JPS6051658B2 - Automatic rate analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for automated rate analysis of blood samples.

Although there have been remarkable developments in clinical biochemical analysis in recent years,
This is largely due to the practical use of various automatic analyzers.

The advent of these various automatic analyzers has had a remarkable effect on both the number of specimens processed and the accuracy of measurement results compared to previous manual methods. However, considering the current state of clinical biochemistry analysis that makes extensive use of such automated analyzers, from the perspective of accuracy of measurement results,
There are many issues that need to be reflected on.

In biochemical analysis using conventional automatic analyzers, the biggest problem in terms of accuracy is endogenous errors in colorimetric analysis. Recently, for the purpose of reducing such endogenous errors, automatic analyzers that adopt a reaction rate measurement method (hereinafter referred to as the rate method) instead of colorimetric analysis have been put into practical use, and have become popular in the field of biochemical analysis. Future development is expected.

However, even in the ι route method, which has few endogenous errors and is theoretically highly accurate, depending on how it is applied to an automatic analyzer, it may produce errors greater than those of conventional colorimetric analysis methods.

The rate method can only be used by measuring the rate of a chemical reaction involving the target substance in a steady state (a state in which there are no side reactions and the target reaction is proceeding at a constant rate).
This allows for more accurate measurements than conventional colorimetric methods. However, with the various automatic analyzers currently in use, there is no way to confirm that the measurement was performed in a steady state, so depending on the sample, they may give significantly erroneous results than conventional colorimetry. There are quite a few. In other words, in the early stages when the rate method began to be used for research purposes, the measurement method was close to a manual method, and the entire reaction process from the start of the reaction to the end of the reaction was observed in an analog manner. Errors were not a problem. However, as mentioned above, various automatic analyzers are now required to have high processing speeds, and the target substance is measured without confirming that the reaction is occurring in a steady state. It started to cause errors. Figure 1 shows diphosphopyridine nucleotide (reduced form) (N
．． This figure schematically shows the change in absorbance of the reaction solution in the case of rate measurement based on the decrease in ADH.

That is, the sample serum is first mixed with a first reagent (usually consisting of a buffer and a reactant other than one of the substrates for the reaction of the target substance) (time T). This mixture is then incubated at a suitable temperature. During this incubation, various side reactions (causing endogeneous errors) proceed, N and ADH are consumed, and the absorbance is A.
decreases from Sufficient incubation (time
As soon as the side reactions have almost finished in step 1), the second reaction initiation liquid (one of the substrates for the reaction of the target substance) is added to start the target reaction, and as the reaction progresses, N
As ADH is consumed, the absorbance decreases from A1 to A3 to a constant value. In other words, the reaction of the target substance starts at a certain rate, but the rate of this reaction is not constant from the beginning and becomes almost constant after a certain time (T2 in the figure) (the reaction reaches a steady state and the change in absorbance becomes linear). Become). The reaction has started! How long does it take for the reaction to become stable? is generally called ragtime. A steady state occurs when the amount of NADH is sufficient ~T
5, however, as time passes, NADH becomes insufficient and the reaction gradually slows down, and when NADH is completely consumed at T5 to T6, the reaction stops and the absorbance reaches a constant value ~ (after !). In order to obtain correct measurement results in rate measurement, it is necessary to measure the change in absorbance over a certain period of time (for example, T3 to T4) between T2 and T5, which is a steady state.

If the rate is measured manually, it is easy to obtain the correct absorbance change at time T3 to T4 by observing the absorbance change in FIG. 1 in an analog manner in the entire region after ζ. However, in order to observe the entire area after the reaction has started, it takes at least several minutes to complete, but automated methods require rapid processing of a large number of samples. In the analyzer, the observation time is limited to about 3 times and 2 times.

That is, when the rate method is applied to an automatic analyzer, it is no exaggeration to say that the largest error in the measurement results is due to the short observation time of the automatic analyzer. In other words, the rate method error caused by an automatic analyzer is due to the fact that the optimal lag time ζ ~ T2 from the start of the reaction to the start of observation differs significantly depending on the concentration (activity) of the target substance in the sample. First, this is due to the nature of the automatic analyzer, which has to take a fixed amount of time. This will be explained in detail with reference to FIG. In other words, in Fig. 2, A1 is the reaction starting point (second reagent addition time), A1 is the absorbance at that point, T3 to T4 are the observation times of absorbance changes in the automatic analyzer, and ~ is the lack of NADH as mentioned above. Reaction curves 1, 2, and 3 are reaction curves of simulated serum samples at low, medium, and high concentrations using the pure substance of interest, respectively.

As shown in the figure, if the observation time is set from T3 to T4, the reaction will be linear (steady state) between T3 and T4 in the case of samples with a medium or lower concentration of the target substance, such as 1 and 2. Therefore, by measuring the change in absorbance during that time, the correct concentration of the target substance can be determined. However, in a sample with a high concentration of the target substance as in 3.
At T3, the reaction is already close to stopping due to lack of NADH, and the change in absorbance between T3 and T4 is extremely small, indicating that although it is an abnormal sample with a high concentration, it is a normal sample with a seemingly low concentration. It will be measured as . If such a measurement error were to occur, this would not be an endogenous error ratio in the colorimetric analysis method, but would be a serious problem that could affect the patient's life. As a fundamental solution to these drawbacks of the rate method in automatic analyzers, the best method is to observe changes in absorbance over the entire region after the start of the reaction, as in the case of the above-mentioned manual method.

However, for this purpose, it takes several minutes to one hour to process one sample.
This method is complicated and the processing speed is limited to about 1 throat body per hour, so it cannot be applied to automatic analyzers that require a high processing speed. A method conventionally used as the next best solution is to check the absolute value of the final absorbance at the observation end point ζ as well as the absorbance change within the observation time T3 to ζ. In other words, if the final absorbance of the test solution is larger than the theoretical limit absorbance A2 at which the reaction rate begins to decrease due to lack of N and ADH, there is no mistake as mentioned above, and conversely, if it is small, then there is no such mistake. There is a device that outputs and detects that the sample is an abnormally high concentration sample that cannot be measured. In this case, the operator can prevent the above-mentioned serious mistake by diluting the abnormal specimen with physiological saline or the like and retesting. However, in actual samples, it has been found that even by checking the final absorbance, it is not possible to completely prevent measurement errors for high-concentration samples as described above.

This is because, unlike the simulated sample synthesized from pure substances shown in Figure 2, the actual sample contains various interfering substances such as chyle, hemolysis, and jaundice, so the above-mentioned reaction rate begins to decrease. This is because it is difficult to accurately grasp the target absorbance limit A2. That is, in the case of an ideal specimen as shown in FIG. 2, the test liquid is 340r1WL. The absorbance at is NA
Since it depends only on the reactants in the reagent such as DH and substrate, it can be determined from the final absorbance alone whether the remaining amount of NADH is sufficient, that is, whether the serum has a high concentration as described above. On the other hand, in actual samples containing interfering substances as mentioned above, the absorption due to these interfering substances is added to the absorption of the reagent, so if the final absorbance at ζ of the test solution is larger than the theoretical limit absorbance mentioned above, However, the above-mentioned measurement error for high-concentration serum often occurs. For example, the third
Absorption spectra 4 and 5 in the figure show the respective absorption spectra of an ideal non-turbid serum sample measurement solution and a strong chyle serum sample measurement solution, but the chyle components in the sample are 340r1Tn. It has been found that the conventional method of checking the absolute value of the final absorbance is almost completely ineffective when chyle serum is used as a specimen. Also, Figure 4 shows 2 in Figure 3.
This shows the reaction curve (340n7T1.) of a high-concentration simulated sample in which the same amount of the target pure substance is added to the seed serum. However, in the case of high-concentration serum 6 that does not become cloudy, the final absorbance is less than the theoretical limit absorbance A2. There is no problem because it is output as a high concentration abnormal sample, but in case of cloudy high concentration serum, N
Even when ADH is completely consumed (after t1), the absorbance of the measurement solution is higher than the above-mentioned theoretical limit absorbance, so it is output as a high concentration abnormal sample, which is significantly mistaken as a normal low concentration normal sample. It will give the measurement results. The purpose of the present invention is to improve the drawbacks of the conventional methods described above and to provide a highly accurate automatic rate analysis method that does not cause the above-mentioned serious measurement errors even for cloudy and highly concentrated serum. . In order to achieve the above object, the present invention first utilizes the characteristics of a multi-wavelength photometer to measure the degree of turbidity in a sample from the visible spectrum of the sample liquid, and from this measurement value, calculates the theoretical limit absorbance as described above. is corrected to obtain the true limit absorbance, and this is compared with the aforementioned final absorbance to determine whether or not the measurement of the analyte in the test solution was performed in a constant speed region. .

That is, the greatest feature of the present invention is that, unlike the conventional method of determining a fixed theoretical limit absorbance, the present invention determines a true limit absorbance that differs for each specimen. The degree of turbidity is corrected in the present invention because the spectrum due to turbidity of serum has a relatively simple shape as shown in Fig.
! ．． This method utilizes the fact that most of the absorption of the test liquid in the above long wavelength range is due to turbidity in the sample.

That is, when we examined the spectra of test solutions prepared by diluting serum with various degrees of turbidity with a neutral buffer solution for transaminase rate measurement, we found that the 600 and 70
It was found that the ratio of the absorbance at 0r17T1 and the absorbance at 340r1m converged to a certain constant value K1.

A (Oh.

) is 340r17TL. of each test liquid. Absorbance of A(60
0-7 (1X)) is the same test solution of 600 r1m and 70 (9
) K1 is a constant. Similarly, it was found that the following relationship also holds true.

Since the shape of the spectrum differs slightly depending on the state of the turbid particles and the influence of other interfering substances, K1~\ is a constant that converges within a certain range, but in any case, the above-mentioned theoretical limit absorbance value It has been experimentally confirmed that this method has sufficient accuracy for applications such as determining true limit absorbance values. The state of convergence is best for K2, followed by K1.
is good, and K3 and K4 have slightly poor convergence. Therefore, if the theoretical limit absorbance when performing rate measurement at 340 nrrL is AR and the true limit absorbance is AO, the following relationship holds true.

Also 340r17TL. When rate measurement is performed using the two-wavelength method of 376r17n and 376r17n, the following relationship is applied.

As is clear from the above, by using the true limit absorbance determined by equations (5) to (7) instead of the conventional theoretical limit absorbance, measurement errors for cloudy, high-concentration serum can be prevented. can do.

Next, Table 1 shows the results of measurements of several types of simulated samples prepared by adding known concentrations of pure LDH to normal serum and chyle serum, using both the conventional method and the method of the present invention. Samples 1-4 are normal serum and samples 5 and 6 are chyle serum. That is, in the case of non-turbid serum like Samples 1 to 4, both the conventional method and the method of the present invention give the same results, but in the case of cloudy and highly concentrated serum like Samples 5 to 6, the conventional method gives lower results. In contrast, the method of the present invention correctly outputs high-concentration abnormal serum (marked with *), whereas the serum is output as a normal serum with a high concentration.

Generally, LDH, . GOT. ．． When testing for enzymes such as GPT, there are often thousands of abnormal serum titers, and considering that the number of cloudy samples has been increasing in recent years,
The effect of the present invention, which can reliably detect abnormal specimens, is remarkable. In the present invention, the wavelength for measuring turbidity is not limited to the aforementioned 600r1WL1700r1m, and the wavelength for measuring the target substance is also 340r17T.
1. , 376n7n, and other measurement wavelengths can be used depending on the properties of the sample to be tested.

As described above, according to the present invention, there is no measurement error due to the degree of turbidity of a blood sample, and highly accurate automatic rate analysis can be performed.

[Brief explanation of the drawing]

Figure 1 shows a general reaction curve for the rate response of serum;
The figure shows similar reaction curves for samples with three concentrations: low, medium, and high. Figure 3 shows the absorption spectra of normal high-concentration serum and cloudy high-concentration serum. Figure 4 shows the absorption spectra of the above two types of serum. It is a reaction curve of a sample.

Claims (1)

[Claims] 1. In an automatic rate analysis method in which a reagent is added to a blood sample, the change in absorbance over time in a constant rate region due to the reaction is measured, and the concentration of the analyte in the sample is determined from the amount of change. Measure the visible spectrum related to turbidity, correct the theoretical limit absorbance in the constant speed range set in advance using this measurement value to find the true limit absorbance, and compare this absorbance with the final absorbance of the measurement. 1. An automatic rate analysis method characterized by determining whether or not measurement of a sample was performed in a constant rate region of reaction.