JPH0635948B2 - Absorption type automatic analyzer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a change in absorbance of a reaction solution prepared in a reaction vessel by circulating a plurality of reaction vessels that also serve as measurement cells, and an enzyme activity unit based on the absorbance. Also, the present invention relates to a technique for preventing the calculation error of the concentration conversion coefficient based on the optical path length difference of the measurement cell or the calculation coefficient of the enzyme activity unit conversion coefficient in the absorption type automatic analyzer for measuring the concentration of the target component.

(Prior Art) In an analyzer using absorbance as a measurement parameter, a mixed solution of a sample and a reagent is housed in a reaction container, and the intensity of light transmitted through the reaction container that also serves as the measurement cell is measured. The intensity of the emitted light depends not only on the concentration of the mixed solution but also on the optical path length of the reaction container, that is, the total optical path length including the liquid thickness and the size in the optical path direction.

Therefore, a plurality of samples while circulating the plurality of reaction vessels and reusing the reaction vessels, in an absorbance analyzer that processes different analysis items, the same amount of pure water as the sample prior to the analysis work, Alternatively, a reagent blank solution prepared by mixing a zero-concentration standard solution containing no target component and an analytical reagent, and a standard prepared by mixing a standard solution containing the target component of a known concentration in the same amount as the sample and an analytical reagent. using the sample reaction mixture, the absorbance in 1 group of the group 1 comprising a plurality of reaction vessels - as shown in FIG. 5 was prepared a predetermined amount of reagent blank solution a _b1, a _b2 ‥‥ a _b4 In addition, a certain amount of standard sample reaction solution was prepared in the group 2 consisting of the following 1 to a plurality of reaction vessels, and the absorbances A _s5 , A _s6 ...
Measuring the A _s8, the average absorbance of the reagent blank solution And average absorbance of standard sample reaction solution Difference from Was calculated, and the concentration (enzyme activity unit) was calculated from the ratio of this to the concentration of the target component in the standard solution.

According to such a method, since the reaction vessel is made with high accuracy so that the optical path lengths are as uniform as possible, it is possible to analyze a component having a large difference in absorbance per unit concentration of the target component. Indicates that the measurement accuracy is practically sufficient.

However, even though there is a slight difference between the individual reaction vessels, there are variations in the optical path length, so the reagent blank solution and the standard sample reaction solution have high absorbance and a small difference in absorbance per unit concentration, for example, GOT and GPT
In the determination of the transaminase enzyme activity unit conversion factor such as, there was a case in which a non-negligible error occurred.

That is, when 80 reaction vessels made of hard glass (nominal optical path length 6 mm) used for practical analysis were sampled, 0.8 ml of the same dye solution was dispensed into each sample and the absorbance was measured for each. Became:

Average value (): 1.644 (ABS) Coefficient of variation (CV): 0.227% Minimum absorbance: 1.637 ABS Maximum absorbance: 1.651 ABS From this result, the ratio of the difference from the average value, that is, the variation coefficient (CV) is 0.227%. The difference between the minimum absorbance and the maximum absorbance, that is, the maximum fluctuation range is
[(1.651-1.637) /1.644] x 100 = 0.85%.

The effect of this maximum fluctuation range on the measurement of enzyme activity units will be discussed using GOT analysis as an example.

GSCC (German Society of Clinical Chemistry), a typical GOT analysis method
Method and IFCC (International Society for Clinical Chemistry) method, the final absorbance of the reagent blank solution is 1.3 to 1. at the measurement wavelength of 340 nm.
Since the range of 4ABS is adopted, 1.4ABS as a calculated value
To adopt.

The ratio of the amount of reagent solution (TV) to the amount of sample (SV) is not always specified, but since the range of SV / TV = 1/25 to 1/40 is generally used, it is a calculated value. As SV / TV = 1/30 is adopted.

Furthermore, as the concentration of the pyruvine standard solution used for obtaining the enzyme activity unit conversion coefficient (K) of GOT, 2 mmoles,
1 millimol and 0.5 millimol were adopted, and the influence on the enzyme activity unit conversion coefficient (K) at each of the above concentrations was calculated by setting the fluctuation range of the optical path length of the reaction vessel to 1%, that is, ± 0.5%.

I. When the concentration of pyruvic acid in the standard solution is 2 millimolar: Fluctuation range of the absorbance when measuring the absorbance of the reagent blank solution (absorbance 1.4 ABS) 1.4 × (100 ± 0.5) /100=1.393 〜 1.407 (ABS) Fluctuation range of absorbance during absorbance measurement 1.4- △ C × ε × (SV / TV) × l = 1.4- (2 × 10 ^-3 ) × (6.30 × (10 ³ ) × 1.0 = 0.980 (ABS) However, △ C corresponds to the decreasing concentration of NADH caused by the LDH reaction of pyrupic acid and NADH in the GOT reagent, that is, the molar concentration of pyruvic acid.

ε is the molecular extinction coefficient of NADH SV / TV is the volume ratio of the reagent blank solution to the standard sample reaction solution l is the optical path length of the reaction container, 1.0 cm in this calculation example Fluctuation range of absorbance in this standard sample reaction solution Is 0.98 x (100 ± 0.5) / 100 = 0.975 to 0.985 (ABS) Effect of fluctuation range of optical path length of reaction vessel on enzyme activity unit conversion coefficient (K) K = [concentration of standard solution C (micromol / liter )] / [Absorbance of standard sample reaction solution-absorbance of reagent blank solution] = (2 x 10 ³ ) / [(0.975 to 0.985)-(1.393 to 1.407)] = (2 x 10 ³ ) / (-0.408 to 0.432) = -4.902 to -4.630 x 10 ³ Comparing this value with K (theoretical value) = (2 x 10 ³ ) /0.42 = -4.762 x 10 ³ [(4.902-4.630) /4.762] x 100 = The fluctuation range is 5.71%.

II. When the concentration of pyruvic acid in the standard solution is 1 mmol, the fluctuation range of the absorbance when measuring the absorbance of the standard reaction solution The absorbance of the standard sample reaction solution is 1.4- (1 × 10 ^-3 ) × (6.30
× 10 ³ × (1/30) × 1.0 = 1.19 (ABS) The absorbance fluctuation range (range) is 1.19 × (100 ± 0.5) / 100 =
1.184 to 1.196 (ABS) The fluctuation range of the optical path length of the reaction vessel is the enzyme activity conversion coefficient (K)
Effect on K = 1 x 10 ³ /[(1.184 ~ 1.196)-(1.393 ~ 1.407)] = 1 x 10 ³ /(0.197 ~ 0.223) = -5.076 ~ -4.484 x 10 ³ K (theoretical value) =- Compared with 4.762, [(5.076-4.484) /
4.762] × 100 = 12.43% fluctuation range.

III. When the concentration of pyruvic acid in the standard sample solution is 0.5 mmol, the absorbance of the standard reaction solution is 1.4- (1 × 10 ³ ) × (6.30 × 10
³ x (1/30) x 1.0 = 1.295 (ABS) The absorbance fluctuation range (range) is 1.295 x (100 ± 0.5) / 1
00 = 1.289-1.301 (ABS) Effect of fluctuation range of optical path length of reaction vessel on enzyme activity unit conversion coefficient (K) K = 0.5 × 10 ³ /[(1.289-1.301)-(1.393-1.407)]=0.5 × 10 ³ /(-0.092 to -0.118) = − 5.435 to −4.237 × 10 ³ Comparing this with K (theoretical value), the fluctuation range is [(5.435-4.237) /4.762] × 100 = 25.16%. .

Thus, there is a problem that the thinner the concentration of the standard solution, the more marked the influence of the difference in optical path length between the reaction vessels on the enzyme activity unit conversion coefficient (K).

The present invention has been made in view of such a problem, and its object is that the absorbance of both the sample blank solution and the reaction solution is high because the influence of the optical path length existing between the reaction vessels is likely to be manifested. In the analysis of items with a small difference in absorbance per unit concentration, for example, in determining the transaminase enzyme activity value conversion factor (K) of GOT, GPT, etc., it is possible to eliminate the factor of K variation due to the variation of the optical path length as much as possible. An object is to provide an absorption type automatic analyzer.

(Means for Solving the Problem) In order to solve such a problem, in the present invention, a plurality of reaction vessels that also serve as measurement cells are accommodated and the reaction vessels are used as a sample dispensing station, a reagent dispensing station, and an absorbance. A reaction container transfer means for circulating the measurement station and the washing station, a reaction container detection means for detecting that the reaction container has reached the sample dispensing station, and a first calibration container for calibration during the first transfer of the reaction container. The first liquid and the analytical reagent, and the second liquid for calibration and the analytical reagent during the second transfer are respectively injected, and the absorbance is stored in association with each reaction container of the first liquid / analytical reagent reaction liquid. When the absorbance of each reaction container due to the second liquid / analytical reagent reaction liquid is measured, the absorbance of the first liquid / analytical reagent reaction liquid stored in the storage device is read out. A means for calculating the difference in absorbance and calculating the concentration conversion coefficient is provided.

(Function) Since the difference in absorbance between the standard solutions of the first solution and the second solution measured in the same reaction vessel is used, an extreme combination of optical path length differences existing between the reaction vessels, that is, the maximum optical path length It is possible to measure with the original accuracy of the device, because it is possible to prevent the difference in absorbance that has a large error between the one having the minimum optical path difference and the one having the minimum optical path difference from being included in the calculation of the concentration (enzyme activity unit) conversion coefficient. Becomes

(Example) Therefore, the details of the present invention will be described below based on an illustrated example.

FIG. 1 shows an embodiment of the present invention, in which reference numeral 1 is a reaction disk, and a plurality of reaction vessels 2, 2, ...
Are accommodated, and each of the reaction vessels 2, 2, 2, ... Is cyclically moved through the sample dispensing station 3, the reagent dispensing station 4, the absorbance measuring station 5, and the washing station 6. Between the injection station 3 and the absorbance measurement station 5, the reaction vessel 2,
.. are kept at a constant temperature.

Reference numeral 7 is a reaction container detector arranged at the sample dispensing position, and the signal from this is input to a control device 8 constituted by a microcomputer. Reference numeral 9 denotes the above-mentioned data storage device, which has at least the same storage area as the number of the reaction vessels 2, 2, 2, ... Stored in the reaction disk 1 as shown in FIG. It is configured to store the measured absorbance, for example, the absorbance of the reagent blank solution.

Next, the operation of the apparatus thus configured will be described based on the flow chart shown in FIG.

When the device is set to the calibration mode, the control device 7 confirms that it is now facing the reaction container detector 7 (step a), confirms that this reaction container 2 is the first one, and sequentially, The addresses of the second and subsequent reaction vessels 2, 2, 2, ... Are assigned to the data storage device 9.

In this way, following the identification work for each of the reaction vessels 2, 2, 2, ...
The control device 8 distributes the respective predetermined amounts of the first standard for calibration and the analytical reagent, which are installed in advance in the sample dispensing station 3, into the identified reaction vessels 2, 2, 2 ... (From FIG. 4). It is poured (steps C and C), and these reaction vessels 2, 2, 2, ... Are moved to the absorbance measurement station 5 while maintaining a constant temperature (Step E). First reaction container 2 containing this first standard sample standard reaction solution
Arrives at the absorption measurement station (step e),
The absorbance A _{b1 of} this is measured (step F). The absorbance A _b1 is stored in association with the first area of the data storage device, that is, the order of the reaction vessels (step
G). Hereinafter, the second step by steps (a) to (h),
The same process is performed on the third reaction vessel
Absorbance A _b2, A _b3 ‥‥ A _bn for the standard reaction solution is measured and stored in the data storage device 9 Te with corresponding transfer order of the reaction vessel 2,2,2 ‥‥.

In this way, the reaction vessels 2, 2, 2, ... Out of the absorbance measurement station are washed and dried in the washing station 6 and then put into a reusable state.
The reusable reaction vessels 2, 2, 2 ... Return to their original positions and face the reaction vessel detector 7. Of course, the reaction vessels 2, 2 housed in the reaction disc 1
Since the number of 2 ... Is known, the control device 8 recognizes that the first reaction container 2 is located at the sample dispensing station 3 (steps, re).

At this stage, the control device 8 prepares a predetermined amount of each of the second standard solution for calibration and the analytical reagent in the first reaction container 2 (FIG. 4) in the sample dispensing station 3 (step, (2), and is again transferred to the absorbance measuring station 4 (step E). This operation is also performed on the second and subsequent reaction vessels 2, 2, ... (FIG. 4). Absorbance A _s1 for the first reaction vessel 2
When is measured (stepwa, power), control device 8
Is the absorbance A _b1 of the first standard sample reaction solution of the first reaction container measured last time from the data storage device 9.
Is read out and the difference between them (A _s1 −A _b1 ) is calculated (step yo).

In this way, the difference (A _s2
-A _b2 ), (A _s3 -A _b3 ), ... (A _sn -A _bn ) at the stage when the measurement is completed (step), the average value (As-Ab) = [(A _s1 -A _b1 ) + (A _s2 −A _b2 ) ⁺ ‥‥
⁺ (A _sn −A _bn )] / n is calculated (step), and based on this average value, the concentration (enzyme activity unit) conversion coefficient K = C /
(AS-Ab) is calculated (step S).

According to this, the calculated absorbance difference (A
Since S-Ab) is within the fluctuation range of the reaction container, the fluctuation range of the concentration (enzyme activity unit) conversion coefficient (K) also falls within the fluctuation range of the optical path length of the reaction container itself. The error due to the difference in optical path length is not remarkably increased, and the measurement accuracy of the device itself and that of the reaction container can be exhibited.

In this example, the difference in absorbance in each reaction container is determined, and the concentration (enzyme activity unit) conversion coefficient is calculated based on the average value thereof. It is clear that the same effect can be obtained even if the conversion coefficient of is calculated and the average value thereof is used.

(Effects of the Invention) As described above, in the present invention, a plurality of reaction vessels that also serve as measurement cells are accommodated, and the reaction vessels are circulated through a sample dispensing station, a reagent dispensing station, an absorbance measuring station, and a washing station. Reaction container transfer means, a reaction container detection means for detecting that the reaction container has reached the sample dispensing station, a first liquid for calibration and an analytical reagent at the first transfer of the reaction container, and At the time of the second transfer, the second liquid for calibration and the analytical reagent are separately dispensed, and the absorbance is stored in the storage means in association with each reaction container for the reaction liquid of the first liquid / analytical reagent, and the second liquid. When the absorbance of each reaction container due to the analysis reagent reaction solution is measured, the absorbance of the first solution / analysis reagent reaction solution stored in the storage means is read to obtain the difference in absorbance, and the concentration is changed. Since the means for calculating the arithmetic coefficient is provided, the absorbance difference between the first and second liquids is measured in the same reaction container, and the concentration (enzyme activity within the range of the optical path length difference caused by the manufacturing error of the reaction container is measured). (Unit) A conversion coefficient can be obtained, an unexpected large error can be prevented from being mixed, and an analysis result having high measurement accuracy and reliability can be obtained.

[Brief description of drawings]

FIG. 1 is a block diagram of an apparatus showing an embodiment of the present invention, FIG. 2 is a schematic view showing an embodiment of a data storage device used in the apparatus of FIG. 1, and FIGS. FIG. 5 is an explanatory diagram showing a conventional calibration method for the absorption type automatic analyzer, which is a flowchart and an explanatory diagram showing the operation. 1 ... Reaction disk, 2 ... Reaction container 3 ... Sample dispensing station 4 ... Reagent dispensing station 5 ... Absorbance measuring station 6 ... Washing station 7 ... Reaction vessel detector 9 ... Data storage device

Claims (1)

[Claims] 1. A reaction container transfer means for accommodating a plurality of reaction containers also serving as measurement cells and circulating the reaction container through a sample dispensing station, a reagent dispensing station, an absorbance measuring station, and a washing station, and a reaction container. A reaction container detecting means for detecting arrival at the sample dispensing station, a first liquid and an analytical reagent for calibration during the first transfer of the reaction container, and a first solution for calibration during the second transfer. Two liquids and an analytical reagent are separately dispensed, and the absorbance is stored in the storage means in association with each reaction container for the first liquid / analytical reagent reaction liquid, and each reaction container for the second liquid / analytical reagent reaction liquid is stored. When the absorbance of is measured, the absorbance of the first liquid / analytical reagent reaction solution stored in the storage means is read to obtain the difference in absorbance and the concentration conversion coefficient is calculated. Dynamic analysis apparatus.