Patent Description:
As the automatic analysis device, there are a biochemical automatic analysis device or the like which performs a quantitative and qualitative analysis of the concentration of a component of a biological sample such as blood or urine in the field of a biochemical test or a hematological test, and the like, a blood clotting time automatic analysis device (hereinafter sometimes referred to as "blood clotting time measuring device") or the like which measures a blood clotting time, and a nucleic acid amplification test device or the like which measures a cycle time involved in nucleic acid amplification.

In the former biochemical automatic analysis device or the like, at the start of an analysis in a day or in the case where a reagent is used up, and therefore, the reagent is replaced by a reagent in a reagent vessel with a different lot number, or the like, a standard sample is measured to create a standard curve, and then, a control sample is measured, whereby an operator confirms the validity of the measured values based on the analysis result. Thereafter, a sample to be tested (which refers to a sample with an unknown concentration such as a patient specimen ordered to be tested, and is hereinafter referred to as "sample with an unknown concentration") is analyzed. In an analysis of a sample with an unknown concentration, a standard curve is created beforehand using a standard sample, and the concentration is calculated using the created standard curve. By doing this, an analysis result with no difference between facilities or no difference between reagent lots is obtained by reflecting the conditions of the device and the conditions of the reagent.

However, in the measurement of a blood clotting time by deposition of fibrin in a blood clotting test, mainly an electrical resistance detection system, an optical detection system, a mechanical system, or the like is used, and a mainstream system is an optical detection system (detection of transmitted light or detection of scattered light) or a mechanical system (detection of viscosity) having an excellent processing ability. In this manner, since the measurement system differs, even if the same item is analyzed for the same specimen, the measurement result of a blood clotting time differs. In addition, in a test reagent for a blood clotting time, a biological component is contained, and therefore, the reactivity varies depending on each lot, and therefore, the measures value of a blood clotting time varies.

As a conventional technique related to the accuracy control of sample measurement, for example, PTL <NUM> (<CIT>) proposes a method in which a measured blood clotting time of a specimen is converted to a standardized blood clotting time by using a standard curve plotted by assigning a standard blood clotting time having been determined beforehand and the blood clotting time of a calibration substance measured in a test system.

Patent document <CIT> discloses an automatic analysis device and a sample analysis method with the features in the precharacterizing portion of the independent claims. Further related automatic analysis devices are disclosed in <CIT>, <CIT> and <CIT>.

As described above, in a biochemical automatic analysis device or the like, when the concentration of a component is measured, standardization is generally performed such that a difference in the conditions of the device, the lot of the reagent, or the like is absorbed using a calibrated standard curve.

However, in a blood clotting time measuring device or the like, for example, in the measurement of an APTT (activated partial thromboplastin time) or the like, a measurement result is reported by a blood clotting time (sec), and therefore, reflection of the conditions of the reagent by creating a standard curve again using a standard sample as described above could not be performed. Due to this, it is more important to ascertain the conditions of the reagent using a control sample. In addition, in the case of a lyophilized reagent to be used in several items for the blood clotting test reagent, the reagent is dissolved by a user, and therefore, the conditions of the reagent differ due to a variation in the dissolving conditions even if the reagent of the same lot is used. In other words, a difference in the measurement result may sometimes occur depending on the dissolving conditions of the reagent, and therefore, also in such a case, it is more important to ascertain the conditions of the reagent using a control sample. Further, also in a nucleic acid amplification test device or the like, there is an item to report a measurement result by a reaction cycle number without creating a standard curve, and therefore, it has the same problem.

The invention has been made in view of such circumstances, and has its object to provide an automatic analysis device and an analysis method capable of improving the reliability of the measurement result by ascertaining the conditions of the reagent and the conditions of the device using a sample having a known blood clotting time (hereinafter referred to as "blood clotting time reference sample") and adjusting the measurement conditions.

In order to achieve the above object, the invention provides the automatic analysis device and the sample analysis method defined in the independent claims. Further advantageous features are set out in the dependent claims.

According to the invention, the reliability of the measurement result can be improved by ascertaining the conditions of a reagent using a blood clotting time reference sample and setting an appropriate signal reference value for each reagent vessel.

A first embodiment useful to understand the invention will be described with reference to the drawings.

In this embodiment, as one example of an automatic analysis device, a blood clotting time measuring device which measures a time from when a reagent and a biological sample (hereinafter simply referred to as "sample") such as blood or urine are mixed to when fibrin is deposited as a blood clotting time in accordance with the amount of optical change will be described.

<FIG> is a view schematically showing the entire structure of an automatic analysis device according to this embodiment.

In <FIG>, an automatic analysis device <NUM> is roughly composed of a sample dispensing probe (sample dispensing mechanism) <NUM>, a sample disk <NUM>, a reagent dispensing probe (reagent dispensing mechanism) <NUM>, a reagent disk <NUM>, a reaction vessel container <NUM>, a gripper <NUM>, a detection unit <NUM>, a waste box <NUM>, an operation section <NUM>, a storage section <NUM>, and a control section <NUM>.

The sample dispensing probe <NUM> sucks a sample or a blood clotting time reference sample held in a sample vessel <NUM> disposed on the sample disk <NUM> which rotates clockwise and counterclockwise, and discharges the sample into a reaction vessel <NUM>. The sample dispensing probe <NUM> performs a sample suction action and a sample discharge action by the action of a sample syringe pump <NUM> controlled by the control section <NUM>.

The reagent dispensing probe <NUM> sucks a reagent held in a reagent vessel <NUM> disposed on the reagent disk <NUM> and discharges the reagent into the reaction vessel <NUM>. The reagent dispensing probe <NUM> performs a reagent suction action and a reagent discharge action by the action of a reagent syringe pump <NUM> controlled by the control section <NUM>.

Inside the reagent dispensing probe <NUM>, a reagent heating mechanism <NUM> is included, and the temperature of the reagent sucked by the reagent dispensing probe <NUM> is increased to a suitable temperature (predetermined temperature) by the reagent heating mechanism <NUM> controlled by the control section <NUM>.

The gripper <NUM> transports and places the reaction vessel <NUM>. The gripper <NUM> transports and places the reaction vessel <NUM> from the reaction vessel container <NUM> to a reaction vessel placing section <NUM> of the detection unit <NUM> by rotating in a horizontal direction while gripping the reaction vessel <NUM>.

The detection unit <NUM> has one or more reaction vessel placing sections <NUM> for placing the reaction vessel <NUM> (in this embodiment, a case where the detection unit <NUM> has one reaction vessel placing section <NUM> is shown), and performs the measurement of an optical intensity of the reaction vessel <NUM> inserted into the reaction vessel placing section <NUM>. Incidentally, in this embodiment, a case where one detection unit <NUM> is disposed is shown, however, it is not limited thereto, and the device may be configured to include a plurality of detection units <NUM>. A light source <NUM> of the detection unit <NUM> irradiates light onto the reaction vessel <NUM>. The light irradiated from the light source <NUM> is scattered by the reaction mixture in the reaction vessel <NUM>. A detection section (light sensor) <NUM> is constituted by a photodiode or the like. The detection section <NUM> receives scattered light, which is scattered by the reaction mixture in the reaction vessel <NUM>, converts the light to a current, and outputs the measured light signal indicating the intensity of the received scattered light to an A/D converter <NUM>. The measured signal of the scattered light A/D-converted by the A/D converter <NUM> is input to the control section <NUM> through an interface <NUM>. The action of the detection unit <NUM> is controlled by the computer <NUM> for control.

The gripper <NUM> grips the reaction vessel <NUM> after completion of the measurement and disposes of the reaction vessel <NUM> in the waste box <NUM>.

An analysis item for a sample to be analyzed by the automatic analysis device <NUM> is input to the control section <NUM> from the operation section <NUM> through a keyboard 118b as an input unit or an operation screen displayed on a display section 118c. Incidentally, the device may be configured to use a GUI (Graphical User Interface) for inputting an analysis item by operating an analysis item displayed on the display section 118c with a pointer or the like using a mouse 118a.

The control section <NUM> includes an overall control section 120a, a measurement control section 120b, and a signal reference value setting control section 120c.

The overall control section 120a controls the action of the automatic analysis device such as dispensing of the sample or the reagent, transfer of the reaction vessel <NUM>, disposal of the reaction vessel <NUM>, or the like.

The measurement control section 120b performs a measurement process for measuring the reaction time of a sample based on the result of comparison between a signal reference value having been determined beforehand and a light intensity (signal value) which changes over time in accordance with the degree of the mixing reaction of the sample and the reagent. Incidentally, the signal value in this embodiment is a scattered light intensity, and the blood clotting time of the sample is calculated based on the measured light signal from the detection unit <NUM>. The calculated blood clotting time is output to the display section 118c and also stored in the storage section <NUM>. Incidentally, the clotting time as the calculation result may be printed and output by a printer <NUM> through the interface <NUM>.

The signal reference value setting control section 120c performs a signal reference value setting process (which will be described later) for setting a signal reference value such that the blood clotting time measured based on the result of comparison between the signal reference value and the signal value (scattered light intensity) which changes over time in accordance with the mixing reaction of a blood clotting time reference sample or the like and a reagent corresponds to the expected value of the blood clotting time having been determined beforehand as corresponding thereto. Here, the signal reference value in the calculation of the blood clotting time refers to the ratio on the basis of which blood clotting is determined when normalization is performed by taking the amount of light at the start of the reaction as <NUM> and the optical change until completion of the reaction as <NUM>. That is, the time when the amount of light exceeds the ratio (signal reference value) having been determined beforehand is defined as the blood clotting time. The determined signal reference value set by the signal reference value setting control section 120c and the identification information having been set beforehand for the reagent are stored in the storage section <NUM> as the corresponding information.

Here, a method for calculating the blood clotting time in the measurement process in this embodiment will be described.

<FIG> is a view showing one example of the change over time in the amount of scattered light detected by the detection unit <NUM> in the mixing reaction of the sample and the reagent.

In this embodiment, the blood clotting time is calculated from the blood clotting reaction curve of the signal value measured over time by the detection section. In the blood clotting reaction, when a sample and a given reagent are discharged into the reaction vessel <NUM> placed in the reaction vessel placing section <NUM> by the reagent dispensing probe <NUM>, a blood clotting reaction starts as the mixing reaction. That is, the action of discharge of the reagent by the reagent dispensing probe <NUM> serves as the starting point, and the blood clotting reaction is started (time: t = t0).

In the blood clotting reaction curve shown in <FIG>, the scattered light intensity E reaches a fixed minimum value Eb from the start of the measurement (time: t = t0) to the time t = t1, the scattered light intensity E increases from the time t = t1 to the time t = t2, and the scattered light intensity E reaches a fixed maximum value Ep when the time t is t2 and thereafter.

In such a blood clotting reaction curve, a region in which the scattered light intensity E reaches the fixed minimum value Eb refers to a base line region, and a region in which the scattered light intensity E reaches the fixed maximum value Ep refers to a plateau region.

In the measurement process of the blood clotting reaction, when normalization is performed with respect to the change in the amount of scattered light between the time t = t1 and the time t = t2 by taking the scattered light intensity in the base line region as <NUM>% and the scattered light intensity in the plateau region as <NUM>%, the time when the ratio (the signal reference value S) having been determined beforehand is exceeded is defined as the blood clotting time T.

Next, a method for calculating a blood clotting time Ta when the measurement is performed using the reagent of a lot A and a blood clotting time Tb when the measurement is performed using the reagent of a lot B will be described with reference to <FIG>. When the blood clotting reaction curve is normalized by taking the scattered light intensity in the base line region as <NUM>% and the scattered light intensity in the plateau region as <NUM>%, and the signal reference value S is set to <NUM>%, the blood clotting time Ta is <NUM> sec. Here, the reactivity differs when the lot of the reagent is changed, and therefore, even in the case where the same sample is measured, the blood clotting reaction curves do not necessarily overlap each other. For example, in <FIG>, in the case where the measurement is performed using the reagent of a lot B, the blood clotting time Tb calculated from the blood clotting reaction curve is <NUM> sec. This indicates that a difference in the measurement result occurs depending on the lot of the reagent even in the case where the same sample is measured, and in this embodiment, a method for decreasing the variation in the measured value by minimizing the difference.

Next, a method for setting the signal reference value in the signal reference value setting process of this embodiment will be described.

<FIG> is a flow chart showing the outline of a processing procedure of a signal reference value setting process. Further, <FIG> are views illustrating part of the procedure of the signal reference value setting process in more detail using a blood clotting reaction curve.

In <FIG>, it is necessary to confirm whether or not the conditions of the device or the reagent are favorable before the measurement. As a method for confirming the conditions of the device or the reagent, for example, after confirming the temperature of the detector or the reagent probe with a heating function (S401), the measurement is performed using a control sample (S402). In the case where the conditions of either or both of the device and the reagent are not favorable, it is necessary to improve the conditions so that favorable conditions are obtained by performing the maintenance of the device or replacing the reagent (S403). On the other hand, in the case where the conditions of both are favorable as a result of confirmation in S401 and S402, it is determined whether or not the measured value of the blood clotting time reference sample calculated using the current signal reference value SLocal which is the initial value having been set beforehand is in a predetermined range in Step <NUM> (S404). In the case where the measured value is in the predetermined range, in Step <NUM>, setting is performed so that the current signal reference value SLocal is continued to be used (S405). On the other hand, in the case where the measured value is outside the predetermined range, in Step <NUM>, the signal reference value setting control section 120c reads the range of variation and the expected value (Te) of the blood clotting time in the case where the blood clotting time reference sample is measured using the reagent to be used in the signal reference value setting process in the blood clotting time measuring device to serve as a reference (Step S406).

Here, in the case where the measured value of the blood clotting time reference sample obtained using the current signal reference value SLocal in S404 is in the allowable range, it is not necessary to reset the signal reference value.

Subsequently, in Step <NUM>, in the blood clotting time measuring device to be used for the signal reference value setting process, the measurement of the blood clotting time reference sample is performed using the reagent to be used for setting the signal reference value, whereby a blood clotting reaction curve (see <FIG>) is obtained (Step S407).

Subsequently, in Step <NUM>, the signal reference value SLocal at which the blood clotting time calculated by applying the expected value Te of the blood clotting time with respect to the blood clotting reaction curve (see <FIG>) obtained in S407 corresponds to the expected value Te is set (see <FIG>, S408).

Then, in Step <NUM>, the signal reference value SLocal obtained in S408 is stored in the storage section <NUM> as the information corresponding to the reagent used in the signal reference value setting process (S409).

In an example which is not part of the claimed invention, in the measurement of the sample with an unknown concentration thereafter, in the case where the reagent of the same lot as that of the reagent used in the signal reference value setting process is used, the signal reference value SLocal in the storage section <NUM> is read and used. Incidentally, in this embodiment, with respect to the reagent of the same lot, the same signal reference value SLocal is used, however, the corresponding signal reference value SLocal may be determined each time by performing the signal reference value setting process with respect to each reagent vessel. In this case, the reliability of the measurement result can be further improved. The method for controlling the signal reference value for each reagent vessel will be described later.

Incidentally, in the blood clotting time measuring device to serve as a reference, as for the calculation of the range of variation and the expected value Te of the blood clotting time in the case where the blood clotting time reference sample to be used in the signal reference value setting process is measured, by performing the same measurement a plurality of times, a mean, a median, or the like of the calculated blood clotting time in combination of a specific reagent and the blood clotting time reference sample is defined as the expected value Te, and a standard deviation, a dispersion, or the like thereof is given as the range of variation. Incidentally, in the calculation of the expected value Te and the range of variation in this measurement, it is desired to perform the calculation in consideration of a diurnal or day-to-day variation. Further, it is preferred that with respect to the blood clotting time measuring device to serve as a reference, in consideration of the machine difference between specified device types, the status of accuracy control for each facility is controlled by a network as to whether the measurement is repeated by a plurality of devices, and the measurement results of the devices in a plurality of facilities are aggregated and reflected.

According to the procedure as described above, the difference between devices with respect to an item (for example, APTT: activated partial thromboplastin time) that treats the calculated blood clotting time as the measurement result as it is without creating a standard curve is corrected.

In general, in the field of blood testing, a value is determined by a combination of the reagent of the same lot and the sample. In particular, in the blood clotting test, a reagent containing a biological component is used as the reagent in some cases, and in such a case, a lot-to-lot difference is large, the sensitivity to a chemical is greatly varies for each reagent, and so on. Therefore, standardization is not achieved. However, even in the case of such an item, if the combination is a combination of the reagent of the same lot and the sample, the expected value of the result measured in the specified device can be uniquely determined. That is, the reagent sufficiently tested at the time of shipping and the blood clotting time reference sample can define the expected value and the range of variation of the blood clotting time by the combination of the sample with a variety of reagents.

Here, the control of the signal reference value SLocal for each reagent vessel will be described.

<FIG> is a view showing one example of a list of signal reference values corresponding to a plurality of reagent vessels.

In <FIG>, with respect to the reagent vessel <NUM> placed at each position of the reagent disk <NUM>, the item, the lot number of the reagent, and the lot number of the blood clotting time reference sample are listed, and a case where an intrinsic signal reference value is made to correspond to each reagent vessel is shown. In the case where a plurality of reagent vessels are placed for the same item, the signal reference value is shown independently. The item which has not been measured can be made identifiable on the operation screen. Preferably, when the measurement is requested, the number of requests and the number of remaining tests are confirmed, and in the case where a reagent bottle for which the signal reference value has not been set is planned to be used, a warning that the signal reference value has not been set is issued from the control section <NUM>. Further, it is also considered that even in the case where the signal reference value has not been set, when an operator gives permission, the same signal reference value as the signal reference value of another reagent vessel is set. In the case of APTT reagents at position <NUM> and position <NUM>, the reagent vessels are different, however, the lot numbers are both A00001, and therefore, a common signal reference value for each lot can be set. In the case of PT reagents at position <NUM> and position <NUM>, the lot numbers are B00001 and B00002 and are different, and therefore, different signal reference values are set. However, in the case where even if the lot numbers are the same, different signal reference values are desired to be set, it is possible to set the signal reference values for the reagent vessels. For example, the reagents at position <NUM> and position <NUM> show a case where although the lot numbers are the same, different signal reference values are set for each reagent vessel is shown. This is because even in the case of the reagents of the same lot, the reactivity sometimes differs depending on the storage conditions of the reagent or the like, and therefore, it is effective that the signal reference value is controlled for each reagent vessel. Further, position <NUM> and position <NUM> show a case where the signal reference value has not been set.

The action in this embodiment configured as described above will be described.

In the signal reference value setting process in the blood clotting time measuring device which is the automatic analysis device according to this embodiment, first, an operator obtains the expected value Te and the range of variation of the blood clotting time through a bar code attached to the reagent, a network, or a method of reading or the like in the package insert of the reagent (Step S406 in <FIG>). Preferably, it is desired that the identification information such as a bar code attached to the reagent is read and the information corresponding to the reagent is obtained through a network, whereby the information is directly incorporated in the storage section <NUM> of the blood clotting time measuring device to be used for the signal reference value setting process. However, in the case where the direct incorporation cannot be performed, the operator can manually input the information from the operation section <NUM>.

Here, the error of the measurement result in a clinical test leads to a diagnostic error, and therefore, it is an essential requirement that the validity of the measurement result is confirmed. In the automatic analysis device or the like, it is a general rule that the validity of the measurement result is confirmed by performing measurement using a control sample periodically in addition to when the reagent lot is changed. The variation using the control sample in the biochemical automatic analysis device or the like is, for example, controlled as shown in <FIG> shows an example of periodically measuring a control sample. Here, the "Mean" indicates a case where a mean determined for each device type is defined as the expected value, and as for the range of variation with respect to the Mean, ± 3SD is defined as the allowable range. Here, the "Mean" and the "SD" are preferably the results calculated by a plurality of reference devices or the values obtained by aggregating the measurement results in a plurality of facilities.

On the other hand, with respect to an item to report the measurement result by the blood clotting time (sec) without creating a standard curve such as the measurement of an APTT (activated partial thromboplastin time), the difference between devices is not corrected by calibration, and therefore, a difference between devices occurs even if the device type is the same, and the blood clotting time does not necessarily match the mean determined for each device type. <FIG> shows an example of periodically measuring a control sample in a blood clotting time measuring device or the like to which this embodiment is not applied. In such a case, it is not suitable to set the range of variation of the mean determined for each device type to ± 3SD, and therefore, it is necessary to correct the difference between devices of the mean.

The correction of the difference between devices can be performed by setting the signal reference value for calculating the blood clotting time in the blood clotting reaction curve for each facility and for each reagent vessel.

Further, as for the timing of the signal reference value setting process, the signal reference value setting process is preferably performed when a new reagent vessel is placed and for each item when the blood clotting time reference sample is measured. At this time, the signal reference value SLocal is set (Steps S407 and S408 in <FIG>).

Subsequently, in an example which is not part of the present invention, when an operator performs an analysis of a sample with an unknown concentration, in the control section <NUM>, the content of the measurement is confirmed, and a sample and a reagent to be used, and a position where the measurement is performed are allocated. A time when the change in the light intensity exceeds SLocal is calculated as the blood clotting time from the obtained measurement result.

The effect of this embodiment configured as described above will be described.

As described above, for example, in the measurement of an APTT (activated partial thromboplastin time), reflection of the conditions of the reagent by creating a standard curve again cannot be performed, and therefore, it is more important to ascertain the conditions of the reagent using a blood clotting time reference sample. In addition, in the case of a lyophilized reagent to be used in several items for the blood clotting test reagent, the reagent is dissolved by a user, and therefore, the conditions of the reagent differ due to a variation in the dissolving conditions even in the case of the reagent of the same lot. In other words, a difference in the measurement result may sometimes occur depending on the dissolving conditions of the reagent, and therefore, it is more important to ascertain the conditions of the reagent using a control sample for each reagent vessel.

On the other hand, this embodiment is configured such that the difference between devices is corrected by performing the setting of the signal reference value that corresponds to the expected value of the blood clotting time having been determined beforehand as corresponding to the blood clotting time reference sample using a blood clotting reaction curve which changes over time in accordance with the mixing reaction of a blood clotting time reference sample and a reagent, and therefore, the conditions of the reagent are more easily ascertained using the blood clotting time reference sample, and the reliability of the measurement result can be improved.

A modification of the first embodiment useful to understand the invention will be described.

In the first embodiment, a change in scattered light intensity is illustrated as the blood clotting reaction curve, however, in this modification, a case where a change in transmitted light is used as the blood clotting reaction curve is shown.

<FIG> is a view showing one example of the change over time in the amount of transmitted light detected by the detection unit <NUM> in the mixing reaction of a sample and a reagent in the modification.

In the modification of this embodiment, a blood clotting time is calculated from a blood clotting reaction curve measured over time by the detection section. In a blood clotting reaction, when a given reagent is discharged into the reaction vessel <NUM> which held a sample and is placed in the reaction vessel placing section <NUM> by the reagent dispensing probe <NUM>, a blood clotting reaction starts as the mixing reaction. That is, the action of discharge of the reagent by the reagent dispensing probe <NUM> serves as the starting point, and the blood clotting reaction is started (time: t = t0).

In the blood clotting reaction curve shown in <FIG>, the transmitted light intensity E reaches a fixed maximum value Es from the start of the measurement (time: t = t0) to the time t = t1, the transmitted light intensity E decreases from the time t = t1 to the time t = t2, and the transmitted light intensity E reaches a fixed minimum value Ee when the time t is t2 and thereafter.

In the measurement process of this blood clotting reaction, when the change in the amount of transmitted light between the time t = t1 and the time t = t2 is taken as <NUM>%, the time when the ratio (the signal reference value S) having been determined beforehand is exceeded is defined as the blood clotting time T.

The other configuration is the same as that of the first embodiment.

Also in this modification configured as described above, the same effect as that of the first embodiment can be obtained.

Incidentally, also in a genetic testing device represented by a real-time PCR device, the basic structure is the same as that of the first embodiment or this modification, and this technique can be applied by replacing the light intensity to be measured over time by a fluorescence intensity, and representing the cycles on the horizontal axis.

That is, in the field of genetic testing, by a nucleic acid amplification method, a very small amount of a virus or a bacterium is amplified so as to be able to be detected, whereby a disease is determined. A most common example of the nucleic acid amplification method is a PCR (Polymerase Chain Reaction) method. In a common PCR method, a DNA is amplified two-fold by changing the temperature in one cycle consisting of three steps of thermal denaturation, annealing, and extension. When a double-stranded DNA is denatured into single strands in the thermal denaturation step, a primer having a sequence complementary to a target nucleic acid can bind to the target nucleic acid in the annealing step. In the subsequent extension step, a double-stranded cDNA is synthesized by the action of a DNA synthase. By repeating this cycle, the target DNA is amplified exponentially.

In recent years, among the PCR methods, a real-time PCR method which is simple and has high accuracy has been widely used. The real-time PCR method is a method in which a probe or the like attached with a fluorescent dye is used when performing PCR and a DNA is detected by measuring a fluorescence intensity while amplifying the DNA, and the fluorescence intensity is enhanced as the DNA is amplified. Here, the determination of amplification in the real-time PCR method is performed by calculating the cycles (Ct value: Threshold Cycle Value) when a signal reference value having been determined is exceeded. In particular, in a quantitative test, a standard curve is created from a relationship between the Ct value and the concentration of a sample with a known concentration, for example, a standard sample, and, in an example which is not part of the claimed invention, the sample with an unknown concentration is quantitatively determined.

However, in the real-time PCR method, other than quantitative measurement, genotyping, qualitative analysis, and the like can be performed, and for these items, the Ct value is reflected in the measurement result as it is without creating a standard curve.

<FIG> is a view illustrating a method for calculating a Ct value in a real-time PCR method, and shows an example of a PCR reaction curve in the case where the measurement is completed after <NUM> cycles. In <FIG>, the start of the measurement is defined as a 1st cycle, the end of the measurement is defined as a 60th cycle, and when normalization is performed by taking the amount of fluorescence at the start of the measurement as <NUM>% and the change in the amount of fluorescence until the completion of the measurement as <NUM>%, the cycle number at the time when the ratio (the signal reference value S) having been determined beforehand is exceeded is defined as the Ct value.

<FIG> is a view illustrating a method for calculating Cta which is a Ct value when the measurement is performed using the reagent of a lot A and Ctb which is a Ct value when the measurement is performed using the reagent of a lot B. When normalization is performed by taking the fluorescence intensity in the base line region of the PCR reaction curve as <NUM>% and the fluorescence intensity in the plateau region as <NUM>%, and the signal reference value S is set to <NUM>, Cta is <NUM>. Here, the reactivity differs when the lot of the reagent is changed, and therefore, even in the case where the same sample is measured, the PCR reaction curves do not necessarily overlap each other. For example, in <FIG>, in the case where the measurement is performed using the reagent of a lot B, Ctd calculated from the PCR reaction curve is <NUM>. In such a case, when the expected Ct value (Cte) having been defined beforehand is used, the variation in the measured value depending on the low can be reduced. Specifically, the method is the same as the flow chart shown in <FIG>, and therefore, a detailed description will be omitted, however, the flow chart can be applied by replacing the Te value which is the expected value of the blood clotting time in <FIG> by the Cte value which is the expected value in the PCR reaction. In the example in <FIG>, in the case where Cte is <NUM>, the signal reference values SLocala and SLocalb obtained from the PCR reaction curves when measurement is performed using the reagent of a lot A and the reagent of a lot B can be set to <NUM>% and <NUM>%, respectively. On the other hand, as another example of the nucleic acid amplification method, there is a method in which a reaction is allowed to proceed at a fixed temperature without actively changing the temperature unlike the PCR method. Examples thereof include constant temperature amplification methods such as a LAMP (loop-mediated isothermal amplification) method and a TRC (transcription-reverse transcription concerted) method. The amplification in the constant temperature amplification method can be detected by measuring a fluorescence intensity or a turbidity at fixed intervals concurrently with amplification in the same manner as real-time PCR. In the detection, a method in which a fluorescent dye having a primer or a specific sequence which binds to a target nucleic acid, a fluorescent dye which directly intercalates into a DNA, or the like is added, and fluorescence is detected is generally used, however, as the LAMP method, there is also a method in which a byproduct produced accompanying the amplification of a nucleic acid is detected by turbidity or fluorescence. In the constant temperature amplification method, a result is often calculated by a qualitative analysis, and in such a case, a standard curve is not created in some cases. Since a time T when a given turbidity or fluorescence intensity is exceeded is used in place of the Ct value, it is possible to set the signal reference value by replacing the cycle number in <FIG> by the amplification start time T.

In this manner, the invention can be applied in the same manner as the first embodiment also to an analysis item for which a time or a cycle number at which the signal reference value is exceeded is calculated as the measurement result from the amount of change in the measured signal value (the amount of transmitted light, the amount of scattered light, the amount of fluorescence, or the turbidity).

In the first embodiment, a method for setting a signal reference value using a blood clotting time reference sample at one concentration is shown, however, in the present invention, a case where the measurement is performed using a plurality of blood clotting time reference samples such as a high-concentration sample and a low-concentration sample having different reactivity is shown.

<FIG> is a view showing one example of the change over time in the amount of scattered light detected by the detection unit <NUM> in the respective mixing reactions of a reagent and a sample at a high concentration and a low concentration.

In <FIG>, when the expected value of the blood clotting time for the high-concentration sample is represented by T1 and the expected value of the blood clotting time for the low-concentration sample is represented by T2, a signal reference value (SLocal_high) which matches the expected value T1 and a signal reference value (SLocal_low) which matches the expected value T2 are determined from a blood clotting reaction curve obtained in the automatic analysis device which measures the blood clotting time. At this time, as an example which is not part of the claimed invention, the value of the signal reference value SLocal to be used in the calculation of the blood clotting time is used as the mean of the respective signal reference values (<FIG>).

Further, according to an embodiment of the present invention, an approximate straight line is formed with respect to a plurality of signal reference values and blood clotting times obtained from the reaction curves of a plurality of blood clotting time reference samples, and a signal reference value which is different for each concentration can also be set.

Examples of an approximate formula include a formula obtained by linear approximation, polynomial approximation, logarithmic approximation, or exponential approximation of a plurality of calculated signal reference values (<FIG>, and <FIG>). Further, there is also a method in which a plurality of calculated signal reference values are connected with broken lines, each of which is approximated by a linear function (<FIG>). Here, an approximation method using the linear function: Y = aT + b will be described in detail with reference to <FIG>.

In <FIG>, with respect to the expected value T1 (<NUM> sec) of the blood clotting time for the high-concentration sample and the expected value T2 (<NUM> sec) of the blood clotting time for the low-concentration sample, the intersection points thereof with the respective blood clotting reaction curves are <NUM>% and <NUM>%, respectively. At this time, as shown in <FIG>, from a straight line obtained by connecting the above two intersection points, a slope a and an intercept b are obtained, and the formula: SLocal = <NUM>. 4T + <NUM> can be derived.

<FIG> is a view illustrating a method for calculating the concentration of an unknown sample at this time. That is, a value on the X axis of the intersection point A between a reaction curve (a reaction curve (a) in <FIG>) obtained by measuring the unknown sample and the SLocal = <NUM>. 4T + <NUM> calculated in <FIG> is the blood clotting time T.

As described above, when the measurement is performed using a device in each facility, in a combination of a reagent having been sufficiently tested and a blood clotting time reference sample, by setting an intrinsic signal reference value SLocal for each reagent vessel, an accurate blood clotting time can be calculated without difference between devices or difference between reagent vessels.

Incidentally, the allowable range of the signal reference value in this embodiment is limited to a range in which the amount of light changes (a range of the amount of light from <NUM>% to <NUM>%), more preferably limited to a range from <NUM>% to <NUM>%. According to this embodiment, by setting the allowable range of the signal reference value SLocal and the allowable range of the blood clotting time reference sample for each item beforehand, it can be determined whether or not the measurement result of the blood clotting time reference sample is in the allowable range, and in the case where the measurement result largely exceeds the allowable range, a system alarm is issued, and it is possible to propose a retest or to make the reagent unusable.

The procedure for issuing a system alarm is shown in a flow chart in <FIG>. First, in Step <NUM>, the expected value Te and the range of variation of the blood clotting time are obtained by a method such as reading or the like of a bar code attached to the reagent, a network, or the description of the package insert of the reagent (S2001). Subsequently, in Step <NUM>, by measuring a blood clotting time reference sample, a blood clotting reaction curve is obtained (S2002). Here, in Step <NUM>, the blood clotting times Tmax and Tmin for the upper limit and the lower limit of the allowable signal reference value having been set beforehand are calculated (S2003), and in Step <NUM>, it is determined whether or not the expected value Te of the blood clotting time satisfies the following relationship: Tmin < Te < Tmax (S2004). Here, in the case where Te satisfies the conditions of the range, in Step <NUM>, the signal reference value SLocal at which the blood clotting time becomes Te is set (S2005), and in Step <NUM>, the signal reference value SLocal is stored in the storage section (S2006), whereby it is used for the calculation of the blood clotting time of a sample with an unknown concentration.

On the other hand, in the case where the expected value Te of the blood clotting time does not satisfy the following relationship: Tmin < Te < Tmax, in Step <NUM>, a system alarm that the signal reference value cannot be set is issued (S2007).

Specific examples are shown in <FIG> shows an example in which the signal reference value can be set. Here, when the lower limit of the signal reference value is set to <NUM>%, the upper limit of the signal reference value is set to <NUM>%, and the expected value Te is set to <NUM> sec, the blood clotting times when the signal reference value is <NUM>% and <NUM>% are as follows: Tmin = <NUM> sec and Tmax = <NUM> sec. This case satisfies the following relationship: Twin < Te < Tmax, and therefore, the signal reference value at which the blood clotting time becomes Te can be calculated, and the signal reference value SLocal is <NUM>%.

<FIG> shows an example in which the setting of the signal reference value is failed. Here, the lower limit of the signal reference value is set to <NUM>%, the upper limit of the signal reference value is set to <NUM>%, and the expected value Te is set to <NUM> sec. The blood clotting times when the signal reference value is <NUM>% and <NUM>% are as follows: Twin = <NUM> sec and Tmax = <NUM> sec, and therefore, Te is smaller than Tmin, and thus, the relationship in S2004 is not satisfied. Accordingly, the signal reference value at which the blood clotting time becomes Te cannot be calculated. In this case, in the reaction curve of the blood clotting time reference sample, Te is outside the variable range of the signal reference value, and therefore, any of the blood clotting time reference sample, the reagent, and the device may have a problem, and thus, a system alarm is issued to notify of abnormality.

In this manner, by setting the allowable range of the signal reference value beforehand, it is possible to notify of abnormality in measurement.

A second embodiment of the invention will be described with reference to the drawings.

This embodiment is configured to apply the first embodiment to the confirmation of the validity of the preparation of a reagent in the case of a lyophilized reagent.

For example, in a reagent for measuring a prothrombin time (hereinafter referred to as "PT"), a tissue thromboplastin derived from an animal is contained, and in order to maintain stability, a lyophilized reagent is usually used. Conventionally, the lyophilized reagent is dissolved manually by a laboratory technician, and therefore, there was a problem that a variation in the measured value due to the preparation error cannot be controlled in the device. On the other hand, in order to mount a function to prepare a reagent on the device, a mechanism for dispensing a solution and a mechanism for stirring the solution are needed, and therefore, there was a problem that the size of the device is increased and also the cost is increased. Therefore, by setting a signal reference value SLocal for each reagent vessel as described above even if the lot is the same, a measurement result can be obtained without creating a standard curve by setting a signal reference value for each reagent vessel without measuring a standard sample with respect to the reagent prepared by the same method.

Here, a procedure when a calibration result obtained by measurement in a reagent vessel <NUM> using a reagent of a lot A and a blood clotting time reference sample of a lot B is taken over to a reagent vessel <NUM> will be described. <FIG> is a view illustrating a method for calculating the blood clotting time of the blood clotting time reference sample from a signal reference value having been set beforehand in the case where the analysis item is a PT, and <FIG> is an explanatory view showing a method for creating a standard curve from the calculated blood clotting time.

First, with respect to the measurement result obtained using the reagent of a lot A and the blood clotting time reference sample of a lot B, the information of the expected value Te and the range of variation of the blood clotting time is read and obtained from the bar code of the reagent, a network, or the package insert of the reagent. Subsequently, by using the reagent of the lot A in the reagent vessel <NUM>, the blood clotting time reference sample of the lot B is measured, and a signal reference value SLocal_1 is set (see <FIG>). Subsequently, the blood clotting time reference sample at three concentrations is measured in the reagent vessel <NUM>, and blood clotting times T1 to T3 calculated using a blood clotting reaction curve and the signal reference value SLocal_1 are obtained (see <FIG>). Plotting is performed on a graph using the obtained T1 to T3, whereby a standard curve is created (see <FIG>).

In the case where a sample with an unknown concentration is measured in the reagent vessel <NUM>, a blood clotting time Tx is calculated using the signal reference value SLocal_1 (<FIG>), and Tx is applied to the standard curve, and the concentration Cx of the sample with an unknown concentration is calculated (<FIG>). Subsequently, the residual amount of the sample with an unknown concentration in the reagent vessel <NUM> is decreased, and it is taken into account that the reagent vessel <NUM> will be replaced by a reagent vessel <NUM> of the same lot (lot A), and an analysis is performed.

In the past, in the case where the labor and cost for performing calibration again were considered, when the measurement result of a sample were in the allowable range, the calibration result of the reagent vessel <NUM> was taken over without performing calibration. However, in the reagent vessel <NUM>, the reactivity differs from that in the reagent vessel <NUM> in a strict sense due to a difference in the amount of water for dissolving the lyophilized reagent or a difference in the storage stability, and therefore, a small difference may occur in some cases.

According to this embodiment, a more accurate measurement result can be obtained by setting a signal reference value which is different for each reagent vessel from the measurement result of a blood clotting time reference sample even without performing calibration. Also in this case, it is a prerequisite that the range of variation of the signal reference value is in a predetermined range in which the measured value of the blood clotting time reference sample calculated using the same is allowable, and therefore, with respect to the value outside this predetermined range, the signal reference value SLocal should not be set. In such a case, according to the flow chart in <FIG>, it is desired to issue a system alarm.

According to this embodiment, by setting the allowable range of SLocal for each item beforehand, it can be determined whether or not the measurement result of the blood clotting time reference sample is in the allowable range, and in the case where the measurement result is outside the allowable range, an alarm is issued, and it is possible to propose the confirmation of the conditions of the reagent and the conditions of the device.

Here, a case where the signal reference value obtained from the blood clotting reaction curve of the blood clotting time reference sample is in the allowable range of variation is assumed, and the creation of a standard curve for the reagent vessel <NUM> and the calculation of the concentration of a sample with an unknown concentration will be described with reference to <FIG>. First, the blood clotting time reference sample of the lot B is measured using the reagent of the lot A in the reagent vessel <NUM>, and a signal reference value SLocal_2 for the reagent vessel <NUM> is set (see <FIG>). Subsequently, as shown in <FIG>, SLocal_2 is applied to the reaction curve of the blood clotting time reference sample at three concentrations, and blood clotting times T1', T2', and T3' are calculated (<FIG>). Subsequently, the concentration of the standard sample and the obtained T1', T2', and T3' are plotted, whereby the standard curve for the reagent vessel <NUM> is created (<FIG>). Here, the blood clotting reaction curve of the sample with an unknown concentration is obtained as a blood clotting reaction curve (a) in <FIG>, the blood clotting time Tx obtained using SLocal_2 is applied to the standard curve created in <FIG>, and the concentration Cx of the sample with an unknown concentration is calculated (see <FIG>).

That is, since the difference between the reagent vessels is corrected by setting the signal reference values intrinsic to each of the reagent vessel <NUM> and the reagent vessel <NUM>, a more accurate result can be provided for the quantitative determination of the sample with an unknown concentration by creating a standard curve without performing new calibration in the reagent vessel <NUM>.

Incidentally, when the standard curve in the PT analysis is created, there is a case where the standard curve for each facility is not created and used, but the result of the standard curve provided by the manufacturer of the reagent is obtained through a network, a bar code, or the like and used. The standard curve provided by the manufacturer of the reagent refers to a standard curve created based on the result measured using a reagent having been sufficiently tested and a sample with a known concentration, and it is not necessary to perform calibration in a facility, and therefore, the burden on the examiner and the running cost can be reduced, however, it is also considered that an error derived from a difference between devices often leads to the occurrence of an error in the measurement result. However, also in this case, an accurate measurement result can be obtained without newly creating a standard curve in a facility by setting the signal reference value according to the procedure in <FIG> to reduce the difference between devices.

A third embodiment of the invention will be described with reference to the drawings.

In this embodiment, the signal reference value setting process in the first embodiment is performed by reflecting the status of degradation of the reagent and the conditions of the device (such as a change in the amount of light).

<FIG> is an explanatory view showing a day-to-day variation in the measurement result of the control sample in the case where the analysis item is a PT.

In the automatic analysis device, a method in which measurement using a sample with a known concentration, for example, a control sample is periodically performed, and in the case where the measurement result is outside the range of variation of the expected value determined using a specific lot and a sample, correction is performed by performing calibration is generally used. For example, as shown in <FIG>, with respect to the accuracy control of the PT, the accuracy control is periodically performed, and the conditions of the reagent are checked. In the case where the measurement result is outside the range of variation of 3SD, calibration is performed. However, accompanying this operation, the cost of the reagent, standard sample, and consumables is increased. Therefore, by resetting the signal reference value for calculating the blood clotting time, the same effect as in the case of performing calibration is obtained even without actually measuring the standard sample. It does not matter whether the reagent to be used in this case is a lyophilized product or a liquid reagent.

Claim 1:
An automatic analysis device (<NUM>) for analyzing the blood clotting time of a sample, comprising:
a sample vessel placing section (<NUM>) in which a sample vessel (<NUM>) containing the sample to be analyzed is to be placed;
a reagent vessel placing section (<NUM>) in which a reagent vessel (<NUM>) containing a reagent to be used for measuring the sample is to be placed;
a reaction vessel (<NUM>) in which the reagent and the sample are to be mixed and reacted;
a sample dispensing mechanism (<NUM>) adapted to dispense the sample into the reaction vessel from the sample vessel;
a reagent dispensing mechanism (<NUM>) adapted to dispense the reagent into the reaction vessel from the reagent vessel;
a detector (<NUM>) adapted to detect a signal value that changes over time in accordance with the degree of the mixing reaction of the sample and the reagent; and
a control section (<NUM>) adapted to analyze the sample based on the result of the detection,
wherein
the signal value is any one of the amount of transmitted light, the amount of scattered light, the amount of fluorescence, and the turbidity; and
the control section includes:
a signal reference value setting control section (120c) adapted to create a blood clotting reaction curve obtained from the signal value changing over time in a mixing reaction of a blood clotting time reference sample having a known expected blood clotting time and the reagent, and to set the value of the blood clotting reaction curve at the known expected blood clotting time as a signal reference value;
a storage section (<NUM>) adapted to store the set signal reference value together with a lot number of the reagent as identification information for the reagent, and
a processing section adapted to determine the blood clotting time of the sample as the time when the signal value detected by the detector exceeds the stored signal reference value,
characterized in that the signal reference value setting section is adapted to set a plurality of signal reference values based on the reaction curve of the blood clotting time reference sample at a plurality of known concentrations, to determine an approximate formula from a relationship between the set plurality of signal reference values and the blood clotting time, and to set a signal reference value function which varies depending on the concentration of the blood clotting time reference sample based on said approximate formula.