JP3553691B2 - Reticulocyte measuring reagent and measuring method - Google Patents
Reticulocyte measuring reagent and measuring method Download PDFInfo
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- JP3553691B2 JP3553691B2 JP15347295A JP15347295A JP3553691B2 JP 3553691 B2 JP3553691 B2 JP 3553691B2 JP 15347295 A JP15347295 A JP 15347295A JP 15347295 A JP15347295 A JP 15347295A JP 3553691 B2 JP3553691 B2 JP 3553691B2
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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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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
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Description
【０００１】
【産業上の利用分野】
本発明は、臨床検査分野において、血液中の網状赤血球を測定する試薬および測定する方法に関する。
【０００２】
【従来の技術】
網状赤血球とは、骨髄中で造血幹細胞から分化成熟した赤芽球系細胞が脱核し、骨髄から末梢血液中に放出された直後の若い赤血球であり、１〜２日後に成熟赤血球となる。網状赤血球は、細胞成熟の名残として、成熟赤血球には含まれていない少量のＲＮＡあるいはリボソーム、ミトコンドリアなどの細胞内小器官を有している。
【０００３】
臨床検査分野において、網状赤血球を分類・計数することは、患者の骨髄内での造血状態を把握する上で極めて重要な検査である。正常人においては、網状赤血球は、全赤血球中の０．５〜２．０％を占めており、骨髄造血が抑制された状態では減少し、骨髄造血が亢進した状態では増加する。例えば、再生不良性貧血、悪性腫瘍の化学療法時では減少し、溶血性貧血等では増加する。
【０００４】
さて、網状赤血球を測定するためには、従来より血液とニューメチレンブルー（ＮＭＢ）、ブリリアントクレシルブルー（ＢＣＢ）などの塩基性色素を含有する染色液とを混合する（超生体染色）ことによって、網状赤血球中に含まれる上記残存物質を網状に析出させ、塗抹標本を作製し、１個１個の細胞を顕微鏡で観察することにより、成熟赤血球と網状赤血球とを弁別し計数する方法（用手法）が行われている。
【０００５】
しかしながら、用手法は操作が煩雑であり、検査技師間で細胞判別の個人差や細胞計数数の少なさに由来する統計学的誤差が大きいことが知られている。
【０００６】
これらの問題を解決するために、前述の塩基性色素のかわりに、蛍光性の塩基性色素で網状赤血球を蛍光染色し、フローサイトメータで細胞の前方散乱光強度、蛍光強度を測定し、主に蛍光強度の差で成熟赤血球と網状赤血球を弁別し、網状赤血球を計数する方法が行われている。さらに、網状赤血球の蛍光強度によって、網状赤血球を成熟度別に分類計数すること（成熟指数の計算）も可能である。例えば、蛍光強度の強い網状赤血球の比率、すなわち最も若い網状赤血球の比率は、骨髄造血能の回復の指標として有用であることが知られている。
【０００７】
この方法に用いられる色素としては、オーラミンＯが良く知られている。オーラミンＯは、実質的に３０秒以内で網状赤血球を染色でき、迅速かつ精度よく網状赤血球を分析することができる。オーラミンＯ以外にもアクリジンオレンジ、チアゾールオレンジなども使用できるが、５〜３０分の染色時間を要する。
【０００８】
一方、網状赤血球を特徴づける物質の１つであるＲＮＡを特異的に染色する色素が特公昭６３−６１６２２号、米国特許第４，９５７，８７０号に開示されている。
【０００９】
【発明が解決しようとする課題】
オーラミンＯ、アクリジンオレンジやチアゾールオレンジを用いた網状赤血球の分析方法では、極めて有用な方法であるにもかかわらず、蛍光色素を励起するための光源として非常に高価なアルゴンレーザを必要とするため、装置が非常に高価なものになってしまう。また、上記公報には、励起光源としてアルゴンレーザ以外の光源を用いた網状赤血球を測定する技術は開示されていない。
【００１０】
さらには色素の細胞膜透過性の問題が存在する。上記公報に開示されているＲＮＡを特異的に染色する色素は、ＲＮＡと溶液中で混合することによって、飛躍的に蛍光強度が増大する。従って、原理的には網状赤血球の検出のための優れた色素となるはずである。しかしながら、実際に網状赤血球を検出しようとした場合、ＲＮＡ水溶液と混合する場合とは異なり、まず色素は細胞膜を透過しなければならない。しかし、色素の細胞膜透過性は色素によって異なり、全ての色素が速やかに細胞膜を透過し、ＲＮＡと結合するわけではない。そのため、全ての色素が網状赤血球の検出に使用できるわけではない。
【００１１】
アルゴンレーザの波長４８８ｎｍで励起できる色素は、分子の大きさが比較的小さく、比較的容易に細胞膜を透過するため、細胞中のＲＮＡと容易に結合できる。そのため、染色のために特別な工夫をしなくても、例えば、通常良く用いられるＰＢＳ緩衝液に色素を溶解しただけの染色液で網状赤血球の測定が可能である。
【００１２】
青領域よりも長波長の励起光を発するレーザはアルゴンレーザよりも安価であるが、そのような励起光源を使用して網状赤血球の測定を行うためには、青領域よりも長波長で励起可能な色素を使用しなければならない。しかし、そのような色素は、青領域（例えば４８８ｎｍ）で励起できる色素に比べて、分子内各部の炭化水素鎖長が長い、あるいは大きな芳香環を有する等のために、大半の物質は極性が低い。このような色素で赤血球を染色した場合、多くの色素は細胞膜の脂質二重層あるいはヘモグロビンに結合し、非特異蛍光を発しやすい。あるいは、細胞膜に対して透過性が悪い。例えば、本明細書の参考例に記載するように、上述の色素の１種を用いて血液試料を染色し、赤半導体レーザを光源とするフローサイトメータで測定した場合、図１に示すように赤血球の非特異蛍光が強く、網状赤血球を正確に測定することはできない。さらに、細胞膜透過性が悪く、染色のために少なくとも３０分以上の時間が必要である。
【００１３】
なお、非特異蛍光の問題は、何も青領域よりも長波長で励起可能な色素に特有の問題ではなく、青領域で励起可能な色素についても起こりうる。
【００１４】
一般に疎水性分子は、親水性分子より速やかに赤血球細胞膜を透過し、小さな分子は大きな分子よりも速やかに膜を透過するとされているが、どのような色素が網状赤血球を迅速にかつ特異的に染色することができるかを予測するのは困難である（特開平６−１８０３１５号、４頁、カラム６、３８行目〜５頁、カラム７、６行目参照）。
【００１５】
本発明は、Ｈｅ／Ｎｅレーザや赤半導体レーザなどの安価な励起光源を用いたフローサイトメータを用いて、網状赤血球を正確かつ迅速に測定するための試薬ならびに測定方法を提供することを目的とする。
【００１６】
【課題を解決するための手段】
本発明者らは上述した目的を達成するために鋭意研究した結果、本発明の色素が網状赤血球を正確かつ迅速に染色し、安価な励起光源を用いるフローサイトメータによっても網状赤血球を精度よく測定しうることを発見して本発明を完成した。
【００１７】
すなわち、本発明は、以下の構造式：
【化２】
ケニルまたはハロゲン化アルキルである］
からなる群から選ばれる少なくとも１つの色素を含有することを特徴とする網状赤血球測定用試薬を提供する。
【００１８】
本発明において使用可能な色素の例としては、以下のものが挙げられる。
【００１９】
（１）１，１’，３，３，３’，３’−ヘキサメチルインドジカルボシアニン アイオダイド（ＨＩＤＣＩ、ＬＡＭＢＤＡ ＰＨＹＳＩＫ社）
（１，１’，３，３，３’，３’−ｈｅｘａｍｅｔｈｙｌｉｎｄｏｄｉｃａｒｂｏｃｙａｎｉｎｅ ｉｏｄｉｄｅ）
【化３】
（１，１’−ｄｉｅｔｈｙｌ−２，４’−ｑｕｉｎｏｃａｒｂｏｃｙａｎｉｎｅ
ｉｏｄｉｄｅ）
【化４】
（Ｐｉｎａｃｙａｎｏｌｅ ｃｈｌｏｒｉｄｅ）
【化５】
【化６】
【００２０】
色素濃度がこの範囲を越えた場合、色素にもよるが、一般的には、赤血球の非特異蛍光が増加し、成熟赤血球と網状赤血球との弁別が困難になる。
【００２１】
色素濃度がこの範囲以下の場合、使用不可能ではないが、使用の容器壁面への吸着、分解等により色素濃度の減少が発生しやすくなる。
【００２２】
本発明の試薬中には、赤血球の非特異蛍光を抑制するための多価アニオンを含有することができる。一般に、赤血球系細胞を超生体染色する場合、溶血を防ぐために、染色液の浸透圧は、生理的浸透圧付近（２８０ｍＯｓｍ／ｋｇ程度）に調整される。この目的には、塩化ナトリウムを主成分（通常１００ｍＭ以上）とするＰＢＳが汎用されている。
【００２３】
ところが染色液中に塩化ナトリウム、とくに塩化物イオンが多量に存在する場合、赤血球の非特異蛍光が強く、成熟赤血球と網状赤血球の弁別が困難になる。しかし、塩化物イオンを多価アニオンで置換した場合、赤血球の非特異蛍光が著しく抑制され、成熟赤血球と網状赤血球の弁別が容易になることを本発明者らは見いだした。使用できる多価アニオンとしては、クエン酸、シュウ酸、フタル酸、琥珀酸等の多価カルボン酸イオン、燐酸イオン、硫酸イオン、炭酸イオン等が好適である。濃度は、試薬中の全アニオン成分に占める割合がモル比で５０％以上が使用可能であり、好ましくは７０％以上が好適である。
【００２４】
ハロゲン等の単価アニオンは速やかに細胞膜を通過して、ヘモグロビン分子と結合してヘモグロビンの立体構造を変化させ、色素に対する親和性を増加させ、結果として、赤血球の非特異蛍光が増加すると考えられる。単価アニオンの大部分を多価アニオンに置換することにより赤血球の非特異蛍光は抑制される。
【００２５】
なお、多価アニオンの対イオンはアルカリ金属イオンが好適である。
【００２６】
さらに本発明において、多価アニオンを組み合わせて用いるとさらに好適である。例えば、燐酸イオンを主成分とし（８０〜２００ｍＭ）、多価カルボン酸を組み合わせる（５〜８０ｍＭ）。燐酸とシュウ酸との組合せは特に好適であり、赤血球の非特異蛍光を抑制するだけでなく網状赤血球の蛍光強度を増加させる効果がある。
【００２７】
本発明においては、浸透圧補償剤を添加することもできる。浸透圧補償剤は赤血球の低張溶血を防ぐために添加するものであり、通常１５０ｍＯｓｍ／ｋｇ以上の浸透圧があればよい。浸透圧が高すぎることの弊害は特に認めないが、通常６００ｍＯｓｍ／ｋｇ程度までで使用される。上述の多価アニオンで十分な浸透圧が得られる場合は必須の成分ではない。浸透圧補償剤として特に制限されるものはない。例えば、プロピオン酸などのアルカリ金属塩、グルコース、マンノースなどの糖類が好適に使用される。なお、全アニオン成分に占める割合が５０％未満であれば、ハロゲン化物も使用できる。
【００２８】
さらに本発明においては、緩衝剤を含有させることもできる。緩衝剤は、安定した染色結果を得られるように、ｐＨを一定に保つために加えられるものである。通常使用される緩衝剤、例えば、カルボン酸類、燐酸、グッド緩衝剤等が使用でき、数ｍＭ〜１００ｍＭ程度の濃度で使用される。ｐＨは染色に好適なｐＨが選ばれる。好適なｐＨは、色素によって異なるが、４．０〜１１．０の範囲から選ばれる。ｐＨがこの範囲よりも低すぎる場合、あるいは高すぎる場合、赤血球が溶血しやすくなり、正確な測定ができなくなる。さらに、ｐＨが高すぎる場合、赤血球膜上の酸性官能基が解離してマイナスチャージを持ち、カチオン性である色素と結合することにより、赤血球の非特異蛍光が増加して、成熟赤血球と網状赤血球の弁別が困難になる傾向がある。
【００２９】
さらに好適には４．５〜９．０のｐＨ範囲、より好適には４．５〜７．０の範囲が選ばれる。なお、前述の多価アニオンの塩類によって、ｐＨを染色に適した値に安定に保つことができるのであれば必須の成分ではない。
【００３０】
また、本発明の試薬には、赤血球球状化剤として界面活性剤を赤血球が溶血しない濃度でかつ網状赤血球の染色を阻害しない濃度で含有させることもできる。
【００３１】
さらに、色素が水溶液中で不安定な場合、色素を適当な水溶性の非水溶媒、例えば、エタノール、ジメチルスルホキシド、エチレングリコール等に溶解して保存し、使用時に希釈液と混合して使用することができる。希釈液の成分としては、前述の多価アニオンの塩類、浸透圧補償剤、緩衝剤あるいは赤血球球状化剤を含有させることができる。
【００３２】
本発明の試薬を用い、フローサイトメータで網状赤血球を測定するには、
（ａ）網状赤血球測定用試薬と血液学的試料とを混合して測定用試料を作製し、
（ｂ）測定用試料をフローサイトメータに流し、
（ｃ）細胞からの蛍光と、大きさ情報を反映するパラメータを測定し、
（ｄ）大きさ情報あるいは、大きさ情報と蛍光強度で赤血球系細胞と血小板を弁別し、
（ｅ）蛍光強度で赤血球と網状赤血球と白血球を弁別し、網状赤血球数、網状赤血球比率を測定する。
【００３３】
測定用試料の作製にあたっては、網状赤血球測定用試薬と血液学的試料との混合比は、１００：１〜１０００：１、反応時間は１０〜１２０秒、反応温度は２５〜５０℃の範囲で作製することができる。
【００３４】
大きさ情報を反映するパラメータとしては、前方散乱光強度、電気抵抗信号が挙げられる。また、蛍光は、前方蛍光または側方蛍光のいずれを測定してもよい。
【００３５】
本発明の方法で使用するフローサイトメータの光源は、色素によって異なるが、Ｈｅ／Ｎｅレーザ、赤半導体レーザ等安価なものが好適である。
【００３６】
【実施例】
参考例
次の構造式を有する色素（米国特許第４，９５７，８７０号に開示された範囲に含まれる）：
【化７】
【００３７】
上記の染色液２ｍｌに抗凝固処理血液１０μｌを加え、室温で３０分間インキュベートした後、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで、前方散乱光および６６０ｎｍ以上の側方蛍光を測定した。
【００３８】
図１に示したように、赤血球の非特異蛍光が強く、網状赤血球と成熟赤血球の弁別が困難である。
【００３９】
本参考例の試薬で処理した検体の網状赤血球比率は、０．７８％であった。一方、同じ検体を対照としてアルゴンレーザを光源とするＲ−２０００システム（東亜医用電子株式会社製、染色液はオーラミンＯを使用）で測定した場合、１．５％であり、本参考例の試薬では、網状赤血球を正確に測定できないことが判明した。
【００４０】
実施例１
ＨＩＤＣＩ １ｍｇをＰＢＳ（ＮａＨ２ＰＯ４ １０ｍＭ、ＮａＣｌ １４０ｍＭ、ＮａＯＨでｐＨ７．０に調整）１ｌ に溶解し染色液とした。
【００４１】
上記の染色液２ｍｌに抗凝固処理血液１０μｌを加え、３５℃で２０秒間インキュベートした後、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで、前方散乱光および６６０ｎｍ以上の側方蛍光を測定した。
【００４２】
図２に示したように、網状赤血球と成熟赤血球との間に十分な蛍光強度の差があり、網状赤血球を測定できる。
【００４３】
本実施例の試薬で処理した検体の網状赤血球比率は、１．７６％であった。一方、同じ検体を参考例と同様にＲ−２０００システムで測定した場合、１．６５％であり、本実施例の試薬を用いると、網状赤血球を正確に測定できることが判明した。
【００４４】
実施例２
ＨＩＤＣＩ １ｍｇを１５０ｍＭ ＮａＨ２ＰＯ４水溶液（ＮａＯＨでｐＨ７．０に調整）１ｌ に溶解し、 染色液とした。測定は実施例１と同様に行った。
【００４５】
図３に示したように、成熟赤血球の非特異蛍光が抑制され、さらに良好に網状赤血球を測定できる。
【００４６】
本実施例の試薬で処理した検体の網状赤血球比率は、１．３０％であった。一方、同じ検体を参考例と同様にＲ−２０００システムで測定した場合、１．３３％であり、本実施例の試薬を用いても、網状赤血球を正確に測定できることが判明した。
【００４７】
実施例３
シュウ酸 ３０ｍＭ
ＮａＨ２ＰＯ４ １２０ｍＭ
ＮａＯＨでｐＨ５．０に調整
上記の水溶液に色素を溶解し、染色液を調製した。
【００４８】
色素として、ＨＩＤＣＩ １ｍｇ／ｌ用いた場合を図４に、１，１’−ジエチル−２，４’−キノカルボシアニン アイオダイド３ｍｇ／ｌを用いた場合を図５に、ピナシアノール クロライド１ｍｇ／ｌを用いた場合を図６に示す。いずれの場合でも、網状赤血球と成熟赤血球との間に十分な蛍光強度の差があり、網状赤血球を測定できる。
【００４９】
本実施例で用いた検体を参考例と同様にＲ−２０００システムで測定したとき、その網状赤血球比率は、２，０１％であった。同じ検体を本実施例の色素で染色し、赤半導体レーザを光源とするフローサイトメータを用いて測定したとき、
色素ＨＩＤＣＩ：１．９８％
色素１，１’−ジエチル−２，４’−キノカルボシアニン アイオダイド：
２．０５％
色素ピナシアノール クロライド：１．８９％
であり、本実施例のいずれの色素で染色して赤半導体レーザで測定する場合にもアルゴンレーザと同様に網状赤血球を正確に測定できることが判明した。
【００５０】
実施例４
実施例３でＨＩＤＣＩ １ｍｇ／ｌを用いた場合と、対照法としてＲ−２０００システム（東亜医用電子株式会社製、染色液はオーラミンＯを使用）を用いた場合との相関図を図７に示す。
【００５１】
直線回帰式Ｙ＝０．９３＋０．１８、相関係数ｒ＝０．９１５１（ｎ＝３７）であり、Ｒ−２０００システムと比較して、良好な相関が得られた。
【００５２】
【発明の効果】
本発明によれば、安価な光源を用いたフローサイトメータで、網状赤血球を測定することができる。また、色素と多価アニオンを組み合わせることによって、さらに精度を向上させることができる。
【図面の簡単な説明】
【図１】参考例に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。本図ならびに以下の図において、ＲＢＣは赤血球を、Ｒｅｔは網状赤血球を、ＰＬＴは血小板を表す。なお、白血球は蛍光強度が非常に強く、信号がオーバーフローしている。
【図２】実施例１に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。
【図３】実施例２に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。
【図４】色素としてＨＩＤＣＩを用いる実施例３に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。
【図５】色素として１，１’−ジエチル−２，４’−キノカルボシアニン アイオダイドを用いる実施例３に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。
【図６】色素としてピナシアノール クロライドを用いる実施例３に記載の染色液で染色した血液を、赤半導体レーザ（波長６３３ｎｍ）を光源とするフローサイトメータで前方散乱光および６６０ｎｍ以上の側方蛍光を測定した結果を示す。
【図７】実施例３でＨＩＤＣＩを用いた場合と、Ｒ−２０００システム（染色液はオーラミンＯ）を用いた場合の相関図を示す。[0001]
[Industrial applications]
The present invention relates to a reagent and a method for measuring reticulocytes in blood in the field of clinical testing.
[0002]
[Prior art]
Reticulocytes are young erythrocytes immediately after erythroid cells differentiated and matured from hematopoietic stem cells in bone marrow are enucleated and released from the bone marrow into peripheral blood, and become mature erythrocytes 1-2 days later. Reticulocytes have a small amount of RNA or organelles such as ribosomes and mitochondria not contained in mature red blood cells, as a remnant of cell maturation.
[0003]
In the clinical examination field, classification and counting of reticulocytes is a very important test for understanding the hematopoietic state in the bone marrow of a patient. In a normal person, reticulocytes account for 0.5 to 2.0% of all red blood cells, decrease in a state where myelopoiesis is suppressed, and increase in a state where myelopoiesis is enhanced. For example, it decreases during chemotherapy for aplastic anemia and malignant tumor, and increases for hemolytic anemia and the like.
[0004]
By the way, in order to measure reticulocytes, conventionally, blood is mixed with a staining solution containing a basic dye such as new methylene blue (NMB) or brilliant cresyl blue (BCB) (super vital staining). A method of discriminating mature red blood cells from reticulocytes by counting the remaining substances contained in reticulocytes in a reticulated form, preparing a smear, and observing each cell with a microscope (method for use) ) Has been done.
[0005]
However, it is known that the operation method is complicated, and that there is a large statistical error due to individual differences in cell discrimination between test technologists and a small number of cell counts.
[0006]
In order to solve these problems, reticulocytes are fluorescently stained with a fluorescent basic dye instead of the aforementioned basic dye, and the forward scattered light intensity and the fluorescence intensity of the cells are measured with a flow cytometer. In addition, a method of discriminating mature red blood cells from reticulocytes based on a difference in fluorescence intensity and counting reticulocytes is performed. Furthermore, it is also possible to classify and count reticulocytes by maturity according to the fluorescence intensity of the reticulocytes (calculation of the maturation index). For example, it is known that the ratio of reticulocytes having a high fluorescence intensity, that is, the ratio of the youngest reticulocytes, is useful as an indicator of the recovery of bone marrow hematopoiesis.
[0007]
Auramine O is well known as a dye used in this method. Auramine O can stain reticulocytes substantially within 30 seconds, and can analyze reticulocytes quickly and accurately. Besides auramine O, acridine orange, thiazole orange and the like can be used, but a dyeing time of 5 to 30 minutes is required.
[0008]
On the other hand, dyes that specifically stain RNA, which is one of the substances that characterize reticulocytes, are disclosed in JP-B-63-61622 and U.S. Pat. No. 4,957,870.
[0009]
[Problems to be solved by the invention]
In the method of analyzing reticulocytes using auramine O, acridine orange or thiazole orange, although an extremely useful method, an extremely expensive argon laser is required as a light source for exciting a fluorescent dye. The equipment becomes very expensive. Further, the above publication does not disclose a technique for measuring reticulocytes using a light source other than an argon laser as an excitation light source.
[0010]
Furthermore, there is a problem of the permeability of the dye to the cell membrane. The dye that specifically stains RNA disclosed in the above publication dramatically increases the fluorescence intensity when mixed with RNA in a solution. Therefore, in principle, it should be an excellent dye for the detection of reticulocytes. However, when actually trying to detect reticulocytes, unlike when mixed with an aqueous RNA solution, the dye must first penetrate the cell membrane. However, dyes have different cell membrane permeability, and not all dyes penetrate cell membranes quickly and bind to RNA. Therefore, not all dyes can be used to detect reticulocytes.
[0011]
Dyes that can be excited at the wavelength of 488 nm of an argon laser have a relatively small molecular size and penetrate cell membranes relatively easily, so that they can easily bind to RNA in cells. Therefore, for example, reticulocytes can be measured using a staining solution in which only a dye is dissolved in a commonly used PBS buffer solution without any special device for staining.
[0012]
Lasers that emit excitation light at longer wavelengths than the blue region are cheaper than argon lasers, but to measure reticulocytes using such an excitation light source, they can be excited at longer wavelengths than the blue region. Must be used. However, most of such dyes have polarities due to a longer hydrocarbon chain length in each part of the molecule or a larger aromatic ring than dyes that can be excited in the blue region (for example, 488 nm). Low. When erythrocytes are stained with such dyes, many dyes tend to bind to the lipid bilayer of cell membrane or hemoglobin and emit nonspecific fluorescence. Alternatively, permeability to cell membranes is poor. For example, as described in Reference Example of the present specification, when a blood sample is stained with one of the above-described dyes and measured with a flow cytometer using a red semiconductor laser as a light source, as shown in FIG. The non-specific fluorescence of erythrocytes is so strong that reticulocytes cannot be measured accurately. Further, the cell membrane permeability is poor, and a time of at least 30 minutes is required for staining.
[0013]
The problem of non-specific fluorescence is not a problem specific to a dye that can be excited at a wavelength longer than that of the blue region, but can occur with a dye that can be excited at the blue region.
[0014]
Generally, hydrophobic molecules penetrate red blood cell membranes more quickly than hydrophilic molecules, and small molecules penetrate membranes more quickly than large molecules.However, any dye can rapidly and specifically reticulate red blood cells. It is difficult to predict whether it can be stained (see JP-A-6-180315,
[0015]
An object of the present invention is to provide a reagent and a measuring method for accurately and quickly measuring reticulocytes using a flow cytometer using an inexpensive excitation light source such as a He / Ne laser or a red semiconductor laser. I do.
[0016]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above-mentioned object, and as a result, the dye of the present invention stains reticulocytes accurately and quickly, and accurately measures reticulocytes even with a flow cytometer using an inexpensive excitation light source. The present invention was completed by discovering that the present invention can be performed.
[0017]
That is, the present invention provides the following structural formula:
Embedded image
A reticulocyte measurement reagent, characterized by containing at least one dye selected from the group consisting of:
[0018]
Examples of dyes that can be used in the present invention include the following.
[0019]
(1) 1,1 ′, 3,3,3 ′, 3′-Hexamethylindodicarbocyanine iodide (HIDCI, LAMBDA PHYSIK)
(1,1 ′, 3,3,3 ′, 3′-hexamethylindodicarbocyanine iodide)
Embedded image
(1,1'-diethyl-2,4'-quinocarbocyanine
iodide)
Embedded image
Embedded image
Embedded image
[0020]
If the dye concentration exceeds this range, non-specific fluorescence of erythrocytes generally increases, depending on the dye, and it becomes difficult to distinguish mature erythrocytes from reticulocytes.
[0021]
When the dye concentration is below this range, it is not impossible to use the dye, but the dye concentration tends to decrease due to adsorption and decomposition on the wall surface of the used container.
[0022]
The reagent of the present invention may contain a polyvalent anion for suppressing nonspecific fluorescence of erythrocytes. Generally, when erythroid cells are subjected to super-viable staining, the osmotic pressure of the staining solution is adjusted to near physiological osmotic pressure (about 280 mOsm / kg) in order to prevent hemolysis. For this purpose, PBS containing sodium chloride as a main component (usually 100 mM or more) is widely used.
[0023]
However, when a large amount of sodium chloride, particularly chloride ions, is present in the staining solution, non-specific fluorescence of erythrocytes is strong, and it becomes difficult to distinguish mature erythrocytes from reticulocytes. However, the present inventors have found that when the chloride ion is replaced with a polyvalent anion, non-specific fluorescence of erythrocytes is remarkably suppressed, and it becomes easy to discriminate mature erythrocytes from reticulocytes. Suitable polyvalent anions include polyvalent carboxylate ions such as citric acid, oxalic acid, phthalic acid, and succinic acid, phosphate ions, sulfate ions, and carbonate ions. The concentration may be 50% or more, preferably 70% or more, of the total anion component in the reagent in a molar ratio.
[0024]
It is considered that a monovalent anion such as a halogen quickly passes through a cell membrane, binds to a hemoglobin molecule, changes the three-dimensional structure of hemoglobin, and increases affinity for a dye. As a result, nonspecific fluorescence of erythrocytes is thought to increase. Non-specific fluorescence of erythrocytes is suppressed by replacing most of the monovalent anions with polyvalent anions.
[0025]
The counter ion of the polyvalent anion is preferably an alkali metal ion.
[0026]
Furthermore, in the present invention, it is more preferable to use a polyvalent anion in combination. For example, a phosphate ion is a main component (80 to 200 mM), and a polyvalent carboxylic acid is combined (5 to 80 mM). The combination of phosphoric acid and oxalic acid is particularly preferred, and has the effect of not only suppressing the nonspecific fluorescence of red blood cells but also increasing the fluorescence intensity of reticulocytes.
[0027]
In the present invention, an osmotic pressure compensating agent can be added. The osmotic pressure compensating agent is added to prevent hypotonic hemolysis of erythrocytes, and usually has an osmotic pressure of 150 mOsm / kg or more. The adverse effect of an excessively high osmotic pressure is not particularly recognized, but it is usually used up to about 600 mOsm / kg. It is not an essential component when a sufficient osmotic pressure can be obtained with the above-mentioned polyvalent anion. There is no particular limitation on the osmotic pressure compensating agent. For example, alkali metal salts such as propionic acid, and sugars such as glucose and mannose are preferably used. In addition, as long as the proportion of the total anion component is less than 50%, a halide can be used.
[0028]
Further, in the present invention, a buffer may be contained. The buffer is added to keep the pH constant so as to obtain a stable staining result. Commonly used buffers such as carboxylic acids, phosphoric acid, and good buffers can be used, and are used at a concentration of about several mM to 100 mM. As the pH, a pH suitable for dyeing is selected. The preferred pH depends on the dye, but is selected from the range of 4.0 to 11.0. If the pH is too low or too high, the erythrocytes will be easily lysed, making accurate measurement impossible. Furthermore, when the pH is too high, the acidic functional groups on the erythrocyte membrane dissociate and have a negative charge, and bind to a cationic dye, thereby increasing the nonspecific fluorescence of the erythrocytes, and causing mature erythrocytes and reticulocytes. Discrimination tends to be difficult.
[0029]
More preferably, a pH range of 4.5 to 9.0, more preferably a range of 4.5 to 7.0 is selected. It is not an essential component if the above-mentioned salts of polyvalent anions can stably maintain the pH at a value suitable for dyeing.
[0030]
Further, the reagent of the present invention may contain a surfactant as a red blood cell sphering agent at a concentration at which red blood cells do not lyse and at a concentration which does not inhibit reticulocyte staining.
[0031]
Furthermore, when the dye is unstable in an aqueous solution, the dye is dissolved and stored in a suitable water-soluble non-aqueous solvent, for example, ethanol, dimethyl sulfoxide, ethylene glycol, etc., and used by mixing with a diluent at the time of use. be able to. As components of the diluent, the above-mentioned salts of polyvalent anions, osmotic pressure compensating agents, buffers or erythrocyte sphering agents can be contained.
[0032]
Using the reagent of the present invention, to measure reticulocytes with a flow cytometer,
(A) preparing a measurement sample by mixing a reticulocyte measurement reagent and a hematological sample,
(B) flowing the measurement sample through a flow cytometer,
(C) measuring the fluorescence from the cells and the parameters reflecting the size information,
(D) discriminating erythroid cells and platelets by size information or size information and fluorescence intensity,
(E) Red blood cells, reticulocytes and white blood cells are discriminated based on the fluorescence intensity, and the reticulocyte count and the reticulocyte ratio are measured.
[0033]
In preparing the measurement sample, the mixing ratio of the reticulocyte measurement reagent and the hematological sample is 100: 1 to 1000: 1, the reaction time is 10 to 120 seconds, and the reaction temperature is 25 to 50 ° C. Can be made.
[0034]
The parameters reflecting the magnitude information include forward scattered light intensity and electric resistance signal. The fluorescence may be measured as either forward fluorescence or side fluorescence.
[0035]
The light source of the flow cytometer used in the method of the present invention varies depending on the dye, but an inexpensive light source such as a He / Ne laser or a red semiconductor laser is preferable.
[0036]
【Example】
Reference Examples Dyes having the following structural formula (included in the range disclosed in US Pat. No. 4,957,870):
Embedded image
[0037]
10 μl of anticoagulated blood was added to 2 ml of the above staining solution, incubated at room temperature for 30 minutes, and then forward scattered light and lateral fluorescence of 660 nm or more were measured with a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source. did.
[0038]
As shown in FIG. 1, non-specific fluorescence of erythrocytes is strong, and it is difficult to discriminate between reticulocytes and mature erythrocytes.
[0039]
The reticulocyte ratio of the sample treated with the reagent of this reference example was 0.78%. On the other hand, when the same sample was measured using an R-2000 system using an argon laser as a light source (manufactured by Toa Medical Electronics Co., Ltd., the staining solution used was Auramine O), it was 1.5%, Found that reticulocytes could not be measured accurately.
[0040]
Example 1
1 mg of HIDCI was dissolved in 1 l of PBS (10 mM of NaH 2 PO 4 , 140 mM of NaCl, adjusted to pH 7.0 with NaOH) to obtain a staining solution.
[0041]
10 μl of anticoagulated blood was added to 2 ml of the above-mentioned staining solution, incubated at 35 ° C. for 20 seconds, and forward scattered light and lateral fluorescence of 660 nm or more were measured with a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source. It was measured.
[0042]
As shown in FIG. 2, there is a sufficient difference in fluorescence intensity between reticulocytes and mature red blood cells, and reticulocytes can be measured.
[0043]
The reticulocyte ratio of the sample treated with the reagent of this example was 1.76%. On the other hand, when the same sample was measured by the R-2000 system in the same manner as in the reference example, it was 1.65%, and it was found that the use of the reagent of this example allowed accurate measurement of reticulocytes.
[0044]
Example 2
1 mg of HIDCI was dissolved in 1 liter of a 150 mM NaH 2 PO 4 aqueous solution (adjusted to pH 7.0 with NaOH) to obtain a staining solution. The measurement was performed in the same manner as in Example 1.
[0045]
As shown in FIG. 3, nonspecific fluorescence of mature red blood cells is suppressed, and reticulocytes can be measured more favorably.
[0046]
The reticulocyte ratio of the sample treated with the reagent of this example was 1.30%. On the other hand, when the same sample was measured by the R-2000 system in the same manner as in the Reference Example, it was 1.33%, and it was found that reticulocytes could be accurately measured even by using the reagent of this example.
[0047]
Example 3
Oxalic acid 30 mM
NaH 2 PO 4 120 mM
The pH was adjusted to 5.0 with NaOH, and the dye was dissolved in the above aqueous solution to prepare a staining solution.
[0048]
FIG. 4 shows the case where 1 mg / l of HIDCI was used as a dye, and FIG. 5 shows the case where 3 mg / l of 1,1′-diethyl-2,4′-quinocarbocyanine iodide was used, and FIG. 5 shows the case where 1 mg / l of pinacyanol chloride was used. FIG. In any case, there is a sufficient difference in fluorescence intensity between reticulocytes and mature red blood cells, and reticulocytes can be measured.
[0049]
When the sample used in this example was measured by the R-2000 system in the same manner as in the Reference Example, the reticulocyte ratio was 2.01%. When the same sample was stained with the dye of the present example and measured using a flow cytometer using a red semiconductor laser as a light source,
Dye HIDCI: 1.98%
2.05%
Dye pinacyanol chloride: 1.89%
It was found that the reticulocytes can be accurately measured in the same manner as in the case of the argon laser when the dye is stained with any of the dyes of this example and measured with a red semiconductor laser.
[0050]
Example 4
FIG. 7 shows a correlation diagram between the case where
[0051]
The linear regression equation was Y = 0.93 + 0.18, and the correlation coefficient r was 0.9151 (n = 37), indicating that a better correlation was obtained as compared with the R-2000 system.
[0052]
【The invention's effect】
According to the present invention, reticulocytes can be measured with a flow cytometer using an inexpensive light source. Further, the accuracy can be further improved by combining a dye and a polyvalent anion.
[Brief description of the drawings]
FIG. 1 shows the results of measurement of forward scattered light and lateral fluorescence of 660 nm or more of blood stained with the staining solution described in Reference Example with a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source. In this figure and the following figures, RBC represents red blood cells, Ret represents reticulocytes, and PLT represents platelets. Note that the white blood cells have a very strong fluorescence intensity, and the signal overflows.
FIG. 2 shows the results of measuring forward scattered light and lateral fluorescence of 660 nm or more of blood stained with the staining solution described in Example 1 with a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source.
FIG. 3 shows the results of measuring forward scattered light and lateral fluorescence of 660 nm or more of blood stained with the staining solution described in Example 2 using a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source.
FIG. 4 Measures forward scattered light and lateral fluorescence of 660 nm or more of blood stained with the staining solution described in Example 3 using HIDCI as a dye with a flow cytometer using a red semiconductor laser (wavelength 633 nm) as a light source. The results obtained are shown.
FIG. 5 is a flow chart of blood stained with the staining solution described in Example 3 using 1,1′-diethyl-2,4′-quinocarbocyanine iodide as a dye, using a red semiconductor laser (wavelength: 633 nm) as a light source. The results of measuring forward scattered light and side fluorescence of 660 nm or more with a cytometer are shown.
FIG. 6 is a flow cytometer using a red semiconductor laser (wavelength: 633 nm) as a light source to measure forward scattered light and side fluorescence at 660 nm or more using blood stained with the staining solution described in Example 3 using pinacyanol chloride as a dye. The result of the measurement is shown.
FIG. 7 shows a correlation diagram between the case where HIDCI was used in Example 3 and the case where an R-2000 system (staining solution was Auramine O) was used.
Claims (7)
からなる群から選ばれる少なくとも１つの色素を含む、網状赤血球と成熟赤血球を弁別可能に染色する網状赤血球測定用試薬。The following structural formula:
A reagent for measuring reticulocyte , which comprises at least one dye selected from the group consisting of:
（ａ）網状赤血球測定用試薬と血液学的試料とを混合して測定用試料を作製し、（ｂ）測定用試料をフローサイトメータに流し、
（ｃ）細胞からの蛍光と、大きさ情報を反映するパラメータを測定し、
（ｄ）大きさ情報あるいは、大きさ情報と蛍光強度で赤血球系細胞と血小板を弁別し、
（ｅ）蛍光強度で赤血球と網状赤血球と白血球を弁別し、網状赤血球数、網状赤血球比率を測定する。The method of claim 6, wherein the measurement is performed by the following steps:
(A) mixing a reticulocyte measurement reagent and a hematological sample to prepare a measurement sample, and (b) flowing the measurement sample through a flow cytometer.
(C) measuring the fluorescence from the cells and the parameters reflecting the size information,
(D) discriminating erythroid cells and platelets by size information or size information and fluorescence intensity,
(E) Red blood cells, reticulocytes and white blood cells are discriminated based on the fluorescence intensity, and the reticulocyte count and the reticulocyte ratio are measured.
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| JP15347295A JP3553691B2 (en) | 1995-04-12 | 1995-06-20 | Reticulocyte measuring reagent and measuring method |
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| Application Number | Priority Date | Filing Date | Title |
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| JP8703795 | 1995-04-12 | ||
| JP7-87037 | 1995-04-12 | ||
| JP15347295A JP3553691B2 (en) | 1995-04-12 | 1995-06-20 | Reticulocyte measuring reagent and measuring method |
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| JP3425830B2 (en) * | 1995-10-06 | 2003-07-14 | シスメックス株式会社 | New compounds and their uses |
| TW379284B (en) * | 1996-04-12 | 2000-01-11 | Toa Medical Electronics | Agent for detecting reticulocyte |
| JP3783808B2 (en) * | 1997-05-19 | 2006-06-07 | シスメックス株式会社 | Leukocyte classification and counting reagent |
| US5874311A (en) * | 1997-11-21 | 1999-02-23 | Coulter International Corp. | Method for differentiation of reticulocytes in blood |
| 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 |
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