JP2002207034A - Anomalous cell detecting method - Google Patents
Anomalous cell detecting methodInfo
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- JP2002207034A JP2002207034A JP2001002880A JP2001002880A JP2002207034A JP 2002207034 A JP2002207034 A JP 2002207034A JP 2001002880 A JP2001002880 A JP 2001002880A JP 2001002880 A JP2001002880 A JP 2001002880A JP 2002207034 A JP2002207034 A JP 2002207034A
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- scattered light
- dimensional distribution
- abnormal cells
- distribution
- detecting
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明はフローサイトメトリ
による異常細胞の検出に関する。The present invention relates to the detection of abnormal cells by flow cytometry.
【０００２】[0002]
【従来の技術】一般に白血病などの血液における腫瘍化
の診断は、細胞表面に出現する抗原（表面マーカー）の
測定や腫瘍化を特定する遺伝子の同定、顕微鏡観察によ
る形態学的判定などを組み合わせて行われる。例えば、
ＡＴＬ（成人Ｔ細胞白血病）症例において血液中の腫瘍
化細胞を検出するには、色素で標識したモノクローナル
抗体を使用して疾患特異的に出現する表面抗原を検出す
る方法や特定の原因遺伝子（ＨＴＬＶ−Ｉ遺伝子）を検
出する方法などがある。2. Description of the Related Art In general, diagnosis of tumor formation in blood such as leukemia is performed by a combination of measurement of antigens (surface markers) appearing on the cell surface, identification of genes specifying tumor formation, and morphological judgment by microscopic observation. Done. For example,
To detect neoplastic cells in blood in ATL (adult T-cell leukemia) cases, a method of detecting a surface antigen that appears specifically in a disease using a monoclonal antibody labeled with a dye, or a specific causative gene (HTLV) -I gene).
【０００３】近年、フローサイトメータの普及により、
色素で標識したモノクローナル抗体を用いて、リンパ球
などの細胞表面マーカーの測定が頻繁に行われている。
この方法は、測定結果が得られるまでに時間を要する、
種々の抗体を使用するためコストがかかるという欠点が
ある。また、顕微鏡による形態学的観察では、種々の染
色法と細胞形態とを組み合わせて判定するが、一部の腫
瘍細胞は、正常細胞と区別ができない、観察者によって
結果が異なるなどの欠点が挙げられる。In recent years, with the spread of flow cytometers,
Measurement of cell surface markers such as lymphocytes is frequently performed using a monoclonal antibody labeled with a dye.
This method takes time to get the measurement result,
There is a disadvantage in that it is costly to use various antibodies. In addition, morphological observation by microscopy determines using a combination of various staining methods and cell morphology, but some tumor cells have drawbacks such as indistinguishable from normal cells and different results depending on the observer. Can be
【０００４】[0004]
【発明が解決しようとする課題】本発明は、短時間、低
コストで、精度良く腫瘍細胞を検出する方法を提供する
ことを目的とする。SUMMARY OF THE INVENTION An object of the present invention is to provide a method for detecting tumor cells with high accuracy in a short time and at low cost.
【０００５】[0005]
【課題を解決するための手段】本発明は以下の工程： （１）試料を、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、白血球および異常細胞を測定に好適
な状態にする溶血剤と混合する工程 （２）（１）で調製した試料をフローサイトメータで測
定し、少なくとも２つの散乱光を測定する工程 （３）（２）で測定した個々の細胞の散乱光信号から異
なる散乱光を２軸として、２次元分布を得る工程、 （４）２次元分布から、正常な白血球を少なくとも４つ
に分類計数する工程 （５）２次元分布から、リンパ球系腫瘍細胞の分布領域
を設定し、リンパ球系腫瘍細胞を検出する工程 （６）２次元分布から、血小板凝集または大型血小板の
分布領域を設定し、血小板凝集または大型血小板を検出
する工程 （７）２次元分布から、芽球の分布領域を設定し、芽球
を検出する工程 からなる異常細胞検出方法を提供する。The present invention provides the following steps: (1) dissolving a sample to such an extent that erythrocytes in a blood sample are not hindered in the measurement, so that leukocytes and abnormal cells are in a state suitable for measurement; Step of mixing with a hemolytic agent (2) Step of measuring the sample prepared in (1) with a flow cytometer and measuring at least two scattered lights (3) From the scattered light signal of each cell measured in (2) A step of obtaining a two-dimensional distribution with different scattered light as two axes; (4) a step of classifying and counting normal leukocytes into at least four from the two-dimensional distribution; and (5) a distribution of lymphoid tumor cells from the two-dimensional distribution. Step of setting area and detecting lymphoid tumor cells (6) Step of setting platelet aggregation or large platelet distribution area from two-dimensional distribution and detecting platelet aggregation or large platelet (7) Two-dimensional distribution , Blast The present invention provides a method for detecting abnormal cells, which comprises the steps of: setting a distribution region of the cells; and detecting blasts.
【０００６】[0006]
【発明の実施の形態】本発明でいう血液試料とは、末梢
血液、骨髄液、尿、アフェレーシスで採取した血液試料
など、白血球を含む体液試料をいう。BEST MODE FOR CARRYING OUT THE INVENTION The blood sample referred to in the present invention refers to a body fluid sample containing leukocytes, such as peripheral blood, bone marrow fluid, urine, and a blood sample collected by apheresis.
【０００７】本発明でいうリンパ球系腫瘍細胞とは、白
血病によって腫瘍化したリンパ球のことをいう。[0007] The term "lymphocyte tumor cells" as used in the present invention refers to lymphocytes that have become tumors due to leukemia.
【０００８】本発明でいう、血液試料中の赤血球を測定
の障害とならないように溶解し、白血球及び異常細胞を
測定に好適な状態にする溶血剤と混合する工程とは、血
液試料を適当な溶血剤と混合する工程である。In the present invention, the step of lysing erythrocytes in a blood sample so as not to hinder the measurement and mixing the erythrocytes with a hemolyzing agent to bring white blood cells and abnormal cells into a state suitable for the measurement includes the steps of: This is a step of mixing with a hemolytic agent.
【０００９】本工程の目的は、赤血球を測定の障害とな
らないように溶解するだけである。この目的に使用する
溶血剤は、少なくとも一つのカチオン性界面活性剤、少
なくとも一つのノニオン性界面活性剤、ｐＨを一定に保
つための緩衝剤を含むｐＨ４．５〜１１．０の水溶液で
ある。The purpose of this step is only to lyse the red blood cells without interfering with the measurement. The hemolytic agent used for this purpose is an aqueous solution of pH 4.5 to 11.0 containing at least one cationic surfactant, at least one nonionic surfactant and a buffer for keeping the pH constant.
【００１０】カチオン性界面活性剤としては、４級アン
モニウム塩型界面活性剤又はピリジニウム塩型界面活性
剤が好ましい。４級アンモニウム塩型およびピリジニウ
ム塩型界面活性剤は、The cationic surfactant is preferably a quaternary ammonium salt type surfactant or a pyridinium salt type surfactant. The quaternary ammonium salt type and pyridinium salt type surfactants include
【００１１】[0011]
【化１】 Embedded image
【００１２】〔Ｒ1は炭素数６〜１８のアルキル又はア
ルケニル基、Ｒ2およびＲ3は炭素数１〜４のアルキル又
はアルケニル基、Ｒ4は炭素数１〜４のアルキルおよび
アルケニル基又はベンジル基、Ｘはハロゲン原子であ
る。〕で表される全炭素数９〜３０の界面活性剤が挙げ
られる。Ｒ1の炭素数６〜１８のアルキル又はアルケニ
ル基としてはへキシル、オクチル、デシル、ドデシル、
テトラデシル等を挙げることができるが、とりわけオク
チル、デシル、ドデシル等の直鎖のアルキル基が好まし
い。また、Ｒ2およびＲ3の炭素数１〜４のアルキル、ア
ルケニル基としては、メチル、エチル、プロピル、ブチ
ル等を挙げることができるが、とりわけ、メチル、エチ
ル、プロピル等の炭素数１〜３のアルキル基が好まし
い。さらに、Ｒ 4の炭素数１〜４のアルキルおよびアル
ケニル基としては、メチル、エチル、プロピル、ブチル
等を挙げることができるが、とりわけ、メチル、エチ
ル、プロピル等の炭素数１〜３のアルキル基が好まし
い。[R1Is an alkyl or a
Lucenyl group, RTwoAnd RThreeIs an alkyl having 1 to 4 carbon atoms or
Is an alkenyl group, RFourIs an alkyl having 1 to 4 carbons and
An alkenyl group or a benzyl group, X is a halogen atom
You. And a surfactant having 9 to 30 carbon atoms represented by
Can be R1Alkyl or alkenyl having 6 to 18 carbon atoms
Hexyl, octyl, decyl, dodecyl,
Tetradecyl, etc.
Linear alkyl groups such as tyl, decyl and dodecyl are preferred.
No. Also, RTwoAnd RThreeAlkyl having 1 to 4 carbon atoms,
Lucenyl groups include methyl, ethyl, propyl,
And methyl, ethyl, etc.
Alkyl groups having 1 to 3 carbon atoms, such as
No. Further, R FourAlkyl and alk having 1 to 4 carbon atoms
As the kenyl group, methyl, ethyl, propyl, butyl
And the like.
Alkyl groups having 1 to 3 carbon atoms, such as
No.
【００１３】ノニオン性界面活性剤としては以下の式の
ポリオキシエチレン系ノニオン界面活性剤が好ましい：
Ｒ1−Ｒ2−（ＣＨ2ＣＨ2Ｏ）n−Ｈ〔式中、Ｒ1は炭素数
８〜２５のアルキル、アルケニル又はアルキニル基；Ｒ
2はＯ、As the nonionic surfactant, a polyoxyethylene-based nonionic surfactant of the following formula is preferred:
R 1 -R 2- (CH 2 CH 2 O) n-H wherein R 1 is an alkyl, alkenyl or alkynyl group having 8 to 25 carbon atoms;
2 is O,
【００１４】[0014]
【化２】 またはＣＯＯ；ｎは１０〜５０の整数を表す。〕Embedded image Or COO; n represents an integer of 10 to 50. ]
【００１５】溶血剤の組成は特に限定されるものではな
いが、例えば、特開平６−２０７９２号、特開平７−１
８１１７７号に記載の溶血剤などが好適に使用される。Although the composition of the hemolytic agent is not particularly limited, for example, Japanese Patent Application Laid-Open Nos.
No. 81177 is preferably used.
【００１６】さらに、少なくとも１つの、分子内に１つ
の芳香環を有する有機酸を含有することは、白血球およ
び異常細胞を分類に好適なように調整するために好適で
ある。例えば、安息香酸、フタル酸、馬尿酸、サリチル
酸、ｐ−アミノベンゼンスルホン酸、ヘンゼンスルホン
酸などが好適に使用できる。好ましくは0.1〜１００ｍ
Ｍ、より好ましくは１〜５０ｍＭ、さらに好ましくは１
０〜３０ｍＭの濃度範囲で使用できる。Furthermore, the inclusion of at least one organic acid having one aromatic ring in the molecule is suitable for adjusting leukocytes and abnormal cells to be suitable for classification. For example, benzoic acid, phthalic acid, hippuric acid, salicylic acid, p-aminobenzenesulfonic acid, benzene sulfonic acid and the like can be suitably used. Preferably 0.1-100m
M, more preferably 1 to 50 mM, even more preferably 1
It can be used in a concentration range of 0 to 30 mM.
【００１７】これらの有機酸を含有することにより、特
に好酸球の散乱光強度が増加し、結果として、好中球と
好酸球の分離が改善される。By containing these organic acids, the scattered light intensity of eosinophils is particularly increased, and as a result, the separation of neutrophils and eosinophils is improved.
【００１８】ｐＨが４．５よりも低い場合、好酸球の分
離が悪くなり、正常白血球を分画することが困難にな
る。ｐＨ１１．０よりも高い場合は、白血球が損傷を受
けやすくなり、好ましくない。When the pH is lower than 4.5, the separation of eosinophils becomes poor, and it becomes difficult to fractionate normal leukocytes. When the pH is higher than 11.0, leukocytes are easily damaged, which is not preferable.
【００１９】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ーで測定できる散乱光であり、側
方散乱光、前方低角散乱光（受光角度０〜５度付近）、
前方高角散乱光（５〜２０度付近）等をいい、白血球の
大きさもしくは内部構造情報を反映する散乱角度が選ば
れる。The scattered light referred to in the present invention is scattered light that can be measured by a commercially available flow cytometer, and includes side scattered light, forward low-angle scattered light (light receiving angle of about 0 to 5 degrees),
It refers to forward high-angle scattered light (around 5 to 20 degrees) and the like, and a scattering angle that reflects the size of white blood cells or internal structure information is selected.
【００２０】異なる２つの散乱光の１つに前方散乱光
（低角でも高角でもどちらでも良い）を使用すると、細
胞の大きさに関する情報が得られる。The use of forward scattered light (either low or high angle) as one of the two different scattered lights provides information on cell size.
【００２１】フローサイトメーターの光源は、特に限定
されず、例えば、アルゴンイオンレーザー、He／Neレー
ザー、赤色半導体レーザー、水銀アークランプなどが使
用される。特に半導体レーザーは気体レーザーに比べ非
常に安価であり、装置コストを大幅に下げることができ
るため、好適である。The light source of the flow cytometer is not particularly limited, and for example, an argon ion laser, a He / Ne laser, a red semiconductor laser, a mercury arc lamp and the like are used. In particular, a semiconductor laser is very inexpensive as compared with a gas laser, and can greatly reduce the apparatus cost, and is therefore preferable.
【００２２】「測定した個々の細胞の散乱光信号から異
なる散乱光を２軸として２次元分布を得る工程」とは、
例えば、Ｘ軸に側方散乱光、Ｙ軸に前方散乱光をとって
２次元分布を描くことである。"A step of obtaining a two-dimensional distribution from the measured scattered light signals of individual cells using two different scattered lights as two axes"
For example, drawing a two-dimensional distribution using side scattered light on the X axis and forward scattered light on the Y axis.
【００２３】「２次元分布から、正常な白血球を少なく
とも４つに分類計数する工程」とは、例えば、Ｘ軸に側
方散乱光、Ｙ軸に前方散乱光をとって、２次元分布を描
くと、図１に示すように、各白血球細胞は細胞毎に集団
を形成する。この集団を適当な解析ソフトで解析するこ
とにより、各白血球集団の数と割合を算出する。The "step of classifying and counting at least four normal white blood cells from the two-dimensional distribution" means, for example, drawing a two-dimensional distribution by taking side scattered light on the X axis and forward scattered light on the Y axis. As shown in FIG. 1, each white blood cell forms a population for each cell. The number and ratio of each leukocyte population are calculated by analyzing this population with appropriate analysis software.
【００２４】「２次元分布から、リンパ球系腫瘍細胞の
領域を設定し、リンパ球腫瘍細胞を検出する工程」と
は、例えば、Ｘ軸に側方散乱光、Ｙ軸に前方散乱光をと
って、２次元分布を描くと、図１に示すように、リンパ
球系腫瘍細胞は集団を形成する。この集団を適当な解析
ソフトで解析することにより、リンパ球系腫瘍細胞集団
の数を算出する。血小板凝集、大型血小板、芽球も同様
にして、各集団の数を算出する。各集団の数にはそれぞ
れ閾値を設定しておき、その設定値を超えたらメッセー
ジを表示する。The "step of setting a lymphocyte tumor cell region from the two-dimensional distribution and detecting the lymphocyte tumor cell" means, for example, that side scattered light is taken on the X axis and forward scattered light is taken on the Y axis. If a two-dimensional distribution is drawn, as shown in FIG. 1, lymphoid tumor cells form a population. The number of lymphocyte tumor cell populations is calculated by analyzing this population with appropriate analysis software. The number of each group is calculated in the same manner for platelet aggregation, large platelets, and blasts. A threshold value is set for the number of each group, and a message is displayed when the threshold value is exceeded.
【００２５】[0025]
【実施例】実施例１ リンパ球腫瘍細胞出現例 以下の組成の試薬を調製した。 HEPES（市販品） 10mM フタル酸2Na（市販品） 20mM BC30TX（ポリオキシエチレン(30)セチルエーテル） 1500ppm （日光ケミカルズ（株）） ドデシルトリメチルアンモニウムクロライド（市販品） 550ppm NaOHでpHを7.0に調整。Example 1 Example of appearance of lymphocyte tumor cells A reagent having the following composition was prepared. HEPES (commercially available) 10 mM 2Na phthalate (commercially available) 20 mM BC30TX (polyoxyethylene (30) cetyl ether) 1500 ppm (Nikko Chemicals Co., Ltd.) Dodecyltrimethylammonium chloride (commercially available) 550 ppm pH adjusted to 7.0 with NaOH.
【００２６】上記試薬1.0mlに抗凝固剤処理した健常者
およびＡＴＬ患者の血液をそれぞれ30μｌずつ加え、35
℃で40秒間反応させた後、フローサイトメータで前方低
角散乱光、側方散乱光を測定した。光源は633nmの赤色
半導体レーザを使用した。To each of the above reagents (1.0 ml), 30 μl each of blood of a healthy person and an ATL patient treated with an anticoagulant was added.
After reacting at 40 ° C. for 40 seconds, forward low-angle scattered light and side scattered light were measured with a flow cytometer. The light source used was a 633 nm red semiconductor laser.
【００２７】図２にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった健常人のスキャッタグラムを示す。FIG. 2 shows a scattergram of a healthy person with side scattered light on the X axis and forward low angle scattered light on the Y axis.
【００２８】図３にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとったＡＴＬ患者のスキャッタグラムを示す。
リンパ球領域(Lymph)よりも前方散乱光強度の低い位置
に集団が認められ、本法によりリンパ球系腫瘍細胞であ
るＡＴＬ細胞が検出できることが確認された。FIG. 3 shows a scattergram of an ATL patient with side scattered light on the X axis and forward low angle scattered light on the Y axis.
A population was observed at a position where the forward scattered light intensity was lower than the lymphocyte region (Lymph), and it was confirmed that ATL cells, which are lymphoid tumor cells, could be detected by this method.
【００２９】実施例２ 血小板凝集出現例 実施例１の試薬1.0mlに抗凝固剤処理した血小板凝集が
出現した血液をそれぞれ30μｌずつ加え、35℃で40秒間
反応させた後、フローサイトメータで前方低角散乱光、
側方散乱光を測定した。光源は633nmの赤色半導体レー
ザを使用した。Example 2 Example of Platelet Aggregation Appearance To 1.0 ml of the reagent of Example 1 was added 30 μl of anticoagulant-treated blood in which platelet aggregation had appeared, and the mixture was reacted at 35 ° C. for 40 seconds. Low angle scattered light,
The side scattered light was measured. The light source used was a 633 nm red semiconductor laser.
【００３０】図４にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった血小板凝集のスキャッタグラムを示す。
リンパ球領域(Lymph)とゴースト領域(Ghost)の間に集団
が認められ(PLT Clumps)、本法により血小板凝集が検出
できることが確認された。FIG. 4 shows a scattergram of platelet aggregation with side scattered light on the X axis and forward low angle scattered light on the Y axis.
A population was observed between the lymphocyte area (Lymph) and the ghost area (Ghost) (PLT Clumps), confirming that platelet aggregation could be detected by this method.
【００３１】実施例３ 大型血小板出現例 実施例１の試薬1.0mlに抗凝固剤処理した大型血小板が
出現した血液をそれぞれ30μｌずつ加え、35℃で40秒間
反応させた後、フローサイトメータで前方低角散乱光、
側方散乱光を測定した。光源は633nmの赤色半導体レー
ザを使用した。Example 3 Example of appearance of large platelets 30 μl of blood in which large platelets treated with an anticoagulant appeared were added to 1.0 ml of the reagent of Example 1 and reacted at 35 ° C. for 40 seconds. Low angle scattered light,
The side scattered light was measured. The light source used was a 633 nm red semiconductor laser.
【００３２】図５にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった大型血小板のスキャッタグラムを示す。
リンパ球領域(Lymph)とゴースト領域の間に集団(Ghost)
が認められ(Large PLT)、本法により大型血小板が検出
できることが確認された。FIG. 5 shows a scattergram of large platelets with side scattered light on the X-axis and forward low angle scattered light on the Y-axis.
Population between lymphocyte area (Lymph) and ghost area (Ghost)
(Large PLT), confirming that large platelets can be detected by this method.
【００３３】実施例４ 芽球出現例 実施例１の試薬1.0mlに抗凝固剤処理した芽球が出現し
た血液をそれぞれ30μｌずつ加え、35℃で40秒間反応さ
せた後、フローサイトメータで前方低角散乱光、側方散
乱光を測定した。光源は633nmの赤色半導体レーザを使
用した。Example 4 Example of appearance of blast cells [0033] To 1.0 ml of the reagent of Example 1 was added 30 µl of blood in which blast cells treated with an anticoagulant appeared, and reacted at 35 ° C for 40 seconds. Low angle scattered light and side scattered light were measured. The light source used was a 633 nm red semiconductor laser.
【００３４】図６にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった芽球のスキャッタグラムを示す。単球領
域（Mono）の左上方に集団が認められ（Blast）、本法
により芽球が検出できることが確認された。FIG. 6 shows a scattergram of blast cells with side scattered light on the X axis and forward low angle scattered light on the Y axis. A population was observed above and to the left of the monocyte region (Mono) (Blast), confirming that blasts could be detected by this method.
【００３５】[0035]
【発明の効果】本発明によれば、短時間、低コストで、
精度良く腫瘍細胞などの異常細胞を検出することができ
る。According to the present invention, in a short time and at low cost,
Abnormal cells such as tumor cells can be accurately detected.
【図１】白血球及び異常細胞のスキャッタグラムの模式
図である。FIG. 1 is a schematic diagram of a scattergram of white blood cells and abnormal cells.
【図２】本発明の実施例１における健常人のスキャッタ
グラムである。FIG. 2 is a scattergram of a healthy person in Example 1 of the present invention.
【図３】本発明の実施例１におけるＡＴＬ患者のスキャ
ッタグラムである。FIG. 3 is a scattergram of an ATL patient in Example 1 of the present invention.
【図４】本発明の実施例２における血小板凝集のスキャ
ッタグラムである。FIG. 4 is a scattergram of platelet aggregation in Example 2 of the present invention.
【図５】本発明の実施例３における大型血小板のスキャ
ッタグラムである。FIG. 5 is a scattergram of large platelets in Example 3 of the present invention.
【図６】本発明の実施例４における芽球のスキャッタグ
ラムである。FIG. 6 is a scattergram of blasts in Example 4 of the present invention.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｇ０１Ｎ 33/49 Ｇ０１Ｎ 33/49 Ｈ (72)発明者 小国 振一郎 神戸市中央区脇浜海岸通１丁目５番１号 シスメックス株式会社内 Ｆターム(参考） 2G045 BB01 BB29 BB34 CA11 CA17 CA23 CA24 FA12 FA29 GC11 2G059 AA06 BB13 BB14 CC20 DD03 EE02 FF08 GG01 GG10 HH02 HH06 4B063 QA01 QA19 QQ03 QQ08 QR52 QS36 QX02 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme court ゛ (Reference) G01N 33/49 G01N 33/49 H (72) Inventor Shinichiro Oguni 1-5 Wakihama Kaigandori, Chuo-ku, Kobe-shi No. 1 Sysmex Corporation F-term (reference) 2G045 BB01 BB29 BB34 CA11 CA17 CA23 CA24 FA12 FA29 GC11 2G059 AA06 BB13 BB14 CC20 DD03 EE02 FF08 GG01 GG10 HH02 HH06 4B063 QA01 QA19 QQ03 QQ08 QR52
Claims (4)
ない程度に溶解し、白血球および異常細胞を測定に好適
な状態にする溶血剤と混合する工程 （２）（１）で調製した試料をフローサイトメーターで
測定し、少なくとも２つの散乱光を測定する工程 （３）（２）で測定した個々の細胞の散乱光信号から異
なる散乱光を２軸として、２次元分布を得る工程、 （４）２次元分布から、正常な白血球を少なくとも４つ
に分類計数する工程 （５）２次元分布から、リンパ球系腫瘍細胞の分布領域
を設定し、リンパ球系腫瘍細胞を検出する工程 （６）２次元分布から、血小板凝集または大型血小板の
分布領域を設定し、血小板凝集または大型血小板を検出
する工程 （７）２次元分布から、芽球の分布領域を設定し、芽球
を検出する工程。1. A method for detecting abnormal cells comprising the following steps: (1) Hemolysis of a sample so that erythrocytes in a blood sample are not disturbed in the measurement and leukocytes and abnormal cells are in a state suitable for measurement. (2) Step of measuring the sample prepared in (1) with a flow cytometer and measuring at least two scattered lights (3) Different from individual cell scattered light signals measured in (2) A step of obtaining a two-dimensional distribution with scattered light as two axes; (4) a step of classifying and counting at least four normal leukocytes from the two-dimensional distribution; (5) a distribution area of lymphoid tumor cells from the two-dimensional distribution. And setting a platelet aggregation or large platelet distribution region from the two-dimensional distribution, and detecting platelet aggregation or large platelets (7) From the two-dimensional distribution, Set the distribution area of the sphere, detecting the blasts.
ない程度に溶解し、白血球および異常細胞を測定に好適
な状態にする溶血剤が、少なくとも１つのノニオン性界
面活性剤と少なくとも１つのカチオン性界面活性剤であ
って、ｐＨが４．５〜１１．０の間にある、請求項１記
載の異常細胞検出方法。2. A hemolytic agent which lyses erythrocytes in a blood sample to such an extent that it does not interfere with the measurement and renders leukocytes and abnormal cells suitable for the measurement, comprises at least one nonionic surfactant and at least one cation. 2. The method for detecting abnormal cells according to claim 1, wherein the method is a surfactant, and the pH is between 4.5 and 11.0.
する有機酸もしくはその塩を含有する請求項２に記載の
異常細胞検出方法。3. The method for detecting abnormal cells according to claim 2, wherein the hemolytic agent contains an organic acid having at least one aromatic ring or a salt thereof.
前方高角散乱光、側方散乱光から選ばれる少なくとも２
つである請求項１〜３記載の異常細胞検出方法。4. The light receiving angle of the scattered light is forward low-angle scattered light,
At least 2 selected from forward high-angle scattered light and side scattered light
The method for detecting abnormal cells according to claim 1, wherein:
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