JP7129732B2 - Serum sample testing device and serum sample testing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a serum sample testing device and a method for testing a serum sample. It also relates to a method for obtaining a Raman spectrum of a serum sample and a cell for Raman spectroscopic analysis of a serum sample.
This application claims priority based on Japanese Patent Application No. 2019-170596 filed in Japan on September 19, 2019, the content of which is incorporated herein.

Cancer is an important disease that accounts for most of the causes of death in the world. Early detection of cancer and early initiation of appropriate treatment improves treatment results. Currently, most cancer diagnoses are definitively diagnosed by histopathological diagnosis by microscopic observation of tissue specimens after image diagnosis by ultrasonography, CT, MRI, or the like.

A blood test is one of the most basic medical tests, and is widely used because it is useful and minimally invasive. Methods for diagnosing cancer by blood tests include methods for measuring tumor markers (for example, Patent Document 1).

WO2018/047793

Tumor marker tests are widely used for cancer screening because they are minimally invasive and simple. However, tumor marker tests often result in false positives or false negatives, and it cannot be said that appropriate screening can be performed.
Therefore, development of a new cancer test method using a blood sample is required.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a serum sample testing apparatus and a serum sample testing method capable of simply and quickly determining the presence or absence of cancer in a subject using a blood sample. Another object of the present invention is to provide a method for obtaining a Raman spectrum of a serum sample and a cell for Raman spectroscopic analysis of a serum sample, which can be applied to the apparatus and method for testing a serum sample.

The present invention includes the following aspects.
[1] With regard to the Raman spectrum acquisition unit that acquires the Raman spectrum of the serum sample of the subject, and the first peak, the second peak, and the third peak that are set according to the cancer type to be examined, An intensity acquisition unit that acquires the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3) from the Raman spectrum, and the first peak The intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity (P1) of the and the intensity ratio of the intensity of the third peak (P3) to the intensity of the first peak (P1) an intensity ratio calculator that calculates (P3/P1); a determination unit that determines the presence or absence of cancer in the subject based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1); A serum sample test device comprising:
[2] The serum sample test apparatus according to [1], wherein the Raman spectrum acquisition unit acquires the Raman spectrum of the serum sample while the serum sample forms a convex curved surface.
[3] A step of obtaining a Raman spectrum of a serum sample of a subject; , obtaining the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3), and the intensity of the first peak (P1) The intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity of the first peak (P1), and the intensity ratio (P3/P1) of the intensity of the third peak (P3) to the intensity of the first peak (P1) and determining the presence or absence of cancer in the subject based on the intensity ratio (P2/P2) and the intensity ratio (P3/P1).
[4] The method for testing a serum sample according to [3], wherein the Raman spectrum of the serum sample is obtained while the serum sample forms a convex curved surface.
[5] A method for acquiring a Raman spectrum of a serum sample, comprising the steps of forming a convex curved surface with the serum sample, and acquiring a Raman spectrum of the serum sample while maintaining the convex curved surface.
[6] Raman spectroscopic analysis of a serum sample, comprising: a convex curved surface forming part for forming a convex curved surface on a serum sample; and a supply channel connected to the convex curved surface forming part and supplying the serum sample to the convex curved surface forming part. cell for.

ADVANTAGE OF THE INVENTION According to this invention, the serum sample test|inspection apparatus which can determine the presence or absence of cancer in a test object simply and rapidly with a blood sample, and the test|inspection method of a serum sample are provided. Also provided are a method for acquiring a Raman spectrum of a serum sample and a cell for Raman spectroscopic analysis of a serum sample, which are applicable to the serum sample testing device and testing method.

1 is a schematic diagram showing an example of the configuration of a serum sample testing device; FIG. FIG. 2 is a schematic diagram showing an example of the configuration of a processing device included in the serum sample testing device; 1 shows an example of a Raman spectrum acquired by a Raman spectrum acquisition unit; 3B shows the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3) in the Raman spectrum of FIG. 3A. An example of a Raman shift table is shown. An example of a peak information table is shown. An example of a threshold information table is shown. FIG. 4 is a flow diagram showing an example of the operation of the serum sample test device; FIG. 4 is a flowchart showing an example of an operation for setting first to third Raman peaks and thresholds corresponding to cancer types; It is a side view which shows an example of the cell for Raman spectroscopy. FIG. 8 is a vertical cross-sectional view of the Raman spectroscopic analysis cell shown in FIG. 7, including a part of the convex surface forming portion and the serum sample supply path; FIG. 8A is a longitudinal cross-sectional view showing a state in which the serum sample forms a convex curved surface at the convex curved surface forming portion. FIG. 10 is a photograph showing a state in which a convex curved surface is formed with a serum sample; FIG. FIG. 4 is a diagram showing an example of Raman spectra of serum samples; 1064 nm laser, output 200 mW, objective lens magnification 20 times. An example of an XY graph (Raman plot) with the intensity ratio (P3/P1) on the X axis and the intensity ratio (P2/P1) on the Y axis is shown. Phenylalanine C—C bending vibration (twist), Amide I, and CH ₃ bending vibration or CH bending motion were set as the first to third peaks corresponding to gastric cancer, respectively. An example of a Raman plot is shown. Cholesterol, Amide I, and phenylalanine benzene ring respiratory vibration were set as the first to third peaks corresponding to colorectal cancer, respectively. The metal plate used in Reference Example 1 is shown.

<Definition, etc.>
A "subject" means an animal whose serum sample is used to determine the presence or absence of cancer. Subjects include humans or non-human mammals. Mammals other than humans include primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, hamsters, rats, etc.), rabbits, dogs, cats, cows, goats, sheep, horses, and the like. The subject is preferably human.

A "serum sample" means a sample from which blood cell components have been removed from blood. A serum sample can be prepared from blood by a known method such as centrifugation. A "subject serum sample" means a serum sample prepared using blood drawn from a subject. In this specification, a subject from whom blood used for preparation of a serum sample is collected may be referred to as a "subject from which the serum sample is derived."

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and overlapping descriptions are omitted. The dimensional ratios in each drawing are exaggerated for explanation and do not necessarily match the actual dimensional ratios.

<Serum sample testing device>
In one embodiment, the present invention includes a Raman spectrum acquisition unit that acquires a Raman spectrum of a serum sample of a subject, a first peak, a second peak, and a and an intensity acquisition unit that acquires the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3) from the Raman spectrum for 3 peaks. , the intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity of the first peak (P1), and the intensity of the third peak to the intensity of the first peak (P1) an intensity ratio calculation unit that calculates an intensity ratio (P3/P1) of (P3), and the presence or absence of cancer in the subject based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1) and a determination unit for determining.

FIG. 1 is a schematic diagram showing an example of the configuration of the serum sample testing apparatus of this embodiment. The serum sample test device 100 comprises a Raman spectrum acquisition section 110 and a processing device 190 .

The Raman spectrum acquisition unit 110 acquires the Raman spectrum of the serum sample of the subject. The Raman spectrum acquisition unit 110 includes a stage 120, a light source device 130, a dichroic filter 140, a beam splitter 141, a long-pass filter 142, lenses 143 and 144, an objective lens 150, a spectroscope 160, a cooled CCD camera 170, and an observation CCD camera 171. It has The Raman spectrum acquisition unit 110 is, for example, a microscopic Raman spectrometer such as a Raman microscope.

A Raman spectroscopic analysis cell 1 holding a serum sample 50 is mounted on the stage 120 . Stage 120 is coupled to stage scanner 121 . The stage scanner 121 drives the stage 120 parallel and perpendicular to the plane on which the Raman spectroscopic analysis cell 1 is placed, as indicated by arrows xyz in the drawing. This allows the focus of the excitation light to be adjusted to a desired position on the serum sample 50 . A serum sample 50 is held in the Raman spectroscopic analysis cell 1 while forming a convex curved surface.

The light source device 130 generates excitation light with which the serum sample is irradiated. The light source device 130 is preferably a laser light source that generates laser light with a wavelength of 785 nm or 1064 nm, and more preferably a laser light source that generates laser light with a wavelength of 1064 nm. A clearer Raman spectrum can be obtained by using a laser light source with the above wavelength. The light source device 130 may include two or more types of laser light sources (eg, a 785 nm laser light source and a 1064 nm laser light source), and these laser light sources may be switchable. A maximum output of the laser light source is 50 to 500 mW. Specific examples of the laser light source include a laser light source capable of generating a laser light with a wavelength of 1064 nm and a maximum output of 200 mW, or a laser light source capable of generating a laser light with a wavelength of 785 nm and a maximum output of 50 mW. etc.

The dichroic filter 140 reflects excitation light from the light source device 130 and guides the excitation light to the beam splitter 141 . Dichroic filter 140 also transmits Raman scattered light from serum sample 50 and guides the Raman scattered light to long-pass filter 142 .

The beam splitter 141 transmits the excitation light from the dichroic filter 140 . Beam splitter 141 also splits the Raman scattered light from serum sample 50 and guides it to dichroic filter 140 , lens 143 and CCD camera 171 for observation.

Objective lens 150 focuses the excitation light transmitted through beam splitter 141 onto serum sample 50 . The objective lens 150 is preferably a lens that enables observation in the near-infrared region corresponding to the excitation light from the light source device 130 . The magnification of the objective lens 150 is preferably 10 to 50 times, more preferably 10 to 30 times, still more preferably 10 times or 20 times, and particularly preferably 20 times. The numerical aperture (NA) of the objective lens 150 is preferably 0.2 or more, more preferably 0.2 to 0.65, and further preferably 0.3 to 0.6. Preferably, it is particularly preferably 0.4 to 0.5. A clearer Raman spectrum can be obtained by setting the magnification and NA of the objective lens within the above ranges.

The long-pass filter 142 cuts the short wavelength region of the Raman scattered light from the serum sample 50 and transmits the long wavelength region. The lens 144 collects the Raman scattered light that has passed through the long-pass filter 142 and directs it to the spectroscope 160 .

A spectroscope 160 disperses the Raman scattered light. The cooled CCD camera 170 detects the light of each wavelength split by the spectroscope 160 . Thereby, a Raman spectrum of the serum sample 50 is obtained.

Observation CCD camera 171 detects Raman scattered light from serum sample 50 split by beam splitter 141 and guided to lens 143 . The observation CCD camera 171 is used to focus the excitation light on the desired position of the serum sample 50 . The excitation light is preferably focused on the surface of the convex surface of the serum sample 50, and more preferably on the vertex of the convex surface (point a in FIG. 8B).

The configuration of the Raman spectrum acquisition section is not limited to the above, and a configuration commonly used in microscopic Raman spectroscopic devices can be appropriately adopted.

FIG. 2 is a schematic diagram showing an example of the configuration of the processing device 190. As shown in FIG. The processing device 190 includes an intensity acquisition unit 191 , an intensity ratio calculation unit 192 , a determination unit 193 , an output unit 194 , a storage unit 195 and a control unit 196 . The processing unit 190 is, for example, a computer having a processor such as a central processing unit (CPU).

The intensity acquisition unit 191 acquires the first peak intensity (P1), the second peak intensity ( P2), and the intensity of the third peak (P3), respectively.

The first to third peaks are manually or automatically set according to the type of cancer to be examined.

Here, a case where the set cancer type is stomach cancer will be described as an example. FIG. 3A is an example of a serum Raman spectrum acquired by the Raman spectrum acquisition unit 110. FIG. FIG. 4A is an example of a Raman shift table that stores correspondence information between known Raman peaks and Raman shifts. The first to third peaks set corresponding to gastric cancer are respectively (1) phenylalanine C—C bending vibration (twist), (15) Amide I, and (13) CH ₃ bending vibration or C—H In the case of bending motion (numbers in parentheses indicate numbers in the Raman shift table of FIG. 4A), these peaks are identified from the Raman shift table of FIG. 4A as shown in FIG. 3B. Next, obtain the first peak intensity (P1), the second peak intensity (P2), and the third peak intensity (P3) by reading the height of each peak from the serum Raman spectrum. be able to.

When there are multiple cancer types to be detected, the intensity acquisition unit 191 first identifies each peak of the serum Raman spectrum and acquires the intensity of each peak, and then obtains the intensity (P1 ) to (P3) may be obtained for each cancer type.

The intensity ratio calculator 192 calculates the intensity ratio (P2/P1) and the intensity ratio (P3/P1) from the intensities (P1) to (P3) acquired by the intensity acquirer 191 . The intensity ratio (P2/P1) is the intensity ratio of the intensity of the second peak (P2) to the intensity of the first peak (P1) (intensity of the second peak (P2)/intensity of the first peak (P1 )). The intensity ratio (P3/P1) is the intensity ratio of the intensity of the third peak (P3) to the intensity of the first peak (P1) (intensity of the third peak (P3)/intensity of the first peak (P1 )).

Based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1) acquired by the intensity ratio calculation unit 192, the determination unit 193 determines the presence or absence of cancer in the subject from which the serum sample 50 is derived. The lower threshold (hereinafter referred to as "lower threshold (P2/P1)") and upper threshold (hereinafter referred to as "upper threshold (P2/P1)") of the normal range of the intensity ratio (P2/P1), and the intensity ratio (P3 /P1) of the normal range (hereinafter referred to as “lower threshold (P3/P1)”) and upper threshold (hereinafter referred to as “upper threshold (P3/P1)”) are manually or automatically. The determination unit 193 calculates the intensity ratio (P2/P1) and the intensity ratio (P3/P1) obtained by the intensity ratio calculation unit 192 as the lower threshold (P2/P1), the upper threshold (P2/P1), and the lower threshold (P3/P1) and the upper threshold (P3/P1) respectively. Based on the comparison, the determining unit 193 determines that (i) the intensity ratio (P2/P1) is equal to or lower than the lower threshold (P2/P1) and the intensity ratio (P3/P1) is equal to or lower than the lower threshold (P3/P1). or (ii) when the intensity ratio (P2/P1) is greater than or equal to the upper threshold (P2/P1) and the intensity ratio (P3/P1) is greater than or equal to the upper threshold (P3/P1), A specimen is determined to have cancer.

For example, if the test subject is gastric cancer, the first to third peaks are (1) phenylalanine C—C bending vibration (twist), (15) Amide I, and (13) CH ₃ bending vibration or C- H bending motion can be set, and the lower threshold (P2/P1) and upper threshold (P2/P1), and the lower threshold (P3/P1) and upper threshold (P3/P1) are 2.2 to 2.2. 7 (preferably 2.2 to 2.5) and 2.7 to 3.2 (preferably 2.7 to 3.0), and 2.2 to 2.7 (preferably 2.3 to 2.6 ) and 2.7 to 3.2 (preferably 2.8 to 3.1). Further, for example, the test object is colon cancer, the first to third peaks can be set as (2) cholesterol, (15) Amide I, and (5) phenylalanine benzene ring respiratory vibration, respectively, and the lower threshold (P2/P1 ) and upper threshold (P2/P1), and lower threshold (P3/P1) and upper threshold (P3/P1) are 1.6 to 2.4 (preferably 1.7 to 2.0) and 2 .4 to 3.0 (preferably 2.4 to 2.7), and 2.2 to 2.7 (preferably 2.2 to 2.5) and 2.5 to 3.0 (preferably 2.5); 6 to 2.9).

The output unit 194 outputs the determination result of the determination unit 193 to a display device or the like.

The storage unit 195 stores various information necessary for the operation of the serum sample test device 100 . Information stored in the storage unit 195 includes, for example, a Raman shift table (see FIG. 4A), a peak information table (see FIG. 4B) including correspondence information between cancer types and first to third peaks, and an intensity ratio (see FIG. 4B). P2/P1) and the intensity ratio (P3/P1), a threshold information table (see FIG. 4C) for setting lower and upper thresholds for each cancer type. The storage unit 195 is, for example, a storage device such as a hard disk drive or memory.

The control unit 196 controls the overall operation of the serum sample test device 100 and the operation of each unit.

<<Execution operation of serum sample test processing>>
Next, an example of the operation of the serum sample test processing in the serum sample test device 100 will be described with reference to FIG.

(Step S10)
A Raman spectrum acquisition unit 110 acquires the Raman spectrum of the serum sample 50 . When obtaining the Raman spectrum, the serum sample 50 is held in the Raman spectroscopic analysis cell 1 and placed on the stage 120 while forming a convex curved surface. Excitation light (eg, laser light with a wavelength of 1064 nm) generated from light source device 130 is reflected by dichroic filter 140 , transmitted through beam splitter 141 , condensed by objective lens 150 , and applied to serum sample 50 . At this time, it is preferable to focus the excitation light on the surface (preferably, the vertex) of the convex surface formed by the serum sample 50 . Raman scattered light generated from the serum sample 50 by excitation light irradiation passes through the objective lens 150, the beam splitter 141, and the dichroic filter 140, the short wavelength region is cut by the long-pass filter 142, and the light is collected by the lens 144. Then, the light is split by the spectroscope 160 . The separated light of each wavelength is detected by the cooled CCD camera 170 to obtain the Raman spectrum (serum Raman spectrum) of the serum sample 50 (see FIG. 3A).

The light source device 130 preferably generates excitation light with an output of 50 to 500 mW, more preferably 100 to 300 mW, even more preferably 150 to 250 mW. 200 mW is particularly preferred. When the output is within the above range, it is possible to obtain a clearer Raman spectrum while suppressing thermal denaturation of components in the serum sample. The excitation light irradiation time is preferably 10 to 30 seconds, more preferably 10 to 20 seconds, and even more preferably 15 seconds. When the irradiation time is within the above range, it is possible to obtain a clearer Raman spectrum while suppressing thermal denaturation of components in the serum sample.

(Step S20)
A cancer type to be detected is set. The cancer type may be manually set by the operator via an input device connected to the processing device 190 . Alternatively, the cancer type may be automatically set from the cancer types stored in the peak information table.

(Step S30)
The intensity acquisition unit 191 obtains the first peak intensity (P1), the second peak intensity (P2), and the second peak intensity (P2) from the serum Raman spectrum for the first peak, the second peak, and the third peak. 3 peak intensities (P3) are obtained respectively. The first peak, second peak, and third peak are set according to the cancer type set in step S20. The first to third peaks corresponding to cancer types may be manually set by an operator via an input device, or may be automatically set from information in a peak information table. The intensity acquisition unit 191 refers to the Raman shift table to identify each peak in the serum Raman spectrum (see FIG. 3B), and obtains the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the second peak (P2). 3 peak intensity (P3) is obtained.

(Step S40)
The intensity ratio calculator 192 calculates the intensity ratio (P2/P1) and the intensity ratio ( P3/P1) is calculated. Specifically, the intensity ratio (P2/P1) is calculated from the intensity of the first peak (P1) and the intensity of the second peak (P2), and the intensity of the first peak (P1) and the third peak The intensity ratio (P3/P1) is calculated from the intensity (P3) of .

(Step S50)
The determination unit 193 determines the presence or absence of cancer in the subject from which the serum sample 50 is derived based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1). The determination unit 193 refers to the threshold information table, and determines the lower threshold (P2/P1), the upper threshold (P2/P1), the lower threshold (P3/P1), and the upper threshold (P3/P1) corresponding to the cancer type set in step S20. Get the threshold (P3/P1). Alternatively, the lower threshold (P2/P1) and upper threshold (P2/P1), and the lower threshold (P3/P1) and upper threshold (P3/P1) are manually input by the operator according to the cancer type. may Next, the determination unit 193 compares the intensity ratio (P2/P1) with the lower threshold (P2/P1) and the upper threshold (P2/P1). Also, the intensity ratio (P3/P1) is compared with a lower threshold (P3/P1) and an upper threshold (P3/P1). The determination unit 193 determines that (i) the intensity ratio (P2/P1) is equal to or less than the lower threshold (P2/P1) and the intensity ratio (P3/P1) is equal to or less than the lower threshold (P3/P1) from the comparison result. or (ii) the intensity ratio (P2/P1) is greater than or equal to the upper threshold (P2/P1) and the intensity ratio (P3/P1) is greater than or equal to the upper threshold (P3/P1), then the serum sample Subjects from whom 50 are derived are determined to have cancer.

(Step S60)
The output unit 194 outputs the result of determination in step S50 to the display device, and the operation of the serum sample test device 100 ends.

If there are multiple cancer types to be detected, steps S20 to S60 may be repeated for each cancer type. Thereby, the presence or absence of cancer in the subject can be examined for each cancer type to be detected.

<<Method for selecting the first to third peaks>>
Next, a method for selecting the first to third peaks corresponding to cancer types will be described with reference to FIG.

(Step S100)
First, a target cancer type is set, and a group of subjects known to have the cancer type (hereinafter referred to as "cancer subject group") and a group of subjects known not to have the cancer type Blood is collected from a group of subjects (hereinafter referred to as "non-cancer subject group") to prepare serum samples. The target cancer type can be arbitrarily selected according to the subject group. Cancer types include, for example, stomach cancer, colon cancer, liver cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, lung cancer, pancreatic cancer, head and neck cancer, brain tumor, Examples include, but are not limited to, malignant mesothelioma and sarcoma. The target cancer type may be selected from cancer types for which the first to third peaks have not yet been set.

(Steps S110, S120)
A Raman spectrum (hereinafter referred to as a "non-cancer Raman spectrum group") of a serum sample of a non-cancer subject group (hereinafter referred to as a "non-cancer serum group") is obtained. Also, a Raman spectrum (hereinafter referred to as a "cancer Raman spectrum group") of serum samples of a cancer subject group (hereinafter referred to as a "cancer serum group") is acquired. Acquisition of the Raman spectrum can be performed in the same manner as in step S10.

(Step S130)
Select a combination of 1st to 3rd peaks to be examined (hereinafter referred to as "provisional 1st to 3rd peaks") from the peaks commonly present in the non-cancer Raman spectrum group and the cancer Raman spectrum group. do. The provisional first to third peak combinations are randomly selected as an example.

(Step S140)
For the tentative first to third peaks selected in step S130, from the non-cancer Raman spectrum group and the cancer Raman spectrum group, the first peak intensity (P1), the second peak intensity (P2), and The intensity of the third peak (P3) is obtained respectively.

(Step S150)
From the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3) obtained in step S140, the intensity ratio (P2/P1) and Calculate the intensity ratio (P3/P1).

(Step S160)
It is determined whether there is a significant difference between the non-cancer Raman spectrum group and the cancer spectrum group with respect to the intensity ratio (P2/P1) and the intensity ratio (P3/P1) calculated in step S150. Specifically, there is a significant difference between the intensity ratio (P2/P1) group obtained from the non-cancer Raman spectrum group and the intensity ratio (P2/P1) group obtained from the cancer Raman spectrum group. determine whether Also, it is determined whether there is a significant difference between the intensity ratio (P3/P1) group obtained from the non-cancer Raman spectrum group and the intensity ratio (P3/P1) group obtained from the cancer Raman spectrum group. do. A known significance test method can be used to determine the significance. A significant difference test method includes, for example, a t-test.

(Step S170)
It is checked whether there is another combination of the temporary first to third peak combinations, and if there is another combination (step S170: YES), the process returns to step S130. If there is no other combination (step S170: NO), the process proceeds to step S180.

(Step S180)
In step S160, it is determined that there is a significant difference between the non-cancer Raman spectrum group and the cancer Raman spectrum group for both the intensity ratio (P2/P1) and each intensity ratio (P3/P1). A combination of the first to third peaks to be set for the serum test of the target cancer type is selected from the combination group of the first to third peaks. For example, from the group of temporary first to third peak combinations determined to have a significant difference, a combination that can set a lower threshold value and an upper threshold value that both sensitivity and specificity are good, the first to third can be selected as a peak.

Selection of the first to third peaks is performed using an XY graph (hereinafter also referred to as "Raman plot") with the intensity ratio (P2/P1) as the Y axis and the intensity ratio (P3/P1) as the X axis. may For example, a Raman plot is created for each combination of tentative first to third peaks. A Raman plot is selected from the Raman plots in which the existing regions of the plots of the cancer Raman spectrum group and the existing regions of the plots of the non-cancer Raman spectrum group are least overlapped. The tentative 1st to 3rd peaks used in the Raman plot are selected as the 1st to 3rd peaks.

Also, for the selected first to third peaks, so that both sensitivity and specificity are good, the lower and upper thresholds of the intensity ratio (P2/P1), and the lower and lower thresholds of the intensity ratio (P3/P1) Set the upper threshold.

Selection of the first to third peaks corresponding to cancer types may be performed by the serum sample test device 100 . In this case, the serum sample test apparatus 100 can have a configuration for executing each process of steps S100 to S180 in addition to the above-described units. For example, a temporary peak selection unit that selects a combination of the first to third temporary peaks, and acquires the intensity (P1), the intensity (P2), and the intensity (P3) for the temporary first to third peaks from each Raman spectrum. a second intensity acquisition unit that calculates the intensity ratio (P2/P1) and the second intensity calculation unit that calculates the intensity ratio (P2/P1); A significant difference determination unit that determines a significant difference between a cancer group and a cancer group, and a peak selection unit that selects the first to third peaks and threshold values may be provided.

The first to third peaks selected in step S180 may be stored in the peak information table (see FIG. 4B) of the storage unit 195 together with information on the target cancer type. Further, the lower and upper thresholds of the intensity ratio (P2/P1) and the lower and upper thresholds of the intensity ratio (P3/P1) selected in step S180 are stored in the threshold information table of the storage unit 195 (see FIG. 4C). ) may be stored in These pieces of information newly stored in the storage unit 195 can be used when the serum sample test device 100 executes the serum test processing.

According to the serum sample testing apparatus described above, the presence or absence of cancer in a subject can be quickly determined by a simple method of measuring Raman scattered light of a serum sample of the subject.

The reason why the presence or absence of cancer in a subject can be determined from the Raman spectrum of a serum sample is that, in a subject with cancer, changes occur in the metabolic balance, and cancer-specific changes occur in the component composition in the blood. is presumed to be Since the mode of change in metabolic balance differs depending on the cancer type, it is presumed that the first to third peaks should be set according to the cancer type.

<Test method for serum sample>
In one embodiment of the present invention, the serum sample testing device comprises a step of obtaining a Raman spectrum of a serum sample of a subject, a first peak set according to the type of cancer to be tested, a second peak Obtaining the intensity of the first peak (P1), the intensity of the second peak (P2), and the intensity of the third peak (P3) from the Raman spectrum for a peak and a third peak and the intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity of the first peak (P1), and the third peak to the intensity of the first peak (P1). and determining the presence or absence of cancer in the subject based on the intensity ratio (P2/P2) and the intensity ratio (P3/P1). and a method for testing a serum sample.

The serum sample testing method of the present embodiment can be performed by the serum sample testing device. Alternatively, the processing of steps S10 to S50 may be performed without using the serum sample test device. In this case, a commercially available micro-Raman spectrometer can be used to obtain the Raman spectrum of the serum sample. The microscopic Raman spectroscopy device preferably includes a light source device and an objective lens similar to those of the serum sample test device 100 .

<Method for Acquisition of Raman Spectrum of Serum Sample>
In one embodiment, the present invention comprises a step of forming a convex curved surface with a serum sample (convex curved surface forming step), and a step of acquiring a Raman spectrum of the serum sample while maintaining the convex curved surface (Raman spectrum acquisition step), and a method for obtaining a Raman spectrum of a serum sample.

Serum samples contain a large number of components, making it difficult to obtain clear Raman spectra. However, by using the method of this embodiment, a clear Raman spectrum of a serum sample can be obtained.

Each step of the method of this embodiment will be described below.

(Convex curved surface forming step)
A convex curve is formed with the serum sample. A convex curved surface can be formed, for example, by a droplet of a serum sample. A cell for Raman spectroscopic analysis, a syringe, and the like, which will be described later, can be used to form the convex curved surface. When a syringe is used, the serum sample is drawn with the syringe and then the plunger is returned to form a droplet of the serum sample at the tip of the needle. Since the droplet is in contact with the serum sample in the syringe, the droplet maintains its convex curved surface without drying in a short period of time.

Although the size of the convex curved surface formed by the serum sample is not particularly limited, it can have a radius of curvature of 1000 to 5000 μm, for example. A droplet forming a convex curve can be formed with, for example, 5-30 μL of serum sample. The amount of serum sample forming droplets is preferably 5 to 20 μL, more preferably 10 to 15 μL, even more preferably 10 μL.

(Raman spectrum acquisition step)
A Raman spectrum of the serum sample is acquired while the convex curved surface is maintained. By constantly supplying the serum sample to the droplet of the serum sample that forms the convex curved surface, the convex curved surface can be maintained while the Raman spectrum is being acquired.

Acquisition of Raman spectra can be performed by the aforementioned serum sample testing device or other micro-Raman spectroscopy devices. As for the excitation light irradiation conditions, the same conditions as those mentioned for the serum sample test device 100 can be used. The excitation light is focused on the droplet forming a convex curve. The excitation light is preferably focused on the surface of the convex surface, more preferably on the vertex of the convex surface. By focusing the excitation light on the vertex of the convex curved surface, stable Raman spectra can be acquired under similar conditions among a plurality of serum samples.

According to the Raman spectrum acquisition method of the present embodiment, a clear Raman spectrum can be acquired in a serum sample. Further, by obtaining the Raman spectrum while maintaining the convex curved surface, drying of the serum sample is suppressed, thereby suppressing denaturation of components in the serum sample. This makes it possible to detect changes in serum components in cancer-bearing subjects, and to perform cancer tests using serum samples.

The reason why a clear Raman spectrum can be obtained by maintaining the convex curved surface is not clear. , the excitation light is converged by the lens effect of the convex curved surface.

The method for obtaining a Raman spectrum according to this embodiment can be applied to the serum sample testing device and the method for testing a serum sample.

<Cell for Raman Spectroscopic Analysis of Serum Sample>
In one embodiment, the present invention comprises a convex curved surface forming part for forming a convex curved surface on a serum sample, and a supply path connected to the convex curved surface forming part and supplying the serum sample to the convex curved surface forming part. A cell for Raman spectroscopy of serum samples is provided.

The Raman spectroscopic analysis cell of this embodiment will be described below with reference to FIGS. 7, 8A and 8B.

FIG. 7 is a side view showing an example of the Raman spectroscopic analysis cell of this embodiment. The Raman spectroscopic analysis cell 1 includes a hollow tube 10 , an outer cylinder 20 , a plunger 30 and a support portion 40 .

The hollow tube 10 has a convex surface forming portion 11 and a supply channel 12 . The hollow tube 10 has a hollow interior, and a serum sample can be retained therein. The hollow tube 10 is preferably made of a material that does not generate autofluorescence and does not generate Raman scattered light when irradiated with excitation light (eg, laser light of 1064 nm). Such materials include, for example, metals. Among metals, stainless steel is preferable.

FIG. 8A is a vertical cross-sectional view including part of the convex surface forming portion 11 and the supply path 12. FIG. FIG. 8B shows a state in which the serum sample 50 forms a convex curved surface in the convex curved surface forming portion 11 in the vertical cross-sectional view of FIG. 8A. The hollow tube 10 is cut obliquely at its tip to form an oblique side 10c. The convex curved surface forming portion 11 is arranged at the tip portion (between the longest side 10a and the shortest side 10b) of the hollow tube 10 where the oblique side 10c is formed. When the serum sample 50 is filled in the Raman spectroscopic analysis cell 1 , the serum sample 50 can form a convex curved surface in the convex curved surface forming part 11 .

The inner diameter Di and the outer shape Do of the hollow tube 10 are not particularly limited as long as the serum sample 50 can form a convex curved surface in the convex curved surface forming portion 11 . The inner diameter Di can be, for example, 500 to 2500 μm, preferably 600 to 2000 μm, more preferably 700 to 1500 μm, even more preferably 800 to 1200 μm, particularly preferably 1000 μm. The outer diameter Do can be, for example, 600 to 3000 μm, preferably 700 to 2500 μm, more preferably 800 to 1700 μm, even more preferably 900 to 1500 μm, and particularly preferably 1200 μm. If the inner diameter Di is too large, the droplet forming the convex surface in the convex surface forming portion 11 becomes large, and the convex surface may sway during the acquisition of the Raman spectrum. Also, if the inner diameter Di is too large, the size of the droplet forming the convex curved surface in the convex curved surface forming portion 11 becomes small, and the droplet may dry during acquisition of the Raman spectrum.

An angle θ formed by the oblique side 10c and the longest side 10a can be, for example, 5 to 40°. The angle θ is preferably 10 to 30°, more preferably 15 to 25°. The difference in length between the longest side 10a and the shortest side 10b (the length of the convex curved surface forming portion 11) can be, for example, 1000 to 10000 μm. The difference in length between the longest side 10a and the shortest side 10b is preferably 2000-8000 μm, more preferably 2500-7000 μm.

The supply path 12 is connected to the convex curved surface forming part 11 and supplies the serum sample 50 to the convex curved surface forming part 11 .

The outer cylinder 20 is formed of a hollow cylinder, and includes a cylindrical portion 20a and a hollow tube holding portion 20b. The hollow tube holding portion 20b holds the hollow tube 10, and the internal space of the outer cylinder 20 and the internal space of the hollow tube 10 are connected at the holding portion. The size of the outer cylinder 20 is not particularly limited as long as it can hold the hollow tube 10 . The internal space of the outer cylinder 20 may have a capacity of, for example, approximately 100 to 1000 μL. The material of the outer cylinder 20 is not particularly limited, and glass, resin, or the like can be used.

The outer cylinder 20 may have a scale portion 21 on the cylinder portion 20a. In the example of FIG. 7, the scale portion 21 is composed of scales 21a and 21b. Scale 21b can be used as a measure of the amount of serum sample to be collected. The scale 21 a can be used as a measure of the amount of convex surface formation by the convex surface forming portion 11 . The volume difference between the scale 21a and the scale 21b can be, for example, 5 to 20 μL or 10 to 15 μL. A specific example is 10 μL.

The plunger 30 is slidably arranged inside the outer cylinder 20 . The plunger 30 is not particularly limited as long as it has a size that allows it to slide inside the outer cylinder 20 . The plunger 30 may have a gasket 31 at its tip that is inserted into the outer cylinder 20 .

The supporting portion 40 stably holds the Raman spectroscopic analysis cell 1 on the stage of the microscopic Raman spectroscopic device during acquisition of the Raman spectrum. In the example of FIG. 7 , the support portion 40 is composed of a support piece 41 , a support piece 42 and a support piece 43 . The support piece 41 is arranged at a position near the hollow tube holding portion 20b of the outer cylinder 20, the support piece 42 is arranged at the opening of the outer cylinder 20, and the support piece 43 is at the tip of the plunger 30 opposite to the gasket 31. are placed in The support piece 42 and the support piece 43 also function as a grip when taking a serum sample or forming a convex curved surface. The number of support pieces forming the support portion 40 is not limited to three, and may be two or four or more. If the outer cylinder 20 has a shape that can be stably held on the stage of the microscopic Raman spectroscopic device (for example, a polygonal prism shape, a half-cylindrical shape, etc.), the support part 40 may be omitted.

A method of using the Raman spectroscopic analysis cell 1 having the configuration as described above will be described.

First, the plunger 30 is pushed into the outer cylinder 20 until the gasket 31 of the plunger 30 comes into contact with the hollow tube holding portion 20b, and the hollow tube 10 is introduced into the serum sample to the middle of the supply channel 12. As shown in FIG. In this state, by pulling the plunger 30 to the position of the scale 21b, a serum sample can be collected up to the position of the scale 21b.

Next, the Raman spectroscopic analysis cell 1 is placed on the stage of the microscopic Raman spectrometer, and the plunger 30 is slowly returned to the position of the scale 21a. As a result, a droplet of the serum sample is formed on the convex curved surface forming portion 11, and the droplet forms a convex curved surface. The convex curved surface formed at this time has a distance r of about 500 to 4000 μm between the intersection point o and the tip point b of the shortest side 10b when the intersection point of the perpendicular line from the vertex a and the extension line of the shortest side 10b is the intersection point o. , the distance h between the intersection point o and the vertex a is preferably about 300 to 2000 μm (see FIG. 8B). The distance r is more preferably 1000 to 3000 μm, even more preferably 1500 to 2500 μm. The distance h is more preferably 500-2000 μm, even more preferably 800-1500 μm.

During acquisition of the Raman spectrum of the serum sample, the convex curved surface forming section 11 is constantly supplied with the serum sample via the supply path 12 . As a result, the droplets of the serum sample formed by the convex curved surface forming part 11 are not dried, and the change in component concentration is suppressed. Therefore, a clear Raman spectrum can be obtained stably.

The Raman spectroscopic analysis cell of the present embodiment can be used for the above-described serum sample testing device, the method for testing a serum sample, and the method for obtaining a Raman spectrum of a serum sample.

Although the present invention will be described below with reference to experimental examples, the present invention is not limited to the following experimental examples.

<Preparation of serum sample>
144 patients, including 3 gastric cancer patients (stage I-II: 2, stage III-IV: 1) and 9 colorectal cancer patients (stage I-II: 7, stage III-IV: 2) Blood was obtained by blood collection from the subjects. The blood of each subject was centrifuged (1500 g, 5 minutes), the supernatant was obtained, and the serum sample of each subject was prepared.

<Acquisition of Raman spectrum>
A microscopic Raman spectrometer (manufactured by Bayspec) was used for the Raman spectroscopy. Specifications of the microscopic Raman apparatus are as follows.
Excitation light source: 785 nm (maximum output 50 mW), 1064 nm (maximum output 200 mW)
Detection system: CCD camera (1.4 pixels/~22 fps)
Specimen stage: Upright type, motorized/computer controlled

The objective lens is LMPLN10XIR (10x magnification, NA0.3, WD18, FN22, no glass thickness correction, no silicon thickness correction), LMPLN20XIR (20x magnification, NA0.45, WD8.3, FN22, glass thickness correction 0- 1.2, silicon thickness correction 0-1.2), or MPLN50XIR (50x magnification, NA 0.65, WD 4.5, FN22, glass thickness correction 0-1.2, silicon thickness correction 0-1.2) ( Both were manufactured by Olympus Corporation).

A 1 mL serum sample was collected using a syringe equipped with an 18-gauge needle (inner diameter 1000 μm, outer diameter 1200 μm; Flowmax (registered trademark), Nipro), and the serum sample in the syringe was slowly extruded until the tip of the needle protruded. Droplets with curved surfaces were formed (see FIG. 9). The size of the droplet was adjusted so that the distance r and the distance h shown in FIG. 8B were about 2000 μm and about 1000 μm, respectively.

A syringe with a serum sample droplet formed on the needle tip was placed on the sample stage of a microscopic Raman spectrometer, and a 785 nm or 1064 nm laser was irradiated focusing on the apex of the convex curved surface of the droplet. The output was 50 mV when a 785 nm wavelength laser was used, and the output was 200 mV when a 1064 nm wavelength laser was used. The laser irradiation time in one measurement was 15 seconds, baseline correction was performed using measurement software (Pathological System Version 1.0.1.0, manufactured by Bayspec), and the Raman spectrum was recorded. Three measurements were performed for one serum sample, and the average value was obtained as the Raman spectrum of the serum sample.

A clear Raman spectrum could be obtained by using either an excitation light source of 785 nm or 1064 nm, but a clearer Raman spectrum could be obtained by using a laser light source of 1064 nm. As for the objective lens, a clearer Raman spectrum could be obtained when an objective lens with a magnification of 10 times or a magnification of 20 times was used.

FIG. 10 shows an example of a Raman spectrum of a serum sample obtained by irradiating a 1064 nm laser with an output of 200 mW and using an objective lens with a magnification of 20 times. The figure shows the positions of the peaks described in the Raman shift table of FIG. 4A.

<Evaluation of gastric cancer>
Regarding the Raman spectrum obtained from the serum sample of each subject, the parameter showing a significant difference between the gastric cancer subject and the non-gastric cancer subject was examined. As a result, (1) phenylalanine C—C bending vibration (twist) as the first peak, (15) Amide I as the second peak, and (13) CH ₃ bending vibration or C—H as the third peak. It was confirmed that the use of bending motion could discriminate serum samples from gastric cancer subjects based on the intensity ratio (P2/P1) and intensity ratio (P3/P1).

FIG. 11 shows Raman plots when the first to third peaks are set as described above. The plots for non-gastric cancer patients tended to concentrate in the center of the graph, while the plots for gastric cancer patients tended to scatter to the lower left or upper right. By setting cut-off lines (P2/P1 ≤ 2.2 and P3/P1 ≤ 2.45, P2/P1 ≥ 2.8 and P3/P1 ≥ 2.95) at the lower right and upper left of the Raman plot, A serum sample from a gastric cancer subject could be discriminated with a sensitivity of 100% and a specificity of 80.8%.

<Evaluation of colorectal cancer>
Regarding the Raman spectrum obtained from the serum sample of each subject, parameters showing a significant difference between colon cancer subjects and non-stomach cancer subjects were examined. As a result, from the Raman peaks shown in FIG. 4A, by using (2) cholesterol as the first peak, (15) Amide I as the second peak, and (5) phenylalanine benzene ring breathing vibration as the third peak, , based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1), it was confirmed that serum samples from gastric cancer subjects could be discriminated.

FIG. 12 shows Raman plots when the first to third peaks are set as described above. The plots for non-colorectal cancer patients tended to converge in the center of the graph, while the plots for colorectal cancer patients tended to scatter to the lower left or upper right. By setting cut-off lines (P2/P1 ≤ 1.8 and P3/P1 ≤ 2.2, P2/P1 ≥ 2.4 and P3/P1 ≥ 2.8) at the bottom right and top left of the Raman plot, the sensitivity It was possible to discriminate serum samples from colon cancer subjects with a specificity of 88% and a specificity of 71.3%.

From the above results, by setting the first peak, the second peak, and the third peak corresponding to the cancer type, based on the intensity ratio (p2/P1) and the intensity ratio (P3/P1) , it was shown that the presence or absence of cancer in a subject can be determined.

<Reference example 1>
A Raman spectrum of the serum sample was obtained in the same manner as described above, except that the stainless steel metal plate shown in FIG. 13 was used instead of the syringe. A serum sample was placed in the wells of the metal plate, and the liquid surface of the serum sample was focused and irradiated with a laser.

The obtained Raman spectrum was extremely unstable, and peak analysis was difficult.

<Reference example 2>
Air-drying of serum samples was performed to prepare semi-dried serum samples. A Raman spectrum was obtained in the same manner as described above, except that the semi-dried serum sample was used.

A clear Raman spectrum could be obtained, but no spectral difference was observed between cancer patients and non-cancer patients. This was presumed to be due to the modification of the components in the serum sample by drying.

<Reference example 3>
Heat drying of serum samples was performed to prepare heat dried serum samples. A Raman spectrum was obtained in the same manner as described above, except that the heat-dried serum sample was used.

A clear Raman spectrum could be obtained, but no spectral difference was observed between cancer patients and non-cancer patients. This was presumed to be due to the modification of components in the serum sample due to heat denaturation.

<Reference example 4>
A glass fiber-absorbed serum sample was prepared by absorbing the serum sample onto a glass fiber. A Raman spectrum was obtained in the same manner as described above, except that the glass fiber-absorbed serum sample was used.

A clear Raman spectrum could be obtained, but no spectral difference was observed between cancer patients and non-cancer patients. It was presumed that this was because the components in the serum sample were modified by drying due to absorption on the glass fiber.

ADVANTAGE OF THE INVENTION According to this invention, the serum sample test|inspection apparatus and the test|inspection method of a serum sample which can determine the presence or absence of cancer in a test object simply and rapidly with a blood sample are provided. Also provided are a method for obtaining a Raman spectrum of a serum sample and a cell for Raman spectroscopic analysis of a serum sample, which are applicable to the serum sample testing device and testing method.

1 Raman Spectroscopic Analysis Cell 10 Hollow Tube 11 Convex Curved Surface Forming Part 12 Supply Path 20 Outer Tube 21 Scale Part 20a Cylinder Part 20b Hollow Tube Holding Part 20b
21a, 21b Scale 30 Plunger 31 Gasket 40 Supporting Part 41, 42, 43 Supporting Piece 50 Serum Sample 51 Convex Curved Surface 100 Serum Sample Inspection Device 110 Raman Spectrum Acquisition Unit 120 Stage 130 Light Source Device 140 Dichroic Filter 141 Beam Splitter 142 Long Pass Filter 143, 144 lens 150 objective lens 160 spectrometer 170 cooled CCD camera 171 observation CCD camera 190 processing device 191 intensity acquisition unit 192 intensity ratio calculation unit 193 determination unit 194 output unit 195 storage unit 196 control unit

Claims (3)

a Raman spectrum acquisition unit that acquires a Raman spectrum of the serum sample of the subject;
For the first peak, the second peak, and the third peak set according to the cancer type to be examined, from the Raman spectrum, the intensity (P1) of the first peak, the second peak an intensity acquisition unit that acquires the intensity (P2) of and the intensity (P3) of the third peak;
The intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity of the first peak (P1), and the intensity of the third peak to the intensity of the first peak (P1) ( an intensity ratio calculator that calculates the intensity ratio (P3/P1) of P3);
a determination unit that determines the presence or absence of cancer in the subject based on the intensity ratio (P2/P1) and the intensity ratio (P3/P1);
with
The Raman spectrum acquisition unit acquires the Raman spectrum of the serum sample by focusing the excitation light on the vertex of the convex curved surface in a state where the droplet of the serum sample forms a convex curved surface.
Serum sample testing device. A step of acquiring a Raman spectrum of a serum sample of a subject, wherein the droplet of the serum sample forms a convex curved surface, and excitation light is focused on the vertex of the convex curved surface to obtain the Raman spectrum of the serum sample. obtaining a spectrum;
For the first peak, the second peak, and the third peak set according to the cancer type to be examined, from the Raman spectrum, the intensity (P1) of the first peak, the second peak obtaining the intensity (P2) of and the intensity (P3) of the third peak;
The intensity ratio (P2/P1) of the intensity of the second peak (P2) to the intensity of the first peak (P1), and the intensity of the third peak to the intensity of the first peak (P1) ( A step of calculating the intensity ratio (P3/P1) of P3);
determining the presence or absence of cancer in the subject based on the intensity ratio (P2/P 1 ) and the intensity ratio (P3/P1);
A method for testing a serum sample, comprising: forming a convex curved surface with a droplet of the serum sample;
obtaining a Raman spectrum of the serum sample by focusing excitation light on the apex of the convex surface while maintaining the convex surface of the droplet;
A method of obtaining a Raman spectrum of a serum sample, comprising:

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