JP4107441B2 - Reagent for classification counting of erythroblasts - Google Patents
Reagent for classification counting of erythroblasts Download PDFInfo
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- JP4107441B2 JP4107441B2 JP15233997A JP15233997A JP4107441B2 JP 4107441 B2 JP4107441 B2 JP 4107441B2 JP 15233997 A JP15233997 A JP 15233997A JP 15233997 A JP15233997 A JP 15233997A JP 4107441 B2 JP4107441 B2 JP 4107441B2
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- erythroblasts
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
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- Physics & Mathematics (AREA)
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明はフローサイトメトリーによる赤芽球の分類計数に用いる試薬に関する。
【０００２】
【従来の技術】
臨床検査の分野において、赤芽球の分類計数を行うことは、疾患の診断を行う上で極めて有用な情報を得ることができる。
【０００３】
通常、赤芽球は骨髄中に存在し末梢血液中には存在しない。末梢血への赤芽球の出現は、急性骨髄性白血病、溶血性貧血、鉄欠乏性貧血、悪性貧血等の疾患が存在する可能性を示しており、赤芽球の分類計数を行うことは、これらの疾患を診断する上で非常に有用である。
【０００４】
従来、赤芽球の分類計数を行うには、血液の塗抹標本を作製し、適当な染色を施した後に顕微鏡で観察しながら分類計数するのが一般的であった。
【０００５】
一方、近年、フローサイトメータの原理を応用した種々の全自動白血球分類計数装置が提供されている。しかしながら、これらの装置は、赤芽球が出現した場合に、アブノーマルフラッグなどによって、赤芽球の存在する可能性があることを示唆するのみで、赤芽球を正確に分類計数する事はできなかった。
【０００６】
また、これとは別に特開平４−２６８４５３号、米国特許第５，５５９，０３７号に、赤芽球を分類計数する方法が開示されている。
【０００７】
これらの方法はいずれも、適当な溶血剤で赤血球系細胞の細胞膜のみを傷害し（色素の細胞膜透過性を付与する）、白血球系細胞の細胞膜を傷害しない（色素の細胞膜透過性を付与しない）溶解剤で処理した後に（あるいは同時に）、蛍光色素で細胞膜を傷害された赤芽球のみを染色し、その蛍光強度を測定することによって、赤芽球と白血球を弁別し、赤芽球を測定する方法である。
【０００８】
これらの方法は、採血直後の新鮮血液を用いる場合は正確な測定が可能であるが、採血後時間の経過とともに、赤芽球のみならず、白血球の細胞膜が傷害され易くなり、あるいは、溶血剤と混合する以前に細胞膜が傷害されるために、白血球の一部が蛍光色素によって染色されてしまう。特にリンパ球系細胞が傷害された場合、傷害されたリンパ球と赤芽球を明瞭に弁別することは困難であり、赤芽球を正確に分類計数することができなくなるという問題がある。また、一部のリンパ芽球出現検体、あるいは化学療法などによって、白血球系細胞の細胞膜が溶血剤による障害を受けやすくなった検体では採血直後でも正確に赤芽球を分類計数する事は困難である。
【０００９】
【発明が解決すべき課題】
本発明は、採血後時間の経過した検体、あるいはダメージを受けやすい白血球が存在する場合でも、高精度で赤芽球を正確に分類計数するための試薬を提供することを目的とする。
【００１０】
【課題を解決するための手段】
本発明は、特定の溶血剤及び特定の蛍光色素を組み合わせた試薬によって、白血球と赤芽球を弁別可能に染色して、赤芽球を分類計数し得るようにしたものである。
【００１２】
【発明の実施の態様】
本発明に係る試薬が適用される血液試料とは、末梢血液、骨髄液、尿、アフェレーシス等で採取した試料など、白血球、赤芽球を含む体液試料をいう。
【００１３】
本発明に用いる溶血剤は、血液試料中の赤血球を測定の障害とならない程度に溶解し、白血球並びに赤芽球を染色に好適な状態にするものである。このような溶血剤とは、浸透圧１００ｍＯｓｍ／ｋｇ以下、ｐＨ２．０〜５．０の水溶液である。
【００１４】
溶血剤を使用する目的は、通常白血球細胞の１０００倍の濃度で存在し、白血球、赤芽球を測定する上で障害となる赤血球を溶血することである。そして、これにより、赤芽球と白血球の間に蛍光強度の差異を生じさせることである。
【００１５】
赤血球は、若干の個体差はあるが通常１５０ｍＯｓｍ／ｋｇ以下の浸透圧で細胞膜に細孔を生じ、細胞内部のヘモグロビンを流出し、光学的に透明となる（溶血する）。光学的に透明となった赤血球は、測定の障害とはならなくなる。赤血球の溶血は浸透圧の低いほど、ｐＨの低いほど速やかに進行する。本発明で使用する溶血剤では、個体差を考慮して１００ｍＯｓｍ／ｋｇ以下の浸透圧を使用する。この浸透圧を達成するためには、例えば、ＮａＣｌ、ＫＣｌなどの電解質、糖類又は後述の緩衝剤濃度などにより浸透圧を調整することができる。
【００１６】
ｐＨが低すぎる場合、赤血球のみならず、白血球、赤芽球にも過度の傷害を与える為、後述する蛍光強度の差異が得にくくなる。
【００１７】
溶液のｐＨは、赤血球の溶血が効率よく行われるため、酸性側にすることが好適である。したがって、２．０〜５．０のｐＨが使用される。さらに好適には２．５〜４．５のｐＨが選ばれる。
【００１８】
また、ｐＨを一定に保つためには、緩衝剤を使用することが好適であり、設定するｐＨ±２．０の付近にｐＫａを有する緩衝剤が使用できる。例えば、クエン酸、リンゴ酸、ジグリコール酸、マロン酸などの緩衝剤を含むことができる。
【００１９】
さらに、溶血剤中に少なくとも１つの、分子内に少なくとも１つの芳香環を有する有機酸もしくはその塩を含有することにより、より効果的に（短時間に）赤血球を溶血することができる。好ましい有機酸としては、例えばサリチル酸、フタル酸などが挙げられる。
【００２０】
本条件下では赤芽球の細胞膜も赤血球と同様に細孔を生じ溶血するが、赤芽球細胞核の性状は、ほぼ生きた細胞と同様に保たれる。
【００２１】
一方、白血球細胞の細胞膜への傷害は明確ではないが、光学的顕微鏡による観察では、生細胞と顕著な差異は認められない。
【００２２】
本発明で使用される溶血剤は、低張浸透圧で赤血球系細胞を溶血するもので、他の溶血剤で使用されるような各種界面活性剤、サポニン、アルコール類などの溶解剤、溶解補助剤は本質的に不要である。
【００２３】
しかしながら、難溶性の色素の可溶化剤や、赤血球ゴーストの凝集防止、ゴースト収縮、赤血球溶血促進などの目的で添加する界面活性剤、アルコール、サポニンなどを、含有しても良い。
【００２４】
しかしながら、多量の界面活性剤の存在は、特に赤芽球の核の性状を変化させ、後述する赤芽球と白血球の蛍光強度の差異を小さくするという問題があり好ましくない。
【００２５】
従って、本発明に使用する溶血剤は、従来の溶血剤とは異なり、本質的に細胞成分を溶解する界面活性剤などの成分を含まない方が好ましい。
【００２６】
この結果、予期せぬことに、従来不可能であると考えられていた赤芽球と白血球間の明瞭な蛍光強度の差異を生じさせることができた。
【００２７】
少なくとも白血球と赤芽球を弁別可能に染色する蛍光色素とは、以下の群からなる色素のうち少なくとも１種類が使用される。
【００２８】
【化１７】
【化１８】
【化１９】
【化２０】
【００２９】
上記に記載した色素のうちＮＫシリーズは日本感光色素研究所（株）より、ＬＤＳ７３０、ＬＤ７００はＥｘｃｉｔｏｎ社より、その他のものは市販品を購入することができる。
【００３０】
蛍光色素は、溶血剤に溶解させ、溶血剤と同時に血液に作用させても（混合させても）良いし、溶解処理（工程の）後、適当な溶媒（水、低級アルコール、エチレングリコール,ＤＭＳＯ、等）に溶解したものを添加しても良い。
【００３１】
色素の濃度は使用する色素により異なるが、一般に０．０１−１００ｍｇ／Ｌ、好ましくは０．１−１０ｍｇ／Ｌ、より好ましくは０．３−３．０ｍｇ／Ｌである。なお、この濃度は溶血剤と色素溶液とを混合した状態での濃度である。
【００３２】
前述の溶血剤で処理した血球を上述の色素で染色した場合、白血球細胞は強く染色され、フローサイトメータで測定した場合強い蛍光を発する。一方赤芽球は弱く染色され、弱い蛍光を発する。白血球と赤芽球の蛍光強度に差異が生じる作用機序は明確ではないが、おそらく赤芽球の核（ＤＮＡ）が凝縮しているために、色素の細胞核への取り込みが阻害されていると考えられる。
【００３３】
通常の溶血剤では、界面活性剤等が使用されているために、赤芽球の細胞核の凝集構造がゆるみ、白血球細胞と同程度の染色性を示してしまい、赤芽球と白血球を弁別することが困難になると考えられる。
【００３４】
本発明の好ましい態様では、サリチル酸などの有機酸、色素、及び所望によりポリオキシエチレン系ノニオン界面活性剤などの界面活性剤を精製水に溶解し、ＮａＯＨなどを用いてｐＨを調整して得られる簡単な組成の試薬を用いることができる。試薬を試料と混合し、１５−５０℃、好ましくは２０−４０℃で、５−１２０秒間、好ましくは１０−４０秒間反応させる。
【００３５】
このようにして調製した測定用試料をフローサイトメータで測定し、少なくとも１つの散乱光と、少なくとも１つの蛍光を測定する。
【００３６】
本発明でいう散乱光とは、一般に市販されるフローサイトメータで測定できる散乱光をさし、前方低角散乱光（受光角度０〜５度付近）、前方高角散乱光（受光角度５〜２０度付近）等をいい、白血球の大きさ情報を反映する散乱角度が選ばれる。このうち低角前方散乱光がより好適である。
【００３７】
蛍光とは、前述の細胞成分と結合した色素から発せられるもので、使用する色素によって好適な受光波長が選択される。蛍光信号は、細胞化学的特性を反映するものである。
【００３８】
フローサイトメータの光源は、特に限定されず、色素の励起に好適な波長の光源が選ばれる。例えば、アルゴンイオンレーザ、Ｈｅ−Ｎｅレーザ、赤色半導体レーザなどが使用される。特に半導体レーザは気体レーザに比べ非常に安価であり、装置コストを大幅に下げることができる。
【００３９】
次いで、測定した散乱光と蛍光の強度差を用いて赤芽球を分類計数する。すなわち、測定した散乱光と蛍光の強度差を用いて赤芽球を分類計数するには、（１）例えばＸ軸に前方低角散乱光、Ｙ軸に蛍光をとってスキャッタグラムを描いた場合、例えば図１に示すように、各細胞は集団（クラスター）を形成して分布する；そして（２）この集団を、適当な解析ソフトで解析することにより、赤芽球の数と割合を算出する、ことによって容易に行うことができる。
【００４０】
本発明を以下の実施例によってさらに詳しく説明するが、本発明には種々の変更、修飾が可能であり、従って、本発明の範囲は以下の実施例によって限定されるものではない。
【００４１】
【実施例】
実施例１
以下の組成の試薬を調製した。
上記試薬１．０ｍｌに抗凝固剤処理した赤芽球が末梢血に出現した患者の血液３０μｌを加え、３５℃で１０秒間反応させたのちフローサイトメータで、前方低角散乱光、蛍光を測定した。光源は６３３ｎｍの赤色半導体レーザを使用した。蛍光は６６０ｎｍ以上の波長の蛍光を測定した。
【００４３】
図２にＸ軸に前方低角散乱光強度、Ｙ軸に赤蛍光強度をとったスキャッタグラムを示す。血球は単核球（リンパ球,単球）、顆粒球（好中球,好酸球,好塩基球）、赤芽球の３つの集団を形成する。
【００４４】
実施例２
以下の組成の試薬を調製した。
上記試薬１．０ｍｌに抗凝固剤処理した赤芽球が末梢血に出現した患者の血液３０μｌを加え、３５℃で１０秒間反応させたのちフローサイトメータで、前方低角散乱光、蛍光を測定した。光源は６３３ｎｍの赤色半導体レーザを使用した。蛍光は６６０ｎｍ以上の波長の蛍光を測定した。
【００４６】
図３にＸ軸に前方低角散乱光強度、Ｙ軸に赤蛍光強度をとったスキャッタグラムを示す。血球は単核球（リンパ球,単球）、顆粒球（好中球,好酸球,好塩基球）、赤芽球の３つの集団を形成する。
【００４７】
実施例３
以下の組成の試薬を調製した。
上記試薬１．０ｍｌに抗凝固剤処理した赤芽球が末梢血に出現した患者の血液３０μｌを加え、３５℃で１０秒間反応させたのちフローサイトメータで、前方低角散乱光、蛍光を測定した。光源は６３３ｎｍの赤色半導体レーザを使用した。蛍光は６６０ｎｍ以上の波長の蛍光を測定した。
【００４９】
図４にＸ軸に前方低角散乱光強度、Ｙ軸に赤蛍光強度をとったスキャッタグラムを示す。血球は単核球（リンパ球,単球）、顆粒球（好中球,好酸球,好塩基球）、赤芽球の３つの集団を形成する。
【００５０】
実施例４
以下の組成の試薬を調製した。
上記試薬１．０ｍｌに抗凝固剤処理した赤芽球が末梢血に出現した患者の血液３０μｌを加え、３５℃で１０秒間反応させたのちフローサイトメータで、前方低角散乱光、蛍光を測定した。光源は６３３ｎｍの赤色半導体レーザを使用した。蛍光は６６０ｎｍ以上の波長の蛍光を測定した。
【００５２】
図５にＸ軸に前方低角散乱光強度、Ｙ軸に赤蛍光強度をとったスキャッタグラムを示す。血球は単核球（リンパ球,単球）、顆粒球（好中球,好酸球,好塩基球）、赤芽球の３つの集団を形成する。
【００５３】
各々の集団にウインドウを設けウィンドウ内の細胞数の計数、細胞比率の算出を行う。
【００５４】
実施例５
以下の組成の試薬を調整した。
上記試薬１．０ｍＬに抗凝固剤処理した赤芽球が末梢血に出現した患者の血液３０μｌを加え、３５℃で１０秒間反応させたのちフローサイトメータで、前方低角酸乱光、蛍光を測定した。光源は６３３ｎｍ赤色半導体レーザを使用した。蛍光は６６０ｎｍ以上の波長の蛍光を測定した。
【００５６】
図６にＸ軸に前方低角散乱光強度、Ｙ軸に赤蛍光強度をとったスキャッタグラムを示す。血球は単核球（リンパ球、単球）、顆粒球（好中球、好酸球、好塩基球）、赤芽球の３つの集団を形成する。
【００５７】
各々の集団にウインドウを設けウインドウ内の細胞数の計数、細胞比率の算出を行う。
【００５８】
図７に用手法（メイーグリュンワルド−ギムザ染色、５００カウント）と本法を用いた場合の測定結果の相関図を示す。
【００５９】
本発明に係る試薬を用いると、採血後時間の経過した検体、あるいはダメージを受けやすい白血球が存在する場合でも、高精度で赤芽球を正確に分類計数することができる。また、本発明に係る試薬を採用すれば、フローサイトメータを用いることにより短時間で効率よく赤芽球の分類計数を行うことができ、各種疾患の診断に有用な情報を提供することができる。
【図面の簡単な説明】
【図１】 本発明に係る試薬を用いて測定した血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムの一例である。
【図２】 実施例１の試薬で測定した赤芽球が末梢血に出現した患者の血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムである。
【図３】 実施例２の試薬で測定した赤芽球が末梢血に出現した患者の血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムである。
【図４】 実施例３の試薬で測定した赤芽球が末梢血に出現した患者の血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムである。
【図５】 実施例４の試薬で測定した赤芽球が末梢血に出現した患者の血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムである。
【図６】 実施例５の試薬で測定した赤芽球が末梢血に出現した患者の血液試料の前方低角散乱強度−赤蛍光強度スキャッタグラムである。
【図７】 用手法と本発明に係る試薬を用いて得られた測定結果の相関図を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a reagent used for classification and counting of erythroblasts by flow cytometry.
[0002]
[Prior art]
In the field of clinical examination, classification and counting of erythroblasts can provide extremely useful information for diagnosing diseases.
[0003]
Normally, erythroblasts are present in the bone marrow and not in the peripheral blood. The appearance of erythroblasts in the peripheral blood indicates the presence of diseases such as acute myeloid leukemia, hemolytic anemia, iron deficiency anemia, and pernicious anemia. It is very useful in diagnosing these diseases.
[0004]
Conventionally, in order to perform classification and counting of erythroblasts, it has been common to prepare a blood smear, apply appropriate staining, and then perform classification and counting while observing with a microscope.
[0005]
On the other hand, in recent years, various fully automatic white blood cell classification and counting apparatuses that apply the principle of a flow cytometer have been provided. However, these devices can accurately classify and count erythroblasts only by suggesting that erythroblasts may exist due to abnormal flags etc. when erythroblasts appear. There wasn't.
[0006]
Separately, JP-A-4-268453 and US Pat. No. 5,559,037 disclose a method for classifying and counting erythroblasts.
[0007]
Both of these methods impair wound only cell membranes of erythroid cells by an appropriate lysing agent (to impart the cell membrane permeability of the dye), not injure cell membranes of leukocytic cells (not confer cell membrane permeability of the dye) After treatment with a lysing agent (or at the same time), only erythroblasts whose cell membranes have been damaged with a fluorescent dye are stained, and the fluorescence intensity is measured to discriminate between erythroblasts and leukocytes and measure erythroblasts. It is a method to do.
[0008]
These methods allow accurate measurement when using fresh blood immediately after blood collection. However, not only erythroblasts but also leukocyte cell membranes are easily damaged over time after blood collection, or a hemolytic agent. Since the cell membrane is damaged prior to mixing with, a part of the white blood cell is stained with the fluorescent dye. In particular, when lymphocyte cells are damaged, it is difficult to clearly distinguish damaged lymphocytes from erythroblasts, and there is a problem that erythroblasts cannot be accurately classified and counted. In addition, it is difficult to accurately classify and count erythroblasts even immediately after blood collection in some lymphoblastic specimens or specimens whose cell membranes of leukocyte cells are susceptible to damage by hemolytic agents due to chemotherapy or the like. is there.
[0009]
[Problems to be Solved by the Invention]
An object of the present invention is to provide a reagent for accurately classifying and counting erythroblasts with high accuracy even when a specimen whose time has passed after blood collection or a leukocyte susceptible to damage exists.
[0010]
[Means for Solving the Problems]
In the present invention , leukocytes and erythroblasts can be discriminated by a reagent combining a specific hemolytic agent and a specific fluorescent dye so that erythroblasts can be classified and counted.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
The blood sample to which the reagent according to the present invention is applied refers to a body fluid sample containing leukocytes and erythroblasts, such as a sample collected by peripheral blood, bone marrow fluid, urine, apheresis and the like.
[0013]
The hemolytic agent used in the present invention dissolves red blood cells in a blood sample to an extent that does not hinder measurement, and makes white blood cells and erythroblasts suitable for staining . Such a hemolytic agent is an aqueous solution having an osmotic pressure of 100 mOsm / kg or less and a pH of 2.0 to 5.0.
[0014]
The purpose of using a hemolytic agent is to hemolyze erythrocytes, which usually exist at a concentration 1000 times that of white blood cells, and are an obstacle to the measurement of leukocytes and erythroblasts . And this is to produce a difference in fluorescence intensity between erythroblasts and leukocytes.
[0015]
Although there are slight individual differences, erythrocytes usually produce pores in the cell membrane at an osmotic pressure of 150 mOsm / kg or less, and the hemoglobin inside the cells flows out and becomes optically transparent (hemolyzed). Red blood cells that have become optically clear no longer interfere with the measurement. Red blood cell hemolysis proceeds more rapidly as the osmotic pressure is lower and the pH is lower. In the hemolytic agent used in the present invention, an osmotic pressure of 100 mOsm / kg or less is used in consideration of individual differences. In order to achieve this osmotic pressure, for example, the osmotic pressure can be adjusted by electrolytes such as NaCl and KCl, sugars, or a buffer concentration described later.
[0016]
If the pH is too low, not red blood cells only, white blood cells, in order to give undue injury to red blasts, the difference in fluorescence intensity to be described later becomes difficult to obtain.
[0017]
The pH of the solution is preferably set to the acidic side because red blood cells are efficiently hemolyzed. Therefore, a pH of 2.0 to 5.0 is used. More preferably, a pH of 2.5 to 4.5 is selected.
[0018]
In order to keep the pH constant, it is preferable to use a buffer, and a buffer having a pKa in the vicinity of the set pH ± 2.0 can be used. For example, a buffering agent such as citric acid, malic acid, diglycolic acid, malonic acid can be included.
[0019]
Furthermore, by containing at least one organic acid or salt thereof having at least one aromatic ring in the molecule in the hemolytic agent, it is possible to hemolyze red blood cells more effectively (in a short time). Preferred examples of the organic acid include salicylic acid and phthalic acid.
[0020]
Under these conditions, the cell membrane of erythroblasts forms pores and hemolyzes like erythrocytes, but the properties of erythroblast cell nuclei are maintained almost the same as those of living cells.
[0021]
On the other hand, although damage to the cell membrane of white blood cells is not clear, observation with an optical microscope shows no significant difference from living cells.
[0022]
The hemolytic agent used in the present invention hemolyzes erythroid cells with hypotonic osmotic pressure. Various surfactants used in other hemolytic agents, solubilizing agents such as saponins and alcohols, and solubilizing aids Agents are essentially unnecessary.
[0023]
However, it may contain a solubilizing agent for a hardly soluble dye, a surfactant added for the purpose of preventing aggregation of erythrocyte ghost, ghost contraction, erythrocyte hemolysis, alcohol, saponin, and the like.
[0024]
However, the presence of a large amount of a surfactant is not preferable because there is a problem that the properties of erythroblast nuclei are changed and the difference in fluorescence intensity between erythroblasts and leukocytes described later is reduced.
[0025]
Therefore, it is preferable that the hemolytic agent used in the present invention does not contain components such as surfactants that essentially dissolve cell components, unlike conventional hemolytic agents.
[0026]
As a result, it was possible to unexpectedly produce a clear difference in fluorescence intensity between erythroblasts and leukocytes, which was previously considered impossible.
[0027]
As the fluorescent dye that stains at least leukocytes and erythroblasts in a distinguishable manner , at least one kind of dyes consisting of the following groups is used.
[0028]
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[0029]
NK series of the dye described above are from Nippon photosensitive dye Laboratories (Inc.), LDS730, LD700 than Ex c iton the company, while others can be purchased commercially.
[0030]
The fluorescent dye may be dissolved in a hemolytic agent, and may act on the blood simultaneously with the hemolytic agent (mixed), or after dissolution treatment (step), an appropriate solvent (water, lower alcohol, ethylene glycol, DMSO) , Etc.) may be added.
[0031]
The concentration of the dye varies depending on the dye used, but is generally 0.01-100 mg / L, preferably 0.1-10 mg / L, more preferably 0.3-3.0 mg / L. This concentration is a concentration in a state where the hemolytic agent and the dye solution are mixed.
[0032]
When blood cells treated with the above-mentioned hemolytic agent are stained with the above-described dye, white blood cells are strongly stained and emit strong fluorescence when measured with a flow cytometer. On the other hand, erythroblasts are weakly stained and emit weak fluorescence. Although the mechanism of action that causes a difference in the fluorescence intensity of leukocytes and erythroblasts is not clear, the uptake of pigment into the cell nucleus is probably inhibited due to condensation of erythroblast nuclei (DNA). Conceivable.
[0033]
In normal hemolytic agents, surfactants etc. are used, so the aggregation structure of nuclei of erythroblasts loosens and shows the same level of staining as white blood cells, and discriminates erythroblasts from leukocytes. It will be difficult.
[0034]
In a preferred embodiment of the present invention, an organic acid such as salicylic acid, a dye, and optionally a surfactant such as a polyoxyethylene nonionic surfactant are dissolved in purified water, and the pH is adjusted using NaOH or the like. A reagent having a simple composition can be used. The reagent is mixed with the sample and reacted at 15-50 ° C., preferably 20-40 ° C., for 5-120 seconds, preferably 10-40 seconds.
[0035]
The measurement sample thus prepared is measured with a flow cytometer, and at least one scattered light and at least one fluorescence are measured.
[0036]
Scattered light as used in the present invention refers to scattered light that can be generally measured with a commercially available flow cytometer, and includes forward low-angle scattered light (light reception angle of about 0 to 5 degrees) and forward high-angle scattered light (light reception angle of 5 to 20). A scattering angle that reflects white blood cell size information is selected. Of these, low-angle forward scattered light is more suitable.
[0037]
Fluorescence is emitted from a dye combined with the aforementioned cell component, and a suitable light receiving wavelength is selected depending on the dye used. The fluorescent signal reflects the cytochemical properties.
[0038]
The light source of the flow cytometer is not particularly limited, and a light source having a wavelength suitable for excitation of the dye is selected. For example, an argon ion laser, a He—Ne laser, a red semiconductor laser, or the like is used. In particular, a semiconductor laser is very cheap compared to a gas laser, and the apparatus cost can be greatly reduced.
[0039]
Next, erythroblasts are classified and counted using the measured difference in intensity between scattered light and fluorescence. That is, the classified and counted erythroblasts use of differences in the scattered light and the fluorescence measured, when painted scattergram taking fluorescence forward low angle scattered light, the Y-axis (1) For example X-axis For example, as shown in FIG. 1, each cell forms a population (cluster) and is distributed; and (2) the number and ratio of erythroblasts are calculated by analyzing this population with an appropriate analysis software. This can be done easily.
[0040]
The present invention will be described in more detail with reference to the following examples. However, various changes and modifications can be made to the present invention, and therefore the scope of the present invention is not limited by the following examples.
[0041]
【Example】
Example 1
A reagent having the following composition was prepared.
Add 30 μl of blood from patients with anticoagulant-treated erythroblasts to 1.0 ml of the above reagent and react for 10 seconds at 35 ° C., then measure forward low-angle scattered light and fluorescence with a flow cytometer did. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or longer.
[0043]
FIG. 2 shows a scattergram in which the X-axis represents the forward low-angle scattered light intensity and the Y-axis represents the red fluorescence intensity. Blood cells form three populations: mononuclear cells (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.
[0044]
Example 2
A reagent having the following composition was prepared.
Add 30 μl of blood from patients with anticoagulant-treated erythroblasts to 1.0 ml of the above reagent and react for 10 seconds at 35 ° C., then measure forward low-angle scattered light and fluorescence with a flow cytometer did. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or longer.
[0046]
FIG. 3 shows a scattergram with the forward low angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations: mononuclear cells (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.
[0047]
Example 3
A reagent having the following composition was prepared.
Add 30 μl of blood from patients with anticoagulant-treated erythroblasts to 1.0 ml of the above reagent and react for 10 seconds at 35 ° C., then measure forward low-angle scattered light and fluorescence with a flow cytometer did. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or longer.
[0049]
FIG. 4 shows a scattergram with the forward low angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations: mononuclear cells (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.
[0050]
Example 4
A reagent having the following composition was prepared.
Add 30 μl of blood from patients with anticoagulant-treated erythroblasts to 1.0 ml of the above reagent and react for 10 seconds at 35 ° C., then measure forward low-angle scattered light and fluorescence with a flow cytometer did. The light source used was a 633 nm red semiconductor laser. The fluorescence was measured at a wavelength of 660 nm or longer.
[0052]
FIG. 5 shows a scattergram with the forward low angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations: mononuclear cells (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.
[0053]
A window is provided for each group, and the number of cells in the window is counted and the cell ratio is calculated.
[0054]
Example 5
Reagents having the following compositions were prepared.
Add 30 μl of blood of the patient whose erythroblast treated with anticoagulant to peripheral blood to 1.0 mL of the above reagent, react for 10 seconds at 35 ° C., and then use a flow cytometer to detect forward low-angle acid random light and fluorescence. It was measured. A 633 nm red semiconductor laser was used as the light source. The fluorescence was measured at a wavelength of 660 nm or longer.
[0056]
FIG. 6 shows a scattergram with the forward low angle scattered light intensity on the X axis and the red fluorescence intensity on the Y axis. Blood cells form three populations: mononuclear cells (lymphocytes, monocytes), granulocytes (neutrophils, eosinophils, basophils), and erythroblasts.
[0057]
A window is provided for each group, and the number of cells in the window is counted and the cell ratio is calculated.
[0058]
FIG. 7 shows a correlation diagram of measurement results when using the method (May-Grunwald-Giemsa staining, 500 counts) and this method.
[0059]
When the reagent according to the present invention is used, it is possible to accurately classify and count erythroblasts with high accuracy even in the presence of a specimen whose time has passed after blood collection or leukocytes which are easily damaged. In addition, if the reagent according to the present invention is employed, erythroblast classification and counting can be performed efficiently in a short time by using a flow cytometer, and information useful for diagnosis of various diseases can be provided. .
[Brief description of the drawings]
FIG. 1 is an example of a forward low angle scattering intensity-red fluorescence intensity scattergram of a blood sample measured using a reagent according to the present invention.
FIG. 2 is a forward low angle scattering intensity-red fluorescence intensity scattergram of a blood sample of a patient in which erythroblasts appearing in peripheral blood as measured with the reagent of Example 1.
FIG. 3 is a forward low-angle scattering intensity-red fluorescence intensity scattergram of a blood sample of a patient in which erythroblasts appear in peripheral blood as measured with the reagent of Example 2.
FIG. 4 is a forward low angle scattering intensity-red fluorescence intensity scattergram of a blood sample of a patient whose erythroblasts measured in the peripheral blood as measured with the reagent of Example 3;
FIG. 5 is a forward low angle scattering intensity-red fluorescence intensity scattergram of a blood sample of a patient in which erythroblasts measured in the reagent of Example 4 appear in peripheral blood.
6 is a forward low angle scattering intensity-red fluorescence intensity scattergram of a blood sample of a patient whose erythroblasts measured in the peripheral blood measured with the reagent of Example 5. FIG.
FIG. 7 shows a correlation diagram of the measurement results obtained using the method and the reagent according to the present invention.
Claims (2)
分子内に少なくとも１つの芳香環を有する有機酸又はその塩を含有し、浸透圧１００ｍＯｓｍ／ｋｇ以下でｐＨ２．０〜５．０の水溶液である溶血剤、及び
少なくとも白血球と赤芽球を上記弁別可能に染色する以下の群から選ばれる少なくとも１つの蛍光色素を含むことを特徴とする赤芽球分類計数用試薬。
A hemolytic agent containing an organic acid having at least one aromatic ring in the molecule or a salt thereof, and an aqueous solution having an osmotic pressure of 100 mOsm / kg or less and a pH of 2.0 to 5.0, and at least the discrimination between leukocytes and erythroblasts A reagent for erythroblast classification counting, comprising at least one fluorescent dye selected from the following group capable of staining.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15233997A JP4107441B2 (en) | 1997-06-10 | 1997-06-10 | Reagent for classification counting of erythroblasts |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15233997A JP4107441B2 (en) | 1997-06-10 | 1997-06-10 | Reagent for classification counting of erythroblasts |
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|---|---|---|---|
| JP2006118603A Division JP4338206B2 (en) | 2006-04-23 | 2006-04-23 | Classification and counting method of erythroblasts |
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| Publication Number | Publication Date |
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| JPH10339729A JPH10339729A (en) | 1998-12-22 |
| JP4107441B2 true JP4107441B2 (en) | 2008-06-25 |
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| JP4796443B2 (en) * | 2006-06-08 | 2011-10-19 | シスメックス株式会社 | Reagent for sample analysis, reagent kit for sample analysis, and sample analysis method |
| JP4914656B2 (en) | 2006-06-26 | 2012-04-11 | シスメックス株式会社 | Reagent for sample analysis, reagent kit for sample analysis, and sample analysis method |
| CN101743469B (en) | 2007-09-27 | 2013-05-29 | 希森美康株式会社 | Reagent kit for sample analysis and sample analysis method |
| WO2016106688A1 (en) * | 2014-12-31 | 2016-07-07 | 深圳迈瑞生物医疗电子股份有限公司 | Nucleated red blood cell warning method and device, and flow cytometer |
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