Method for determining plasma fractionation conditions, plasma fractionation device, and DNA extraction device equipped with same

Abstract

This method for determining fractionation conditions for plasma 105 includes: a step for measuring a blood sample by means of a blood condition confirmation unit; a step for obtaining a converted number of elapsed days from the blood collection date of the blood sample on the basis of the measurement results; a step for calculating the amount of gDNA in the blood sample on the basis of the converted number of elapsed days; and a step for determining the plasma fractionation conditions on the basis of the calculated amount of gDNA. The present invention thus provides a method for determining plasma fractionation conditions, a plasma fractionation device, and a DNA extraction device equipped with the same, with which it is possible to perform a plasma fractionation step under more appropriate conditions than in the past.

Description

Method for determining plasma fractionation conditions, plasma fractionation device, and DNA extraction device equipped with the same

[0001] The present invention relates to a method for determining plasma fractionation conditions, which enables setting appropriate plasma fractionation conditions by determining the state of plasma in a blood collection tube, a plasma fractionation device, and a DNA extraction device equipped with the same.

[0002] Patent Document 1 describes an example of a specimen information detection device that can detect the state of a specimen with high accuracy and high efficiency, which includes an imaging device that images a specimen container containing a specimen, an illumination device that irradiates the specimen container with light from a direction intersecting the imaging direction during the imaging, and an information processing unit that detects the state inside the specimen container by image processing based on image information of the specimen container obtained by imaging the specimen container.

[0003] Japanese Patent Application Laid-Open No. 2022-15923

[0004] In recent years, it has become clear that the peripheral blood of cancer patients contains DNA derived from cancer cells, and this DNA has attracted attention as a target for detecting genetic information about cancer.

[0005] When cells in the body are destroyed by apoptosis or other processes, the genomic DNA (gDNA) present in the nucleus is fragmented, resulting in short DNA called cell-free DNA (cfDNA). DNA released from cells originating from diseases such as cancer also circulates in the blood, and is called cell-tumor DNA (ctDNA) to distinguish it from cfDNA.

[0006] This new technology, which extracts ctDNA from bodily fluids such as blood and uses it as a test subject to diagnose diseases such as cancer, is called liquid biopsy testing.

[0007] Compared to biopsy tissue testing, which involves directly collecting tissue during surgery, liquid biopsy testing has the advantage of being relatively easy to obtain test samples using minimally invasive methods such as blood sampling. In other words, by detecting ctDNA derived from cancer cells in the blood, it is expected that cancer can be detected early without analyzing the cancer cells themselves at the disease site.

[0008] The workflow of liquid biopsy testing begins with centrifuging whole blood collected from a patient to separate plasma and buffy coat. Plasma components are typically obtained by performing two centrifugations. Next, ctDNA is extracted from the separated plasma, and gDNA is extracted from the buffy coat. The DNA is then sized and quantified in a quality check (QC) step.

[0009] The DNA obtained in the pre-processing steps from centrifugation to QC is used to analyze mutations in gene sequences using next generation sequencing (NGS) and digital PCR (dPCR: d Polymerase Chain Reaction) to diagnose cancer and select therapeutic drugs.

[0010] Genetic testing using NGS is a test that detects mutations and mutation levels in gene sequences by reading DNA base sequences in a massively parallel manner. One example of genetic testing using NGS is a method for diagnosing cancer by separately analyzing and comparing the sequences of gDNA and ctDNA derived from cancer cells, which have normal base sequences. On the other hand, genetic testing using dPCR is a test that divides the DNA into thousands of parallel PCR reactions and measures mutation detection and mutation levels by amplifying specific gene sequences.

[0011] In this workflow, the plasma separation step requires that plasma containing a large amount of ctDNA, the target of gene mutation analysis, be collected without waste while preventing contamination with other components, making this a very important step for proceeding with subsequent processes.

[0012] The state of the plasma components obtained after centrifugation varies depending on the number of days since blood collection and the type of blood collection tube. Furthermore, the buffy coat, which is a white blood cell fraction, also varies in terms of its fragility and viscosity, just like the plasma. Therefore, if plasma is collected under uniform collection conditions, the amount of collected plasma is small, and if other components, particularly the buffy coat, are mixed in, ctDNA will be contaminated with white blood cell-derived gDNA during nucleic acid extraction. This can lead to problems such as reduced sensitivity in mutation detection using NGS and dPCR.

[0013] For these reasons, it is important to determine the state of plasma in a sample, taking into account the number of days since blood collection or the type of blood collection tube, and to optimize the separation conditions according to the plasma state.

[0014] The hemolysis state of plasma is often used as an index for distinguishing the state of plasma. In Patent Document 1, the hemolysis state of plasma or serum is determined from the hue using the HSV method. If the hemolysis state is determined to exceed a threshold, it is determined that the test is unsuitable due to sample test inhibition, and the steps after fractionation are not carried out.

[0015] However, the state of plasma components after centrifugation varies depending on the number of days since blood collection and the type of blood collection tube.Furthermore, the state (friability) of the buffy coat, which is a white blood cell fraction, also varies, just like plasma.

[0016] Therefore, if plasma is separated under uniform separation conditions, there is a problem that leukocytes derived from the buffy coat may be mixed in. That is, gDNA may be mixed in during ctDNA extraction, which may reduce the sensitivity in gene mutation analysis such as next-generation sequencing (NGS) or digital PCR (dPCR), and therefore it is desirable to avoid this.

[0017] To avoid this issue, it is necessary to standardize the type of blood collection tube, the number of days since blood collection, and even the storage conditions to some extent, but adopting such measures is extremely difficult. Therefore, it is desirable and required to take measures when performing the plasma separation process on the analytical side.

[0018] The present invention provides a method for determining plasma fractionation conditions, a plasma fractionation device, and a DNA extraction device equipped with the same, which enable the plasma fractionation step to be carried out under more appropriate conditions than conventional methods.

[0019] The present invention includes multiple means for solving the above-mentioned problems, and one example thereof includes the steps of determining the number of days that have passed since the date of collection of a blood sample, calculating the amount of gDNA in the blood sample based on the number of days that have passed, and determining plasma separation conditions based on the calculated amount of gDNA.

[0020] According to the present invention, the plasma separation step can be carried out under more suitable conditions than in the past. Objects, configurations and effects other than those described above will become apparent from the following description of the examples.

[0021] 1 is a schematic diagram of a plasma fractionation apparatus and a DNA extraction apparatus equipped with the same according to Example 1. FIG. 2 is a flowchart showing an example of the flow of a method for determining plasma fractionation conditions according to Example 1. FIG. 3 is a diagram showing the relationship between absorbance and the number of days elapsed since the date of blood collection. FIG. 4 is a diagram showing the relationship between hue and saturation. FIG. 5 is a diagram showing the relationship between hue and brightness. FIG. 6 is a diagram showing the relationship between gDNA amount and the number of days elapsed since the date of blood collection. FIG. 7 is a diagram showing details of fractionation conditions set for the threshold and range of gDNA amount set in FIG. 6. FIG. 8 is a diagram showing the results of examining gDNA contamination in plasma, the presence or absence of precipitate after second centrifugation, and gDNA contamination in the precipitate for two samples with different numbers of days elapsed since the date of blood collection according to Example 2. FIG. 9 is a flowchart showing an example of the flow of a method for determining plasma fractionation conditions according to Example 3.

[0022] Hereinafter, examples of the method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped with the same of the present invention will be described with reference to the drawings.

[0023] In the following examples, the configurations (including the steps in the flowcharts) are not necessarily essential unless otherwise specified or considered to be clearly essential in principle.

[0024] Furthermore, in the drawings used in this specification, identical or corresponding components are denoted by the same or similar reference numerals, and repeated description of these components may be omitted.

[0025] Example 1 A method for determining plasma fractionation conditions, a plasma fractionation device, and a DNA extraction device including the same according to Example 1 of the present invention will be described with reference to FIGS. 1 to 7. FIG.

[0026] First, the overall configuration of the plasma fractionation device and DNA extraction device, and the samples to be fractionated will be described with reference to Fig. 1. Fig. 1 is a schematic diagram of the plasma fractionation device of Example 1 and the DNA extraction device equipped with the same.

[0027] The DNA extraction device 500 shown in FIG. 1 includes a plasma fractionation device 100 and a nucleic acid extraction unit 200 .

[0028] The plasma collection device 100 is a device for collecting plasma 105 from a blood sample, and includes a collection machine 101, an actuator 102, a camera 108A, a light receiving unit 108B, a light 109, a motor driver 111, and a controller 110.

[0029] The sample to be separated is a blood specimen in a blood collection tube 104 obtained by centrifuging a whole blood specimen obtained by drawing blood from a patient. The blood cell components obtained after centrifuging the blood collection tube 104 containing the blood specimen are, from the top down, plasma 105, buffy coat 106, and red blood cells 107.

[0030] The fractionator 101 has a function for collecting plasma 105 from a blood sample in a blood collection tube 104, and is designed so that the user can collect an appropriate amount of plasma 105 using a controller 110. A fractionator tip 103 is detachably provided at the tip of the fractionator.

[0031] The actuator 102 is a mechanism that is operated by driving a motor driver 111, and can appropriately adjust the height of the fractionator 101 so that the fractionator 101 starts aspirating plasma 105 from an appropriate height and ends the aspirating at a height just before the buffy coat 106 layer.

[0032] The camera 108A is positioned so as to be able to capture an image of the entire blood collection tube 104. When capturing an image of the blood collection tube 104, the image is captured in the presence of light irradiated from the illumination 109. The light receiving unit 108B is a device that detects transmitted light emitted from the illumination 109 and transmitted through the blood collection tube 104 or reflected light from the blood collection tube 104. The camera 108A and the light receiving unit 108B may be integrated together.

[0033] The illumination 109 irradiates the blood collection tube 104 with light when the camera 108A captures an image of the blood collection tube 104 or when the light receiving unit 108B acquires transmitted light or reflected light from the blood collection tube 104. The light irradiated onto the blood collection tube 104 is preferably transmitted light or reflected light, but is not limited to either. The color of the light is preferably white light or blue light, but is not limited to either, as with the type of irradiated light.

[0034] The camera 108A, the light receiving unit 108B, and the lighting 109 constitute a blood condition checking unit that measures the condition of the blood sample.

[0035] The controller 110 is a part that controls the operations of the fractionator 101, actuator 102, camera 108A, light receiving unit 108B, lighting 109, motor driver 111, etc., and is composed of a display device such as a liquid crystal display, an input device, a storage device composed of a recording medium such as an HDD or SSD and its controller, a CPU, a memory, etc. The control of the operation of each device by the controller 110 is executed based on various programs recorded in the storage device.

[0036] The control processes for the operations executed by the controller 110 may be integrated into a single program, or may be divided into multiple programs, or may be a combination of these. Furthermore, some or all of the programs may be implemented by dedicated hardware or may be modularized.

[0037] In this embodiment, the controller 110 determines the converted number of days elapsed since the date of collection of the blood sample based on the results of measurements taken by the camera 108A, the light receiving unit 108B, and the illumination 109, calculates the amount of gDNA in the blood sample based on the converted number of days elapsed, and determines the plasma fractionation conditions based on the calculated amount of gDNA. Details of this will be described later.

[0038] The nucleic acid extraction unit 200 is a mechanism for extracting nucleic acids from the plasma 105 fractionated from the blood sample by the plasma fractionation device 100 .

[0039] Next, the steps for collecting plasma from a whole blood sample, which are preferably performed by the plasma collection device 100 shown in FIG. 1, and the method for determining the collection conditions for plasma 105 in Example 1 of the present invention will be described with reference to FIGS. 2 to 7.

[0040] FIG. 2 is a flowchart showing an example of the flow of the method for determining plasma separation conditions in Example 1, FIG. 3 is a diagram showing the relationship between the number of days elapsed since the date of blood collection and absorbance, FIG. 4 is a diagram showing the relationship between hue and saturation, FIG. 5 is a diagram showing the relationship between hue and brightness, FIG. 6 is a diagram showing the relationship between the number of days elapsed since the date of blood collection and the amount of gDNA, and FIG. 7 is a diagram showing details of the separation conditions set for the thresholds and ranges of gDNA amount set in FIG. 6.

[0041] 2, first, the user operates the controller 110 to set a threshold value for the amount of gDNA mixed in the plasma 105 (S201). This step S201 corresponds to the step of setting the threshold value for the amount of gDNA.

[0042] Next, the type of the blood collection tube 104 is confirmed (S202). In this step S202, the user confirms the type of the blood collection tube 104 and inputs it into the controller 110. Alternatively, the controller 110 recognizes the type of the blood collection tube 104 using a barcode attached to the blood collection tube 104. Alternatively, other methods may be used.

[0043] The controller 110 has data showing the relationship between the number of days elapsed since the blood collection date and the amount of gDNA for each type of blood collection tube 104, as shown in FIG. 3, and the selected data will be used in the subsequent steps.

[0044] Next, the hue, saturation, brightness or absorbance of the plasma 105 obtained by first centrifuging the whole blood sample is measured (S203). This step S203 corresponds to the step of measuring the blood sample by the blood condition confirmation unit.

[0045] The method for measuring hue, saturation, and brightness involves first acquiring an image of the blood collection tube 104 after the centrifugation process using the camera 108A. The type of image acquired at this time is preferably an RGB image. This RGB image is then converted to HSV, and the boundaries between the plasma 105 layer, the buffy coat 106 layer, and the red blood cell 107 layer are detected, and only the plasma 105 layer is extracted. Figure 4 shows a plot of data for each type of plasma diagnosed as high hemolysis, low hemolysis, jaundice, normal, and chyle according to the hue and saturation values, and Figure 5 shows a plot of data for each type of plasma diagnosed as high hemolysis, low hemolysis, jaundice, normal, and chyle according to the hue and brightness values.

[0046] Here, it is possible to determine not only the region of the plasma 105 layer, but also the amount of plasma 105 and the region of the interface between the plasma 105 layer and the buffy coat 106 layer. Detecting the boundary between the buffy coat 106 layer and the plasma 105 layer is an important step from the viewpoint of preventing gDNA derived from leukocytes in the buffy coat 106 from being mixed into the plasma 105. Based on the region of the plasma 105 layer obtained by extraction, color information including the hue, saturation, and brightness of the plasma 105 layer is obtained.

[0047] In the method for measuring absorbance, incident light is irradiated from the illumination 109 onto the layer of plasma 105 in the blood collection tube 104, which is the measurement target, and the intensities of the transmitted light are measured by the light-receiving unit 108B. The absorbance is calculated from the intensities of the incident light and the transmitted light measured by the light-receiving unit 108B.

[0048] As shown in Figure 3, the longer the number of days since the blood collection date, the higher the absorbance of the plasma 105 layer at a wavelength of 414 nm. It is known that an absorbance peak is observed at 414 nm when hemoglobin contained in red blood cells is bound to oxygen, and light of this wavelength can be used to determine the blood condition of the plasma 105 layer and the amount of gDNA contamination. Therefore, the higher the absorbance, the better the blood condition of the plasma 105 and the longer the number of days since the blood collection date.

[0049] At this time, it is preferable to measure the hue, saturation, and brightness using white light, and the absorbance using blue light with a wavelength of 414 [nm].

[0050] In this way, preferably, a blue light source is used as the illuminator 109, and a light-receiving unit 108B that measures the absorbance of the blood sample is used as the blood condition confirmation unit, and in the measurement step, the absorbance of the blood sample is measured by the illuminator 109 that irradiates the blood sample and the light-receiving unit 108B, or preferably, a white light source is used as the illuminator 109, and a camera 108A that captures an image of the appearance of the blood sample is used as the blood condition confirmation unit, and in the measurement step, any one or more of the hue, saturation, and brightness of the blood sample can be measured by the illuminator 109 that irradiates the blood sample and the camera 108A.

[0051] Next, the converted number of days elapsed since the blood collection date is calculated (S204).

[0052] 3, the controller 110 stores data showing the relationship between the number of days elapsed since the date of blood collection and the absorbance of the plasma 105. If the absorbance is measured using blue light in step S203, the converted number of days elapsed since the date of blood collection is calculated based on the measured absorbance. Similarly, if the hue, saturation, or brightness is measured using white light in step S203, the converted number of days elapsed since the date of blood collection is calculated based on the measured values ​​of the hue, saturation, or brightness.

[0053] At this time, the number of days since the actual blood collection date is not taken into consideration because the state of the plasma 105, such as the degree of hemolysis of the blood sample in the blood collection tube 104, varies depending on the storage condition, and the number of days since the actual blood collection date is unreliable, so the converted number of days since the actual blood collection date is calculated.

[0054] This step S204 corresponds to the step of determining the converted number of days elapsed since the date of collection of the blood sample based on the measurement results. In the step of determining the converted number of days elapsed, the converted number of days can be calculated using data showing the relationship between the absorbance value of the blood sample and the converted number of days elapsed, or the converted number of days can be calculated using data showing the relationship between one or more values ​​of hue, saturation, and brightness and the converted number of days elapsed.

[0055] Next, the amount of gDNA is calculated from the calculated converted number of days elapsed (S205). This step S205 corresponds to the step of calculating the amount of gDNA in the blood sample based on the converted number of days elapsed. Furthermore, in this step of calculating the amount of gDNA, the amount of gDNA can be calculated using data showing the relationship between the converted number of days elapsed and the amount of gDNA mixed in for each type of blood collection tube 104 containing the blood sample.

[0056] In addition to the data selected in step S202 showing the relationship between the number of days elapsed since the blood collection date and the amount of gDNA as shown in Figure 3, the controller 110 is assumed to have recorded data showing the relationship between the number of days elapsed since the blood collection date and the amount of gDNA as shown in Figure 6, and the amount of gDNA predicted to actually be mixed in the plasma 105 is calculated using the converted number of days elapsed since the blood collection date calculated in the previous step S204.

[0057] 6 shows the calculated gDNA amount data for each number of days elapsed since the blood collection date, plotted with n=5. It also shows each region when a threshold value set for gDNA amount in step S201 is set. However, this threshold value can be freely set by the user, and is not limited to the values ​​shown in the figure.

[0058] Next, the calculated amount of gDNA is compared with a set threshold value for the amount of gDNA (S206).

[0059] Based on the threshold value of the gDNA amount set by the user in step S201, the controller 110 compares whether the gDNA amount calculated in the previous step S205 exceeds the threshold value. When the comparison is complete, the regions for each threshold value shown in Figure 6, that is, the fractionation conditions shown in the next step S207, are set. Figure 6 is an example, and the present invention is not limited to this.

[0060] Next, the plasma fractionation conditions are set (S207). This step S207 corresponds to the step of determining the plasma fractionation conditions based on the calculated gDNA amount.

[0061] For example, the controller 110 sets the fractionation conditions based on the graph of Fig. 6. Parameters of the fractionation conditions include the linear velocity (aspiration flow rate) of the plasma 105, the plasma aspiration stop position, the liquid surface tracking descent speed of the fractionation tip 103, and the insertion distance of the tip of the fractionation tip 103 into the liquid, as shown in Fig. 7.

[0062] For example, it is assumed that the user sets the threshold for the amount of gDNA mixed in the plasma 105 to 1.0 [ng / μL] in step S201.

[0063] In fact, if the calculated amount of gDNA is 1.0 [ng / μL] or more (area C in FIG. 6), the sample is excluded from the sample processing, and the user is instructed to collect blood from the patient again.

[0064] Next, if the calculated amount of gDNA is 0.1 [ng / μL] or more and less than 1.0 [ng / μL] (region B in Figure 6), in order to prevent contamination with gDNA derived from the buffy coat 106, the separation conditions are set to be milder than the usual conditions (condition A below) (aspiration speed 500 [μL / s], aspiration stop position 8 [mm] above the buffy coat 106 layer).

[0065] Furthermore, when the calculated amount of gDNA is less than 0.1 [ng / μL] (area A in Figure 6), the procedure is performed under normal fractionation conditions (aspiration speed of 1,000 [μL / s], aspiration stop position 5 [mm] above the buffy coat 106 layer).

[0066] In this way, in step S207, which is the step of determining the plasma fractionation conditions, the set threshold value and the calculated gDNA amount are compared to determine the plasma fractionation conditions.

[0067] The threshold value of the gDNA amount and the number of regions to be set shown in this step are merely examples and are not limited to these. In addition, the user can also set the details of the sorting conditions.

[0068] Next, the plasma 105 is collected in accordance with the collection conditions (see FIG. 7) set in the previous step S207 (S208). When collecting the plasma 105, the image is captured by the camera 108A, or the amount of the plasma 105 has been calculated in step S203, and the plasma 105 may be collected based on this information.

[0069] Next, the effects of this embodiment will be described. The method for determining separation conditions for plasma 105 according to the first embodiment of the present invention described above includes the steps of determining the number of days elapsed since the date of collection of a blood sample, calculating the amount of gDNA in the blood sample based on the number of days elapsed, and determining plasma separation conditions based on the calculated amount of gDNA. In particular, the method further includes the step of measuring the blood sample using a blood condition confirmation unit. In the step of determining the number of days elapsed, a converted number of days elapsed since the date of collection of the blood sample is determined based on the measurement results, and in the step of calculating the amount of gDNA, the amount of gDNA is calculated based on the converted number of days elapsed.

[0070] In this way, by determining the plasma fractionation conditions on the analysis side, taking into account the blood condition of the plasma components obtained after centrifugation, the number of days since blood collection, and the type of blood collection tube 104, it is possible to avoid plasma fractionation that would result in poor test results due to the inclusion of large amounts of components such as gDNA that hinder analysis, without standardizing the type of blood collection tube, the number of days since blood collection, and even the storage conditions to a certain extent. Therefore, it is possible to reduce the number of unnecessary nucleic acid extractions and NGS performed compared to the past, avoiding costs, wasting test materials, and the time required for retesting, i.e., it is possible to improve test efficiency compared to the past.

[0071] Furthermore, in the step of calculating the amount of gDNA, the amount of gDNA is calculated using data showing the relationship between the converted number of days elapsed for each type of blood collection tube 104 containing the blood sample and the amount of gDNA mixed in. This allows the amount of mixed gDNA to be calculated with higher accuracy, making it possible to set more appropriate plasma separation conditions.

[0072] Furthermore, the blood condition confirmation unit uses a camera 108A that captures an image of the appearance of the blood sample, and in the measurement step, one or more of the hue, saturation, and brightness of the blood sample are measured using a light 109 that illuminates the blood sample and the camera 108A, and in the step of determining the converted number of days elapsed, the converted number of days elapsed is calculated using data that shows the relationship between the value of one or more of the hue, saturation, and brightness and the converted number of days elapsed. In particular, by using a white light source as the light 109, it is possible to make a judgment using data that is highly correlated with the amount of gDNA contamination.

[0073] Furthermore, the blood condition confirmation unit uses a light receiving unit 108B that measures the absorbance of the blood sample, and in the measurement step, the absorbance of the blood sample is measured using an illumination 109 that irradiates the blood sample and the light receiving unit 108B, and in the step of determining the converted number of days, the converted number of days is calculated using data that shows the relationship between the absorbance value of the blood sample and the converted number of days. In particular, by using a blue light source as the illumination 109, the amount of gDNA contamination can be determined using data that is highly correlated with the amount of gDNA contamination.

[0074] Furthermore, the method further includes a step of setting a threshold value for the amount of gDNA, and in the step of determining the plasma separation conditions, the set threshold value is compared with the calculated amount of gDNA to determine the plasma separation conditions. This makes it possible to set separation conditions that can accommodate changes in the allowable amount of gDNA contamination depending on the subsequent nucleic acid extraction and analysis method, thereby enabling more appropriate sample testing.

[0075] Example 2 A method for determining plasma fractionation conditions, a plasma fractionation device, and a DNA extraction device including the same according to Example 2 of the present invention will be described with reference to FIG.

[0076] Figure 8 is a graph showing the results of examining gDNA contamination in plasma, the presence or absence of precipitate after the second centrifugation, and gDNA contamination in the precipitate for two samples collected on different days from the date of blood collection.

[0077] Considering the results of Figure 8, it is thought that plasma 105 on the day of blood collection is likely to be contaminated with leukocytes, while plasma 105 1 to 7 days after blood collection is not contaminated with leukocytes. One possible reason for this is the fragility of the buffy coat 106 layer, which varies depending on the number of days that have passed since the blood collection date. That is, fibrin is generated as the number of days that have passed since the blood collection date passes, and after the first centrifugation, fibrin accumulates mainly in the buffy coat 106 layer, which is thought to be related to the viscosity and fragility of the buffy coat 106 layer.

[0078] From this result, a condition is assumed in which the amount of gDNA calculated in step S206 shown in FIG. 2 of Example 1 falls below the threshold value of the amount of gDNA set by the user.

[0079] In this embodiment, in step S207 of FIG. 2, the number of days from the actual blood collection date is also taken into consideration. For samples collected on the day of blood collection, the buffy coat 106 layer is fragile, and there is a possibility that leukocyte-derived gDNA may be mixed in during plasma collection. Therefore, collection is performed under conditions that are milder than the usual collection conditions, for example, condition B instead of condition A in FIG. 7.

[0080] On the other hand, for samples for which one day or more has passed since the blood collection date, the buffy coat 106 layer has not become brittle, so plasma is separated under normal separation conditions.

[0081] However, the "number of days from the actual blood collection date" for allocating the separation conditions described in this example is only an example, and is not limited to this.

[0082] The other configurations and operations are substantially the same as those of the method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith in the first embodiment, and therefore details are omitted here.

[0083] The method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith according to Example 2 of the present invention also provide substantially the same effects as those of the method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith according to Example 1 described above.

[0084] A method for determining plasma separation conditions, a plasma separation device, and a DNA extraction device including the same according to Example 3 of the present invention will be described with reference to Fig. 9. Fig. 9 is a flowchart showing an example of the flow of the method for determining plasma separation conditions according to Example 3.

[0085] In this example, the steps up to the collection of plasma will be described.

[0086] 9, the specimen (blood collection tube 104) is placed on the information reading unit, and a threshold value for the amount of gDNA is set (S301). This step S301 is substantially the same as step S201 shown in FIG.

[0087] Next, the sample information is read and information on the type of the blood collection tube 104 is obtained (S302). This step S302 is substantially the same as step S202 shown in FIG.

[0088] Next, a first centrifugation is performed (S303), and the blood collection tube 104 is transferred to a holder (S304). After that, an image of the blood collection tube 104 is taken (S305), and the plasma 105 region is identified, and the amount of plasma 105 and the interface with the buffy coat 106 layer are determined (S306). These steps S303 to S306 are not shown as corresponding steps in Figure 2, but are also implicitly performed in the flow of Figure 2.

[0089] Next, the hue, saturation, and brightness are measured using white light (S307A), and the absorbance (414 nm) is measured using blue light (S307B). These steps S307A and S307B are the same as step S203 shown in FIG.

[0090] Next, the converted number of elapsed days is calculated (S308). This step S308 is substantially the same as step S204 shown in FIG.

[0091] Next, the amount of gDNA is calculated from the calculated converted number of days elapsed (S309). This step S309 is substantially the same as step S205 shown in FIG.

[0092] Next, the calculated gDNA amount is compared with a set threshold value for the gDNA amount (S310), plasma fractionation conditions are set (S311), and plasma 105 is fractionated (S312). Step S310 is substantially the same as step S206 shown in Figure 2, step S311 is substantially the same as step S207 shown in Figure 2, and step S312 is substantially the same as step S208 shown in Figure 2.

[0093] Next, a solution made of, for example, PBS (phosphate buffered saline) is added to the blood collection tube 104, and additional plasma 105 is collected (S313).

[0094] Specifically, this step is performed in addition to the sample that has undergone plasma collection (see FIG. 7) performed in the previous step S312. It is expected that performing this step will lead to the recovery of plasma 105 remaining in the blood collection tube 104 and the recovery of ctDNA during the nucleic acid extraction step.

[0095] In step S313, first, an additional solution is dispensed into the blood collection tube 104 after the plasma has been collected. The solution used at this time is preferably PBS (phosphate buffered saline) (pH 7.4). It is known that the general pH of the plasma 105 is around 7, and the density of phosphate buffered saline is about 1 [g / cm ３ Therefore, even if this solution is added, it remains in the plasma 105 layer located at the top layer and does not affect the physiological activity of the plasma 105 or the degradation of the ctDNA. However, the solution is not limited to phosphate buffered saline, as long as it remains in the plasma 105 layer and does not affect the physiological activity of the plasma 105 or the degradation of the ctDNA.

[0096] When dispensing the solution, the dispensing speed is reduced while the fractionation tip 103 is in contact with the wall of the blood collection tube 104 or while the tip of the fractionation tip 103 is in contact with the liquid surface, so that the buffy coat 106 layer and the red blood cell 107 layer, which may become impurities, do not fly up.

[0097] Theoretically, the larger the amount of solution added, the more recovery of plasma 105 is possible, which is preferable. However, since the amount depends on the amount of plasma 105 used in the subsequent nucleic acid extraction step, it is necessary to adjust the amount to match the amount of plasma 105 to be additionally collected.

[0098] After the addition of the solution, plasma is collected again under the same conditions as those used to collect the plasma 105 in the previous step S312.

[0099] Next, the plasma 105 is collected, and the collected tube is transferred to the centrifugal module (S314), where it is centrifuged again (S315). This step is performed to further increase the degree of purification of the plasma 105 collected after the first centrifugation.

[0100] Next, the plasma 105 in the recovery tube that has been subjected to the second centrifugation in the previous step S315 is transferred to a holder for collection (S316), and the plasma 105 is re-collected (S317) to prevent the precipitate generated during the second centrifugation from being mixed into the plasma 105. The precipitate generated by the second centrifugation includes blood cells such as white blood cells and platelets.

[0101] The conditions for the second fraction collection in step S313 and the conditions for the re-fraction collection in step S317 are preferably the same as the conditions for the first fraction collection in step S312 (S208), i.e., the conditions are determined for each sample according to the converted number of days elapsed. In this way, the conditions for aspiration and discharge speed, aspiration stop position, and solution addition can be made uniform.

[0102] The other configurations and operations are substantially the same as those of the method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith in the first embodiment, and therefore details are omitted here.

[0103] The method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith according to Example 3 of the present invention also provide substantially the same effects as those of the method for determining plasma fractionation conditions, the plasma fractionation device, and the DNA extraction device equipped therewith according to Example 1 described above.

[0104] Furthermore, in the step of determining the plasma collection conditions, by adjusting the suction and discharge speed, suction stop position, and solution addition conditions, it is possible to achieve appropriate collection and re-collection of plasma 105 according to the state of the blood sample.

[0105] <Others> The present invention is not limited to the above-described examples, and includes various modifications. The above-described examples have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those having all of the described configurations.

[0106] It is also possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of one embodiment to the configuration of another embodiment.It is also possible to add, delete, or replace part of the configuration of each embodiment with the configuration of another embodiment.

[0107] DESCRIPTION OF SYMBOLS 100: Plasma fractionation device 101: Fractionation machine 102: Actuator 103: Fractionation tip 104: Blood collection tube 105: Plasma 106: Buffy coat 107: Red blood cells 108A: Camera (blood condition confirmation unit, imaging device) 108B: Light receiving unit (blood condition confirmation unit) 109: Lighting (blood condition confirmation unit, light source) 110: Controller 111: Motor driver 200: Nucleic acid extraction unit 500: DNA extraction device

Claims

1. A method for determining plasma separation conditions, comprising: a step of obtaining the number of days elapsed since the blood sample was collected; a step of calculating the amount of gDNA in the blood sample based on the number of days elapsed; and a step of determining plasma separation conditions based on the calculated amount of gDNA.

2. The method for determining plasma separation conditions according to claim 1, further comprising a step of measuring the blood sample by a blood state confirmation unit. In the step of obtaining the number of days elapsed, based on the result of the measurement, a converted number of days elapsed since the blood sample was collected is obtained as the number of days elapsed. In the step of calculating the amount of gDNA, the amount of gDNA is calculated based on the converted number of days elapsed.

3. The method for determining plasma separation conditions according to claim 2, wherein in the step of calculating the amount of gDNA, the amount of gDNA is further obtained using data indicating the relationship between the converted number of days elapsed and the amount of gDNA mixed for each type of blood collection tube containing the blood sample.

4. The method for determining plasma separation conditions according to claim 2, wherein as the blood state confirmation unit, an imaging device for imaging the appearance of the blood sample is used. In the step of measuring, one or more of hue, saturation, and lightness of the blood sample are measured by a light source for irradiating the blood sample and the imaging device. In the step of obtaining the converted number of days elapsed, the converted number of days elapsed is calculated using data indicating the relationship between one or more values of hue, saturation, and lightness and the converted number of days elapsed.

5. The method for determining plasma separation conditions according to claim 4, wherein a white light source is used as the light source.

6. The method for determining plasma separation conditions according to claim 2, wherein as the blood state confirmation unit, an absorbance measurement device for measuring the absorbance of the blood sample is used. In the step of measuring, the absorbance of the blood sample is measured by a light source for irradiating the blood sample and the absorbance measurement device. In the step of obtaining the converted number of days elapsed, the converted number of days elapsed is calculated using data indicating the relationship between the absorbance value of the blood sample and the converted number of days elapsed.

7. The method for determining plasma collection conditions according to claim 6, wherein a blue light source is used as the light source. A method for determining plasma collection conditions.

8. The method for determining plasma collection conditions according to claim 2, wherein in the step of determining the plasma collection conditions, the suction and discharge rate, the suction stop position, and the conditions for adding a solution are made consistent. A method for determining plasma collection conditions.

9. The method for determining plasma collection conditions according to claim 2, further comprising the step of setting a threshold value for the amount of gDNA, and in the step of determining the plasma collection conditions, comparing the set threshold value with the calculated amount of gDNA to determine the plasma collection conditions. A method for determining plasma collection conditions.

10. An apparatus for collecting plasma from a blood sample, comprising: a collection machine for collecting the plasma from the blood sample; and a controller for obtaining the number of days elapsed from the blood collection date of the blood sample, calculating the amount of gDNA in the blood sample based on the number of days elapsed, and determining plasma collection conditions based on the calculated amount of gDNA. A plasma collection apparatus.

11. The plasma collection apparatus according to claim 10, further comprising a blood state confirmation unit for measuring the state of the blood sample, wherein the controller obtains a converted number of days elapsed from the blood collection date of the blood sample as the number of days elapsed based on the result of the measurement by the blood state confirmation unit, and calculates the amount of gDNA in the blood sample based on the converted number of days elapsed. A plasma collection apparatus.

12. A DNA extraction apparatus comprising the plasma collection apparatus according to claim 10.

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