JPS62153758A - Method for measuring reticulocyte by flow sight meter - Google Patents
Method for measuring reticulocyte by flow sight meterInfo
- Publication number
- JPS62153758A JPS62153758A JP60293700A JP29370085A JPS62153758A JP S62153758 A JPS62153758 A JP S62153758A JP 60293700 A JP60293700 A JP 60293700A JP 29370085 A JP29370085 A JP 29370085A JP S62153758 A JPS62153758 A JP S62153758A
- Authority
- JP
- Japan
- Prior art keywords
- light
- fluorescence
- exciting
- reticulocytes
- optical
- 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
- 210000001995 reticulocyte Anatomy 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims description 16
- 230000003287 optical effect Effects 0.000 claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims abstract description 17
- 210000003743 erythrocyte Anatomy 0.000 claims description 13
- 230000005284 excitation Effects 0.000 claims description 12
- 210000001772 blood platelet Anatomy 0.000 claims description 8
- 230000000638 stimulation Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 2
- 210000000601 blood cell Anatomy 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 7
- 238000007796 conventional method Methods 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000004159 blood analysis Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000005375 photometry Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- YDSPVZISFWHZIL-UHFFFAOYSA-N 9-(4-aminophenyl)-7-methylacridin-3-amine;nitrate Chemical compound [O-][N+]([O-])=O.C12=CC(C)=CC=C2N=C2C=C(N)C=CC2=C1C1=CC=C(N)C=C1 YDSPVZISFWHZIL-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- JPIYZTWMUGTEHX-UHFFFAOYSA-N auramine O free base Chemical compound C1=CC(N(C)C)=CC=C1C(=N)C1=CC=C(N(C)C)C=C1 JPIYZTWMUGTEHX-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000004298 light response Effects 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
- G01N2015/012—Red blood cells
- G01N2015/014—Reticulocytes
Landscapes
- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
不発明は血液分析時に血液中の網状赤血球のフローサイ
トメータによる測定技術に関するものである。DETAILED DESCRIPTION OF THE INVENTION The invention relates to a technique for measuring reticulocytes in blood using a flow cytometer during blood analysis.
不発明のフローサイトメータによる網状赤血球測定方法
は臨床検査の分野において検体血液中の幼若な網状赤血
球をはじめ他の血球等有形成分ン光学的に分類測定する
際にきわめて■益である。The uninvented method for measuring reticulocytes using a flow cytometer is extremely useful in the field of clinical testing when optically classifying and measuring formed components such as immature reticulocytes and other blood cells in blood samples.
従来の技術
染色試薬ヶ用いて予め特定細胞や粒子を染色して検体の
血液分析χ行う光学的かつフローサイトメトリーによる
血液分析測定技術はこれまで昧々に具体比されている。BACKGROUND OF THE INVENTION Conventional techniques Blood analysis and measurement techniques using optical and flow cytometry methods, in which specific cells and particles are stained in advance using staining reagents and blood analysis of a specimen is performed, have been described in detail up to now.
血液試料については、螢光染色した血球か放出する螢光
の水平・垂直方向の異方性比馨測定するもの(特開昭5
9−102159号公報参照)、細胞の螢光強度分布よ
り細胞の分類計数を行うもの(特公昭54−14957
号公報参照)、細胞の螢光染色試薬として網状赤血球に
対する選択性の強いチオフラピンT乞用いているもの(
特開昭59−142465号公報参照)、下、″a出願
人による三角フローセルで挟角散乱光についてこれを高
効率で受光しようとするもの(待筑昭60−66169
号)、同じく不願出、願人による別状赤血球に対して極
めて選択性の強いオーラミンQを螢光染色試薬とするも
の(特願昭60−123128号)、レーザ光源乞用い
螢光と散乱光乞同時測光して網状赤血球を同定計数する
もの(米国特許第4525706号)などを挙げること
ができる。For blood samples, the anisotropy ratio in the horizontal and vertical directions of the fluorescence emitted from fluorescently stained blood cells is measured (Japanese Patent Laid-Open No. 5
9-102159), a method for classifying and counting cells based on the fluorescence intensity distribution of cells (Japanese Patent Publication No. 54-14957)
Thiofurapine T, which has strong selectivity for reticulocytes, is used as a fluorescent staining reagent for cells.
(Refer to Japanese Patent Application Laid-Open No. 142465/1982), below, a device by the applicant "a" which attempts to receive narrow angle scattered light with high efficiency using a triangular flow cell (Machichiku Sho 60-66169)
(No. 60-123128), also unfiled by the applicant, using auramine Q, which is extremely selective for differentiated red blood cells, as a fluorescent staining reagent (Patent Application No. 123128/1983), and a method using a laser light source for fluorescence and scattered light. Examples include one that simultaneously measures light to identify and count reticulocytes (US Pat. No. 4,525,706).
フローサイトメータによる上記の如き代表的な血液分析
には例えば゛第4図に示す基本構成の測定系が用いられ
ている。血液試料を流動させるフローセルろの細胃耶に
励起光Eをレーザ光源1からコンデンサレンズ2を介し
て入射し、この光学的刺激域からの側方散乱光及び側方
螢光を散乱光除去フィルタソ、コンデンサレンズ10.
フィールドアパーチャ11.受光フィルタ12乞経て光
検出素子16で側方螢光のみ測光すると共に、フローセ
ル6かも直進し10光のうち励起光Bの一部χビームス
トッパ4で除去しfこ後コンデンサレンズ5、フィール
ドアパーチャ6を経て前方散乱元Sを光検出素子7で測
光し、同一細胞まγこは粒子から発せられ1こものとし
てそれぞれの光強度信号は渭幅器14,8を経て解析部
15に入力され血球種別1粒子ごとに特定され計数され
て表示又は記録器16により表示される。For example, a measurement system having the basic configuration shown in FIG. 4 is used for the above-mentioned typical blood analysis using a flow cytometer. Excitation light E is incident from a laser light source 1 through a condenser lens 2 into the narrow stomach of a flow cell in which a blood sample is made to flow, and side scattered light and side fluorescence from this optical stimulation area are removed by a scattered light removal filter and a condenser. Lens 10.
Field aperture 11. After passing through the light receiving filter 12, only the side fluorescent light is measured by the photodetecting element 16, and at the same time, the flow cell 6 also goes straight, and part of the excitation light B among the 10 lights is removed by the χ beam stopper 4. After that, the condenser lens 5 and the field aperture 6 are removed. Then, the forward scattering source S is photometered by the photodetecting element 7, and each light intensity signal is assumed to be emitted from the same cell particle and is inputted to the analysis unit 15 via the transducers 14 and 8 to determine the blood cell type. Each particle is identified, counted, and displayed on the display or recorder 16.
上記のフローサイトメータによる血球分析では光学的刺
激域からの応答としての散乱光及び螢光が複数の光軸に
沼って配置された測光系に進み。In blood cell analysis using the above-mentioned flow cytometer, scattered light and fluorescent light as a response from the optical stimulation area travels to a photometric system arranged along a plurality of optical axes.
それぞれの光軸上で受光するようにされている。Light is received on each optical axis.
従って、第2図(b)及び第4図に見る如(、V−ザ光
源1から真直ぐに(Loで調整後固定しである)フロー
セル乙に向かう励起光Eの光軸Aに宿ってフローセル?
法んで配置され1こコンデンサレンズ2,5については
、それらのmノし−及び焦点位置稠整乞矢印し。+ L
1 + L2 のそれぞれに対し終了したとしても
、上記調整に伴って、光@Aと置父する光軸Bに溜つ1
こ側方螢光Fの測光系についてもコンデンサレンズ10
乞矢印L3方向にコンデンサレンズ2,5とのずれを解
消するように調整しなければならず、この元@B上での
ずれの修正は再び光軸AK沼った光学系のずれを生むの
でLL+L2を再修正する必要が生ずる。かくして、光
軸A 、 B Kl”oつた焦点及び細心のずれの修正
後にも。Therefore, as shown in FIG. 2(b) and FIG. ?
As for the single condenser lenses 2 and 5, which are arranged horizontally, the arrows indicate their m and focal positions. +L
Even if it is completed for each of 1 + L2, due to the above adjustment, 1 accumulated on the optical axis B, which is the same as the light @A.
Condenser lens 10 is also used for the photometry system of this lateral fluorescence F.
Adjustment must be made to eliminate the misalignment between the condenser lenses 2 and 5 in the direction of arrow L3, and correcting this misalignment on the base @B will again cause misalignment of the optical system. It becomes necessary to modify LL+L2 again. Thus, even after the optical axes A and B have been corrected for focus and minute deviations.
さらにこれらの再修正が少なくとも数回要求されるのみ
ならず、散乱光と螢光とて家光軸か異なるので経時変[
ヒが異質となり、第6図左側の従来り1」に示すように
調整時の実線信号に対して点線のよ5な4怒度ずれ波形
か螢光信号(場合によっては散乱元信号)に偏って現わ
れる。この1こめ、特定の血球細胞又は粒子に対応した
複数の元学的特性乞同時に、相対的に解析部べぎフロー
サイトメータでは定時的に感度のずれを再調整しないと
血球又は粒子の種類別の同定及びそれらの計数に相当の
誤差の発生が避けられなかった。特に、この偏りは2次
元分布解析乞行う従来技術において問題であつ1こ。Furthermore, not only are these re-corrections required at least several times, but the optical axes of scattered light and fluorescent light are different, so changes over time [
As shown in the conventional method 1 on the left side of Figure 6, the waveform is biased toward a 5 degree deviation waveform (as shown by the dotted line) or a fluorescent signal (in some cases, the scattering source signal) with respect to the solid line signal during adjustment. appears. In this case, multiple chemical characteristics corresponding to specific blood cells or particles are required, and at the same time, it is necessary to periodically readjust the sensitivity deviation of the flow cytometer in the analysis section. Considerable errors were unavoidable in the identification and counting of these. In particular, this bias is a problem in conventional techniques that perform two-dimensional distribution analysis.
次に、複数の検出情報によって成熟赤血球及び血小板か
ら網状赤血球の正確な弁別2行うために1i複雑な信号
処理法が採用されてさ1こ。詳しくは。Next, a complex signal processing method is employed to perform accurate discrimination of reticulocytes from mature red blood cells and platelets by multiple detection information. For more information.
前記米国特許第11325706号乞参照され1C2゜
これは、1)散乱光強度と螢光強度の2次元分布図を作
成し、11)赤血球と血小板と’&[号上分離するため
に升別基準直顧を作り、111)血小板乞取除い1こ血
球群の螢光強度を散乱光強度信号で補正し、IV)補正
された螢光強度に対して頻度分布曲線を求めこの分布曲
線の半分で成熟赤血球の分布曲線ケ正規分布として推定
しこの正規分布曲線からすれ1こ信号群を網状赤血球群
として算定する。という手順である。上記iV)は特に
複雑であるだけでな(、赤血球の螢元分布乞正規分布と
して仮定しているので、これに伴って成熟赤血球と(幼
若)網状赤血球のそれぞれの螢光強度に相関関係かある
ものと擬制しなければならず、これン前提とした血球分
析結果が無視し難い誤差乞発生することがあつ1こ。こ
のことは現実の検体の螢光特性かこうした正規分布や相
関関係にそれほど捉われない多様なものであるところか
ら派生しており、特にこの傾向は網状赤血球比率が多い
検体はど大きい等から染色時の染色量も関係し壬いるも
のと見られる。Please refer to the above-mentioned US Pat. No. 1,132,5706. This involves 1) creating a two-dimensional distribution map of the scattered light intensity and fluorescent light intensity, and 11) using the square separation criteria to separate red blood cells and platelets. 111) Correct the fluorescence intensity of one blood cell group from which platelets are removed using the scattered light intensity signal, and IV) find a frequency distribution curve for the corrected fluorescence intensity and use half of this distribution curve. The distribution curve of mature red blood cells is estimated as a normal distribution, and a group of signals that fall within the normal distribution curve is calculated as a reticulocyte group. This is the procedure. Not only is the above iV) particularly complicated, but it also assumes a normal distribution of the red blood cell fluorescence distribution, so there is a correlation between the fluorescence intensity of mature red blood cells and (immature) reticulocytes. This may lead to a non-negligible error in the blood cell analysis results based on this assumption. In particular, this tendency is thought to be related to the amount of staining at the time of staining, such as how large the specimens with a high proportion of reticulocytes are.
問題?解決する1こめの手段
本発明は、フローサイトメータによる網状赤血球の測定
に際し網状赤血球比率の大小に代表される検体の特質に
左右されろことなく、多様な検俸乞同時に処理しても検
体相互の待機ン正しく挾出し計数誤差の発生を最小[ヒ
すること並ひに測光光学系を簡素[ヒすることを可能に
したものであり。problem? One Means to Solve the Problem The present invention provides a method for measuring reticulocytes using a flow cytometer, regardless of the characteristics of the specimen, such as the size of the reticulocyte ratio. This makes it possible to minimize the occurrence of counting errors and to simplify the photometry optical system.
二の1こめに不発明は新しい螢光染色染料を使用し。The second invention was to use a new fluorescent dye.
倹(1)信号を改善して弁別処理乞簡素で安定なものと
すると共に、励起光の光軸に沿って配置され1こフロー
セルの光学的刺激域から放出される出射光について励起
光χ事実上除去し1こうえで励起光の光軸に沿つ1こ同
軸上の前方散乱光及び前方螢光ン測元して網状赤血球を
測定するフローサイトメータによる網状赤血球測定方法
ヶ特色とするものである。(1) In addition to improving the signal and making the discrimination process simple and stable, the excitation light A method for measuring reticulocytes using a flow cytometer that removes the top and measures reticulocytes by measuring forward scattered light and forward fluorescence on the same axis along the optical axis of excitation light. It is.
実 施 列
不発明によるフローサイトメータによる網状赤血球測定
方法では先ずフラボホスフィンR4主剤とする血球測定
用試薬500に対して抗凝固性全血液試料1の比率で混
合して螢光染色7行う。試薬について詳しくは特願昭5
9−201273号ケ参照のこと、次に試料を測定する
。第1図罠示す如くフローサイトメータのフローセル2
6に対してv −サ光源20からフィルタ21.コンデ
ンサレンズ22χ介して励起光E′が入射される。フロ
ーチャンネル中の試料液ヲ泣むよ5にして流れるシース
族の中心部ン光学的刺激域として上記励起元暑入射集束
し、上記光学的刺激域から出射されγこ光のうち励起光
を主成分とする光馨ビームストッパ24で遮断する。前
方螢光F′と前方散乱光S′とはコンデンサレンズ25
で平行光勝に戻し1こ後スリット板26を通して小径の
ビームに絞り、これをダイクロイックミラー27で削方
散乱元S、削万螢光rの2成分に分離する。前方散乱光
はダイクロイックミラー27の背後で光検出素子2Bで
受光され、増幅器29で増幅され1こ前方散乱光強度信
号はA/D変換器60ケ経て解析部66に入り表示m3
7で表示される。−万、ダイクロイックミラー27で反
射された前方螢光fはフィルタ′51で迷光等を除去し
1こ後光侠出素子32で受光され、増幅器64で増幅さ
れ1こ前号螢光強度信号はA/L)変換器35乞経て解
析部ろ6に入り表示部67で表示される。なお、前方散
乱光強度13号の増幅度と前方螢光強度の増幅度は必要
な場合それぞれ独立して又は相関連させてゲインコント
ロール3乙により制御することかできる。In the method for measuring reticulocytes using a flow cytometer according to the invention, first, 500 parts of a blood cell measurement reagent containing flavophosphine R4 as a main ingredient are mixed in a ratio of 1 part of an anticoagulant whole blood sample, and fluorescent staining 7 is performed. For details about reagents, please refer to the patent application in 1973.
9-201273, the sample is then measured. Flow cell 2 of flow cytometer as shown in Figure 1
6 from the v-sa light source 20 to the filter 21 . Excitation light E' is incident through the condenser lens 22χ. As the sample liquid in the flow channel flows, the central part of the sheath group serves as an optical stimulation region where the excitation source heat is focused, and the excitation light of the γ light emitted from the optical stimulation region is mainly focused. The light component is blocked by a beam stopper 24. The forward fluorescent light F' and the forward scattered light S' are transmitted through the condenser lens 25.
The beam is returned to a parallel beam, and after one step, it is narrowed down to a small diameter beam through a slit plate 26, and this is separated into two components, a beam scattering source S and a beam r, by a dichroic mirror 27. The forward scattered light is received by the photodetecting element 2B behind the dichroic mirror 27, amplified by the amplifier 29, and the forward scattered light intensity signal passes through 60 A/D converters, enters the analysis section 66, and is displayed on m3.
7 is displayed. - 10,000, the forward fluorescent light f reflected by the dichroic mirror 27 is filtered to remove stray light etc. by the filter '51, and then received by the light output element 32 and amplified by the amplifier 64, and the previous fluorescent light intensity signal is A/L) The signal enters the analyzer 6 through the converter 35 and is displayed on the display 67. Incidentally, the amplification degree of the forward scattered light intensity No. 13 and the amplification degree of the forward fluorescent light intensity can be controlled by the gain control 3B independently or in conjunction with each other, if necessary.
作 用 上記フローサイトメータを用いる測定方法では。For production In the above measurement method using a flow cytometer.
第2図(a)に示す如<、Loの設定−1,、、L、2
の調整後フローセル2ろへの入射励起光の光軸と同軸上
で前方螢光と前方散乱光とが集光され波長域でそれぞれ
分離し測光される。励起光E′は励起光波長選択フィル
タ21馨透過すると光源20の発光波長から特定の波長
帯に絞られているが、フローセル260通過後にビーム
ストッパ24でカットされる。スリット板26?通じて
点光源になされ1こ3答元は、ダイクロイックミラー2
7で吸光波長に達しない波長光を主取分とする前方螢光
強度射し又吸光改良を越える波長光を主成分とする前方
散乱光を透過させて二分割する。光検出累子28.32
は検知し1こ散乱光、螢光ン強度信号として谷血球毎の
元応答馨光電変換し、これらの信号は瑠幅、A/D変換
された後解析部66でデータ記録解析され、公知の知見
に従って通常(成熟)赤血球、血小板、網状(幼若)赤
血球、白血球などに分類計数され得るか、螢光染色し1
こ場合これらの血球のうち網状赤血球の計数テークは臨
床検査上置も重要である。1個の網状赤血球による光応
答は、第2図に示すように、従来方法では測光光学系の
調整をフローセルを中心とする6個のレンズにつき異な
る、光軸に市って行っていたので光学的な偏りが相違し
ており、第6図(a)、(b)の右側の散乱光及び螢光
の対応ピークで表わされるように理想的に調整されてい
たものか1時間経過により1例えば第6図左$111(
a)のように散乱光の信号は変比しないが同図(b)の
ように螢光の信号のみ理想からかなりずれて2Etjl
ll(b)の点線のように低値乞とり易くなる。従って
、螢光が減弱し1こ網状赤血球の強度信号は血小板や一
部の通常赤血球との混同2生じ弁別が不安定であり結果
的に計数誤差編与えろことになる。不発明によれば。As shown in Fig. 2(a), <,Lo setting-1,,,L,2
After adjustment, the forward fluorescent light and the forward scattered light are collected on the same axis as the optical axis of the excitation light incident on the flow cell 2, separated into wavelength ranges, and photometered. When the excitation light E' passes through the excitation light wavelength selection filter 21, it is narrowed down to a specific wavelength band from the emission wavelength of the light source 20, but after passing through the flow cell 260, it is cut off by the beam stopper 24. Slit plate 26? The dichroic mirror 2 is used as a point light source through
At step 7, the forward fluorescent light intensity is emitted, the main component of which is light with a wavelength that does not reach the absorption wavelength, and the forward scattered light, which is mainly composed of light with a wavelength that exceeds the absorption wavelength, is transmitted and divided into two parts. Photodetector 28.32
After detecting the scattered light and fluorescence, the original response of each blood cell is photoelectrically converted as an intensity signal, and these signals are A/D converted and then data recorded and analyzed in the analysis section 66, using a known method. Depending on the findings, they can be classified and counted as normal (mature) red blood cells, platelets, reticulated (immature) red blood cells, white blood cells, etc., or they can be fluorescently stained and counted.
In this case, counting reticulocytes among these blood cells is also important in clinical examination. As shown in Figure 2, the photoresponse of a single reticulocyte is determined by the conventional method, in which the photometric optical system is adjusted on different optical axes for six lenses centered on the flow cell. As shown by the corresponding peaks of scattered light and fluorescence on the right side of Figures 6(a) and (b), the bias was different, and whether it was ideally adjusted or not after 1 hour, for example, Figure 6 left $111 (
As shown in (a), the signal of the scattered light does not change the ratio, but as shown in (b) of the same figure, only the signal of the fluorescent light deviates considerably from the ideal and becomes 2Etjl.
As shown by the dotted line in ll(b), it becomes easier to beg for a lower price. Therefore, the fluorescence is attenuated and the intensity signal of the reticulocytes is confused with platelets and some normal red blood cells, making the discrimination unstable and resulting in a counting error. According to non-invention.
第2図(a)に示す様に唯一の元@への同軸上において
集束系馨しl + L2 r LOで位置及び則・し・
ル、fl整するため、同一血球起源の散乱光と螢光とは
事央上対応した最大ピーク及び減弱傾回乞も1こらし、
この対応ピークは第6図(a)(b) 中央の芙紛同
士。As shown in Figure 2 (a), the focusing system is focused on the same axis to the only element @.
In order to adjust the light and fl, the scattered light and fluorescence originating from the same blood cells have corresponding maximum peaks and attenuation slopes.
The corresponding peaks are the two in the center of Figure 6 (a) and (b).
点1嵌同士の様に対口、して検知される。They are detected as two points that fit together.
発明の効果 本発明は次の9口き特有の効果2有する。Effect of the invention The present invention has the following 9-bit unique effects 2.
(1) 血球の弁別安定性が著しく改善された。(1) Blood cell discrimination stability was significantly improved.
同一血球起源の螢光と散乱光とを一軸で同時測定でるの
で、集束光学系を最高感反になるように1こや丁く、短
時間に調整し優るのみならず。Since fluorescent light and scattered light originating from the same blood cell can be measured simultaneously on one axis, it is not only possible to adjust the focusing optical system minutely and quickly to obtain the best sensitivity.
このルー整に多少の不足があっても、或いは経時的に調
整が少しすね、Cさても一軸上の散乱光と螢光か同時に
対応して強度変動するので、前しい試薬θ〕封状1ヒ作
用による恢lf1倍号の改善と相俟って1、布状赤血球
についてその弁別レベル乞固定しても極めて安定な弁別
が可能である。Even if there is some deficiency in this Lu adjustment, or if there is a slight adjustment over time, the intensity of scattered light and fluorescent light on one axis will fluctuate at the same time. Coupled with the improvement in the ratio of lf1 due to the effect of hypersensitivity, extremely stable discrimination of cloth red blood cells is possible even if the discrimination level is fixed.
(2) 試料処理速度が高い。(2) High sample processing speed.
フローセルの元刺激域乞成熟赤血球と血小板とが同時通
過し1こ場合、この時の光応答は網状赤血球に類似する
即ち網状赤血球による散乱光。When mature red blood cells and platelets simultaneously pass through the source stimulation area of the flow cell, the light response at this time is similar to that of reticulocytes, that is, light scattered by reticulocytes.
蛍光強度信号と紛られしい信号χ発し、この紛れは特に
網状赤血球比率が小さくフローチャンネルに名ける試薬
の流動径か太さいと増大する。従来法ではこれ乞避ける
1こめ、70−チヤンイ・ル馨細く絞り細胞通過を1個
つつにして同時通過乞防止しようとしたが、これに伴っ
て試料処理スピード乞格段に落とさざる乞イ身なかった
。不発明方法によれば、弁別レベルの安定性が尚く、こ
の1こめ成熟赤血球数及び血小板数の監視ン導入して同
時通過乞補償しやてい1こめ試料処理スピード馨犠牲に
することな(高能率で試料処理を遂行し得る。A signal χ that is confused with the fluorescence intensity signal is generated, and this confusion increases especially when the reticulocyte ratio is small and the flow diameter of the reagent in the flow channel is large. In order to avoid this problem in the conventional method, one attempt was made to narrow down the 70-cell tube to allow each cell to pass through one at a time to prevent simultaneous passage. Ta. According to the uninvented method, the stability of the discrimination level is improved, and the monitoring of mature red blood cell and platelet counts can be implemented to compensate for simultaneous passage without sacrificing sample processing speed. Sample processing can be performed with high efficiency.
(3) 光学系の調整工数か低減され1こ。(3) The number of man-hours required for adjusting the optical system has been reduced by one.
特定の血球起源の散乱光と螢光と2異なる光軸において
検知すると、−万の光軸上の測光系の修正が他方の光軸
上σ連軸光系の修正を誘発させ。When scattered light and fluorescent light originating from specific blood cells are detected on two different optical axes, a modification of the photometric system on the -10,000 optical axis induces a modification of the σ-linked optical system on the other optical axis.
このため両党強度の正確な測定を可能にする集束位置と
光軸の細密な調整に至るまでには何回もの修正を反覆し
なければならす、この手間と所要時間は相当大ぎなもの
かあった。本発明では一軸に沼って測光糸7a−調整丁
れは足りるので調整工数が最小限でしかも簡単に行われ
ることになっTこ。For this reason, it is necessary to repeat many corrections in order to reach the fine adjustment of the focusing position and optical axis that enables accurate measurement of the strength of both parties. Ta. In the present invention, since the photometric thread 7a is adjusted in one axis, the number of adjustment steps is minimized and the adjustment can be easily performed.
第1図は本発明によるフローサイトメーク2用い1こ網
状赤血球の測定方法を実施する際の基本構成の説明図、
第2図は光軸?中心として見1こ従来法と不発明方法に
おける測光光学系の比較のための概略図、第6図は検知
される光強度信号の同様な比較の1こめのビーク波形図
、第4図は従来の代表的なフローサイトメータの基本構
成を示す。
特許出願人 東亜医用電子株式会社
(従来 )
3図
時間FIG. 1 is an explanatory diagram of the basic configuration when carrying out the method for measuring reticulocytes using flow cytomake 2 according to the present invention;
Is the optical axis in Figure 2? 1 is a schematic diagram for comparison of photometric optical systems in the conventional method and the uninvented method, FIG. 6 is a peak waveform diagram for a similar comparison of detected light intensity signals, and FIG. 4 is a diagram of the conventional method. The basic configuration of a typical flow cytometer is shown below. Patent applicant Toa Medical Electronics Co., Ltd. (previously) Figure 3 Time
Claims (1)
激域から放出される出射光について前記励起光を事実上
除去したうえで、前記光学的刺激域から放出される前記
光軸に沿つた同軸上の前方散乱光及び前方螢光を測光し
て測光データより成熟赤血球及び血小板と網状赤血球と
を弁別し網状赤血球数を測定することを特徴とするフロ
ーサイトメータによる網状赤血球測定方法。After substantially removing the excitation light emitted from the optical stimulation area of the flow cell disposed along the optical axis of the excitation light, the output light is emitted from the optical stimulation area along the optical axis. A method for measuring reticulocytes using a flow cytometer, characterized in that the number of reticulocytes is measured by photometrically measuring forward scattered light and forward fluorescent light on the same axis, distinguishing mature red blood cells and platelets from reticulocytes based on the photometric data.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60293700A JPS62153758A (en) | 1985-12-27 | 1985-12-27 | Method for measuring reticulocyte by flow sight meter |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP60293700A JPS62153758A (en) | 1985-12-27 | 1985-12-27 | Method for measuring reticulocyte by flow sight meter |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS62153758A true JPS62153758A (en) | 1987-07-08 |
JPH0464590B2 JPH0464590B2 (en) | 1992-10-15 |
Family
ID=17798106
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP60293700A Granted JPS62153758A (en) | 1985-12-27 | 1985-12-27 | Method for measuring reticulocyte by flow sight meter |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS62153758A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0767382A3 (en) * | 1995-10-06 | 1998-02-25 | Toa Medical Electronics Co., Ltd. | Fluorescent compounds and their use for measuring reticulocytes |
EP0806664A3 (en) * | 1996-04-12 | 1998-04-08 | Toa Medical Electronics Co., Ltd. | A reagent for measuring reticulocytes and a method of measuring them |
US20210033592A1 (en) * | 2018-04-28 | 2021-02-04 | Shenzhen Mindray Bio-Medical Electronics Co. | Blood analyzer and analysis method |
-
1985
- 1985-12-27 JP JP60293700A patent/JPS62153758A/en active Granted
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0767382A3 (en) * | 1995-10-06 | 1998-02-25 | Toa Medical Electronics Co., Ltd. | Fluorescent compounds and their use for measuring reticulocytes |
EP0806664A3 (en) * | 1996-04-12 | 1998-04-08 | Toa Medical Electronics Co., Ltd. | A reagent for measuring reticulocytes and a method of measuring them |
US20210033592A1 (en) * | 2018-04-28 | 2021-02-04 | Shenzhen Mindray Bio-Medical Electronics Co. | Blood analyzer and analysis method |
Also Published As
Publication number | Publication date |
---|---|
JPH0464590B2 (en) | 1992-10-15 |
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