JPH0666808A - Chromogen measurement method - Google Patents

Abstract

PURPOSE:To compute chyle, hemoglobin and bilirubin by measuring absorbance of a specimen blank liquid by means of four kinds of wavelengths, and since one of these wavelengths is absorbed only by the chyle, adopting the absorbance of the chyle to an exponential function of the wavelength to find a regression function in relation to the chyle, while solving equations of absorbance in the measurement wavelengths in relation to the hemoglobin and bilirubin. CONSTITUTION:A fixed quantity of reagent for blank reaction is mixed with a specimen to prepare a specimen blank liquid and absorbance, when it is measured with measurement wavelengths lambda1-lambda4 is denoted by A(lambda1)-A(lambda4), thus establishing equations. The wavelength lambda4 is adapted to the measurement wavelength for the specimen having only the absorbance because of the chyle. Ah(lambda), Ab(lambda), and At(lambda) respectively represent the absorbance of the hemoglobin, bilirubin and chyle at the wavelength lambda. The absorbance by the chyle is expressed by At(lambda)=alambda<b>, wherein (a), (b) are constants based on the number of particles and a diameter of the particle respectively. In relation to the hemoglobin and bilirubin, absorbance conversion between wavelengths from absorbance at one wavelength to absorbance at another wavelengtn can be performed by multiplucation of a predetermined coefficient. By multiplying the absorbance by concentration conversion coefficient and dilution magnification, the hemoglobin and bilirubin can be quantitatively determined.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying chyle, hemoglobin and bilirubin in a multi-item biochemical analysis of a sample containing chromogens such as chyle (turbidity), hemoglobin (hemolysis) and bilirubin (jaundice). It is a thing.

[0002]

2. Description of the Related Art As a method for quantifying three types of chromogens of chyle, hemoglobin and bilirubin contained in a sample to be analyzed and measured in a clinical test from the same sample at the same time,
There is an influence of chyle in the visible light wavelength range, but the influence of hemoglobin and bilirubin is not substantially measured, and the degree of chyle is obtained by measuring the absorbance at an appropriate wavelength in a long wavelength range, The degree of hemoglobin is determined based on the absorbance value at a proper wavelength in the medium wavelength range where the effect of bilirubin is not present and the measured value in the long wavelength range,
It has been proposed to determine the degree of bilirubin based on the value of absorbance at an appropriate wavelength in the short wavelength region where the influence of hemoglobin is present in the visible light region and the measured value in the intermediate wavelength region (Japanese Patent Publication No. 61- (See Japanese Patent Publication No. 19933).

[0003]

In the above cited example, in order to obtain the milkiness, the absorbance is calculated at two wavelengths in the long wavelength range where the effects of hemoglobin and bilirubin are not substantially obtained, and the difference in absorbance is calculated. Hemoglobin is obtained by multiplying the chyle degree in the mid-wavelength range by multiplying a constant constant that represents the difference in absorbance per unit turbidity obtained with the reference solution, and subtracting the chyle degree from the difference in the two-wavelength absorbance in the mid-wavelength range. The degree is calculated. However, with respect to chyle, the constant representing the absorbance conversion between wavelengths is a value related to the size of particles in the sample and varies depending on the sample. Therefore, if the constant is used, it is said that it is constant even if the sample changes, even though it has to be calculated for each sample.Therefore, there is an error in the values of hemoglobin and bilirubin calculated using the constant. Is included. Also, in the method of the cited example, since it is necessary to measure two wavelengths for each of the three types of chromogens,
A total of 6 wavelengths must be measured.

Therefore, the first object of the present invention is to consider that the absorbance of the chyle is a function of the particle size,
Correct calculation of hemoglobin and bilirubin. A second object of the present invention is to reduce the error as the number of measurement wavelengths increases, but to shorten the measurement time by simplifying the measurement device by reducing the number of measurement wavelengths required for chromogen calculation. .

[0005]

According to the present invention, a sample blank solution is prepared by mixing a blank reaction reagent with a sample in which suspended substances are mixed, and the absorbance of this sample blank solution is determined by absorption of chyle. Absorbance of bilirubin and the absorbance is measured by at least four types of wavelengths including wavelengths that do not substantially exist, and for chyle, the absorbance is calculated by assuming that the absorbance is represented by an exponential function of wavelength, and a regression function of the absorbance is obtained, For hemoglobin and bilirubin, assuming that there is a predetermined relationship between the absorbances at different wavelengths, make a simultaneous linear equation for the absorbances at the measurement wavelengths and solve it to determine the extent of chyle, hemoglobin and bilirubin. To calculate. A blank reaction reagent is one obtained by removing reaction components from various known reaction reagents used for measurement of various analytical items and having no absorption in the above measurement wavelength range, for example, used as a solvent for reaction reagents. Buffer solution, physiological saline,
For example, water.

[0006]

[Function] Absorbance of a sample blank solution prepared by mixing a certain amount of sample with a certain amount of blank reaction reagent, and measuring this sample blank solution at measurement wavelengths λ ₁ , λ ₂ , λ ₃ , and λ _4. Are A (λ ₁ ), A (λ ₂ ), A (λ ₃ ), A
(λ ₄ ), the following equation holds. A (λ ₁ ) = Ah (λ ₁ ) + Ab (λ ₁ ) + At (λ ₁ ) A (λ ₂ ) = Ah (λ ₂ ) + Ab (λ ₂ ) + At (λ ₂ ) A (λ ₃ ) = Ah ( λ ₃ ) + Ab (λ ₃ ) + At (λ ₃ ) A (λ ₄ ) = At (λ ₄ ), where Ah (λ), Ab (λ), At (λ) are hemoglobin, bilirubin at wavelength λ, and It is the absorbance due to chyle, and the wavelength λ ₄ is the measurement wavelength at which the sample has only the absorbance due to chyle. The absorption spectrum of each chromogen is shown in FIG. H represents hemoglobin, B represents bilirubin, and t represents chyle. Measurement wavelength λ ₁ , λ ₂ ,
λ ₃ and λ ₄ are, for example, 340, 410, 450, 660 nm
And

The absorbance due to chyle can be expressed as At (λ) = aλ ^b (see JP-A-62-179639). Here, a is a constant resulting from the number of particles, b is a constant resulting from the average particle diameter, and both a and b differ depending on the sample. On the other hand, regarding hemoglobin and bilirubin, it has been experimentally confirmed that the shape of the absorption spectrum does not change even if the sample is different, so by multiplying the absorbance of one wavelength to the absorbance of another wavelength by a previously determined coefficient. It is possible to convert absorbance between wavelengths.

Using the above relationships, the above equation can be modified according to the absorbance at 340 nm as follows. A (340) = Ah (340) + Ab (340) + At (340) A (410) = K ₁ · Ah (340) + K ₂ · Ab (340) + At (410) A (450) = K ₃ · Ah (340) + K ₄ · Ab (340) + At (450) A (660) = At (660) where K ₁ is the measurement wavelength of the absorbance of hemoglobin 340
The conversion coefficient for converting the absorbance at nm to the absorbance at the measurement wavelength of 410 nm, K ₃ is the conversion coefficient for converting the absorbance of hemoglobin to the absorbance at the measurement wavelength of 450 nm from the absorbance at the measurement wavelength of 340 nm, and K ₂ is the absorbance of bilirubin. From absorbance at measurement wavelength 340 nm to measurement wavelength 410 nm
A conversion coefficient for converting the absorbance at ₄ to K ₄ is the absorbance of bilirubin from the absorbance at the measurement wavelength of 340 nm to the measurement wavelength of 45.
It is a conversion coefficient for converting the absorbance at 0 nm.

The above equations can be summarized as follows using the relationship of At (λ) = aλ ^b . {(K ₂・ K ₃ -K ₁・ K ₄ ) A (340)-(K ₃ -K ₄ ) A (410) + (K ₁ -K ₂ ) A (450)} / A (660) = { (K ₂・ K ₃ -K ₁・ K ₄ ) ・ 340 ^b − (K ₃ -K ₄ ) ・ 410 ^b + (K ₁ -K ₂ ) ・ 450 ^b } / 660 ^b

In this equation, the left side is a constant value, b can be obtained by approximate calculation, and a can be obtained by a = A (660) / 660 ^b . Further, Ah (410) = A ( 410) -K 5 · Ab (450) -a410 b Ab (450) = A (450) -K 6 · Ah (410) becomes -a450 ^b. Here, K ₅ is a conversion coefficient for converting the absorbance of bilirubin from the absorbance at the measurement wavelength of 410 nm to the absorbance at the measurement wavelength of 450 nm, and K ₆ is the absorbance of hemoglobin from the absorbance at the measurement wavelength of 450 nm to the measurement wavelength of 410 nm.
It is a conversion coefficient for converting the absorbance at.

As a result, Ah (410) = {A (410) -K ₅ .A (450) -a.410 ^b + K ₅ .a.450 ^b } / (1-K ₅ .K ₆ ) Ab ( 450) = {K ₆ · A (410) -A (450) -K ₆ · a · 410 ^b + a · 450 ^b } / (K ₅ · K ₆ -1), which is the maximum absorption wavelength of hemoglobin and bilirubin. The absorbance of each component alone at near wavelengths is determined. Hemoglobin and bilirubin can be quantified by multiplying the obtained absorbance by the concentration conversion coefficient and the dilution rate.

[0012]

[Examples] FIG. 2 shows an example of measurement of a real sample containing hemoglobin and chyle diluted 31 times with physiological saline.
Absorption was measured every 10 nm and the measurement points (black circles) were connected to each other to form the absorption spectrum shown as Sample in the figure. Of the measurement points, the measurement wavelengths 340, 410,
By applying the above calculation using the absorbances at 450 and 660 nm, t is shown as a spectrum of turbidity due to chyle, and H is the spectrum obtained by subtracting the expected absorbance of turbidity from the measured absorbance. Calculated a = 6.2
68 × 10 ⁸ , b = −2.693, and hemoglobin concentration was 52 mg / dl.

FIG. 3 shows an example in which an actual sample containing hemoglobin, bilirubin and chyle was diluted 31 times with physiological saline and measured. The measurement is the same as in FIG. 2, and absorption is measured every 10 nm and the measurement points (black circles) are connected to each other to form an absorption spectrum shown as Sample in the figure. Also in this case, among the measurement points, the measurement wavelengths 340, 410,
By applying the above calculation using the absorbances at 450 and 660 nm, t is shown as the spectrum of turbidity due to chyle, and the spectrum obtained by subtracting the expected absorbance of turbidity from the measured absorbance is H + B. Calculated a =
4.184 × 10 ⁷ , b = −2.089, hemoglobin concentration 11 mg / dl, bilirubin concentration 19 mg /
It became dl. Separately, in an analysis in which a reaction reagent is added to measure the absorbance, if the absorbance conversion coefficient between each wavelength is obtained for hemoglobin and bilirubin, for chyle, the relation between these chromogens at the measurement wavelength is determined from the relation of A = aλ ^b . Since the absorbance can be predicted, the true absorbance due to the reaction alone can be found by subtracting it from the measured absorbance taking into account the sample amount / reagent amount ratio.
It is possible to correct the quantitative value and the limit absorbance.

[0014]

In the present invention, regarding the chyle, the wavelength-absorbance regression function is obtained on the assumption that the absorbance is represented by the exponential function of the wavelength. Therefore, the measurement of the chyle depends on the particle size in the sample. Can be done without. Then, the degree of chyle is accurately obtained, and the calculated values of hemoglobin and bilirubin derived therefrom are also accurate. Since each chromogen can be accurately obtained, the absorbance correction for the analysis item can be accurately performed.

[Brief description of drawings]

FIG. 1 is a diagram showing the absorbance of each chromogen.

FIG. 2 is a diagram showing absorption spectra of a sample and chromogen in an example in which a sample containing hemoglobin and chyle was measured.

FIG. 3 is a diagram showing absorption spectra of a sample and chromogen in an example in which a sample containing hemoglobin, bilirubin, and chyle was measured.

Claims (1)

[Claims] 1. A sample blank solution is prepared by mixing a blank reaction reagent with a sample in which suspended substances are mixed, and the absorbance of this sample blank solution has absorption of chyle and absorption of hemoglobin and bilirubin is substantially At least 4 including no wavelength
Absorbance is measured by the wavelength of the seed, and for chyle, the wavelength-absorbance regression function is obtained assuming that the absorbance is represented by an exponential function of wavelength, and for hemoglobin and bilirubin, it is determined in advance between the absorbances at different wavelengths. A method for measuring chromogen in which simultaneous linear equations regarding the absorbance at the measurement wavelength are created, and are solved to calculate the degrees of chyle, hemoglobin and bilirubin on the assumption that there is a certain relationship.