JP3064645B2 - Sample dispensing confirmation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing a specimen sample derived from a body fluid such as plasma or serum, such as an infectious disease test by agglutination, a cross-match test, or an allergy test. More specifically, the present invention relates to a method for dispensing these sample samples into a reaction vessel and reacting the sample with an analysis reagent to perform analysis, and confirming whether or not the sample sample has been correctly dispensed.

[0002]

2. Description of the Related Art Conventionally, from the viewpoint of a useful index for prevention or treatment of infectious diseases, biochemical analysis such as infectious disease inspection or cross-compatibility test utilizing agglutination reaction, or enzyme immunoassay ( Immunological analysis such as EIA) has been performed.
Generally, these analyzes are performed by dispensing a specimen sample such as a body fluid and an analysis reagent into a reaction vessel of an analyzer, mixing and reacting. However, no matter how accurate these analytical methods are, if the dispensing of the specimen sample is not performed correctly as expected, a determination error may result. For example, a healthy person is determined to be abnormally low, an infectious disease-positive person (non-healthy person) is determined to be an infectious disease-negative person (normal person), or a blood group is determined to be compatible despite being incompatible. . Frequent occurrence of such a determination error may lead to transmission of an infectious disease or blood transfusion error due to blood group incompatibility. Therefore, in such a test, it is important to confirm whether or not the sample has been accurately dispensed into the reaction container.

[0003] In an analysis using plasma or serum as a sample, the difficulty of dispensing confirmation is particularly problematic. For example, ABO blood group tests include a frontal test for blood cells and a back test for plasma. In the frontal test for blood cells, blood cells are separated when agglutination is determined. It is easy to determine whether or not it has been injected. However, such a plasma dispensing cannot be confirmed by a back test in which plasma is a test target. Similarly, in the case of an infectious disease test using plasma as a sample, the dispensing of the sample cannot be confirmed. Therefore, it is necessary to always perform a test in consideration of the occurrence of false negative. Therefore, especially in the analysis using plasma or serum as a sample, it is desired to develop a method capable of reliably determining whether or not sample dispensing is appropriate.

[0004] As a conventional sample dispensing confirmation method, first,
A method for photoelectrically confirming dispensing described in JP-A-59-17161 is known. This was devised as one of the methods for confirming the dispensing of a specimen at the time of PK7000 series blood group test and infectious disease test.
However, in this method, the amount of the sample to be dispensed is confirmed in the dilution step which is the pretreatment of the sample, and therefore, the amount of the sample actually involved in the reaction cannot be known. Japanese Patent Application No. 3-217736 (not disclosed at the time of filing the present application)
A dispensing confirmation method using the pH buffering ability of the specimen itself is described. This method takes advantage of the relatively large buffering capacity of body fluids, and when a predetermined amount of a diluent of a pH indicator is added to a reaction vessel before the reaction, if the sample has been dispensed, the pH of the diluent will increase. It detects the dispensing failure based on the change and the change of the color of the indicator. However, this method is difficult to apply to an analysis using a diluent having a larger pH buffer capacity than a specimen, such as an infectious disease test.

[0005] On the other hand, based on the fact that the specimen contains protein, it is possible to confirm the dispensed amount using the protein as an index. However, in immunological analysis, it is inappropriate to confirm the dispensed volume using the protein amount as an index after mixing the sample and the reagent because the reagent component contains antigen or antibody protein. is there. Also, it is not economical to separately add an indicator for the purpose of confirming the dispensed amount of the sample.

[0006]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to reduce the influence of a sample or reagent component by using, as an index, a component that is necessarily contained in a sample derived from a body fluid such as serum or plasma. It is an object of the present invention to provide a method for confirming the suitability of sample dispensing without receiving it.

[0007]

Means for Solving the Problems To solve the above-mentioned problems, the present invention uses, as an index, a stabilizing agent which is generally required to be added to a sample sample prepared from a body fluid to prevent its denaturation, After the analysis was completed, it was decided to confirm the dispensing by detecting this stabilizer.

That is, according to the present invention, a sample sample derived from a body fluid is dispensed into a reaction container and reacted with an analysis reagent to analyze an analyte in the sample sample. In the method for confirming the suitability of dispensing, a step of adding a stabilizer to the specimen sample in advance, and the presence or absence and / or amount of the stabilizer using a stabilizer detection reagent after the reaction is completed. A sample dispensing confirmation method, comprising the steps of: Hereinafter, the present invention will be described in detail.

First, the analysis of a specimen sample on which the method for confirming dispensing of the present invention is based will be briefly described. This analysis includes all types of analysis performed by dispensing a sample sample derived from a body fluid and an analysis reagent for analyzing the sample in a reaction container of the analyzer, and mixing and reacting. For example, an infectious disease test by agglutination, a cross-match test and an allergy test, or an immunological test, etc.

[0010] Any of the components present in the body fluid can be used as the specimen sample in the above analysis, and examples thereof include blood cells, plasma, serum, and urine.
However, one of the advantages of the present invention is that it is possible to accurately confirm the dispensing of plasma or serum, which was not possible in the past. Therefore, when plasma or serum is used, the effect of the present invention can be reduced. Notable.

The analytical reagent used in the above analysis is not particularly limited, and various reagents are used depending on the purpose of the analysis. For example, in a cross-matching test which is one of the pre-transfusion tests, an anti-A antibody and an anti-B antibody are used to examine a blood type. Further, as shown in Examples described later, an anti-HB antibody is used in the hepatitis B virus test.

The reaction in the above analysis is not particularly limited.
For example, any conventionally used reaction such as an agglutination reaction or an enzyme immunoassay (EIA) can be used.
A preferred analytical reaction is an agglutination reaction. For the agglutination reaction,
It is mainly classified into three types: direct agglutination reaction, indirect agglutination reaction, and direct passive agglutination reaction. In addition, immunoadhesive hemagglutination reaction and reverse passive agglutination reaction (hereinafter referred to as "RP
HA ”). All of these agglutination reactions are preferable as the analysis reaction used in the present invention.

A feature of the present invention is that, in the above analysis, a stabilizing agent added in advance to the sample sample is used as an index for confirming whether or not the sample sample is correctly dispensed. It is to measure the presence and / or amount of the stabilizer after the completion of the reaction. Hereinafter, the sample dispensing confirmation method of the present invention will be described in detail. First, a stabilizer is added to the sample before dispensing the sample into the reaction container.

The stabilizer in the present invention is added to prevent physical or chemical denaturation of the sample, and has the following stabilizing effect in addition to having the intended stabilizing action. Any one that satisfies the two requirements can be used. First of all, in the body fluid, it is not contained at all, or even if it is contained,
It must be free from variations in the content due to individual differences, or must be much smaller than the amount added.
Second, it must be inert to body fluid components and have no effect on component analysis. And third, there must be a way to specifically analyze that substance.

In view of the above, a blood coagulation inhibitor is preferably used as the stabilizer of the present invention. This is because a blood coagulation inhibitor is a stabilizer that satisfies the above three requirements, and is naturally required to collect blood cells to obtain plasma.

As a blood coagulation inhibitor, heparin, EDTA (ethylenediaminetetraacetic acid) which is a chelator, or various citrates can be used. Especially, it is preferable to use EDTA and citrate which are inexpensive and easy to handle.

In the present invention, citric acid [HO.C. (COOH). (CH ₂ CO 3)
OH) ₂ ], which is preferably used particularly in a sample in which the life of the blood must be considered. In general, citric acid exists widely in nature and is contained in human blood in the form of citrate ion in the form of 13 to 27 μg / ml (three kinds of citrate ions are theoretically considered. However, these are hereinafter collectively referred to as citric acids).

Citric acids are mainly used as an anticoagulant (such as an anticoagulant) such as an ACD solution containing the above as a main component or a CPD solution which is frequently used in pre-transfusion tests such as blood banks and the Red Cross. (Mixture). Citric acids require a much larger amount than blood to prevent blood coagulation. The quantitative ratio of anticoagulant to blood is determined, and the ratio is 14 parts by volume of CPD solution to 100 parts by volume of blood. This ratio is generally recommended, and many clinics adhere to it. Next, after the analysis of the test sample is completed, the presence or absence of a stabilizer is determined using a stabilizer detection reagent, and / or
Or measure the amount.

The method for measuring the presence and / or amount of the stabilizer, and the use of the reagent for detecting the stabilizer, differ depending on the type of the stabilizer used. For example, when using citric acid as a stabilizer,
As described in Examples below, a glycylglycine buffer solution contains malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and reduced nicotinamide dinucleotide (NADH). And a second reagent containing citrate lyase in a glycylglycine buffer.

This reagent for detecting a stabilizer may be used for the following measurement immediately after preparation or may be stored. The storage is preferably performed at a low temperature in consideration of the stability of the reagent.

A method for measuring the presence or absence and the amount of citric acid used as a stabilizer by using the first reagent and the second reagent is to replace the amount of the stabilizer with the amount of NADH and to reduce the amount of NADH. It is to be measured. That is,
When the first and second reagents are added after the analysis reaction and the decomposition reaction of citric acid is performed by the action of citrate-degrading enzyme, the amount of NADH decreases according to the degree of progress of the enzyme reaction. That is, the amount of citric acid contained as a stabilizer is N
It will be replaced by the decrease in ADH. Therefore, before and after the reaction by the decomposing enzyme, 3
The amount of decrease in NADH, that is, the content of citric acid, can be measured by a method of measuring the absorbance value at 40 nm and calculating the amount of change in absorbance to prepare a calibration curve. The reaction with the enzyme is preferably performed at a constant temperature such as 37 ° C. or room temperature. This is because the reaction rate of the enzyme reaction is somewhat affected by the temperature change.

The NADH change amount is determined by diaphorase,
And phenazine methosulfate (PMS)
A method of changing to formazan which is a tetrazolium salt can also be applied. The advantage of this method is that the amount of NADH can be confirmed visually.

In the case of the enzyme immunoassay (EIA), the test solution after completion of the reaction is not subjected to the above-mentioned measurement, but the plasma sample is reacted, and then the labeled antigen bound to the antibody is released. It is necessary to perform measurement using the test solution immediately before the separation of the labeled antigen (B / F separation). By the above method, it is possible to confirm whether or not the specimen sample has been accurately dispensed.

[0024]

According to the method of the present invention, the dispensing of serum or plasma, which could not be confirmed conventionally, can be accurately confirmed. In addition, since the components inevitably contained in the sample are used as an index, the method can be applied to the case where any analysis reagent is used, and is excellent in economical efficiency.

[0025]

The embodiments will be described in detail below. Example 1 RPHA, one of the hepatitis B virus tests
Example 1) Preparation of CPD solution

Citric acid [HO.C. (COOH). (C
H ₂ COOH) ₂ ], 0.33 g, trisodium citrate [HO.C. (COONa). (CH ₂ COON)
a) ₂ ] 2.63 g and glucose [C ₆ H ₁₂ O ₆ ]
2.32 g was dissolved in purified water. After adjusting the total amount to 100 ml, the pH was measured, and it was confirmed that the pH was 5.4 to 5.8. (If the pH is not within this range, the pH is adjusted with citric acid or sodium hydroxide.) 2) Preparation of CPD-containing plasma

1.4 ml of the CPD solution prepared in 1) above
10.0 ml of blood immediately after collection was added to a test tube containing. After thoroughly mixing by inversion, the mixture was centrifuged at 1,500 rpm for 10 minutes to obtain a liquid portion (CPD-containing plasma). 3) Implementation of RPHA method

The CPD-containing plasma obtained in the above 2) is converted to RP
Diluted 4-fold with HA buffer. This diluted plasma 25μ
1 and 25 μl of the anti-HB antibody-sensitized blood cell suspension were dropped on a microplate, and stirred with a plate mixer.
After standing for about 2 hours, a blood cell image of the sensitized blood cells on the bottom of the plate was observed, and it was determined that HB antigen was positive if aggregated, and HB antigen negative if not aggregated. 4) Preparation of citric acid detection reagent

Glycylglycine [CH ₂ (NH ₂ ) CO
NHCH ₂ COOH] was dissolved in 1,000 ml of purified water to prepare a 0.1 M glycylglycine solution. Also, 4 g of sodium hydroxide NaOH was added to 1,000 g
This was dissolved in ml of purified water to prepare a 0.1 M sodium hydroxide solution. Both are mixed so that the pH becomes 7.8,
A 0.1 M glycylglycine buffer was made. About 100 ml of malate dehydrogenase (MDH) is added to 100 ml of this buffer.
Unit, about 780 units of lactate dehydrogenase (LDH), and about 1 unit of reduced nicotinamide dinucleotide (NADH)
6 mg was added to prepare a reagent (i). Also, 0.1
Approximately 400 units of citrate lyase was dissolved in 10 ml of M glycylglycine buffer to prepare reagent (ii). 5) Citric acid determination method a) Measurement method

In 1 ml of the reagent (i) prepared in 4) above,
After completion of the measurement of HB antigen by the RPHA method in 3) above, 25 μl of the supernatant was added, and the mixture was allowed to stand for 1 minute. Then, the absorbance value (E ₁ ) at 340 nm was measured using a UV meter. Further, 10 μl of the reagent (ii) was added, the mixture was rapidly stirred, and the absorbance (E ₂ ) was measured after standing for 5 minutes.
From the absorbance value (E ₁ ) immediately before the addition of the reagent (ii), the reagent (i)
i) The amount of change in absorbance (E ₁ −E ₂ ) was determined by subtracting the absorbance value (E ₂ ) 5 minutes after the addition. (B) Preparation of calibration curve

Similarly, in a) above, the amount of change in absorbance when 5, 10, 15, 20, or 30 μl of the supernatant was added was determined. The results are shown in Table 1 below. The change in the absorbance of a standard solution containing 0.4 g / l of citric acid was 0.3862.

[0032]

[Table 1] Further, as shown in FIG. 1, a calibration curve representing the relationship between the amount of the supernatant liquid and the amount of change in absorbance was created from the above results. By using the calibration curve of FIG. 1, the amount of the diluted sample actually used in the RPHA method can be known from the amount of the supernatant liquid after the RPHA method is performed. Although the present embodiment shows an application example of the present invention in an infectious disease test by the agglutination method, the application of the present invention is not limited to this.

[0033]

The specimen dispensing confirmation method of the present invention can accurately confirm the dispensing of any body fluid, and can generate false negatives in infectious disease tests due to poor dispensing of samples, It is possible to prevent a determination error or the like in the cross matching test. Therefore, it is possible to prevent transmission of an infectious disease or blood transfusion error due to blood group incompatibility. In addition, the method for confirming sample dispensing of the present invention is excellent in economical efficiency because the component necessarily contained in the sample is used as an index.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve obtained from the relationship between the amount of supernatant and the amount of change in absorbance after the RPHA method in one example of the present invention.

Claims (3)

(57) [Claims] When a sample sample derived from a body fluid is dispensed into a reaction vessel and reacted with an analysis reagent to analyze an analyte in the sample sample, it is determined whether or not the sample sample is dispensed. The method for confirming comprises a step of adding a stabilizer in advance to the specimen sample and a step of measuring the presence and / or amount of the stabilizer using a stabilizer detection reagent after the reaction is completed. A method for confirming sample dispensing, comprising: 2. The method according to claim 1, wherein the stabilizer is a blood coagulation inhibitor containing citric acids as a main component, and the sample sample contains at least components constituting blood. 3. The method according to claim 2, wherein blood is used as the sample, and 14 parts by volume of a blood coagulation inhibitor is added to 100 parts by volume of the blood and mixed.