Calculation method, calculation device, and calculation program

Abstract

To provide a calculation method for accurately calculating a starting point of signal rise from fluorescence or chemiluminescence signals obtained from a biological sample.SOLUTION: A calculation method calculates a starting point of signal rise (Xs,Ys) from a fluorescence signal measured using a biological sample. The fluorescence signal is a time series data composed of a data pair (measurement time, fluorescence intensity) of fluorescence intensity with respect to measurement time. The calculation method includes the steps of: calculating a tentative starting point (Xs',Ys'); and calculating the starting point of the signal rise (Xs,Ys) based on the tentative starting point (Xs',Ys').SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a calculation method, a calculation device, and a calculation program, and more particularly to a calculation method, a calculation device, and a calculation program for calculating the starting point of a signal increase and the amount of signal increase from a fluorescent or chemiluminescent signal acquired from a biological sample. [Background technology]

[0002] To evaluate biological reactions, a reagent that induces a biological reaction is added to a biological sample, and changes accompanying the biological reaction are detected by fluorescence, chemiluminescence, etc. For example, a method for evaluating the activity of neutrophil cells is known in which a neutrophil stimulant is added to a biological sample and the myeloperoxidase activity or superoxide production activity is detected by fluorescence or chemiluminescence (e.g., Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2019-213464 [Patent Document 2] Patent No. 6285691 Summary of the Invention [Problem to be solved by the invention]

[0004] When measuring fluorescence, chemiluminescence, etc. using a biological sample (for example, a sample containing whole blood as disclosed in Patent Document 2), the signal intensity of the measured fluorescence, chemiluminescence, etc. tends to be small and distorted due to the influence of impurities contained in the biological sample (for example, red blood cells and other impurities in the case of whole blood). This makes it difficult to accurately determine the starting point of the signal increase and the amount of increase in the signal, which makes it difficult to calculate accurate indicators (see Figures 18 and 19).

[0005] The present invention aims to solve the above-mentioned problems, that is, to provide a calculation method that can accurately calculate the starting point of a signal increase and the amount of signal increase from a fluorescent or chemiluminescent signal acquired from a biological sample. Another object of the present invention is to provide a calculation device and a calculation program for implementing the calculation method. [Means for solving the problem]

[0006] The present invention is based on the method for determining the starting point of signal rise (X s ,Y s ) is a calculation method for calculating The fluorescence signal is time-series data consisting of a data pair (measurement time, fluorescence intensity) of fluorescence intensity versus measurement time, The above calculation method is based on the temporary starting point (X s’ ,Y s’ ), and the temporary starting point (X s’ ,Y s’ ), the start of the signal rise (X s ,Y s ) calculating The above temporary starting point (X s’ ,Y s’ ) is calculated by After adding a reagent that induces a biological reaction to a biological sample, the approximate line f with the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent has settled. min (x) and approximate line f min Calculating the right end X1 value of (x); a step of calculating a right end X2 value at which an approximate line of the fluorescent signal having the X1 value as the left end has a maximum slope; Among the approximation lines of the fluorescence signal data with the X2 value at the right end, the approximation line f with the greatest slope max calculating (x); The above approximate line f min (x) and the above approximation line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) and X of the above temporary reference pointc’ The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) The above starting point (X s ,Y s ) is calculated by X of the above temporary starting point s’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b (x)'; The above approximate line f b (x)' and the above approximation line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) and X of the above reference point c The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) is the starting point of the signal rise.

[0007] The present invention provides a method for calculating an increase in a signal from a fluorescent signal measured using a biological sample, the method comprising the steps of: The starting point of the signal rise (X s ,Y s ) is calculated by the calculation method to find the start point of the signal rise (X s ,Y s ) and the step of calculating the starting point (X s ,Y s Calculating an increase in the fluorescent signal based on the The step of calculating the increase in the fluorescent signal comprises: X of the above starting point s calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as the Yline; The point where the fluorescence intensity of the above fluorescence signal is at its maximum is called the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l a step of calculating the value X of the above starting point s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b calculating (x); The above approximate line f b Peak point X on (x) p Y value at the value b a step of calculating the value Peak point Y p Value and above Y l The difference between the peak Yp value and the above Y value is calculated as the increase in the fluorescent signal (l). b and calculating the difference between the values ​​as the increase in the fluorescent signal (b), The present invention also relates to a calculation method in which the increase in the fluorescent signal (1) or the increase in the fluorescent signal (b) is determined as the increase in the signal.

[0008] According to the calculation method of the present invention described above, it is possible to accurately calculate the starting point of the signal increase and the amount of increase in the signal from the fluorescent signal acquired from the biological sample.

[0009] The present invention also provides a method for determining the start point of signal rise (X) from a chemiluminescence signal measured using a biological sample. s ,Y s ) is a calculation method for calculating The chemiluminescence signal is time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time, The above calculation method is based on the temporary starting point (X s’ ,Ys’ ), the step of calculating the temporary starting point (X s’ ,Y s’ ) based on the initial starting point (X s ,Y s ), the step of calculating the reference point (X c ,Y c ) and the initial starting point and the reference point (X c ,Y c ) to the starting point (X s ,Y s ) calculating The above temporary starting point (X s’ ,Y s’ ) is calculated by After adding a reagent that induces a biological reaction to a biological sample, the peak point (X) at which the chemiluminescence intensity reaches its maximum within the range after the addition of the reagent is determined. p ,Y p ) The time from when the disturbance of the chemiluminescence signal caused by the addition of the reagent settles to the time when the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) X at the peak point above p The moving average calculated from at least two consecutive data pairs going back from the Y value of the bottom point is b The first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) The initial starting point (X s ,Y s ) is calculated by X of the above temporary starting point s’ calculating an average value Y of the chemiluminescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as the Yline; X at the peak point above p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) The reference point (X c ,Y c ) is calculated by X at the peak point above p calculating the left end X1 value at which an approximate line of the chemiluminescence signal having the right end X1 value has the maximum slope; The X2 value at the right end where the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is taken as the X value at the peak point. p calculating a value within a range of Among the chemiluminescence signal approximation lines when the right end is the X2 value and the left end is within the range up to the X1 value, the approximation line f with the greatest slope max calculating (x); The above approximate line f max The intersection of (x) and the above Y line is the reference point (X c ,Y c ) The above starting point (X s ,Y s ) is calculated by X of the above reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the initial starting point is smaller than the recalculation reference value, the initial starting point is changed to the starting point (X s ,Y s ) and X of the above reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) as the starting point of the signal rise.

[0010] The present invention also provides a method for calculating an increase in a signal from a chemiluminescent signal measured using a biological sample, the method comprising: The starting point of the signal rise (X) from the chemiluminescence signal measured using the above biological sample s ,Y s ) is calculated by the calculation method to find the start point of the signal rise (X s ,Y s ) and calculate the end point (X e ,Y e ) and calculating an increase in chemiluminescence signal, The above end point (X e ,Y e ) is calculated by X at the peak point above p The first point where the moving average calculated from at least two consecutive data pairs falls within the range of the Y line at least twice in succession after the value is set as the end point (X e ,Y e ) The step of calculating the increase in the chemiluminescence signal comprises: The above Y line is the baseline, and the above starting point X s Value to the above end point X e calculating an integrated value of chemiluminescence intensity up to a value; The present invention also relates to a calculation method in which the integrated value is used as the amount of increase in the signal.

[0011] According to the calculation method of the present invention described above, it is possible to accurately calculate the starting point of the signal rise and the amount of increase in the signal from the chemiluminescent signal obtained from the biological sample.

[0012] The present invention also relates to a calculation device and a calculation program used in the above-mentioned calculation method. The calculation device and calculation program according to the present invention are, for example, as follows.

[0013] The starting point of the signal rise (X) from the fluorescence signal measured using a biological sample s ,Y s ) is a calculation device that calculates a data acquisition unit that acquires the fluorescent signal as time-series data consisting of a data pair (measurement time, fluorescent intensity) of fluorescent intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain the tentative starting point (X s’ ,Y s’ ) a first calculation unit that calculates The tentative starting point (X s’ ,Y s’ ) data and the time series data acquired by the data acquisition unit are processed to find the start point of the signal rise (X s ,Y s a second calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the approximate line f with the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent has settled. min (x) and approximate line f min Calculating the rightmost X1 value of (x); Calculating the right end X2 value at which the approximate line of the fluorescent signal with the X1 value at the left end has the maximum slope; Among the approximation lines of the fluorescence signal data with the X2 value at the right end, the approximation line f with the greatest slope max Calculating (x), The above approximate line f min (x) and the above approximation line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) X of the above temporary reference point c’ The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) In the second calculation unit, X of the above temporary starting point s’The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculating (x)', The above approximate line f b (x)' and the above approximation line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) X of the above reference point c The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) as the starting point of the signal rise.

[0014] A calculation device for calculating an increase in a signal from a fluorescent signal measured using a biological sample, The starting point of the signal rise from the above-mentioned fluorescent signal (X s ,Y s ) is transmitted to a calculation device that calculates the start point (X s ,Y s a third calculation unit that processes the data of (a) and the time-series data acquired by the data acquisition unit to calculate an increase in the fluorescent signal; In the third calculation unit, X of the above starting point s Calculating the average value Y of the fluorescence intensity from at least two consecutive data pairs, going back from the value, and calculating the horizontal line of the average value Y as the Yline; The point where the fluorescence intensity of the above fluorescence signal is at its maximum is called the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l Calculating it as a value, X of the above starting point sThe approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculating (x), The above approximate line f b Peak point X on (x) p Y value at the value b Calculating it as a value, Peak point Y p Value and above Y l The difference between the peak Yp value and the above Y value is calculated as the increase in the fluorescent signal (l). b Calculating the difference between the values ​​as the increase in the fluorescent signal (b); A calculation device that performs processing including determining the increase in the fluorescent signal (l) or the increase in the fluorescent signal (b) as the increase in the signal.

[0015] The starting point of the signal rise (X) from the fluorescence signal measured using a biological sample s ,Y s ) is a calculation program for calculating Computer, a data acquisition unit that acquires the fluorescent signal as time-series data consisting of data pairs (measurement time, fluorescent intensity) of fluorescent intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain the tentative starting point (X s’ ,Y s’ a first calculation unit that calculates The tentative starting point (X s’ ,Y s’ ) data and the time series data acquired by the data acquisition unit are processed to find the start point of the signal rise (X s ,Y s ) as a second calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the approximate line f with the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent has settled. min (x) and approximate line f minCalculating the rightmost X1 value of (x); Calculating the right end X2 value at which the approximate line of the fluorescent signal with the X1 value at the left end has the maximum slope; Among the approximation lines of the fluorescence signal data with the X2 value at the right end, the approximation line f with the greatest slope max Calculating (x), The above approximate line f min (x) and the above approximation line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) X of the above temporary reference point c’ The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) In the second calculation unit, X of the above temporary starting point s’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculating (x)', The above approximate line f b (x)' and the above approximation line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) X of the above reference point c The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) as the starting point of the signal rise.

[0016] A calculation program for calculating an increase in a signal from a fluorescent signal measured using a biological sample, The starting point of the signal rise from the above-mentioned fluorescent signal (X s ,Y s ) for the calculation program that calculates Computer, The start point of the signal rise calculated by the second calculation unit (X s ,Y s ) and the time-series data acquired by the data acquisition unit, and functioning as a third calculation unit that calculates an increase in the fluorescent signal; In the third calculation unit, X of the above starting point s Calculating the average value Y of the fluorescence intensity from at least two consecutive data pairs, going back from the value, and calculating the horizontal line of the average value Y as the Yline; The point where the fluorescence intensity of the above fluorescence signal is at its maximum is called the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l Calculating it as a value, X of the above starting point s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculating (x), The above approximate line f b Peak point X on (x) p Y value at the value b Calculating it as a value, Peak point Y p Value and above Y l The difference between the peak Yp value and the above Y value is calculated as the increase in the fluorescent signal (l). b Calculating the difference between the values ​​as the increase in the fluorescent signal (b); a calculation program for causing the program to function to execute a process including determining the increase in the fluorescent signal (1) or the increase in the fluorescent signal (b) as the increase in the signal;

[0017] The starting point of the signal rise (X) from the chemiluminescence signal measured using a biological sample s ,Y s ) is a calculation device that calculates a data acquisition unit that acquires the chemiluminescence signal as time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of the chemiluminescence intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain the tentative starting point (X s’ ,Y s’ ) a first calculation unit that calculates The tentative starting point (X s’ ,Y s’ ) data and the time series data acquired by the data acquisition unit are processed to obtain the initial starting point (X s ,Y s ) a second calculation unit that calculates The time series data acquired by the data acquisition unit is processed to obtain the reference point (X c ,Y c ) a third calculation unit that calculates The initial starting point data calculated by the second calculation unit, the reference point (X c ,Y c ) data and the time series data acquired by the data acquisition unit are processed to obtain the starting point (X s ,Y s a fourth calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the peak point (X) at which the chemiluminescence intensity reaches its maximum within the range after the addition of the reagent is determined. p ,Y p ) The time from when the disturbance of the chemiluminescence signal caused by the addition of the reagent settles to the time when the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) X at the peak point above p The moving average calculated from at least two consecutive data pairs going back from the Y value of the bottom point is bThe first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) In the second calculation unit, X of the above temporary starting point s’ Calculating the average value Y of the chemiluminescence intensity from at least two consecutive data pairs, and calculating the horizontal line of the average value Y as the Yline; X at the peak point above p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) In the third calculation unit, X at the peak point above p Calculate the left end X1 value at which the approximate line of the chemiluminescence signal with the value at the right end has the maximum slope; The X2 value at the right end where the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is taken as the X value at the peak point. p Calculation within the range of values, Among the chemiluminescence signal approximation lines when the right end is the X2 value and the left end is within the range up to the X1 value, the approximation line f with the greatest slope max Calculating (x), The above approximate line f max The intersection of (x) and the above Y line is the reference point (X c ,Y c ) In the fourth calculation unit, X of the above reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the initial starting point is smaller than the recalculation reference value, the initial starting point is changed to the starting point (X s ,Y s ) and X of the above reference point c Value and the initial starting point X s The difference between the value (X c Value-X sIf the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) as the starting point of the signal rise.

[0018] A calculation device for calculating an increase in a chemiluminescence signal measured using a biological sample, comprising: The starting point of the signal rise from the chemiluminescence signal (X s ,Y s ) is calculated by the calculation device that processes the time series data acquired by the data acquisition unit to calculate the end point (X e ,Y e a fifth calculation unit that calculates the start point (X s ,Y s ) data, the end point (X e ,Y e a sixth calculation unit that processes the time-series data acquired by the data acquisition unit and calculates an increase in the chemiluminescence signal; In the fifth calculation unit, X at the peak point above p The first point where the moving average calculated from at least two consecutive data pairs falls within the range of the Y line at least twice in succession after the value is set as the end point (X e ,Y e ) In the sixth calculation unit, The above Y line is the baseline, and the above starting point X s Value to the above end point X e Calculating the integrated value of chemiluminescence intensity up to the value; A calculation device that executes a process including determining the integrated value as an increase amount of the signal.

[0019] The starting point of the signal rise (X) from the chemiluminescence signal measured using a biological sample s ,Y s ) is a calculation program for calculating Computer, a data acquisition unit that acquires the chemiluminescence signal as time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain the tentative starting point (X s’ ,Y s’ a first calculation unit that calculates The tentative starting point (X s’ ,Y s’ ) data and the time series data acquired by the data acquisition unit are processed to obtain the initial starting point (X s ,Y s a second calculation unit for calculating The time series data acquired by the data acquisition unit is processed to obtain the reference point (X c ,Y c a third calculation unit for calculating The initial starting point data calculated by the second calculation unit, the reference point (X c ,Y c ) data and the time series data acquired by the data acquisition unit are processed to obtain the starting point (X s ,Y s ) and functioning as a fourth calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the peak point (X) at which the chemiluminescence intensity reaches its maximum within the range after the addition of the reagent is determined. p ,Y p ) The time from when the disturbance of the chemiluminescence signal caused by the addition of the reagent settles to the time when the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) X at the peak point above pThe moving average calculated from at least two consecutive data pairs going back from the Y value of the bottom point is b The first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) In the second calculation unit, X of the above temporary starting point s’ Calculating the average value Y of the chemiluminescence intensity from at least two consecutive data pairs, and calculating the horizontal line of the average value Y as the Yline; X at the peak point above p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) In the third calculation unit, X at the peak point above p Calculate the left end X1 value at which the approximate line of the chemiluminescence signal with the value at the right end has the maximum slope; The X2 value at the right end where the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is taken as the X value at the peak point. p Calculation within the range of values, Among the chemiluminescence signal approximation lines when the right end is the X2 value and the left end is within the range up to the X1 value, the approximation line f with the greatest slope max Calculating (x), The above approximate line f max The intersection of (x) and the above Y line is the reference point (X c ,Y c ) In the fourth calculation unit, X of the above reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the initial starting point is smaller than the recalculation reference value, the initial starting point is changed to the starting point (X s ,Y s ) and X of the above reference point c Value and the initial starting point Xs The difference between the value (X c Value-X s If the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) The above starting point (X s ,Y s ) as the starting point of the signal rise.

[0020] A calculation program for calculating an increase in a chemiluminescence signal measured using a biological sample, The starting point of the signal rise from the chemiluminescence signal (X s ,Y s ) for the calculation program that calculates Computer, The time series data acquired by the data acquisition unit is processed to obtain the end point (X e ,Y e ) and the start point (Xs, Ys) data of the signal rise calculated by the fourth calculation unit, the end point (X e ,Y e ) data and the time-series data acquired by the data acquisition unit, and functioning as a sixth calculation unit that calculates an increase in the chemiluminescence signal; In the fifth calculation unit, X at the peak point above p The first point where the moving average calculated from at least two consecutive data pairs falls within the range of the Y line at least twice in succession after the value is set as the end point (X e ,Y e ) In the sixth calculation unit, The above Y line is the baseline, and the above starting point X s Value to the above end point X e Calculating the integrated value of chemiluminescence intensity up to the value; a calculation program for causing the processing to be performed, including using the integrated value as an increase in the signal; [Effects of the Invention]

[0021] According to the present invention, it is possible to provide a calculation method that can accurately calculate the starting point of a signal increase and the amount of signal increase from a fluorescent or chemiluminescent signal acquired from a biological sample. The present invention also provides a calculation device and a calculation program for implementing the calculation method. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram illustrating a hardware configuration of a calculation device according to an embodiment. [Figure 2] FIG. 1 is a schematic diagram illustrating a functional configuration of a calculation device according to an embodiment. [Figure 3] 10 is a flowchart illustrating a method for calculating the starting point of a signal increase and the amount of signal increase from a fluorescent signal according to one embodiment. [Figure 4] FIG. 1 is a schematic diagram illustrating a functional configuration of a calculation device according to an embodiment. [Figure 5] 10 is a flowchart illustrating a method for calculating the starting point of a signal rise and the amount of signal increase from a chemiluminescent signal according to one embodiment. [Figure 6] FIG. 10 is a diagram illustrating a process for calculating a tentative reference point (Xc', Yc'). [Figure 7] FIG. 10 is a diagram illustrating a process for calculating a tentative starting point (Xs', Ys'). [Figure 8] FIG. 10 is a diagram illustrating a process for calculating a reference point (Xc, Yc). [Figure 9] FIG. 10 is a diagram illustrating a process for calculating a starting point (Xs, Ys). [Figure 10] FIG. 10 is a diagram illustrating a process for calculating the Yl value. [Figure 11] FIG. 10 is a diagram illustrating a process for calculating a Yb value. [Figure 12]FIG. 1 is a diagram showing a schematic process for calculating the increase in fluorescent signal (l) or (b). [Figure 13] FIG. 10 is a diagram illustrating a process for calculating a peak point (Xp, Yp) and a bottom point (Xb, Yb). [Figure 14] FIG. 10 is a diagram illustrating a process for calculating a tentative starting point (Xs', Ys'). [Figure 15] FIG. 10 is a diagram illustrating a process for calculating Yline. [Figure 16] FIG. 10 is a diagram illustrating a process for calculating the initial starting point (Xs, Ys). [Figure 17] FIG. 10 is a diagram illustrating a process for calculating an approximation line fmax(x). [Figure 18] 10A and 10B are diagrams illustrating a process for calculating a reference point (Xc, Yc) and a process for calculating a starting point (Xs, Ys). [Figure 19] 10A and 10B are diagrams illustrating a process for calculating a reference point (Xc, Yc) and a process for calculating a starting point (Xs, Ys). [Figure 20] FIG. 10 is a diagram illustrating a process for calculating an integrated value of chemiluminescence intensity. [Figure 21] 1 is a graph showing an example of fluorescent and chemiluminescent signals measured using a sample containing whole blood. [Figure 22] 1 is a graph showing an example of fluorescent and chemiluminescent signals measured using a sample containing whole blood. DETAILED DESCRIPTION OF THE INVENTION

[0023] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0024] [Method for calculating the starting point of signal increase and the amount of signal increase from the fluorescent signal] The method for calculating the start point of a signal rise and the amount of signal increase from a fluorescent signal according to this embodiment includes calculating the start point of a signal rise from a fluorescent signal measured using a biological sample, and calculating the amount of signal increase based on the calculated start point of the signal rise. The former can be regarded as an independent calculation method for calculating the start point of a signal rise from a fluorescent signal measured using a biological sample.

[0025] FIG. 3 is a flowchart showing a method for calculating the start point of a signal rise and the amount of signal increase from a fluorescent signal according to one embodiment. The method for calculating the start point of a signal rise and the amount of signal increase from a fluorescent signal according to one embodiment is a flowchart showing a method for calculating the start point of a signal rise and the amount of signal increase from a fluorescent signal according to one embodiment. s’ ,Y s’ ) and the process of searching (calculating) the start point of the signal rise (X s ,Y s ) and calculating the increase in fluorescent signal (total amount of light emitted).

[0026] The fluorescence signal that is the target of the calculation method according to this embodiment is time-series data consisting of a data pair (X, Y) of fluorescence intensity versus measurement time = (measurement time, fluorescence intensity).

[0027] Furthermore, the fluorescent signal that is the subject of the calculation method according to this embodiment is not particularly limited, as long as it is a fluorescent signal measured by adding a reagent that induces a biological reaction to a biological sample and detecting a change accompanying the biological reaction using a fluorescent reagent, etc. A specific example of the fluorescent signal is a fluorescent signal measured by adding a neutrophil stimulant (e.g., formylmethionylleucylphenylalanine, phorbol 12-myristate 13-acetate, opsonized zymon) to a biological sample containing neutrophil cells (e.g., a sample containing whole blood) and detecting increased production of hypochlorous acid (or its halogen equivalent) accompanying activation of myeloperoxidase using a fluorescent reagent (e.g., aminophenylfluorescein).

[0028] The fluorescence signal can be any fluorescence signal measured using a known fluorescence measuring device, such as a fluorometer, a fluorescence lifetime photometer, a fluorescence and luminescence simultaneous measuring device, etc. The excitation wavelength and observation wavelength of the fluorescence can be set appropriately depending on the fluorescence to be measured, and the calculation method according to this embodiment can be applied to any excitation wavelength and observation wavelength.

[0029] The calculation method according to the present invention can be suitably applied to a fluorescence signal that includes, for example, measurement data of fluorescence intensity at one point every 0.1 to 100 seconds. The fluorescence signal may include, for example, measurement data of fluorescence intensity at 0.01 to 10 points per second.

[0030] The fluorescence signal may be time-series data. The fluorescence signal may be time-series data including, for example, measurement data of the fluorescence intensity before adding a reagent that induces a biological reaction to the biological sample, measurement data of the fluorescence intensity at the time when the reagent that induces a biological reaction is added to the biological sample, and measurement data of the fluorescence intensity at the time when the change accompanying the biological reaction peaks (takes a maximum value). As the fluorescence intensity data of the fluorescence signal, for example, the measured fluorescence intensity data itself may be used, or fluorescence intensity data that has been subjected to a smoothing process (e.g., smoothed data using a moving average) may be used.

[0031] The calculation method for calculating the starting point of a signal rise from a fluorescent signal measured using a biological sample according to this embodiment (hereinafter also simply referred to as the "starting point calculation method") is to calculate a tentative starting point (X s’ ,Y s’ ) (temporary starting point calculation process), and the temporary starting point (X s’ ,Y s’ ), the start of the signal rise (X s ,Y s ) (starting point calculation step).

[0032] The tentative starting point calculation process is After adding a reagent that induces a biological reaction to a biological sample, the approximate line f with the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent has settled.min (x) and approximate line f min Step of calculating the right end X1 value of (x) (X1 value and f min (x) calculation step), and a step of calculating the right end X2 value at which the approximate line of the fluorescent signal having the X1 value at the left end has the maximum slope (X2 value calculation step); Among the approximation lines of the fluorescence signal data with the X2 value at the right end, the approximation line f with the greatest slope max Step (f max (x) calculation step), and approximation line f min (x) and approximate line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) (temporary reference point calculation step); Temporary reference point X c’ The moving average calculated from at least two consecutive data pairs from the value of min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) (tentative starting point calculation step).

[0033] X1 value and f min In the (x) calculation step, after adding a reagent that induces a biological reaction to the biological sample, the approximate line f having the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent has settled is calculated. min (x) and approximate line f min Calculate the rightmost X1 value of (x).

[0034] In this specification, an "approximation line" refers to a line derived by linear approximation using the leftmost and rightmost data pairs and all data pairs between the leftmost and rightmost data pairs. The "leftmost" data pair is the data pair with the earliest measurement time (the absolute value of the measurement time is small), and the "rightmost" data pair is the data pair with the latest measurement time (the absolute value of the measurement time is large). Linear approximation can be performed, for example, by least squares method, principal component analysis, etc.

[0035] Adding a reagent that induces a biological reaction to a biological sample causes a disturbance in the fluorescent signal due to physical impact, but this disturbance disappears after a certain period of time (or reaches a state where its effect on data analysis can be ignored). The "point at which the disturbance in the fluorescent signal due to the addition of the reagent settles down" may be determined visually from a graph plotting the data pair (X, Y) = (measurement time, fluorescent intensity) of fluorescence intensity versus measurement time. Alternatively, the mean and standard deviation of the fluorescent intensity data may be calculated for 20 to 40 consecutive data pairs over time from the time of reagent addition (referred to as "injection"), and the left end of the first data pair at which the coefficient of variation (standard deviation / mean value) is 0.03% or less for five or more consecutive times may be determined as the relevant point.

[0036] Furthermore, if the measurement conditions for the fluorescent signal (including the composition of reagents, etc.; the same applies below) are the same, the time elapsed until the disturbance in the fluorescent signal due to the addition of a reagent disappears (or the effect on data analysis becomes negligible) is approximately constant. Therefore, the "point at which the disturbance in the fluorescent signal due to the addition of a reagent settles down" can be set in advance. That is, for example, the parameter [F1] can be used to set Injection+[F1] as the "point at which the disturbance in the fluorescent signal due to the addition of a reagent settles down." Here, Injection+[F1] indicates the data pair [F1] (point) after the addition of the reagent.

[0037] The right-end X1 value is preferably determined from among data pairs from the point at which the fluorescent signal begins to rise after the minimum width sufficient to obtain a straight line along the signal before the rise. This eliminates the possibility of the right-end X1 value being determined from a point very close to the point at which the fluorescent signal begins to rise. Furthermore, the right-end X1 value is preferably determined from among data pairs from the point at which the fluorescent signal begins to rise after the point at which the fluorescent signal begins to rise. The width sufficient to obtain a straight line along the signal before the rise and the point at which the fluorescent signal begins to rise after the point at which the fluorescent signal begins to rise after the point at which the fluorescent signal begins to rise are nearly constant under the same measurement conditions for the fluorescent signal, and therefore can be set in advance. For example, the parameter [F2] (the number of data pairs corresponding to the point at which the fluorescent signal begins to rise after the point at which the fluorescent signal begins to rise after the point at which the fluorescent signal begins to rise) and the parameter [F3] (the number of data pairs corresponding to the width at which a straight line along the signal before the rise) can be used to search for the right-end X1 value within the range from Injection + [F1] + [F3] and before Injection + [F1] + [F2].

[0038] In the X2 value calculation step, the right end X2 value at which the approximate line of the fluorescent signal with the X1 value at the left end has the maximum slope is calculated.

[0039] The right-end X2 value is preferably determined by searching from among the data pairs from the X1 value onward, with a minimum width sufficient to obtain a straight line following the increasing signal. This prevents a point very close to the X1 value from becoming the right-end X2 value. The width sufficient to obtain a straight line following the increasing signal is nearly constant if the measurement conditions for the fluorescent signal are the same, so it can be set in advance. That is, for example, the parameter [F5] (the number of data pairs corresponding to a width sufficient to obtain a straight line following the increasing signal) can be used to search for the right-end X2 value in the range from the X1 value + [F5] onward.

[0040] The right-end X2 value is preferably searched within the range of the maximum possible right-end X2 value, which is the final point of the fluorescence signal data (the data pair with the latest measurement time). The maximum possible right-end X2 value may be set to a value before the final point of the fluorescence signal data, if necessary. For example, in the case of fluorescence signal data containing two points of fluorescence intensity measurement data per second, the maximum possible right-end X2 value may be set as Injection + [F1] + [F3] + [F4] using parameter [F4] (the number of data pairs specifying a time point before the final point of the fluorescence signal data). By setting the maximum possible right-end X2 value in this way, it is possible to eliminate the influence of, for example, the rare occurrence of two or more peaks in the fluorescence signal.

[0041] f max In the (x) calculation step, the approximate line f having the maximum slope among the approximate lines of the fluorescence signal data with the X2 value at the right end is calculated. max Calculate (x).

[0042] The range for searching for the left end of the approximation line is not particularly limited, but the search may be limited to a certain range. Specifically, for example, if the fluorescence signal contains measurement data of fluorescence intensity at two points per second, the search can be performed within the following range. (i) From the point when the fluorescent signal disturbance due to the addition of the reagent has settled down to the X1 value (when the X2 value - X1 value < 1000) (ii) From the point at which the fluorescent signal disturbance caused by the addition of the reagent has settled down to the point at which a sufficient width is subtracted from the X2 value to obtain a straight line along the increasing signal (when the X2 value - X1 value ≥ 1000). The width sufficient to obtain a straight line along the increasing signal in (ii) is almost constant if the measurement conditions of the fluorescent signal are the same, so it can be set in advance. That is, for example, by using the parameter [F6] (the number of data pairs corresponding to the width sufficient to obtain a straight line along the increasing signal), (ii)' From the point when the disturbance of the fluorescent signal due to the addition of the reagent has settled down to the X2 value - [F6] (when the X2 value - X1 value ≥ 1000) It can also be done as follows.

[0043] In the tentative reference point calculation step, the approximate line f min (x) and approximate line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) is calculated as

[0044] In the temporary starting point calculation step, the X of the temporary reference point c’ The moving average calculated from at least two consecutive data pairs from the value of min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) is calculated as

[0045] Temporary reference point X c’ "Going back from a value" means moving from a tentative reference point toward earlier measurement times in time-series data. In this specification, "moving average" refers to an average value calculated from at least two consecutive data pairs. The number of data pairs used to calculate the moving average is preferably a number that can cancel spike noise. The number of points that can cancel spike noise can be confirmed by actually calculating a moving average for the target fluorescent signal. Since the number of points that can cancel spike noise is nearly constant if the measurement conditions for the fluorescent signal are the same, it can also be set in advance. For example, the parameter [F8] (the number of data pairs that corresponds to the number of points that can cancel spike noise) can be used to calculate the moving average to consecutive [F8] points.

[0046] Moving average is approximated by line f min The point that is close enough to (x) can be calculated as the tentative starting point. The moving average is the approximate line f min Whether the moving average is close enough to (x) can be determined by, for example, the approximation line f min It can be judged by whether it is within the noise width of (x) ± the fluorescence signal. Note that the tentative starting point is used to calculate the reference point, so a looser criterion may be adopted. For example, if the moving average is closer to the approximation line fmin (x) It may also be determined whether or not the signal falls within a range wider than the noise width of the ±fluorescence signal.

[0047] The noise width of the fluorescent signal can be set, for example, as the standard deviation of the fluctuation in the baseline of the fluorescent signal. The baseline may be, for example, the fluorescent signal observed in the biological sample before the addition of a reagent, or the fluorescent signal observed after the addition of a reagent, in a range that does not include fluorescence associated with biological reactions. A width wider than the noise width of the fluorescent signal may be set, for example, within the range of 1.1 times the noise width of the fluorescent signal to 6.0 times the noise width of the fluorescent signal. A width wider than the noise width of the fluorescent signal can also be set in advance. That is, for example, the moving average can be set to the approximation line f using the parameter [F7] (width wider than the noise width of the fluorescent signal). min It may be determined whether or not the value is within the range of (x)±[F7].

[0048] Also, the moving average is approximated by the line f min To ensure that the moving average is close enough to (x), the approximation line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) is preferably calculated as the number of consecutive entries that can cancel out the data variability. The number of times that the data variability can be canceled out can be confirmed by actually calculating the tentative starting point for the target fluorescent signal. The number of times that the data variability can be canceled out is almost constant if the fluorescent signal measurement conditions are the same, so it can also be set in advance. That is, for example, by using parameter [F9] (the number of times that the data variability can be canceled out), the moving average can be calculated as the number of times that the moving average can be canceled out by the approximation line f min (x) ± The first point that falls within the range wider than the noise width of the fluorescence signal [F9] times consecutively is taken as the tentative starting point (X s’ ,Y s’ ) may also be calculated.

[0049] Figure 6 shows the tentative reference point (X c’ ,Y c’6 is a diagram showing a process of calculating the X1 value and f min (x) calculation step, X2 value calculation step, f max FIG. 7 shows the results of the calculation step (x) and the calculation step of the tentative reference point. s’ ,Y s’ 7 is a diagram showing a process of calculating the tentative starting point. FIG. 7 shows the result of performing the tentative starting point calculation step of the tentative starting point calculation process.

[0050] The starting point calculation process is Temporary start point X s’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Step (f b (x)' calculation step) and approximate line f b (x)' and approximate line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) (reference point calculation step); Reference point X c The moving average calculated from at least two consecutive data pairs from the value of b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) (starting point calculation step).

[0051] f b In the (x)' calculation step, the temporary starting point X s’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculate (x)'.

[0052] The range for searching for the left end of the approximation line is not particularly limited as long as it is within the range after the point at which the disturbance in the fluorescent signal due to the addition of the reagent has settled, but the range may be limited for the search. Specifically, for example, if the fluorescent signal contains measurement data of the fluorescent intensity at two points per second, the search can be performed within the following range. (i) When the fluorescent signal disturbance caused by the addition of the reagent has settled down ~ (X s’ value -500) (X s’ (When the value is greater than or equal to the value at the time of reagent addition + 700) (ii) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down, ~ (X s’ value -400) (between the time of reagent addition +700>X s’ (When the value is greater than or equal to the value at the time of reagent addition + 600) (iii) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down ~ (X s’ Between (value -300) (time of reagent addition +600) > X s’ (When the value is greater than or equal to the value at the time of reagent addition + 500) (iv) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down, ~ (X s’ Between (value -200) (time of reagent addition +500) > X s’ value) However, X s’ If the value -200< the time when the disturbance of the fluorescent signal due to the addition of the reagent has settled, then the time when the disturbance of the fluorescent signal due to the addition of the reagent has settled ~ X s’ Between the values.

[0053] As mentioned above, the "point at which the disturbance in the fluorescent signal due to the addition of the reagent settles down" can also be set in advance. That is, for example, the parameter [F11] can be used to set Injection+[F11] as the "point at which the disturbance in the fluorescent signal due to the addition of the reagent settles down." Here, Injection+[F11] indicates the data pair [F11] (point) after the reagent is added.

[0054] approximate line f bIt is preferable that (x)' is calculated as an approximation line having a minimum slope and is equal to or greater than the slope of the fluorescent signal obtained when no reagent is added. The slope of the fluorescent signal obtained when no reagent is added can be determined, for example, by calculating an approximation line of the fluorescent signal observed in the biological sample before the addition of the reagent. The slope of the fluorescent signal obtained when no reagent is added is almost constant if the measurement conditions for the fluorescent signal are the same, so it can also be set in advance. That is, for example, the approximation line f can be calculated using the parameter [F10] (slope of the fluorescent signal obtained when no reagent is added). b (x)' can also be calculated as an approximate line with a gradient equal to or greater than [F10] and minimum.

[0055] In the reference point calculation step, the approximate line f b (x)' and approximate line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) is calculated as

[0056] In the starting point calculation step, the X of the reference point c The moving average calculated from at least two consecutive data pairs from the value of b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) is calculated as

[0057] Reference point X cGoing back from a value means moving from a reference point toward earlier measurement times in time-series data. As mentioned above, a moving average is an average value calculated from at least two consecutive data pairs. The number of data pairs used to calculate the moving average is preferably a number that can cancel spike noise. The number of points that can cancel spike noise can be confirmed by actually calculating a moving average for the target fluorescent signal. The number of points that can cancel spike noise is almost constant if the measurement conditions for the fluorescent signal are the same, so it can also be set in advance. That is, for example, by using the parameter [F8] (the number of data pairs that corresponds to the number of points that can cancel spike noise), the number of data pairs used to calculate the moving average can be set to consecutive [F8] points.

[0058] Moving average approximate line f b The point that is close enough to (x)' can be used as the starting point. The moving average is the approximate line f b Whether the moving average is close enough to (x)' can be determined by, for example, the approximation line f b This can be determined by whether or not (x)' is within the noise width of the fluorescence signal. The noise width of the fluorescence signal is almost constant if the measurement conditions for the fluorescence signal are the same, so it can be set in advance. That is, for example, by using parameter [F12] (noise width of the fluorescence signal), the moving average can be determined by the approximation line f b It may be determined whether or not the value is within the range of (x)'±[F12].

[0059] Also, the moving average is approximated by the line f b To ensure that the moving average is close enough to (x)', the approximation line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s) is preferably calculated as the number of consecutive entries that can cancel out the data variability. The number of times that the data variability can be canceled out can be confirmed by actually calculating the tentative starting point for the target fluorescent signal. The number of times that the data variability can be canceled out is almost constant if the fluorescent signal measurement conditions are the same, so it can also be set in advance. That is, for example, by using parameter [F9] (the number of times that the data variability can be canceled out), the moving average can be calculated as the number of times that the moving average can be canceled out by the approximation line f b The first point that falls within the noise width of (x)' ± the fluorescence signal [F9] times in succession is taken as the starting point (X s ,Y s ) may also be calculated.

[0060] Figure 8 shows the reference point (X c ,Y c 8 is a diagram showing the process of calculating f b FIG. 9 shows the results of the (x)' calculation step and the reference point calculation step. s ,Y s 9 is a diagram showing a process for calculating the starting point. FIG. 9 shows the result of performing the starting point calculation step of the starting point calculation process.

[0061] The starting point (X s ,Y s ) accurately captures the starting point of the signal rise of the fluorescence signal measured using a biological sample, and the starting point (X s ,Y s ) can be taken as the starting point of the signal rise.

[0062] The method for calculating the signal increase amount from a fluorescent signal measured using a biological sample according to this embodiment (hereinafter also simply referred to as the "method for calculating the increase amount") is to calculate the start point of the signal increase (X s ,Y s ) and the process of calculating the starting point (X s ,Y s ) based on the result of the measurement (increase calculation step).

[0063] The increase amount calculation step includes: Starting point X s a step of calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs going back from the value, and calculating a horizontal line of the average value Y as the Yline (Yline calculation step); The point where the fluorescence intensity of the fluorescent signal is maximum is the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l a step of calculating the Y1 value; Starting point X s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Step (f b (x) calculation step), and approximation line f b Peak point X on (x) p Y value at the value b Step (Y b value calculation step), Peak point Y p Value and Y l The difference between the peak Yp value and the Y value is calculated as the increase in the fluorescent signal (l). b and a step of calculating the difference between the values ​​as the increase (b) in the fluorescent signal (increase calculation step).

[0064] In the Y line calculation step, the X of the starting point s Working backward from the value, the average value Y of the fluorescence intensity is calculated from at least two consecutive data pairs, and the horizontal line of the average value Y is calculated as the Yline.

[0065] Starting point X s"Going back from the starting point" means moving from the starting point toward earlier measurement times in the time series data. The number of data pair points used to calculate the average value is preferably a number sufficient to reflect the average signal level immediately before the starting point. The number of points sufficient to reflect the average signal level immediately before the starting point can be confirmed by actually calculating and comparing the average value for the target fluorescent signal. The number of points sufficient to reflect the average signal level immediately before the starting point is nearly constant if the fluorescent signal measurement conditions are the same, and can therefore be set in advance. That is, for example, the parameter [F13] (the number of data pair points corresponding to the number sufficient to reflect the average signal level immediately before the starting point) can be used to set the number of data pair points used to calculate the average value to consecutive [F13] points.

[0066] In the Y1 value calculation step, the point where the fluorescence intensity of the fluorescence signal is maximum is taken as the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l Calculate the value as Y l The value is X=X p is the Y value of the intersection of the line represented by and the Y line.

[0067] f b (x) In the calculation step, the starting point X s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b Calculate (x).

[0068] The range for searching for the left end of the approximation line is not particularly limited as long as it is within the range after the point at which the disturbance in the fluorescent signal due to the addition of the reagent has settled, but the range may be limited for the search. Specifically, for example, if the fluorescent signal contains measurement data of the fluorescent intensity at two points per second, the search can be performed within the following range. (i) When the fluorescent signal disturbance caused by the addition of the reagent has settled down ~ (X s value -500) (X s (When the value is greater than or equal to the value at the time of reagent addition + 700) (ii) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down, ~ (X s value -400) (between the time of reagent addition +700>X s (When the value is greater than or equal to the value at the time of reagent addition + 600) (iii) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down ~ (X s Between (value -300) (time of reagent addition +600) > X s (When the value is greater than or equal to the value at the time of reagent addition + 500) (iv) When the disturbance in the fluorescent signal due to the addition of the reagent has settled down, ~ (X s Between (value -200) (time of reagent addition +500) > X s value) However, X s If the value -200< the time when the disturbance of the fluorescent signal due to the addition of the reagent has settled, then the time when the disturbance of the fluorescent signal due to the addition of the reagent has settled ~ X s Between the values.

[0069] As mentioned above, the "point at which the disturbance in the fluorescent signal due to the addition of the reagent settles down" can also be set in advance. That is, for example, the parameter [F11] can be used to set Injection+[F11] as the "point at which the disturbance in the fluorescent signal due to the addition of the reagent settles down." Here, Injection+[F11] indicates the data pair [F11] (point) after the reagent is added.

[0070] approximate line f b It is preferable that (x) is calculated as an approximation line having a minimum slope and is equal to or greater than the slope of the fluorescent signal obtained when no reagent is added. The slope of the fluorescent signal obtained when no reagent is added can be determined, for example, by calculating an approximation line of the fluorescent signal observed in a biological sample before the addition of a reagent. The slope of the fluorescent signal obtained when no reagent is added is almost constant if the measurement conditions for the fluorescent signal are the same, so it can also be set in advance. That is, for example, the approximation line f can be calculated using the parameter [F10] (slope of the fluorescent signal obtained when no reagent is added). b (x) can also be calculated as an approximate line with a gradient equal to or greater than [F10] and minimum.

[0071] Y b In the value calculation step, the approximation line f b Peak point X on (x) p Y value at the value b Calculate the value as Y b The value is X=X p The line and approximate line f b This is the Y value of the intersection with (x).

[0072] In the increase calculation step, the peak point Y p Value and Y l The difference between the values ​​is calculated as the increase in the fluorescent signal (l), or the peak point Y p value and the Y b The difference between these values ​​is calculated as the increase in the fluorescent signal (b).

[0073] The calculated increase in fluorescent signal (l) (total luminescence (l)) reflects the increase in fluorescent signal obtained from the entire reaction system used in the measurement. On the other hand, the calculated increase in fluorescent signal (b) (total luminescence (b)) is considered to be the fluorescent signal obtained from the reaction system used in the measurement, excluding fluorescent signals that are not derived from biological reactions, and therefore may enable more accurate evaluation of biological reactions. Whether the increase in fluorescent signal (l) or (b) is used may be selected appropriately depending on the purpose of the measurement, etc.

[0074] Figure 10 shows the Y l 10 shows the results of the Yline calculation step and the Y1 value calculation step of the increase amount calculation process. b FIG. 11 is a diagram showing a process for calculating the f value in the increment calculation step. b (x) Calculation step and Y b 12 is a diagram showing a process for calculating the increase (l) or (b) of the fluorescent signal. FIG. 12 shows the result of performing the increase calculation step of the increase calculation process.

[0075] The increase (l) or (b) calculated by the above-mentioned method for calculating the increase accurately captures the increase in the signal of a fluorescent signal measured using a biological sample, and the increase (l) or (b) can be used as the signal increase.

[0076] The parameters [F1] to [F13] described above can be set appropriately depending on the specific aspects of the fluorescent signal that is the target of the calculation method according to this embodiment. Examples of factors that affect the setting of the parameters [F1] to [F13] include the type of biological sample, the type of reagent added, the type of biological reaction to be detected, the type of fluorescent reagent, the number of measurement data of fluorescent intensity per unit time, and whether or not smoothing processing is performed on the measurement data.

[0077] For example, in the case of a fluorescent signal (including measurement data of fluorescence intensity at two points per second) measured by adding a neutrophil stimulant (formylmethionylleucylphenylalanine) to a biological sample containing neutrophil cells (a sample including whole blood) and detecting the increased production of hypochlorous acid due to the activation of myeloperoxidase with a fluorescent reagent (aminophenylfluorescein), the following specific numerical values ​​can be exemplified as each parameter indicated by [F1] to [F13]. [F1] 100-200 points, preferably 150 points [F2] 2000-3100 points, preferably 2550 points [F3] 200-400 points, preferably 300 points [F4] 2000-2500 points, preferably 2250 points [F5] 600-1000 points, preferably 800 points [F6] 800 to 1200 points, preferably 1000 points [F7] 200 to 240, preferably 220 [F8] 4 to 6 points, preferably 5 points [F9] 3 to 5 times, preferably 4 times [F10] 0.070 to 0.090, preferably 0.080 [F11] 80 to 120 points, preferably 100 points [F12] 30 to 50, preferably 40 [F13] 15 to 25 points, preferably 20 points

[0078] [Method for calculating the starting point of signal increase and the amount of signal increase from chemiluminescence signal] The method for calculating the start point of a signal rise and the amount of signal increase from a chemiluminescent signal according to this embodiment includes calculating the start point of a signal rise from a chemiluminescent signal measured using a biological sample, and calculating the amount of signal increase based on the calculated start point of the signal rise. The former can be regarded as an independent calculation method for calculating the start point of a signal rise from a chemiluminescent signal measured using a biological sample.

[0079] 5 is a flowchart showing a method for calculating the starting point of a signal rise and the amount of signal increase from a chemiluminescent signal according to one embodiment. The method for calculating the starting point of a signal rise and the amount of signal increase from a chemiluminescent signal according to one embodiment is carried out by calculating a tentative starting point (X s’ ,Y s’ ) search (calculation), the initial starting point (X s ,Y s ) search (calculation), the reference point (X c ,Y c ) calculation process, the initial starting point (X s ,Y s ) and the reference point (X c ,Y c ) and recalculate if necessary, and s ,Y s ) and calculate the end point (X e ,Y e ) and calculating the increase (integrated value) of the chemiluminescence signal.

[0080] The chemiluminescence signal that is the subject of the calculation method according to this embodiment is time-series data consisting of a data pair of chemiluminescence intensity versus measurement time (X, Y)=(measurement time, chemiluminescence intensity).

[0081] Furthermore, the chemiluminescent signal that is the subject of the calculation method according to this embodiment is not particularly limited, as long as it is a chemiluminescent signal measured by adding a reagent that induces a biological reaction to a biological sample and detecting the changes associated with the biological reaction using a chemiluminescent reagent or the like. A specific example of a chemiluminescent signal is a chemiluminescent signal measured by adding a neutrophil stimulant (e.g., formylmethionylleucylphenylalanine, phorbol 12-myristate 13-acetate, opsonized zymon) to a biological sample containing neutrophil cells (e.g., a sample containing whole blood), and detecting the increase in superoxide production associated with the activation of superoxide-producing activity with a chemiluminescent reagent (e.g., 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, 2-methyl-6-p-methoxyphenylethynylimidazopyrazinone, or an indocyanine-type imidazopyranodine compound).

[0082] The chemiluminescence signal can be any chemiluminescence signal measured using a known luminescence measuring device, such as a chemiluminescence measuring device, a bioluminescence measuring device, a simultaneous fluorescence and luminescence measuring device, etc. The observation wavelength of the chemiluminescence can be appropriately set depending on the chemiluminescence to be measured, and the calculation method according to this embodiment can be applied to any observation wavelength.

[0083] The calculation method according to the present invention can be suitably applied to a chemiluminescent signal that includes, for example, one measurement data point of chemiluminescent intensity every 0.1 to 25 seconds. The chemiluminescent signal may include, for example, 0.04 to 10 measurement data points of chemiluminescent intensity per second.

[0084] The chemiluminescent signal may be time-series data. The chemiluminescent signal may be time-series data including, for example, measurement data of chemiluminescent intensity before adding a reagent that induces a biological reaction to a biological sample, measurement data of chemiluminescent intensity at the time when the reagent that induces a biological reaction is added to the biological sample, and measurement data of chemiluminescent intensity at the time when the change accompanying the biological reaction peaks (takes a maximum value). As the chemiluminescent intensity data of the chemiluminescent signal, for example, the measured chemiluminescent intensity data itself may be used, or chemiluminescent intensity data that has been subjected to a smoothing process (e.g., smoothed data using a moving average) may be used.

[0085] The method for calculating the starting point of a signal rise from a chemiluminescence signal measured using a biological sample according to this embodiment (hereinafter simply referred to as a "method for calculating the starting point of a chemiluminescence signal") is to calculate a tentative starting point (X s’ ,Y s’ ) (temporary starting point calculation step), s’ ,Y s’ ) based on the initial starting point (X s ,Y s ) calculation process (initial starting point calculation process), the reference point (X c ,Y c ) (reference point calculation step), and c ,Y c ) to the starting point (X s ,Y s ) (starting point calculation step).

[0086] The tentative starting point calculation process is After adding a reagent that induces a biological reaction to a biological sample, the peak point (X) at which the chemiluminescence intensity reaches its maximum within the range after the addition of the reagent is determined. p ,Y p ) (peak point calculation step); The time from the point where the disturbance of the chemiluminescence signal due to the addition of the reagent has settled down to the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) (bottom point calculation step); Peak point X p The moving average calculated from at least two consecutive data pairs going back from the bottom point is the Y b The first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) (tentative starting point calculation step).

[0087] In the peak point calculation step, after adding a reagent that induces a biological reaction to the biological sample, the peak point (X p ,Y p ) is calculated.

[0088] The range after the reagent addition is not particularly limited as long as it is from the time of reagent addition (injection) onwards, but it may be a range excluding a predetermined number of data pairs immediately after injection. This makes it possible to eliminate the effects of disturbances in the chemiluminescence signal due to the addition of reagent. Specifically, for example, if the chemiluminescence signal contains measurement data of chemiluminescence intensity at two points per second, the range after the reagent addition may be set to the range after injection + 10 (the number of data pairs).

[0089] In the bottom point calculation step, the time from the point where the disturbance of the chemiluminescence signal due to the addition of the reagent has settled down to the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) is calculated.

[0090] Adding a reagent that induces a biological reaction to a biological sample causes a disturbance in the chemiluminescence signal due to physical impact, but this disturbance disappears after a certain period of time (or reaches a state where its effect on data analysis can be ignored). The "point at which the disturbance in the chemiluminescence signal due to the addition of the reagent settles down" may be determined visually from a graph plotting the chemiluminescence intensity data pair (X, Y) = (measurement time, chemiluminescence intensity) against measurement time. Alternatively, the mean and standard deviation of the chemiluminescence intensity data may be calculated for 10 to 20 consecutive data pairs over time from the time of reagent addition (referred to as "injection"), and the left end of the first data pair at which the coefficient of variation (standard deviation / mean value) is 0.2% or less for five or more consecutive times may be determined as the relevant point of time.

[0091] If the measurement conditions for the chemiluminescent signal (including the composition of reagents, etc.; the same applies below) are the same, the time elapsed until the disturbance in the chemiluminescent signal due to the addition of a reagent disappears (or until the effect on data analysis becomes negligible) is approximately constant. Therefore, the "point at which the disturbance in the chemiluminescent signal due to the addition of a reagent settles down" can be set in advance. That is, for example, the parameter [C1] can be used to set Injection+[C1] as the "point at which the disturbance in the chemiluminescent signal due to the addition of a reagent settles down." Here, Injection+[C1] indicates the data pair [C1] (point) after the addition of the reagent.

[0092] In the tentative starting point calculation step, the X of the peak point p The moving average calculated from at least two consecutive data pairs going back from the bottom point is the Y b The first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) is calculated as

[0093] Peak point X p Going back from the value means that in time series data, the X value at the peak point pThis means moving from the previous value toward earlier measurement times. As mentioned above, a moving average is an average value calculated from at least two consecutive data pairs. The number of data pairs used to calculate the moving average is preferably a number that can cancel spike noise. The number of data pairs that can cancel spike noise can be confirmed by actually calculating a moving average for the target chemiluminescence signal. The number of data pairs that can cancel spike noise is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, the number of data pairs used to calculate the moving average can be set to consecutive [C3] points using the parameter [C3] (the number of data pairs that corresponds to the number of data pairs that can cancel spike noise).

[0094] Y at the bottom of the moving average b The point that is close enough to the moving average Y value can be calculated as the tentative starting point. b For example, whether the moving average is close enough to the bottom point Y b This can be determined by whether the value is within the range of the noise width of the chemiluminescence signal.

[0095] The noise width of the chemiluminescent signal can be set, for example, as the standard deviation of the fluctuation in the baseline of the chemiluminescent signal. The baseline may be, for example, the chemiluminescent signal observed in the biological sample before the addition of the reagent, or the chemiluminescent signal observed after the addition of the reagent, in a range that does not include chemiluminescence associated with biological reactions. The noise width of the chemiluminescent signal is almost constant if the measurement conditions for the chemiluminescent signal are the same, so it can also be set in advance. That is, for example, the moving average can be calculated by using the parameter [C2] (noise width of the chemiluminescent signal) to determine the Y value of the bottom point of the approximation line. b It may also be determined whether the value is within a range equal to or less than the value + [C2].

[0096] Also, the moving average is the bottom point Y b To ensure that the moving average is close enough to the bottom point, bThe first point that falls within the range of the value + the noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) is preferably calculated as the number of consecutive entries that can cancel out the data variability. The number of times that the data variability can be cancelled out can be confirmed by actually calculating the tentative starting point for the target chemiluminescence signal. The number of times that the data variability can be cancelled out is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, by using parameter [C4] (the number of times that the data variability can be cancelled out), the moving average is calculated as the number of times that the data variability can be cancelled out at the bottom point Y b The first point that falls within the range of the value + the noise width of the chemiluminescence signal [C4] times consecutively is set as the tentative starting point (X s’ ,Y s’ ) may also be calculated.

[0097] Figure 13 shows the peak point (X p ,Y p ) and bottom point (X b ,Y b 13 shows the results of the peak point calculation step and bottom point calculation step of the tentative starting point calculation process. s’ ,Y s’ 14 is a diagram showing a process of calculating the tentative starting point. FIG. 14 shows the result of performing the tentative starting point calculation step of the tentative starting point calculation process.

[0098] The initial starting point calculation process is Temporary start point X s’ a step of calculating an average value Y of the chemiluminescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as the Yline (Yline calculation step); Peak point X p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) (initial starting point calculation step).

[0099] In the Y line calculation step, the X of the temporary starting point s’ Working backward from the value, the average value Y of the chemiluminescence intensity is calculated from at least two consecutive data pairs, and the horizontal line of the average value Y is calculated as the Yline.

[0100] Temporary start point X s’ Going back from the value means that in time series data, the tentative starting point X s’ This means that the average value moves from the previous value toward earlier measurement times. The number of data pairs used to calculate the average value is preferably a number sufficient to reflect the average signal level before the increase. The number of data pairs sufficient to reflect the average signal level before the increase can be confirmed by actually calculating and comparing the average value for the chemiluminescence signal of interest. The number of data pairs sufficient to reflect the average signal level before the increase is almost constant if the measurement conditions for the chemiluminescence signal are the same, and can therefore be set in advance. That is, for example, the number of data pairs used to calculate the average value Y can be set to consecutive [C5] points using the parameter [C5] (the number of data pairs sufficient to reflect the average signal level before the increase).

[0101] In the Yline calculation step, it is preferable to exclude data pairs from the time of reagent addition until the time when the disturbance of the chemiluminescence signal due to the reagent addition has settled from the data pairs used to calculate the average value Y. This makes it possible to eliminate the influence of the disturbance of the chemiluminescence signal due to the reagent addition. Specifically, for example, the average value Y may be calculated by excluding data pairs within the range from "Injection" to "Injection+[C6]" using the parameter [C6]. Here, "Injection+[C6]" indicates the data pairs from the time when the reagent was added until point [C6] later.

[0102] In the first starting point calculation step, the peak point X p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) is calculated as

[0103] Peak point Xp Going back from the value means that in time series data, the X value at the peak point p This means that the standard deviation moves from the average value to earlier measurement times. The standard deviation is a value calculated from the chemiluminescence intensity data of the data pair used to calculate the average value Y. The standard deviation is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can be set in advance. That is, for example, by using the parameter [C7] (standard deviation), the first point where the chemiluminescence intensity falls within the range of the average value Y + [C7] or less is set as the initial starting point (X s ,Y s ) may also be calculated.

[0104] FIG. 15 is a diagram showing the outline of the process of calculating Yline. FIG. 15 shows the result of performing the Yline calculation step of the initial starting point calculation process. FIG. 16 shows the result of performing the initial starting point (X s ,Y s 16 is a diagram showing a process of calculating the initial starting point. FIG. 16 shows the result of performing the initial starting point calculation step of the initial starting point calculation process.

[0105] The reference point calculation step includes: Peak point X p a step of calculating the left end X1 value at which the approximate line of the chemiluminescence signal with the value at the right end has the maximum slope (X1 value calculation step); The X1 value at the left end of the approximate line of the chemiluminescence signal is the X2 value at the right end where the slope is greatest, and the X value at the peak point is the X p a step of calculating the X2 value within the range of the X2 value; The approximate line f with the greatest slope among the approximate lines of the chemiluminescence signal when the right end is the X2 value and the left end is within the range up to the X1 value. max Step (f max (x) calculation step), and approximate line f max The intersection of (x) and Y line is the reference point (X c ,Y c ) (reference point calculation step).

[0106] In the X1 value calculation step, the X value of the peak point is calculated.p The left end X1 value at which the approximate line of the chemiluminescence signal with the value at the right end has the maximum slope is calculated.

[0107] The range for searching for the left end X1 value is not particularly limited, but the search may be limited to a certain range. Specifically, for example, if the chemiluminescence signal contains measurement data of chemiluminescence intensity at two points per second, the search can be performed within the following range. (i) When the disturbance of the chemiluminescence signal due to the addition of the reagent has settled down ~ (X p Between (X) and (wide enough to get a straight line along the signal after the rise) p value ≥ the point where the peak is expected to be obtained) (ii) When the disturbance in the chemiluminescence signal due to the addition of the reagent has settled down, ~ (X p Between (X) and (wide enough to get a straight line along the signal after the rise) p value < the point where the peak is expected to be obtained)

[0108] The point where the peak is expected to be obtained can be confirmed by actually measuring the chemiluminescence signal of interest. The point where the peak is expected to be obtained is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, by using parameter [C8] (the number of average data pairs from the time of adding the reagent to the point where the peak is expected to be obtained), X p It is also possible to select either (i) or (ii) by comparing the value with Injection+[C8].

[0109] The "point at which the chemiluminescence signal disturbance due to the addition of the reagent settles down" can also be set in advance. For example, using the parameter [C9], Injection+[C9] can be set as the "point at which the chemiluminescence signal disturbance due to the addition of the reagent settles down." Here, Injection+[C9] indicates the data pair [C9] (point) after the addition of the reagent.

[0110] The widths (i) and (ii) sufficient to obtain a straight line along the increasing signal are almost constant if the measurement conditions of the chemiluminescence signal are the same, and can therefore be set in advance. That is, for example, the parameters [C10] and [C11] (the number of data pairs corresponding to the widths sufficient to obtain a straight line along the increasing signal) are used to set (i)'(Injection+[C9])~(X p Value - [C10]) (X p (when value ≧ (injection+[C8])) (ii)' (Injection+[C9])~(X p Value - [C11]) (X p When value < (injection+[C8]) It can also be done as follows.

[0111] In the X2 value calculation step, the right-end X2 value at which the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is calculated as the X2 value at the peak point. p Calculate within the range of values.

[0112] The right-end X2 value is preferably determined by searching from among the data pairs from the X1 value onward, with a minimum width sufficient to obtain a straight line along the signal after the increase. This makes it possible to prevent a point very close to the X1 value from becoming the right-end X2 value. The width sufficient to obtain a straight line along the signal after the increase is nearly constant if the measurement conditions for the chemiluminescence signal are the same, and therefore can be set in advance. That is, for example, the parameter [C12] (the number of data pairs corresponding to a width sufficient to obtain a straight line along the signal after the increase) can be used to search for the right-end X2 value in the range from the X1 value + [C12] onward.

[0113] f max In the (x) calculation step, the approximate line f having the greatest slope among the approximate lines of the chemiluminescence signal when the right end is the X2 value and the left end is within the range up to the X1 value is selected. max Calculate (x).

[0114] The left end is preferably searched within a range going back from the point where the minimum width is subtracted from the X2 value, with the minimum width being set as the minimum width required to obtain a straight line following the signal after the rise. This makes it possible to eliminate the possibility of a point very close to the X2 value being the left end. The width sufficient to obtain a straight line following the signal after the rise is as explained in the X2 value calculation step.

[0115] In the reference point calculation step, the approximate line f max The intersection of (x) and Y line is the reference point (X c ,Y c ) is calculated as

[0116] Figure 17 shows the approximate line f max 17 is a diagram showing a process for calculating X1 value, X2 value and f in the reference point calculation process. max 18 and 19 show the results of the calculation step (x). c ,Y c ) and the process of calculating the starting point (X s ,Y s 18 and 19 show the results of performing the reference point calculation step and the start point calculation step of the reference point calculation process.

[0117] In the starting point calculation process, (A) Reference point X c Value and initial starting point X s The difference between the value (X c Value-X s If the initial start point is smaller than the recalculation reference value, the initial start point is set to the start point (X s ,Y s ) and (a) Reference point X c Value and initial starting point X s The difference between the value (X c Value-X s If the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Ys ) is calculated as

[0118] In the starting point calculation process, X c Value and X s The difference between the value (X c Value-X s First, it is determined whether the value of the recalculation reference value is equal to or greater than the recalculation reference value.

[0119] difference(X c Value-X s If the initial start point is smaller than the recalculation reference value, the initial start point is set to the start point (X s ,Y s ) (in the case of (a) above).

[0120] difference(X c Value-X s If the value) is equal to or greater than the recalculation reference value, the start point (X s ,Y s ) is recalculated (in the case of (a) above). The recalculation is performed by recalculating the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) is calculated as follows.

[0121] The number of data pairs used to calculate the average value is preferably a number that can cancel spike noise. The number of data pairs that can cancel spike noise can be confirmed by actually calculating a moving average for the target chemiluminescence signal. The number of data pairs that can cancel spike noise is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, by using the parameter [C3] (the number of data pairs that corresponds to the number of data pairs that can cancel spike noise), the number of data pairs used to calculate the average value can be set to consecutive [C3] points.

[0122] To ensure that the starting point is close enough to the rising point of the data, the first point where the mean value is greater than or equal to the previous mean value for at least two consecutive times is taken as the starting point (Xs ,Y s ) is calculated. The number of times that the average value is continuously greater than or equal to the previous average value is preferably set to the number of times that the variability in the data can be cancelled. The number of times that the variability in the data can be cancelled can be confirmed by actually calculating the average value multiple times for the target chemiluminescence signal. The number of times that the variability in the data can be cancelled is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, by using the parameter [C14] (the number of times that the variability in the data can be cancelled), the first point at which the average value is greater than or equal to the previous average value [C14] times in a row can be set as the starting point (X s ,Y s ) may also be calculated.

[0123] The recalculation reference value can be, for example, the width required from when the signal starts to rise until the signal slope reaches its maximum. The width required from when the signal starts to rise until the signal slope reaches its maximum is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can be set in advance. That is, for example, parameter [C13] (the number of data pairs corresponding to the width required from when the signal starts to rise until the signal slope reaches its maximum) is used to calculate X c Value and X s The difference between the value (X c Value-X s value).

[0124] The starting point (X s ,Y s ) is calculated as follows: c Value-X s The value is smaller than the recalculation reference value (recalculation is not required). s ,Y s ) is calculated as follows: c Value-X s This is the case when the value is equal to or greater than the recalculation reference value (when recalculation is necessary).

[0125] The starting point (X) calculated by the above-mentioned method for calculating the starting point of the chemiluminescence signal s ,Y s) accurately captures the starting point of the rise in the chemiluminescence signal measured using a biological sample, and this starting point (X s ,Y s ) can be taken as the starting point of the signal rise.

[0126] The method for calculating the amount of increase in a chemiluminescent signal measured using a biological sample according to this embodiment (hereinafter also simply referred to as the "method for calculating the amount of increase in a chemiluminescent signal") calculates the start point of the signal rise (X s ,Y s ) and calculate the end point (X e ,Y e ) (end point calculation step), and a step of calculating the increase in the chemiluminescence signal (increase calculation step).

[0127] The endpoint calculation process is the peak point X p The first point where the moving average calculated from at least two consecutive data pairs after the value falls below the Y line for at least two consecutive times is set as the end point (X e ,Y e ) is calculated as follows.

[0128] The number of data pairs used to calculate the moving average is preferably a number that can cancel spike noise. The number of data pairs that can cancel spike noise can be confirmed by actually calculating a moving average for the target chemiluminescence signal. The number of data pairs that can cancel spike noise is almost constant if the measurement conditions for the chemiluminescence signal are the same, so it can also be set in advance. That is, for example, the parameter [C15] (the number of data pairs that corresponds to the number of data pairs that can cancel spike noise) can be used to calculate the moving average to consecutive [C15] data pairs.

[0129] To ensure that the moving average is close enough to the baseline, the endpoint (X) is the first point at which the moving average falls within the range below the Y line at least twice in a row. e ,Y e) is preferably calculated as the number of times that the moving average falls within the range below Yline consecutively [C16] times. The number of times that the moving average falls within the range below Yline consecutively [C16] times is preferably set as the number of times that the moving average falls within the range below Yline consecutively [C16] times. The number of times that the moving average falls within the range below Yline consecutively [C16] times is preferably set as the end point (X e ,Y e ) may also be calculated.

[0130] The increase calculation process uses Yline as the baseline and the starting point X s Value to End Point X e This is a step of calculating the integrated value of the chemiluminescence intensity up to the value.

[0131] Fig. 20 is a diagram showing the process of calculating the integrated value of chemiluminescence intensity, and shows the results of the end point calculation step and the increase amount calculation step.

[0132] The integrated value of chemiluminescence intensity calculated by the above-mentioned method for calculating the increase in chemiluminescence signal accurately captures the increase in the signal of a chemiluminescence signal measured using a biological sample, and the integrated value of chemiluminescence intensity can be used as the increase in the signal.

[0133] The parameters [C1] to [C16] described above can be set appropriately depending on the specific aspect of the chemiluminescent signal that is the target of the calculation method according to this embodiment. Examples of factors that affect the setting of the parameters [C1] to [C16] include the type of biological sample, the type of reagent added, the type of biological reaction to be detected, the type of chemiluminescent reagent, the number of measurement data of chemiluminescent intensity per unit time, and whether or not smoothing processing is performed on the measurement data.

[0134] For example, in the case of a chemiluminescence signal (including measurement data of chemiluminescence intensity at two points per second) measured by adding a neutrophil stimulant (formylmethionylleucylphenylalanine) to a biological sample containing neutrophil cells (a sample including whole blood) and detecting the increase in superoxide production associated with the activation of superoxide production activity using a chemiluminescence reagent (2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one), the following specific numerical values ​​can be exemplified as each of the parameters indicated by [C1] to [C16]. [C1] 80 to 120 points, preferably 100 points [C2] 8 to 12, preferably 10 [C3] 4 to 6 points, preferably 5 points [C4] 2 to 4 times, preferably 3 times [C5] 80 to 120 points, preferably 100 points [C6] 60 to 100 points, preferably 80 points [C7] Standard deviation (SD) of mean value Y [C8] 600 to 900 points, preferably 750 points [C9] 40 to 60 points, preferably 50 points [C10] 600 to 800 points, preferably 700 points [C11] 200 to 300 points, preferably 250 points [C12] 180 to 220 points, preferably 200 points [C13] 120-180 points. Preferably 150 points [C14] 2 to 4 times, preferably 3 times [C15] 4 to 6 points, preferably 5 points [C16] 2 to 4 times, preferably 3 times

[0135] 21 and 22 are graphs showing examples of fluorescent and chemiluminescent signals measured using a sample containing whole blood. As can be seen from FIGS. 21 and 22, the influence of impurities (e.g., red blood cells and other impurities) contained in whole blood tends to cause signal disturbances, such as small signal intensities for fluorescent and chemiluminescent signals, unstable baselines, and fluctuations in measurement data. This tendency is particularly pronounced with fluorescent signals, which are susceptible to scattering.

[0136] FIG. 21 shows the starting point (X s ,Y s ) and the starting point (X s ,Y s ) calculated by the calculation method according to this embodiment. s ,Y s ) can be seen to capture the starting point of the signal rise more accurately than the starting point obtained by the conventional method.

[0137] In addition, the starting point (X s ,Y s The calculation of the peak point (X) where the chemiluminescence intensity is maximum was carried out as follows: p ,Y p (a) The average value Y of the chemiluminescence intensity between any two points (A and B) before adding the reagent was calculated, and the horizontal line of the average value Y was calculated as the Yline. (c) The X of the peak point p Starting from the value, the first point where the average value of every 5 consecutive points falls within the range of Y line for 3 consecutive times is set as the starting point (X s ,Y s ) was calculated as

[0138] FIG. 22 shows the starting point (X s ,Y s ), approximation line f max (x) and approximate line f b (x)' is shown. Approximation line f max (x) is a straight line that approximates the fluorescent signal that rises near the starting point. b (x)' is a straight line that approximates the baseline near the starting point. s ,Y s ), approximation line f max (x) and approximate line f b It can be seen that (x)' captures the starting point of the signal rise and the amount of signal increase more accurately.

[0139] [Calculation device and calculation program for calculating the starting point of signal increase and the amount of signal increase from a fluorescent signal] Fig. 1 is a schematic diagram showing the hardware configuration of a calculation device according to an embodiment, and Fig. 2 is a schematic diagram showing the functional configuration of a calculation device according to an embodiment. The calculation device according to an embodiment can be used in combination with a fluorescence measurement device (not shown).

[0140] 1, the computing device 1 is physically configured as a computer including a computational storage device 3 including a main storage device such as a CPU 21, a ROM 22, and a RAM 23, a communication module 24 such as a network card for transmitting and receiving data to and from other devices, an auxiliary storage device 25 such as a hard disk, an input device 4 such as a keyboard and a mouse, and a display device 5 such as a monitor. Each function of the computing device described below is realized by loading predetermined computer software onto the hardware such as the CPU 21, ROM 22, and RAM 23, thereby operating the input device 4, the display device 5, and the communication module 24 under the control of the CPU 21 and reading and writing data from and to the main storage devices 22 and 23 and the auxiliary storage device 25.

[0141] The arithmetic and memory device 3 is connected to the fluorometer so as to be able to communicate with it, or is electrically connected to it. The arithmetic and memory device 3 controls the output of measurement light from the light source of the fluorometer and acquires detection signals from the fluorescence detector of the fluorometer. The arithmetic and memory device 3 also acquires fluorescence signals output from the fluorescence detector that detects fluorescence from the biological sample, and acquires time-series data consisting of data pairs (measurement time, fluorescence intensity) of fluorescence intensity versus measurement time. Furthermore, the arithmetic and memory device 3 derives a tentative starting point (X s’ ,Y s’ ) and calculate the temporary starting point (X s’ ,Y s’ ) data and time series data, the start point of the signal rise (X s ,Y s When the calculation storage device 3 is made to function as a calculation device for calculating the amount of increase in the signal, the calculation storage device 3 calculates the start point (Xs ,Y s ) and calculates the increase in the fluorescent signal from the time-series data. An input device 4 such as a keyboard and a mouse, and a display device 5 such as a display, are connected to the calculation and storage device 3. The calculation and storage device 3 is a computer such as a personal computer; a smart device such as a smartphone or a tablet; or a cloud server to which the smart device is connected via a network. When the calculation and storage device 3 is a smart device, the input device 4 and the display device 5 are built into the smart device. When the calculation and storage device 3 is a cloud server, the input device 4 and the display device 5 may be built into the smart device or may be electrically connected to the personal computer.

[0142] As shown in Figure 2, the calculation device 1 includes, as functional components, a data acquisition unit F1, a first data calculation unit F2, a second data calculation unit F3, a third data calculation unit F4, and a data display unit F5. The first data calculation unit F2, the second data calculation unit F3, and the third data calculation unit F4 may be shared. The data display unit F4 may be omitted if data display is not required. Furthermore, when the calculation device 1 is connected to a fluorescence measurement device, it may further include a control unit.

[0143] The data acquisition unit F1 acquires a fluorescence signal output from a fluorescence detector that detects fluorescence from a biological sample, and acquires time-series data consisting of data pairs of fluorescence intensity versus measurement time (measurement time, fluorescence intensity). The data acquisition unit F1 may be a device that acquires time-series data from the fluorescence detector, or may be a device that acquires time-series data directly input from an input device. The time-series data acquired by the data acquisition unit F1 may be stored in a data storage unit such as the auxiliary storage device 25.

[0144] The first data calculation unit F2 calculates the tentative starting point (X s’ ,Y s’ ) is calculated. s’ ,Y s’) is the approximate line f with the smallest slope among the approximate lines of the fluorescence signal, whose left end is the point at which the disturbance of the fluorescence signal due to the addition of a reagent that induces a biological reaction to the biological sample settles down after the reagent is added to the time series data. min (x) and approximate line f min Calculating the right end X1 value of (x), calculating the right end X2 value at which the approximate line of the fluorescence signal with the X1 value as the left end has the maximum slope, and calculating the approximate line f having the maximum slope among the approximate lines of the fluorescence signal data with the X2 value as the right end. max (x) and calculate the approximation line f min (x) and the above approximation line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) and calculate the X of the above temporary reference point. c’ The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) can be calculated by executing a process including the calculation of the time series data stored in a data storage unit such as the auxiliary storage device 25. In the first data calculation unit F2, the time series data stored in a data storage unit such as the auxiliary storage device 25 can be read and used. In addition, in the first data calculation unit F2, the calculated tentative starting point (X s’ ,Y s’ ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0145] The second data calculation unit F3 calculates the tentative starting point (X s’ ,Y s’ ) data and the start point of the signal rise (X s ,Y s ) is calculated. s ,Y s ) is the temporary starting point (X s’ ,Y s’ ) data and time series data, the above tentative starting point X s’The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b (x)', and the approximation line f b (x)' and the above approximation line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) and calculate the X of the reference point c The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) and calculate the starting point (X s ,Y s ) stored in a data storage unit such as the auxiliary storage device 25 is used to calculate the signal rise. s’ ,Y s’ ) data and / or time series data may be read out and used. In addition, in the second data calculation unit F3, the calculated starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0146] The first data calculation unit F2 and the second data calculation unit F3 may be the same data calculation unit.

[0147] When the calculation device 1 is made to function as a calculation device for calculating the amount of increase in a signal, the third data calculation unit F4 calculates the starting point (X s ,Y s The increase in the fluorescent signal is calculated from the data of the start point (X s ,Y s When the third data calculation unit F4 is used as a calculation device for calculating the X sCalculate the average value Y of the fluorescence intensity from at least two consecutive data pairs, and calculate the horizontal line of the average value Y as the Y line. The point where the fluorescence intensity of the fluorescence signal is maximum is called the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l Calculate the value of the starting point X s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b (x) and calculate the approximation line f b Peak point X on (x) p Y value at the value b The value calculated as the peak Y p Value and above Y l The difference between the peak Yp value and the above Y value is calculated as the increase in the fluorescent signal (l). b The third data calculation unit F4 calculates the difference between the value of the starting point (X s ,Y s ) data and / or time-series data may be read out and used. In addition, in the third data calculation unit F4, data on the calculated increase in the fluorescent signal may be stored in a data storage unit such as the auxiliary storage unit 25.

[0148] The first data calculation unit F2, the second data calculation unit F3, and the third data calculation unit F4 may be the same data calculation unit.

[0149] The data display section F5 shows the calculated start point of the signal rise (X s ,Y s ) and / or data on the increase in fluorescent signal.

[0150] When the calculation device 1 is connected to a fluorescence measurement device, the calculation device 1 may further include a control unit. The control unit, for example, controls the output of measurement light from the light source of the fluorescence measurement device and acquires detection signals from the fluorescence detector of the fluorescence measurement device.

[0151] The calculation program according to this embodiment causes a computer to function as the above-mentioned data acquisition unit F1, first data calculation unit F2, second data calculation unit F3, and third data calculation unit F4. The calculation program according to this embodiment may also cause a computer to function as the above-mentioned data display unit F5 and control unit. By loading the calculation program into a computer, the computer can calculate the starting point of the signal rise (X s ,Y s The calculation program according to the present embodiment operates as a calculation device that calculates the increase in signal from a fluorescent signal measured using a biological sample, or as a calculation device that calculates the increase in signal from a fluorescent signal measured using a biological sample. The calculation program according to the present embodiment is provided, for example, by being recorded on a computer-readable recording medium. The recording medium may be a non-transitory recording medium. Examples of the recording medium include recording media such as flexible disks, CDs, and DVDs, recording media such as ROMs, and semiconductor memories.

[0152] 3 is a flowchart showing a method for calculating the start point of a signal increase and the amount of signal increase from a fluorescent signal according to one embodiment. The method for calculating the start point of a signal increase and the amount of signal increase from a fluorescent signal, performed by the calculation device 1, allows automatic and highly accurate calculation of the start point of a signal increase and the amount of signal increase. Furthermore, as a specific embodiment of the method for calculating the start point of a signal increase and the amount of signal increase from a fluorescent signal, performed by the calculation device 1 described below, the specific embodiment of the above-mentioned [Method for calculating the start point of a signal increase and the amount of signal increase from a fluorescent signal] can be applied.

[0153] First, the data acquisition unit F1 acquires a fluorescence signal output from a fluorescence detector that detects fluorescence from a biological sample, and acquires time-series data consisting of data pairs of fluorescence intensity versus measurement time (measurement time, fluorescence intensity). Then, the data acquisition unit F1 acquires the above-mentioned parameters [F1] to [F13] as needed (data input, parameter setting step). The data input, parameter setting step may include storing the acquired time-series data and data of each parameter [F1] to [F13] in a data storage unit such as the auxiliary storage device 25. The acquisition of the time-series data and the acquisition of data of each parameter [F1] to [F13] may be performed simultaneously or at separate times.

[0154] Next, the first data calculation unit F2 calculates, for the time-series data, an approximation line f having the smallest slope among the approximation lines of the fluorescence signal, the left end of which is the point at which the disturbance in the fluorescence signal due to the addition of a reagent that induces a biological reaction to the biological sample has settled down after the addition of the reagent. min (x) and approximate line f min a step of calculating the right end X1 value of (x); a step of calculating the right end X2 value at which the approximate line of the fluorescence signal with the X1 value as the left end has the maximum slope; and a step of calculating the approximate line f having the maximum slope among the approximate lines of the fluorescence signal data with the X2 value as the right end. max (x), the approximation line f min (x) and the above approximation line f max The intersection of (x) is the temporary reference point (X c’ ,Y c’ ) and calculate the X of the above temporary reference point. c’ The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f min The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescence signal at least twice consecutively is taken as the tentative starting point (X s’ ,Y s’ ) (Step i: Temporary starting point (X s’ ,Y s’Step i may include reading out necessary parameters from the time series data stored in a data storage unit such as the auxiliary storage device 25 and the parameters [F1] to [F13]. Step i also includes searching for the calculated tentative starting point (X s’ ,Y s’ ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0155] Next, the second data calculation unit F3 calculates the tentative starting point (X s’ ,Y s’ ) data and time series data, the above tentative starting point X s’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b (x)', the approximation line f b (x)' and the above approximation line f max The intersection of (x) and (x) is the reference point (X c ,Y c ) the step of calculating the X of the reference point c The moving average calculated from at least two consecutive data pairs from the previous value is the approximate line f b The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice consecutively is taken as the starting point (X s ,Y s ) is calculated as the starting point (X s ,Y s ) is set as the starting point of the signal rise (Step ii: Starting point (X s ,Y s Step ii is a search for the time series data, the tentative starting point (X s’ ,Y s’ ) data and the parameters [F1] to [F13]. Step ii may also include reading out necessary parameters from the calculated starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0156] The calculation device 1 calculates the time from the fluorescent signal to the start point of the signal rise (X s ,Y s ), the data display unit F5 displays the start point (X s ,Y s ) (analysis result output step).

[0157] Next, the third data calculation unit F4 calculates the X s a step of calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs going back from the value, and calculating a horizontal line of the average value Y as the Y line; a step of calculating a point where the fluorescence intensity of the fluorescence signal is maximum as the peak point (X p ,Y p ) and the peak point X on the Y line p Y value at the value l The step to calculate as a value, X of the starting point above s The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled. b (x), the approximation line f b Peak point X on (x) p Y value at the value b Step and peak point Y to be calculated as a value p Value and above Y l The difference between the peak Yp value and the above Y value is calculated as the increase in the fluorescent signal (l). b The process includes a step of calculating the difference between the value of the fluorescent signal (i) and the value of the fluorescent signal (ii) as the increase in the fluorescent signal (b), and a step of determining the increase in the fluorescent signal (i) or the increase in the fluorescent signal (b) as the increase in the signal (step iii: calculation of the total amount of luminescence). s ,Y s ) and the parameters [F1] to [F13]. Step iii may also include storing the calculated data on the increase in the fluorescent signal in a data storage unit such as the auxiliary storage device 25.

[0158] When the calculation device 1 is made to function as a calculation device that calculates the signal increase amount from the fluorescent signal, the data display unit F5 may display the fluorescent signal increase amount (l) calculated in step iii and / or the fluorescent signal increase amount (b) (analysis result output step).

[0159] [Calculation device and calculation program for calculating the starting point of signal increase and the amount of signal increase from a chemiluminescence signal] Fig. 1 is a schematic diagram showing the hardware configuration of a calculation device according to an embodiment, and Fig. 4 is a schematic diagram showing the functional configuration of a calculation device according to an embodiment. The calculation device according to an embodiment can be used in combination with a luminescence measuring device (not shown).

[0160] 1, the computing device 1 is physically configured as a computer including a computational storage device 3 including a main storage device such as a CPU 21, a ROM 22, and a RAM 23, a communication module 24 such as a network card for transmitting and receiving data to and from other devices, an auxiliary storage device 25 such as a hard disk, an input device 4 such as a keyboard and a mouse, and a display device 5 such as a monitor. Each function of the computing device described below is realized by loading predetermined computer software onto the hardware such as the CPU 21, ROM 22, and RAM 23, thereby operating the input device 4, the display device 5, and the communication module 24 under the control of the CPU 21 and reading and writing data from and to the main storage devices 22 and 23 and the auxiliary storage device 25.

[0161] The arithmetic and memory device 3 is connected to the luminescence measuring device so as to be able to communicate with it, or is electrically connected to it. The arithmetic and memory device 3 controls the light detection of the luminescence measuring device and / or acquires a detection signal from a photodetector of the luminescence measuring device. The arithmetic and memory device 3 also acquires a chemiluminescence signal output from a photodetector that detects chemiluminescence from a biological sample, and acquires time-series data consisting of data pairs (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time. Furthermore, the arithmetic and memory device 3 calculates a tentative starting point (Xs’ ,Y s’ ) and calculate the temporary starting point (X s’ ,Y s’ ) data and time series data from the initial starting point (X s ,Y s ) and calculate the reference point (X c ,Y c ) and calculate the initial starting point data, the reference point (X c ,Y c ) data and time series data from the starting point (X s ,Y s When the calculation device 3 is used as a calculation device for calculating the amount of increase in the signal, the calculation device 3 calculates the end point (X e ,Y e ) and calculate the starting point of the signal rise (X s ,Y s ) data, end point (X e ,Y e The increase in the chemiluminescence signal is calculated from the time-series data and the time-series data. An input device 4 such as a keyboard and a mouse, and a display device 5 such as a display, are connected to the calculation and storage device 3. The calculation and storage device 3 is a computer such as a personal computer; a smart device such as a smartphone or a tablet; or a cloud server to which the smart device is connected via a network. When the calculation and storage device 3 is a smart device, the input device 4 and the display device 5 are built into the smart device. When the calculation and storage device 3 is a cloud server, the input device 4 and the display device 5 may be built into the smart device or may be electrically connected to the personal computer.

[0162] As shown in FIG. 4, the calculation device 1 includes, as functional components, a data acquisition unit C1, a first data calculation unit C2, a second data calculation unit C3, a third data calculation unit C4, a fourth data calculation unit C5, a fifth data calculation unit C6, a sixth data calculation unit C7, and a data display unit C8. The first data calculation unit C2, the second data calculation unit C3, the third data calculation unit C4, the fourth data calculation unit C5, the fifth data calculation unit C6, and the sixth data calculation unit C7 may be shared. The data display unit C8 may be omitted if data display is not required. Furthermore, when the calculation device 1 is connected to a luminescence measuring device, it may further include a control unit.

[0163] The data acquisition unit C1 acquires a chemiluminescence signal output from a photodetector that detects chemiluminescence from a biological sample, and acquires time-series data consisting of data pairs of chemiluminescence intensity versus measurement time (measurement time, chemiluminescence intensity). The data acquisition unit C1 may be a device that acquires time-series data from a photodetector, or may be a device that acquires time-series data directly input from an input device. The time-series data acquired by the data acquisition unit C1 may be stored in a data storage unit such as the auxiliary storage device 25.

[0164] The first data calculation unit C2 calculates a tentative starting point (X s’ ,Y s’ ) is calculated. s’ ,Y s’ ) is the peak point (X ) at which the chemiluminescence intensity reaches its maximum after the addition of a reagent that induces a biological reaction to the biological sample. p ,Y p ) and calculate the time from the time when the disturbance of the chemiluminescence signal due to the addition of the reagent settles down to the time when the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) and calculate the X of the peak point p The moving average calculated from at least two consecutive data pairs going back from the Y value of the bottom point is bThe first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) can be calculated by executing a process including calculating the time series data stored in a data storage unit such as the auxiliary storage device 25 in the first data calculation unit C2. In addition, the first data calculation unit C2 can calculate the time series data by executing a process including calculating the time series data as the calculated tentative starting point (X s’ ,Y s’ ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0165] The second data calculation unit C3 calculates the tentative starting point (X s’ ,Y s’ ) data and the initial starting point (X s ,Y s ) is calculated. s ,Y s ) is the temporary starting point (X s’ ,Y s’ ) data and time series data, the above tentative starting point X s’ Calculate the average value Y of the chemiluminescence intensity from at least two consecutive data pairs, and calculate the horizontal line of the average value Y as the Yline. p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) stored in a data storage unit such as the auxiliary storage device 25. s’ ,Y s’ ) data and / or time series data may be read out and used. In addition, in the second data calculation unit C3, the calculated initial starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0166] The third data calculation unit C4 calculates the reference point (X c ,Yc ) is calculated. c ,Y c ) is the X of the peak point above for the time series data. p The left end X1 value at which the approximate line of the chemiluminescence signal with the X1 value at the right end has the maximum slope is calculated, and the right end X2 value at which the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is calculated as the X2 value at the peak point. p The approximate line f with the greatest slope among the approximate lines of the chemiluminescence signal when the right end is the X2 value and the left end is within the range of the X1 value. max (x) and calculate the approximation line f max The intersection of (x) and the above Y line is the reference point (X c ,Y c ) can be calculated by executing a process including calculating the reference point (X c ,Y c ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0167] The fourth data calculation unit C5 calculates the initial starting point data calculated by the second data calculation unit C3 and the reference point (X c ,Y c ) data and time series data from the starting point (X s ,Y s ) is calculated. s ,Y s ) is the initial starting point data, the reference point (X c ,Y c ) data and time series data, the X of the reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the initial starting point is smaller than the recalculation reference value, the initial starting point is changed to the starting point (X s ,Y s ) and calculate the X of the reference point above. c Value and the initial starting point Xs The difference between the value (X c Value-X s If the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) and calculate the starting point (X s ,Y s ) as the starting point of the signal rise. The fourth data calculation unit C5 calculates the initial start point data, the reference point (X c ,Y c ) data and / or time series data may be read out and used. In addition, the fourth data calculation unit C5 may read out and use the calculated starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0168] The first data calculation unit C2, the second data calculation unit C3, the third data calculation unit C4, and the fourth data calculation unit C5 may be the same data calculation unit.

[0169] When the calculation device 1 is made to function as a calculation device for calculating the amount of increase in the signal, the calculation device 1 further includes a fifth data calculation unit C6 and a sixth data calculation unit C7. s ,Y s ), the fifth data calculation unit C6 and the sixth data calculation unit C7 are unnecessary.

[0170] The fifth data calculation unit C6 calculates the end point (X e ,Y e ) is calculated. e ,Y e ) is the X of the peak point above for the time series data. pThe first point where the moving average calculated from at least two consecutive data pairs falls within the range of the Y line at least twice in succession after the value is set as the end point (X e ,Y e ) can be calculated by executing a process including the calculation of the end point (X e ,Y e ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0171] The sixth data calculation unit C7 calculates the start point (X s ,Y s ) data, the end point (X e ,Y e The increase in the chemiluminescence signal is calculated from the data of the start point (Xs, Ys) of the signal rise and the end point (X e ,Y e ) data and time series data, the above Y line is used as the baseline, and the above starting point X s Value to the above end point X e The sixth data calculation unit C7 calculates the signal intensity from the start point (X s ,Y s ) data, end point (X e ,Y e The sixth data calculation unit C7 may read out and use the calculated integrated value data of the chemiluminescence intensity in a data storage unit such as the auxiliary storage unit 25.

[0172] The first data calculation unit C2, the second data calculation unit C3, the third data calculation unit C4, the fourth data calculation unit C5, the fifth data calculation unit C6, and the sixth data calculation unit C7 may be the same data calculation unit.

[0173] The data display section C8 shows the calculated start point of the signal rise (X s ,Y s ) data and / or data on the integrated value of chemiluminescence intensity.

[0174] When the calculation device 1 is connected to a luminescence measuring device, the calculation device 1 may further include a control unit. The control unit, for example, controls the light detection of the luminescence measuring device and acquires the detection signal from the photodetector of the luminescence measuring device.

[0175] The calculation program according to this embodiment causes a computer to function as the above-mentioned data acquisition unit C1, first data calculation unit C2, second data calculation unit C3, third data calculation unit C4, fourth data calculation unit C5, fifth data calculation unit C6, and sixth data calculation unit C7. The calculation program according to this embodiment may also cause a computer to function as the above-mentioned data display unit C8 and control unit. By loading the calculation program into a computer, the computer can calculate the starting point of the signal rise (X s ,Y s The calculation program according to the present embodiment operates as a calculation device that calculates the increase in a signal from a chemiluminescent signal measured using a biological sample, or as a calculation device that calculates the increase in a signal from a chemiluminescent signal measured using a biological sample. The calculation program according to the present embodiment is provided, for example, by being recorded on a computer-readable recording medium. The recording medium may be a non-transitory recording medium. Examples of the recording medium include recording media such as flexible disks, CDs, and DVDs, recording media such as ROMs, and semiconductor memories.

[0176] 5 is a flowchart showing a method for calculating the start point of a signal rise and the amount of signal increase from a chemiluminescent signal according to one embodiment. The method for calculating the start point of a signal rise and the amount of signal increase from a chemiluminescent signal, performed by the calculation device 1, allows automatic and highly accurate calculation of the start point of a signal rise and the amount of signal increase. Furthermore, as a specific embodiment of the method for calculating the start point of a signal rise and the amount of signal increase from a chemiluminescent signal, performed by the calculation device 1 described below, the specific embodiment of the above-mentioned "Method for calculating the start point of a signal rise and the amount of signal increase from a chemiluminescent signal" can be applied.

[0177] First, the data acquisition unit C1 acquires a chemiluminescence signal output from a photodetector that detects chemiluminescence from a biological sample, and acquires time-series data consisting of data pairs of chemiluminescence intensity versus measurement time (measurement time, chemiluminescence intensity). Then, the data acquisition unit C1 acquires the above-mentioned parameters [C1] to [C16] as needed (data input, parameter setting step). The data input, parameter setting step may include storing the acquired time-series data and data of each parameter [C1] to [C16] in a data storage unit such as the auxiliary storage device 25. The acquisition of the time-series data and the acquisition of data of each parameter [C1] to [C16] may be performed simultaneously or at separate times.

[0178] Next, the first data calculation unit C2 calculates, for the time series data, a peak point (X p ,Y p ) the time from the time when the disturbance of the chemiluminescence signal due to the addition of the reagent settles down to the time when the peak point X p The bottom point (X) where the chemiluminescence intensity is the minimum within the range b ,Y b ) the step of calculating X at the peak point p The moving average calculated from at least two consecutive data pairs going back from the Y value of the bottom point is bThe first point that falls within the range of the value + noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X s’ ,Y s’ ) (Step i: Temporary starting point (X s’ ,Y s’ Step i may include reading out necessary parameters from the time series data stored in a data storage unit such as the auxiliary storage device 25 and the parameters [C1] to [C16]. Step i also includes searching for the calculated tentative starting point (X s’ ,Y s’ ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0179] Next, the second data calculation unit C3 calculates the tentative starting point (X s’ ,Y s’ ) data and time series data, the above tentative starting point X s’ Calculate the average value Y of the chemiluminescence intensity from at least two consecutive data pairs, and calculate the horizontal line of the average value Y as the Yline. p Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X s ,Y s ) (Step ii: Initial starting point (X s ,Y s Step ii is a search for the time series data stored in the data storage unit such as the auxiliary storage device 25, the tentative starting point (X s’ ,Y s’ ) and the parameters [C1] to [C16]. Step ii may also include reading out necessary parameters from the calculated initial starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0180] Next, the third data calculation unit C4 calculates the X of the peak point for the time series data. pThe left end X1 value at which the approximate line of the chemiluminescence signal with the X1 value at the right end has the maximum slope is calculated, and the right end X2 value at which the approximate line of the chemiluminescence signal with the X1 value at the left end has the maximum slope is calculated as the X2 value at the peak point. p The approximate line f with the greatest slope among the approximate lines of the chemiluminescence signal when the right end is the X2 value and the left end is within the range of the X1 value. max (x) and calculate the approximation line f max The intersection of (x) and the above Y line is the reference point (X c ,Y c ) (Step iii: Reference point (X c ,Y c Step iii may include reading out necessary parameters from the time series data stored in a data storage unit such as the auxiliary storage device 25 and the parameters [C1] to [C16]. Step iii may also include reading out necessary parameters from the time series data stored in a data storage unit such as the auxiliary storage device 25 and the parameters [C1] to [C16]. c ,Y c ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0181] Next, the fourth data calculation unit C5 calculates the initial start point data, the reference point (X c ,Y c ) data and time series data, the X of the reference point c Value and the initial starting point X s The difference between the value (X c Value-X s If the initial starting point is smaller than the recalculation reference value, the initial starting point is changed to the starting point (X s ,Y s ) and calculate the X of the reference point above. c Value and the initial starting point X s The difference between the value (X c Value-X s If the value) is equal to or greater than the recalculation reference value, the initial starting point X s The first point where the average value calculated for each pair of at least two consecutive data points is equal to or greater than the previous average value for at least two consecutive times is taken as the starting point (X s ,Y s ) and calculate the starting point (X s ,Ys ) is set as the starting point of the signal rise (Step iv: Calculation of the starting point). In Step iv, the initial starting point data, the reference point (X c ,Y c ) data, time series data, and parameters [C1] to [C16]. Step iv may also include reading out necessary parameters from the calculated starting point (X s ,Y s ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0182] The calculation device 1 is set to calculate the time from the chemiluminescence signal to the start point of the signal rise (X s ,Y s ), the data display unit C8 displays the start point (X s ,Y s ) (analysis result output step).

[0183] Next, the fifth data calculation unit C6 calculates the X of the peak point for the time series data. p The first point where the moving average calculated from at least two consecutive data pairs falls within the range of the Y line at least twice in succession after the value is set as the end point (X e ,Y e ) (Step v: Calculating the end point (X e ,Y e Step v may include reading out necessary parameters from the time series data stored in a data storage unit such as the auxiliary storage device 25 and the parameters [C1] to [C16]. Step v also includes searching for the calculated end point (X e ,Y e ) may be stored in a data storage unit such as the auxiliary storage device 25.

[0184] Next, the sixth data calculation unit C7 calculates the start point (Xs, Ys) data of the signal rise, the end point (X e ,Y e) data and time series data, the above Y line is used as the baseline, and the above starting point X s Value to the above end point X e Step vi: Calculation of an integrated value. Step vi is a process of calculating an integrated value of the chemiluminescence intensity up to the value, and using the integrated value as the amount of increase in the signal (Step vi: Calculation of an integrated value). Step vi is a process of calculating an integrated value. e ,Y e ) data, time-series data, and parameters [C1] to [C16]. Step vi may also include storing the calculated integrated value data of chemiluminescence intensity in a data storage unit such as auxiliary storage device 25.

[0185] When the calculation device 1 is made to function as a calculation device that calculates the signal increase amount from the chemiluminescence signal, the data display unit C8 may display the integrated value of the chemiluminescence intensity calculated in step vi (analysis result output step).

Claims

1. The starting point of the signal rise (X ｓ , Y ｓ ) is a calculation method for calculating The fluorescence signal is time-series data consisting of a data pair (measurement time, fluorescence intensity) of fluorescence intensity versus measurement time, The calculation method is to calculate the tentative starting point (X ｓ’ , Y ｓ’ ), and calculating the temporary starting point (X ｓ’ , Y ｓ’ ) based on the start point of the signal rise (X ｓ , Y ｓ ) calculating The temporary starting point (X ｓ’ , Y ｓ’ ) is calculated by After adding a reagent that induces a biological reaction to a biological sample, the approximate line f having the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent settles down is ｍｉｎ (x), and the approximation line f ｍｉｎ Right end of (x) １ calculating a value; The X １ The right end X where the approximate line of the fluorescence signal with the value at the left end is at its maximum slope ２ calculating a value; The X ２ Among the approximation lines of the fluorescence signal data with the value at the right end, the approximation line f with the greatest slope ｍａｘ (x) The approximate line f ｍｉｎ (x) and the approximation line f ｍａｘ The intersection of (x) is the temporary reference point (X ｃ’ , Y ｃ’ ) and X of the temporary reference point ｃ’ A moving average calculated from at least two consecutive data pairs going back from the value is ｍｉｎ The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescent signal at least twice consecutively is defined as the tentative starting point (X ｓ’ , Y ｓ’ ) and The starting point (X ｓ , Y ｓ ) is calculated by The temporary starting point X ｓ’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ (x)'; The approximate line f ｂ (x)' and the approximation line f ｍａｘ The intersection of (x) is the reference point (X ｃ , Y ｃ ) and The reference point X ｃ A moving average calculated from at least two consecutive data pairs going back from the value is ｂ The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) and The time point at which the disturbance of the fluorescent signal due to the addition of the reagent settles is determined by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.03% or less for five or more consecutive data pairs. the noise width of the fluorescent signal is the standard deviation of the baseline fluctuation of the fluorescent signal; the width wider than the noise width of the fluorescent signal is 1.1 times the noise width of the fluorescent signal to 6.0 times the noise width of the fluorescent signal, The starting point (X ｓ , Y ｓ ) is the starting point of the signal rise.

2. The starting point of the signal rise (X ｓ , Y ｓ ) a calculation device for calculating a data acquisition unit that acquires the fluorescent signal as time-series data consisting of data pairs (measurement time, fluorescent intensity) of fluorescent intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain a tentative starting point (X ｓ’ , Y ｓ’ a first calculation unit that calculates The tentative starting point (X ｓ’ , Y ｓ’ ) data and the time series data acquired by the data acquisition unit to determine the start point (X ｓ , Y ｓ a second calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the approximate line f having the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent settles down is ｍｉｎ (x), and the approximation line f ｍｉｎ Right end of (x) １ calculating the value, The X １ The right end X where the approximate line of the fluorescence signal with the value at the left end is at its maximum slope ２ calculating the value, The X ２ Among the approximation lines of the fluorescence signal data with the value at the right end, the approximation line f with the greatest slope ｍａｘ Calculating (x); The approximate line f ｍｉｎ (x) and the approximation line f ｍａｘ The intersection of (x) is the temporary reference point (X ｃ’ , Y ｃ’ ) X of the temporary reference point ｃ’ A moving average calculated from at least two consecutive data pairs going back from the value is ｍｉｎ The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescent signal at least twice consecutively is defined as the tentative starting point (X ｓ’ , Y ｓ’ ) In the second calculation unit, The temporary starting point X ｓ’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ Calculating (x)'; The approximate line f ｂ (x)' and the approximation line f ｍａｘ The intersection of (x) is the reference point (X ｃ , Y ｃ ) The reference point X ｃ A moving average calculated from at least two consecutive data pairs going back from the value is ｂ The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) The starting point (X ｓ , Y ｓ ) as the start of the signal rise, The time point at which the disturbance of the fluorescent signal due to the addition of the reagent settles is determined by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.03% or less for five or more consecutive data pairs. the noise width of the fluorescent signal is the standard deviation of the baseline fluctuation of the fluorescent signal; The width wider than the noise width of the fluorescent signal is 1.1 times the noise width of the fluorescent signal to 6.0 times the noise width of the fluorescent signal.

3. The starting point of the signal rise (X ｓ , Y ｓ ) is a calculation program for calculating Computer, a data acquisition unit that acquires the fluorescent signal as time-series data consisting of data pairs (measurement time, fluorescent intensity) of fluorescent intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain a tentative starting point (X ｓ’ , Y ｓ’ a first calculation unit that calculates The tentative starting point (X ｓ’ , Y ｓ’ ) data and the time series data acquired by the data acquisition unit to determine the start point (X ｓ , Y ｓ ) and In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the approximate line f having the smallest slope among the approximate lines of the fluorescent signal whose left end is the point at which the disturbance of the fluorescent signal due to the addition of the reagent settles down is ｍｉｎ (x), and the approximation line f ｍｉｎ Right end of (x) １ calculating the value, The X １ The right end X where the approximate line of the fluorescence signal with the value at the left end is at its maximum slope ２ calculating the value, The X ２ Among the approximation lines of the fluorescence signal data with the value at the right end, the approximation line f with the greatest slope ｍａｘ Calculating (x); The approximate line f ｍｉｎ (x) and the approximation line f ｍａｘ The intersection of (x) is the temporary reference point (X ｃ’ , Y ｃ’ ) X of the temporary reference point ｃ’ A moving average calculated from at least two consecutive data pairs going back from the value is ｍｉｎ The first point that falls within the range of (x) ± the width wider than the noise width of the fluorescent signal at least twice consecutively is defined as the tentative starting point (X ｓ’ , Y ｓ’ ) In the second calculation unit, The temporary starting point X ｓ’ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ Calculating (x)'; The approximate line f ｂ (x)' and the approximation line f ｍａｘ The intersection of (x) is the reference point (X ｃ , Y ｃ ) The reference point X ｃ A moving average calculated from at least two consecutive data pairs going back from the value is ｂ The first point that falls within the noise width of the (x)' ± fluorescence signal at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) The starting point (X ｓ , Y ｓ ) as the start point of the signal rise, The time point at which the disturbance of the fluorescent signal due to the addition of the reagent settles is determined by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.03% or less for five or more consecutive data pairs. the noise width of the fluorescent signal is the standard deviation of the baseline fluctuation of the fluorescent signal; The width wider than the noise width of the fluorescent signal is 1.1 times to 6.0 times the noise width of the fluorescent signal.

4. A method for calculating an increase in a signal from a fluorescent signal measured using a biological sample, comprising: The calculation method according to claim 1 calculates the start point of the signal rise (X ｓ , Y ｓ ), and calculating the starting point (X ｓ , Y ｓ Calculating an increase in the fluorescent signal based on the The step of calculating the increase in the fluorescent signal comprises: The starting point X ｓ calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs going back from the value, and calculating a horizontal line of the average value Y as Yline; The point where the fluorescence intensity of the fluorescence signal is maximum is called the peak point (X ｐ , Y ｐ ) and the peak point X on the Y line ｐ Y value at the value ｌ a step of calculating the value The starting point X ｓ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ (x) The approximate line f ｂ (x) Peak point X on ｐ Y value at the value ｂ a step of calculating the value Peak point Y ｐ value and the Y ｌ The difference between the peak point Yp value and the Y value is calculated as the increase in the fluorescent signal (l). ｂ and calculating the difference between the values ​​as the increase in the fluorescent signal (b), The time point at which the disturbance of the fluorescent signal due to the addition of the reagent settles is determined by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.03% or less for five or more consecutive data pairs. The calculation method is such that the increase in the fluorescent signal (l) or the increase in the fluorescent signal (b) is taken as the increase in the signal.

5. A calculation device for calculating an increase in a signal from a fluorescent signal measured using a biological sample, 3. The calculation device according to claim 2, wherein the start point (X ｓ , Y ｓ a third calculation unit that processes the time-series data acquired by the data acquisition unit and calculates an increase in the fluorescent signal; In the third calculation unit, The starting point X ｓ Calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs, going back from the value, and calculating the horizontal line of the average value Y as Yline; The point where the fluorescence intensity of the fluorescence signal is maximum is called the peak point (X ｐ , Y ｐ ) and the peak point X on the Y line ｐ Y value at the value ｌ Calculating it as a value, The starting point X ｓ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ Calculating (x); The approximate line f ｂ (x) Peak point X on ｐ Y value at the value ｂ Calculating it as a value, Peak point Y ｐ value and the Y ｌ The difference between the peak point Yp value and the Y value is calculated as the increase in the fluorescent signal (l). ｂ Calculating the difference between the values ​​as the increase in the fluorescent signal (b); a process is performed that includes determining the increase in the fluorescent signal (l) or the increase in the fluorescent signal (b) as a signal increase; The calculation device determines the point at which the disturbance in the fluorescent signal due to the addition of the reagent has settled down by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the point at which the reagent was added, and determining that the point at which the coefficient of variation (standard deviation / average value) has become 0.03% or less for five or more consecutive data pairs is the left end of the first data pair.

6. A calculation program for calculating an increase in a signal from a fluorescent signal measured using a biological sample, The calculation program according to claim 3, Computer, The start point of the signal rise calculated by the second calculation unit (X ｓ , Y ｓ ) and the time-series data acquired by the data acquisition unit to calculate an increase in the fluorescent signal, In the third calculation unit, The starting point X ｓ Calculating an average value Y of the fluorescence intensity from at least two consecutive data pairs, going back from the value, and calculating the horizontal line of the average value Y as Yline; The point where the fluorescence intensity of the fluorescence signal is maximum is called the peak point (X ｐ , Y ｐ ) and the peak point X on the Y line ｐ Y value at the value ｌ Calculating it as a value, The starting point X ｓ The approximate line f with the smallest slope among the approximate lines of the fluorescent signal when the right end is the value and the left end is the range after the disturbance of the fluorescent signal due to the addition of the reagent has settled down. ｂ Calculating (x); The approximate line f ｂ (x) Peak point X on ｐ Y value at the value ｂ Calculating it as a value, Peak point Y ｐ value and the Y ｌ The difference between the peak point Yp value and the Y value is calculated as the increase in the fluorescent signal (l). ｂ Calculating the difference between the values ​​as the increase in the fluorescent signal (b); the increase in the fluorescent signal (l) or the increase in the fluorescent signal (b) is set as a signal increase, The calculation program determines the point at which the disturbance in the fluorescent signal due to the addition of the reagent has settled down by calculating the average value and standard deviation of the fluorescent intensity data for 20 to 40 consecutive data pairs over time from the point at which the reagent was added, and determining that the point at which the coefficient of variation (standard deviation / average value) has become 0.03% or less for five or more consecutive data pairs is the left end of the first data pair.

7. The starting point of the signal rise (X ｓ , Y ｓ ) is a calculation method for calculating The chemiluminescence signal is time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time, The calculation method is to calculate the tentative starting point (X ｓ’ , Y ｓ’ ), calculating the temporary starting point (X ｓ’ , Y ｓ’ ) based on the initial starting point (X ｓ , Y ｓ ), calculating the reference point (X ｃ , Y ｃ ), and calculating the initial starting point and the reference point (X ｃ , Y ｃ ) to the starting point (X ｓ , Y ｓ ) calculating The temporary starting point (X ｓ’ , Y ｓ’ ) is calculated by After adding a reagent that induces a biological reaction to a biological sample, the peak point (X ｐ , Y ｐ ) and The time from the time when the disturbance of the chemiluminescence signal due to the addition of the reagent settled down to the time when the peak point X ｐ The bottom point (X) at which the chemiluminescence intensity is minimum within the range ｂ , Y ｂ ) and X of the peak point ｐ A moving average calculated from at least two consecutive data pairs going back from the bottom point is ｂ The first point that falls within the range of the value + the noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X ｓ’ , Y ｓ’ ) and The initial starting point (X ｓ , Y ｓ ) is calculated by The temporary starting point X ｓ’ calculating an average value Y of the chemiluminescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as Yline; X of the peak point ｐ Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X ｓ , Y ｓ ) and The reference point (X ｃ , Y ｃ ) is calculated by X of the peak point ｐ The left end X where the approximate line of the chemiluminescence signal with the value at the right end is at its maximum slope １ calculating a value; The X １ The right end X where the approximate line of the chemiluminescence signal with the value at the left end is at its maximum slope ２ The value is the X ｐ calculating a value within a range of The X ２ The value is the right end, and the left end is the X １ Among the chemiluminescence signal approximation lines when the value is within the range, the approximation line f with the greatest slope is ｍａｘ (x) The approximate line f ｍａｘ The intersection of (x) and the Y line is the reference point (X ｃ , Y ｃ ) and The starting point (X ｓ , Y ｓ ) is calculated by The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the initial starting point is smaller than the recalculation reference value, the initial starting point is set to the starting point (X ｓ , Y ｓ ) and The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the value) is equal to or greater than the recalculation reference value, the X ｓ The first point where the average value calculated for each pair of at least two consecutive data points after that value is equal to or greater than the previous average value at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) The time point at which the disturbance in the chemiluminescence signal due to the addition of the reagent settles down is determined by calculating the average value and standard deviation of the chemiluminescence intensity data for 10 to 20 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.2% or less for 5 or more consecutive data pairs; the noise width of the chemiluminescence signal is the standard deviation of the baseline fluctuation of the chemiluminescence signal; The starting point (X ｓ , Y ｓ ) is the starting point of the signal rise.

8. The starting point of the signal rise (X ｓ , Y ｓ ) a calculation device for calculating a data acquisition unit that acquires the chemiluminescence signal as time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain a tentative starting point (X ｓ’ , Y ｓ’ a first calculation unit that calculates The tentative starting point (X ｓ’ , Y ｓ’ ) data and the time series data acquired by the data acquisition unit are processed to obtain the initial starting point (X ｓ , Y ｓ a second calculation unit that calculates The time series data acquired by the data acquisition unit is processed to obtain a reference point (X ｃ , Y ｃ a third calculation unit that calculates The initial starting point data calculated by the second calculation unit, the reference point (X ｃ , Y ｃ ) data and the time series data acquired by the data acquisition unit are processed to obtain the starting point (X ｓ , Y ｓ a fourth calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the peak point (X ｐ , Y ｐ ) The time from the time when the disturbance of the chemiluminescence signal due to the addition of the reagent settled down to the time when the peak point X ｐ The bottom point (X) at which the chemiluminescence intensity is minimum within the range ｂ , Y ｂ ) X of the peak point ｐ A moving average calculated from at least two consecutive data pairs going back from the bottom point is ｂ The first point that falls within the range of the value + the noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X ｓ’ , Y ｓ’ ) In the second calculation unit, The temporary starting point X ｓ’ Calculating an average value Y of chemiluminescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as Yline; X of the peak point ｐ Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X ｓ , Y ｓ ) In the third calculation unit, X of the peak point ｐ The left end X where the approximate line of the chemiluminescence signal with the value at the right end is at its maximum slope １ calculating the value, The X １ The right end X where the approximate line of the chemiluminescence signal with the value at the left end is at its maximum slope ２ The value is the X ｐ Calculation should be made within the range of values. The X ２ The value is the right end, and the left end is the X １ Among the chemiluminescence signal approximation lines when the value is within the range, the approximation line f with the greatest slope is ｍａｘ Calculating (x); The approximate line f ｍａｘ The intersection of (x) and the Y line is the reference point (X ｃ , Y ｃ ) In the fourth calculation unit, The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the initial starting point is smaller than the recalculation reference value, the initial starting point is set to the starting point (X ｓ , Y ｓ ) and The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the value) is equal to or greater than the recalculation reference value, the X ｓ The first point where the average value calculated for each pair of at least two consecutive data points after that value is equal to or greater than the previous average value at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) The starting point (X ｓ , Y ｓ ) as the start of the signal rise, The time point at which the disturbance in the chemiluminescence signal due to the addition of the reagent settles down is determined by calculating the average value and standard deviation of the chemiluminescence intensity data for 10 to 20 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.2% or less for 5 or more consecutive data pairs; The noise width of the chemiluminescent signal is the standard deviation of the baseline fluctuation of the chemiluminescent signal.

9. The starting point of the signal rise (X ｓ , Y ｓ ) is a calculation program for calculating Computer, a data acquisition unit that acquires the chemiluminescence signal as time-series data consisting of a data pair (measurement time, chemiluminescence intensity) of chemiluminescence intensity versus measurement time; The time series data acquired by the data acquisition unit is processed to obtain a tentative starting point (X ｓ’ , Y ｓ’ a first calculation unit that calculates The tentative starting point (X ｓ’ , Y ｓ’ ) data and the time series data acquired by the data acquisition unit are processed to obtain the initial starting point (X ｓ , Y ｓ a second calculation unit for calculating The time series data acquired by the data acquisition unit is processed to obtain a reference point (X ｃ , Y ｃ a third calculation unit for calculating The initial starting point data calculated by the second calculation unit, the reference point (X ｃ , Y ｃ ) data and the time series data acquired by the data acquisition unit are processed to obtain the starting point (X ｓ , Y ｓ ) and functioning as a fourth calculation unit that calculates In the first calculation unit, After adding a reagent that induces a biological reaction to a biological sample, the peak point (X ｐ , Y ｐ ) The time from the time when the disturbance of the chemiluminescence signal due to the addition of the reagent settled down to the time when the peak point X ｐ The bottom point (X) at which the chemiluminescence intensity is minimum within the range ｂ , Y ｂ ) X of the peak point ｐ A moving average calculated from at least two consecutive data pairs going back from the bottom point is ｂ The first point that falls within the range of the value + the noise width of the chemiluminescence signal at least twice consecutively is set as the tentative starting point (X ｓ’ , Y ｓ’ ) In the second calculation unit, The temporary starting point X ｓ’ Calculating an average value Y of chemiluminescence intensity from at least two consecutive data pairs, going back from the value, and calculating a horizontal line of the average value Y as Yline; X of the peak point ｐ Going back from the value, the first point that falls within the range of the average value Y + standard deviation is the initial starting point (X ｓ , Y ｓ ) In the third calculation unit, X of the peak point ｐ The left end X where the approximate line of the chemiluminescence signal with the value at the right end is at its maximum slope １ calculating the value, The X １ The right end X where the approximate line of the chemiluminescence signal with the value at the left end is at its maximum slope ２ The value is the X ｐ Calculation should be made within the range of values. The X ２ The value is the right end, and the left end is the X １ Among the chemiluminescence signal approximation lines when the value is within the range, the approximation line f with the greatest slope is ｍａｘ Calculating (x); The approximate line f ｍａｘ The intersection of (x) and the Y line is the reference point (X ｃ , Y ｃ ) In the fourth calculation unit, The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the initial starting point is smaller than the recalculation reference value, the initial starting point is set to the starting point (X ｓ , Y ｓ ) and The reference point X ｃ value and the initial starting point X ｓ The difference between the value (X ｃ Value - X ｓ If the value) is equal to or greater than the recalculation reference value, the X ｓ The first point where the average value calculated for each pair of at least two consecutive data points after that value is equal to or greater than the previous average value at least twice in succession is taken as the starting point (X ｓ , Y ｓ ) The starting point (X ｓ , Y ｓ ) as the start point of the signal rise, The time point at which the disturbance in the chemiluminescence signal due to the addition of the reagent settles down is determined by calculating the average value and standard deviation of the chemiluminescence intensity data for 10 to 20 consecutive data pairs over time from the time point at which the reagent was added, and the time point at which the coefficient of variation (standard deviation / average value) becomes 0.2% or less for 5 or more consecutive data pairs; A calculation program, wherein the noise width of the chemiluminescent signal is the standard deviation of the baseline fluctuation of the chemiluminescent signal.

10. A method for calculating an increase in a chemiluminescence signal measured using a biological sample, comprising: The calculation method according to claim 7 calculates the start point of the signal rise (X ｓ , Y ｓ ), calculating the end point (X ｅ , Y ｅ ) and calculating an increase in chemiluminescence signal, The end point (X ｅ , Y ｅ ) is calculated by X of the peak point ｐ The first point where the moving average calculated from at least two consecutive data pairs after the value falls within the range of Y line at least twice consecutively is set as the end point (X ｅ , Y ｅ ) The step of calculating the increase in the chemiluminescence signal comprises: The Y line is the baseline, and the starting point X ｓ value to the end point X ｅ calculating an integrated value of chemiluminescence intensity up to a value; A calculation method in which the integrated value is used as the amount of increase in the signal.

11. A calculation device for calculating an increase in a chemiluminescence signal measured using a biological sample, comprising: The calculation device according to claim 8, further comprising: a data acquisition unit that processes the time series data acquired by the data acquisition unit to obtain an end point (X ｅ , Y ｅ a fifth calculation unit that calculates the start point (X ｓ , Y ｓ ) data, the end point (X ｅ , Y ｅ a sixth calculation unit that processes the time-series data acquired by the data acquisition unit and calculates an increase in the chemiluminescence signal; In the fifth calculation unit, X of the peak point ｐ The first point where the moving average calculated from at least two consecutive data pairs after the value falls within the range of Y line at least twice consecutively is set as the end point (X ｅ , Y ｅ ) In the sixth calculation unit, The Y line is the baseline, and the starting point X ｓ value to the end point X ｅ Calculating the integrated value of chemiluminescence intensity up to the value; A calculation device that performs processing including determining the integrated value as an increase in a signal.

12. A calculation program for calculating an increase in a chemiluminescence signal measured using a biological sample, The calculation program according to claim 9, Computer, The time series data acquired by the data acquisition unit is processed to obtain an end point (X ｅ , Y ｅ a fifth calculation unit for calculating the start point (Xs, Ys) of the signal rise calculated by the fourth calculation unit, and the end point (X ｅ , Y ｅ ) data and the time-series data acquired by the data acquisition unit, and functioning as a sixth calculation unit that calculates an increase in a chemiluminescence signal; In the fifth calculation unit, X of the peak point ｐ The first point where the moving average calculated from at least two consecutive data pairs after the value falls within the range of Y line at least twice consecutively is set as the end point (X ｅ , Y ｅ ) In the sixth calculation unit, The Y line is the baseline, and the starting point X ｓ value to the end point X ｅ Calculating the integrated value of chemiluminescence intensity up to the value; a calculation program for causing the processing to be performed, the processing including setting the integrated value as an increase in the signal;

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