that the digital signals are converted into digital data, i.e. second measurement data. The converted 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 side fluorescence signal (side fluorescence intensity) can be obtained as feature parameters contained in the second measurement data. In this way, the second measurement process is completed. In addition, as will be illustrated hereinafter, the CPU 51a of the information processing unit 5 performs a given analysis processing on the second measurement data. Accordingly, the analysis result data is generated including numerical data of NRBC, and the analysis result data is recorded in the hard disk 51d.
Data Processing Process
Next, a data processing process will be described.
In Step S302, the CPU 51a performs the classification of a lymphoblast/nucleated red blood cell group from other blood cell groups using the first measurement data, and the counting of blood cells contained in the lymphoblast/nucleated red blood cell group (Step S302). This processing will be described in more detail.
Next, in Step S303, the CPU 51a performs the classification of the nucleated red blood cell group from other blood cell groups, using the second measurement data, and the counting of nucleated red blood cells (Step S303). This processing will be described in more detail.
Next, in Step S304, the CPU 51a determines whether or not a difference between the number of blood cells (NL) contained in the lymphoblast/nucleated red blood cell group obtained in Step S302 and the number of nucleated red blood cells (NN) obtained in Step S303 is equal to or higher than a given base value T (Step S304). The base value T is a value such that when an NL−NN value is equal to or higher than the base value T, it can be determined that lymphoblasts are contained in the blood sample, and when an NL−NN value is less than the base value T, it can be determined that lymphoblasts are not contained in the blood sample. The base value T is previously set taking into consideration an error in the number of blood cells (NL) contained in the lymphoblast/nucleated red blood cell group and the number of nucleated red blood cells (NN). When NL−NN≧T in this processing (“YES” in Step S304), it can be determined that lymphoblasts are contained in the blood sample. Therefore, in this case, the CPU 51a sets “1” into a lymphoblast flag provided in the RAM 51c (Step S305). In this connection, the lymphoblast flag is a flag reflecting the existence and nonexistence of lymphoblasts in the blood sample. When “1” is set into the lymphoblast flag, this represents the presence of lymphoblasts. When “0” is set into the lymphoblast flag, this represents the absence of lymphoblasts. Thereafter, the CPU 51a switches to a processing of Step S307.
On the other hand, when NL−NN<T in Step S304 (“NO” in Step S304), it can be determined that lymphoblasts are not contained in the blood sample. Therefore, in this case, the CPU 51a sets “0” into the lymphoblast flag (Step S306). Thereafter, the CPU 51a switches to a processing of Step S307.
In Step S307, the CPU 51a stores the thus obtained analysis results (including the nucleated red blood cell count, and the lymphoblast flag) in the hard disk 51d (Step S307). Then, the CPU 51a displays an analysis result screen displaying the analysis results stored in the hard disk 51d on an image display section 52 (Step S308), and then terminates the processing.
By using a specific example of the scattergram, the detection of lymphoblasts will be described in more detail.
As can be seen from
As can be seen from
In this manner, by referring to the scattergrams that can be obtained from the first measurement data and the second measurement data, grounds of the detection results for the existence and nonexistence of lymphoblasts by the blood analyzer 1 can be more accurately grasped. Further, the operator can determine the validity of the detection results for the existence and nonexistence of lymphoblasts by the blood analyzer 1.
According to the configuration as described above, the blood analyzer 1 can detect the lymphoblast/nucleated red blood cell group through the measurement of an L-Blast assay sample prepared by mixing a blood sample with a first reagent containing a nucleic acid-staining fluorescent dye by means of an optical detector D, and can measure the number of blood cells contained in the lymphoblast/nucleated red blood cell group. Further, based on the number of blood cells and the number of nucleated red blood cells obtained by the second measurement process, it is possible to detect whether or not lymphoblasts are contained in the blood sample. According to the above-mentioned measurement of the blood analyzer 1, it is possible to detect lymphoblasts without the use of a fluorescence-labeled antibody. As a consequence, it is possible to detect lymphoblasts while reducing measurement costs.
In the sample preparation section 22, there is no particular limit to the reaction temperature and the reaction time, upon mixing of the blood sample and the first reagent. Therefore, the reaction temperature and time may be appropriately established depending on the damaged or stained state of blood cells in the blood sample. Specifically, if the reaction temperature is high, the reaction time may be shortened. If the reaction temperature is low, the reaction time may be adjusted to be longer. More specifically, mixing of the blood sample and the reagent is preferably performed at a temperature of 20° C. to 40° C. for 3 to 20 seconds.
In the above-mentioned embodiment, even though there has been described the configuration in which the first measurement process is performed using a first reagent containing a hemolytic agent and a nucleic acid-staining fluorescent dye, the present invention is not limited thereto. Alternatively, the first measurement process may also be configured to include separately preparing a reagent containing a hemolytic agent and a reagent containing a nucleic acid-staining dye, mixing these two reagents with a blood sample to prepare an L-Blast assay sample, and detecting a lymphoblast/nucleated red blood cell group and counting the number of blood cells in the lymphoblast/nucleated red blood cell group. In this case, concentrations of a surfactant, a solubilizing agent and a fluorescent dye are adjusted to the above-specified concentration range when the above-mentioned two reagents were mixed. Here, a mixing ratio of the hemolytic agent-containing reagent and the nucleic acid-staining dye-containing reagent is preferably in the range of 1000:1 to 10:1.
Even though there is no particular limit to the order of mixing individual reagents of the reagent kit with the blood sample when it is desired to use the above-mentioned reagent kit, it is preferred that two reagents are mixed, and then the blood sample is mixed to the mixed reagents.
In the above-mentioned embodiment, even though there has been described the configuration which includes performing the first measurement process and the second measurement process, and detecting whether or not lymphoblasts are contained in the blood sample, using first measurement data that can be obtained by the first measurement process and second measurement data that can be obtained by the second measurement process, the present invention is not limited thereto. As shown in
In the above-mentioned embodiment, even though there has been described the configuration which includes detecting a lymphoblast/nucleated red blood cell group and counting blood cells contained in the lymphoblast/nucleated red blood cell group, based on the first measurement data, detecting nucleated red blood cells and counting nucleated red blood cells, based on the second measurement data, calculating a difference between the number of blood cells in the lymphoblast/nucleated red blood cell group and the number of nucleated red blood cells, and comparing the calculated difference and the base value T to determine the existence and nonexistence of lymphoblasts, the present invention is not limited thereto. For example, there may be a configuration in which the detection of the lymphoblast/nucleated red blood cell group is performed based on the first measurement data, and the detection of nucleated red blood cells is performed based on the second measurement data, it is determined that lymphoblasts are present in the blood sample if there are particles being detected as the lymphoblast/nucleated red blood cell group and there are no particles being detected as nucleated red blood cells, and it is determined that there are no lymphoblasts in the blood sample for other cases than the above-mentioned cases. Further, a difference between the number of blood cells in the lymphoblast/nucleated red blood cell group and the number of nucleated red blood cells, in terms of the number of lymphoblasts, can be displayed on the analysis result screen. Further, with regard to the difference between the number of blood cells in the lymphoblast/nucleated red blood cell group and the number of nucleated red blood cells, the resulting numerical value obtained from deduction of a given numerical value from the calculated difference, in terms of the number of lymphoblasts, can be displayed on the analysis result screen.
In the above-mentioned embodiment, there has been described the configuration in which if a difference (NL−NN value) between the number of blood cells (NL) contained in the lymphoblast/nucleated red blood cell group and the number of nucleated red blood cells (NN) is equal to or higher than a base value T, it is determined that there are lymphoblasts in the blood sample, and if an NL−NN value is less than the base value T, it is determined that there are no lymphoblasts in the blood sample. However, the present invention is not limited thereto. For example, there may be a configuration in which if the number of blood cells (NL) contained in the lymphoblast/nucleated red blood cell group is larger than the number of nucleated red blood cells (NN), it is determined that there are lymphoblasts in the blood sample, and if the number of blood cells (NL) is equal to or less than the number of nucleated red blood cells (NN), it is determined that there are no lymphoblasts in the blood sample.
As described above, on the scattergram of side-scattered light intensity and side fluorescence intensity in the first measurement data shown in
In addition, there may also be a configuration in which mature white blood cells are classified into lymphocytes, basophils, monocytes, and a blood cell group composed of neutrophils and eosinophils, using side-scattered light intensity and side fluorescence intensity in the first measurement data, individual blood cells are counted for lymphocytes, basophils, monocytes, and blood cells contained in the blood cell group composed of neutrophils and eosinophils, and the number of individual blood cells is displayed on the analysis result screen. Further, there may also be a configuration in which, assuming that basophils are also contained in the blood cell group composed of neutrophils and eosinophils, mature white blood cells are classified into lymphocytes, monocytes and granulocytes, distinctively from lymphocytes and monocytes as granulocytes, blood cells contained in each cluster are counted, and the number of lymphocytes, monocytes, and granulocytes is displayed on the analysis result screen.
In the above-mentioned embodiment, even though there has been described the configuration which includes detecting the lymphoblast/nucleated red blood cell group, using side-scattered light intensity and side fluorescence intensity in the first measurement data, the present invention is not limited thereto. As shown in
Further, there may be a configuration which includes classification of mature white blood cells into lymphocytes, monocytes and granulocytes, using forward-scattered light intensity and side fluorescence intensity in the first measurement data, counting of lymphocytes, monocytes, and granulocytes, and display of the number of individual blood cells on the analysis result screen.
Further, as shown in
In the above-mentioned embodiment, even though there has been described the configuration in which the second measurement process is performed by the blood analyzer 1, and the nucleated red blood cell group is detected in the data processing process, the present invention is not limited thereto. That is, there may also be a configuration in which the information on the detection of the nucleated red blood cell group is obtained by other blood analyzers different from the blood analyzer 1 or by manual manipulations, and the thus obtained information is input using an input device of the blood analyzer 1. Here, as the input device of the blood analyzer 1, mention may be made of the above-exemplified input section 53 and communication interface 51g. More specifically, the information on the detection of the nucleated red blood cell group can be input to the blood analyzer 1, using the input section 53 composed of a keyboard and a mouse, which is connected to the I/O interface 51f. In addition, the blood analyzer 1 can be connected to the above-mentioned other blood analyzers using the communication interface 51g, and the information on the detection of the nucleated red blood cell group can be input to the blood analyzer 1 through the medium of the communication interface 51g.
In the above-mentioned embodiment, even though there has been described the configuration in which controlling of the measurement unit 2 and processing of the measurement data are performed through the execution of the computer program 54a by the CPU 51a, the present invention is not limited thereto. There may also be a configuration in which controlling of the measurement unit 2 and processing of the measurement data are performed by using special hardware, such as FPGA or ASIC, which can perform the same processes carried out by the computer program 54a.
Further, in the above-mentioned embodiment, the configuration has been described such that all the processes of the computer program 54a are performed by the single computer 5a, but the invention is not limited thereto. The same processes carried out by the above-mentioned computer program 54a may be implemented by a distributed system in which the processes are distributed on and performed by a plurality of apparatuses (computers).
As discussed above, the sample examination system of the present invention is useful as a blood analyzer which performs the optical measurement of a blood sample, and the classification of cell groups contained in the blood sample into plural populations.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-087609 | Mar 2009 | JP | national |
