WO2025141773A1 - Specimen testing method and specimen testing system - Google Patents

Abstract

The present invention comprises: a plasma isolation step for isolating plasma from blood; a blood condition checking step for checking the condition of the blood; a nucleic acid extraction step for extracting nucleic acids from the plasma; a measurement step for measuring the nucleic acids; a nucleic acid quality checking step for checking the quality of the nucleic acids and determining the amount of nucleic acids in the blood from the quality of the nucleic acids; a prediction step for predicting the amount of gDNA from data indicating the relationship between the result obtained in the blood condition checking step for each type of blood collection tube and the amount of gDNA contained in the blood; and a nucleic acid amount calculation step for 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 acids determined in the nucleic acid quality checking step. In this way, the time until when the test result is obtained and the amount of substances required for the testing are reduced as compared with conventional methods, thereby providing a specimen testing method and a specimen testing system that allow for obtaining results more accurately and more rapidly as compared with conventional ones.

Description

Sample testing method and sample testing system

The present invention relates to a specimen testing method and a specimen testing system for recovering nucleic acids from blood and analyzing the nucleic acids.

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

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.

In recent years, information obtained through nucleic acid analysis, such as cancer genome testing using next-generation sequencing (NGS) systems and digital PCR (Polymerase Chain Reaction), is being used in a variety of fields, including medicine, clinical testing, and the pharmaceutical and food industries.

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

Nucleic acid extraction methods generally do not use harmful organic solvents such as phenol or chloroform, but are based on the property of nucleic acids binding to silica in the presence of chaotropic agents or the property of nucleic acids binding to silica in the presence of organic solvents.

Using these methods, nucleic acid extraction methods using a nucleic acid capture chip that incorporates a silica-containing solid phase as a nucleic acid capture carrier, and methods using magnetic beads (nucleic acid capture carriers) whose surfaces are covered with silica have been reported. These methods include a step of binding nucleic acid to the nucleic acid capture carrier, and an elution step of eluting the nucleic acid from the nucleic acid capture carrier using an eluent.

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

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).

Specifically, the concentrations of free hemoglobin and potassium ions in the blood may increase, and it has been reported 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.

As a quality control process after nucleic acid extraction from blood, the concentration and amount of nucleic acid and the amount of impurities are measured by measuring absorbance using a spectrophotometer or by staining the nucleic acid with a fluorescent dye and measuring the fluorescence intensity. 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.

In addition, if the solvent in which the DNA is dissolved contains EDTA (ethylenediaminetetraacetic acid), ethanol, phenol, etc., it 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 specified to be avoided from being included in the solvent.

In some cases, the state of nucleic acid decomposition can be confirmed by observing the length and length distribution of the nucleic acids using electrophoresis.

Cell-free DNA (cfDNA) is extracted from plasma or serum, and may be present in the blood and may be derived from cells destroyed by the immune system or cells that have undergone apoptosis (cell death). Most extracted cfDNA is 140-200 bp in length. If high molecular weight DNA is found, it is suspected that gDNA (genome DNA) derived from nucleated cells may have been mixed in, and the sample is not suitable for cfDNA analysis.

Since high molecular weight DNA can be fragmented by physical impact, care must be taken when mixing the solution, such as by gently tapping with a finger. In addition, repeated freezing and thawing should be avoided whenever possible. If blood is not stored for an appropriate period or under appropriate conditions after collection (vibration, long period of time), the blood cells will break down and gDNA will leak out.

A variety of specialized blood collection tubes are manufactured and sold to prevent blood cells from breaking down. However, at present, all of them have a limit of how long they can preserve blood cells, at around two weeks.

Generally, 1-50 ng of cfDNA can be extracted from 1 mL of plasma. Although there is a range depending on the progression of the disease, if the yield is excessive, it is suspected that gDNA derived from nucleated cells may have been mixed in, and it is therefore unsuitable for cfDNA analysis. When using frozen DNA stock solution to prepare a library for NGS, it is advisable to quantify it immediately before use.

Here, if tests were to be conducted by collecting samples from various regions at a testing center, it would be difficult to conduct tests on the same day blood was collected, and a certain storage period and transportation would be required after blood was collected. As a result, the presence of gDNA contamination would only be noticed by performing QC after nucleic acid extraction, but 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 costs involved. In addition, there would be a significant loss of time due to the need to retest, during which the disease could progress.

The present invention provides a specimen 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.

The present invention includes multiple means for solving the above problems, and one example is a method for testing a sample consisting of blood, comprising a plasma separation step of separating plasma from the blood, a blood condition confirmation step of confirming the condition of the blood, a nucleic acid extraction step of extracting nucleic acid from the plasma, a measurement step of measuring the nucleic acid, a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and determining the amount of nucleic acid in the blood from the quality of the nucleic acid, 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 acid 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.

The present invention can provide 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. Problems, configurations, and effects other than those described above will become clear from the description of the examples below.

FIG. 1 is a diagram showing an overview of a specimen testing system according to a first embodiment. 4 is a flowchart illustrating a process performed by a management system in the sample testing system of the first embodiment. FIG. 1 is a graph showing the relationship between the gDNA concentration in plasma and the number of days since blood was collected from a sample in a certain blood collection tube. FIG. 4 is a graph showing the relationship between the gDNA concentration in plasma and the number of days elapsed since blood was collected from a sample in a blood collection tube different from that in FIG. 3 . FIG. 5 is a graph showing the relationship between the gDNA concentration in plasma and the number of days elapsed since blood was collected from a sample in a blood collection tube different from that in FIGS. 3 and 4 . FIG. 4 is a graph showing the relationship between the number of days elapsed since blood was collected from the blood collection tube in FIG. 3 and the absorbance of plasma. FIG. 5 is a graph showing the relationship between the number of days elapsed since blood was collected from the blood collection tube in FIG. 4 and the absorbance of plasma. FIG. 6 is a graph showing the relationship between the number of days elapsed since blood was collected from the blood collection tube in FIG. 5 and the absorbance of plasma. FIG. 13 is a graph showing an example of the correlation between absorbance and the amount of gDNA mixed in for each type of blood collection tube. FIG. 13 is a graph showing the relationship between saturation and gDNA amount of a plasma sample in a blood collection tube. FIG. 13 is a graph showing the relationship between the brightness of a plasma sample in a blood collection tube and the amount of gDNA. FIG. 13 is a diagram showing an overview of a specimen testing system according to a second embodiment. FIG. 1 shows sample conditions used to investigate the effect of gDNA contamination on mutation detection. A figure showing the results of evaluating the difference in mutation rate (VAF: Variant Allele Frequency) relative to the amount of gDNA contamination. FIG. 1 shows the results of evaluating the difference in mutation rate (VAF) relative to the amount of gDNA contamination. FIG. 1 shows the results of evaluating the difference in mutation rate (VAF) relative to the amount of gDNA contamination. FIG. 13 is a diagram showing an overview of a specimen testing system according to a third embodiment. FIG. 13 is a diagram showing an example of a consumables order screen in the sample testing system of the third embodiment.

Below, examples of the specimen testing method and specimen testing system of the present invention will be described with reference to the drawings. Note that in the drawings used in this specification, identical or corresponding components are given the same or similar reference numerals, and repeated explanations of these components may be omitted.

Example 1
A sample testing method and a sample testing system according to a first embodiment of the present invention will be described with reference to Figs. 1 to 11 .

First, the overall configuration of a specimen testing system for testing blood specimens will be described with reference to FIG. 1. FIG. 1 is a diagram showing an overview of the specimen testing system of the first embodiment.

The specimen testing system 1 shown in FIG. 1 is a system for testing specimens made of blood, and includes a plasma fractionation 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.

This specimen testing system 1 solves the problem of unnecessary NGS and nucleic acid extraction being performed because gDNA is mixed in during cfDNA extraction, reducing the sensitivity of mutation detection and making it impossible to detect.

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

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. This plasma collection unit 11 is preferably the entity that performs the plasma collection step of collecting plasma from blood.

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 lighting that irradiates light onto the blood collection tube when capturing an image of the blood collection tube. The lighting is preferably, but not limited to, a white light or blue light source. This blood condition confirmation unit 14 is preferably the entity that executes the blood condition confirmation step that confirms the condition of the blood.

The nucleic acid extraction unit 17 is a mechanism for extracting nucleic acid from plasma, and its configuration can be a known configuration. This nucleic acid extraction unit 17 preferably performs the nucleic acid extraction step of extracting nucleic acid from plasma.

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 quality of nucleic acids, and includes a configuration for performing fluorescence detection. In addition to the configuration for fluorescence detection, 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 quality of nucleic acids, 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 quality of nucleic acids.

The assay setup unit 23 is a mechanism for setting up the reagents used for measurement.

The post-setup QC unit 26 is a mechanism that performs checks before measurement by the measurement unit 29.

The measurement unit 29 is a mechanism that performs nucleic acid measurement, specifically, at least one of NGS and dPCR, and this measurement unit 29 is preferably the entity that performs the measurement step of measuring nucleic acid.

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

The management system 35 is a part that manages the specimen testing system 1 by controlling the operation of the plasma collection 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 displays the request screen 350 in FIG. 18 described later), an input device (preferably capable of operating the request screen 350 in FIG. 18 described later), a storage device consisting of a recording medium such as an HDD or SSD and its controller, a CPU, memory, etc. The management system 35 controls the operation of each device based on various programs recorded in the storage device.

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

In this embodiment, 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 since blood collection, depending on the type of at least one blood collection tube, and determines the processing step in either or both of the plasma collection unit 11 and the nucleic acid extraction unit 17 based on the predicted amount of gDNA that will be mixed in. Note that the "processing step" in this invention includes the plasma collection process in the plasma collection unit 11 and the nucleic acid extraction process in the nucleic acid extraction unit 17.

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.

These details will be given later.

This management system 35 preferably executes, depending on at least one type of blood collection tube, 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, a decision step of determining a processing step for the collected blood based on the amount of mixed gDNA 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 acid 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.

Next, the flow of estimating the gDNA contamination state based on the state of whole blood or plasma and determining whether or not nucleic acid extraction is required, 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 flow chart explaining the processing performed by the management system 35 in the specimen testing system 1 of Example 1.

As shown in Figure 2, the first preparation step is to build and record known data in the management system 35 (S100).

More specifically, data on the number of days since blood collection and the amount of gDNA contamination after nucleic acid extraction are obtained as known data for each type of blood collection tube. Since the recommended storage conditions differ depending on the type of blood collection tube due to differences in the ingredients of the additives that protect each blood cell component, it is necessary to accumulate data for each type of blood collection tube in advance. This data is accumulated by the manufacturer that provides this management system 35 and may be provided to the user, or the user may perform it independently. Since the storage conditions, transportation conditions of the blood collection tube, and the performance of the nucleic acid extraction reagent differ depending on each user, more accurate management is possible by performing it by the user himself.

Then, the management system 35 acquires information on the type of blood collection tube (S105). After that, the process proceeds to one of steps S110, S115, or S120 depending on the detection process settings.

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 to 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 by which the type of blood collection tube can be determined are not limited to these.

Next, the management system 35 calculates the amount of gDNA from the known data and the number of days since blood collection.
1) Calculate the amount of gDNA from the data on the relationship between the number of days since blood collection and the amount of gDNA (S110)
2) Confirm the blood condition, and calculate the amount of gDNA from the data on the relationship between 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 flow is divided into two parts. The following description will be given with reference to Figures 3 to 8. Figures 3 to 5 are diagrams showing the relationship between the DNA concentration and the number of days elapsed since blood collection of a specimen in a certain blood collection tube, and Figures 6 to 8 are diagrams showing the relationship between the absorbance and the number of days elapsed since blood collection of a specimen in the blood collection tube in Figures 3 to 5.

Note that 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 at least one type of 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.

In addition, 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 will be described later.

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 on the relationship between the recorded amount of DNA and the number of days since blood collection (data such as those in Figures 3, 4, and 5), and proceeds to step S140.

Specifically, Figure 3 shows the data on the number of days since blood collection and the amount of gDNA linked to the blood collection tube (the amount of gDNA is calculated using the data on the number of days since blood collection and the amount of gDNA).

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

Here, since the amount of gDNA to be suppressed differs depending on the various assays used in the 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 timing coinciding with the start of step S100 described above and step S140 described below.

By using an approximation formula such as linear or exponential approximation for the data shown in Figure 3 or Figure 4, which are dotted lines, it is possible to estimate the amount of gDNA obtained the number of days after blood collection.

In contrast, 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 the plasma absorbance (S115), or measures the saturation, hue, and brightness (S120). This step S115 or step S120 corresponds to the blood condition checking step for checking the blood condition.

First, we will explain the process of measuring using absorbance (S115).

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 components of the supernatant using the plasma separation unit 11.

Then, the management system 35 calculates the pseudo number of days since blood collection in the known data from the newly measured absorbance value of the analysis sample using an approximation equation obtained from data such as 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 equation such as Figure 6 to calculate the amount of gDNA (S130), and the process proceeds to step S140.

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

In this way, it is possible to calculate the amount of contaminated gDNA based on the absorbance obtained.

Next, we will explain the process (S125) using saturation, hue, and brightness.

In this case, the blood condition confirmation unit 14 also measures one or more of the 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 components of the supernatant using the plasma separation unit 11.

Then, 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 gDNA amount of the specimen in the blood collection tube, and Figure 11 shows the relationship between the brightness and gDNA amount of the specimen in the blood collection tube.

For example, assuming that it is desired to suppress the amount of gDNA contamination to 1 [ng] or less per 1 [mL] of plasma, then, for example, from the data in FIG. 10, a hue of 45 or less is set as gDNA contamination of 1 [ng/mL] or more. For more strict implementation, the data in FIG. 11 may also be used in combination, with a hue of 42 or less and a brightness of 0.6 or less. The calculation is one example, and a threshold value may also be set based on the contribution rate by performing principal component analysis.

In addition, 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 values for each measurement environment.

Alternatively, the management system 35 may calculate the pseudo number of days since blood collection from the known data and 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 calculate the amount of gDNA by applying this calculated pseudo number of days to an approximation formula such as that shown in Figure 6 (S130).

Then, the amount of gDNA determined by any one of steps S110, S130, or S135 is compared to a specified amount (e.g., whether it is 1 ng or less) to determine the process step (S140). This step S140 corresponds to a decision step that determines the processing step for the collected blood based on the amount of gDNA present predicted in the prediction step, and in this decision step, the amount of gDNA present predicted in the prediction step is compared to a threshold value to determine the processing step.

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

Next, in the management system 35, the plasma fractionation unit 11 fractionates or re-fractions plasma from the blood (plasma fractionation step), and the nucleic acid extraction unit 17 extracts nucleic acid from the plasma (nucleic acid extraction step).

There is no specific method for extracting nucleic acid. 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 acid binding to silica in the presence of chaotropic agents or the property of nucleic acid binding to silica in the presence of organic solvents have become common.

Using these methods, nucleic acid extraction methods using a nucleic acid capture chip that incorporates a silica-containing solid phase as a nucleic acid capture carrier, and methods using magnetic beads (nucleic acid capture carriers) with a silica-covered surface have been reported. These methods include a step of binding nucleic acid to the nucleic acid capture carrier, and an elution step of eluting the nucleic acid from the nucleic acid capture carrier using an eluent.

In the method using magnetic beads, after the elution step, a magnet is used to recover the magnetic beads from the eluent. 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 ejected 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.

In the management system 35, as a quality control step after nucleic acid extraction, the nucleic acid extraction QC section 20 measures 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 the fluorescence intensity. Indicators for nucleic acid quality control can include quantity, length, purity, and structural integrity. This step corresponds to the nucleic acid quality confirmation step, in which the quality of the nucleic acid is confirmed and the amount of nucleic acid in the blood is calculated from the quality of the nucleic acid.

Good indicators of DNA chemical purity are an A260/A280 value of 1.8-2.0 and an A260/A230 value of greater than 1.0. In addition, 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. Please refer to the manufacturer's instructions for use, and take care to avoid any substances that are specified to be avoided from being included in the solvent.

In addition, 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.

The amount of gDNA contamination can be confirmed by, for example, using electrophoresis to look at the length and length distribution of the nucleic acid. 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 decomposition, the distribution may be lower than that. In other words, detection by electrophoresis makes it possible to confirm the state of decomposition of nucleic acids.

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

The above describes several methods for checking the quality of nucleic acids, but the method is not limited to these. Also, gDNA is used as the quality to be checked, but this is not limited to these. The quantity, length, purity, and structural integrity may be used as indicators for judgment. If the amount of nucleic acid is insufficient, the length is short, the purity is low, and the structural integrity is low, the expected results will not be obtained in the subsequent steps. Therefore, appropriate values for such indicators are determined in step 0).

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, etc., shown in Figures 3 to 11, and correlated information can be accumulated to accumulate data and update existing data.

For example, in preparation for the case where there is an update to the blood collection tubes themselves that the management system 35 does not handle (such as an extension of the storage period due to improvements in additives), since the correlation between absorbance and the amount of mixed gDNA will change, new data showing examples of the correlation between absorbance and the amount of mixed gDNA 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 is also referenced by the customer side, making it possible to handle updates to the blood collection tubes themselves.

In such a nucleic acid quality confirmation step, it is desirable to calculate the amount of gDNA contained in the sample. Furthermore, regardless of the presence or absence of a nucleic acid amount calculation step, it is desirable to further have an improvement step of 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 desirable to further have an accumulation step of executing the improvement step on a test community basis and accumulating data, which uses the data to calculate a correlation and makes it possible to calculate the presence of gDNA even in unknown cfDNA collection tubes.

Here, the calculated gDNA amount is compared with the gDNA amount predicted in the prediction step, and if it is determined that the difference is equal to or exceeds a certain threshold, it is possible that inappropriate processing may have occurred in the steps following the plasma collection step.

For example, when collecting blood and centrifuging it to separate the components of the supernatant, if nucleic acid extraction is performed after aspirating the buffy coat, which is mainly composed of white blood cells, or the layer of red blood cells, which is the lower layer, gDNA can be mixed in from the white blood cells. Possible causes of this include, for example, a fault in the CCD camera used as the liquid level detection mechanism, the lighting used for image detection, or a fault in the pressure sensor or capacitance sensor. In addition, even if the liquid level is detected correctly, there may be a problem with the mechanism for separating plasma or serum, such as it not working in the correct position, the dispensing tip not being inserted correctly, or the aspirating speed being inappropriate.

If this process is being carried out using automated equipment, the management system 35 can raise a flag and inform the user that there is an abnormality in the equipment, such as the imaging system that checks the dispensing or liquid phase height, and can instruct the user to check the location of the abnormality as appropriate. Alternatively, the manufacturer of the automated equipment can be contacted directly. Contacting the manufacturer makes it possible to carry out repairs remotely or to prepare repair parts in advance, allowing for a rapid repair response.

This process corresponds to an output step of comparing the calculated gDNA amount with the gDNA amount predicted in the prediction step, and outputting the occurrence of an abnormality if it is determined that the difference is equal to or greater than a certain threshold, or a step of comparing the gDNA amount predicted from the number of days since blood collection with the gDNA amount predicted using the number of days since pseudo-blood collection, and outputting the occurrence of an abnormality if it is determined that the difference is equal to or greater than a certain threshold. This output step can be executed regardless of the presence or absence of the prediction step and the nucleic acid amount calculation step.

Then, the management system 35 executes assay setup and measurement using NGS and dPCR by the assay setup unit 23, post-setup QC unit 26, measurement unit 29, and data analysis unit 32 (measurement step for measuring nucleic acids).

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

For example, when analyzing cancer, one or more portions of a gene sequence related to cancer are amplified and adapters consisting of DNA sequences are added to both ends. These adapters are used when measuring with a next-generation DNA sequencer (NGS) or digital PCR. Therefore, the type and number of genes in the gene sequence to be amplified will vary depending on the analysis being performed, and will also vary depending on the measuring device used for the analysis.

In addition, when preparation is performed, appropriate confirmation is made through steps performed by the above-mentioned nucleic acid extract QC unit 20 to ensure that the preparation has been performed appropriately. For example, it is confirmed whether there is an appropriate amount of nucleic acid before the amplification of the gene sequence, whether there is an appropriate amount of nucleic acid after amplification, whether there is an appropriate amount of nucleic acid when an adapter is attached and brought into various measuring devices, etc. If the amount is not appropriate, the previous process may be repeated as appropriate or the process may be interrupted, and the management system may be used to control the process so that the analysis is reliably performed by the measuring device.

Next, we will explain the effects of this embodiment.

The method for testing a blood specimen according to the first embodiment of the present invention described above includes a plasma separation step of separating plasma from blood, a blood condition confirmation step of confirming the condition of the blood, a nucleic acid extraction step of extracting nucleic acid from plasma, a measurement step of measuring the nucleic acid, a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and determining the amount of nucleic acid in the blood from the quality of the nucleic acid, 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 acid 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.

The specimen testing system 1 for testing specimens consisting of blood according to the above-mentioned embodiment 1 of the present invention includes a plasma collection unit 11 for collecting plasma from blood, a blood condition confirmation unit 14 for confirming the condition of the blood, a nucleic acid extraction unit 17 for extracting nucleic acid from the plasma, a measurement unit 29 for measuring the nucleic acid, a quality confirmation unit for confirming the quality of the nucleic acid and determining the amount of nucleic acid in the blood from the quality of the nucleic acid, a data analysis unit 32 for analyzing the measurement results from the measurement unit 29, and a management system 35 for managing the specimen testing system. 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 acid 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.

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 the amount of gDNA contamination can be determined before QC is performed after nucleic acid extraction, unnecessary nucleic acid extraction and NGS can be reduced compared to conventional methods, and costs, wasted testing materials, and the time required for retesting can be avoided.

In addition, the nucleic acid quality confirmation step 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. This makes it possible to improve blood collection tubes and accommodate new types of blood collection tubes without having to replace or update the specimen testing system 1, making it easier to improve the testing procedure and easily further improve testing accuracy. Furthermore, it also becomes possible to reduce the economic burden on users for system updates.

Furthermore, in the nucleic acid quality confirmation step, the amount of gDNA contained in the sample is calculated, and the calculated amount of gDNA is compared with the amount of gDNA predicted in the prediction step. If it is determined that the difference is equal to or exceeds a certain threshold, an output step is further included to output an abnormality, allowing for quicker action to be taken in the area where the abnormality has occurred, and further shortening the time until results are obtained.

In addition, by further including an accumulation step in which the improvement step is executed for each testing community and data is accumulated, the amount of data that can be used as a judgment criterion can be dramatically acquired and accumulated, which makes it possible to further improve the accuracy of judgment.

Example 2
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.

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 blood condition information, but also calculates the correct mutation detection rate by subtracting the effect of gDNA from the mutation detection value calculated based on data obtained by NGS or dPCR in the subsequent process. Figure 12 is a diagram showing the overview of the specimen testing system of Example 2.

In the management system 35A of the specimen testing system 1A of this embodiment, regardless of the presence or absence of a prediction step and a nucleic acid amount calculation step, the influence of the gDNA amount predicted using the number of days since the pseudo blood collection can be subtracted from the mutation detection value calculated by NGS or dPCR after processing in the processing step (correction step). In addition, the influence of the gDNA amount predicted in the prediction step can be subtracted from the measured result (measurement step).

For this purpose, data on the effect of gDNA contamination on mutation detection is first obtained. For example, as shown in Figure 13, standard sample cfDNA is mixed with varying amounts or ratios of gDNA, and prepared using an NGS analysis preparation reagent, and data on the change in mutation detection rate when there is gDNA contamination compared to when there is no gDNA contamination is obtained using a sequence decoding device.

Figure 13 shows the preparation conditions for samples used to investigate the effect of gDNA contamination on mutation detection, and Figures 14 to 16 show the evaluation results of the difference in mutation rate depending on the amount of gDNA contamination.

In Figure 13, two samples are prepared with 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.

Figures 14 to 16 are divided into three graphs, each showing the mutation rate already contained in the standard sample cfDNA. The horizontal axis shows the gDNA mixing conditions described in Figure 13, and the vertical axis shows the mutation rate detected by analysis using a sequence decoding device.

As shown in Figures 14 to 16, the mutation detection rate decreases as the amount of gDNA mixed in increases. Of particular note is 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.

For example, mutations that are driver genes for cancer can be detected at a mutation rate of 1%, 2% or less, allowing for early detection of cancer, post-operative testing, and monitoring during treatment; however, the presence of gDNA contamination makes it impossible to carry out appropriate testing. The data shown in Figure 14 shows that at a mutation site 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.

The effect of such gDNA contamination on the mutation rate differs depending on the analysis preparation reagent. Therefore, when repeatedly using the same analysis preparation reagent for analysis, by obtaining such data in advance, it is possible to estimate the amount of gDNA from the state of the blood, such as hemolysis, and determine whether nucleic acid extraction or NGS analysis should be performed, thereby avoiding unnecessary work. The above example shows an analysis preparation reagent for NGS, but is not limited to this. Analysis using digital PCR or capillary electrophoresis can also be applied to all analysis devices that are affected by the decrease in mutation rate due to gDNA contamination.

This data will be collected in advance and used to analyze unknown samples.

In this embodiment, the process from obtaining blood collection tube information to the measurement step of measuring nucleic acids using a measuring device such as a next-generation DNA sequencer (NGS) or digital PCR is the same as that shown in embodiment 1.

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

Next-generation DNA sequencers (NGS) map the base sequence read to a reference genome sequence, making it possible to comprehensively measure where mutations occur and the number of molecules with and without mutations.

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, PCR is performed in these wells, and the presence or absence of the target gene is detected by fluorescence. Some wells contain one or more target genes, while others contain no target genes at all. Since the target genes in the sample are distributed randomly, there is no certainty that only one gene was distributed. Therefore, in addition to calculating the possibility of multiple targets being distributed, a correction coefficient is applied using a Poisson model to make corrections rather than simply counting the presence or absence, and by quantifying the number of target genes contained in the sample, the amount of mutations and mutation rate of cancer genes are calculated.

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, in comparison with data on the gDNA contamination rate and its effect on the mutation rate previously obtained as known data.

If the amount of mutations detected is halved from the known data results due to the presence of an equal amount of gDNA relative to the amount of cfDNA, then the amount of mutation or mutation rate calculated in the assay setup and measurement steps is corrected to a result that is halved. The calculation may be performed by calculating an approximation formula for the above data. The approximation formula may be either linear or exponential.

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 and blood condition confirmation unit 14, etc.

The rest of the configuration and operation are substantially the same as those of the specimen testing method and specimen testing system of the first embodiment described above, and details are omitted.

The specimen testing method and specimen testing system of the second embodiment of the present invention also provide substantially the same effects as those of the specimen testing method and specimen testing system of the first embodiment described above.

In addition, in the measurement step, by subtracting the effect of the gDNA amount predicted in the prediction step from the measured results, the accuracy of the decision as to whether or not to perform subsequent processing steps and what type of processing steps to perform can be improved in order to perform the test, making it possible to construct a testing method and testing system that can achieve daily improvements in testing accuracy.

Example 3
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.

In the specimen testing systems 1 and 1A and specimen testing methods of Examples 1 and 2, examples were shown in which a series of testing steps were performed in a system located within a single analytical facility, but 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.

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

In the configuration shown in FIG. 17, the management system 35B shares the start and results of work at sample sites 43, 46, and 49 in multiple hospitals, etc., over a network for the process up to nucleic acid extract QC, and executes consumables management such as analysis planning and forecasting of consumables to be used at the test site 40.

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

For example, by configuring management system 35B in a 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 test parts.

This executes a sharing step in which the work status and work results of the processing step are shared over the 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 amount calculation step are shared over the network.

In addition, this sharing step allows for analysis planning and consumable management, including forecasting of consumables to be used.

Specifically, by sharing the experimental preparation status at sample sites 43, 46, and 49, if the experiment passes QC, a message such as an email is sent to test site 40, which is the next process account, via cloud or server-type management system 35B. By receiving such a message, it becomes possible to prepare appropriate reagents and consumables for dsDNA, ssDNA, and RNA analysis, and to create an efficient analysis plan in advance.

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

FIG. 18 is a diagram showing an example of an order screen for consumables 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 FIG. 18, the user selects the selection area 363 of target display area 360 such as the target reagent or container 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 making it possible to order 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, 49 before they are needed.

The management system 35B can also set flags based on trends in the results (QC results) of each process. This allows for the execution of a warning step in which a flag is set based on trends in 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.

For example, it is possible to link patient information (clinical information, results of other tests (tumor markers), blood collection tube, blood collection date, blood collection tube transportation date) with QC results (nucleic acid extraction results) and set a flag.

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. This will enable further improvements in testing accuracy.

Alternatively, if a correlation is found between a blood collection tube and poor QC results, it is possible to detect poor compatibility between the blood collection tube and the nucleic acid extraction reagent, or a faulty lot number for the nucleic acid extraction reagent or blood collection tube lot number. This allows the testing of the blood specimen in question to be suspended or monitored closely, and resampling, etc. to be performed promptly.

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

The rest of the configuration and operation are substantially the same as those of the specimen testing method and specimen testing system of the first embodiment described above, and details are omitted.

The specimen testing method and specimen testing system of the third embodiment of the present invention also provide substantially the same effects as those of the specimen testing method and specimen testing system of the first embodiment described above.

In addition, by further including a sharing step for sharing the work status and work results in 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 amount calculation step over a network, it becomes possible to handle cases where each step is divided among multiple facilities.

Furthermore, by further including a warning step that raises 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, various findings obtained by repeated testing can be quickly reflected in the testing system, and the accuracy of the test can be improved at an accelerated rate.

＜Other＞
The present invention is not limited to the above-mentioned embodiment, but includes various modified examples. The above-mentioned embodiment has been described in detail to explain the present invention in an easily understandable manner, and the present invention is not necessarily limited to the embodiment having all of the described configurations.

It is also possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. It is also possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

1, 1A, 1B... Sample testing system 11... Plasma fractionation 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 (13)

A method for testing a sample consisting of blood, comprising:
A plasma separation step of separating plasma from the blood;
a blood condition confirmation step for confirming the condition of the blood;
A nucleic acid extraction step of extracting nucleic acid from the plasma;
a measurement step of measuring the nucleic acid;
a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and determining the amount of nucleic acid in the blood based on the quality of the nucleic acid;
a prediction step of predicting the amount of gDNA from data showing a relationship between the result 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 acid 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. The specimen testing method according to claim 1,
In the nucleic acid quality confirmation step, the amount of gDNA contained in the sample is calculated,
The specimen testing method further comprises an improvement step of adding and correcting the data used in the prediction step, the data indicating the relationship between the result obtained in the blood condition confirmation step and the amount of gDNA contained in the blood. The specimen testing method according to claim 1,
In the nucleic acid quality confirmation step, the amount of gDNA contained in the sample is calculated,
The specimen testing method further comprises an output step of comparing the calculated gDNA amount with the gDNA amount predicted in the prediction step, and outputting the occurrence of an abnormality when it is determined that the difference is equal to or greater than a certain threshold. The specimen testing method according to claim 1,
In the measuring step, the effect of the gDNA amount predicted in the predicting step is subtracted from the measured result, and then a subsequent processing step is determined. The specimen testing method according to claim 2,
The sample testing method further comprises a storage step of performing the improvement step for each testing community and storing the data. The specimen testing method according to claim 1,
The specimen testing method further comprises a sharing step of sharing, via a network, the operation status and operation results of one or more of the plasma fractionation step, the blood condition confirmation step, the nucleic acid extraction step, the measurement step, the nucleic acid quality confirmation step, the prediction step, and the nucleic acid amount calculation step. The specimen testing method according to claim 1,
The specimen testing method further comprises a warning step of raising a flag based on a trend of the relationship between any two of the plasma fractionating step, the blood condition confirming step, the nucleic acid extracting step, the measuring step, the nucleic acid quality confirming step, the prediction step, and the nucleic acid amount calculating step. A method for testing a sample consisting of blood, comprising:
A plasma separation step of separating plasma from the blood;
a blood condition confirmation step for confirming the condition of the blood;
A nucleic acid extraction step of extracting nucleic acid from the plasma;
a measurement step of measuring the nucleic acid;
a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and calculating the amount of gDNA contained in the blood based on the quality of the nucleic acid;
a prediction step of predicting the amount of gDNA from data showing a relationship between the result obtained in the blood condition confirmation step for each type of blood collection tube and the amount of gDNA contained in the blood;
and an improvement step of adding and correcting the data used in the prediction step, the data indicating the relationship between the result obtained in the blood condition confirmation step and the amount of gDNA contained in the blood. A method for testing a sample consisting of blood, comprising:
A plasma separation step of separating plasma from the blood;
A nucleic acid extraction step of extracting nucleic acid from the plasma;
a measurement step of measuring the nucleic acid;
a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and calculating the amount of gDNA contained in the blood based on the quality of the nucleic acid;
A sample testing method comprising: a correction step of subtracting the effect of the gDNA amount calculated in the nucleic acid quality confirmation step from a mutation detection value calculated by NGS or dPCR after processing in a subsequent processing step. A method for testing a sample consisting of blood, comprising:
A plasma separation step of separating plasma from the blood;
A nucleic acid extraction step of extracting nucleic acid from the plasma;
a measurement step of measuring the nucleic acid;
a nucleic acid quality confirmation step of confirming the quality of the nucleic acid and calculating the amount of gDNA contained in the blood based on the quality of the nucleic acid;
and an output step of outputting the occurrence of an abnormality when the amount of gDNA calculated in the nucleic acid quality confirmation step is determined to be equal to or greater than a certain threshold value. The specimen testing method according to claim 9 or 10,
The specimen testing method further comprises a sharing step of sharing, via a network, the operation status and operation results of any one or more of the nucleic acid extraction step, the measurement step, and the nucleic acid quality confirmation step. The specimen testing method according to claim 9 or 10,
The specimen testing method further comprises a warning step of raising a flag based on a trend of a relationship between any two of the nucleic acid extraction step, the measurement step, and the nucleic acid quality confirmation step. A system for testing a sample made of blood, comprising:
A plasma fractionation unit that fractionates plasma from the blood;
a blood condition confirmation unit for confirming the condition of the blood;
A nucleic acid extraction unit that extracts nucleic acid from the plasma;
A measurement unit that measures the nucleic acid;
a quality confirmation unit that confirms the quality of the nucleic acid and determines the amount of nucleic acid in the blood based on the quality of the nucleic acid;
a data analysis unit that analyzes the measurement results of the measurement unit;
A management system that manages the sample testing system,
The management system predicts the amount of gDNA from data showing the relationship between the results obtained by the blood condition confirmation unit 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.

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