Specimen inspection method and specimen inspection system

Abstract

This specimen inspection method comprises: a prediction step for predicting the amount of gDNA mixed in collected blood on the basis of the number of days elapsed since blood collection and data indicating the relationship between the number of days elapsed since blood collection and the amount of gDNA included in blood in accordance with the type of at least one blood collection tube; and a determination step for determining a processing step of the collected blood on the basis of the amount of gDNA to be mixed predicted in the prediction step. Thus, it is possible to provide a specimen inspection method and a specimen inspection system that achieve accurate and rapid results as compared to prior art by reducing the time until an inspection result is obtained and the amount of substances required for inspection as compared to prior art.

Description

Sample testing method and sample testing system

[0001] The present invention relates to a sample testing method and a sample testing system for recovering nucleic acids from blood and analyzing the nucleic acids.

[0002] Non-Patent Document 1 describes the results of examining the preservation ability of blood cell collection tubes to prevent gDNA contamination due to white blood cell rupture, and evaluating the performance when combined with highly sensitive mutation detection technology.

[0003] Yunlong Zhao et al., “Performance comparison of blood collection tubes as liquid biopsy storage system for minimizing cfDNA contamination from genomic DNA”, J Clin Lab Anal. 2019 Feb;33(2):e22670.

[0004] In recent years, information obtained by nucleic acid analysis, such as cancer genome testing using next-generation sequencing (NGS) systems and digital polymerase chain reaction (PCR), has been utilized in various fields, including medical care, clinical testing, the pharmaceutical industry, and the food industry.

[0005] Nucleic acid extraction from various biological samples such as blood, tissue, and cultured cells is an essential pretreatment step for nucleic acid analysis, and it is known that the quality of the extracted nucleic acid has a significant impact on the analytical process.

[0006] Common nucleic acid extraction methods do not use harmful organic solvents such as phenol or chloroform, but rather rely on the property of nucleic acids to bind to silica in the presence of a chaotropic agent or an organic solvent.

[0007] Using these methods, nucleic acid extraction methods using a nucleic acid capture chip incorporating a silica-containing solid phase as a nucleic acid capture carrier, and methods using magnetic beads (nucleic acid capture carriers) with silica-coated surfaces have been reported. These methods include a step of binding nucleic acids to the nucleic acid capture carrier and an elution step of eluting the nucleic acids from the nucleic acid capture carrier using an eluent.

[0008] For example, non-invasive prenatal genetic testing (NIPT) in obstetrics is a useful method for extracting nucleic acids from blood and detecting chromosomal abnormalities in the fetus.

[0009] However, it has been reported that the quality of the blood sample in NIPT may affect the accuracy of the test (see, for example, Non-Patent Document 1).

[0010] Specifically, it has been reported that the concentrations of free hemoglobin and potassium ions in the blood may increase, and that these changes may affect the accuracy and reliability of NIPT, suggesting that quality control of blood samples is important when performing NIPT in clinical laboratories.

[0011] As a quality control step after nucleic acid extraction from blood, the concentration and amount of nucleic acid and the content of impurities are measured by measuring absorbance using a spectrophotometer or by measuring fluorescence intensity after staining the nucleic acid with a fluorescent dye. Indicators of good chemical purity of DNA are an A260 / A280 value of 1.8-2.0 and an A260 / A230 value of greater than 1.0.

[0012] Furthermore, if the solvent in which the DNA is dissolved contains EDTA (ethylenediaminetetraacetic acid), ethanol, phenol, etc., these may inhibit the reaction of the NGS library preparation reagent. Please refer to the manufacturer's instructions for use, and take care to avoid any substances that are recommended to be avoided from being included in the solvent.

[0013] In some cases, the state of degradation of nucleic acids can be confirmed by observing the length and length distribution of the nucleic acids using electrophoresis.

[0014] Cell-free DNA (cfDNA) is sometimes extracted from plasma or serum to analyze DNA derived from cells destroyed by the immune system or from cells undergoing apoptosis (self-death). Most extracted cfDNA is 140-200 bp in length. If high-molecular-weight DNA is detected, it is suspected to be contaminated with genome DNA (gDNA) derived from nucleated cells, making it unsuitable for cfDNA analysis.

[0015] High molecular weight DNA can be fragmented by physical impact, so care must be taken when mixing the solution, such as by gently tapping with your fingers. Repeated freezing and thawing should also be avoided whenever possible. Improper storage conditions (such as vibration or prolonged storage) after blood collection can cause blood cells to break down, resulting in the loss of gDNA.

[0016] A variety of specialized blood collection tubes are manufactured and sold to prevent blood cells from breaking down, but at present, they can only preserve blood cells for around two weeks, which is a limit.

[0017] Generally, 1-50 ng of cfDNA can be recovered from 1 mL of plasma. Although this varies depending on the progression of the disease, excessive yields are unsuitable for cfDNA analysis because they may contain gDNA derived from nucleated cells. When using frozen DNA stock solutions for NGS library preparation, it is recommended that they be quantified immediately before use.

[0018] However, if we were to collect samples from various regions at a testing center and conduct testing, it would be difficult to conduct testing on the same day as the blood was collected, and a certain storage period and transportation would be required after collection. As a result, the presence of gDNA contamination would only be detected by performing QC after nucleic acid extraction, and unnecessary nucleic acid extraction and NGS would be performed, resulting in a waste of reagents used for nucleic acid extraction and testing, as well as the associated costs. In addition, there is a significant loss of time due to the need to retest, during which the patient's condition could progress.

[0019] The present invention provides a sample testing method and system that achieves more accurate and rapid results than conventional methods by reducing the time required to obtain test results and the amount of material required for testing.

[0020] The present invention includes multiple means for solving the above-mentioned problems, and one example thereof includes a prediction step of predicting the amount of gDNA that will be mixed into the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood and the number of days that have passed since blood collection, depending on the type of at least one blood collection tube, and a determination step of determining a processing step for the collected blood based on the amount of gDNA that will be mixed in predicted in the prediction step.

[0021] According to the present invention, it is possible to obtain more accurate and rapid results than in the past by reducing the time required to obtain test results and the amount of material required for the test compared to the past. Objects, configurations, and effects other than those described above will become apparent from the following description of the examples.

[0022] 4 and 5. A diagram showing an overview of the specimen testing system of Example 1. A flowchart explaining the processing performed by the management system in the specimen testing system of Example 1. A diagram showing the relationship between the gDNA concentration contained in plasma and the number of days elapsed since blood collection of a specimen in a certain blood collection tube. A diagram showing the relationship between the gDNA concentration contained in plasma and the number of days elapsed since blood collection of a specimen in a blood collection tube different from that of FIG. 3. A diagram showing the relationship between the gDNA concentration contained in plasma and the number of days elapsed since blood collection of a specimen in a blood collection tube different from that of FIGS. 3 and 4. A diagram showing the relationship between the absorbance of plasma and the number of days elapsed since blood collection of a specimen in the blood collection tube of FIG. 3. A diagram showing the relationship between the absorbance of plasma and the number of days elapsed since blood collection of a specimen in the blood collection tube of FIG. 4. A diagram showing the relationship between the absorbance of plasma and the number of days elapsed since blood collection of a specimen in the blood collection tube of FIG. 5. A diagram showing an example of the correlation between the absorbance and the amount of gDNA mixed in for each type of blood collection tube. A diagram showing the relationship between the saturation and the amount of gDNA of a plasma specimen in a blood collection tube. A diagram showing the relationship between the brightness and the amount of gDNA of a plasma specimen in a blood collection tube. A diagram showing an overview of the specimen testing system of Example 2. FIG. 1 is a diagram showing sample conditions used to investigate the effect of gDNA contamination on mutation detection. FIG. 2 is a diagram showing evaluation results of differences in mutation rate (VAF: Variant Allele Frequency) relative to the amount of gDNA contamination. FIG. 3 is a diagram showing evaluation results of differences in mutation rate (VAF) relative to the amount of gDNA contamination. FIG. 4 is a diagram showing evaluation results of differences in mutation rate (VAF) relative to the amount of gDNA contamination. FIG. 5 is a diagram showing an overview of the specimen testing system of Example 3. FIG. 6 is a diagram showing an example of a consumables ordering screen in the specimen testing system of Example 3.

[0023] The following describes examples of the sample testing method and sample testing system of the present invention with reference to the drawings. In the drawings used in this specification, identical or similar reference numerals are used to designate identical or corresponding components, and repeated explanations of these components may be omitted.

[0024] Example 1 A sample testing method and sample testing system according to Example 1 of the present invention will be described with reference to FIGS. 1 to 11. FIG.

[0025] First, the overall configuration of a specimen testing system for testing specimens made of blood will be described with reference to Fig. 1. Fig. 1 is a diagram showing an outline of the specimen testing system of the first embodiment.

[0026] The specimen testing system 1 shown in Figure 1 is a system for testing specimens consisting of blood, and includes a plasma collection section 11, a blood condition confirmation section 14, a nucleic acid extraction section 17, a nucleic acid extraction QC section 20, an assay setup section 23, a post-setup QC section 26, a measurement section 29, a data analysis section 32, and a management system 35.

[0027] This specimen testing system 1 solves the problem of unnecessary NGS and nucleic acid extraction being performed because gDNA contamination during cfDNA extraction reduces the sensitivity of mutation detection and makes it impossible to detect.

[0028] More specifically, the state of hemolysis, in which red blood cells in the blood are destroyed and hemoglobin, the blood pigment, is eluted, is measured using an image of color information such as red, green, and blue, or color information such as saturation, hue, and brightness, and the absorbance of hemoglobin at around 410 to 430 nm.This indirectly detects the leakage of gDNA due to the destruction of white blood cells based on the state of hemoglobin elution and the state of red blood cells being destroyed, and calculates the amount of unnecessary gDNA contained during nucleic acid extraction and the unnecessary components that will affect the enzymatic reaction after nucleic acid extraction, thereby determining whether or not to perform the nucleic acid extraction step and NGS step, thereby solving the above-mentioned problems.

[0029] The plasma collection unit 11 is a mechanism for collecting plasma from blood, and is composed of, for example, various mechanisms capable of collecting liquids. The plasma collection unit 11 preferably performs the plasma collection step of collecting plasma from blood.

[0030] The blood condition confirmation unit 14 is a mechanism for confirming the condition of the blood in the blood collection tube, and is composed of, for example, an image sensor such as a CCD camera arranged so as to be able to capture an image of the entire blood collection tube, and an illuminator that irradiates the blood collection tube with light when capturing an image of the blood collection tube. The illuminator is preferably, but not limited to, a white light or blue light source. This blood condition confirmation unit 14 preferably executes a blood condition confirmation step that confirms the condition of the blood.

[0031] The nucleic acid extraction unit 17 is a mechanism for extracting nucleic acids from plasma, and a known configuration can be adopted for the configuration thereof. The nucleic acid extraction unit 17 preferably performs the nucleic acid extraction step of extracting nucleic acids from plasma.

[0032] The nucleic acid extraction QC unit 20 is a mechanism for confirming the quality of nucleic acids and determining the amount of nucleic acids in blood from the nucleic acid quality, and includes a configuration for performing fluorescence detection. In addition to the fluorescence detection configuration, it also includes a configuration for confirming the length of nucleic acids by performing electrophoresis or the like. This nucleic acid extraction QC unit 20 preferably performs a nucleic acid quality confirmation step for confirming the quality of nucleic acids by fluorescence detection, electrophoresis, or real-time PCR and determining the amount of nucleic acids in blood from the nucleic acid quality, or a nucleic acid quality confirmation step for confirming the quality of nucleic acids and calculating the amount of gDNA contained in blood from the nucleic acid quality.

[0033] The assay setup section 23 is a mechanism for setting up the reagents used in the measurement.

[0034] The post-setup QC section 26 is a mechanism for performing checks before measurement by the measurement section 29 .

[0035] The measurement unit 29 is a mechanism for performing nucleic acid measurement, specifically at least one of NGS and dPCR, and this measurement unit 29 is preferably the main body that performs the measurement step of measuring nucleic acids.

[0036] The data analysis unit 32 is a mechanism for analyzing measurement results of at least one of NGS and dPCR. The data analysis unit 32 may be part of the management system 35.

[0037] The management system 35 is a part that manages the specimen testing system 1 by controlling the operations of the plasma fractionation unit 11, blood condition confirmation unit 14, nucleic acid extraction unit 17, nucleic acid extraction QC unit 20, assay setup unit 23, post-setup QC unit 26, measurement unit 29, and data analysis unit 32, and is composed of a display device such as an LCD display (preferably displaying a request screen 350 in FIG. 18 , which will be described later), an input device (preferably capable of operating the request screen 350 in FIG. 18 , which will be described later), a storage device consisting of a recording medium such as an HDD or SSD and its controller, a CPU, memory, etc. The control of the operation of each device by the management system 35 is executed based on various programs recorded in the storage device.

[0038] The control processes for the operations executed by the management system 35 may be integrated into one program, or may be divided into multiple programs, or may be a combination of these. Furthermore, some or all of the programs may be realized by dedicated hardware or may be modularized.

[0039] In this embodiment, the management system 35 predicts the amount of gDNA that will be present in the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood, and the number of days since blood collection, for at least one type of blood collection tube, and determines the processing step in either or both of the plasma fractionation unit 11 and the nucleic acid extraction unit 17 based on the predicted amount of gDNA. Note that the "processing step" in the present invention includes the plasma fractionation process in the plasma fractionation unit 11 and the nucleic acid extraction process in the nucleic acid extraction unit 17.

[0040] In addition, in this embodiment, the management system 35 predicts the amount of gDNA from data showing the relationship between the results obtained by the blood condition confirmation unit 14 for each type of blood collection tube and the amount of gDNA contained in the blood, and calculates the amount of nucleic acids other than gDNA contained in the blood by subtracting the predicted amount of gDNA from the amount of nucleic acid determined by the quality confirmation unit.

[0041] These will be explained in more detail below.

[0042] This management system 35 preferably executes, depending on the type of at least one blood collection tube, a prediction step of predicting the amount of gDNA to be mixed into the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood and the number of days since blood collection; a determination step of determining a processing step for the collected blood based on the amount of gDNA to be mixed in predicted in the prediction step; a prediction step of predicting the amount of gDNA from data showing the relationship between the results obtained in the blood condition confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood; and a nucleic acid amount calculation step of calculating the amount of nucleic acids other than gDNA contained in the blood by subtracting the amount of gDNA predicted in the prediction step from the amount of nucleic acid determined in the nucleic acid quality confirmation step.

[0043] Next, a process for estimating the state of gDNA contamination based on the state of whole blood or plasma and determining whether or not nucleic acid extraction is necessary, which is preferably executed by the management system 35 of the specimen testing system 1 shown in Fig. 1, will be described with reference to Fig. 2. Fig. 2 is a flowchart illustrating the process performed by the management system 35 in the specimen testing system 1 of Example 1.

[0044] As shown in FIG. 2, first, as a preparation step, known data is constructed and recorded in the management system 35 (S100).

[0045] More specifically, data on the number of days since blood collection and the amount of gDNA contamination after nucleic acid extraction are acquired as known data for each type of blood collection tube. Because the additives used to protect each blood cell component differ depending on the type of blood collection tube, and therefore the recommended storage conditions vary, data must be accumulated in advance for each type of blood collection tube. This data accumulation may be performed by the manufacturer of the management system 35 and provided to the user, or the user may perform it independently. Because storage conditions, blood collection tube transportation conditions, and nucleic acid extraction reagent performance vary from user to user, more accurate management can be achieved by performing the data themselves.

[0046] Next, the management system 35 acquires information on the type of blood collection tube (S105), after which the process proceeds to step S110, step S115, or step S120 depending on the setting of the detection process.

[0047] The information on the type of blood collection tube in step S105 can be obtained by the user visually inspecting the blood collection tube and inputting the information into the system, or by using an image sensor used in the step of checking the blood condition in step S115 or step S120 described below; methods for determining the type of blood collection tube are not limited to these.

[0048] Next, the management system 35 calculates the amount of gDNA from the known data and the number of days since blood collection. In this embodiment, the process is divided into the following steps: 1) calculate the amount of gDNA from data relating the number of days since blood collection and the amount of gDNA (S110); and 2) confirm the blood condition and calculate the amount of gDNA from data relating the number of days since blood collection calculated from the blood condition and the amount of gDNA (S115 or S120 → S125 → S130, or S115 or S120 → S135). The following description will be given with reference to Figures 3 to 8. Figures 3 to 5 show the relationship between the DNA concentration and the number of days since blood collection for a sample in a certain blood collection tube, and Figures 6 to 8 show the relationship between the absorbance and the number of days since blood collection for the sample in the blood collection tube shown in Figures 3 to 5.

[0049] Both 1) and 2) correspond to a prediction step of predicting the amount of gDNA mixed into the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood and the number of days since blood collection, depending on the type of at least one blood collection tube, or predicting the amount of gDNA from data showing the relationship between the results obtained in the blood condition confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood.

[0050] In this prediction step, the number of days since the pseudo-blood collection is calculated from data showing the relationship between the results obtained in the blood condition confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood, and the number of days since the pseudo-blood collection is used to predict the amount of gDNA. Details of this will be described later.

[0051] First, in the case of processing 1) (S110), the management system 35 calculates the amount of gDNA in the blood in the target blood collection tube from the data relating to the recorded amount of DNA and the number of days elapsed since blood collection (data such as those in Figures 3, 4, and 5), and proceeds to step S140.

[0052] Specifically, the data on the number of days since blood collection and the amount of gDNA linked to the blood collection tube are shown in Figure 3 (the data on the number of days since blood collection and the amount of gDNA are used to calculate the amount of gDNA).

[0053] For example, assuming that the amount of gDNA contamination is to be kept below 1 ng per 1 mL of plasma, a dotted line is drawn at the threshold value in FIG. 3 or 4.

[0054] Here, since the amount of gDNA to be suppressed differs depending on the various assays used in subsequent steps, such as NGS and dPCR, a step of setting a threshold for the amount of gDNA contained in the blood (setting step) can be executed between the start of step S100 described above and step S140 described below.

[0055] For the data shown in FIG. 3 or 4, which are indicated by dotted lines, it is possible to estimate the amount of gDNA obtained at certain days after blood collection using an approximation formula such as linear or exponential approximation.

[0056] On the other hand, in the case of process 2) (S115 or S120 → S125 → S130, or S115 or S120 → S135), the management system 35 checks the condition of the blood in the target blood collection tube using the blood condition checking unit 14. To check the blood condition, the management system 35 measures plasma absorbance (S115) or measures saturation, hue, and brightness (S120). Step S115 or step S120 corresponds to the blood condition checking step for checking the blood condition.

[0057] First, the flow of measurement using absorbance (S115) will be described.

[0058] The blood condition confirmation unit 14 measures the absorbance of the plasma or serum to be analyzed (S115). Prior to the measurement, the plasma or serum is obtained by centrifuging the collected blood and separating the supernatant components using the plasma separation unit 11.

[0059] Thereafter, the management system 35 calculates the pseudo number of days since blood collection for the known data from the newly measured absorbance value of the sample to be analyzed, using an approximation formula obtained from data such as any one of Figures 6 to 8, which shows the relationship between the known number of days since blood collection and the known number of days since blood collection for plasma or serum, etc., and absorbance (S125). The calculated pseudo number of days since blood collection is applied to the approximation formula such as Figure 6 to calculate the amount of gDNA (S130), and the process proceeds to step S140.

[0060] Alternatively, the management system 35 calculates the amount of gDNA from the absorbance measurement results based on the data described above in S110, using known data and the blood condition measurement results (S135), and proceeds to step S140.

[0061] In this way, it becomes possible to calculate the amount of contaminated gDNA based on the absorbance obtained.

[0062] Next, the flow (S125) using saturation, hue, and brightness will be described.

[0063] In this case, the blood condition confirmation unit 14 measures one or more values ​​of saturation, hue, and brightness of the plasma or serum to be analyzed (S120). Prior to the measurement, the plasma or serum is obtained by centrifuging the collected blood and separating the supernatant components using the plasma separation unit 11.

[0064] Thereafter, the management system 35 calculates the amount of gDNA contamination from the relationship between the known "saturation, hue, and brightness" and the gDNA amount data shown in Figures 10 and 11 (S135), and proceeds to step S140. Figure 10 shows the relationship between the saturation and the gDNA amount of the specimen in the blood collection tube, and Figure 11 shows the relationship between the brightness and the gDNA amount of the specimen in the blood collection tube.

[0065] For example, assuming that the amount of gDNA contamination is to be kept to 1 ng or less per mL of plasma, a hue of 45 or less would be considered to indicate a gDNA contamination of 1 ng / mL or more, based on the data in Figure 10. For more strict implementation, the data in Figure 11 may also be used, with a hue of 42 or less and a brightness of 0.6 or less. The calculation is an example, and a threshold may also be set based on the contribution rate by performing principal component analysis.

[0066] Incidentally, since "saturation, hue, and brightness," including the relationships shown in Figures 10 and 11, depend on the measurement environment, such as lighting and background color, it is desirable to standardize the measurement environment or to obtain them for each measurement environment.

[0067] Alternatively, the management system 35 may calculate the number of days since the pseudo-blood sampling from the known data and the blood condition measurement results based on the data described above in S125 and S130 from the measurement results of "saturation, hue, and brightness" (S125), and then apply this calculated number of days since the pseudo-blood sampling to an approximation formula such as that shown in Figure 6 to calculate the amount of gDNA (S130).

[0068] Thereafter, the amount of gDNA determined by any one of steps S110, S130, and S135 is compared with a predetermined amount (e.g., whether it is 1 ng or less) to determine the process step (S140). This step S140 corresponds to a determination step for determining the processing step for the collected blood based on the amount of gDNA contaminating the collected blood predicted in the prediction step. In this determination step, the amount of gDNA contaminating the collected blood predicted in the prediction step is compared with a threshold value to determine the processing step.

[0069] For example, if it is determined that the level is less than 1 ng, the plasma is fractionated and re-fractionated and then the procedure moves to nucleic acid extraction; if it is determined that the level is 1 ng or more, abnormal treatment can be performed, such as not extracting nucleic acid, extracting nucleic acid but using it for another purpose, or drawing blood again.

[0070] Next, in the management system 35, the plasma fractionating unit 11 fractionates or re-fractions plasma from the blood (plasma fractionating step), and the nucleic acid extracting unit 17 extracts nucleic acid from the plasma (nucleic acid extracting step).

[0071] There is no particular restriction on the method for extracting nucleic acids, and for example, harmful organic solvents such as phenol and chloroform have been used in the past. In recent years, methods based on the property of nucleic acids to bind to silica in the presence of a chaotropic agent or an organic solvent have become common.

[0072] Using these methods, nucleic acid extraction methods using a nucleic acid capture chip incorporating a silica-containing solid phase as a nucleic acid capture carrier, and methods using magnetic beads (nucleic acid capture carriers) with silica-coated surfaces have been reported. These methods include a step of binding nucleic acids to the nucleic acid capture carrier and an elution step of eluting the nucleic acids from the nucleic acid capture carrier using an eluent.

[0073] In the method using magnetic beads, after the elution step, the magnetic beads are recovered from the eluent using a magnet. One example is a method in which the eluent containing the magnetic beads is drawn into a dispensing tip, the magnetic beads are retained in the dispensing tip using a magnet, and only the eluent is discharged from the dispensing tip. Another example is a method in which a rod-shaped magnet (which may be covered) is inserted into the eluent containing the magnetic beads and the magnetic beads are recovered from the eluent.

[0074] As a quality control step after nucleic acid extraction, the management system 35 uses the nucleic acid extraction QC unit 20 to measure the concentration and amount of nucleic acid and the amount of impurities by measuring absorbance using a spectrophotometer or by staining the nucleic acid with a fluorescent dye and measuring fluorescence intensity. Nucleic acid quality control indicators can include quantity, length, purity, and structural integrity. This step corresponds to the nucleic acid quality confirmation step, which confirms the quality of the nucleic acid and determines the amount of nucleic acid in the blood from the nucleic acid quality.

[0075] Indicators of good chemical purity of DNA are an A260 / A280 ratio of 1.8-2.0 and an A260 / A230 ratio of greater than 1.0. Furthermore, if the solvent in which the nucleic acids are dissolved contains EDTA, ethanol, phenol, etc., it may inhibit the reaction of the NGS library preparation reagent. Refer to the manufacturer's instructions for use and take care to avoid any substances recommended for avoidance in the solvent.

[0076] Furthermore, since the amount of nucleic acid measured here is the combined amount of cfDNA and gDNA, the amount of cfDNA may be calculated by subtracting the amount of gDNA predicted in the prediction step. This step corresponds to the nucleic acid amount calculation step in which the amount of nucleic acid other than gDNA contained in the blood is calculated by subtracting the amount of gDNA predicted in the prediction step from the amount of nucleic acid determined in the nucleic acid quality confirmation step.

[0077] The amount of gDNA contamination can be confirmed by observing the length and length distribution of nucleic acids using, for example, electrophoresis. More specifically, cfDNA has a distribution with a peak around 140-180 bp, while gDNA has a distribution with a peak around several tens of kbp. Depending on the state of degradation, the length distribution may be lower than that. In other words, detection by electrophoresis makes it possible to confirm the state of degradation of nucleic acids.

[0078] There is also a method for evaluating nucleic acid quality using the Ct value (or Cq value) obtained by real-time PCR as an indicator of structural integrity. Specifically, primers are prepared and reacted to generate two PCR amplification products of different lengths. For example, a method is used in which the difference in Ct value (ΔCt value or ΔCq value) obtained from amplicon size (e.g., a short amplicon of approximately 50 to 100 bp and a long amplicon of approximately 100 to 300 bp) is used as an indicator. This method confirms that the obtained nucleic acid will not be amplified if it has been decomposed for some reason or if the double-stranded DNA has been partially nicked (partially fragmented), thereby confirming the structural integrity of the nucleic acid to determine whether it can be used in subsequent steps.

[0079] Although several methods for confirming the quality of nucleic acids have been mentioned above, they are not limited to these. Furthermore, although gDNA has been mentioned as the quality to be confirmed, this is not limited to these. Quantity, length, purity, and structural integrity may also be used as indicators for judgment. If the amount of nucleic acid is insufficient, the length is short, the purity is low, or the structural integrity is low, the expected results will not be obtained in subsequent steps. Therefore, appropriate values ​​for such indicators are determined in step 0).

[0080] Furthermore, the quality information of the nucleic acid obtained here can be combined with information on the type of blood collection tube and blood condition such as hemolysis shown in Figures 3 to 11, and correlated information can be accumulated to accumulate data and update existing data.

[0081] For example, in preparation for a case where the blood collection tubes themselves are updated in a way that the management system 35 does not support (such as an extension of the storage period due to improvements in additives), the correlation between absorbance and the amount of gDNA mixed in will change, so new data showing examples of the correlation between absorbance and the amount of gDNA mixed in for each type of blood collection tube, as shown in Figure 9, can be created and recorded in a database within the management system 35 that can also be referenced by the customer, making it possible to respond to updates to the blood collection tubes themselves.

[0082] The nucleic acid quality confirmation step preferably further includes an improvement step of calculating the amount of gDNA contained in the sample and adding and correcting data used in the prediction step that indicates the relationship between the results obtained in the blood condition confirmation step and the amount of gDNA contained in the blood. It is also preferable to further include an accumulation step of performing the improvement step for each test community and accumulating data, using the data to calculate correlations and enable calculation of gDNA contamination even in unknown cfDNA collection tubes.

[0083] Here, the calculated amount of gDNA is compared with the amount of gDNA predicted in the prediction step, and if it is determined that the difference is greater than a certain threshold, it is possible that inappropriate processing has been performed in the steps after the plasma collection step.

[0084] For example, when collecting blood and centrifuging it to separate the supernatant, if nucleic acid extraction is performed after aspirating the lower layer, buffy coat, which is primarily composed of white blood cells, or the red blood cell layer, gDNA can be contaminated from the white blood cells. Possible causes of this include defects in the CCD camera used as the liquid level detection mechanism, the lighting used for image detection, or the pressure or capacitance sensor. Even if the liquid level is detected correctly, there may be problems with the mechanism used to separate plasma or serum, such as not operating in the correct position, not inserting the dispensing tip correctly, or using an inappropriate aspiration speed.

[0085] Therefore, if this process is performed using an automated device, the management system 35 can raise a flag and notify the user that there is an abnormality in the device, such as the imaging system that checks the dispensing or liquid phase height, and can instruct the user to check the abnormality as appropriate. Alternatively, the management system 35 can directly contact the manufacturer of the automated device. Contacting the manufacturer allows for remote repairs and the preparation of repair parts in advance, enabling a prompt repair response.

[0086] This process corresponds to an output step in which the calculated gDNA amount is compared with the gDNA amount predicted in the prediction step, and if it is determined that the difference is greater than a certain threshold, an abnormality is detected, or a step in which the gDNA amount predicted from the number of days elapsed since blood collection is compared with the gDNA amount predicted using the number of days elapsed since pseudo-blood collection, and if it is determined that the difference is greater than a certain threshold, an abnormality is detected.

[0087] Thereafter, the management system 35 executes assay setup and measurement using NGS and dPCR using the assay setup section 23, post-setup QC section 26, measurement section 29, and data analysis section 32 (measurement step for measuring nucleic acids).

[0088] The quality-confirmed nucleic acids are prepared for analysis using measuring devices such as next-generation DNA sequencers (NGS) and digital PCR.

[0089] For example, when analyzing cancer, one or more gene sequences related to cancer are amplified and adapters consisting of DNA sequences are attached to both ends. These adapters are used for measurement using next-generation DNA sequencers (NGS) or digital PCR. Therefore, the types and number of genes in the gene sequences to be amplified vary depending on the target analysis, and also vary depending on the measurement device used.

[0090] Furthermore, during preparation, appropriate preparation is checked by the steps performed by the nucleic acid extract QC unit 20. For example, it is checked whether there is an appropriate amount of nucleic acid before amplifying the gene sequence, whether there is an appropriate amount of nucleic acid after amplification, whether there is an appropriate amount of nucleic acid when adapters are attached and brought into various measuring devices, etc. If the amount is not appropriate, the previous step may be re-performed or the process may be interrupted as appropriate, and the management system may be used to ensure that the analysis is performed reliably by the measuring device.

[0091] Next, the effects of this embodiment will be described.

[0092] The specimen testing method of Example 1 of the present invention described above includes a prediction step of predicting the amount of gDNA to be mixed into the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood and the number of days since blood collection, depending on the type of at least one blood collection tube, and a determination step of determining a processing step for the collected blood based on the amount of gDNA to be mixed in predicted in the prediction step.

[0093] Furthermore, the specimen testing system 1 for testing specimens consisting of blood in Example 1 of the present invention described above comprises a plasma fractionation unit 11 that fractionates plasma from the blood, a nucleic acid extraction unit 17 that extracts nucleic acids from the plasma, a measurement unit 29 that measures the nucleic acids, a data analysis unit 32 that analyzes the measurement results from the measurement unit 29, and a management system 35 that manages the specimen testing system, and the management system 35 predicts the amount of gDNA that will be mixed into the collected blood based on data showing the relationship between the number of days since blood collection and the amount of gDNA contained in the blood and the number of days that have passed since blood collection, depending on the type of at least one blood collection tube, and determines a processing step in at least one of the plasma fractionation unit 11 and the nucleic acid extraction unit 17 based on the predicted amount of gDNA that will be mixed in.

[0094] This makes it possible to estimate the impact on nucleic acid extraction or the process after nucleic acid extraction based on information such as the appearance information obtained from the blood sample, the type of blood collection tube, and the number of days since blood collection.As a result, the amount of gDNA contamination can be determined before QC is performed after nucleic acid extraction, which reduces the number of unnecessary nucleic acid extractions and NGS performed compared to conventional methods, thereby avoiding costs, wasted test materials, and the time required for retesting.

[0095] The method also includes a blood condition confirmation step for confirming the condition of the blood, and in the prediction step, the number of days since the pseudo-blood collection is calculated from data showing the relationship between the results obtained in the blood condition confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood, and the number of days since the pseudo-blood collection is used to predict the amount of gDNA.This means that the amount of gDNA can be predicted with higher accuracy, making it possible to make more appropriate decisions for subsequent processes.

[0096] Furthermore, the method further includes a setting step for setting a threshold value for the amount of gDNA contained in the blood, and in the determination step, the processing step is determined by comparing the amount of gDNA predicted in the prediction step with the threshold value, thereby making it possible to make decisions that are tailored to the processing in the subsequent measurement step, and to realize measurements that are suited to the user's environment.

[0097] Furthermore, by further including a step of comparing the amount of gDNA predicted from the number of days elapsed since blood collection with the amount of gDNA predicted using the number of days elapsed since simulated blood collection, and outputting an abnormality if it is determined that the difference is greater than a certain threshold, it is possible to take action more quickly in response to the abnormality, thereby further reducing the amount of testing that goes to waste and further shortening the time until results are obtained.

[0098] Second Embodiment A sample testing method and a sample testing system according to a second embodiment of the present invention will be described with reference to FIGS. 12 to 16. FIG.

[0099] The specimen testing system 1A of this embodiment shown in Figure 12 is equipped with a management system 35A that not only calculates the amount of gDNA contamination from information on the blood condition, but also calculates a correct mutation detection rate by subtracting the influence of gDNA from the mutation detection value calculated based on data obtained by NGS or dPCR in a subsequent process. Figure 12 is a diagram showing an overview of the specimen testing system of Example 2.

[0100] In the management system 35A of the specimen testing system 1A of this embodiment, the influence of the gDNA amount predicted using the number of days since pseudo-blood collection can be subtracted from the mutation detection value processed in the processing step and calculated by NGS or dPCR (correction step).Furthermore, the influence of the gDNA amount predicted in the prediction step can be subtracted from the measured result (measurement step).

[0101] For this purpose, first, data on the effect of gDNA contamination on mutation detection is obtained. For example, as shown in Figure 13, standard sample cfDNA is mixed with varying amounts or ratios of gDNA, and the mixture is prepared using an NGS analysis preparation reagent, and data on the change in mutation detection rate when gDNA is present and when it is not present is obtained using a sequence decoding device.

[0102] FIG. 13 shows the preparation conditions of samples used to investigate the effect of gDNA contamination on mutation detection, and FIGS. 14 to 16 show the evaluation results of the difference in mutation rate relative to the amount of gDNA contamination.

[0103] 13, two samples are prepared at one mixing ratio. In addition, under conditions No. 7 to No. 10, a portion of the mixed solution is used as an analytical preparation reagent for NGS.

[0104] 14 to 16 are divided into three graphs, each showing the mutation rate initially contained in the standard sample cfDNA. The horizontal axis shows the gDNA mixing conditions described in Fig. 13, and the vertical axis shows the mutation rate detected by analysis using a sequence decoder.

[0105] 14 to 16, it can be seen that the mutation detection rate decreases as the amount of gDNA mixed in increases. Of particular note is the fact that in the EGFR gene shown in Figure 14, sites (bases) with a mutation rate of 1% or 2% are no longer detected depending on the amount of gDNA mixed in.

[0106] For example, mutations that are driver genes for cancer can be detected at a mutation rate of 1%, 2%, or even lower, allowing for early detection of cancer, postoperative testing, and monitoring during treatment, but the presence of gDNA contamination makes it impossible to perform appropriate testing.The data shown in Figure 14 shows that at mutation sites with a mutation rate of 3%, the mutation detection rate is roughly halved when an amount of gDNA equivalent to the amount of cfDNA is mixed in.

[0107] The impact of gDNA contamination on the mutation rate varies depending on the analytical preparation reagent. Therefore, when repeatedly using the same analytical preparation reagent for analysis, obtaining such data in advance allows for estimation of the amount of gDNA based on blood conditions such as hemolysis, and determining whether nucleic acid extraction or NGS analysis is necessary, thereby avoiding unnecessary work. While the above example uses analytical preparation reagents for NGS, this is not limiting. Analysis using digital PCR or capillary electrophoresis can also be applied to all analytical devices that are affected by the reduction in mutation rate due to gDNA contamination.

[0108] Such data will be acquired in advance and then analysis of unknown samples will be carried out.

[0109] In this embodiment, the process from obtaining blood collection tube information to the measurement step of measuring nucleic acids using a measurement device such as a next-generation DNA sequencer (NGS) or digital PCR is the same as that shown in the first embodiment.

[0110] After the measurement step, the management system 35A calculates the amount of mutation.

[0111] Next-generation DNA sequencers (NGS) can map the read base sequence to a reference genome sequence, thereby determining where mutations occur and comprehensively measuring mutations based on the number of mutated and unmutated molecules.

[0112] Digital PCR counts the number of molecules of each gene contained in a prepared sample. Specifically, the sample is distributed into multiple wells used for analysis, and PCR is performed in these wells to fluorescently detect the presence or absence of the target gene. Some wells contain one or more target genes, while others contain no target genes at all. Because target genes in a sample are distributed randomly, there is no guarantee that only one gene was distributed. Therefore, in addition to calculating the possibility of multiple distribution of targets, a Poisson model is used to apply a correction coefficient, and rather than simply counting the presence or absence, correction is made to quantify the number of target genes contained in the sample, thereby calculating the mutation amount and mutation rate of cancer genes.

[0113] Furthermore, the management system 35A can calculate the extent to which the mutation detection rate will decrease based on the amount of gDNA estimated in the process prior to measurement or the amount of gDNA actually measured in the quality control process, by comparing it with data on the gDNA contamination rate and its impact on the mutation rate obtained in advance as known data.

[0114] If the presence of an equal amount of gDNA relative to the amount of cfDNA reduces the detected mutations by half compared to the results of known data, the mutation amount and mutation rate calculated in the assay setup and measurement steps are corrected to the result of reducing them by half. The calculation may be performed by calculating an approximation formula for the above data. The approximation formula may be either linear or exponential.

[0115] Furthermore, the management system 35A can also review the separation conditions in the plasma separation unit 11, and make maintenance calls for the plasma separation unit 11, the blood condition confirmation unit 14, and the like.

[0116] The other configurations and operations are substantially the same as those of the specimen testing method and specimen testing system of the first embodiment described above, and details thereof will be omitted.

[0117] The sample testing method and sample testing system according to the second embodiment of the present invention also provides substantially the same effects as those of the sample testing method and sample testing system according to the first embodiment described above.

[0118] Furthermore, by further including a correction step in which the treatment is carried out through a treatment process including plasma collection in the plasma collection unit 11 and nucleic acid extraction in the nucleic acid extraction unit 17, and the influence of the gDNA amount predicted using the number of days since the pseudo-blood collection is subtracted from the mutation detection value calculated by NGS or dPCR, the accuracy of determining whether or not to carry out subsequent treatment processes and what type of treatment process to carry out can be improved with each test, making it possible to construct a testing method and testing system that can achieve daily improvements in testing accuracy.

[0119] Third Embodiment A sample testing method and a sample testing system according to a third embodiment of the present invention will be described with reference to FIGS. 17 and 18. FIG.

[0120] The specimen testing systems 1, 1A and specimen testing methods of Examples 1 and 2 show examples in which a series of testing steps are performed in a system located within a single analytical facility, whereas the specimen testing system 1B and specimen testing method of this embodiment show a form in which a series of testing steps are performed across multiple facilities.

[0121] Figure 17 is a diagram showing an overview of a specimen testing system of Example 3. In the specimen testing system 1B of this example shown in Figure 17, a plasma fractionation unit 11, a blood condition confirmation unit 14, a nucleic acid extraction unit 17, a nucleic acid extraction QC unit 20, and part of a management system 35B are provided at a sample site 43 of a hospital or the like, and an assay setup unit 23, a post-setup QC unit 26, a measurement unit 29, a data analysis unit 32, and part of a management system 35B are provided at a test site 40 of a testing business or the like, consolidating some of the processes at the test site 40. Sample sites 46 and 49 have the same configuration as sample site 43 or have more or fewer components.

[0122] In the form shown in Figure 17, the management system 35B shares the start of work and work results at sample sites 43, 46, 49 of multiple hospitals, etc. over a network for the process up to nucleic acid extract QC, and performs consumable management such as analysis planning and prediction of consumables to be used at the test site 40.

[0123] In the case of a testing network in which samples are collected at test site 40 in this way, managing information about each sample (patient information, information about nucleic acid extraction results) becomes complicated. Therefore, the start of work and work results at sample sites 43, 46, and 49 are shared over the network using the functions of management system 35B, and this is linked to analysis planning and consumables management (prediction of consumables to be used) at test site 40.

[0124] For example, by configuring the management system 35B in cloud or server format, it can be connected via a network to sample sites 43, 46, 49, test site 40, and manufacturer site 52 that manufactures and sells consumables such as reagents and various testing parts.

[0125] This allows a sharing step to be executed in which the work status and work results of the processing step are shared over a network, or the work status and work results of one or more of the plasma fractionation step, blood condition confirmation step, nucleic acid extraction step, measurement step, nucleic acid quality confirmation step, prediction step, and nucleic acid quantity calculation step are shared over a network.

[0126] In addition, in this sharing step, consumables management including analysis planning and prediction of consumables to be used can be performed.

[0127] Specifically, by sharing the experimental preparation status at sample sites 43, 46, and 49, if the test passes QC, a notification such as email is sent to test site 40, which is the account for the next process, via cloud or server-based management system 35B. This notification makes it possible to prepare appropriate reagents and consumables for dsDNA, ssDNA, and RNA analysis, and to create efficient analysis plans in advance.

[0128] In other words, consumables can be checked when work begins at sample sites 43, 46, and 49 or when the work results are obtained, and in some cases, an alarm can be set in advance if there is a shortage of reagent consumables, allowing analysis to proceed without delay.

[0129] Figure 18 is a diagram showing an example of a consumables order screen in the specimen testing system of Example 3. For example, when "Consumables" is selected in tag area 355 of request screen 350 as shown in Figure 18, the user selects the target reagent, container, or other item in selection area 363 of target display area 360 while checking the number in remaining amount display area 366, enters the required number in order amount display area 370, and presses "Order" in instruction area 373, thereby placing an order for consumables on the cloud. Based on the received order, manufacturer site 52 can deliver the ordered consumables to test site 40 and sample sites 43, 46, and 49 before they are needed.

[0130] The management system 35B can also set a flag based on the trend of the results (QC results) in each process, thereby executing a warning step of setting a flag based on the trend of the relationship between any two of the plasma fractionation step, blood condition confirmation step, nucleic acid extraction step, measurement step, nucleic acid quality confirmation step, prediction step, and nucleic acid amount calculation step.

[0131] For example, it is possible to link patient information (clinical information, other test (tumor marker) results, blood collection tube, blood collection date, blood collection tube transportation date) with QC results (nucleic acid extraction results) and set a flag.

[0132] More specifically, if a correlation is found between tumor marker values ​​and blood conditions, or information on the amount and length of nucleic acid extracts, a flag can be raised and the analyst informed, which will enable further improvements in testing accuracy.

[0133] Alternatively, if a correlation is found between the blood collection tube and poor QC results, it is possible to detect incompatibility between the blood collection tube and the nucleic acid extraction reagent, or a faulty lot number for the nucleic acid extraction reagent or the blood collection tube lot number.This allows the testing of the blood specimen in question to be suspended or monitored closely, and re-sampling or other measures to be taken promptly.

[0134] Furthermore, based on the information on the blood collection date, the date of transport of the blood collection tube, the blood condition, and the amount and length of the nucleic acid extract, it is possible to flag any correlations with poor results such as poor blood collection conditions (inadequate mixing after collection) or poor transport conditions (temperature, vibration, etc.). This will lead to the adoption of more appropriate blood collection conditions, transport, etc.

[0135] The other configurations and operations are substantially the same as those of the specimen testing method and specimen testing system of the first embodiment described above, and details thereof will be omitted.

[0136] The sample testing method and sample testing system according to the third embodiment of the present invention also provides substantially the same effects as those of the sample testing method and sample testing system according to the first embodiment described above.

[0137] Furthermore, by further including a sharing step for sharing the work status and work results in the processing steps over a network, it becomes possible to cope with cases where each step is divided among a plurality of facilities.

[0138] Furthermore, in the shared step, by performing consumable management including analysis planning and forecasting of consumables to be used, it is possible to minimize the possibility of the test being interrupted due to a shortage of consumables, thereby enabling test results to be obtained more smoothly.

[0139] <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.

[0140] 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.

[0141] DESCRIPTION OF SYMBOLS 1, 1A, 1B... Sample testing system 11... Plasma fraction collection section 14... Blood condition confirmation section 17... Nucleic acid extraction section 20... Nucleic acid extraction QC section (quality confirmation section) 23... Assay setup section 26... Post-setup QC section 29... Measurement section 32... Data analysis section 35, 35A, 35B... Management system 40... Test site 43, 46, 49... Sample site 52... Manufacturer site 350... Request screen 355... Tag area 360... Target display area 363... Selection area 366... ​​Remaining amount display area 370... Order amount display area 373... Instruction area

Claims

1. A sample inspection method comprising: a prediction step of predicting the amount of gDNA mixed into the collected blood based on data showing the relationship between the number of days elapsed since blood collection and the amount of gDNA contained in the blood, and the number of days elapsed since blood collection, according to the type of at least one blood collection tube; and a determination step of determining a treatment process for the collected blood based on the amount of gDNA mixed predicted in the prediction step.

2. The sample inspection method according to claim 1, further comprising a blood state confirmation step of confirming the state of the blood, wherein in the prediction step, a pseudo blood collection elapsed days is obtained from data showing the relationship between the result obtained in the blood state confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood, and the amount of gDNA is predicted using the pseudo blood collection elapsed days.

3. The sample inspection method according to claim 2, further comprising a setting step of setting a threshold value for the amount of gDNA contained in the blood, wherein in the determination step, the treatment process is determined by comparing the amount of gDNA mixed predicted in the prediction step with the threshold value.

4. The sample inspection method according to claim 2, further comprising a step of comparing the amount of gDNA predicted from the number of days elapsed since blood collection with the amount of gDNA predicted using the pseudo blood collection elapsed days, and outputting an abnormal occurrence when it is determined that the difference is equal to or greater than a certain threshold value.

5. The sample inspection method according to claim 2, further comprising a correction step of subtracting the influence of the amount of gDNA predicted using the pseudo blood collection elapsed days from the mutation detection value calculated by NGS or dPCR after being processed by the treatment process.

6. The sample inspection method according to claim 1, further comprising a sharing step of sharing the working state and working results in the treatment process via a network.

7. The sample inspection method according to claim 6, wherein in the sharing step, consumable management including an analysis plan and prediction of consumables to be used is performed.

8. A system for examining a specimen consisting of blood, comprising: a plasma separation unit that separates plasma from the blood; a nucleic acid extraction unit that extracts nucleic acids from the plasma; a measurement unit that measures the nucleic acids; a data analysis unit that analyzes the measurement results obtained by the measurement unit; and a management system that manages the specimen examination system, wherein the management system predicts the amount of gDNA contaminating the collected blood based on data showing the relationship between the number of days elapsed since blood collection and the amount of gDNA contained in the blood and the number of days elapsed since blood collection, according to the type of at least one blood collection tube, and determines a processing step in one or more of the plasma separation unit and the nucleic acid extraction unit based on the predicted amount of contaminating gDNA. Specimen examination system.

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