JPH05264414A - Specimen-dispensing confirming method - Google Patents
Specimen-dispensing confirming methodInfo
- Publication number
- JPH05264414A JPH05264414A JP4058422A JP5842292A JPH05264414A JP H05264414 A JPH05264414 A JP H05264414A JP 4058422 A JP4058422 A JP 4058422A JP 5842292 A JP5842292 A JP 5842292A JP H05264414 A JPH05264414 A JP H05264414A
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- JP
- Japan
- Prior art keywords
- sample
- dispensing
- specimen
- reaction
- stabilizer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、凝集反応による感染症
検査や交差適合試験、或いはアレルギー試験のように血
漿または血清等の体液に由来する検体試料の分析方法に
関する。より具体的には、これら検体試料を反応容器に
分注し、分析用試薬と反応させて分析を行う際に、前記
検体試料の分注が正しく行われたか否かを確認する方法
に関する。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 an agglutination reaction, a cross-match test, or an allergy test. More specifically, the present invention relates to a method for confirming whether or not the sample sample is dispensed correctly when the sample sample is dispensed into a reaction container and reacted with an analysis reagent to perform analysis.
【０００２】[0002]
【従来の技術】従来、感染症の予防上或いは治療上の有
用な指標とする観点から、凝集反応を利用した感染症検
査又は交差適合性試験等の生化学的分析、或いは酵素免
疫測定法（ＥＩＡ）等の免疫学的分析が行われている。
一般に、これらの分析は、分析装置の反応容器内に体液
等の検体試料及び分析用試薬を分注し、混合して反応さ
せることによって行われる。しかし、これらの分析法自
体がいかに高精度であっても、検体試料の分注が期待通
り正しく行われなければ、結果としては判定ミスを生ず
ることになる。例えば健常者が低値異常と判定された
り、感染症陽性者（非健常者）が感染症陰性者（健常
者）と判定されたり、或いは血液型が不適合にもかかわ
らず適合と判定されたりする。このような判定ミスが頻
繁に発生すると、感染症の伝播、或いは血液型不適合に
よる輸血ミス等を引き起こすことになる。従って、この
ような検査においては、検体が反応容器中に正確に分注
されたか否かを確認することが重要である。2. Description of the Related Art Conventionally, from the viewpoint of being a useful index for preventing or treating infectious diseases, biochemical analysis such as infectious disease test or cross-compatibility test utilizing agglutination reaction, or enzyme immunoassay ( Immunological analysis such as EIA) has been performed.
Generally, these analyzes are carried out by dispensing a specimen sample such as a body fluid and an analytical reagent into a reaction container of an analyzer, mixing and reacting them. However, no matter how highly accurate these analytical methods themselves are, if the dispensing of the specimen sample is not performed correctly as expected, as a result, a judgment error will occur. For example, a healthy person may be determined to have low abnormalities, an infectious disease-positive person (non-healthy person) may be determined to be an infectious disease-negative person (healthy person), or a blood type may be determined to be compatible despite incompatibility. .. If such a judgment error occurs frequently, it may cause a transmission of an infectious disease or a blood transfusion error due to blood group mismatch. Therefore, in such a test, it is important to confirm whether or not the sample is accurately dispensed into the reaction container.
【０００３】血漿或いは血清を検体とする分析において
は、分注確認の困難性が特に問題となっている。例え
ば、ＡＢＯ式血液型検査には、血球を検査対象としたオ
モテ検査と血漿を検査対象としたウラ検査とがあり、血
球を対象としたオモテ試験では、凝集反応の結果の判定
時に血球が分注されているか否かの判断は容易にでき
る。しかし、血漿が検査対象であるウラ検査では、この
ような血漿分注の確認はできない。また、同様に血漿を
検体として行われる感染症等の検査でも、検体分注の確
認ができないために、常に偽陰性の発生を考えて検査を
する必要がある。従って、特に血漿または血清を検体と
する分析においては、検体分注の適否の判別を確実に行
い得る方法の開発が望まれている。In analysis using plasma or serum as a sample, the difficulty of confirming dispensing is a particular problem. For example, in the ABO blood group test, there are a front test for blood cells and a back test for plasma. In the front test for blood cells, blood cells are separated when determining the result of agglutination reaction. It can be easily judged whether or not it has been poured. However, such a plasma dispensing cannot be confirmed by the back test in which plasma is the test target. Similarly, in the case of an infectious disease test performed using plasma as a sample, it is necessary to always consider the occurrence of false negatives because the sample dispensing cannot be confirmed. Therefore, especially in the analysis using plasma or serum as a sample, it is desired to develop a method capable of surely determining the suitability of sample dispensing.
【０００４】従来の検体分注確認方法としては、まず、
特開昭５９−１７１６１号に記載された、光電的に分注
を確認する方法が公知である。これはＰＫ７０００シリ
ーズの血液型検査及び感染症検査時に、検体の分注を確
認するための方法の１つとして考案されたものである。
しかし、この方法は、検体の前処理である希釈工程にお
いて検体の分注量を確認するものであるため、実際に反
応に関わった検体量を知ることはできない。また、特願
平３−２１１７３６号（本願出願時には未公開）には、
検体自身の持つｐＨ緩衝能を利用する分注確認方法が記
載されている。この方法は体液の緩衝能が比較的大きい
ことを利用したもので、ｐＨ指示薬の希釈液を反応前の
反応容器中に所定量加えると、検体が分注されていれ
ば、希釈液のｐＨが変化し、指示薬の色が変化すること
に基づいて分注不良を検知するものである。しかし、こ
の方法は、例えば感染症検査等のように、検体よりも大
きなｐＨ緩衝能を有する希釈液を用いる分析には適用が
困難である。The conventional sample dispensing confirmation method is as follows.
A method for photoelectrically confirming the 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 blood group examination and infectious disease examination of PK7000 series.
However, since this method confirms the amount of the sample dispensed in the dilution step which is the pretreatment of the sample, the amount of the sample actually involved in the reaction cannot be known. In addition, Japanese Patent Application No. 3-212736 (unpublished at the time of filing of this application)
A method for confirming dispensing using the pH buffering capacity of the sample itself is described. This method utilizes the fact that the buffering capacity of body fluid is relatively large, and if a predetermined amount of a diluent of pH indicator is added to the reaction vessel before the reaction, the pH of the diluent is adjusted if the sample is dispensed. This is to detect a defective dispensing based on the change and the color of the indicator. However, this method is difficult to apply to an analysis using a diluting liquid having a pH buffering capacity larger than that of a sample, such as an infectious disease test.
【０００５】一方検体が蛋白質を含んでいることに基づ
けば、蛋白質を指標として分注量を確認することも可能
ではある。しかし、免疫学的分析にはおいては、試薬成
分中に抗原ないし抗体蛋白を含んでいるため、検体と試
薬とを混合した後に、蛋白量を指標として分注量を確認
するのは不適当である。また、検体の分注量を確認する
目的で、別途指標物を添加するのも経済的ではない。On the other hand, based on the fact that the sample contains a protein, it is also possible to confirm the dispensed amount using the protein as an index. However, in immunological analysis, it is not appropriate to confirm the dispensed amount using the protein amount as an index after mixing the sample and the reagent because the reagent component contains the antigen or antibody protein. is there. Further, it is not economical to add a separate indicator substance for the purpose of confirming the dispensed amount of the sample.
【０００６】[0006]
【発明が解決しようとする課題】従って、本発明の課題
は、血清または血漿等の体液に由来する検体試料中に必
然的に含まれる成分を指標とすることにより、検体また
は試薬成分の影響を受けずに検体分注の適否を確認する
方法を提供することにある。SUMMARY OF THE INVENTION Therefore, the object of the present invention is to determine the influence of a sample or reagent component by using, as an index, a component necessarily contained in a sample sample derived from a body fluid such as serum or plasma. It is to provide a method of confirming the adequacy of sample dispensing without receiving.
【０００７】[0007]
【課題を解決するための手段】上記課題を解決するため
に、本発明は、体液から調製した検体試料では、一般に
その変性を防止するために添加が必要とされる安定化剤
を指標とし、分析が終了した後に、この安定化剤を検出
することで分注確認を行うこととした。In order to solve the above-mentioned problems, the present invention uses, as an index, a stabilizer which is generally required to be added in order to prevent the denaturation of a specimen sample prepared from a body fluid, After the analysis was completed, it was decided to confirm the dispensing by detecting this stabilizer.
【０００８】すなわち、本発明によれば、体液に由来す
る検体試料を反応容器に分注し、これを分析用試薬と反
応させることによって検体試料中の分析対象物を分析す
るに際し、前記検体試料の分注の適否を確認する方法に
おいて、前記検体試料に予め安定化剤を添加する工程
と、前記反応が終了した後に安定化剤検出試薬を用いて
前記安定化剤の有無及び／又は量を測定する工程とを具
備することを特徴とする検体分注確認方法が提供され
る。以下、本発明を詳細に説明する。That is, according to the present invention, when a sample sample derived from a body fluid is dispensed into a reaction container and reacted with an analytical reagent to analyze an analyte in the sample sample, the sample sample In the method for confirming the suitability of dispensing, the step of adding a stabilizer to the sample sample in advance, and the presence and / or amount of the stabilizer using a stabilizer detection reagent after the reaction is completed. A method for confirming the dispensing of a sample is provided, which comprises a step of measuring. Hereinafter, the present invention will be described in detail.
【０００９】まず、本発明の分注確認方法を行う前提と
なる検体試料の分析について簡単に説明する。この分析
には、分析装置の反応容器内に体液由来の検体試料及び
これを分析するための分析用試薬を分注し、混合して反
応させることによって行われるすべての種類の分析が含
まれる。例えば、凝集反応による感染症検査、交差適合
試験及びアレルギー試験、或いは免疫学的試験等であ
る。First, a brief description will be given of the analysis of a specimen sample, which is a prerequisite for carrying out the dispensing confirmation method of the present invention. This analysis includes all kinds of analysis performed by dispensing a specimen sample derived from a body fluid and an analytical reagent for analyzing the specimen sample in a reaction container of an analyzer, mixing and reacting them. For example, infectious disease test by agglutination reaction, cross-match test and allergy test, immunological test and the like.
【００１０】前記分析における検体試料としては、体液
中に存在する成分のいずれも用いることができ、その例
としては、血球、血漿、血清及び尿などが挙げられる。
しかし、本発明の利点の１つとして、従来は不可能であ
った血漿或いは血清の分注確認を正確に行い得ることが
挙げられるから、血漿或いは血清を用いた場合に、本発
明の効果が顕著である。Any of the components present in body fluids 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 is It is remarkable.
【００１１】前記分析における分析用試薬は特に限定さ
れず、分析の目的に応じて種々の試薬が用いられる。例
えば、輸血前検査の１つである交差適合試験において
は、血液型を調べるために抗Ａ抗体、抗Ｂ抗体が用いら
れる。また、後述の実施例に示すようには、Ｂ型肝炎ウ
イルス検査では抗ＨＢ抗体を用いる。The analytical reagent 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-match test, which is one of the pre-transfusion tests, anti-A antibody and anti-B antibody are used to check the blood group. Further, as shown in Examples described later, an anti-HB antibody is used in the hepatitis B virus test.
【００１２】前記分析における反応は特に限定されず、
例えば凝集反応或いは酵素免疫測定法（ＥＩＡ）等の従
来用いられている反応はいずれも用いることができる。
好ましい分析反応は、凝集反応である。凝集反応には、
主に直接凝集反応、間接凝集反応、又は直接受身凝集反
応の３つに分類され、その他にも免疫粘着血球凝集反応
や、後述の実施例で用いた逆受身凝集反応（以下「ＲＰ
ＨＡ」と呼ぶ）等がある。これらの凝集反応は、いずれ
も本発明に用いる分析反応として好ましいものである。The reaction in the above analysis is not particularly limited,
For example, any conventionally used reaction such as agglutination reaction or enzyme immunoassay (EIA) can be used.
The preferred analytical reaction is an agglutination reaction. For the agglutination reaction,
It is mainly classified into three types of direct agglutination reaction, indirect agglutination reaction, and direct passive agglutination reaction. In addition, immunoadhesive hemagglutination reaction and reverse passive agglutination reaction used in Examples described later (hereinafter referred to as “RP
"HA"). All of these agglutination reactions are preferable as the analytical reaction used in the present invention.
【００１３】本発明の特徴は、上記分析において、検体
試料の分注が正確に行われているか否かを確認するため
の指標として、前記検体試料に予め添加されている安定
化剤を用い、前記反応が終了した後に該安定化剤の有無
及び／又は量を測定することにある。以下、本発明の検
体分注確認方法を詳細に説明する。まず、検体試料を反
応容器に分注する前に、該検体試料に安定化剤を添加す
る。A feature of the present invention is that, in the above analysis, a stabilizer previously added to the sample sample is used as an index for confirming whether or not the sample sample is accurately dispensed. It is to measure the presence and / or amount of the stabilizer after the reaction is completed. Hereinafter, the sample dispensing confirmation method of the present invention will be described in detail. First, before dispensing the specimen sample into the reaction container, a stabilizer is added to the specimen sample.
【００１４】本発明における安定化剤は、検体試料の物
理的或いは化学的変性を防止するために添加されるもの
であるが、所期の安定化作用を有することに加えて、以
下に述べる３つの要求を満たすものであればいずれも用
いることが可能である。まず第一に、体液中には、全く
含有されていないか、或いは含有されているとしても、
個体差による含有量の変動がないもの、又はその添加量
と比較して遥かに微量であるものでなければならない。
第二に、体液成分に対し、不活性であり、かつ成分分析
に対しても影響を与えないものでなければならない。そ
して第三に、その物質を特異的に分析できる方法が存在
しなければならない。The stabilizer in the present invention is added in order to prevent physical or chemical denaturation of the specimen sample, and in addition to having the desired stabilizing effect, it will be described below. Any one can be used as long as it satisfies one requirement. First of all, in body fluids, at all, or even if not,
The content should not change due to individual differences, or should be much smaller than the amount added.
Secondly, it should be inert to body fluid components and should not affect the component analysis. And third, there must be a method that can specifically analyze the substance.
【００１５】以上の見地から、本発明の安定化剤とし
て、血液凝固阻害剤が好ましく用いられる。血液凝固阻
害剤は、上記３つの要求を満たす安定化剤であり、血球
を採取して血漿を得るために通常は当然に必要とされる
ものだからである。From the above viewpoint, a blood coagulation inhibitor is preferably used as the stabilizer of the present invention. This is because the blood coagulation inhibitor is a stabilizer that satisfies the above-mentioned three requirements, and is usually naturally required to collect blood cells and obtain plasma.
【００１６】血液凝固阻害剤としては、ヘパリン、キレ
ーターであるＥＤＴＡ（エチレンジアミン四酢酸）、或
いは種々のクエン酸塩類などを用いることができる。中
でも、安価で、かつ取扱いが容易なＥＤＴＡやクエン酸
塩類を用いることが好ましい。As the blood coagulation inhibitor, heparin, a chelator EDTA (ethylenediaminetetraacetic acid), or various citrates can be used. Above all, it is preferable to use EDTA and citrates which are inexpensive and easy to handle.
【００１７】本発明において特に好ましく用いられるの
は、クエン酸［ＨＯ・Ｃ・（ＣＯＯＨ）・（ＣＨ2 ＣＯ
ＯＨ）2 ］であり、特にその血液の寿命をも考慮しなけ
ればならない検体において好ましく用いられる。クエン
酸は一般に、自然界に広く存在し、ヒトの血液中にはク
エン酸イオンの形で１３〜２７μｇ／ｍｌ含有されてい
る（クエン酸イオンには、理論的には３種類のものが考
えられるが、以下これを総称してクエン酸類と呼ぶ）。Particularly preferably used in the present invention is citric acid [HO.C. (COOH). (CH 2 CO
OH) 2 ], and is preferably used particularly in a sample in which the life of the blood must also be considered. Citric acid generally exists widely in nature and is contained in human blood in the form of citrate ions of 13 to 27 μg / ml (there are three theoretically possible citrate ions). However, hereinafter this is collectively referred to as citric acid).
【００１８】クエン酸類は、主に、これを主成分とする
ＡＣＤ液、或いは現在血液銀行や赤十字等の輸血前検査
において頻繁に用いられているＣＰＤ液等の抗凝固液
（血液凝固阻害剤の混合液）の形態で用いられる。クエ
ン酸類は、血液凝固防止のために、血液中の含有量より
遥かに多い量が必要である。抗凝固液の血液に対する量
的割合は決められており、その割合は血液１００容量部
に対してＣＰＤ液１４容量部である。この割合は一般的
に推奨されており、多くの診療機関がその比率を厳守し
ているものである。次に、検査試料の分析が終了した
後、安定化剤検出試薬を用いて安定化剤の有無、及び／
又は量を測定する。Citric acids are mainly anticoagulant liquids (such as blood coagulation inhibitors) such as ACD liquids containing them as a main component or CPD liquids which are frequently used in pre-transfusion tests of blood banks and the Red Cross at present. It is used in the form of a mixed solution). The amount of citric acid is required to be much higher than the content in blood in order to prevent blood coagulation. The quantitative ratio of the 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 / absence of the stabilizer and / or
Or measure the amount.
【００１９】安定化剤の有無、及び／又は量を測定する
ための方法、並びに安定化剤検出試薬を用いる場合の当
該試薬は、用いられた安定化剤の種類に依存して異な
る。例えば、安定化剤としてクエン酸を用いる場合は、
後述の実施例に記載されているように、グリシルグリシ
ン緩衝液に、リンゴ酸脱水素酵素（ＭＤＨ）、乳酸脱水
素酵素（ＬＤＨ）、及び還元型のニコチンアミドジヌク
レオチド（ＮＡＤＨ）を含有させた第一試薬、及びグリ
シルグリシン緩衝液にクエン酸リアーゼを含有させた第
二試薬の２つが用いられる。The method for measuring the presence and / or the amount of the stabilizer and the reagent for using the stabilizer detection reagent differ depending on the kind of the stabilizer used. For example, when using citric acid as a stabilizer,
As described in Examples below, a glycylglycine buffer solution was made to contain malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and reduced nicotinamide dinucleotide (NADH). And a second reagent in which citrate lyase is contained in a glycylglycine buffer solution.
【００２０】この安定化剤検出試薬は、調製後直ぐに以
下の測定に用いてもよく、また保存しておくことも可能
である。保存は、試薬の安定性を考慮し、低温で行われ
ることが好ましい。This stabilizing agent detection reagent 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.
【００２１】上記第一試薬及び第二試薬を用いることに
より、安定化剤として用いたクエン酸の有無及び量を測
定する方法は、安定化剤の量をＮＡＤＨ量に置き換え
て、このＮＡＤＨ量を測定するものである。すなわち、
分析反応後に第一及び第二試薬を加え、クエン酸分解酵
素の作用によりクエン酸の分解反応を行うと、この酵素
反応の進行度に応じてＮＡＤＨの量が減少する。すなわ
ち、安定化剤として含有されているクエン酸の量は、Ｎ
ＡＤＨの減少量に置き換わることになる。従って、分解
酵素による反応の前後においてＵＶメーターを用いて３
４０ｎｍの吸光度値を測定し、吸光度変化量を求めて検
量線を作成する方法によってＮＡＤＨの減少量、すなわ
ち、クエン酸の含有量を測定することができる。前記酵
素による反応は、３７℃、或いは室温等一定の温度で行
われることが好ましい。酵素反応の反応速度が温度変化
により多少影響を受けるためである。A method for measuring the presence and amount of citric acid used as a stabilizer by using the above-mentioned first reagent and second reagent is to replace the amount of the stabilizer with the amount of NADH and It is something to measure. That is,
When the first and second reagents are added after the analytical reaction and the citric acid decomposing reaction is carried out by the action of the citric acid decomposing enzyme, the amount of NADH decreases depending on the progress of this enzymatic reaction. That is, the amount of citric acid contained as a stabilizer is N
It will be replaced by the reduced amount of ADH. Therefore, using a UV meter before and after the reaction with the degrading enzyme,
The NADH decrease amount, that is, the citric acid content can be measured by the method of measuring the absorbance value at 40 nm and obtaining the absorbance change amount to prepare a calibration curve. The reaction with the enzyme is preferably carried out at a constant temperature such as 37 ° C. or room temperature. This is because the reaction rate of the enzymatic reaction is somewhat affected by the temperature change.
【００２２】また、ＮＡＤＨ変化量を、ジアホラーゼ、
やフェナジンメトサルフェート（ＰＭＳ）等を用いて、
テトラゾリウム塩であるホルマザンに変化させる方法を
適用することもできる。この方法の利点は、ＮＡＤＨ量
を目視で確認できることである。In addition, the amount of NADH change can be calculated by using diaphorase,
Or 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 visually confirmed.
【００２３】なお、酵素免疫測定法（ＥＩＡ）における
測定の場合は、反応終了後の検液をを上記測定に供する
のではなく、血漿サンプルを反応させた後、抗体と結合
した標識抗原と遊離の標識抗原の分離（Ｂ／Ｆ分離）を
行う直前の検液を用いて測定を行う必要がある。以上の
方法により、検体試料が正確に分注されているか否かを
確認することができる。In the case of the enzyme immunoassay (EIA), the test liquid 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 and freed. It is necessary to carry out the measurement using the test liquid immediately before the separation of the labeled antigen (2) (B / F separation). By the above method, it is possible to confirm whether or not the specimen sample is accurately dispensed.
【００２４】[0024]
【作用】本発明の方法により、従来確認が不可能であっ
た血清或いは血漿の分注を正確に確認できる。また、検
体中に必然的に含まれる成分を指標としているため、い
ずれの分析用試薬を用いる場合にも適用でき、かつ経済
性にも優れている。By the method of the present invention, it is possible to accurately confirm the dispensing of serum or plasma, which could not be confirmed conventionally. In addition, since the component that is necessarily contained in the sample is used as an index, it can be applied to any of the analytical reagents and is excellent in economic efficiency.
【００２５】[0025]
【実施例】以下実施例を詳細に説明する。 実施例１ Ｂ型肝炎ウイルス検査の１つであるＲＰＨＡ
法に適用した例 １）ＣＰＤ液の調製EXAMPLES Examples will be described in detail below. Example 1 RPHA, one of hepatitis B virus tests
Example applied to the method 1) Preparation of CPD solution
【００２６】クエン酸［ＨＯ・Ｃ・（ＣＯＯＨ）・（Ｃ
Ｈ2 ＣＯＯＨ）2 ］０．３３ｇ、クエン酸三ナトリウム
［ＨＯ・Ｃ・（ＣＯＯＮａ）・（ＣＨ2 ＣＯＯＮ
ａ）2 ］２．６３ｇ、及びグルコース［Ｃ6 Ｈ12Ｏ6 ］
２．３２ｇを精製水に溶解した。全量を１００ｍｌにし
た後、ｐＨを測定し、ｐＨ５．４〜５．８であることを
確認した。（ここで、ｐＨがこの範囲内でなければ、ク
エン酸、または水酸化ナトリウムでｐＨの調整を行
う。） ２）ＣＰＤ含有血漿の調製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. (Here, if the pH is not within this range, the pH is adjusted with citric acid or sodium hydroxide.) 2) Preparation of CPD-containing plasma
【００２７】上記１）で調製したＣＰＤ溶液１．４ｍｌ
を含む試験管に、採取直後の血液１０．０ｍｌを添加し
た。十分転倒混和した後、１，５００ｒｐｍ、１０分で
遠心し、液体部分（ＣＰＤ含有血漿）を得た。 ３）ＲＰＨＡ法の実施1.4 ml of the CPD solution prepared in 1) above
Immediately after the collection, 10.0 ml of blood was added to the test tube containing. After thoroughly inverting and mixing, centrifugation was performed at 1,500 rpm for 10 minutes to obtain a liquid portion (CPD-containing plasma). 3) Implementation of RPHA method
【００２８】上記２）で得られたＣＰＤ含有血漿をＲＰ
ＨＡ用緩衝液で４倍に希釈した。この希釈血漿２５μ
ｌ、及び抗ＨＢ抗体感作血球浮遊液２５μｌをマイクロ
プレートに滴下し、プレートミキサーにより攪拌した。
約２時間放置した後、感作血球のプレート底面での血球
像を観察し、凝集していればＨＢ抗原陽性、凝集してい
なければＨＢ抗原陰性と判断した。 ４）クエン酸検出試薬の調製The CPD-containing plasma obtained in 2) above was treated with RP.
It was diluted 4-fold with HA buffer. 25μ of this diluted plasma
1 and 25 μl of anti-HB antibody-sensitized hemocyte suspension were added dropwise to the microplate and stirred with a plate mixer.
After standing for about 2 hours, the blood cell image on the bottom surface of the plate of the sensitized blood cells was observed, and if aggregated, it was determined to be HB antigen positive, and if not aggregated, HB antigen negative. 4) Preparation of citric acid detection reagent
【００２９】グリシルグリシン［ＣＨ2 （ＮＨ2 ）ＣＯ
ＮＨＣＨ2 ＣＯＯＨ］１３．２ｇを１，０００ｍｌの精
製水に溶解し、０．１Ｍグリシルグリシン溶液を作成し
た。また、水酸化ナトリウムＮａＯＨ４ｇを１，０００
ｍｌの精製水に溶解し、０．１Ｍ水酸化ナトリウム溶液
を作成した。両者をｐＨが７．８になるように混合し、
０．１Ｍグリシルグリシン緩衝液を作成した。この緩衝
液１００ｍｌにリンゴ酸脱水素酵素（ＭＤＨ）約３８０
単位、乳酸脱水素酵素（ＬＤＨ）約７８０単位、及び還
元型のニコチンアミドジヌクレオチド（ＮＡＤＨ）約１
６ｍｇを添加し、試薬（ｉ）を調製した。また、０．１
Ｍグリシルグリシン緩衝液１０ｍｌに、約４００単位の
クエン酸リアーゼを溶解し、試薬（ｉｉ）を調製した。 ５）クエン酸の定量方法 ａ）測定方法Glycylglycine [CH 2 (NH 2 ) CO
NHCH 2 COOH] 13.2 g was dissolved in 1,000 ml of purified water to prepare a 0.1 M glycylglycine solution. In addition, 1,000g of sodium hydroxide NaOH 4g
It was dissolved in ml of purified water to prepare a 0.1 M sodium hydroxide solution. Both were mixed so that the pH was 7.8,
A 0.1 M glycylglycine buffer was prepared. Approximately 380 malate dehydrogenase (MDH) in 100 ml of this buffer solution
Unit, lactate dehydrogenase (LDH) about 780 units, and reduced nicotinamide dinucleotide (NADH) about 1
Reagent (i) was prepared by adding 6 mg. Also, 0.1
Reagent (ii) was prepared by dissolving about 400 units of citrate lyase in 10 ml of M glycylglycine buffer. 5) Quantitative method of citric acid a) Measuring method
【００３０】上記４）で調製した試薬（ｉ）１ｍｌに、
上記３）のＲＰＨＡ法によるＨＢ抗原測定終了後の上清
液２５μｌを加え、１分間放置した後、ＵＶメーターを
用いて３４０ｎｍにおける吸光度値（Ｅ1 ）を測定し
た。更に、試薬（ｉｉ）１０μｌを添加して素早く攪拌
し、５分間放置した後の吸光度値（Ｅ2 ）を測定した。
試薬（ｉｉ）添加直前の吸光度値（Ｅ1 ）から試薬（ｉ
ｉ）添加５分後の吸光度値（Ｅ2 ）を減算することによ
って吸光度変化量（Ｅ1 −Ｅ2 ）を求めた。 （ｂ）検量線の作成To 1 ml of the reagent (i) prepared in 4) above,
After adding 25 μl of the supernatant liquid after the HB antigen measurement by the RPHA method described in 3) above and allowing it to stand for 1 minute, 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 value (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 absorbance change amount (E 1 -E 2 ) was obtained by subtracting the absorbance value (E 2 ) 5 minutes after the addition. (B) Preparation of calibration curve
【００３１】同様に、上記ａ）において、上清液をそれ
ぞれ５、１０、１５、２０、又は３０μｌ添加した場合
の吸光度変化量を求めた。その結果を下記の表１に示
す。なお、クエン酸０．４ｇ／ｌを含有する標準液の吸
光度変化量を測定したところ０．３８６２であった。Similarly, in the above a), the amount of change in absorbance was determined when 5, 10, 15, 20, or 30 μl of the supernatant was added. The results are shown in Table 1 below. The amount of change in absorbance of a standard solution containing 0.4 g / l of citric acid was 0.3862.
【００３２】[0032]
【表１】 また、図１に示すように、上記結果より、上清液量と吸
光度変化量の関係を表す検量線を作成した。図１の検量
線を用いると、ＲＰＨＡ法実施後の上清液量から、この
ＲＰＨＡ法において実際に使用された希釈検体量を知る
ことができる。本実施例は凝集法による感染症検査にお
ける本発明の適用例を示したが、本発明の適用はこれに
限られるものではない。[Table 1] Further, as shown in FIG. 1, a calibration curve showing the relationship between the amount of supernatant and the change in absorbance was prepared from the above results. By using the calibration curve of FIG. 1, the amount of diluted sample actually used in this RPHA method can be known from the amount of the supernatant liquid after the RPHA method. Although the present example 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】[0033]
【発明の効果】本発明の検体分注確認方法は、いずれの
体液の分注をも正確に確認することができるため、サン
プルの分注不良に起因する感染症検査での偽陰性発生
や、交差適合試験における判定ミス等を防止することが
できる。従って、感染症の伝播或いは血液型不適合によ
る輸血ミスなどを防ぐことができる。また、本発明の検
体分注確認方法は、検体中に必然的に含まれる成分を指
標としているため、経済性にも優れている。EFFECTS OF THE INVENTION The sample dispensing confirmation method of the present invention can accurately confirm the dispensing of any body fluid, so that false negative occurrence in an infectious disease test due to poor dispensing of a sample, It is possible to prevent erroneous judgments in the cross matching test. Therefore, it is possible to prevent a transfusion error due to transmission of an infectious disease or blood group mismatch. In addition, the sample dispensing confirmation method of the present invention is excellent in economic efficiency because it uses the components that are necessarily contained in the sample as indicators.
【図１】本発明の一実施例において、ＲＰＨＡ法実施後
の上清液量及び吸光度変化量の関係から得られた検量線
を示す図。FIG. 1 is a diagram showing a calibration curve obtained from the relationship between the amount of supernatant liquid and the amount of change in absorbance after carrying out the RPHA method in one example of the present invention.
Claims (3)
注し、これを分析用試薬と反応させることによって検体
試料中の分析対象物を分析するに際し、前記検体試料の
分注の適否を確認する方法において、前記検体試料に予
め安定化剤を添加する工程と、前記反応が終了した後に
安定化剤検出試薬を用いて前記安定化剤の有無及び／又
は量を測定する工程とを具備することを特徴とする検体
分注確認方法。1. When analyzing an analyte in a sample sample by dispensing a sample sample derived from a body fluid into a reaction container and reacting the sample with a reagent for analysis, the suitability of dispensing of the sample sample is checked. The method for confirmation comprises the steps of adding a stabilizer to the sample sample in advance, and 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, which comprises:
する血液凝固阻害剤であり、前記検体試料が、少なくと
も血液を構成する成分を含む請求項１記載の検体分注確
認方法。2. The sample dispensing confirmation method according to claim 1, wherein the stabilizer is a blood coagulation inhibitor containing citric acid as a main component, and the sample sample contains at least a component constituting blood.
１００容量部に対して血液凝固阻害剤１４容量部を添加
混合する請求項２記載の検体分注確認方法。3. The sample dispensing confirmation method according to claim 2, wherein blood is used as the sample sample, and 14 parts by volume of a blood coagulation inhibitor is added and mixed with 100 parts by volume of the blood.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4058422A JP3064645B2 (en) | 1992-03-16 | 1992-03-16 | Sample dispensing confirmation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4058422A JP3064645B2 (en) | 1992-03-16 | 1992-03-16 | Sample dispensing confirmation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05264414A true JPH05264414A (en) | 1993-10-12 |
| JP3064645B2 JP3064645B2 (en) | 2000-07-12 |
Family
ID=13083949
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4058422A Expired - Fee Related JP3064645B2 (en) | 1992-03-16 | 1992-03-16 | Sample dispensing confirmation method |
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| Country | Link |
|---|---|
| JP (1) | JP3064645B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003004753A (en) * | 2001-06-18 | 2003-01-08 | Aloka Co Ltd | Dispensing conformity-determining apparatus |
-
1992
- 1992-03-16 JP JP4058422A patent/JP3064645B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003004753A (en) * | 2001-06-18 | 2003-01-08 | Aloka Co Ltd | Dispensing conformity-determining apparatus |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3064645B2 (en) | 2000-07-12 |
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