JPS604929B2 - Colorimetric analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to improvements in colorimetric analysis methods in biochemistry that utilize changes in absorbance for specimens containing interfering chromogens.

The recent trend toward automation in the field of biochemical testing is extremely remarkable.

Automatic analyzers have also been developed in a wide variety of models, from the early flow type to the recent various discrete types. These various automatic analyzers have shown remarkable effects in both expanding the overall number of tests and improving the accuracy of measurement results. In other words, most of the automatic analyzers currently in use have reached a stage where they almost satisfy the needs of their users in terms of sample processing speed and precision. be. However, when considering the accuracy of measurement results, the various automatic analyzers currently in use still have many problems and are far from meeting the needs of users. In particular, various chromogens, including hemolysis (hemoglobin), bilirubin (bilirubin), and chyle (turbidity), impair the measurement accuracy of automatic analyzers. The influence of such interfering chromogens is particularly noticeable in colorimetric analysis and nephelometric analysis using the end point method, and is almost non-existent in the rate method (reaction rate measuring method). However, although it is possible in principle to use the rate method for all substances used in biochemical tests, there are problems with the cost of reagents, ease of operation, processing speed, etc. Therefore, at present, the ratio of colorimetric analysis to the total number of tests is still extremely high. Therefore, if a colorimetric analysis method (including nephelometric analysis method) for automatic equipment without deterioration of accuracy due to chromogen as described above could be developed, it would play an extremely important role.

The most basic method conventionally used to prevent such chromogen interference is to measure sample blanks for every sample and every test item.

Of course, the composition of the reagent for measuring the sample blank, etc.
There are many issues to consider, but basically, a sample blank is measured using a reagent that removes appropriate substances involved in the measurement reaction, and the difference between this and the reaction solution of the sample is used to determine the purpose. By calculating the substances, accurate analysis values close to the true values can be obtained without the influence of interfering substances as mentioned above. However, when such a sample blank correction method is used in an automatic analyzer, each sample needs to be measured twice.
It inevitably reduces the sample processing speed of the device by half, and it also has disadvantages such as increasing the number of types of reagents required, so it is currently not being applied to anything other than testing a few special test items. . In addition, air bubbles and solid matter are another factor that reduces the accuracy of analytical values in colorimetric analysis. This is fu. This occurs both in colorimetric analysis using a cell and in direct colorimetry using a test tube. There are more than 4 types of target substances that are measured by colorimetric analysis in biochemical tests, and conventionally, the single wavelength method or the two wavelength method has been used as the colorimetric analysis method. .

That is, the former measures the absorbance at a specific wavelength near the absorption center of a reactant or reaction product corresponding to the target substance, and the wavelength near the center and another appropriate wavelength are measured. The latter measures the difference in absorbance at However, both methods have the problem of being affected by various interfering substances as mentioned above. FIG. 1 schematically shows the relationship between each interfering substance and the absorption spectrum.

In the figure, 10' is the absorption spectrum of an ideal serum reaction solution that does not contain any interfering substances, and 12 is
Absorption spectrum of a serum reaction solution containing interfering substances such as chyle, yellowing, hemolysis, etc. 13 is the absorption spectrum when air bubbles or solid matter is mixed into the reaction solution shown by spectrum 10 . In FIG. 1, considering the one-wavelength colorimetric method at wavelength input 2, spectrum 12 is about twice that of spectrum 10, even though the concentrations of the target substances in these three samples are essentially the same. Spectrum 13 provides approximately four times as many measurements as spectrum 10. In the case of the two-wavelength colorimetric method, minute bubbles and solid objects generally only shift the spectrum in parallel in the vertical direction, and do not cause as large an error as in the one-wavelength colorimetric method.

In reality, the influence on the long and short wavelength sides differs depending on the position and size of bubbles and solids, so the error due to bubbles and solids is not as small as expected from spectrum 13, but it is It is significantly smaller than that of the colorimetric method. In the case of the two-wavelength colorimetric method, the above-mentioned chromogen interference is spectrum 1
As shown in Figure 2, it is complicated, with a positive error of 50% in the case of wavelength ^2-^3, and 30% in the case of wavelength input 21.
gives a negative error of

An object of the present invention is to provide a method for determining the degree of influence caused by the above-mentioned interfering substances. A feature of the present invention is that colorimetric analysis of a single sample is performed using both a one-wavelength colorimetric method and a two-wavelength colorimetric method, and the ratio of the analytical values of these two methods is determined.

According to a preferred embodiment of the present invention, the three major interference factors, namely, chyle, hemolysis, and yellow value, and interference due to air bubbles and solid matter can be corrected, and accurate measurement values can be obtained.

In a preferred embodiment of the present invention, in the colorimetric method,
Perform colorimetric analysis on a single sample using both the one-wavelength colorimetric method and the two-wavelength colorimetric method, and the ratio of the two analytical values obtained is 1.
When the ratio is within a predetermined range close to the above, that analytical value is adopted as the analysis result, and when the ratio of both analytical values is outside the predetermined range, both analytical values are corrected according to the amount of interfering substances in the sample. However, when the ratio of the two corrected analysis values obtained is within a predetermined range close to 1, that corrected analysis value is adopted as the analysis result, and when the ratio of the two corrected analysis values is outside the predetermined range. adopts the corrected analysis value of the two-wavelength colorimetric method as the analysis result, and/or re-examines.

As is clear from FIG. 1, the present invention provides true values for measurements made by the one-wavelength method and the two-wavelength method only for ideal specimens that do not contain interfering substances, bubbles, or solid matter. This method takes advantage of the fact that when the values match, but conversely, the sample contains interfering substances, bubbles, or solids, the two measured values do not match.

Hereinafter, a method according to an embodiment of the present invention will be described in detail using an example in which six items A to F are measured simultaneously.

First, a colorimetric analysis is performed by reacting in the same manner as a normal multi-item automatic analyzer, but measurements are performed using both the one-wavelength colorimetric method and the two-wavelength colorimetric method, and the ratio is calculated. Now, the analytical values obtained by the single wavelength colorimetric method for items A to F are 2,
~f, and the analytical value obtained by the two-wavelength analysis method is a2
Based on ~, the ratio K^~KF of both is expressed by the following formula. K^=Ki--・--...Skin. ．．・．．・KB = Misaki Hadashu KC = Gohada ---.・・・．・・・．・KD=Poison...
……………(41 KB=Sa・‐・‐‐‐・・‐‐‐‐‐‐.・‐・‐‐'
KF: Number.........'6' The ratio K^~KF obtained by each of the above formulas is within a predetermined range close to 1, for example 0.95.
~1.09, the average of one or both of the analysis values is adopted as the analysis result.

In addition, at the time of the above-mentioned analysis, from the spectrum of the analysis item whose spectrum is easiest to analyze, for example, the chylity level x, hemolysis level Y, and yellow apex level Z, which are interfering substances in the serum sample, are determined. FIG. 2 shows the absorption spectrum of GOT under ultraviolet light.

In the figure, 14 shows the spectrum of the ideal normal serum reaction solution containing no interfering chromogen mentioned above, using a water target, and 16 shows the spectrum of the high chyle serum reaction solution using a reagent blank target. Figure 18 shows the spectrum of the reaction solution of high yellowing serum, also using a reagent blank. Also, in the figure, the wavelength ^, . is 34 enemies,
Enter, 2 is 37 touches, ^, 3 is 41 rule m, entry, 4 is 45 blood, ^, 5 is 48 blood, entry, 6 is 505n, ^, 7 is 54 verses, ^18 is 57 Enemy skin, entering, 9 is 60 touches, entering 2
o is 60 enemies, ^2 is 70 skins, and entering 22 is 85 bloods. As is clear from the figure, the amount of the interfering chromogen can be determined by analyzing the visible wavelength spectrum of the reaction solution for GOT measurement. FIG. 3 shows an absorption spectrum obtained when analyzed using a multi-wavelength automatic analyzer.

In the figure, 20 is the spectrum of the floating standard solution diluted with the GOT measurement solution, 22 is the spectrum of the hemolytic standard solution also diluted with the GOT measurement solution, and 24 is the spectrum of the yellow top standard solution also diluted with the GOT measurement solution. It is. The floating standard solution is made by diluting and emulsifying fine polystyrene powder equivalent to 20 Kunkel units with GOT reagent, and the hemolytic standard solution is 1.
The hemoglobin standard solution of 9/00 was diluted and dissolved in GOT measurement solution under the same conditions as the sample serum. It was diluted and dissolved with
The spectra 20, 22, and 24 are all spectra for reagent blanks. Moreover, each wavelength is the same as in FIG. As is clear from Figure 3, among the specimen blank absorptions that appear in the visible wavelength range in the GOT reagent, the long wavelength range after ^sha is due to the chyle, and the intermediate wavelength range between 1, 7 and 9 is due to the chyle. This is due to hemolysis, and it can be seen that the short wavelengths before 6, 6 are due to three components: nipple, hemolysis, and yellow stain.

Therefore, it is possible to differentially measure these three components by the method described below. That is, the absorption spectrum of the sample reaction solution for GOT measurement is measured over all wavelengths using a multi-wavelength photometer, and the GOT of the target substance is measured based on the ultraviolet absorption. Determine the degree of chylosis, hemolysis, and yellow pox in the sample serum.

First, select two appropriate wavelengths after ^■ (for example, ^2o and input 2,
), calculate the chyle x using the following formula.

X=person; nose………………” [71 Here, no−a is the difference in absorbance between wavelength ^2o and illusion in the specimen, and T2o− is the unit turbidity determined in advance from spectrum 20. It is a constant representing the unit absorbance difference per unit.

Next, select two appropriate wavelengths of the medium wavelength castle (for example, ^, 8 and enter, 9
), the degree of hemolysis Y is determined using the following formula from the absorbance differences A, 8-, and 9.

Y=A, 8-1, 9-x. T, 8-・9…”””…’8
}day 18-19 where T,8-,9 is spectrum 2
The constants, 8-, 9, which were previously determined from 0 and which indicate the absorbance difference per unit turbidity, are constants which were also previously determined from the spectrum 22 and which indicate the absorbance difference per unit hemolytic degree.

Next, from the absorbance difference A, 5-, 6 at two appropriate wavelengths in the shorter wavelength range (for example, I, 5 and I, 6), the yellow value Z is determined using the following formula.

Z=A, 5-, 6-x. T,5-,6-Y. day, 5-,
6...[9}B,5-16 Here, T,5-,6 is a constant indicating the absorbance difference per unit turbidity determined in advance from spectrum 20, and T,5-,6 is also calculated from spectrum 22. A constant indicating the difference in absorbance per unit degree of hemolysis determined in advance, B
, 5-, and 6 are constants indicating the absorbance difference per unit degree of yellow pox, which was similarly determined in advance from the spectrum 24.

Table 1 shows the results of measuring the high chyle serum and high bilirubin serum shown in FIG. 2 using formulas '7' to [9].

Table 1 Next, the values of the ratios KA to KF that are outside the above ranges are corrected using the values of the chylity degree x, hemolysis degree Y, and yellow pox degree Z.

For example, if the ratio Kc is outside the above range, the corrected analysis values C,', C2' are determined by the following OQ and OU equations, respectively. C,'=C,1Q×-8Y-yZ...
……00C2′〒C2-Q′×-8′Y-y′Z……
Here, the six constants Q, Q', 8, 8', y, and y' are constants determined in advance depending on the reagent and apparatus used.

Next, the corrected analysis value ratio Kc' is determined using the corrected analysis values C,' and C2'. KC'=zero...--
・--.・・・． ...If this Kc' falls within a predetermined range close to 1, for example, within 0.95 to 1.05, the measured value of analysis item C of this test serum is C,', C2',
adopts the average of both.

Even if the above-mentioned side is corrected by the 0-bo formula, the formula, 02
Items where the value of the ratio Kc' of the corrected analysis value obtained by , report the measured values a2~ etc. of the two-wavelength colorimetric method, which are considered to be highly reliable, but at the same time request re-examination.

By measuring the milkiness, hemolysis degree, and yellowing degree of each specimen as described above, and performing data processing,
The accuracy of colorimetric analysis is significantly improved, and solid matter such as air bubbles and dust can also be distinguished, so the reliability of the measurement results is significantly increased.

In addition, in the above example, the ratio of analysis values is 0.95 to
Although it is possible as long as it is within the range of 1.05, this range is not limited to the above range. Further, the method for obtaining the corrected analysis values is not limited to the above embodiment, and, for example, simultaneous equations may be solved from the measured values at four wavelengths. In addition, although the present invention is applied to a colorimetric analysis method for serum in the above example, it is clear that the scope of application of the present invention is not limited to this and can be applied to general colorimetric analysis methods. .

In the above-mentioned example, in the colorimetric analysis method, a single sample is subjected to colorimetric analysis using both the one-wavelength colorimetric method and the two-wavelength colorimetric method, and the ratio of the two calculated analytical values is close to 1 at a predetermined value. When it is within the range, that analysis value is adopted as the analysis result, and when the ratio of both analysis values is outside the predetermined range, both analysis values are corrected according to the amount of interfering substances in the sample and calculated. When the ratio of both corrected analysis values is within a predetermined range close to 1, that corrected analysis value is adopted as the analysis result, and when the ratio of both corrected analysis values is outside the predetermined range, 2. Since the corrected analysis value of the wavelength colorimetric method is adopted as the analysis result and/or re-examined, the accuracy of the colorimetric analysis is significantly improved and the reliability of the measurement results is increased. Has excellent effects.

According to the present invention, the influence of interfering substances can be easily known by determining the ratio of the measured values of the one-wavelength method and the two-wavelength method, and the influence can be corrected as necessary.

[Brief explanation of the drawing]

Figure 1 is a diagram schematically showing the error factors in the colorimetric analysis method, Figure 2 is a diagram showing an example of the absorption spectrum of a typical sample serum in the GOT reaction solution, and Figure 3 is a diagram showing an example of the absorption spectrum of a typical sample serum in the GOT reaction solution. , same GOT
FIG. 2 is a diagram showing reference spectra of floating, hemolysis, and jaundice in a reaction solution. Multi figure Hair 2 figure Kaya 3 figure

Claims (1)

[Scope of Claims] 1. A colorimetric method characterized in that a colorimetric analysis of a single sample is performed using both a one-wavelength colorimetric method and a two-wavelength colorimetric method, and the ratio of the analytical values of these two methods is determined. Analysis method. 2 Perform colorimetric analysis on a single sample using both the one-wavelength colorimetric method and the two-wavelength colorimetric method, and if the ratio of the two analytical values obtained is within a predetermined range close to 1, the analytical value is When the ratio of the two analysis values is outside the predetermined range, the two analysis values are corrected according to the amount of interfering substances in the sample, and the ratio of the two corrected analysis values is 1. When it is within a predetermined range close to , the corrected analysis value is adopted as the analysis result, and when the ratio of both corrected analysis values is outside the predetermined range, the corrected analysis value of the two-wavelength colorimetry method is used as the analysis result. A colorimetric analysis method characterized in that the colorimetric analysis method is adopted and/or re-examined.