JPS647343B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an automated chemical analysis method, and particularly to an automated chemical analysis method suitable for quantifying a specific component in a biological sample such as serum.

Conventionally, automatic chemical analyzers mainly used for clinical tests have performed two measurement methods. One method is to mix the sample and reagent, conduct a reaction, and then measure the absorbance after a certain period of time.
It is called a colorimetric analysis method, a one-point method, an end-point method, etc., and typical analysis items include the enzymatic method for cholesterol and the Buillet method for total protein. The second measurement method is a method that continuously tracks the change in absorbance after adding a trigger reagent or a starting solution called a starter to the sample solution.
It is called a reaction rate measurement method, initial rate method, rate analysis method, kinetic method, etc., and typical analysis items include glutamate oxaloacetate transaminase and lactate dehydrogenase.

In both the first method (hereinafter referred to as the endpoint method) and the second method (hereinafter referred to as the rate method), the concentration of a specific analysis item or the enzyme activity value is measured using a photometer of an analyzer. When the absorbance was so high that it exceeded the range, the sample had to be diluted with physiological saline, etc., and the sample had to be transferred to the reaction vessel, reagents added, and observed again.
Particularly in patient serum used in clinical tests, the concentration or activity value of a specific component often becomes abnormally high due to illness or the like, and the above-mentioned problems often occur even in routine analysis. Furthermore, samples such as patient serum are often obtained in only the minimum amount required for one analysis, and once the sample has been subjected to a chemical reaction, it may be necessary to dilute the remaining sample depending on the result. There were some things that were possible.

In addition, there are the following problems particularly with the rate method. Lactate dehydrogenase (LDH), which is a typical analysis item using the rate method, is measured using the following reaction.

Pyruvate + NADH ₂ LDH --→ Lactic acid + NAD During the above reaction activated by lactate dehydrogenase, the process in which NADH ₂ , which absorbs at 340 nm, changes to NAD, which does not have absorption at 340 nm, is monitored using a photometer. can determine the activity value of lactate dehydrogenase, but if the sample is serum with a very high activity value of lactate dehydrogenase, pyruvate or NADH ₂ will be consumed within a short time. However, there were times when I lost observation opportunities. In this case as well, the sample had to be diluted with physiological saline or the like and analyzed again, which resulted in a long analysis time and a shortage of samples.

As mentioned above, when measuring a highly concentrated or highly active sample, it is necessary to dilute the sample or reagent concentration depending on the performance of the photometer being used (linearity of the calibration curve, measurement range, etc.). Although it is essential in routine testing to dilute a sample according to There are problems in that there is a risk of sample waste and sample mix-up.

An object of the present invention is to provide an automatic chemical analysis method that allows automatic re-measurement without using a new reaction vessel.

The present invention reduces the time required for testing by making it possible to dilute samples in the reaction container of an automatic chemical analyzer, whereas conventionally high concentration and high unit samples were diluted manually and retested. The aim is to shorten the time, reduce the amount of sample required, and prevent sample mix-ups. That is, the present invention focuses on the fact that samples of automatic chemical analyzers often have high concentrations and high units.After measuring a reaction solution, if dilution and remeasurement are necessary, the reaction solution is Water or reagents are added to the sample to enable measurement to be carried out again within an appropriate absorbance range.

Examples of the present invention will be described below.

FIG. 1 shows an embodiment of the invention.

In the figure, sample container 1 on sample table 14
2, the sample table rotates around the rotation axis 13, and when it reaches the sampling position,
It is pipetted by the probe 10 and discharged into the reaction container 7 on the reaction table 1. The reaction table 1 rotates to the right around the rotating shaft 2 while repeating clockwise and counterclockwise rotations for removal. When the reaction container 7 containing the sample comes to a specific position, the reagent 4 is added by the dispenser 3 and the reaction is started. When the reaction container 7 comes to the measurement position, the light from the lamp 5 passes through the reaction container 7 which also serves as a light absorption cell, enters the photometer 6, and the absorbance data is determined as the absorbance value stored in the absorbance determination device 33. In the following cases,
It is calculated by the data processing device 34 and printed out by the printer 35 as an analysis result. After the measurement has been completed, the reaction vessel 7 is drained of unnecessary reaction liquid by the draining device 8, washed by adding pure water from the washer 9, sucked in pure water again, and left in a dry state. Subjected to the next analysis.

Since it is configured like this, now,
In the analysis of inorganic phosphorus in urine, if the concentration of inorganic phosphorus is extremely high, as shown in Figure 2,
The absorbance value at the first measurement point 17 is the absorbance limit value 20, which is stored in the absorbance judgment device in advance.
In comparison, when the absorbance value measured for the first time is equal to or higher than the absorbance limit value, the reaction container 7 is not cleaned, the reaction table 1 rotates counterclockwise, and when the reaction container comes to the diluent addition point 18, the absorbance increases. A diluent 16 is added from the dilution dispenser 15 in response to a signal from the determination device. In the case of this inorganic phosphorus, distilled water was used as the diluent. The concentration of the diluted reaction solution is measured for absorbance again at the second measurement point 19, and the absorbance at that time is judged by the absorbance judgment device 33 as to whether it is above the set value, and if it is below the absorbance limit value, the data is processed. It is printed out after calculation by the device. The concentration of the analyte in the sample determined from the diluted reaction solution is determined in the data processing device using the following calculation formula.

C _D = K × A _D × (V _A + V _I ) / V _I ... (1) C _D : Concentration (mg/dl) K: Concentration conversion constant before dilution (mg/dl) Abs A _D : After dilution Absorbance of V _I : Volume of reaction solution before dilution V _A : Volume of diluted solution added The following constants were used for the actual input data.

K = 87.3 (mg/dl)/Abs V _I = 1.26 ml _VA = 0.6 ml A _D value was 0.709.

From the above, C _D = 192 mg/dl. The absorbance A _I before dilution is determined as follows.

A _I =C _D /K (2) The absorbance before dilution was 2.20, indicating that it exceeded the absorbance limit of 1.8 of the photometer used in this example.

Another embodiment of the invention is shown in FIG. This embodiment differs from the embodiment shown in FIG. 1 in that a reaction liquid suction pipette 27 is provided. This third
A case where a high level of lactate dehydrogenase (hereinafter referred to as LDH) in serum is explained using an illustrated example.

Pyruvate + NADH ₂ LDH ---→ Lactic acid + NAD Samples of serum and NADH ₂ are mixed and incubated at 37°C. The reaction is started by adding pyruvate, which is called a trigger, and the reaction is measured per unit time using a photometer. Measure the amount of decrease in _NADH2 and LDH
Convert to activity value. As shown in Figure 4, in the case of high unit LDH serum measurement, after trigger addition 21.
NADH ₂ rapidly decreases and is completely consumed before reaching _the first rate measurement range 22, making it impossible to accurately measure the activity value. Conventionally, a reaction limit absorbance 26 is set, and when the measured absorbance is below this absorbance, an absorbance judgment device determines that
It started issuing a warning that it could not be measured. In this embodiment, when the absorbance is below the reaction limit, the reaction table 1 is rotated clockwise by a signal from the absorbance judgment device 33, and all but one part of the reaction liquid is discharged from the reaction container 7 through the reaction liquid suction pipette 27. Thereafter, the reaction table 1 is rotated counterclockwise and NADH ₂ is added again from the dispenser 3 at the second reagent addition point 23 as shown in FIG.
At the second trigger addition point 24, the diluent 16 is added from the dilution dispenser 15.
(in this case the diluent will be the pyruvate reagent). By this operation, the reaction starts again, but this time the sample serum is diluted, so even in the second rate measurement area 25 shown in Figure 4, it is possible to measure at a reaction limit absorbance or higher. .
Further, when the absorbance in the absorbance determination device 33 is equal to or higher than the reaction limit absorbance, the activity value of LDH in the sample determined from the diluted reaction solution is determined in the data processing device using the following calculation formula.

U _D =K ¹ ×ΔA _D × {(Vi−Vo)+V _R +V _T }/Vi−
Vo (2) U _D : Activity value (IU) K ¹ : Unit conversion constant before dilution
IU/(ΔAbs/min) ΔA _D : Change in absorbance per minute ΔAbs/min Vi: Volume of reaction solution before dilution Vo:
Amount of reaction liquid discharged by reaction liquid suction pipette _VR :
Amount of NADH ₂ reagent added after discharging the reaction solution V _T : Amount of trigger reagent added for the second time Actual experimental data is as follows: K ¹ = 6429 IU/(ΔAbs/min) Vi = 1.35 ml Vo = 1.0 ml V _R = 1.0 ml V _T = 0.3 ml ΔA _D was 0.107 ΔAbs/min.

From the above, U _D =3250IU.

According to this example, even in enzyme activity analysis such as LDH analysis, the sample can be diluted without changing the reagent concentration, making it possible to follow the enzyme reaction under optimal reaction conditions. .

Further, FIG. 5 shows an example in which inorganic phosphorus in urine is measured using the embodiment shown in FIG. In other words, if the absorbance at the first measuring point 28 exceeds the absorbance limit value 20C in the measurement of inorganic phosphorus in urine, after discharging the reaction liquid with the reaction liquid suction pipette, the diluting liquid is added at the dilution liquid addition point 29. Add distilled water using the dispenser 15 and measure the absorbance at the second measurement point 30. If the absorbance exceeds the limit value again, repeat the above-mentioned draining and dilution.
Using the data when the absorbance falls within the absorbance limit value, the data processing device calculates the concentration from the number of dilutions and the absorbance value and prints it out.
The concentration of the analyte in the sample determined from the diluted reaction solution can be expressed as follows.

C _D = K x A _D x {(Vi-Vo) + V _A / (Vi-Vo)} ⁿ (3) C _D : Concentration (mg/dl) K: Concentration conversion constant before dilution (mg/dl)/ Abs A _D : Absorbance after dilution Vi:
Volume of reaction solution before dilution Vo: Volume of reaction solution discharged V _A : Volume of diluted solution added n: Number of dilutions The actual experimental data is as follows: K = 87.3 (mg/dl) Abs A _D = 1.7 V _I = 1.41ml Vo=0.4ml V _A =0.4ml n=3 From the above, C _D =404mg/dl. The absorbance A _I before dilution is determined as follows.

A _I =C _D /K The absorbance before dilution was 4.63, which far exceeded the absorbance limit of 1.8 used in this experiment. According to this example, even a reaction solution with extremely high absorbance can be measured within the measurement range of a photometer by repeated dilution.

Therefore, according to this example, high concentrations, which conventionally could only be analyzed by diluting the sample and re-measuring,
A high-unit sample can be automatically diluted within a single analysis process to a range where absorbance can be measured and converted into concentration or units.

As explained above, according to the present invention, re-measurement can be automatically performed without using a new reaction vessel, which not only shortens the time required for re-measurement, but also makes it possible to automatically perform re-measurement without using a new reaction vessel. This has the advantage that it is not necessary to supply a reaction vessel.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing an embodiment of the present invention, FIG. 2 is a diagram showing absorbance changes according to the embodiment shown in FIG. 1,
FIG. 3 is a configuration diagram showing another embodiment of the present invention, and FIG.
The figures are diagrams showing changes in absorbance according to the embodiment shown in FIG. 3, and FIG. 5 is a diagram showing changes in absorbance according to the embodiment shown in FIG. 3. 1... Reaction table, 3... Dispenser, 4... Reagent, 5... Lamp, 6... Photometer, 7... Reaction container, 8... Inhaler, 9... Washer, 15... Dilution dispenser, 27...Reaction liquid suction pipette, 33...
...Absorbance determination device.

Claims (1)

[Claims] 1 In an automatic chemical analysis method in which a reaction solution of a sample and a reagent is generated in a reaction container and the reaction solution is photometered with a photometer to quantify specific components in the sample, photometry of the reaction solution contained in the reaction container is used. A judgment device determines whether the value exceeds a preset limit value, and when the photometric value of the reaction solution exceeds the above limit value, the reaction device after photometry is filled with diluent or reagent solution. Transfer to the addition position, add a predetermined amount of the diluent or reagent liquid to the reaction container containing the reaction liquid at the diluent or reagent addition position, and then position the reaction container at the photometry position again. An automatic chemical analysis method characterized by photometry.