(19)
(11)EP 2 824 444 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
18.04.2018 Bulletin 2018/16

(21)Application number: 13758079.1

(22)Date of filing:  05.03.2013
(51)International Patent Classification (IPC): 
G01N 21/64(2006.01)
G01N 37/00(2006.01)
G06K 9/46(2006.01)
G01N 33/53(2006.01)
G06K 9/00(2006.01)
G06T 7/00(2017.01)
(86)International application number:
PCT/JP2013/056020
(87)International publication number:
WO 2013/133283 (12.09.2013 Gazette  2013/37)

(54)

DETERMINATION METHOD, DETERMINATION DEVICE, DETERMINATION SYSTEM, AND PROGRAM

BESTIMMUNGSVERFAHREN, BESTIMMUNGSVORRICHTUNG, BESTIMMUNGSSYSTEM UND PROGRAMM

PROCÉDÉ DE DÉTERMINATION, DISPOSITIF DE DÉTERMINATION, SYSTÈME DE DÉTERMINATION ET PROGRAMME


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 08.03.2012 JP 2012052311

(43)Date of publication of application:
14.01.2015 Bulletin 2015/03

(73)Proprietor: Toray Industries, Inc.
Tokyo 103-8666 (JP)

(72)Inventors:
  • NAGINO, Kunihisa
    Kamakura-shi, Kanagawa 248-8555 (JP)
  • SASAMOTO, Hiromichi
    Otsu-shi, Shiga 520-8558 (JP)
  • SUZUE, Shigeru
    Otsu-shi, Shiga 520-8558 (JP)

(74)Representative: Kador & Partner 
Corneliusstraße 15
80469 München
80469 München (DE)


(56)References cited: : 
JP-A- 2004 340 574
JP-A- 2009 058 356
JP-A- 2011 004 737
JP-A- 2006 084 281
JP-A- 2009 236 626
US-A1- 2010 130 372
  
  • REIMERS MARK ET AL: "Quality assessment of microarrays: Visualization of spatial artifacts and quantitation of regional biases", BMC BIOINFORMATICS, BIOMED CENTRAL, LONDON, GB, vol. 6, no. 1, 1 July 2005 (2005-07-01), page 166, XP021000761, ISSN: 1471-2105, DOI: 10.1186/1471-2105-6-166
  • ZHAO YINGDONG ET AL: "Evaluation of normalization methods for two-channel microRNA microarrays", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, LONDON, GB, vol. 8, no. 1, 21 July 2010 (2010-07-21), page 69, XP021078893, ISSN: 1479-5876, DOI: 10.1186/1479-5876-8-69
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

Field



[0001] The present invention relates to a determination method, a determination device, a determination system, and a computer program.

Background



[0002] In recent years, a massive amount of genes, proteins, and the like have been enabled to be comprehensively analyzed according to development in technologies such as a microarray experiment or a macroarray experiment. For example, in a DNA microarray, several hundreds to several tens of thousands of DNAs may be arrayed in a matrix to be fixed as spots on a carrier such as a slide glass, and mRNA or cDNA extracted and labeled from a cell to be examined may be hybridized with the spots to measure a gene expression level.

[0003] That is, a substance to be examined such as labeled cDNA selectively binds to a complementary DNA on the carrier, so that the gene expression level can be estimated by acquiring detection intensity of a label. Although reliability as data is required for a selective binding of an amount of the substance to be examined such as the gene expression level, the detection intensity may vary depending on non-biological factors such as a case in which selective binding substances such as complementary DNAs fixed as spots on the carrier are unevenly distributed or a case in which dust is attached to a spot portion.

[0004] Accordingly, a method has been developed for determining uniformity of spots in a DNA microarray and the like. For example, a method for evaluating the uniformity disclosed in Patent Literature 1 includes following steps: (1) background data corresponding to each of the spots is obtained by adapting analyzing software to an image obtained by scanning a monochromatic light emitting image of the DNA microarray, (2) plate-to-plate No. and a plate position of a target DNA corresponding to each of the spots are calculated, (3) the plate-to-plate No. and the plate position are allowed to correspond to each piece of the background data, and (4) the pieces of background data are arranged in the order of the plate No. and the plate position to obtain a sequence BG and a periodicity rule is detected by extracting a sub-sequence from the entire sequence.

[0005] Patent Literature 2 discloses that unevenness is evaluated with reference to a value of a coefficient of variation (CV). Herein, the CV of the spot means a proportion (%) of a standard deviation (SD) to an average value of fluorescence intensity of each spot that is obtained when the detection intensity of a spot of the DNA microarray and the like on the carrier is scanned and measured.

[0006] Regarding an analyzing software of microarray GenePix Pro (manufactured by Molecular Devices, LLC.), each intensity value of pixels included in the spot is compared with an average intensity value of pixels around the spot, and the spot is determined to be defective if a predetermined proportion of the pixels included in the spot satisfies the condition as follows: "(intensity of each pixel included in the spot) - (average intensity value of the pixels around the spot) < 0" (refer to Non Patent Literature 1). According to this, it is detected that a background around the spot becomes high due to certain abnormality, for example, dust is attached thereto or a chip is contaminated.

Citation List


Patent Literature



[0007] 

Patent Literature 1: Japanese Patent Application Laid-open No. 2004-340574

Patent Literature 2: Japanese Patent Application Laid-open No. 2008-039584



[0008] Non Patent Literature 1: Inter Medical co., ltd., "GenePix Pro 7.0 Microarray Image Analysis", [online], Copyright 2006 InterMedical co., ltd., [retrieved on March 6, 2012], Internet <URL: http://www.intermedical.co.jp/homepage/products/axon/genepi xpro7.html>

[0009] US 2010/0130372 A1 refers to methods for generating a normalized expression signal for microarray data based on a theoretical distribution at the unit level to produce a normalized expression signal for the single microarray that is independent of other microarrays.

Summary


Technical Problem



[0010] However, the reliability of the selective binding of an amount of the substance to be examined cannot be appropriately determined by the method for determining the uniformity of spots in the related art.

[0011] Specifically, although the uniformity among a plurality of spots can be evaluated by the method for evaluating the uniformity disclosed in Patent Literature 1, uniformity within single spot cannot be evaluated.

[0012] In the evaluating method with the CV value disclosed in Patent Literature 2, when intensity of some pixels in a pixel group configuring the spot is extremely high or low due to attachment of dust or the like, the standard deviation becomes large and the CV value exceeds a threshold. Accordingly, even though intensity data of the pixel group except for some pixels can be used, the data is eliminated, which causes excessive detection.

[0013] That is, in general, a median (median value) of the pixel group within the spot in an image is used as a representative value of signal intensity of a spot on a DNA chip and the like. For example, when alignment is performed in a circular spot having a diameter of 100 micrometers in the image, a median (spot median) of intensity is obtained for a pixel group of about 70 pixels (pixel size: 10 micrometers square) included in the circle.

[0014] This is because the median hardly vary depending on outliers as compared to a case of using an average value of intensity of the pixel group. That is, when there are outliers such as an extremely large value and an extremely small value of the detection intensity of the pixel group in the spot, an overall average is skewed by the outliers.

[0015] FIG. 1 is a scatter plot illustrating detection intensity when the same substance to be examined is hybridized to two DNA chips. The horizontal axis represents the detection intensity of each spot in one of the DNA chips, and the vertical axis represents the detection intensity of each spot in the other DNA chip. That is, coordinates (X, Y) of one point represents detection intensity (X) measured in one DNA chip and detection intensity (Y) measured in the other DNA chip for the spot to which the same selective binding substance is fixed.

[0016] In this example, the same substance to be examined is hybridized to the two DNA chips, so that ideally Y = X should be satisfied. However, as illustrated in FIG. 1, intensity plots of spots 1 and 2 are largely deviated from a line of Y = X, so that it is considered that there is a defective spot. FIG. 2 is a diagram illustrating an intensity image of the spot 1 (left figure) and a variation graph of the detection intensity along the longitudinal line of the spot 1 (right figure). FIG. 3 is a diagram illustrating an intensity image of the spot 2 (left figure) and a variation graph of the detection intensity along the longitudinal line of the spot 2 (right figure). In the intensity image, a dashed-line circle indicates a spot portion, and the detection intensity is represented with a white gradation value.

[0017] As illustrated in the left figures of FIG. 2 and FIG. 3, the detection intensity is not represented as a uniform circle, but as a non-uniform gradation, in the intensity image of the spot portion. The variation graphs (right figures) represent a variation of the detection intensity along the longitudinal line of the spot portion in a range of one pixel in the horizontal direction and more than ten pixels in the longitudinal direction. Ideally, the detection intensity should be constant, but it largely varies. A main cause of the defect is a defective spot (a defect caused when the selective binding substance is fixed) and attachment of dust.

[0018] In the related art such as disclosed in Patent Literature 2, to eliminate such a defective spot, quality of the spot is determined using the CV value obtained as follows:



[0019] That is, the data has been eliminated as a defective spot when the CV value is equal to or larger than a predetermined reference value. In this case, when 5 pixels in the pixel group including 70 pixels indicate extremely large values in the above example because dust and the like is attached to part of the spot, the CV value becomes equal to or larger than the reference value and the data is eliminated.

[0020] However, the median of the pixel group in the spot is usually used as the representative value of the signal intensity of the spot, so that the value hardly varies even when the outlier is included in part of the spot. Accordingly, the data may be sufficiently usable. That is, in the conventional evaluating method using the CV value, sufficiently usable data has been eliminated by excessive detection of defective spot.

[0021] The present invention is made in view of such a situation, and provides a determination method, a determination device, a determination system, and a computer program that can appropriately evaluate reliability of a selective binding of an amount of a substance to be examined considering non-biological effect for data obtained from a microarray experiment and the like. Solution to Problem

[0022] To achieve the above-described object, a determination method according to the present invention is a determination method for determining reliability of a selective binding of an amount of a substance to be examined obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier includes: a pixel group extracting step of determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot; a median calculating step of calculating a ratio or a difference between a median value of the detection intensity of the pixel group extracted at the pixel group extracting step and a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and a reliability determining step of determining the reliability based on the ratio or the difference calculated at the median calculating step and a certain reference value.

[0023] Moreover, in the above-described determination method according to the present invention, a value of a ratio obtained from expression (1) and/or expression (2) is calculated at the median calculating step, the expressions (1) and (2) being as follows:



(where X is a median value of the detection intensity of the pixel group, Xt is a median value of the detection intensity of the pixel group excluding the certain top proportion of pixels, and Xb is a median value of the detection intensity of the pixel group excluding the certain bottom proportion of pixels), and the reliability determining step determines that the reliability is defective when the value of the ratio calculated at the median calculating step is equal to or larger than the reference value.

[0024] Moreover, in the above-described determination method according to the present invention, the reference value is a value obtained from expression (3) as follows:

(where S is the reference value, C is a constant, Z is an offset value corresponding to a sensitivity setting of a device for detecting the detection intensity of the label, and X is the median value of the detection intensity of the pixel group).

[0025] Moreover, in the above-described determination method according to the present invention, the device for detecting the detection intensity of the label is a photomultiplier, and the offset value is a value obtained from expression (4) as follows:

(where Z is the offset value, X is a gain voltage of the photomultiplier, and A and B are constants).

[0026] Moreover, in the above-described determination method according to the present invention, the carrier is a microarray, the label is a fluorescent label, the detection intensity is a fluorescence amount, and the reliability determining step determines quality of the spot as the the reliability.

[0027] Moreover, a determination device according to the present invention includes at least a control unit determining reliability of a selective binding of an amount of a substance to be examined obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, and the control unit includes: pixel group extracting means for determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot; median calculating means for calculating a ratio or a difference between a median value of the detection intensity of the pixel group extracted by the pixel group extracting means and a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and reliability determining means for determining the reliability based on the ratio or the difference calculated by the median calculating means and a certain reference value.

[0028] Moreover, a determination system according to the present invention is a determination system configured by connecting a detection device for reading detection intensity of a label, which is obtained when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, to a determination device comprising at least a control unit for determining reliability of the selective binding of an amount of the substance to be examined obtained as the detection intensity, wherein the control unit of the determination device includes: image data acquisition means for acquiring, as imaged image data, the detection intensity in the carrier read via the detection device; pixel group extracting means for determining a position of the spot in the image data obtained by the image data acquisition means and extracting a pixel group corresponding to the spot; median calculating means for calculating a ratio or a difference between a median value of the detection intensity of the pixel group extracted by the pixel group extracting means and a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and reliability determining means for determining quality of the reliability based on the ratio or the difference calculated by the median calculating means and a certain reference value.

[0029] Moreover, a program according to the present invention is a computer program that causes a computer comprising at least a control unit to execute a method for determining reliability of a selective binding of an amount of a substance to be examined obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, wherein the control unit is caused to execute the method including: a pixel group extracting step of determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot; a median calculating step of calculating a ratio or a difference between a median value of the detection intensity of the pixel group extracted at the pixel group extracting step and a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and a reliability determining step of determining the reliability based on the ratio or the difference calculated at the median calculating step and a certain reference value.

[0030] The present invention is related to a recording medium and the computer program described above is recorded therein.

Advantageous Effects of Invention



[0031] With the determination method, the determination device, the determination system, and the computer program according to the present invention, the reliability of the selective binding of an amount of the substance to be examined can be appropriately evaluated considering the non-biological effect in the data obtained from the microarray experiment and the like.

Brief Description of Drawings



[0032] 

FIG. 1 is a scatter plot illustrating detection intensity when the same substance to be examined is hybridized to two DNA chips.

FIG. 2 is a diagram illustrating an intensity image of a spot 1 (left figure) and a variation graph of detection intensity along the longitudinal line of the spot 1 (right figure).

FIG. 3 is a diagram illustrating an intensity image of a spot 2 (left figure) and a variation graph of detection intensity along the longitudinal line of the spot 2 (right figure).

FIG. 4 is a block diagram illustrating an example of the entire structure of a determination system according to an embodiment.

FIG. 5 is a flowchart illustrating an example of basic processing of a determination device 100 according to the embodiment.

FIG. 6 is a diagram illustrating a relation among a spot median (X), a median excluding a predetermined top proportion (Xt: Top Cut Median), and a median excluding a predetermined bottom proportion (Xb: Bottom Cut Median).

FIG. 7 is a diagram illustrating a correction curve and white noise in a region having weak signal intensity.

FIG. 8 is a diagram illustrating a result obtained by measuring the same carrier to which the same substance to be examined is provided when a photomultiplier tube (PMT) gain voltage (%) is set to "40" (40% x 1 V).

FIG. 9 is a diagram illustrating a result obtained by measuring the same carrier to which the same substance to be examined is provided when the PMT gain voltage (%) is set to "55" (55% × 1 V).

FIG. 10 is a graph plotting an offset value and the PMT gain voltage (sensitized control voltage).

FIG. 11 is a scatter plot illustrating detection intensity when the same substance to be examined is hybridized to two DNA chips.

FIG. 12 is a diagram illustrating an intensity image of a spot of which inside is non-uniform (upper figure) and a variation graph of the detection intensity along a transverse line of the spot (lower figure).

FIG. 13 is a diagram illustrating a result obtained by sorting values of expression (1) obtained for each spot in ascending order.

FIG. 14 is a diagram illustrating a result obtained by sorting values of expression (2) obtained for each spot in descending order.

FIG. 15 is a scatter plot illustrating a result when the PMT gain voltage is set to 40%.

FIG. 16 is a scatter plot illustrating a result when the PMT gain voltage is set to 55%.

FIG. 17 is a scatter plot illustrating a result when the PMT gain voltage is set to 70%.

FIG. 18 is a logarithmic graph illustrating a correlation between a set value of the PMT gain voltage and an offset value.


Description of Embodiments



[0033] The following describes an embodiment of a determination method, a determination device, a determination system, and a computer program according to the present invention in detail with reference to the drawings. Specifically, the embodiment described below exemplifies a determination system for embodying a technical idea of the present invention, and does not intend to limit the invention to be made with the determination system. The embodiment may be equally applied to a determination method and a determination device according to other embodiments included in the scope of claims. For example, although the following embodiment may describe a determination method performed in the determination system, the embodiment is not limited thereto. The determination method may be manually performed by a person. Although the following embodiment describes an example in which the determination device is connected to an input device of a measuring device and the like that acquires image data and the like, the present invention is not limited thereto. The determination device may store the image data in a storage unit in advance without being connected to the input device and may acquire the image data from the outside via communication.

Overview of the present embodiment



[0034] The following describes an overview of the present embodiment. Subsequently, a configuration, processing, and the like of the present embodiment will be described in detail with reference to the drawings. As a result of keen examination, the present inventors have developed a determination method according to the embodiment described below as an example.

[0035] That is, the present embodiment generally has a basic characteristic as follows. The present embodiment determines reliability of a selective binding of an amount of a substance to be examined obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier.

[0036] Herein, the "substance to be examined" means a sample directly or indirectly obtained from a cell, a tissue, and the like. Examples thereof include genomic DNA, RNA, cDNA, aRNA (RNA that is amplified using cDNA or a complementary sequence thereof as a template), protein, a sugar chain, and lipid. The "label" means a substance that can be detected by a detection unit. Examples thereof include a fluorescent label, a bioluminescent label, and a radioactive isotope label. The "selective binding substance" means a substance that selectively binds to a certain substance. Examples thereof include complementary DNA to DNA, complementary RNA to DNA, an antibody to an antigen, and an enzyme to a chemical substance.

[0037] The "carrier" may be a common substrate such as a DNA chip or a microarray. In addition, it may be a DNA chip substrate composed of polymethyl methacrylate having a rugged structure (3D-Gene (product name), manufactured and sold by Toray Industries, Inc. (corporate name), refer to Japanese Patent Application Laid-open No. 2004-264289). In this case, a projecting portion is spotted.

[0038] First, in the present embodiment, a position of the spot is determined in image data obtained by imaging detection intensity in the carrier and a pixel group corresponding to the spot is extracted. That is, a group of pixels constituting one spot portion on the image is extracted for each spot.

[0039] Subsequently, in the present embodiment, a ratio or a difference is calculated between a median value (X) of the detection intensity of the extracted pixel group and a median value (Xt/Xb) of the detection intensity of the pixel group excluding a predetermined top proportion and/or a predetermined bottom proportion of pixels. For example, in the present embodiment, the ratio may be calculated based on the expression (1) and/or expression (2) as follows:



(where X is the median value of the detection intensity of the extracted pixel group, Xt is the median value of the detection intensity of the pixel group excluding a predetermined top proportion of pixels, and Xb is the median value of the detection intensity of the pixel group excluding a predetermined bottom proportion of pixels).

[0040] In examples described below, each of the predetermined top proportion and predetermined bottom proportion excluded in calculating Xt and Xb is 30% of the extracted pixel group. However, the proportion is not limited thereto. For example, the proportion may be an arbitrary value in a range of 20% to 40%.

[0041] In the present embodiment, reliability is determined based on the calculated ratio or difference and a predetermined reference value S. For example, in the present embodiment, the reliability may be determined to be defective when a value of the calculated ratio or difference is equal to or larger than the reference value S. In the spot 1 of FIG. 2, a value (%) of expression (1) was 34%, and a value (%) of expression (2) was 22.2%. In the spot 2 of FIG. 3, the value (%) of expression (1) was 30%, and the value (%) of expression (2) was 22.3%. Sufficient data can be obtained in both of the spots 1 and 2, the reference value S may be about 25% to 30% so as not to eliminate the data.

[0042] Herein, the reference value S may be a value obtained from expression (3) as follows:

(where S is a reference value, C is a constant, Z is an offset value corresponding to a sensitivity setting of a device for detecting detection intensity of a label, and X is a median value of the detection intensity of the pixel group).

[0043] Herein, the offset value Z may be a value obtained from expression (4) as follows:

(where Z is an offset value, X is a gain voltage of a photomultiplier, and A and B are constants).

[0044] The overview of the embodiment has been described above. According to the embodiment, it is possible to appropriately evaluate the reliability of the selective binding of an amount of the substance to be examined considering the non-biological effect in the data obtained from the microarray experiment and the like. Specifically, it is possible to prevent sufficiently usable data from being eliminated by excessive detection, even though the data includes an outlier in part of the spot.

Configuration of determination system



[0045] First, the following describes the configuration of the determination system according to the present embodiment. FIG. 4 is a block diagram illustrating an example of the entire structure of the determination system according to the present embodiment.

[0046] As illustrated in FIG. 4, the determination system according to the present embodiment generally includes an input device 112 that functions as a detection unit for reading detection intensity of a label, an output device 114, and a determination device 100.

[0047] The determination device 100 generally includes a control unit 102 such as a central processing unit (CPU) that integrally controls the entire determination device 100, a communication control interface unit 104 that is connected to a communication device (not illustrated) such as a router connected to a communication line and the like, an input/output control interface unit 108 that is connected to the input device 112 or the output device 114, and a storage unit 106 that stores therein various databases or tables. These units are communicably connected to each other via an arbitrary channel. Herein, as illustrated in FIG. 4, the determination device 100 may be connected to a network 300 via a communication device such as a router and a wired or wireless communication line such as a dedicated line. The determination device 100 may further be connected to an external system 200 via the network 300.

[0048] The various databases or tables stored in the storage unit 106 (an image data file 106a and a pixel group file 106b) may be storage means such as a fixed disk device. For example, the storage unit 106 may store therein various computer programs, tables, files, databases, Web pages, and the like that are used in various processes.

[0049] Among these components of the storage unit 106, the image data file 106a is an image data storage means that stores therein image data obtained by imaging the detection intensity in the carrier. For example, the image data stored in the image data file 106a may be image information obtained by scanning the detection intensity on a carrier plane by the detection unit. The image data file 106a may store therein the image data in advance, may be image data input from the input device 112 that functions as a detection unit described later, and may be image data received from the external system 200 via the network 300. By way of example, the image data file 106a stores therein, as the image data, gray-scale image data that uses the detection intensity as a gradation value.

[0050] The pixel group file 106b is a pixel group storage unit that stores therein information about the pixel group (such as intensity information) for each spot. For example, information to be stored in the pixel group file 106b is a value of the detection intensity (such as a gradation value) of each pixel, a median value of the pixel group, and the like associated with identification information that uniquely specifies the spot (a block number, a row number, a column number and the like of the carrier). For example, assuming that a carrier (biochip) is used, the carrier including four blocks in one biochip and being capable of detecting expression patterns of 64 (8 x 8) spots per block, that is, total of 256 genes, the pixel group file 106b stores therein a position at which a selective binding substance (such as a DNA fragment) corresponding to each of the genes (the block number, and the row number and the column number of the spot) is arranged in association with the gradation value and the like of the pixel group in the spot.

[0051] In FIG. 4, the input/output control interface unit 108 controls the input device 112 and the output device 114. As the output device 114, a printer, a recording medium output device, and the like may be used herein in addition to a monitor (including a home television). As the input device 112, a detection device (detection unit) that reads the detection intensity of the label may be used in addition to a keyboard, a mouse, and the like.

[0052] Herein, the "detection unit" means a unit that reads the detection intensity of the label obtained when the labeled substance to be examined binds to the selective binding substance fixed as the spot on the carrier. For example, the detection unit may be an examination unit for specifying the position at which the selective binding substance is spotted and acquiring the detection intensity, for example, a fluorescence microscope camera. When the label is a fluorescent label or a bioluminescent label, the detection unit may be a photomultiplier (photomultiplier tube). The detection unit is not limited to a unit that images the detection intensity such as the fluorescence microscope camera, and may be a unit that reads the detection intensity. The detection intensity may be imaged by the determination device 100 by scanning along the carrier plane. When the selective binding substance is DNA, a minute amount of double-stranded DNA binding fluorescent substance may be captured to detect the selective binding amount. The detection unit may also detect an absorption wavelength specific to the DNA. Also when the selective binding substance is protein, a sugar chain, and the like, detection may be performed using the absorption wavelength, the fluorescent substance, a radioactive isotope (radioisotope), hybridization, a technique such as antigen-antibody reaction, and the like, corresponding to a property of the selective binding substance.

[0053] In FIG. 4, the control unit 102 includes a control program such as an operating system (OS), a computer program specifying various processing procedures, and an internal memory for storing required data, and performs information processing for executing various processes with these computer programs and the like. The control unit 102 conceptually includes an image data acquisition unit 102a, a pixel group extracting unit 102b, a median calculation unit 102c, a reliability determination unit 102d, and a reference value determination unit 102e.

[0054] Among these, the image data acquisition unit 102a is image data acquisition means that acquires image data obtained by imaging the detection intensity in the carrier. For example, the image data acquisition unit 102a may acquire, as the imaged image data, the detection intensity in the carrier that is read via the input device 112 functioning as the detection unit. The image data acquisition unit 102a may directly acquire the image data from the input device 112 such as the fluorescence microscope camera, and may image the detection intensity for each pair of coordinates obtained by causing the input device 112 such as the photomultiplier to scan along the carrier plane. The image data acquisition unit 102a may receive the image data from the external system 200 via the network 300. The image data acquisition unit 102a stores the acquired image data in the image data file 106a.

[0055] The pixel group extracting unit 102b is pixel group extracting means that determines the position of the spot in the image data stored in the image data file 106a and extracts the pixel group corresponding to the spot. For example, the pixel group extracting unit 102b may partition each spot portion on the image and extract a pixel group in each compartment based on an arrangement (such as spot center coordinates and a pixel radius) of the position (spot portion) on the carrier at which the selective binding substance is arranged. The pixel group extracting unit 102b may perform positioning by displaying the image data and arrangement pattern data on the output device 114 in a superposed manner and causing a user to perform input operation to move the displayed arrangement pattern via the input device 112 such as a mouse. The pixel group extracting unit 102b stores information about the pixel group (such as intensity information) for each spot in the pixel group file 106b. For example, the pixel group extracting unit 102b may store the value of the detection intensity (such as a gradation value) of each pixel in the pixel group file 106b in association with the identification information (the block number, the row number, the column number and the like of the carrier) that uniquely specifies the spot.

[0056] The median calculation unit 102c is median calculation means that calculates a ratio or a difference between the median value (X) of the detection intensity of the pixel group stored in the pixel group file 106b and the median value (Xt/Xb) of the detection intensity of the pixel group excluding a predetermined top proportion and/or a predetermined bottom proportion of pixels. For example, in the present embodiment, the ratio may be calculated based on the following expression (1) and/or expression (2). For example, the median calculation unit 102c may rearrange the detection intensity (such as a gradation value) of the pixel group in a certain spot stored in the pixel group file 106b by sorting in ascending order or descending order, and may obtain medium detection intensity of the group excluding a predetermined top proportion (x%) and medium detection intensity of the group excluding a predetermined bottom proportion (y%).



(where X is the median value of the detection intensity of the extracted pixel group, Xt is the median value of the detection intensity of the pixel group excluding a predetermined top proportion of pixels, and Xb is the median value of the detection intensity of the pixel group excluding a predetermined bottom proportion of pixels).

[0057] The reliability determination unit 102d is reliability determination means that determines reliability based on the ratio or the difference calculated by the median calculation unit 102c and the predetermined reference value. For example, when the ratio or the difference calculated by the median calculation unit 102c is equal to or larger than the reference value S, the reliability determination unit 102d may determine the reliability to be defective. The reliability determination unit 102d may exclude data of the defective spot from analysis based on the determination result of the reliability, and may output the determination result of the reliability to the output device 114. The control unit 102 may perform processing (such as exclusion of defective spot data) based on the determination result of the reliability by the reliability determination unit 102d and output a data analysis result to the output device 114. An output destination is not limited to a monitor as the output device 114. Alternatively, the result may be output to a printer or a recording medium and the like via a recording medium output device. The control unit 102 such as the reliability determination unit 102d may control the communication control interface 104 and transmit the determination result of the reliability and the analysis result data to the external system 200 via the network 300.

[0058] The reference value determination unit 102e is reference value determination means that determines a reference value as a reference of reliability determination by the reliability determination unit 102d. For example, the reference value determination unit 102e may determine the reference value S based on the following expression (3).

(where S is the reference value, C is the constant, Z is the offset value corresponding to the sensitivity setting of the device for detecting the detection intensity of the label, and X is the median value of the detection intensity of the pixel group).

[0059] Herein, the reference value determination unit 102e may determine the offset value Z in expression (3) based on the following expression (4).

(where Z is the offset value, X is the gain voltage of the photomultiplier, and A and B are constants).

[0060] In FIG. 4, the communication control interface unit 104 controls communication between the determination device 100 and the network 300 (or a communication device such as a router). That is, the communication control interface unit 104 has a function to communicate data to another terminal via a communication line. The network 300 has a function to connect the determination device 100 and the external system 200 to each other. Examples of the network 300 include the Internet. The external system 200 and the determination device 100 are connected to each other via the network 300. The external system 200 has a function to provide an external database or an external computer program related to detection intensity data of the label and the like.

[0061] The external system 200 may be configured as a server device such as a WEB server and an ASP server, or a terminal device. The hardware configuration thereof may include an information processing device such as a workstation and a personal computer being commercially available, and an auxiliary device thereof. Each function of the external system 200 is implemented with a CPU, a disk device, a memory device, an input device, an output device, a communication control device, and the like in the hardware configuration of the external system 200 and with a control program and the like that controls the above devices. The user of the determination device 100 may obtain data of detection intensity value, arrangement data, a computer program, and the like by accessing, via the network 300, an external database such as a detection intensity database related to a DNA chip and the like and a gene arrangement database provided by the external system 200, or a Web site that provides an external program such as a computer program for causing a determination method to be performed. The description about the configuration of the determination system and the determination device 100 according to the present embodiment is finished.

Processing of determination device 100



[0062] The following describes an example of the processing of the determination device 100 according to the embodiment configured as described above in detail with reference to FIG. 5 and FIG. 6. FIG. 5 is a flowchart illustrating an example of basic processing of the determination device 100 according to the embodiment.

[0063] First, as illustrated in FIG. 5, in the determination device 100, the image data acquisition unit 102a acquires image data obtained by imaging the detection intensity in the carrier and stores the acquired image data in the image data file 106a (Step SA-1). For example, the image data acquisition unit 102a may acquire, as imaged image data, the detection intensity in the carrier that is read via the input device 112 functioning as the detection unit. The image data acquisition unit 102a may directly acquire the image data from the input device 112 such as the fluorescence microscope camera, and may image the detection intensity for each pair of coordinates obtained by causing the input device 112 such as the photomultiplier to scan along the carrier plane. The image data acquisition unit 102a may read data from an external recording medium storing therein the detection intensity data or the image data via the input/output control interface unit 108.

[0064] The pixel group extracting unit 102b then determines the position of the spot in the image data stored in the image data file 106a, extracts the pixel group corresponding to the spot, and stores information about the extracted pixel group in the pixel group file 106b (Step SA-2). For example, the pixel group extracting unit 102b may partition each spot portion on the image and extract the pixel group in each compartment based on an arrangement (such as spot center coordinates and a pixel radius) of the position (spot portion) on the carrier at which the selective binding substance is arranged. The pixel group extracting unit 102b stores information about the pixel group (such as intensity information) for each spot in the pixel group file 106b. For example, the pixel group extracting unit 102b may store the value of the detection intensity (such as a gradation value) of each pixel in the pixel group file 106b in association with the identification information (the block number, the row number, the column number and the like of the carrier) that uniquely specifies the spot.

[0065] The median calculation unit 102c acquires the median value (X) of the detection intensity of the pixel group stored in the pixel group file 106b (Step SA-3). For example, the median calculation unit 102c may rearrange the detection intensity (such as a gradation value) of the pixel group in a certain spot by sorting in ascending order or descending order, and may obtain a medium (in the middle of a ranking) detection intensity value to obtain the median value (X).

[0066] The median calculation unit 102c acquires a median value (Xt) of the detection intensity of the pixel group stored in the pixel group file 106b excluding predetermined top proportion (x%) of pixels and/or a median value (Xb) of the detection intensity of the pixel group excluding a predetermined bottom proportion (y%) of pixels (Step SA-4). The predetermined top proportion (x%) and the predetermined bottom proportion (y%) may be, for example, 30%. By way of example, the median calculation unit 102c may rearrange the detection intensity (such as a gradation value) of the pixel group in a certain spot by sorting in ascending order or descending order, and may obtain medium detection intensity (Xt) of the group excluding a predetermined top proportion (x%) and medium detection intensity (Xb) of the group excluding a predetermined bottom proportion (y%). FIG. 6 is a diagram illustrating a relation among a spot median (X), a median excluding a predetermined top proportion (Xt: Top Cut Median), and a median excluding a predetermined bottom proportion (Xb: Bottom Cut Median). Each bar in the bar graph corresponds to one pixel and the length thereof represents the detection intensity corresponding to the gradation value of the pixel. The pixel group in the spot is sorted in descending order according to the intensity along the horizontal axis.

[0067] As illustrated in FIG. 6, the spot median (X) is a medium (middle position) value in the ranking of intensity of the pixel group. The median Xt (Top Cut Median) is a value at the middle position in the pixel group excluding a predetermined top proportion (in this example, 4 pixels). The median Xb (Bottom Cut Median) is a value at the middle position in the pixel group excluding a predetermined bottom proportion (in this example, 4 pixels).

[0068] Returning to FIG. 5 again, the median calculation unit 102c calculates a ratio or a difference between the spot median X and the median Xt excluding the predetermined top proportion and/or a ratio or a difference between the spot median X and the median Xb excluding the predetermined bottom proportion (Step SA-5). Double-headed arrows in FIG. 6 represent a difference between the spot median X and the median Xt excluding the predetermined top proportion and a difference between the spot median X and the median Xb excluding the predetermined bottom proportion. The median calculation unit 102c may calculate the ratio based on the following expression (1) and/or expression (2).



(where X is the median value of the detection intensity of the extracted pixel group, Xt is the median value of the detection intensity of the pixel group excluding a predetermined top proportion of pixels, and Xb is the median value of the detection intensity of the pixel group excluding a predetermined bottom proportion of pixels).

[0069] The reliability determination unit 102d determines reliability based on the ratio or the difference calculated by the median calculation unit 102c and a predetermined reference value (Step SA-6). For example, when the ratio or the difference (such as an absolute value of the difference) calculated by the median calculation unit 102c is equal to or larger than the predetermined reference value S (%), the reliability determination unit 102d may determine the reliability to be defective. The reference value S (%) may be a constant value C (%). When white noise from an electric circuit system and the like cannot be ignored in a region having weak signal intensity, basic C (%) may be corrected by the reference value determination unit 102e (details will be described later).

[0070] An example of the basic processing of the determination device 100 has been described above.

Correction processing of reference value



[0071] In the basic processing of the determination device 100 described above, the reference value S is assumed to be the constant value C. The following describes an example of correction processing in which the reference value determination unit 102e corrects and determines the reference value. FIG. 7 is a diagram illustrating a correction curve and white noise in a region having weak signal intensity. In FIG. 7, the horizontal axis indicates the spot median X representing the signal intensity, and the vertical axis indicates a value of the ratio obtained from expression (1) and expression (2). In this example, the absolute value is not obtained in expression (1) and expression (2). Accordingly, the value obtained from expression (2) is plotted in a positive region along the vertical axis, and the value obtained from expression (1) is plotted in a negative region therealong.

[0072] As illustrated in FIG. 7, the data becomes discrete as the signal intensity is reduced, influence of the white noise cannot be ignored, and the absolute value of the calculated ratio becomes large. Accordingly, it is preferable that the reference value S (%) is appropriately corrected rather than the constant value C (%). As a result of keen examination, the present inventors have found a correction curve for correcting the reference value S as follows: (Y = 10 + 13/X, and Y = -10 - 13/X). A generalized expression thereof will be described below. That is, the reference value determination unit 102e may correct the basic C (%) to determine the reference value S based on the following expression (3).

(where S is the reference value, C is the constant, Z is the offset value corresponding to the sensitivity setting of the device for detecting the detection intensity of the label, and X is the median value of the detection intensity of the pixel group).

[0073] Herein, the offset value Z is a constant value in the same setting. However, the offset value Z is changed when a gain setting of the photomultiplier in scanning is changed. FIG. 8 and FIG. 9 are diagrams each illustrating a result obtained by measuring the same carrier to which the same substance to be examined is provided when the gain voltage (%) is set to "40" (in this case, a control voltage of the photomultiplier tube is as follows: 40% x 1 V = 0.4 V) and when it is set to "55" (55% x 1 V = 0.55 V).

[0074] As illustrated in FIG. 8 and FIG. 9, a distribution state is changed when the gain voltage (sensitized control voltage) is changed. Accordingly, a threshold curve should be changed. The change depends on the offset value Z of a threshold curve as follows: S = C + Z/X. A preferable threshold curve can be obtained by setting the offset value to 13 in the example of FIG. 8, and by setting the offset value to 80 in the example of FIG. 9.

[0075] FIG. 10 is a graph plotting the offset value and the gain voltage (sensitized control voltage). The horizontal axis indicates a logarithmic value of the gain voltage of the photomultiplier (PMT), and the vertical axis indicates the logarithmic value of the offset value.

[0076] As illustrated in FIG. 10, the offset value is changed depending on the gain voltage, and independent of a type of the label (cy3 and cy5). The logarithmic value of the offset value and the logarithmic value of the gain voltage are represented by a linear expression (the expression in FIG. 10). An inclination and an intercept are values specific to a scanner machine (photomultiplier). The following expression (4) is obtained by generalizing a relational expression between the offset value and the gain voltage. That is, the reference value determination unit 102e may determine the offset value Z based on the following expression (4).

where Z is the offset value, X is the gain voltage of the photomultiplier, and A and B are constants. In the example of FIG. 10, the inclination of the graph is about 5.1639 to 5.12552, and the intercept thereof is about - 7.1363 to -7.2711. That is, A ranges from about -7.1363 to -7.2711, and B ranges from about 5.1639 to 5.12552.

[0077] The description about the correction processing of the reference value is finished.

First example



[0078] The following describes a first example in which an appropriate threshold was examined. First, the same substance to be examined was hybridized to two DNA chips to ensure that there is no significant difference in detection intensity thereof. FIG. 11 is a scatter plot illustrating the detection intensity when the same substance to be examined is hybridized to two DNA chips. The horizontal axis indicates the detection intensity of each spot in one DNA chip, and the vertical axis indicates the detection intensity of each spot in the other DNA chip.

[0079] As a result, as illustrated in FIG. 11, there was no significant difference in the detection intensity in the two DNA chips, so that a normal state was ensured. As illustrated in FIG. 12, although there was a spot of which inside is non-uniform, the spot was not eliminated because it can be sufficiently usable as data when a median is used.

[0080] Subsequently, values of the following expression (1) and expression (2) was calculated for a spot of which detection intensity is a gain voltage set value "40" (control voltage of the photomultiplier tube is 40% × 1 V) and the spot median thereof is equal to or larger than 2000 among these spots. The reason why the spot of which spot median is equal to or larger than 2000 was examined is that the white noise from an electric circuit and the like cannot be ignored in a portion having weak signal intensity as described above (for example, refer to FIG. 7 and FIG. 8) .



(where X is the median value of the detection intensity of the pixel group, Xt is the median value of the detection intensity of the pixel group excluding a predetermined top proportion of pixels, and Xb is the median value of the detection intensity of the pixel group excluding a predetermined bottom proportion of pixels).

[0081] FIG. 13 is a diagram illustrating a result obtained by sorting values of expression (1) obtained for each spot in ascending order. FIG. 14 is a diagram illustrating a result obtained by sorting values of expression (2) obtained for each spot in descending order. Block represents the block number, Column represents the row number, and Row represents the column number. The spot is uniquely specified by these items. The item of S_532_Median represents the spot median.

[0082] As illustrated in FIG. 13 and FIG. 14, it can be determined that unevenness due to a defective shape of the spot is not caused when the absolute value is equal to or smaller than 25% in both values of expression (1) and expression (2). Accordingly, it is considered that the absolute value of the reference value for determining reliability is preferably about 25%.

Second example



[0083] The following describes a second example in which an appropriate offset value was examined. In the second example, two chips of 3D-Gene (registered trademark) Human Ver1.1 (manufactured by Toray Industries, Inc.) were used as carriers. A series of procedures to hybridization was performed as described in a protocol using Human Reference RNA (manufactured by Stratagene corporation) as the substance to be examined. As the detection unit, 3D-Gene (registered trademark) Scanner (manufactured by Toray Industries, Inc.) was used.

[0084] Measurement was performed for one of the two hybridized chips by changing a setting of a photomultiplier (PMT) of 3D-Gene (registered trademark) Scanner (manufactured by Toray Industries, Inc.) to 40%, 55%, and 70%, and the values of expression (1) and expression (2) were calculated for each spot. When the PMT setting is changed, a control voltage of the PMT is changed in proportion thereto. Accordingly, a photomultiplier tube gain is also changed. FIG. 15 is a scatter plot illustrating a result when the PMT setting is 40%, FIG. 16 is a scatter plot illustrating a result when the PMT setting is 55%, and FIG. 17 is a scatter plot illustrating a result when the PMT setting is 70%. A cut value is 30%, the spot median is plotted on the horizontal axis, and two values from expression (1) and expression (2) excluding calculation of the absolute value are plotted on the vertical axis.

[0085] For each of FIG. 15 to FIG. 17, a curved line of Y = ±10 ± Z/X (X is the spot median, and Z is the constant) was depicted. The offset value Z in three conditions for the PMT was obtained so that the curved line reaches the bottom of spread of the two values from expression (1) and expression (2). As illustrated in FIG. 15 to FIG. 17, the result shows that the offset value is 13 when the PMT setting is 40%, the offset value is 80 when the PMT setting is 55%, and the offset value is 230 when the PMT setting is 70%.

[0086] Subsequently, a correlation between the PMT value and the offset value was examined. As illustrated in FIG. 18, it was demonstrated that a relation therebetween is represented by a straight line in a logarithmic graph. A correlation coefficient was equal to or larger than 0.99, so that validity of the correction was demonstrated. The following describes expression (4) for correcting the offset value.

where Z is the offset value, and X is the gain voltage of the photomultiplier. In the second example, A was -7.13 and B was 5.16.

[0087] The description about the second example is finished.

[0088] As described above in detail, according to the present embodiment, it is possible to provide the determination method, the determination device, the determination system, and the computer program that can appropriately evaluate the reliability of the selective binding of an amount of the substance to be examined considering non-biological effect for data obtained from the microarray experiment and the like. Accordingly, the present invention is especially available for fields such as medical care, medicine manufacture, development of new drugs, biological research, clinical testing, and for a biotechnology field and the like.

Other embodiments



[0089] The embodiment of the present invention has been described above. Alternatively, the present invention may be implemented by various different embodiments within the scope of technical ideas described in the claims other than the embodiment described above.

[0090] Specifically, the embodiment of the present invention describes an example of using DNA as the selective binding substance. However, the embodiment is not limited thereto. A protein library such as an antibody, a compound library, and the like may be arranged as the selective binding substance. Material of the "carrier" is not limited to glass, and may be membranes or plastics.

[0091] In the above embodiment, a fluorescent chemical substance (for example, Cy3 and Cy5) is used as the label. However, the embodiment is not limited thereto. The label may be made by using a pigment without fluorescence property, a radioactive isotope, protein such as GFP and GRP, a His-tag, biotinylation, and the like.

[0092] In the above example, described is a case in which the determination device 100 performs processing on a stand-alone basis. Alternatively, the determination device 100 may be configured to perform processing in response to a request from a client terminal constituted with a housing separate from the determination device 100 and return the processing result thereof to the client terminal.

[0093] Among the pieces of processing described in the embodiment, the entire or part of the processing described to be automatically performed can be manually performed. Alternatively, the entire or part of the processing described to be manually performed can be automatically performed using a known method. In addition, it is possible to arbitrarily change the processing procedure, the control procedure, the specific name, the information including registration data and the like of each piece of processing, and the database configuration described in the above-described literatures and figures unless otherwise specifically noted.

[0094] Each component of the determination device 100 is only schematically illustrated in the figures, and does not need to be physically configured as illustrated in the figures.

[0095] For example, the entire or arbitrary part of processing functions included in each device of the determination device 100, specifically, each processing function executed by the control unit 102 may be implemented by a CPU and a computer program that is interpreted and executed by the CPU, and may be implemented as hardware by wired logic. The computer program is recorded in a recording medium described later, and mechanically read by the determination device 100 as needed. That is, the storage unit 106 and the like such as a read only memory (ROM) or a hard disk (HD) records therein a computer program for giving a command to the CPU to perform various pieces of processing cooperating as an OS. This computer program is executed by being loaded on a random access memory (RAM), and configures the control device in cooperation with the CPU.

[0096] The computer program may be stored in an application program server connected to the determination device 100 via an arbitrary network 300, and the entire or part thereof can be downloaded as needed.

[0097] The computer program according to the present invention can be stored in a computer-readable recording medium. Herein, the "recording medium" includes any "portable physical medium" such as a memory card, a USB memory, an secure digital (SD) card, a flexible disk, a magneto-optical disc, a ROM, an erasable programmable read only Memory (EPROM), an electrically erasable programmable read only Memory (EEPROM), a compact disc (CD)-ROM, an magneto optical (MO), a digital versatile disc (DVD), and Blu-ray (registered trademark) Disc.

[0098] The "computer program" is a data processing method described using an arbitrary language or description method, and the format thereof such as a source code or a binary code does not matter. The "computer program" is not limited to a singular configuration. The "computer program" may have a distributed configuration as a plurality of modules or libraries, or implement the function thereof in cooperation with a separate computer program represented by the OS. In each device described in the embodiment, a well-known configuration and procedure can be used as a specific configuration for reading the recording medium, a reading procedure, an installation procedure after reading, or the like.

[0099] Various databases and the like (the image data file 106a to the pixel group file 106b) stored in the storage unit 106 are storage means including a memory device such as a RAM and a ROM, a fixed disk device such as a hard disk, a flexible disk, and an optical disc, and store therein various computer programs, tables, databases, and Web page files used for various pieces of processing and for providing a Web site.

[0100] The determination device 100 may also be configured as an information processing device such as a known personal computer, workstation, and the like, or may be configured by connecting any peripheral device to the information processing device. The determination device 100 may be made by installing software (including a computer program, data, and the like) that performs the method of the present invention in the information processing device.

[0101] A specific form of distribution and integration of the devices is not limited to the form illustrated in the figures. The entire or part of the devices may be configured to be functionally or physically distributed/integrated in an arbitrary unit corresponding to various additions and the like, or corresponding to a functional load. That is, the embodiments described above may be arbitrarily combined to be performed, or the embodiments may be selectively performed. Reference Signs List

[0102] 
100
Determination device
102
Control unit
102a
Image data acquisition unit
102b
Pixel group extracting unit
102c
Median calculation unit
102d
Reliability determination unit
102e
Reference value determination unit
104
Communication control interface unit
106
Storage unit
106a
Image data file
106b
Pixel group file
108
Input/output control interface unit
112
Input device
114
Output device
200
External system
300
Network



Claims

1. A determination method for determining reliability of a selective binding of an amount of a substance to be examined, the substance to be examined being a sample directly or indirectly obtained from a cell or a tissue, obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, the carrier being one of a DNA chip or microarray, the determination method comprising:

a pixel group extracting step of determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot;

a median calculating step of calculating a ratio or a difference between a) a median value of the detection intensity of the pixel group extracted at the pixel group extracting step and b) a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and

a reliability determining step of determining the reliability based on the ratio or the difference calculated at the median calculating step and a certain reference value.


 
2. The determination method according to claim 1, wherein
a value of a ratio obtained from expression (1) and/or expression (2) is calculated at the median calculating step, the expressions (1) and (2) being as follows:



(where X is a median value of the detection intensity of the pixel group, Xt is a median value of the detection intensity of the pixel group excluding the certain top proportion of pixels, and Xb is a median value of the detection intensity of the pixel group excluding the certain bottom proportion of pixels), and
the reliability determining step determines that the reliability is defective when the value of the ratio calculated at the median calculating step is equal to or larger than the reference value.
 
3. The determination method according to claim 1 or 2, wherein
the reference value is a value obtained from expression (3) as follows:

(where S is the reference value, C is a constant, Z is an offset value corresponding to a sensitivity setting of a device for detecting the detection intensity of the label, and X is the median value of the detection intensity of the pixel group).
 
4. The determination method according to claim 3, wherein
the device for detecting the detection intensity of the label is a photomultiplier, and
the offset value is a value obtained from expression (4) as follows:

(where Z is the offset value, X is a gain voltage of the photomultiplier, and A and B are constants).
 
5. The determination method according to any one of claims 1 to 4, wherein
the carrier is a microarray,
the label is a fluorescent label,
the detection intensity is a fluorescence amount, and
the reliability determining step determines quality of the spot as the reliability.
 
6. A determination device 100 comprising at least a control unit 102 determining reliability of a selective binding of an amount of a substance to be examined, the substance to be examined being a sample directly or indirectly obtained from a cell or a tissue, obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, the carrier being one of a DNA chip or microarray, the control unit comprising:

pixel group extracting means 102b for determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot;

median calculating means 102c for calculating a ratio or a difference between a) a median value of the detection intensity of the pixel group extracted by the pixel group extracting means 102b and b) a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and

reliability determining means 102d for determining the reliability based on the ratio or the difference calculated by the median calculating means 102c and a certain reference value.


 
7. A determination system configured by connecting a detection device for reading detection intensity of a label, which is obtained when a labeled substance to be examined, the substance to be examined being a sample directly or indirectly obtained from a cell or a tissue, binds to a selective binding substance fixed as a spot on a carrier, the carrier being one of a DNA chip or microarray, to a determination device 100 comprising at least a control unit 102 for determining reliability of the selective binding of an amount of the substance to be examined obtained as the detection intensity, wherein
the control unit 102 of the determination device 100 comprises:

image data acquisition means 102a for acquiring, as imaged image data, the detection intensity in the carrier read via the detection device;

pixel group extracting means 102b for determining a position of the spot in the image data obtained by the image data acquisition means and extracting a pixel group corresponding to the spot;

median calculating means 102c for calculating a ratio or a difference between a) a median value of the detection intensity of the pixel group extracted by the pixel group extracting means 102b and b) a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and

reliability determining means 102d for determining the reliability based on the ratio or the difference calculated by the median calculating means and a certain reference value.


 
8. A computer program that causes a computer comprising at least a control unit to execute a method for determining reliability of a selective binding of an amount of a substance to be examined, the substance to be examined being a sample directly or indirectly obtained from a cell or a tissue, obtained as detection intensity of a label when a labeled substance to be examined binds to a selective binding substance fixed as a spot on a carrier, the carrier being one of a DNA chip or microarray, wherein the control unit is caused to execute the method comprising:

a pixel group extracting step of determining a position of the spot in image data obtained by imaging the detection intensity in the carrier and extracting a pixel group corresponding to the spot;

a median calculating step of calculating a ratio or a difference between a) a median value of the detection intensity of the pixel group extracted at the pixel group extracting step and b) a median value of the detection intensity of the pixel group excluding a certain top proportion of and/or a certain bottom proportion of pixels; and

a reliability determining step of determining the reliability based on the ratio or the difference calculated at the median calculating step and a certain reference value.


 


Ansprüche

1. Bestimmungsverfahren zum Bestimmen von der Verlässlichkeit einer selektiven Bindung von einer Menge einer zu untersuchenden Substanz, wobei die zu untersuchende Substanz eine Probe ist, die direkt oder indirekt aus einer Zelle oder einem Gewebe erhalten wird, welche als Detektions-intensität von einer Markierung erhalten wird, wenn eine markierte, zu untersuchende Substanz an eine selektive Bindungssubstanz, die als ein Spot auf einem Träger fixiert ist, bindet, wobei der Träger einer aus einem DNS-Chip oder einem Microarray ist, wobei das Verstimmungsverfahren das Folgende umfasst:

einen Pixelgruppenextrahierungsschritt zum Bestimmen von einer Position des Spots in Bilddaten, die durch Darstellen von der Detektionsintensität in dem Träger und durch Extrahieren von einer Pixelgruppe, die dem Spot entspricht, erhalten werden;

einen Medianberechnungsschritt zum Berechnen von einem Verhältnis oder einer Differenz zwischen a) einem Medianwert von der Detektionsintensität der extrahierten Pixelgruppe in dem Pixelgruppenextrahierungsschritt und b) einem Medianwert von der Detektionsintensität der Pixelgruppe, die einen bestimmten oberen Anteil und/oder einen bestimmten unteren Anteil der Pixel ausschließt; und

einen Verlässlichkeitsbestimmungsschritt zum Bestimmen von der Verlässlichkeit auf Basis von dem Verhältnis oder der Differenz, die in dem Medianberechnungsschritt berechnet wird, und einem bestimmten Bezugswert.


 
2. Bestimmungsverfahren nach Anspruch 1, wobei
ein Wert von einem Verhältnis, das aus dem Ausdruck (1) und/oder dem Ausdruck (2) erhalten wird, in dem Medianberechnungsschritt berechnet wird, wobei die Ausdrücke (1) und (2) wie folgt sind:



(wobei X ein Medianwert von der Detektionsintensität der Pixelgruppe ist, Xt ein Medianwert von der Detektionsintensität der Pixelgruppe ist, die den bestimmten oberen Anteil der Pixel ausschließt, und Xb ein Medianwert von der Detektionsintensität der Pixelgruppe ist, die den bestimmten unteren Anteil der Pixel ausschließt), und
wobei der Verlässlichkeitsbestimmungsschritt bestimmt, dass die Verlässlichkeit fehlerhaft ist, wenn der Wert des Verhältnisses, welches in dem Medianberechnungsschritt berechnet wird, gleich dem oder größer als der Bezugswert ist.
 
3. Bestimmungsverfahren nach Anspruch 1 oder 2, wobei der Bezugswert ein Wert ist, der aus dem folgenden Ausdruck (3) erhalten wird:

(wobei S der Bezugswert ist, C eine Konstante ist, Z ein Offset-Wert ist, der einer Sensitivitätseinstellung von einer Vorrichtung zum Detektieren von der Detektionsintensität der Markierung entspricht, und X der Medianwert von der Detektionsintensität der Pixelgruppe ist).
 
4. Bestimmungsverfahren nach Anspruch 3, wobei
die Vorrichtung zum Detektieren von der Detektionsintensität der Markierung ein Photovervielfacher ist, und
wobei der Offset-Wert ein Wert ist, der aus dem folgenden Ausdruck (4) erhalten wird:

(wobei Z der Offset-Wert ist, X eine Verstärkungsspannung von dem Photovervielfacher ist und A und B Konstanten sind).
 
5. Bestimmungsverfahren nach einem der Ansprüche 1 bis 4, wobei
der Träger ein Microarray ist,
die Markierung eine Fluoreszenzmarkierung ist,
die Detektionsintensität eine Fluoreszenzmenge ist, und
der Verlässlichkeitsbestimmungsschritt die Qualität von dem Spot als die Verlässlichkeit bestimmt.
 
6. Bestimmungsvorrichtung 100, umfassend mindestens eine Steuereinheit 102, die die Verlässlichkeit von einer selektiven Bindung von einer Menge einer zu untersuchenden Substanz, wobei die zu untersuchende Substanz eine Probe ist, die direkt oder indirekt aus einer Zelle oder einem Gewebe erhalten wird, bestimmt, welche als Detektionsintensität von einer Markierung erhalten wird, wenn eine markierte, zu untersuchende Substanz an eine selektive Bindungssubstanz, die als ein Spot auf einem Träger fixiert ist, bindet, wobei der Träger einer aus einem DNS-Chip oder einem Microarray ist, wobei die Steuereinheit das Folgende umfasst:

Pixelgruppenextrahierungsmittel 102b zum Bestimmen von einer Position des Spots in Bilddaten, die durch Darstellen von der Detektionsintensität in dem Träger und durch Extrahieren von einer Pixelgruppe, die dem Spot entspricht, erhalten werden;

Medianberechnungsmittel 102c zum Berechnen von einem Verhältnis oder einer Differenz zwischen a) einem Medianwert von der Detektionsintensität der extrahierten Pixelgruppe, die durch das Pixelgruppenextrahierungs-mittel 102b extrahiert wird, und b) einem Medianwert von der Detektionsintensität der Pixelgruppe, die einen bestimmten oberen Anteil und/oder einen bestimmten unteren Anteil der Pixel ausschließt; und

Verlässlichkeitsbestimmungsmittel 102d zum Bestimmen von der Verlässlichkeit auf Basis von dem Verhältnis oder der Differenz, die durch das Medianberechnungsmittel 102c berechnet wird, und einem bestimmten Bezugswert.


 
7. Bestimmungssystem, das konfiguriert ist, indem eine Detektionsvorrichtung zum Lesen einer Detektionsintensität von einer Markierung, die erhalten wird, wenn eine markierte, zu untersuchende Substanz an eine selektive Bindungssubstanz, die als ein Spot auf einem Träger fixiert ist, bindet, wobei die zu untersuchende Substanz eine Probe ist, die direkt oder indirekt aus einer Zelle oder einem Gewebe erhalten wird, und wobei der Träger einer aus einem DNS-Chip oder einem Microarray ist, mit einer Bestim-mungsvorrichtung (100), die mindestens eine Steuereinheit 102 umfasst, um die Verlässlichkeit der selektiven Bindung von einer Menge der zu untersuchenden Substanz zu bestimmen, die als die Detektionsintensität erhalten wird, verbunden ist, wobei die Steuereinheit 102 der Bestimmungsvorrichtung 100 das Folgende umfasst:

Bilddatenerfassungsmittel 102a zum Erfassen, als dargestellte Bilddaten, von der Detektionsintensität in dem Träger, die durch die Detektionsvorrichtung gelesen wird;

Pixelgruppenextrahierungsmittel 102b zum Bestimmen von einer Position des Spots in den Bilddaten, die durch das Bilddatenerfassungs-mittel und durch Extrahieren von einer Pixelgruppe, die dem Spot entspricht, erhalten werden;

Medianberechnungsmittel 102c zum Berechnen von einem Verhältnis oder einer Differenz zwischen a) einem Medianwert von der Detektionsintensität der extrahierten Pixelgruppe, die durch das Pixelgruppenextrahierungs-mittel 102b extrahiert wird, und b) einem Medianwert von der Detektionsintensität der Pixelgruppe, die einen bestimmten oberen Anteil und/oder einen bestimmten unteren Anteil der Pixel ausschließt; und

Verlässlichkeitsbestimmungsmittel 102d zum Bestimmen von der Verlässlichkeit auf Basis von dem Verhältnis oder der Differenz, die durch das Medianberechnungsmittel berechnet wird, und einem bestimmten Bezugswert.


 
8. Computerprogramm, das einen Computer veranlasst, der mindestens eine Steuereinheit umfasst, ein Verfahren zum Bestimmen von der Verlässlichkeit einer selektiven Bindung von einer Menge einer zu untersuchenden Substanz auszuführen, wobei die zu untersuchende Substanz eine Probe ist, die direkt oder indirekt aus einer Zelle oder einem Gewebe erhalten wird, welche als Detektionsintensität von einer Markierung erhalten wird, wenn eine markierte, zu untersuchende Substanz an eine selektive Bindungssubstanz, die als ein Spot auf einem Träger fixiert ist, bindet, wobei der Träger einer aus einem DNS-Chip oder einem Microarray ist, wobei die Steuereinheit veranlasst wird, das Verfahren auszuführen, welches das Folgende umfasst:

einen Pixelgruppenextrahierungsschritt zum Bestimmen von einer Position des Spots in Bilddaten, die durch Darstellen von der Detektionsintensität in dem Träger und durch Extrahieren von einer Pixelgruppe, die dem Spot entspricht, erhalten werden;

einen Medianberechnungsschritt zum Berechnen von einem Verhältnis oder einer Differenz zwischen a) einem Medianwert von der Detektionsintensität der extrahierten Pixelgruppe in dem Pixelgruppenextrahierungsschritt und b) einem Medianwert von der Detektionsintensität der Pixelgruppe, die einen bestimmten oberen Anteil und/oder einen bestimmten unteren Anteil der Pixel ausschließt; und

einen Verlässlichkeitsbestimmungsschritt zum Bestimmen von der Verlässlichkeit auf Basis von dem Verhältnis oder der Differenz, die in dem Medianberechnungsschritt berechnet wird, und einem bestimmten Bezugswert.


 


Revendications

1. Procédé de détermination pour déterminer la fiabilité d'une liaison sélective d'une quantité d'une substance à examiner, la substance à examiner étant un échantillon obtenu directement ou indirectement à partir d'une cellule ou d'un tissu, obtenue comme l'intensité de détection d'un marqueur lorsqu'une substance marquée à examiner se lie à une substance de liaison sélective fixée comme un point sur un support, le support étant l'un d'une puce ou d'un microréseau d'ADN, le procédé de détermination comprenant :

une étape d'extraction de groupe de pixels consistant à déterminer une position du point dans les données d'image obtenues en imageant l'intensité de détection dans le support et en extrayant un groupe de pixels correspondant au point ;

une étape de calcul de moyenne consistant à calculer un rapport ou une différence entre a) une valeur moyenne de l'intensité de détection du groupe de pixels extrait à l'étape d'extraction de groupe de pixels et b) une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion d'une certaine proportion supérieure et/ou d'une certaine proportion inférieure de pixels ; et

une étape de détermination de fiabilité consistant à déterminer la fiabilité sur la base du rapport ou de la différence calculée à l'étape de calcul de moyenne et d'une certaine valeur de référence.


 
2. Procédé de détermination selon la revendication 1, dans lequel
une valeur d'un rapport obtenu à partir de l'expression (1) et/ou de l'expression (2) est calculée à l'étape de calcul de moyenne, les expressions (1) et (2) étant les suivantes :



(où X est une valeur moyenne de l'intensité de détection du groupe de pixels, Xt est une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion de la certaine proportion supérieure de pixels, et Xb est une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion de la certaine proportion inférieure de pixels), et
l'étape de détermination de fiabilité détermine que la fiabilité est défectueuse lorsque la valeur du rapport calculée à l'étape de calcul de moyenne est supérieure ou égale à la valeur de référence.
 
3. Procédé de détermination selon la revendication 1 ou 2, dans lequel la valeur de référence est une valeur obtenue à partir de l'expression (3) suivante :

(où S est la valeur de référence, C est une constante, Z est une valeur de décalage correspondant à un réglage de sensibilité d'un dispositif pour détecter l'intensité de détection du marqueur, et X est la valeur moyenne de l'intensité de détection du groupe de pixels).
 
4. Procédé de détermination selon la revendication 3, dans lequel
le dispositif pour détecter l'intensité de détection du marqueur est un photomultiplicateur, et
la valeur de décalage est une valeur obtenue à partir de l'expression (4) comme suit :

(où Z est la valeur de décalage, X est une tension de gain du photomultiplicateur, et A et B sont des constantes).
 
5. Procédé de détermination selon l'une quelconque des revendications 1 à 4, dans lequel
le support est un microréseau,
le marqueur est un marqueur fluorescent,
l'intensité de détection est une quantité de fluorescence, et
l'étape de détermination de fiabilité détermine la qualité du point comme la fiabilité.
 
6. Dispositif de détermination 100 comprenant au moins une unité de commande 102 déterminant la fiabilité d'une liaison sélective d'une quantité d'une substance à examiner, la substance à examiner étant un échantillon obtenu directement ou indirectement à partir d'une cellule ou d'un tissu, obtenue comme l'intensité de détection d'un marqueur lorsqu'une substance marquée à examiner se lie à une substance de liaison sélective fixée comme un point sur un support, le support étant l'un d'une puce ou d'un microréseau d'ADN, l'unité de commande comprenant :

des moyens d'extraction de groupe de pixels 102b pour déterminer une position du point dans les données d'image obtenu en imageant l'intensité de détection dans le support et en extrayant un groupe de pixels correspondant au point ;

des moyens de calcul de moyenne 102c pour calculer un rapport ou une différence entre a) une valeur moyenne de l'intensité de détection du groupe de pixels extrait par les moyens d'extraction de groupe de pixels 102b et b) une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion d'une certaine proportion supérieure et/ou d'une certaine proportion inférieure de pixels ; et

des moyens de détermination de fiabilité 102d pour déterminer la fiabilité sur la base du rapport ou de la différence calculée à l'étape de calcul de moyenne 102c et d'une certaine valeur de référence.


 
7. Système de détermination configuré en connectant un dispositif de détection pour lire l'intensité de détection d'un marqueur, qui est obtenue lorsqu'une substance à examiner, la substance à examiner étant un échantillon obtenu directement ou indirectement à partir d'une cellule ou d'un tissu, se lie à une substance de liaison sélective fixée comme un point sur un support, le support étant l'un d'une puce ou d'un microréseau d'ADN, à un dispositif de détermination 100 comprenant au moins une unité de commande 102 pour déterminer la fiabilité de la liaison sélective d'une quantité de la substance à examiner obtenue comme l'intensité de détection, dans lequel
l'unité de commande 102 du dispositif de détermination 100 comprend :

des moyens d'acquisition de données d'image 102a pour acquérir, comme données d'image imagées, l'intensité de détection dans le support lu via le dispositif de détection ;

des moyens d'extraction de groupe de pixels 102b pour déterminer une position du point dans les données d'image obtenues par les moyens d'acquisition de données d'image et extraire un groupe de pixels correspondant au point ;

des moyens de calcul de moyenne 102c pour calculer un rapport ou une différence entre a) une valeur moyenne de l'intensité de détection du groupe de pixels extrait par les moyens d'extraction de groupe de pixels 102b et b) une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion d'une certaine proportion supérieure et/ou d'une certaine proportion inférieure de pixels ; et

des moyens de détermination de fiabilité 102d pour déterminer la fiabilité sur la base du rapport ou de la différence calculée à l'étape de calcul de moyenne et d'une certaine valeur de référence.


 
8. Programme informatique qui amène un ordinateur comprenant au moins une unité de commande à exécuter un procédé pour déterminer la fiabilité d'une liaison sélective d'une quantité d'une substance à examiner, la substance à examiner étant un échantillon obtenu directement ou indirectement à partir d'une cellule ou d'un tissu, obtenue comme l'intensité de détection d'un marqueur lorsqu'une substance marquée à examiner se lie à une substance de liaison sélective déterminée comme un point sur un support, le support étant l'un d'une puce ou d'un microréseau d'ADN, dans lequel l'unité de commande est amenée à exécuter le procédé comprenant :

une étape d'extraction de groupe de pixels consistant à déterminer une position du point dans les données d'image obtenu en imageant l'intensité de détection dans le support et en extrayant un groupe de pixels correspondant au point ;

une étape de calcul de moyenne consistant à calculer un rapport ou une différence entre a) une valeur moyenne de l'intensité de détection du groupe de pixels extrait à l'étape d'extraction de groupe de pixels et b) une valeur moyenne de l'intensité de détection du groupe de pixels à l'exclusion d'une certaine proportion supérieure et/ou d'une certaine proportion inférieure de pixels ; et

une étape de détermination de fiabilité consistant à déterminer la fiabilité sur la base du rapport ou de la différence calculée à l'étape de calcul de moyenne et d'une certaine valeur de référence.


 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description