JPS62179639A - Multi-item biochemical analysis - Google Patents

Abstract

PURPOSE:To remove errors due to turbid components without use of a number of blank channels or a reference liquid for turbidity, by a method wherein a sample containing suspension material is divided by every item, is mixed with a reaction liquid to measure absorbance and correction of a blank absorbance determined by a specified method is applied. CONSTITUTION:A sample containing suspension material is divided by every item and mixed with a reaction agent for item-wise measurement to make a measuring liquid. Absorbance with a specified wavelength of each measuring liquid is measured. On the other hand, the sample is mixed with a blank reaction reagent excluding reaction components from a reaction reagent to prepare a sample blank liquid. The absorbance of the blank liquid is measured at least about two kinds of wavelengths to obtain a wavelength-absorbance regression function. A blank absorbance at the measuring wavelength is calculated about respective items from the regression function to correct absorbance of each measuring liquid.

Description

[Detailed description of the invention] (b) Industrial application field The present invention relates to a multi-item biochemical analysis method.

More specifically, the present invention relates to a multi-item biochemical analysis method useful for measuring samples containing suspended substances such as chyle serum.

(0) Conventional technology Conventionally, multi-item biochemical analysis of biochemical samples such as serum, plasma, urine, etc. has been carried out using colorimetric analysis or nephelometric analysis. Concentration calculation coefficients obtained by mixing the divided specimens with specified reaction reagents to obtain measurement solutions, measuring the absorbance of each of these measurement solutions at specified wavelengths using the endpoint method, and colorimetrically or turbidimetrically measuring each standard sample. Each item is quantified by multiplying by The analysis items at this time include, for example, TP, ALB,
T-CHO, PL, TG, T-Bi L, D-Bi L
, GLV, Ca, iP, etc., and the absorbance measurement wavelength is usually in the range of 340 to 750 nm.

(c) Problems to be Solved by the Invention However, some specimens include so-called chyle serum, which is cloudy due to a significantly high content of chylomicrons and botanicals. When measuring a certain item using the endpoint method,
In addition to the absorbance changing due to the reaction, such chyle serum has the disadvantage that turbidity causes light scattering, increasing the apparent absorbance and positively affecting the analytical results.

In order to correct this, we prepared a sample blank solution for each item by mixing the sample and a blank reaction reagent (reaction components removed from each reaction reagent), and under the same measurement conditions as the above measurement solution. There is a method of measuring the absorbance and subtracting the absorbance of these sample blank solutions for each item from the absorbance of the above-mentioned measurement solution (sample blank method), or using the blank solution of the actual sample.
The absorbance difference obtained by measuring at the two specified wavelengths was converted into the absorbance per unit turbidity by measuring the turbidity reference solution (polystyrene powder suspension) at the above two wavelengths in advance. This value is multiplied by the conversion p constant to another wavelength (absorbance measurement wavelength of the reaction solution) based on the l single wave, and this is taken as the turbidity of the sample itself at that wavelength, and the reaction is calculated. Correction method by subtracting from the absorbance of the liquid (Machimon Sho 54-6
3785), etc. are known.

However, the former method has a problem in that it requires a large number of tip channels for the blank liquid H side in an automatic analyzer. On the other hand, with Gosho's method, it is not easy to obtain a standard solution for turbidity, and the spectrum differs depending on the particle size of the turbidity component, so a single standard solution is used as a standard that summarizes the turbidity of samples such as chyle serum. It is impossible to make it into a liquid. That is, the particle size of chylomicron, which is the main turbidity component of chyle serum, is about 0.5 pyp, the particle size of lipoprotein β-lipoprotein is about 0.08 pyp, and the particle size of β-lipoprotein is about 0.035 pyp.
α-lipoprotein in the skin is about 0.015. The particle size of each sample is different, and the shape of the spectrum differs depending on the origin of the turbidity of the sample. Therefore, the correction method using the standard solution using polyethylene powder as a single reference material is not accurate. It was difficult to make appropriate corrections.

This invention was made to solve these problems, and in particular, it does not require a large number of blank channels for measuring each sample at different wavelengths, and it also eliminates the need for the turbidity reference solution mentioned above. The purpose of the present invention is to provide a multi-item biochemical analysis method that can eliminate errors caused by turbidity components as accurately as possible without using a turbidity component.

(d) Means for Solving the Problems Thus, according to the present invention, when performing multi-item biochemical analysis of a sample containing suspended solids, a sample for each item measurement can be assigned to the sample divided into each item. The reaction reagents are mixed to form a measurement solution, the absorbance of each of these measurement solutions at a predetermined wavelength is measured, and multiple items are determined based on each absorbance.Furthermore, a blank reaction reagent is mixed with the sample. Prepare one specimen blank solution, measure the absorbance of this blank solution at t1 using at least two different wavelengths, obtain a wavelength-absorbance regression function, and use this regression function to determine the measurement wavelength for each of the above items. A multi-item biochemical analysis method is provided, which is characterized in that the blank absorbance of each measurement solution is corrected by calculating the blank absorbance of each of the blank absorbances.

The most distinctive feature of this invention is that a single sample blank solution is used for one sample when performing multi-item analysis on a sample containing suspended solids such as chyle serum, and For such a single sample blank solution,
The absorbance at two or more wavelengths is measured to obtain a wavelength-absorbance regression function for the sample blank solution, and based on this, the absorbance at the wavelength measured for each item is sieved, and this is calculated as a difference from the absorbance of the reaction solution. The advantages are that chyle can be corrected by subtraction and that no turbidity standard solution is required.

As the reaction reagent used in the method of the present invention, various reaction reagents known in the art that correspond to the intended analysis item are used. These reaction reagents include, for example, antiserum reagents for performing nephelometric analysis by causing an antigen-antibody reaction with antibodies in a specimen.

The blank reaction reagent used in this invention may correspond to any reaction reagent as long as the reaction reagent from which the reaction component is removed has no absorption.
For example, LliWli solution, physiological saline, water, etc., which serve as a solvent for the reaction reagent, can be used.

One specimen blank solution prepared by mixing such a blank reaction reagent with a specimen may be prepared for each specimen.

The absorbance measurement of the sample blank solution is performed using at least two different wavelengths. It is usually appropriate to increase the measurement wavelength to obtain a more accurate regression function. Usually, each item is measured within the range of 340 to 750 nm, so in order to obtain a wavelength-absorbance regression function in this range, it is recommended to appropriately disperse and select 2 to 4 wavelengths within this range. preferable. However, if the intended measurement wavelength range for each item is narrow, the regression function may be determined based on at least two types of wavelengths near both ends.

The method for determining the regression function will be explained below.

First, with the measurement wavelength λ (n■) on the χ axis, the absorbance A (AbS)
If the y-axis is taken as the y-axis, the turbidity spectrum will be as shown in FIG. 1, for example. At the same time, 0 different wavelengths λ1, A2, and
...The absorbance when measured with An is AI and A, respectively.
2...If it is 80, the relationship between wavelength and absorbance is A
It can be approximated to the formula =aλb (a and b are constants). Here, the constant a is determined by the degree of turbidity, and b is determined by the average particle size of the turbidity component. Therefore, the combination of wavelength and absorption (λ, A) = (λs, A
t>, (A2, A2)...(An, An
) by power regression (least squares method in this case), the constant a
, b can be determined, and based on this, the absorbance of the blank liquid at any wavelength can be estimated.

In addition, as mentioned above, the turbid components in chyle serum can be roughly divided into two types: chylomicron and botanical, so in order to perform a more rigorous regression, the relationship between wavelength and absorbance should be expressed as A = at
It is expressed as λbl+a2λto2, where al, bl are constants derived from chylomicrons, a2, b2 are constants derived from lipoproteins, and at, a7,
It is suitable to calculate b, l12 and use it as a regression function of wavelength and absorbance. However, in practical analysis, it has been found that the intended correction can be made sufficiently by power regression from the average particle size as described above.

Note that if the mixing ratios of the analytes in each measurement reaction solution and the sample blank solution are different, volume correction may be performed, and the analyte concentrations in these final reaction solutions do not necessarily have to match -C. Blank liquid ff1vB (〃), specimen fivB (11>) in it, measurement liquid it vA (xl), amount of specimen in it vA (1!), calculated absorbance using the above regression function at the wavelength λ of the specimen blank liquid. If AB is the absorbance of the measurement reaction solution at the wavelength λ, and A△ is the absorbance of the measurement reaction solution at the wavelength λ, then the absorbance A due to the substantial reaction at the wavelength λ of the measurement reaction solution is
c is Ac = AA - (VB ・VA/VA - VB
) -AB can be obtained.

The method of the present invention usually uses a multi-item automatic analyzer equipped with multiple analysis lines corresponding to multiple items, and further includes a dispensing means for adding a blank reaction reagent to the sample, and an absorbance of the sample blank solution. It is preferable that a blank line equipped with a multi-wavelength photometer that measures each wavelength at least at two different wavelengths be provided. Furthermore, based on the absorbance of two or more types measured on the blank line, a wavelength-absorbance regression function is calculated for each sample blank solution containing suspended solids, and the sample blank absorbance at any measurement item/wavelength is calculated. It is preferable to automate the calculation by using a program-controlled microprocessor to calculate the volume, perform the volume correction as necessary, and subtract it from the absorbance of the reaction solution. The configuration of such a multi-item biochemical analyzer is shown in FIG. In Fig. 2, [IH21... is a plurality of analysis lines, (3) is a single blank line, (4) (4)... is a measurement liquid, (
4A) is sample blank solution, f5) (5) (51...
・A sample dispenser! 6H7) is a reaction reagent, (8) is a blank reaction reagent, (9) (to) is a fixed wavelength optical measurement system for measuring each item, 01) is an optical measurement system capable of multi-wavelength photometry (11A '') 111 and 1121 are photoelectric detectors, and 131 is the above-mentioned correction processing unit, respectively.

(E) Effect According to the method of the present invention, a positive error in absorbance due to scattering of suspended matter in a sample present in a measurement solution is corrected.

(F) Example For three types of actual samples (Chyle serum > 504), prepare a blank solution by adding 2.5 xl of physiological saline as a blank reaction reagent to each sample, and measure the absorbance at 340 nm and 700 nm. The constants a and b in the relationship between wavelength and absorbance Δ=a Abl, were calculated by the least squares method.

Based on such a regression function, 400.
450, 500, 550, 600 and 650nm 1.
: The absorbance at On the other hand, actually 340-70
The spectrum obtained by scanning between 0 nm was as shown in FIG.

Table 1 shows the results of a comparison between the calculated absorbance at 400 to 650 nm based on the regression function and the absorbance at 400 to 650 nm based on the measured value (FIG. 3).

(Margins below, continued on next page) Table 1 (Unit: mAbs) In this way, the values obtained by the regression function and the actual absorbance are almost the same, and the absorbance of the blank liquid at different wavelengths is actually measured. It turns out that it is possible to estimate accurately without any errors. Therefore, it has been found that by using the above regression function, it is possible to easily correct turbidity for each item in a multi-item biochemical analysis.

(G) Effects of the Invention According to the method of the present invention, multi-item biochemical analysis using the end point method can be performed without using a special turbid standard solution as in the past, and without requiring a large number of blank channels. This can be done without causing errors due to turbidity. Therefore, it is an extremely useful method especially for biochemical automatic analysis methods and devices that handle Inuyama specimens.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram for determining the wavelength-absorbance regression function in the method of the present invention, FIG. 2 is a configuration explanatory diagram illustrating an apparatus for carrying out the method of the present invention, and FIG. 3 is a diagram for explaining the wavelength-absorbance regression function in the embodiment. It is a graph figure showing the relationship between and absorbance. (4)...Measurement liquid, (4A)...
Sample blank solution, (6) [7...Reaction reagent,
(8)...Blank reaction reagent, 01)...
...Optical 11 measuring system capable of multi-wavelength photometry, OJ...
・Correction calculation section. Figure 2 + 1A 3rd! 21 Appropriate length (nm)

Claims (1)

[Scope of Claims] 1. When performing multi-item biochemical analysis of a sample containing suspended solids, a sample divided into each item is mixed with a reaction reagent for measuring each item to form a measurement solution, The absorbance of each measurement solution at a predetermined wavelength is measured, and multiple items are quantified based on each absorbance.Furthermore, one sample blank solution is prepared by mixing the sample with a blank reaction reagent, and this The absorbance of the blank liquid is measured at at least two wavelengths to obtain a wavelength-absorbance regression function, and from this regression function, the blank absorbance at the measurement wavelength for each of the above items is calculated. A multi-item biochemical analysis method characterized by correcting the absorbance of. 2. The analytical method according to claim 1, wherein the sample containing suspended solids is chyle serum.