Abstract:
The analytical method having both flow cytometery and cytodiagnosis functions comprises the steps of: preparing a sample containing particulate substances such as cells and viruses; injecting the sample into a plate-like sample container; centrifuging the sample container; and using the sample container in which a distribution of the particulate substances has been formed as a preparation for analysis. The preparation is scanned with laser beam to obtain analytical data. Analytical device for this method is also provided.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention relates to a novel analytical method and device for analyzing cells, DNA, antigens and viruses in the field of laboratory test such as clinical examination. In particular, the invention relates to a novel analytical method and device to combine cytometry and cytodiagnosis.  
           [0003]    2. Related Background Art  
           [0004]    A flow cytometer is a popular analytical device for flow cytometry. Flow cytometry is an analytical method where a cellular surface antigen is stained with one or more fluorescence-labeled specific antibodies, or DNA is stained with various dyes that bind to DNA, and obtained parameters, i.e., intensities of fluorescence, respective intensities of forward and side-way scattering light, and the individual numbers, are subjected to various analyses. On the other hand, in the fields of pathology, cytodiagnosis and hemodiagnosis, usually chemically stained smears are prepared and subjected to microscopic judgment.  
           [0005]    In clinical cytodiagnosis, final judgment by an expert is still required in many cases, no matter how instruments have been automated and techniques for judging with image processing have been advanced. On the other hand, in flow cytometry, image observation is not involved. Thus, although flow cytometry has been recognized as a superior clinical test method because of its operability and simplicity, valuable information may be wasted. Under such circumstances, Japanese Patent Application Laid-Open No. 5-119035 (“Imaging Flow Cytometer”) disclosed a flow cytometer having image-pick up function. However, this device cannot provide specimens because cells flow out from the flow cell.  
         SUMMARY OF THE INVENTION  
         [0006]    Accordingly, an object of the invention is to provide a novel analytical device provided with the function of a specimen preparation required for current cytodiagnosis and the function of flow cytometer, and a novel analytical method of flow cytometry with specimen preparation.  
           [0007]    In order to overcome the above described problem, the inventor of this invention conducted an intensive investigation and have finally invented a novel analytical method and device which have both imaging function and specimen-preparation function by combining a centrifugation method and an fluorescence-labeling technique such as flow cytometry. This analytical device is, so to speak, a flow cytometer having imaging and specimen-preparing functions. Here, this analytical device is referred to as a disc cytometer, since it typically uses a disc-shaped sample container, and the method is referred to as flow cytometry. It should be understood, however, that the form of the sample container is not limited to a disc form, as will be evident from the description given later. The sample container must be made up of an optically transparent material since it is cytodiagnosis, immunofluorescence analysis, images of cells etc.  
           [0008]    This disc cytometry adopts differential centrifugation or density-gradient centrifugation which has been used for isolating and purifying biological materials. In density-gradient centrifugation, a sample in a centrifuge tube is centrifuged, and substances in the sample are distributed along a density gradient according to the differences in size, shape, density or specific gravity of the substances. In this invention, a plate-shaped sample container is used instead of a cylindrical centrifuging tube in conventional density-gradient centrifugation, which enables in situ cytodiagnosis as well as analysis and imaging by the fluorescence-labeling method in one container. The sample container is preferably in the form of a disc in view of centrifugation, but it can be in any form as long as it is suitable for centrifugation, for example, a rectangular sample container.  
           [0009]    As the medium for genrating a density gradient during centrifugation, PEG (polyethylene glycol) or sucrose in an appropriate range is used for biological samples. In clinical laboratory tests, commercial solutions such as Ficoll (trade name) and Percoll (trade name) may be used depending on the purpose.  
           [0010]    One aspect of the present invention is an analytical method comprising the steps of: preparing a sample containing particulate substances; injecting the sample into a plate-like sample container; centrifuging the sample container; and using the sample container in which a distribution of the particulate substances has been formed as a preparation for analysis.  
           [0011]    The term “particulate substances” or “cells etc.” herein means biological materials which are subjected to cytodiagnosis or clinical laboratory test, for example, those selected from the group consisting of cells, microbial cells, viruses, DNA and mitochondria.  
           [0012]    Further, in this invention, the sample container-preparation is irradiated and scanned with a laser beam to obtain at least one set of analytical data selected from fluorescence intensity, scattered light intensity and light scattering particle number for particulate substances in the sample. The particulate substances may be subjected to fluorescence labeling reaction before centrifugation. The image data of particulate substances may be obtained on the basis of analytical data.  
           [0013]    In this invention, the same experimental protocols as used in conventional flow cytometry can be used for sample preparation and reaction with florescent antibodies. Accordingly, when a suitably reacted sample is introduced and overlayed on the density gradient solution in a disc container, and centrifuged for a predetermined time at a predetermined rotation speed, the cells etc. are arranged along the density gradient formed. Then the laser scan of the disc container is carried out in the radius direction with rotation of the container, to obtain information such as fluorescence intensity, scattered light intensity and the individual number. Depending on the purpose, image information at a certain position of the disc container can be obtained by using positional information of the disc container from a sensor and an optical system of confocal laser microscope.  
           [0014]    This is the disc-cytometry analysis of the present invention.  
           [0015]    Another aspect of the present invention is an analytical device comprising: means for centrifuging a plate-like sample container; means for generating a laser beam; means for scanning the sample container and irradiating particulate substances having been centrifuged in the sample container with a laser beam; and means for detecting scattered light from the sample container.  
           [0016]    In this invention, the means for detecting scattered light may be means for detecting light scattered from the disc container at different angles; and in such a case, preferably the fluorescence intensities and/or the number of the particulate substances are detected based on the a plurality of scattered light. Preferably, the irradiating means scans the rotating disc container in a radius direction with a laser beam.  
           [0017]    Further, the analytical device of this invention may be provided with means for processing and analyzing data by A/D converting the signals from the scattered light detecting means conversion and using the same as a parameter. In addition, it may be provided with an optical system and image pick-up means for obtaining images of the particulate substances.  
           [0018]    The specimen-providing function of the present invention is very useful for clinical judgement as a supplementary confirmation means; for example, for discrimination of leukemia from malignant lymphoma, acute lymphoblastic leukemia from acute crisis of chronic myelogenous leukemia, and lymphoblastic leukemia from nonlymphoblastic leukemia, all of which are considered to be difficult to discriminate only by current flow cytometry, for identification of immature T cells, and for identification of neoplasm of which identification by DNA Index alone is difficult. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]    [0019]FIG. 1 is a perspective view illustrating the principle of the invention;  
         [0020]    [0020]FIG. 2 is a block diagram illustrating the configuration of an analytical device being one embodiment of the invention;  
         [0021]    [0021]FIG. 3 is a view illustrating the structure for obtaining positional information of a sample container;  
         [0022]    [0022]FIG. 4 is a view illustrating the positional relation in which a sample is disposed;  
         [0023]    [0023]FIG. 5 is a graph showing an analytical result obtained; and  
         [0024]    [0024]FIG. 6 is a graph showing an analytical result obtained. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0025]    In the following preferred embodiments of this invention will be described with reference to the accompanied drawings. First referring to FIG. 1, there is shown a perspective view illustrating the principle of this invention.  
         [0026]    In the example shown in FIG. 1, a transparent plate-like sample container is a disc sample container  1 . The sample container  1  is of a size, for example, 80 mm in radius and 1 mm in height by inside measurement. The container is made of poly(methyl methacrylate) (PMMA) resin which is generally used for disposable sample cells because of uniform transmission properties to visible light and light of which wavelength is 280 to 360 nm. In the center of the upper surface of the sample container  1  a sample inlet  2  is provided and in the center of the lower surface of the same a sample outlet  3  is formed in such a manner as to face the sample inlet  2 .  
         [0027]    The sample container is filled with a gradient solution through the sample inlet  2 , and then a sample solution containing cells etc. is overlaid on the gradient solution, and the sample outlet  3  is capped with a screw cap. The sample solution has already been subjected to intended fluorescence-labeling. Then the sample container  1  is rotated around an axis of rotation  4  for centrifugation. After the centrifugation, the entirety of the sample container  1  is scanned with a laser beam so as to measure the intensities of the forward and sideward scattered light and the fluorescence labeling intensity. Further, the sample at a desired position within the sample container  1  is observed under a microscope or the image is picked up.  
         [0028]    [0028]FIG. 2 is a block diagram illustrating the structure of an analytical device being one embodiment of the invention. This analytical device includes: a disc holder  5  for vacuum mounting a disc sample container  1  on the device; a motor  6  for rotating the disc holder  5 ; a pump  7  and an electromagnetic valve  8  for vacuum mounting of the sample container; a motor controller  9  for controlling the motor  6 ; a movable unit  10  described later; a reflection-type sensor  14  and a binarizing unit  15  for detecting the rotational angle of the sample container  1 ; a single chip microcomputer  23  for controlling the electromagnetic valve  8 , the motor controller  9  and the movable unit  10  and receiving signals from the movable unit  10  and the binarizing unit  15 ; an external memory  24  connected to the single chip microcomputer  23 ; and a USB (universal serial bus) interface  25  for connecting an external personal computer (PC) and the single chip microcomputer  23  with each other. The mechanism of vacuum mounting the disc-like sample container  1  on the device is, in principle, the same as a spin coater used in the semiconductor device manufacturing process, and the sample container is vacuum mounted on the device by evacuating the air in the holder with the pump  7 . Contrarily, when dismounting the sample container  1 , the electromagnetic valve  8  is switched to allow the sample container to communicate with the open air. The single chip microcomputer  23  contains an A-D (analog-to-digital) converter for processing the analog signals input from the movable unit  10  and a timer counter portion for processing the pulse from the binarizing unit  15 .  
         [0029]    In this disc cytometer, its operation is controlled with a software on an external PC. In other words, the PC issues commands to the disc cytometer, the commands are input into the single chip microcomputer  23  through the USB interface  25  and interpreted thereby.  
         [0030]    In the following the movable unit  10  will be described.  
         [0031]    The movable unit  10  is for irradiating the sample container  1  with a laser beam and detecting the light scattered sideward and forward on laser irradiation; thus, it consists of optical systems for laser irradiation and scattered light detection which are integrally housed together.  
         [0032]    The movable unit  10  includes: an XYZ axes controller  11  for moving the movable unit  10  in each axial direction X, Y and Z in 0.1 μm steps; an argon laser (wavelength of 488 nm)  12 ; a laser controller  13  for controlling the modes of argon laser  12  such as on-off and standby modes; a beam expander  26  for making up a confocal microscope optical system using the argon laser  12 ; a solenoid  16  for driving the beam expander  26 ; an object lens  27  for directing the laser beam from the argon laser  12  having passed through the beam expander  26  toward the sample container  1 ; a mirror  28  for bending the forward scattered light having passed through the sample container  1  through, for example, 90°; a condenser lens  29  provided on the light reflecting side of the mirror  28 ; a photomultiplier tube  18  provided on the light reflecting side of the condenser lens  29  for detecting the forward scattered light; a diaphragm unit  30  arranged between the condenser lens  29  and the photomultiplier tube  18 ; a solenoid  17  for driving the diaphragm unit  30 ; a condenser lens  31  for condensing the sideward scattered light from the sample container  1 ; a half-silvered mirror  32  arranged on the light reflecting side of the condenser lens  31 ; a photomultiplier tube  19  into which the light reflected by the half-silvered mirror  32  enters and which detects the intensity of the sideward scattered light; a diaphragm  35  provided between the half-silvered mirror  32  and the photomultiplier tube  19 ; a red-reflecting dichroic mirror  33  into which the light having passed through the half-silvered mirror enters; a photomultiplier tube  20  for detecting the red light reflected by the dichroic mirror  33 ; a diaphragm  36  arranged between the dichroic mirror  33  and the photomultiplier tube  20 ; a band pass filter  38  arranged between the diaphragm  36  and the photomultiplier tube  20 ; a green-reflecting dichroic mirror  34  into which the light having passed through the red-reflecting dichroic mirror  33  enters; a photomultiplier tube  21  for detecting the green light reflected by the dichroic mirror  34 ; a diaphragm  37  arranged between the dichroic mirror  34  and the photomultiplier tube  21 ; a band pass filter  39  arranged between the diaphragm  37  and the photomultiplier tube  21 ; and an amplifier  22  for amplifying the signals from the photomultiplier tubes  18  to  21  and outputting the amplified signals to the single chip microcomputer  23 . In the movable unit  10 , the XYZ axes controller  11 , the laser controller  13 , solenoids  16  and  17 , and the amplifier  22  are controlled by the single chip microcomputer  23 .  
         [0033]    Next, the measurement by using the disc cytometer having the above-described constitution is described.  
         [0034]    The sample container  1  mounted on the disc holder  5  is centrifuged with the motor  6  in accordance with the revolution, acceleration and deceleration, and time given to the motor controller  9  by the single chip microcomputer  23 . After completing the centrifugation, determination is carried out by irradiating the sample container  1  with the argon laser  12  while controlling the rotation speed of the sample container  1  in the tangential direction so as to be almost the same as that of the common flow cytometry, that is, 10 m/sec. At the time of determination, the radial position to be irradiated is controlled by the XYZ axes controller  11  in, for example, 0.1 μm steps. And the revolution of the motor  6  at this point can be monitored with the reflection type sensor  14  directed toward the side surface of the sample container  1  and the binarizing unit  15  for binarizing the signals in such a manner as to introduce the encoder information, which is formed in the form of irregularities on the circumferential side surface of the sample container  1 , to the timer counter portion of the single chip microcomputer  23 . As shown in FIG. 3, a mark notch  41  having a different notch width is provided on the periphery of the sample container  1 , and the absolute position of the sample container  1  is obtained by detecting the mark notch  41 .  
         [0035]    In the movable unit  10 , after completing the centrifugation, the argon laser  12  is switched from the standby mode to the ON mode by the laser controller  13 . This allows the laser to start to scan the sample container  1 . At this point, the beam expander  26  for making up the confocal microscope optical system is removed from the optical path by controlling the solenoid  16 . The laser beam passes through the object lens  27 , irradiates the sample container  1 , and is split into the sideward scattered light scattered at 80° to the sample container  1  and the forward scattered light going straight on. The forward scattered light having gone straight on is reflected by the mirror  28 , is condensed by the condenser lens  29 , passes through the diaphragm unit  30 , and enters into the photomultiplier tube  18 . In the diaphragm unit  30 , pinholes for making up the confocal optical system and the diaphragm for obtaining the scattered light scattered at 1 to 10° to the sample container  1  can be exchanged by operating the solenoid  17 .  
         [0036]    On the other hand, the sideward scattered light is introduced into three detectors (the photomultiplier tubes  19  to  21 ) after passing through the condenser lens  31 . First the sideward scattered light is split by the half-silvered mirror  32 , and the reflected light is introduced into the photomultiplier tube  19 , which is for determining the intensity of the sideward scattered light, via the diaphragm  35 . On the other hand, the light having passed through the half-silvered mirror  32  is introduced into the red-reflecting dichroic mirror  33 . The light reflected by the dichroic mirror  33  is introduced into the photomultiplier tube  20 , which is for determining the intensity of red fluorescence, via the band pass filter  38  and the diaphragm  36 . The light having passed through the red-reflecting dichroic mirror  33  is introduced into the photomultiplier tube  21 , which is for determining the intensity of green fluorescence, by the green-reflecting dichroic mirror  34  via the band pass filter  39  and the diaphragm  37 .  
         [0037]    The light receiving signals from the photomultiplier tubes  18  to  21  are input into the A-D converting portion contained in the single chip microcomputer  23  from the amplifier  22  capable of controlling the gain of the photomultiplier tubes and stored into the external memory  24  via a DMA (Direct Memory Access) controller of the single chip microcomputer  23 .  
         [0038]    For imaging, first, a confocal optical system is formed by operating the solenoids  16  and  17 ; then the origin is calculated from the mark notch  41  having a different notch width from the other notches on the periphery of the container  1 ; the position of the image to incorporate is calculated from the ring counter information that is reset to 0 after every round of the sample container  1  and the radial positional information; to the position, the laser irradiation is moved; and then controlling the XYZ axes controller  11 , an image is obtained. Although a point-by-point scan is made in this embodiment since it adopts a confocal optical microscope system, it is also possible to use an optical system of a CCD (charge coupled device) and a phase contrast microscope.  
         [0039]    In the following, actual experimentation where CD 4  and CD 8 , lymphocyte surface antigens of human peripheral blood, were stained by using the device of the above embodiment is described with experimental protocols.  
         [0040]    Heparinized venous blood collected from a healthy subject was diluted 3-fold with PBS (phosphate-buffered saline), and an 8 ml aliquot of the dilution was added to 2 ml of a Ficoll solution, and centrifuged for 10 minutes at 1100 rpm. Then the lymphocyte layer was removed with a pasteur pipette, washed with 10% FCS (Fetal Bovine Serum) in PBS twice, and adjusted to a lymphocyte concentration of 1×10 7  ml. Two milliliter of an 1:1 mixture of the FITC (fluorescein isothiocyanate)-labeled anti-CD4 monoclonal antibody and the PE (phycoerythrin)labeled anti-CD8 monoclonal antibody was added to the above lymphocyte sample and the mixture was reacted at 4° C. for 30 minutes. After the reaction, the mixture was washed with 10% FCS-PBS three times, to obtain a sample for the next step.  
         [0041]    The sample container  1  was filled with a Ficoll solution  42  in advance. Then the sample  40  prepared as described above was injected into the center portion of the solution  42  (in other words, the center portion of the sample container  1 ) as shown in FIG. 4, and the container was mounted on the above described disc cytometer to make determination. The results are shown in FIG. 5. FIG. 5 shows the analytical results obtained with this experiment. In FIG. 5, both the ordinate and the abscissa are in a logarithmic scale, and the abscissa [FL 1 ] represents the fluorescence intensity of FITC and the ordinate [FL 2 ] the fluorescence intensity of PE.  
         [0042]    The results obtained by changing parameters are plotted in FIG. 6. In FIG. 6, both the ordinate and the abscissa are scaled in a linear scale, and the abscissa [SS] represents the sideward scattered light intensity and the ordinate [FS] the forward scattered light intensity.  
         [0043]    From the results shown in FIGS. 5 and 6, it was confirmed that the disc cytometer according to this invention has analytical functions equivalent to those of a current flow cytometer. Further, when a position where the strongest FITC fluorescence was observed in FIG. 5, thus a bit suspicious position, was imaged by using a confocal laser microscope optical system, a peripherally stained image was obtained. This confirms that the FITC-labeled anti-CD4 monoclonal antibody reacted with the surface antigen of a lymphocyte. In other words, staining was not nonspecific staining, but staining corresponded to proper surface antigens.  
         [0044]    As described above, the advantage of the present invention is that in addition to analytical results equivalent to those by the current flow cytometer, a sample preparation can be also obtained. Further, it has an advantage that it allows more detailed analysis since the actual stained image of cells etc. selected under certain conditions can be checked repeatedly.