JPS6118982B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Application of the Invention] The present invention is applicable to the detection of chyle, hemolysis,
The present invention relates to an analytical method suitable for optically measuring chromogen such as yellow sputum. [Background of the Invention] The recent trend toward automation in the field of biochemical testing is extremely remarkable. Automatic analyzers also
A wide variety of models have been developed, from the early flow type to the more recent 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. 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, chromogens such as hemolysis (hemoglobin), yellow sputum (bilirubin), and chyle (turbidity) are causing a loss of accuracy in the measurement of general test items by automatic biochemical analyzers. In biochemical testing, a common analytical method is to collect a biological sample in a reaction container, react it with a reagent, and optically measure the reaction solution. Among these, a device using a multi-wavelength photometer as a photometer that can disperse light transmitted through a reaction liquid into multiple wavelengths and detect them has the potential to expand the range of application. FIG. 1 schematically shows the relationship between each interfering substance and the absorption spectrum. In the figure, spectrum 10
is the absorption spectrum of the ideal serum reaction solution that does not contain any interfering substances as mentioned above, spectrum 1
2 is an absorption spectrum of an actual serum reaction solution containing interfering substances such as chyle, jaundice, and hemolysis. 1st
In the example shown in the figure, if one-wavelength colorimetry is performed using the wavelength λ ₂ , the spectrum 12 will be spectrum 10
gives an analytical value approximately twice that of On the other hand, although consideration has been given to the influence of chromogen on the measured values of general test items, the test results are based on the inspector's visual observation and indicate the amount of chromogen that is likely to be contained in a symbol (for example, 〓, 〓, +, -
It was displayed in 4 stages). Therefore, test results vary widely among examiners, and it has not been possible to quantitatively determine the amount of interfering chromogen. [Object of the Invention] An object of the present invention is to provide a biochemical analysis method that can measure each chromogen while causing a reaction between a sample and a reagent for the analysis item to be measured. It's about doing. [Summary of the Invention] In the present invention, a blood sample is reacted with a reagent for an analysis item to be measured to obtain a reaction liquid having an absorption in the ultraviolet region in relation to the concentration of the analysis item, and the reaction liquid is exposed to light. to determine the concentration of the above analysis item by irradiating the sample with ultraviolet light and measuring the absorption in the ultraviolet region, to measure chyle, hemolysis, and jaundice in the visible light wavelength range with respect to the reaction solution; The degree of hemolysis should be determined by measuring the long wavelength range in the visible light wavelength range where there is virtually no effect of hemolysis and jaundice. The above-mentioned degree of jaundice is calculated based on the measured value in the medium wavelength range, which is not suitable for the human body, and the measured value in the long wavelength range mentioned above. It is characterized in that it is determined based on the measured value in the wavelength range and the measured value in the medium wavelength range. [Embodiments of the Invention] When the present invention is applied to a clinical automatic analyzer, it is optimal to use a multi-wavelength photometer with one channel. This automatic analyzer includes a sampler that transfers sample containers containing blood samples, a reaction line that transfers a row of reaction containers, a dispenser that distributes the sample from the sample containers to the reaction containers, and a specific analysis for reacting with the sample. A well-known automatic clinical analyzer can be used, which is equipped with a device for adding a reagent for each item into a reaction container, a multi-wavelength photometer that irradiates the reaction solution with light, and measures the absorption characteristics of the reaction solution. In the embodiments of the present invention described below, such an analysis device is used. Before explaining the examples, the concept of chromogen measurement will be explained. The absorption spectra of interfering chromogens such as chyle, hemolysis, and jaundice vary depending on the fluid, and in many cases they overlap, making analysis of their spectra difficult. In particular, when these absorptions are further overlapped with the absorption of general test item substances, the analysis becomes even more difficult, and even if analysis is possible, the precision is significantly reduced. Therefore, even when measuring many items on the same sample at the same time, such as with a multi-item automatic analyzer, rather than analyzing the spectrum for each measurement item, it is better to analyze the analysis item that is easiest to analyze, rather than analyzing the spectrum for each measurement item. In addition to analyzing the spectrum and measuring the analytical items, the amounts of three types of interfering substances in the same reaction solution are also measured. An example of an analysis item that produces a reaction solution in which the interfering chromogen is most easily analyzed is glutamate oxaloacetate transaminase (hereinafter GOT).
), glutamate pyruvate transaminase (hereinafter referred to as GPT), lactate dehydrogenase (hereinafter referred to as LDH), and hydroxybutyrate dehydrogenase (hereinafter referred to as HBDH), which target substances by ultraviolet absorption. The analysis items to be measured are listed. When adding specific reagents to a sample (serum) to obtain a reaction solution and measuring these analytical items,
The absorption of the target substance, reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH), is in the ultraviolet region (340 nm) and not in the visible region.
Among the above-mentioned interfering substances, the absorption spectra of jaundice and hemolysis do not overlap in the visible wavelength range. Moreover, the reagent does not contain substances that react with hemoglobin, yellow sputum, or lipoproteins (one of the causes of cloudiness), and the liquid is a neutral buffer solution (PH
7.4), the spectrum of the above-mentioned interfering substance has a relatively simple shape and is easy to analyze. FIG. 2 shows the absorption spectrum including ultraviolet light of the reaction solution used to measure GOT. In the figure, 14 shows the spectrum of the ideal normal serum reaction solution that does not contain any chromogen as described above, with water as a target;
Reference numeral 16 shows the spectrum of the reaction solution of high chyle serum using a reagent blank target, and 18 shows the spectrum of the reaction solution of high yellow protein serum using the same reagent blank target. Here, the water target spectrum 14 measures the absorption spectrum of a serum reaction solution containing no interfering chromogen contained in a sample cell and the absorption spectrum of water contained in a control cell,
The difference in absorbance between the two at each wavelength point is displayed. In addition, reagent blank target spectrum 16
Measures the absorption spectrum of the high chyle serum reaction solution stored in the sample cell and the absorption spectrum of the measurement reagent (not containing the serum sample) stored in the control cell, and calculates the difference in absorbance between the two at each wavelength. This is what is displayed. Reagent blank target spectrum 18
The method measures the absorption spectrum of the highly icteric serum reaction solution stored in the sample cell and the absorption spectrum of the measurement reagent (not including the blood sample) stored in the control cell, and calculates the difference in absorbance between the two at each wavelength. This is what is displayed. Also, in Figure 2, the wavelength λ ₁₁ is 340n
m, λ ₁₂ is 376nm, λ ₁₃ is 415nm, λ ₁₄ is 450n
m, λ ₁₅ is 480nm, λ ₁₆ is 505nm, λ ₁₇ is 546n
m, λ ₁₈ is 570nm, λ ₁₉ is 600nm, λ ₂₀ is 660n
m, λ ₂₁ is 700 nm, and λ ₂₂ is 850 nm. By analyzing the visible wavelength spectrum of the reaction solution during GOT measurement as shown in the figure, the amount of the interfering chromogen can be determined. Hereinafter, an embodiment of the present invention will be described in detail in the case where a multi-wavelength automatic analyzer equipped with 12 wavelengths is used as the measurement wavelength. FIG. 3 shows an example of an absorption spectrum obtained when analyzed using such an automatic analyzer. In the figure, 20 is the spectrum of the chyle standard solution diluted with the GOT measurement solution, and 22 is
24 is the spectrum of the hemolytic standard solution also diluted with the GOT measurement solution, and 24 is the spectrum of the jaundice standard solution also diluted with the GOT measurement solution. The chyle reference number is obtained by diluting and emulsifying fine polystyrene powder equivalent to 20 Kunkel units with GOT reagent.The hemolysis reference solution is a 1000 mg/dl hemoglobin reference solution, and the GOT measurement solution is prepared under the same conditions as the sample serum. The diluted and dissolved product, yellow sputum standard solution, is obtained by diluting and dissolving 10 mg/dl bilirubin control serum with GOT measurement solution under the same conditions as the sample serum. Said spectrum 2
0, 22, and 24 are all spectra for reagent blanks. In this case, each reagent target spectrum is obtained by measuring the absorption spectrum of each chromogen standard solution stored in each sample cell and the absorption spectrum of the measurement reagent (not including serum sample) stored in a control cell, and The difference in absorbance between each standard solution and the reagent blank solution is displayed. Further, each wavelength is a visible light wavelength similar to that in FIG. As shown in Figure 3, of the specimen blank absorption that appears in the visible wavelength range with the GOT reagent, the long wavelength range after λ ₂₀ is due only to chyle, and the intermediate wavelength range from λ ₁₇ to _{λ 19} is due to chyle and hemolysis. and λ ₁₆
The former short wavelength range is due to the three components of chyle, hemolysis, and yellow sputum. 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 the entire wavelength range using a multi-wavelength photometer, and the GOT of the target substance is measured based on the ultraviolet absorption. Depending on the method, determine the degree of chylity, degree of hemolysis, and degree of yellowness in the sample serum. First, select two suitable wavelengths after λ ₂₀ (for example, λ ₂₀ and λ
₂₁ ) Calculate the chyle X using the following formula from the absorbance difference. X=A _20-21 /T _20-21 ...(1) Here, A _20-21 is the wavelength λ ₂₀ and λ
The absorbance difference T _20-21 of ₂₁ is a constant representing the absorbance difference per unit turbidity, which was determined in advance from spectrum 20. Next, the degree of hemolysis Y is determined from the absorbance difference A _18-19 at two appropriate wavelengths in the medium wavelength range (for example, λ ₁₈ and λ ₁₉ ) using the following equation. Y=A _18-19 -X・T _18-19 /H _18-19
...(2) Here, T _18-19 is a constant indicating the absorbance difference per unit turbidity, which was determined in advance from spectrum 20,
H _18-19 is a constant indicating the absorbance difference per unit degree of hemolysis, which was also determined in advance from the spectrum 22. Next, select two appropriate wavelengths in the shorter wavelength range (for example,
Based on the absorbance difference A _15-16 between λ ₁₅ and λ ₁₆ ), yellowness Z is determined using the following formula. Z=A _15-16 -X・T _15-16 -Y・H _15-1
6 /B _15-16 ... (3) Here, T _15-16 is a constant indicating the absorbance difference per unit turbidity determined in advance from spectrum 20,
H _15-16 is a constant indicating the absorbance difference per unit degree of hemolysis, which was also determined in advance from spectrum 22, and B _15-16
is a constant indicating the absorbance difference per unit yellowness, which was also determined in advance from the spectrum 24. Constants used in each of the above formulas, T _20-21 , T _18-19 ,
T _15-16 , H _18-19 , H _15-16 , and B _15-16 are constant if the automatic analyzer and reagents are the same, so once they are measured, they do not necessarily need to be determined every time.

〔Effect of the invention〕

According to the present invention, it is possible to measure the analytical item by causing a reaction between the sample and the reagent for the analytical item to be measured, and also to measure three types of chromogens: chyle, hemolysis, and yellow sputum. can be done, and
Since the reaction solution for the analysis item to be measured can be used as is for chromogen measurement, there is an advantage that there is no need to provide a special container or analysis channel for chromogen measurement in the biochemical analyzer.

[Brief explanation of the drawing]

Figure 1 is a diagram schematically showing error factors in the colorimetric analysis method, Figure 2 is a diagram showing an example of the absorption spectrum of a typical sample serum as a GOT reaction solution, and Figure 3 is a diagram showing an example of the absorption spectrum of a typical sample serum as a GOT reaction solution. It is a diagram showing each reference spectrum of chyle, hemolysis, and yellow sputum in the same GOT reaction solution.

Claims (1)

[Claims] 1 Reacting a blood sample with a reagent for the analysis item to be measured to obtain a reaction solution that has an absorption in the ultraviolet region in relation to the concentration of the analysis item, and irradiating the reaction solution with light to detect absorption in the ultraviolet region. The concentration of the above analysis item is determined by measuring the concentration of the above analysis item.The above reaction solution is measured for chyle, hemolysis and yellow sputum in the visible light wavelength range.The degree of the above chyle is determined by The degree of hemolysis mentioned above should be determined by measuring the long wavelength range, which is substantially free from the effects of hemolysis and jaundice. It is determined based on the measured value and the measured value in the long wavelength range above, and the degree of jaundice is calculated based on the measured value in the short wavelength range of the visible light wavelength range, the measured value in the long wavelength range above, and the above. A biochemical analysis method characterized by calculation based on measured values in the medium wavelength range.