JP2732448B2 - Automatic rate analysis - Google Patents

Description

The present invention relates to a method for analyzing a sample rate by an automatic analyzer for measuring the same measuring cell a plurality of times. More specifically, the present invention relates to a rate analysis method that can reduce analysis errors due to coloring or turbid components contained in biological fluid samples such as serum, plasma, and urine in clinical biochemical tests, and can measure a wide range of high value samples having a large absorbance change rate. (B) Conventional technology The rate analysis method is superior in principle to the end point method in accuracy, but if applied incorrectly, a large analysis error will be caused. That is, the rate analysis method is a method performed on the assumption that the reaction rate is in a steady state (a state in which the substrate is sufficiently present). If this condition is not satisfied, it is unavoidable that a large analysis error occurs. Conventionally, in order to avoid this, an absorbance range in which rate analysis can be measured is set in advance, and analysis is performed after confirming that the absorbance change rate measurement is within the range. (C) Problems to be Solved by the Invention However, the absorbance range set as described above does not take into account the influence of coloring components and turbidity components in the experimental body (different for each actual sample), so that the There is a risk that the state (substrate shortage state) may be mistaken for a steady state, or the steady state may be mistaken for a non-steady state. Of course, this can be avoided by narrowing the set absorbance range, but in this case, the range that can be measured even for a sample that does not contain a coloring component or a turbid component is narrowed, which is not practical. In addition, the clinical test items to be measured include:
There is a very wide range to be measured. For example, in the measurement of GOT (AST) enzyme activity,
A patient sample having a value of 1000 to several 1000 U / for 3030 U / may be analyzed. Of course 10 to several thousand U /
It is impossible to perform high-precision measurement under the same analysis conditions up to the same.For ultra-high unit samples of several thousand U /, the determination of `` measurement impossible '' is made based on the absorbance check value of an appropriate sample reaction solution, and an appropriate Although the sample is reanalyzed by a method (for example, dilution), some samples may contain components that show absorption at the measurement wavelength or components containing turbid components. Something cannot be determined. The present invention has been made in view of such circumstances, and aims to provide an automatic rate analysis method that maximizes the rate-measurable section that maintains the steady state as described above and that takes into account the influence of turbidity components and the like. Things. (D) Means for Solving the Problems According to the present invention, in a rate analysis method in which an automatic analyzer measures the absorbance of a sample reaction solution at predetermined time intervals within a predetermined time range, a start point and an end point of measurement are determined. It consists of a method of measuring each wavelength by one wavelength measurement and two wavelengths between them, and calculating the concentration of the analyte in the sample reaction solution from a data string of the obtained absorbance values. Comparing with the rate measurement limit absorbance (J ₀ ) set in advance based on the wavelength measurement; and (b) for those determined to be within the rate measurement limit by the above comparison, the data at the end point is further set in advance. when within rate measurement range which is restricted by the rate-measurement limit absorbance (J ₁₎ or reagent blank solution absorbance effective limit absorbance is corrected on the basis of (J ₁ '), 2-wave Data string all calculates the rate of absorbance change from measurements throughout _{(t M ~t N), (} c) when the data of the end point exceeds the rate measurement limit is at least 3 from the start of the two-wavelength measurement (t _M) The change in the absorbance at the point is compared with a substantially allowable change in absorbance (J ₂ ) set in advance based on the two-wavelength measurement. (D) For those determined to be within the allowable range by the above comparison, the following conditions; _{△ a / tn-t M)} ≧ (J 2 / t M ~t N) However, △ a: 2-wavelength measurement starting point (t _M) maximum absorbance change does not exceed the J ₂ from tn: give the △ a automatic rate analysis method is provided to calculate the test concentration, if not satisfied, characterized in that it is determined that the rate analysis not based on the case △ a / tn-t _M which satisfies the two wavelengths measurement points . According to the present invention, in an enzyme activity and substrate rate analysis method in which the rate of increase or decrease of a substrate or a product is measured as a change in absorbance per unit time, a boundary level at which a substrate shortage occurs based on one wavelength measurement. rate measurement limit absorbance (J ₀ and J ₁₎ and said J effective limit absorbance was corrected in consideration of the absorbance of the reagent blank against ₁ (J ₁ ') as well as substantially acceptable absorbance previously set based on the two-wavelength measurement From the change (J ₂ ), the maximum rate measurement section that can maintain a substantially steady state excluding the influence of the coloring and turbid components of the sample reaction solution is determined, and the analyte concentration in the sample reaction solution is determined. It is characterized by a rate analysis method. That is, the limit of the rate measurement is set by the one-wavelength measurement, and the rate measurement is performed based on the data of the two-wavelength measurement that is more accurate than the one-wavelength measurement. The data of the two-wavelength measurement is obtained by subtracting the sub-wavelength measurement value from the main wavelength measurement value. As a result, the influence of the coloring / turbidity components of the sample reaction solution and the blemishes of the measurement cell are eliminated, and a substantial change in absorbance can be obtained. In this method, the main wavelength of the main wavelength and the sub-wavelength used for the two-wavelength measurement can be used as the wavelength for one-wavelength measurement. 2 above
As the wavelength, for example, in the case of GOT enzyme activity measurement, the main wavelength is 340n
m, subwavelength 375 nm. J ₁ above, the intended rate measurement section, in this case two-wavelength measurement section, in the high unit sample having an activity value of about the unsteady state in the non-stationary state, select as clear and colorless as possible,
In this case, the absorbance (Ac ₁ ) at the time of transition from the steady state to the non-steady state in the temporal change of the absorbance measured at one wavelength every predetermined time is set as the rate measurement limit absorbance. The J ₀ is a very rare, because if the sample exhibits a super high unit may be insufficient substrate in the reaction solution immediately after the rate measurement start is determined absorbance this, J ₁ And the value derived from the absorbance of the reagent blank solution after one-wave measurement. J ₁ ′ is the absorbance (Ac ₁ ) when J ₁ is set.
Is subtracted from the absorbance (Ab ₁ ) of the one-wavelength measurement of the reagent blank solution at the measurement position, and is set as the absorbance corresponding to the effective rate measurement limit. As a result, the influence of the coloring / turbid component of the sample reaction solution is offset. The effective permissible absorbance change (J _2), for high unit specimens at the J ₁ setting, the time course of absorbance obtained by the two-wavelength measurement of the main wavelength and sub-wavelength used for rate measurement, unsteady from stationary The absolute absorbance (Ac ₂ ) is determined when the limit absorbance (Ac ₂ ) is determined, and the absorbance change (Ac ₂ −Ab ₂ ) is obtained by subtracting the absorbance (Ab ₂ ) of the reagent blank solution for the two wavelength measurement at the measurement point.
Is converted into a change in absorbance allowed at the two-wavelength measurement start point (t _M ). For the conversion method, reference is made to the description of the embodiment described later. In the method of the present invention, J ₀ , J ₁ , J ₁ ′ and J ₂
In the data sequence of the absorbance values obtained from the measurements at predetermined time intervals, the one-wavelength measured value at the start point and the end point is determined by J ₀ and J ₁ (or J ₁ ′), and the two-wavelength measured value determination by J ₂ is performed with respect to. That is, when the data value at the start point satisfies J ₀ and the data value at the end point satisfies J ₁ or J ₁ ′, the distance from the two-wavelength measurement start point (t _M ) to the two-wavelength measurement end point (t _N ) Absorbance change is J ₂
Therefore, the steady state is maintained over the entire two-wavelength measurement region (t _{M to} t _N ), and the rate analysis is performed using all the data in this region. Become. In the above case, when the data value at the end point measured by one wavelength does not satisfy J ₁ or J ₁ ′, the start point of two-wavelength measurement (t _M )
Absolute value is selected maximum becomes measurement points (tn) without exceeding the J _2, further change in absorbance obtained from the change in absorbance (△ A) of the two-wavelength measurement interval (t _M tn) of absorbance change from Absolute value of rate, | △ A | / tn−t _M is the whole range of two wavelength measurement (t _{M to} t _N )
Rate of absorbance change determined by J ₂ for, when greater than or equal to J ₂ / t _N -t _M is, t _M tn is determined that the rate reaction section was maintained steady state, thus to reduce the rate measurement section rate Will be analyzed. Thus, in the method of the present invention, it is not determined for the ultra-high unit analyte, if absorbance at the start point by 1 wavelength measurement exceeds J _0, the absorbance change of at least three points from the two-wavelength measurement starting point is the J ₂ If and the such absorbance change rate exceeds, | △ a | / tn- t M becomes to be that effect determined in each case when less than J ₂ / t _N -t _M. (E) Function According to the present invention, the data sequence of the absorbance values obtained by measuring the start point and the end point of the measurement within a predetermined time range with one wavelength measurement and measuring the two wavelengths between the start and end points is determined in advance by one wavelength measurement. It is determined from the rate measurement limit absorbance (J ₀ ) set by ( ₁ ) whether or not the starting point is already in an unsteady state due to substrate shortage. Determined data sequence and a steady state with a J ₀ whether a steady state is determined the endpoint with the effective limit absorbance. If the end point is determined to be in the steady state, the data sequence of the entire two-wavelength measurement is used for rate analysis. However, when the end point is determined to be in an unsteady state, the change in absorbance from the start point of the two-wavelength measurement to at least three points is selected by a change within the substantially allowable absorbance change set in advance by the two-wavelength measurement, First, those that have fallen into the unsteady state during this period are excluded.
With respect to the selected one, the change in absorbance from the two-wavelength measurement start point is within the above-described substantially allowable change in absorbance, and the rate of change in absorbance at that time gives an absorbance change rate equal to or greater than the rate of change of absorbance determined by the change in substantially allowable absorbance over the entire two-wavelength measurement range. The data sequence up to the data sequence is determined to be a data sequence under a steady state, and rate analysis is performed using this data sequence. Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. (F) Example An example in which an automatic analyzer shows a constant absorbance change for a relatively long time in an enzyme activity measurement by a two-reagent method within a predetermined time range (ti to t _{N + 1} , but set at equal intervals). explain. (I) Analysis conditions [measurement wavelength] Measurement start point (ti) immediately after dispensing (t ₀ ) of the second reagent (R2): λ ₁ 2 wavelength measurement section (t _{M to} t _N ): λ ₁ / λ ₂ measurement End point (t _{N + 1} )… λ ₁ [Measurement data] Absorbance of sample reaction solution ti …… Ait _{M to} t _N … A _{M to} A _N t _{N + 1} …… A _{N + 1} absorbance ...... Ab ₁ However, λ ₁ is the main wavelength, is λ ₂ which is a by-wavelength. (Ii) rate measurement limit absorbance (J ₀ and J _1), the effective limit absorbance (J ₁ ') and substantially acceptable absorbance change (J ₂₎ for the two-wavelength measurement period as shown in FIG. 1 determining the J ₁ (t _M ~ T _N ), select a high-unit sample that is in an unsteady state and a colorless and transparent sample as possible, and perform one-wavelength (λ ₁ ) measurement on such a sample in the above-mentioned predetermined time range (when a reagent is added: t
_The measurement is performed from ₀ to the end of measurement: t _N ). Based on the absorbance change (mABS / min) (a) appearing on the time series in this range, the boundary absorbance (mAB
Determining S) as J _1. Determination of J _{2 As} shown in FIG. 2, the two-wavelength (λ ₁ / λ ₂ ) measurement of the high unit sample and the reagent blank (for example, using physiological saline as a sample) was performed at the time of the determination of J ₁ above. The change in absorbance over time (b) is determined by measuring in the same manner, and the absorbance (AC ₂ ) at which the reaction process of the high unit sample shifts from the steady state to the unsteady state, and the absorbance (Ab ₂ ) is obtained, reagent addition JP (t _0), the two-wavelength measurement start time (t _M) and the third point of the two-wavelength measurement point (t _{M + 2)} approximately in proportion calculated for at t _M point acceptable absorbance change to the J _1, is calculated by the following equation. J ₂ = (AC ₂ −Ab ₂ ) · [(t _{M + 2} −t _M ) / (t _{M + 2} −t ₀ )] Determination of J ₀ J ₁ previously determined, absorbance of one-wavelength measurement of reagent blank solution (Ab
Since ₁₎ and, considered in the same manner as "determination of J _2", is calculated by the following equation. _{J 0 = (AC 1 -Ab 1} -AC 1) · _{[(t M + 2 -ti) /} (t M + 2 -t 0) ] 'determination of _{J 1' J = J 1 ±} (Ai-Ab 1 (However, +: when observing a change in absorbance focusing on the substrate concentration, −: when observing focusing on the product concentration) Next, J ₀ , J ₁ , J ₁ ′, J ₂ The determination will be described with reference to reaction time courses of various sample reaction solutions (a to e) shown in FIG. These reaction time courses were created based on the data sequence obtained under the above analysis conditions. Respectively two wavelength measurement absorbance, × is _{J 0, J 1, J 1} ', J 2 determined by the absorbance of ultra-high range determined value the. First, the absorbance Ai of the specimen reaction at the starting point (ti) of each data sequence is compared with J _0. As a result, the data string indicating J ₀ ≧ Ai already indicates the unsteady state,
(In other words, the display of the super high price) is made ((a)
). On the other hand, for a data string in which j ₀ <Ai, the change in absorbance from the data from the two-wavelength measurement start point to three points | A _{M + 2} −A _M |
Is compared with the change in real absorbance (J ₂ ). As a result, J _{2
<| A M + 2 -A M} | For data string indicating the interval (t _M ~
Since an unsteady state is indicated within t _{M + 2} ), “measurement impossible” is displayed in the same manner as described above (case (b)). Before the above process, the effective limit absorbance (J ₁ ′) is calculated as follows. J ₁ ′ = J ₁ + (Ai−Ab ₂ ) (However, when Ai <Ab ₁ , J ₁ ′ = J ₁ ) J ₂ ≧ | A _{M + 2} −A _M | The data value (A _{N + 1} ) at the end point (t _{N + 1} ) of the sequence is compared with the already calculated J ₁ ′, and for the data sequence where J ₁ ′ <A _{N + 1} , two-wavelength measurement is performed. throughout (t _M ~t _N) steady state is held for, hence the absorbance change ratio by the following equation,
△ A / △ t = (A N -A M) / specimen concentration based on (t _N -t _M) is calculated. However, since the steady state is not guaranteed for the entire two-wavelength measurement range for the data string satisfying J ₁ ′ ≧ A _{N + 1} , J ₂ ≧ | An−A _M | (M + 2 ≦ n ≦ N) is satisfied. 2 wavelength measurement points to give measurement data an for (tn) is selected, further absorbance change rate from t _M to _{tn, | an-a M |} / (tn-t M)
There the case of more than the absorbance change ratio determined by _{J 2 / (t N -t M} ) is 2 wavelength measurement interval t _M tn is determined that a steady state holding sections, based on the rate of absorbance change at this interval Thus, the analyte concentration is calculated. Also, | An−A _M | / (tn−t _M ) <J ₂ /
In the case of (t _N -t _M) (if the (e)) for this data string is to be made marked "not measurable". In addition, if the above determination process is represented by a flowchart, the fourth determination is performed.
As shown in the figure. However, in FIG.
Each signal in (e) corresponds to each symbol in the above description. According to the above method, the two-wavelength measurement section holding the steady state is accurately selected, and the analyte concentration is calculated for that section based on the rate of change in absorbance. (G) Effects of the Invention According to the present invention, the section in which the steady state is maintained is determined, and the analyte concentration is calculated from the absorbance change rate in this section, so that the rate analysis can be performed with high accuracy. This also eliminates the possibility that the ultra-high unit sample is mistaken for the low unit sample, thereby improving the reliability of the rate analysis method.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph illustrating a method of determining J ₀ and J ₁ , FIG. 2 is a graph illustrating a method of determining J _{2, and} FIG. 3 illustrates a method of the present invention. FIG. 4 is a graph diagram showing various reaction time courses, and FIG. 4 is a flowchart diagram corresponding to the description of FIG.

   ────────────────────────────────────────────────── ─── Continuation of front page    (56) References JP-A-56-155835 (JP, A)                 JP-A-57-72048 (JP, A)                 JP-A-59-56554 (JP, A)                 JP-A-56-108941 (JP, A)

Claims (1)

(57) [Claims] In a rate analysis method in which an automatic analyzer measures the absorbance of a sample reaction solution at predetermined time intervals within a predetermined time range, the start point and the end point of measurement are measured by one wavelength measurement, and the two points are measured by two wavelength measurement in between. The method comprises calculating the analyte concentration in a sample reaction solution from a data sequence of values. (A) The data at the starting point is calculated as a rate measurement limit absorbance (J ₀ ) preset based on one-wavelength measurement. (B) For those determined to be within the rate measurement limit by the above comparison, the data at the end point is further determined based on the preset rate measurement limit absorbance (J ₁ ) or the absorbance of the reagent blank solution. the effective limitation absorbance corrected Te when (J ₁ ') is in the rate measurement range is restricted, the operation rate of absorbance change from all the data strings of the two-wavelength measurement throughout (t _M ~t _N) (C) when the data at the end point exceeds the rate measurement limit, at least three changes in absorbance from the start point (t _M ) of the two-wavelength measurement are determined by the substantially allowable absorbance preset based on the two-wavelength measurement. change (J ₂₎ as compared to, (d) for those determined to be within acceptable by the comparison, the following conditions; (△ a / tn-t M) ≧ (J 2 / t M ~t N) However, ΔA: the maximum absorbance change not exceeding J ₂ from the two-wavelength measurement start point (t _M ) tn: if the two-wavelength measurement point giving the above ΔA is satisfied, based on ΔA / tn−t _M An automatic rate analysis method that calculates the analyte concentration and determines that rate analysis is not possible if the concentration is not satisfied.