JP2009109196A - Dilution ratio deriving method, quantity determination method and analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of deriving an accurate dilution ratio, even when diluting a biological sample containing chyle. <P>SOLUTION: This dilution ratio deriving method includes processes for; measuring an absorbance C1 of an indication material in aqueous solution for dilution to light from a light source A; preparing sample liquid for determination by mixing a biological sample containing an optional component to be determined with the aqueous solution for dilution; measuring an absorbance C2 of the indication material in the sample liquid for determination to the light from the light source A; measuring an absorbance C4 of the indication material in the sample liquid for determination to light from a light source B having a wavelength different from the light from the light source A; and determining a blank absorbance C5 of plasma from the absorbance C4 and a wavelength depending characteristic of each light from the light source A and the light source B of the plasma containing chyle in the biological sample, and determining the dilution ratio D<SB>2</SB>of the biological sample from the absorbance C1 and a value determined by subtracting the absorbance C5 from the absorbance C2. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention utilizes a dilution factor deriving method for accurately deriving the dilution factor of a biological sample, a method for quantifying an arbitrary component in the biological sample using the method, and the dilution factor deriving method The present invention relates to an analyzing apparatus.

  Conventionally, a large-scale automatic analyzer capable of quantifying various components in a biological sample by reacting a biological sample such as blood with an analytical reagent has been put to practical use in the medical field. It has become indispensable. However, not all hospitals have introduced such devices, and outsource analysis of samples for various reasons such as operating costs, especially among small medical institutions such as clinics. There are many things that take this form. When analysis is outsourced, it takes time to obtain an analysis result, and thus the patient is inevitably returned to receive appropriate treatment based on the test result. Furthermore, there arises a problem that it is difficult to quickly respond to emergency cases such as emergency cases. Against this backdrop, in recent years the market is more compact, less costly, shorter in time, more accurate, can measure biological samples with a smaller amount of sample solution, and has a high degree of operational freedom. An analyzer is desired.

  An example of an analyzer having a high degree of freedom in operation is an apparatus that satisfies the condition that a plurality of types of component concentrations can be measured in a short time with high accuracy from a small amount of sample collected by fingertip blood collection or the like. However, the amount of specimen that can be collected without applying stress to the subject by blood collection at the fingertips or the like is at most about a dozen microliters. It is technically difficult to perform component analysis with high accuracy. One solution to this problem is to increase the sensitivity of the analysis system, increase the volume by diluting a small amount of sample, and analyze multiple types of components using a highly sensitive analysis system. .

  In general, when quantifying an arbitrary component in a biological sample, the following reasons, that is, the concentration of the arbitrary component in the biological sample is high, the volume of the biological sample is small, or the analyzer In some cases, the biological sample is diluted with an appropriate aqueous solution. When diluting and analyzing a biological sample as a specimen, the biological sample is diluted to a predetermined dilution factor, then any component in the sample is quantified, and the concentration before dilution is determined based on the dilution factor of the sample. And the concentration of an arbitrary component contained in the biological sample before dilution is determined. For this reason, when accurately quantifying an arbitrary component in a biological sample, accurately knowing the dilution factor is a very important factor in terms of analyzing the biological sample with high accuracy.

  Of the general methods for accurately knowing the dilution ratio, for example, as a method mainly used in large automatic analyzers, a biological sample and an aqueous solution for dilution are quantified with a quantifier and the like. There is a method of accurately adjusting to the dilution ratio. This method has the advantage that the dilution ratio of the sample can be arbitrarily determined in advance, but a mechanism for quantification is required. In addition, as another method, an indicator substance typified by a dye or the like is added in advance to an aqueous solution for dilution, and the optical properties (for example, absorbance) of the indicator substance before and after dilution are measured and compared. There is a method for accurately detecting the dilution ratio. This method has an advantage that a relatively simple configuration can be achieved since a mechanism for quantification is not required, and is advantageous for downsizing of the apparatus. However, since the dilution ratio of the sample is not constant, a mechanism that can cope with it is necessary.

  Conventionally, for example, a blood separation device for diluting blood as a biological sample has been proposed (for example, Patent Document 1). This blood separation device is a dedicated device for mixing an aqueous solution for dilution containing an indicator substance and blood (biological sample).

  FIG. 10 is a diagram illustrating a configuration example of a conventional blood separation instrument. The blood separation device 100 can be inserted into the main body container 101, the filter body 102 connected to the main body container 101, the plasma collection container 103 and the blood cell collection container 104 connected to the main body container 101, and the main body container 101, respectively. It comprises a lid 105, a plasma collection container attaching / detaching portion 106, and a cylindrical blood collection container portion 107 formed on the plasma collection container attaching / detaching portion 106. In blood separation instrument 100, plasma out of blood collected in blood collection container 107 passes through filter body 102 and is mixed with plasma dilution aqueous solution 108 in plasma collection container 103. After mixing, the blood separation device 100 is transported to the examination place, where the dilution factor is detected by a certain qualified person such as a doctor, nurse, clinical laboratory technician, or a specialized engineer. Since the indicator substance (pigment) is mixed in the plasma dilution aqueous solution 108, the indicator substance is measured by an optical method or the like to calculate the dilution rate (dissolution rate) of the solution, and the blood before dilution. The concentration of any component contained in the (biological sample) can be determined.

  Next, a conventional method for deriving a dilution rate of a biological sample and a method for quantifying the concentration of an arbitrary component to be quantified in the biological sample will be described with reference to FIG. FIG. 11 is a step diagram for explaining a method for deriving a dilution rate of a biological sample and a method for quantifying the concentration of an arbitrary component to be quantified in the biological sample.

First, the absorbance (C1) of the indicator substance in the aqueous solution for dilution with respect to the light from the light source (A) is measured (step S111). Absorbance (C1) indicates the concentration of the indicator substance in the aqueous solution for dilution. Next, a biological sample containing an optional component to be quantified is mixed with a dilute aqueous solution containing an indicator substance to prepare a quantification sample solution (step S112). That is, the biological sample is diluted with an aqueous solution for dilution. Next, the absorbance (C2) of the indicator substance in the sample liquid for quantification with respect to the light from the light source (A) is measured (step S113). Absorbance (C2) indicates the concentration of the indicator substance in the sample solution for quantification. Next, the dilution rate (D ₁ ) of the biological sample is derived (step S114). The dilution factor (D ₁ ) is
Dilution factor (D ₁ ) = C1 / (C1-C2) (Formula 1)
It is expressed by the following formula. Next, the concentration (Y) of the arbitrary component to be quantified in the quantification sample solution adjusted in step S112 is quantified (step S115). For example, the concentration of an arbitrary component to be quantified in a quantitative sample solution by optically detecting the reaction state by reacting an arbitrary component with a quantification reagent, for example, detecting the absorbance or turbidity with respect to light of a predetermined wavelength. (Y) is obtained. Next, the concentration (X) of an arbitrary component to be quantified in the biological sample before dilution is derived from the concentration (Y) and the dilution factor obtained in step S114 (step S116). Concentration (X) is
X = D ₁ Y (Formula 2)
It is expressed by the following formula.
Japanese Patent Laid-Open No. 2003-161729

  However, in the conventional method for deriving the dilution rate of a biological sample using the indicator substance, the dilution rate is accurately derived due to the influence of a substance or chromogen generally present in the biological sample itself. There are cases where it is not possible. Since the substance called chromogen itself has light absorption properties, the action of chromogen is measured when measuring the absorbance of the indicator substance after mixing the aqueous solution for dilution containing the indicator substance and the biological sample. As a result, a phenomenon occurs in which an accurate absorbance of the indicator substance after mixing cannot be obtained. As a result, simply comparing the absorbance of the indicator in the diluting aqueous solution before dilution with the absorbance of the indicator in the mixed solution of the diluting aqueous solution and the biological sample is not an exact dilution. It is difficult to calculate the magnification.

  In the conventional method described above, when the dilution ratio is derived by comparing the concentrations of the indicator substance before and after dilution, the absorbance of the indicator substance at the same wavelength is used when obtaining the indicator substance concentration. However, in general, in biological samples, for example, hemolysis (hemoglobin), jaundice (bilirubin), chylomicron, lipoprotein, etc. are so high that the solution becomes cloudy, so-called chyle (serum turbidity) )), And the apparent absorbance when measuring the absorbance of biological samples is increased due to the light absorption effect of chromogen contained in this turbid material, which may adversely affect the analysis results. It has become clear. Therefore, there is a problem that the dilution factor of the biological sample cannot be accurately derived unless the influence of the light absorption action of chromogen is suppressed.

  Theoretically, if these chromogen components can be removed before mixing the dilute aqueous solution containing the indicator substance and the biological sample so that they do not receive light absorption of chromogen in these biological samples, However, in this case, a large-scale equipment is required. Furthermore, it is technically difficult and impractical to completely suppress the effect of chromogen light absorption.

  In addition, the dilution factor measured ignoring the light absorption effect of chromogen contains an error, and the error directly affects the determination of the measurement result. For this reason, the conventional method has a problem that it is difficult to use in a field where derivation of the dilution rate with higher accuracy is required.

  In addition, as the biological sample becomes smaller, it becomes more difficult to maintain the quantitative accuracy of the dilute aqueous solution corresponding to the biological sample, and the quantitative accuracy tends to be adversely affected. Therefore, when the amount of specimen of a biological sample used for analysis is very small, particularly knowing the dilution factor accurately is a very important factor in terms of analyzing the biological sample with high accuracy.

  In addition, when the concentration of an arbitrary component in a biological sample is quantified by a conventional method, the light absorption wavelength of hemolysis (hemoglobin) and jaundice (bilirubin) exists at 600 nm or less, so light is emitted at a wavelength of 600 nm or more. By using the indicator having an absorption action, it is possible to avoid the influence of the light absorption action of hemolysis and jaundice. However, turbidity caused by chylomicron or lipoprotein, so-called chyle, has a light-absorbing action in the visible light region, so the turbidity of the chyle causes light scattering and increases the apparent absorbance. Inconvenience occurs that adversely affects

  From the above, in the present invention, a dilution ratio deriving method capable of deriving a more accurate dilution ratio even when a biological sample containing chyle is diluted, and in the biological sample using the method It is an object of the present invention to provide a quantification method for quantifying an arbitrary component and an analyzer using the dilution factor deriving method.

  The method for deriving a dilution ratio according to the present invention includes a step of measuring the absorbance of an optically detectable indicator contained in an aqueous solution for dilution with respect to light from a first light source, and a biological sample containing components to be quantified And diluting the biological sample to prepare a sample solution for quantification, and the absorbance of the indicator in the sample solution for quantification with respect to the light from the first light source. A step of measuring, a step of measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source having a wavelength different from that of the light of the first light source, and the light of the light of the second light source From the absorbance of the indicator substance in the sample liquid for quantification and the wavelength-dependent characteristics of the light of the first light source and the light of the second light source in the specific component including chyle in the biological sample, the identification Determining the absorbance of the component; and Based on the absorbance of the indicator in the aqueous solution for dilution and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator in the sample liquid for quantification with respect to the light from the first light source. And a step of determining a dilution ratio of the biological sample with an aqueous solution.

  The dilution factor deriving method according to the present invention is characterized in that the light of the second light source has a wavelength larger than a wavelength at which the absorbance of the light absorption characteristic of the indicator substance becomes zero.

  The dilution factor deriving method according to the present invention is characterized in that the specific component is plasma, and the wavelengths of light of the first light source and light of the second light source are 585 to 900 nm.

  The dilution factor deriving method according to the present invention is characterized in that the indicator substance is selected from at least one of the group consisting of a dye, a chromogen, a fluorescent substance, and a luminescent substance.

  In addition, the quantification method according to the present invention includes a step of measuring the absorbance of an optically detectable indicator contained in an aqueous solution for dilution with respect to light from a first light source, and a biological sample containing components to be quantified. And diluting the biological sample to prepare a sample solution for quantification, and the absorbance of the indicator in the sample solution for quantification with respect to the light from the first light source. A step of measuring, a step of measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source having a wavelength different from that of the light of the first light source, and the light of the light of the second light source From the absorbance of the indicator substance in the sample liquid for quantification and the wavelength-dependent characteristics of the light of the first light source and the light of the second light source in the specific component including chyle in the biological sample, the identification Determining the absorbance of the component, and the light from the first light source On the basis of the absorbance of the indicator in the aqueous solution for dilution and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator in the sample liquid for quantification with respect to the light of the first light source A step of obtaining a dilution ratio of the biological sample with an aqueous solution, a step of reacting a component to be quantified in the sample liquid for quantification with a reaction reagent to obtain a concentration of the component to be quantified, and the obtained dilution A step of determining the concentration of the component to be quantified contained in the biological sample from the magnification and the concentration of the component to be quantified in the sample solution for quantification.

  The quantitative method according to the present invention is characterized in that the light of the second light source has a wavelength larger than the wavelength at which the absorbance of the light absorption property of the indicator substance becomes zero.

  The quantitative method according to the present invention is characterized in that the specific component is plasma, and the wavelengths of the light from the first light source and the light from the second light source are 585 to 900 nm.

  In the quantification method according to the present invention, the indicator substance is selected from at least one of the group consisting of a dye, a chromogen, a fluorescent substance, and a luminescent substance.

  The quantification method according to the present invention is characterized in that the biological sample is a blood component and the component to be quantified is a lipid component.

  Further, according to the present invention, an analyzer for quantifying a component to be quantified in a biological sample includes an analysis device into which the biological sample is injected, and a light generation unit that irradiates the analysis device with light. A rotation drive unit that rotates the analysis device around an axis, a light detection unit that detects the transmitted light of the analysis device, and a detection result by the light detection unit to process the biological sample A processing unit that quantifies the components to be quantified, wherein the analysis device includes a first storage unit that stores the biological sample, and an aqueous solution for dilution used for diluting the biological sample. A second storage unit for storing the biological sample and the aqueous solution for dilution, a mixing unit for mixing the biological sample and the aqueous solution for dilution, and the mixing Keep a certain amount of biological sample diluted in And an analysis unit for reacting the biological sample diluted in the mixing unit with a reaction reagent, and when rotated by the rotation driving unit, the biological force is generated by centrifugal force and capillary force of the channel. The biological sample and the diluting aqueous solution are respectively transferred and mixed, and the light generating unit irradiates the diluting aqueous solution with light from a first light source, and the biological sample and the diluting aqueous solution are irradiated. The mixed solution with the aqueous solution is irradiated with light from the first light source and light from a second light source having a wavelength different from that of the light from the first light source, and the light detection unit From the light transmitted through the device, the absorbance of the indicator substance in the aqueous solution for dilution with respect to the light of the first light source, and the indicator substance in the mixed solution with respect to the light of the first light source and the light of the second light source And the processing unit detects the light from the second light source. The specific component based on the absorbance of the indicator in the mixed solution and the wavelength-dependent characteristics of the light from the first light source and the light from the second light source in the specific component including chyle in the biological sample The absorbance of the specific component is calculated from the absorbance of the indicator in the aqueous solution for dilution with respect to the light from the first light source and the absorbance of the indicator in the mixed solution with respect to the light from the first light source. A dilution factor deriving unit for obtaining a dilution factor of the biological sample with the dilution aqueous solution based on the subtracted value is provided.

  According to the dilution ratio deriving method of the present invention, the step of measuring the absorbance of the optically detectable indicator contained in the dilution aqueous solution with respect to the light of the first light source, and the biological including the component to be quantified Mixing the sample with the diluting aqueous solution to prepare a quantifying sample solution, measuring the absorbance of the indicator in the quantifying sample solution with respect to the light from the first light source, and the first Measuring the absorbance of the indicator in the quantification sample solution with respect to the light of the second light source having a wavelength different from that of the light of the light source; and the indicator in the sample solution for quantification with respect to the light of the second light source And determining the absorbance of the specific component from the wavelength-dependent characteristics of the light of the first light source and the light of the second light source for the specific component including chyle in the biological sample; The dilution water for the light of the first light source Based on the absorbance of the indicator substance in the sample solution and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the first light source. The step of determining the dilution rate of the biological sample, so that the influence of the light absorption of the chyle is avoided when determining the dilution rate of the biological sample from the absorbance of the indicator before and after dilution. be able to. As a result, the dilution factor of the biological sample can be derived with higher accuracy.

  Further, according to the quantification method of the present invention, the step of measuring the absorbance of the optically detectable indicator contained in the aqueous solution for dilution with respect to the light of the first light source, and the biological including the component to be quantified Mixing the sample with the diluting aqueous solution to prepare a quantifying sample solution, measuring the absorbance of the indicator in the quantifying sample solution with respect to the light from the first light source, and the first Measuring the absorbance of the indicator in the quantification sample solution with respect to the light of the second light source having a wavelength different from that of the light of the light source; and the indicator in the sample solution for quantification with respect to the light of the second light source And determining the absorbance of the specific component from the wavelength-dependent characteristics of the light of the first light source and the light of the second light source for the specific component including chyle in the biological sample; The aqueous solution for dilution with respect to the light of the first light source Based on the absorbance of the indicator and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator in the sample liquid for quantification with respect to the light of the first light source. A step of obtaining a dilution factor of the sample, a step of reacting a component to be quantified in the sample solution for quantification with a reaction reagent to obtain a concentration of the component to be quantified, the obtained dilution factor, and the sample for quantification And determining the concentration of the component to be quantified contained in the biological sample from the concentration of the component to be quantified in the liquid. From the absorbance of the indicator before and after dilution, the biological When determining the dilution rate of a biological sample, it is possible to avoid the influence of the light absorption effect of the chyle and to obtain a more accurate biological sample dilution rate. Furthermore, based on the dilution factor, the concentration of an arbitrary component in the biological sample can be quantified more accurately.

  The analysis apparatus of the present invention includes an analysis device into which a biological sample is injected, a light generation unit that irradiates light to the analysis device, a rotation drive unit that rotates the analysis device around an axis, A light detection unit for detecting the transmitted light of the analytical device; and a processing unit for processing a detection result by the light detection unit to quantify a component to be quantified in the biological sample. The device for use includes a first storage unit that stores the biological sample, a second storage unit that stores an aqueous solution for dilution used for diluting the biological sample, the biological sample, and the dilution. A flow path for transferring the aqueous solution, a mixing unit for mixing the biological sample and the diluting aqueous solution, a holding unit for holding a certain amount of the biological sample diluted in the mixing unit, The biological sample diluted in the mixing section is reacted with the reaction reagent. The biological sample and the dilute aqueous solution are each transferred and mixed by centrifugal force and capillary force of the flow path when rotated by the rotation drive unit, The light generation unit irradiates the dilution aqueous solution with the light of the first light source, and applies the light of the first light source to the mixed solution of the biological sample and the dilution aqueous solution, The light of the second light source having a wavelength different from that of the light of the first light source is irradiated, and the light detection unit uses the dilution aqueous solution for the light of the first light source from the light transmitted through the analysis device. And detecting the absorbance of the indicator substance in the mixed solution with respect to the light of the first light source and the light of the second light source, and the processing unit detects the light of the second light source. The absorbance of the indicator substance in the mixed solution with respect to The absorbance of the specific component is obtained from the wavelength-dependent characteristics of the light of the first light source and the light of the second light source in the specific component including chyle in the material, and the dilution with respect to the light of the first light source Based on the absorbance of the indicator substance in the aqueous solution and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator substance in the mixed solution with respect to the light of the first light source, the biology by the aqueous solution for dilution In order to determine the dilution rate of a biological sample from the absorbance of the indicator before and after dilution, only two different wavelengths of light are used. Without using a large and complicated apparatus, it is possible to avoid the influence of the light absorption action of the chyle and to obtain a more accurate dilution ratio of the biological sample. Furthermore, based on the dilution factor, the concentration of an arbitrary component in the biological sample can be quantified more accurately.

(Embodiment 1)
A dilution factor deriving method and a quantifying method according to Embodiment 1 of the present invention will be described with reference to FIGS.

  FIG. 1 shows a change in light absorption characteristics of a plasma component containing chyle, and is a graph illustrating an absorption spectrum. This plasma component is a biological sample obtained by extracting only the plasma component after removing the blood cell component by centrifuging whole blood. In FIG. 1, the absorption spectrum of four types of plasma components 1-4 from which the content rate of a chyle differs each is shown. In general, since the content of chyle varies depending on the sample collected from each individual, the absorption spectrum thereof is also different, and as a result, the absorbance is different.

  When deriving the dilution factor, it is desirable that the blank value of the absorbance derived from the specimen is “0”, because an error in deriving the dilution factor does not occur and it is not necessary to consider the influence of light absorption characteristics due to chyle. However, in reality, as shown in FIG. 1, a biological sample having chyle has a light-absorbing action in the entire visible light region having a wavelength of 600 nm or more, and the longer the wavelength is, the stronger the absorption intensity is. It has the property of decreasing. For this reason, it is shown that the absorbance decreases toward the long wavelength side. In this case, the influence of the hazy substance of hemolysis (hemoglobin) and jaundice (bilirubin) exists in the region where the light absorption wavelength is 600 nm or less and can be ignored in the region of 600 nm or more (not shown).

FIG. 2 is a graph showing the relationship between the absorbance of light absorption characteristics of two types of light having different wavelengths in plasma components including chyle, and illustrates the relationship between the absorbance at 620 nm and 770 nm. FIG. 2 shows a graph plotted with respect to four biological samples having different chyle contents used in FIG. 1. As shown in FIG. 2, a good linear relationship was obtained (R ² = 0. 9999). R represents the correlation coefficient of the regression line. In addition, each of the four biological samples having different chyle contents is mixed with PBS (buffer solution) which is an aqueous solution for dilution that does not contain an indicator substance, and is doubled, tripled, four times, A linear relationship was also obtained in the case where double-diluted products were prepared and the relationship between the absorbances of the light absorption characteristics of two types of light with different wavelengths was expressed with respect to those diluted solutions (not shown). ). In other words, the relationship between the absorbance of the light absorption characteristics of two types of light having different wavelengths before and after mixing the biological sample and the aqueous solution for dilution is somewhat different from the slope and y-intercept in comparison with the graph of FIG. Although it does not become the same regression line, it shows that the same linear relationship is obtained. Here, it is not limited to a straight line, and a polynomial may be used to further improve accuracy. As for the selection of the wavelength, any linear relationship is established in the range of 585 nm to 900 nm, and a place suitable for the light absorption characteristics of the indicator used is selected.

  In the dilution factor deriving method according to the first embodiment, a wavelength-dependent characteristic that does not depend on the light absorption characteristic of the indicator substance contained in the aqueous solution for dilution in a certain wavelength region (585 nm to 900 nm) in plasma including chyle. In addition to using light of two wavelengths, the derivation of a highly accurate dilution factor that suppresses an increase in absorbance due to the light absorption effect of chyle was realized using light of two wavelengths.

  FIG. 3 is a graph illustrating the change in the light absorption characteristics of the indicator substance before and after mixing the biological sample and the aqueous solution for dilution. In FIG. 3, an absorption spectrum 5 shows the light absorption characteristics (absorption spectrum) of an aqueous solution for dilution containing brilliant blue FCF, which is an indicator before dilution, and absorption spectra 6 and 7 show brilliant blue FCF, which is an indicator. The light absorption characteristic (absorption spectrum) of the indicator substance in the mixed solution (sample liquid for fixed_quantity | division) after the dilution which mixed the aqueous solution for dilution and the plasma component containing chyle in the ratio of 1: 1 is shown. In particular, the absorption spectrum 6 is a graph when the absorbance of the mixed solution is actually measured. In addition to the absorbance derived from the indicator substance, the absorbance due to the light absorption action of chyle is also added. It is a graph which shows the light absorbency derived from the indicator substance which subtracted the said extra part of the light absorbency by the light absorption effect | action of a chyle.

  In FIG. 3, “C1” represents the peak wavelength of the light absorption characteristic of the indicator substance, and is also the absorbance for deriving the dilution factor. “C2” represents the peak wavelength of the light absorption characteristics of the indicator substance after dilution, and is also the absorbance actually measured. “C3” represents the absorbance derived from the indicator substance, and “C5” represents the absorbance derived from the specimen blank, that is, the absorbance due to the light absorption action of the so-called chyle itself. In order to accurately derive the dilution factor of the biological sample, it is necessary to compare the absorbance C1 and the absorbance C3. However, since the absorbance C2 actually measured after the dilution is added with the absorbance C5 due to the light absorption action of the chyle itself in addition to the absorbance C3 derived from the indicator substance, an accurate dilution ratio cannot be derived. .

  Therefore, in the dilution factor deriving method according to the first embodiment, the dilution factor of the biological sample by the aqueous solution for dilution is derived from the absorbance (C1) and the value obtained by subtracting the absorbance C5 from the absorbance (C2).

  FIG. 5 is a step diagram for explaining the dilution factor deriving method and the method for quantifying an arbitrary component in a biological sample using the dilution factor deriving method according to the first embodiment. Here, a case where blood is used as a biological sample, and the blood is mixed with a dilute aqueous solution containing an indicator substance to prepare a quantitative sample solution will be described as an example.

First, the absorbance (C1) of the indicator substance in the aqueous solution for dilution with respect to the light from the light source (A) is measured (step S511). Next, the biological sample is mixed with the diluting aqueous solution to prepare a quantitative sample solution (mixed solution) (step S512). Next, the absorbance (C2) of the indicator substance in the quantitative sample solution with respect to the light of the light source (A) is measured (step S513), and the absorbance of the indicator substance in the quantitative sample solution with respect to the light of the light source (B) (C4). ). Here, as is clear from the conventional method for deriving the dilution rate, the dilution rate (D ₁ ) of the biological sample in the sample solution for quantification is expressed by the above formula (1). However, as shown in FIG. 3, the absorbance C2 actually measured includes the absorbance C3 derived from the sample blank in addition to the absorbance C3 derived from the indicator substance, as described above. Since it is added as the absorbance, C2 actually measured is represented by the following relational expression.

  C2 = C3 + C5 (Formula 3)

Here, in order to derive a more accurate dilution factor, the dilution factor (D ₂ ) excluding the influence of the absorbance derived from the specimen blank is expressed by the following relational expression.

Dilution factor (D ₂ ) = C1 / (C1-C3) (Formula 4)

Furthermore, the dilution rate (D ₂ ) is expressed by the following relational expression from (Expression 3) and (Expression 4).

Dilution _{(D 2) = C1 / (} C1-C3) = C1 / {C1- (C2-C5)} ( Equation 5)

  If the absorbance C5 derived from the specimen blank can be subtracted from the actually measured absorbance C2, the influence of the amount added as the absorbance due to the light absorption action of the chyle itself is removed, and the accurate absorbance C3 derived from the indicator substance is removed. Can be derived. Here, the absorbance C5 derived from the sample blank in the plasma component including chyle is obtained by using the graph of FIG. 2 showing the relationship between the absorbances of the light absorption characteristics of two light beams having different wavelengths. If it is determined, it is obtained using the characteristic that the absorbance at the other wavelength is derived. As an example, the relational expression of FIG.

  C5 = a × (C4) + b (Formula 6)

  Absorbance C5 represents the absorbance of the sample blank having absorption at the measurement wavelength of the light source (A) used for quantifying the absorbance of the indicator substance in the sample liquid for quantification, and the light of the chyle itself shown in FIG. This is the absorbance due to absorption. The absorbance C4 is a wavelength different from the measurement wavelength of the light of the light source (A), and is the absorbance of the measurement wavelength of the light of the light source (B) whose absorbance value by the indicator is “0”. In actual measurement of absorbance, there is a slight error in the absorbance value of “0” due to the characteristics of the indicator substance, the measurement environment, etc., but generally this error is within a negligible range. It is believed that there is. In Equation 6, “a” represents the slope of the straight line in the graph of FIG. 2, and “b” represents the y-intercept. From this relational expression, the absorbance C5 of the light wavelength of the light source (A) in the sample liquid for quantification is derived from the absorbance of the light wavelength of the light source (B) having a wavelength different from that of the light source (A). From these, from (Equation 5) and (Equation 6), the equation representing the dilution factor of the dilution factor deriving method according to the first embodiment is the following relational equation.

Dilution factor (D ₂ ) = (C1) / [(C1) − {(C2) − (a × (C4) + b)}] (Formula 7)

  Using this (Equation 7), the dilution factor of the biological sample is determined from the absorbance (C2) and absorbance (C4) (step S515). Thereby, the influence by the light absorption action by the chyle derived from the specimen blank is removed, and an accurate dilution factor without error is derived. Further, if necessary, a separate conversion formula may be provided between the derived dilution factor and the theoretical dilution factor.

Next, the concentration (X) of the arbitrary component to be quantified in the biological sample before dilution is quantified from the dilution factor derived as described above. Examples of optional components to be quantified include glucose, total cholesterol, HDL cholesterol, triglyceride, ALT, and AST. In a diluted biological sample, that is, glucose, total cholesterol, HDL cholesterol, triglyceride, ALT, AST, etc. in a sample solution for quantification are reacted with each known reaction reagent for quantifying those substances, The concentration (Y) of an arbitrary component is obtained by detecting the reaction state (step S516). For example, the concentration (Y) of an arbitrary component is obtained by measuring the absorbance and turbidity of the reaction solution with light of a specific wavelength. Next, the concentration (X) of an arbitrary component in the biological sample before dilution is determined from the biological sample dilution rate (D ₂ ) derived from (Equation 7) according to (Equation 8) (step) S517).

X = D ₂ Y (Formula 8)

  An analysis apparatus for realizing the dilution factor deriving method and the quantifying method according to the first embodiment as described above will be described with reference to FIGS. Here, a case where blood is used as a biological sample, and the blood is mixed with a dilute aqueous solution containing an indicator substance to prepare a quantitative sample solution will be described as an example.

  FIG. 6 is a diagram illustrating a configuration example of the analysis apparatus. The analysis apparatus 60 includes an analysis device 61, a rotation drive unit 62 that rotates the analysis device 61, a light generation unit 63, a light detection unit 64, and a processing unit 65 that processes the detection result of the light detection unit 64. Is provided. The processing unit 65 includes a dilution factor deriving unit that performs the dilution factor deriving process and a quantifying unit that performs the quantifying process.

  The analysis device 61 is attached to the rotation drive unit 62. By rotating the analysis device 61 about the axis in the direction of the arrow by the rotation drive unit 62, a centrifugal force is applied to the analysis device 61, and the centrifugal force and the communication channel disposed in the analysis device 61 are applied. By combining the working capillary force, the movement of the fluid stored in the analysis device 61 can be controlled. The flow state or chemical state of the fluid in the analysis device 61 can be detected by measuring the light absorption characteristics of the fluid with respect to the light 66 emitted from the light generation unit 63 by the light detection unit 64.

  FIG. 7 is a view showing a state in which the analysis device 61 is attached to the rotation drive unit 62, and FIG. 8 is a view showing the internal structure of the analysis device 61. The analysis device 61 includes a liquid sample storage chamber 81 for storing a biological sample (liquid sample) containing an arbitrary component to be quantified, and an indicator for diluting the liquid sample and accurately deriving the dilution rate. A diluting aqueous solution storage chamber 82 for storing the diluting aqueous solution containing, and a diluting aqueous solution measuring chamber connected to the diluting aqueous solution storing chamber 82 and measuring and holding a fixed amount of the diluting aqueous solution flowing from the diluting aqueous solution storing chamber 82 83, a mixing chamber that is connected to the liquid sample storage chamber 81 and the diluting aqueous solution measuring chamber 83 and mixes the diluting aqueous solution flowing in from the diluting aqueous solution measuring chamber 83 and the liquid sample flowing in from the liquid sample storing chamber 81. 84, a mixing channel 84 connected to the mixing chamber 84 and holding a certain amount of the liquid sample mixed (diluted) in the mixing chamber 84, and the mixing channel 84 connected to the mixing chamber 84 and transferred from the mixing chamber 84 ( Nozomi The) are arbitrary components in a liquid sample to quantify, and an analysis chamber 86 for measuring absorbance or turbidity of the liquid sample.

  The dilute aqueous solution accommodated in the dilute aqueous solution storage chamber 82 contains an indicator substance. In order to derive an accurate dilution factor, the specimen blank absorbance (C5) of the plasma component in the biological sample is determined. The method of removing is as described above. In addition, in order to enable control of the fluid by the capillary force acting on the connection flow path and the centrifugal force generated by the rotation, the dilution aqueous solution measuring chamber 83 is disposed at a position farther from the axial center than the dilution aqueous solution storage chamber 82. The mixing chamber 84 is disposed farther from the axial center than the dilution aqueous solution measuring chamber 83, and the analysis chamber 86 is disposed farther from the axial center than the mixing chamber 84. The absorbance of the indicator contained in the diluting aqueous solution is measured in the diluting aqueous solution measuring chamber 83 and the mixing chamber 84, and the dilution factor is derived based on the measurement result. Alternatively, the dilution aqueous solution measuring chamber 83 and the mixing chamber 84 may be formed integrally, and the absorbance of the indicator in the dilution aqueous solution before and after mixing may be measured in the same measurement region. If necessary, a separation chamber for separating the liquid sample flowing from the liquid sample storage chamber 81 into a low specific gravity component and a high specific gravity component using centrifugal force is provided between the liquid sample storage chamber 81 and the mixing chamber 84. Further, a liquid sample measuring channel for holding a certain amount of liquid sample may be provided between the separation chamber and the mixing chamber 84 so that a certain amount of liquid sample can be transferred to the mixing chamber 84. Good. The analysis chamber 86 holds reaction reagents that react with arbitrary components to be quantified contained in the liquid sample, and a plurality of them may be arranged for each analysis item as required. In addition, the metering channel 85 is arranged to transfer a certain amount of liquid sample to the analysis chamber 86. Further, when the liquid sample is weighed in the dilute aqueous solution metering chamber 83, the excess liquid amount flows into the overflow chamber 20 connected to the dilute aqueous solution metering chamber 83.

  The operation for deriving the dilution factor and the quantitative operation by the analyzer 60 configured as described above will be described.

First, the analysis device 61 is mounted on the rotation drive unit 62, and the analysis device 61 is rotated about the axis, thereby transferring the dilution aqueous solution to the dilution aqueous solution measuring chamber 83 and stopping the rotation. The biological sample (liquid sample) containing the optional component to be quantified and the diluting aqueous solution in the diluting aqueous solution measuring chamber 83 are respectively transferred to the connecting passage just before the mixing chamber 84. By rotating the analysis device 61 again, the liquid sample and the aqueous solution for dilution are transferred to the mixing chamber 84 and mixed. By stopping the rotation, the mixed solution (quantitative sample solution) is transferred to the connecting passage just before the analysis chamber 86. Thereafter, the analytical device 61 is rotated again to transfer the quantitative sample solution to the analysis chamber 86. In this series of flows, the light detection unit 64 measures the light absorption characteristics of the indicator substance before and after mixing the liquid sample and the aqueous solution for dilution, and based on the measurement result, the dilution factor deriving means of the processing unit 65 The dilution rate (D ₂ ) of the liquid sample is derived using Equation 7 by the method described above. In the process of deriving the dilution factor (D ₂ ), the absorbance (C5) is calculated in order to subtract the absorbance (C5) due to the light absorption action of the chyle itself from the absorbance (C2) actually measured after dilution. The relational expression of the wavelength-dependent characteristics of the two wavelengths of light in plasma components including chyle used in the above is stored in advance in a recording unit (not shown) such as a memory of the analyzer 60, and when necessary, the processing unit 65 dilution rate deriving means reads and uses. In this way, in an analyzer for quantifying the concentration of an arbitrary component to be quantified with high accuracy, it is indispensable to derive the dilution factor with high accuracy. In addition, the process of avoiding the influence of the blank absorbance by subtracting the absorbance (C5) due to the light absorption action of the chyle itself from the absorbance actually measured after dilution (C2) of the present invention is a very important factor. .

Next, the concentration (Y) of the arbitrary component to be quantified in the sample liquid for quantification is quantified by the light detection unit 64. For example, an arbitrary component to be quantified is reacted with a reaction reagent in the analysis chamber 86, the reaction solution is irradiated with light of a specific wavelength from the light generation unit 63, and the absorbance or turbidity is measured by the light detection unit 64. Quantify the concentration (Y). From the measured concentration (Y) of the arbitrary component to be quantified and the dilution rate (D ₂ ) obtained by the dilution rate deriving unit, the quantification unit of the processing unit 65 uses the above formula (8), Determine the concentration (X) of the optional component to be quantified in the liquid sample before mixing (dilution).

  As described above, according to the dilution factor deriving method, the quantifying method, and the analyzer according to the first embodiment, when the biological sample is diluted with the dilute aqueous solution containing the indicator substance, the light source (A) The absorbance of the indicator substance before and after dilution is measured by the light of the light source (B) having a wavelength different from that of the light and the light of the light source (A), and the measured absorbance and the wavelength of the light of the light source (A) Using the wavelength dependent property of the component containing chyle represented by the relational expression with the light wavelength of the light source (B), the blank absorbance (C5) of the component containing chyle in the biological sample is obtained and diluted. The biological sample from the absorbance (C1) of the indicator substance in the previous dilution aqueous solution and the value obtained by subtracting the blank absorbance (C5) from the absorbance (C2) of the indicator substance in the diluted sample solution From the determination of the dilution factor, avoid the effect of light absorption of the chyle, Ri can be determined the dilution factor of precise biological sample, further based on the dilution ratio, it is possible to more accurately quantify the concentration of any component to be quantified in a biological sample.

  Moreover, according to the dilution factor deriving method according to the first embodiment, there is an effect that the measurement accuracy can be improved in any sample as long as it is a biological sample including chyle.

Example 1
In this Example 1, serum as a biological sample is diluted with an aqueous solution for dilution containing brilliant blue FCF as an indicator substance, and the dilution ratio of the serum is derived from the absorbance of brilliant blue FCF before and after dilution. .

  First, powdery brilliant blue FCF is dissolved in a mixed solution of PBS (buffer solution) and 10 mg / ml BSA, and the absorbance is 0 when the optical path length is 1 cm and the peak wavelength is 633 nm (the light wavelength of the light source (A)). The density was adjusted to be .5. This is because the optimum range of the concentration of the brilliant blue FCF, which is the indicator substance, varies depending on the measurement conditions such as the optical path length. This is because it is desirable to keep the absorbance of the substance within a measurable range. Specifically, the absorbance at the measurement wavelength used for deriving the dilution factor is preferably 0.1 to 1.5, and more preferably 0.4 to 0.6. Next, the absorbance (C1) at 633 nm (light wavelength of the light source (A)) of the brilliant blue FCF solution before addition of serum as a biological sample was measured. Next, serum is added to the brilliant blue FCF, and the absorbance (C2) of the mixed solution at 633 nm and the absorbance value of the brilliant blue FCF is “0” at 751 nm (light wavelength of the light source (B)). The absorbance (C4) of was measured. Here, a relational expression (y = 2.143x + 0.0002) of wavelength-dependent characteristics of 633 nm and 751 nm in a specific component (plasma component) including chyle is obtained in advance as shown in FIG. From the C1), the absorbance (C2), and the absorbance (C4), the dilution rate was determined using Equation 7. Table 1 shows the measurement results of test tube numbers 1 to 3 for the sample solution for quantification, which is a mixed solution of serum as a biological sample and an aqueous solution for dilution.

  For test tube number 1, the theoretical dilution factor was 2.0, but when derived by the conventional method, the dilution factor was 2.7. The large deviation from this ideal dilution factor is the effect of chyle. This has been a big problem in the prior art. On the other hand, when derived by the method of the present invention, the dilution factor was 2.0 times. When the deviation from the ideal dilution ratio was calculated in detail, it was 0.6%, and very good results were obtained in view of the 37% deviation in the prior art. Similar results were obtained for test tube numbers 2 and 3. Thus, at a low dilution factor such as a dilution factor of 2.0 to 6.0, a large error has occurred in the derivation of the dilution factor due to the influence of chyle in the conventional technique. In particular, in this experiment, the optical path length was as long as 1 cm, and the deviation from the ideal dilution factor was 25 to 46%, and the effect was remarkable. On the other hand, when the method of the present invention is used, it is possible to derive a highly accurate dilution factor with a deviation of -0.9 to 0.6%, thus demonstrating the effectiveness of the present invention.

(Example 2)
In Example 2, blood as a biological sample is diluted with an aqueous solution for dilution containing amide black 401 as an indicator, and the amount of amide black 401 before and after dilution is quantified in blood. The dilution ratio of triglyceride (TG), which is an optional component to be derived, is derived, and the concentration of triglyceride (TG) contained in a very small amount (10 μl) of blood is measured.

  First, for an aqueous solution for dilution containing Amido Black 401, PBS (buffer solution), and a stabilizer, the absorbance at an optical path length of 1 mm and a peak wavelength of 620 nm (light wavelength of the light source (A)) was measured (C1 = 0). .429). Next, 10 μl of whole blood is mixed with the aqueous solution for dilution, and the mixed solution is centrifuged to remove blood cells. Then, the supernatant has an optical path length of 1 mm, an absorbance (C2) at a wavelength of 620 nm, and a wavelength of 730 nm (light source). Absorbance (C4) at the wavelength of light (B) was measured. The absorbance (C1), absorbance (C2), and absorbance (C4) are preferably measured at the same spot in order to eliminate the influence on measurement accuracy due to variations in optical path length. As shown in FIG. 9, a relational expression (y = 1.745x + 0.00010) of 620 nm and 730 nm in plasma is obtained in advance, and the expression, absorbance (C1), absorbance (C2), and absorbance (C4) From these, the dilution ratio was determined using (Equation 7). Next, the TG concentration of the supernatant is obtained by a known method that can be derived by reacting with a reagent for quantification, etc., and the measured value and the obtained dilution rate are substituted into (Equation 8), thereby diluting. The TG concentration contained in the micro blood before mixing with the aqueous solution was calculated. Table 2 shows the measurement results of test tube numbers 4 to 8 for the sample solution for quantification, which is a mixed solution of plasma in blood as a biological sample and an aqueous solution for dilution. In addition, the amount of the aqueous solution for dilution to be mixed with 10 μl of whole blood was changed under conditions as shown in test tube numbers 4 to 8 in Table 2. The TG concentration in blood used in this experiment was measured with a high-precision large apparatus (Hitachi 7020), and the actual dilution ratio was 164 mg / dl.

  Table 2 shows the dilution ratio (D) in the range from the 2.0-fold dilution to the 10.9-fold dilution derived from the actual concentration in the plasma diluted with the dilution aqueous solution prepared with the above composition. It is calculated by each of the method and the method of the present invention, the concentration of triglyceride (TG) contained in blood is measured from the calculated dilution rate (D), and the results are compared. The actual dilution ratio is 2.0 times for test tube number 4; 4.0 times for test tube number 5; 6.3 times for test tube number 6; 8.2 times for test tube number 7; Although it is 10.9 times, in all the test tubes (4 to 8), the calculated dilution ratio by the method of the present invention is 2.0 times for test tube number 4 and 4.0 times for test tube number 5 The test tube number 6 is 6.1 times, the test tube number 7 is 8.3 times, and the test tube number 8 is 11.0 times. Became. As a result, the calculated concentration quantified by the method of the present invention was calculated as 161 mg / dl, 162 mg / dl, 158 mg / dl, 166 mg / dl, 166 mg / dl in the test tubes (4 to 8). It can be seen that the calculated concentration by the method of the present invention is closer to the measured dilution factor of 164 mg / dl than the calculated concentration by the technique. Further, with respect to the calculated concentration in the range of the calculated 2.0-fold dilution to 10.9-fold, a divergence error of 9.9% at maximum occurs in the conventional method, but 3.8% in the method of the present invention. It can be seen that the deviation error is suppressed within. Compared with the conventional method, it can be seen that the method of the present invention can derive the dilution rate without variation. Thus, the method of the present invention was able to calculate the TG concentration more accurately than the conventional method in any of the test tube numbers 4 to 8.

  The dilution ratio deriving method and quantifying method according to the present invention are suitable for a method and an apparatus for diluting a biological sample containing chyle and quantifying an arbitrary substance contained in the biological sample.

It is a graph showing the example of the absorption spectrum (change of a light absorption characteristic) of human plasma for demonstrating the dilution factor deriving method in Embodiment 1 of this invention. It is a figure showing the wavelength dependence characteristic of plasma for explaining the dilution factor deriving method in Embodiment 1 of the present invention, and a figure showing a relational expression. It is a graph showing the example of the absorption spectrum before and after mixing of a brilliant blue FCF solution and plasma for demonstrating the dilution factor deriving method in Embodiment 1 of this invention. It is a figure showing the wavelength dependence characteristic of plasma for explaining the dilution factor deriving method in Embodiment 1 of the present invention, and a figure showing a relational expression. FIG. 2 is a step diagram for explaining a dilution factor deriving method and a method for quantifying an arbitrary component to be quantified in a biological sample in Embodiment 1 of the present invention. It is a figure which shows the structural example of the analyzer for implement | achieving the dilution rate derivation | leading-out method and quantification method in Embodiment 1 of this invention. It is a top view of the device for analysis which an analyzer for realizing the dilution rate deriving method and quantification method in Embodiment 1 of the present invention has. It is a figure which shows the internal structure of the device for analysis which the analyzer for implement | achieving the dilution factor derivation | leading-out method and quantification method concerning Embodiment 1 of this invention has. It is a figure showing the wavelength dependence characteristic of plasma in Example 2 of the present invention, and a figure showing a relational expression. It is a block diagram of the conventional blood separation instrument. FIG. 10 is a step diagram for explaining a conventional method for deriving a dilution rate and a method for quantifying an arbitrary component to be quantified in a biological sample.

Explanation of symbols

1 Absorption spectrum of specimen 1 Absorption spectrum of specimen 2 3 Absorption spectrum of specimen 3 Absorption spectrum of specimen 4 5 Absorption spectrum of brilliant blue FCF solution 6 Mixed solution in which brilliant blue FCF solution and plasma are mixed at a ratio of 1: 1 Absorption spectrum 7 Absorption spectrum of a mixed solution prepared by mixing a brilliant blue FCF solution and a dilute aqueous solution not containing an indicator substance at a ratio of 1: 1 a slope of graph b y-intercept C1 absorbance of indicator material before mixing Sum of absorbance derived from indicator substance and sample blank after C2 mixing (actual absorbance)
C3 Absorbance derived from the indicator substance after mixing C4 Absorbance of the indicator substance at 0, absorbance at another wavelength (after mixing)
Absorbance from C5 specimen blank (absorbance of chyle component)
D1 Dilution factor D2 derived by the conventional method D2 Dilution factor derived by the method of the present invention X Concentration of the component to be quantified after dilution Y Concentration of the component to be quantified before dilution 60 Analyzer 61 Analytical device 62 Rotation drive Unit 63 light generation unit 64 light detection unit 65 processing unit 66 light 81 liquid sample storage chamber 82 dilution aqueous solution storage chamber 83 dilution aqueous solution measurement chamber 84 mixing chamber 85 measurement channel 86 analysis chamber 87 overflow chamber 100 blood separation instrument 101 Main body container 102 Filter body 103 Plasma collection container 104 Blood cell collection container 105 Lid 106 Plasma collection container attaching / detaching section 107 Blood collection container section 108 Aqueous solution for plasma dilution

Claims (10)

Measuring the absorbance of the optically detectable indicator contained in the aqueous solution for dilution with respect to the light of the first light source;
Mixing a biological sample containing a component to be quantified and the aqueous solution for dilution, diluting the biological sample, and preparing a sample solution for quantification;
Measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the first light source;
Measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source having a wavelength different from that of the light of the first light source;
The absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source, the light of the first light source and the light of the second light source in specific components including chyle in the biological sample Obtaining the absorbance of the specific component from the wavelength-dependent characteristics of
The absorbance of the indicator in the aqueous solution for dilution with respect to the light of the first light source, and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator in the sample liquid for quantification with respect to the light of the first light source; Determining the dilution factor of the biological sample with the diluting aqueous solution based on
A method for deriving a dilution ratio. In the dilution factor deriving method according to claim 1,
The light of the second light source has a wavelength that is greater than the wavelength at which the absorbance of the light absorption characteristic of the indicator substance becomes zero.
A method for deriving a dilution ratio. In the dilution factor deriving method according to claim 2,
The specific component is plasma, and the wavelengths of the light of the first light source and the light of the second light source are 585 to 900 nm.
A method for deriving a dilution ratio. In the dilution factor deriving method according to claim 1,
The indicator is selected from at least one of the group consisting of a dye, a chromogen, a fluorescent substance, and a luminescent substance.
A method for deriving a dilution ratio. Measuring the absorbance of the optically detectable indicator contained in the aqueous solution for dilution with respect to the light of the first light source;
Mixing a biological sample containing a component to be quantified and the aqueous solution for dilution, diluting the biological sample, and preparing a sample solution for quantification;
Measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the first light source;
Measuring the absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source having a wavelength different from that of the light of the first light source;
The absorbance of the indicator substance in the sample liquid for quantification with respect to the light of the second light source, the light of the first light source and the light of the second light source in specific components including chyle in the biological sample Obtaining the absorbance of the specific component from the wavelength-dependent characteristics of
The absorbance of the indicator in the aqueous solution for dilution with respect to the light of the first light source, and the value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator in the sample liquid for quantification with respect to the light of the first light source; Determining the dilution factor of the biological sample with the aqueous solution for dilution, based on
Reacting a component to be quantified in the sample solution for quantification with a reaction reagent to obtain a concentration of the component to be quantified;
Determining the concentration of the component to be quantified contained in the biological sample from the determined dilution factor and the concentration of the component to be quantified in the sample liquid for quantification,
A quantitative method characterized by that. The quantification method according to claim 5, wherein
The light of the second light source has a wavelength larger than the wavelength at which the absorbance of the light absorption characteristic of the indicator substance becomes 0.
A quantitative method characterized by that. The quantification method according to claim 6, wherein
The specific component is plasma, and the wavelengths of the light of the first light source and the light of the second light source are 585 to 900 nm.
A quantitative method characterized by that. The quantification method according to claim 5, wherein
The indicator is selected from at least one of the group consisting of a dye, a chromogen, a fluorescent substance, and a luminescent substance.
A quantitative method characterized by that. The quantification method according to claim 5, wherein
The biological sample is a blood component and the component to be quantified is a lipid component;
It is characterized by that. In an analyzer for quantifying components to be quantified in a biological sample,
An analytical device into which the biological sample is injected;
A light generation unit for irradiating the analysis device with light;
A rotational drive unit for rotating the analytical device about an axis;
A light detection unit for detecting the transmitted light of the analysis device;
A processing unit that processes the detection result by the light detection unit and quantifies the component to be quantified in the biological sample;
The analysis device includes a first storage unit that stores the biological sample, a second storage unit that stores an aqueous solution for dilution used for diluting the biological sample, and the biological sample. A flow path for transferring the aqueous solution for dilution, a mixing unit for mixing the biological sample and the aqueous solution for dilution, and a holding unit for holding a certain amount of the biological sample diluted in the mixing unit And an analysis unit that reacts the biological sample diluted in the mixing unit with a reaction reagent. When rotated by the rotation driving unit, the biological sample is caused by centrifugal force and capillary force of the flow path. Transport and mix the target sample and the aqueous solution for dilution,
The light generation unit irradiates light of the first light source to the aqueous solution for dilution, and the light of the first light source to the mixed solution of the biological sample and the aqueous solution for dilution; Irradiating with light from a second light source having a wavelength different from that of the light from the first light source;
The light detection unit is configured to detect the absorbance of the indicator in the aqueous solution for dilution with respect to the light of the first light source, the light of the first light source, and the light of the second light source from the light transmitted through the analysis device. Detecting the absorbance of the indicator in the mixed solution with respect to light;
The processing unit is configured to absorb the light of the indicator in the mixed solution with respect to the light of the second light source, the light of the first light source in the specific component including chyle in the biological sample, and the second The absorbance of the specific component is obtained from the wavelength-dependent characteristics of the light from the light source, the absorbance of the indicator in the aqueous solution for dilution with respect to the light from the first light source, and the mixed solution with respect to the light from the first light source. A dilution ratio deriving means for determining a dilution ratio of the biological sample with the dilution aqueous solution based on a value obtained by subtracting the absorbance of the specific component from the absorbance of the indicator
An analyzer characterized by that.

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