JP2002207038A - Method for classifying and counting damaged cell - Google Patents
Method for classifying and counting damaged cellInfo
- Publication number
- JP2002207038A JP2002207038A JP2001002883A JP2001002883A JP2002207038A JP 2002207038 A JP2002207038 A JP 2002207038A JP 2001002883 A JP2001002883 A JP 2001002883A JP 2001002883 A JP2001002883 A JP 2001002883A JP 2002207038 A JP2002207038 A JP 2002207038A
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- JP
- Japan
- Prior art keywords
- damaged
- measured
- counting
- classifying
- lymphocytes
- 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
- 238000000034 method Methods 0.000 title claims abstract description 18
- 210000004369 blood Anatomy 0.000 claims abstract description 30
- 239000008280 blood Substances 0.000 claims abstract description 30
- 210000004027 cell Anatomy 0.000 claims abstract description 23
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 18
- 210000001616 monocyte Anatomy 0.000 claims abstract description 15
- 210000000440 neutrophil Anatomy 0.000 claims abstract description 15
- 238000005259 measurement Methods 0.000 claims abstract description 11
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 6
- 210000000265 leukocyte Anatomy 0.000 claims description 28
- 210000003979 eosinophil Anatomy 0.000 claims description 15
- 239000003219 hemolytic agent Substances 0.000 claims description 8
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 239000002736 nonionic surfactant Substances 0.000 claims description 4
- 150000007524 organic acids Chemical class 0.000 claims description 3
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 230000002159 abnormal effect Effects 0.000 claims 1
- 150000001768 cations Chemical class 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000002949 hemolytic effect Effects 0.000 abstract 1
- 125000000217 alkyl group Chemical group 0.000 description 7
- 125000004432 carbon atom Chemical group C* 0.000 description 7
- -1 urine Substances 0.000 description 5
- 125000003342 alkenyl group Chemical group 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000004065 semiconductor Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 2
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical class C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 239000003093 cationic surfactant Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- FDCJDKXCCYFOCV-UHFFFAOYSA-N 1-hexadecoxyhexadecane Chemical compound CCCCCCCCCCCCCCCCOCCCCCCCCCCCCCCCC FDCJDKXCCYFOCV-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 102100033040 Carbonic anhydrase 12 Human genes 0.000 description 1
- 102100033029 Carbonic anhydrase-related protein 11 Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101000867855 Homo sapiens Carbonic anhydrase 12 Proteins 0.000 description 1
- 101000867841 Homo sapiens Carbonic anhydrase-related protein 11 Proteins 0.000 description 1
- 101001075218 Homo sapiens Gastrokine-1 Proteins 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000005466 alkylenyl group Chemical group 0.000 description 1
- 125000000304 alkynyl group Chemical group 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
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
による損傷細胞の分類計数に関する。The present invention relates to the classification and counting of damaged cells by flow cytometry.
【０００２】[0002]
【従来の技術】臨床検査の分野において、白血球の分類
計数を行うことは、疾患の診断を行う上できわめて有用
な情報を得ることができる。例えば、通常、正常な末梢
血中の白血球には、リンパ球、単球、好中球、好酸球、
好塩基球の５つの正常白血球が一定の比率で存在する。
疾患の存在により、これらの白血球の比率は変動される
ことがあり、これらの正常白血球を分類計数することに
より、各白血球の比率を測定することは有用である。2. Description of the Related Art In the field of clinical examination, classification and counting of leukocytes can provide extremely useful information for diagnosing a disease. For example, leukocytes in normal peripheral blood usually include lymphocytes, monocytes, neutrophils, eosinophils,
Five normal leukocytes of basophils are present in a fixed ratio.
The ratio of these leukocytes may fluctuate due to the presence of the disease, and it is useful to measure the ratio of each leukocyte by classifying and counting these normal leukocytes.
【０００３】近年、医療費の削減により、一般病院では
血液検査を検査センターなどに依頼することが多くなっ
ている。従って、検査センターなどでは、採血後時間が
経過した検体や保存状態が悪い検体が送られてくる場合
がある。採血後時間が経過したり、保存状態が悪いと、
白血球細胞は損傷を受けて、非常に細胞膜が壊れやすい
状態になっている。[0003] In recent years, due to the reduction of medical expenses, general hospitals often request blood tests to test centers and the like. Therefore, in an inspection center or the like, a sample whose time has passed since blood collection or a sample whose storage state is poor may be sent. If time has passed since blood collection or storage conditions are poor,
The white blood cells are damaged and the cell membrane is very fragile.
【０００４】従来、全自動白血球分類計数装置では、こ
のような検体の白血球を正確に分類計数することができ
ず、何らかの疾患の存在を認めるフラッグを出力してい
た。この場合、目視による再検を行う必要性が生じるこ
とになる。このようなことが頻繁に発生すると、検査の
非効率化やコスト高を招く恐れがある。Heretofore, a fully automatic leukocyte classification and counting apparatus has not been able to accurately classify and count leukocytes of such a specimen, and has output a flag that recognizes the presence of some disease. In this case, it is necessary to perform a visual reexamination. If this occurs frequently, it may lead to inefficiency in inspection and increase in cost.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、採血後時間
が経過した検体や保存状態が悪い検体でも、正確に白血
球の分類計数が行うことができる方法を提供することを
目的とする。SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for accurately classifying and counting leukocytes even in a sample whose time has elapsed after blood collection or a sample in a poor storage condition.
【０００６】[0006]
【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、白血球および損傷細胞を測定に好適
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、損傷リンパ球の領域を設定し、
損傷リンパ球を分類計数する工程 （６）２次元分布から、損傷単球の領域を設定し、損傷
単球を分類計数する工程 （７）２次元分布から、損傷好中球の領域を設定し、損
傷好中球を分類計数する工程 （８）２次元分布から、損傷好酸球の領域を設定し、損
傷好酸球を分類計数する工程 （９）（４）で測定したリンパ球数と（５）で測定した
損傷リンパ球数を用いて、リンパ球数を補正する工程 （１０）（４）で測定した単球数と（６）で測定した損
傷単球数を用いて、単球数を補正する工程 （１１）（４）で測定した好中球数と（７）で測定した
損傷好中球数を用いて、好中球数を補正する工程 （１２）（４）で測定した好酸球数と（８）で測定した
損傷好酸球数を用いて、好酸球数を補正する工程。から
なる損傷細胞分類計数方法を提供する。The present invention provides the following steps: (1) dissolving a sample so that red blood cells in a blood sample do not disturb the measurement, so that white blood cells and damaged 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 using different scattered lights as two axes; (4) a step of classifying and counting normal leukocytes into at least four from the two-dimensional distribution; and (5) setting an area of damaged lymphocytes from the two-dimensional distribution. And
Step of Classifying and Counting Damaged Lymphocytes (6) Setting the Area of Damaged Monocytes from the Two-Dimensional Distribution and Classifying and Counting Damaged Monocytes (7) Setting the Area of Damaged Neutrophils from the Two-Dimensional Distribution (8) A step of classifying and counting damaged neutrophils (8) Setting a region of damaged eosinophils from the two-dimensional distribution and classifying and counting damaged eosinophils (9) The number of lymphocytes measured in (4) Step of correcting the number of lymphocytes using the number of damaged lymphocytes measured in (5) (10) Monocytes using the number of monocytes measured in (4) and the number of damaged monocytes measured in (6) Step of correcting the number (11) Step of correcting the number of neutrophils using the number of neutrophils measured in (4) and the number of damaged neutrophils measured in (7) (12) Measured in (4) Correcting the number of eosinophils using the obtained number of eosinophils and the number of damaged eosinophils measured in (8). And a method for classifying and counting damaged cells.
【０００７】[0007]
【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.
【０００８】本発明でいう損傷細胞とは、採血後時間が
経過したり、保存状態が悪いために、細胞膜が壊れやす
くなっている白血球のことをいう。The term "damaged cells" as used in the present invention refers to leukocytes whose cell membrane is apt to be broken due to the passage of time after blood collection or poor storage conditions.
【０００９】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、白血球及び損傷細胞を
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 red blood cells in a blood sample so as not to hinder the measurement and mixing the white blood cells and the damaged cells with a hemolytic agent to make the cells suitable for the measurement includes the step of: This is a step of mixing with a hemolytic agent.
【００１０】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 merely 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.
【００１１】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、As the cationic surfactant, a quaternary ammonium salt type surfactant or a pyridinium salt type surfactant is preferable. The quaternary ammonium salt type and pyridinium salt type surfactants include
【００１２】[0012]
【化１】 Embedded image
【００１３】〔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.
【００１４】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 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,
【００１５】[0015]
【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. ]
【００１６】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.
【００１７】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
び損傷細胞の散乱光強度分布を、分類に好適なように調
整するために好適である。Furthermore, the inclusion of at least one organic acid having one aromatic ring in the molecule is suitable for adjusting the scattered light intensity distribution of leukocytes and damaged cells so as to be suitable for classification. .
【００１８】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0.1〜１
００ｍＭ、より好ましくは１〜５０ｍＭ、さらに好まし
くは１０〜３０ｍＭの濃度範囲で使用できる。For example, benzoic acid, phthalic acid, hippuric acid, salicylic acid, p-aminobenzenesulfonic acid, henzensulfonic acid and the like can be suitably used. Preferably 0.1 to 1
It can be used in a concentration range of 00 mM, more preferably 1 to 50 mM, even more preferably 10 to 30 mM.
【００１９】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.
【００２０】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.
【００２１】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 around 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.
【００２２】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 the size of the cells.
【００２３】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。The light source of the flow cytometer is not particularly limited, for example, an argon ion laser, He / Ne
Lasers, red semiconductor lasers, mercury arc lamps and the like are used.
【００２４】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。In particular, semiconductor lasers are very inexpensive as compared with gas lasers, and the cost of the apparatus can be greatly reduced.
【００２５】「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。The “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.
【００２６】「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 using 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.
【００２７】「２次元分布から、損傷リンパ球の領域を
設定し、損傷リンパ球を分類計数する工程」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２に示すように、損傷リンパ球(D
amaged Lymph)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、損傷リンパ球集団の数
と割合を算出する。損傷単球(Damaged Mono)、損傷好中
球(Damaged Neut)、損傷好酸球(Damaged Eo)も同様にし
て、各集団の数と割合を算出する。The "step of setting the area of the damaged lymphocytes from the two-dimensional distribution and classifying and counting the damaged lymphocytes" means, for example, taking the side scattered light on the X axis and the forward scattered light on the Y axis. 2
Drawing the dimensional distribution, as shown in FIG. 2, the damaged lymphocytes (D
amaged Lymph) form a population. The number and ratio of the damaged lymphocyte population are calculated by analyzing this population with appropriate analysis software. The number and ratio of each population are similarly calculated for damaged monocytes (Damaged Mono), damaged neutrophils (Damaged Neut), and damaged eosinophils (Damaged Eo).
【００２８】[0028]
【実施例】実施例１ 採血後時間が経過した場合 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。EXAMPLES Example 1 When a lapse of time after blood collection 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.
【００２９】上記試薬1.0mlに抗凝固剤処理した健常人
の採血直後および採血後4℃で72時間経過した血液をそ
れぞれ30μｌずつ加え、35℃で40秒間反応させた後、FC
Mで前方低角散乱光、側方散乱光を測定した。光源は633
nmの赤色半導体レーザを使用した。To each of the above reagents, 30 μl each of blood immediately after blood collection and 72 hours after blood collection at 4 ° C. was added to 1.0 ml of the above reagent, and reacted at 35 ° C. for 40 seconds.
M was used to measure forward low-angle scattered light and side scattered light. Light source is 633
nm red semiconductor laser was used.
【００３０】図３にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった採血直後のスキャッタグラムを示す。FIG. 3 shows a scattergram immediately after blood collection, with side scattered light on the X axis and forward low angle scattered light on the Y axis.
【００３１】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72時間後のスキャッタグラムを示す。FIG. 4 shows a scattergram 72 hours after taking side scattered light on the X axis and forward low angle scattered light on the Y axis.
【００３２】図５に採血直後、本法による補正前、本法
による補正後の各細胞比率を示す。本法による補正によ
り採血後時間が経過した場合でも、採血直後と同様の結
果が得られた。FIG. 5 shows the cell ratios immediately after blood collection, before correction by this method, and after correction by this method. Even when the time after blood collection had elapsed by the correction according to this method, the same results as those immediately after blood collection were obtained.
【００３３】実施例２ 保存状態が悪い場合 実施例１の試薬1.0mlに抗凝固剤処理した健常人の採血
直後および室温で48時間保存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、FCM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 In the case of poor storage condition 30 μl of blood stored immediately after blood collection and that of a healthy person treated with anticoagulant treated with 1.0 ml of the reagent of Example 1 at room temperature for 48 hours were used.
After the reaction was performed at 35 ° C. for 40 seconds, forward low-angle scattered light and side scattered light were measured by FCM. 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
散乱光をとった採血直後のスキャッタグラムを示す。FIG. 6 shows a scattergram immediately after blood collection, with side scattered light on the X axis and forward low angle scattered light on the Y axis.
【００３５】図７にＸ軸に側方散乱光、Ｙ軸に前方低角
散乱光をとった室温で保存した場合のスキャッタグラム
を示す。FIG. 7 shows a scattergram when stored at room temperature with side scattered light on the X axis and forward low angle scattered light on the Y axis.
【００３６】図８に採血直後、本法による補正前、本法
による補正後の各細胞比率を示す。本法による補正によ
り保存状態が悪い場合でも、採血直後と同様の結果が得
られた。FIG. 8 shows the cell ratios immediately after blood collection, before correction by this method, and after correction by this method. Even when the storage condition was poor due to the correction by this method, the same result as that immediately after blood collection was obtained.
【００３７】[0037]
【発明の効果】本発明によれば、採血後時間が経過した
検体や保存状態が悪い検体でも、正確に白血球の分類計
数が行うことができる。According to the present invention, the classification and counting of leukocytes can be accurately performed even for a sample whose time has elapsed after blood collection or a sample whose storage condition is poor.
【図１】各白血球細胞の２次元分布図の模式図である。FIG. 1 is a schematic diagram of a two-dimensional distribution diagram of each white blood cell.
【図２】損傷細胞が出現した場合の各白血球細胞の２次
元分布図である。FIG. 2 is a two-dimensional distribution diagram of each white blood cell when a damaged cell appears.
【図３】本発明の実施例１における採血直後のスキャッ
タグラムである。FIG. 3 is a scattergram immediately after blood collection in Example 1 of the present invention.
【図４】本発明の実施例１における4℃で72時間保存後
のスキャッタグラムである。FIG. 4 is a scattergram after storage at 4 ° C. for 72 hours in Example 1 of the present invention.
【図５】本発明の実施例１における採血直後、本法によ
る補正前、本法による補正後の各細胞比率である。FIG. 5 shows cell ratios immediately after blood collection, before correction by the present method, and after correction by the present method in Example 1 of the present invention.
【図６】本発明の実施例２における採血直後のスキャッ
タグラムである。FIG. 6 is a scattergram immediately after blood collection in Example 2 of the present invention.
【図７】本発明の実施例２における室温で48時間保存し
た場合のスキャッタグラムである。FIG. 7 is a scattergram when stored at room temperature for 48 hours in Example 2 of the present invention.
【図８】本発明の実施例２における採血直後、本法によ
る補正前、本法による補正後の各細胞比率である。FIG. 8 shows cell ratios immediately after blood collection, before correction by the present method, and after correction by the present method in Example 2 of the present invention.
───────────────────────────────────────────────────── フロントページの続き (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 AA03 AA04 AA07 BB29 BB40 BB51 CA01 CA02 CA11 CA12 CA15 CA17 CA20 CA25 FA14 FA37 GA03 GA10 GC11 JA01 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinichiro Oguni 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe F-term in Sysmex Corporation (Reference) 2G045 AA03 AA04 AA07 BB29 BB40 BB51 CA01 CA02 CA11 CA12 CA15 CA17 CA20 CA25 FA14 FA37 GA03 GA10 GC11 JA01
Claims (4)
法。 （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、白血球および損傷細胞を測定に好適
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、損傷リンパ球の領域を設定し、
損傷リンパ球を分類計数する工程 （６）２次元分布から、損傷単球の領域を設定し、損傷
単球を分類計数する工程 （７）２次元分布から、損傷好中球の領域を設定し、損
傷好中球を分類計数する工程 （８）２次元分布から、損傷好酸球の領域を設定し、損
傷好酸球を分類計数する工程 （９）（４）で測定したリンパ球数と（５）で測定した
損傷リンパ球数を用いて、リンパ球数を補正する工程 （１０）（４）で測定した単球数と（６）で測定した損
傷単球数を用いて、単球数を補正する工程 （１１）（４）で測定した好中球数と（７）で測定した
損傷好中球数を用いて、好中球数を補正する工程 （１２）（４）で測定した好酸球数と（８）で測定した
損傷好酸球数を用いて、好酸球数を補正する工程。1. A method for classifying and counting damaged cells, comprising the following steps: (1) A step of dissolving the sample to such an extent that the red blood cells in the blood sample do not interfere with the measurement, and mixing the sample with a hemolytic agent that renders leukocytes and damaged cells in a state suitable for measurement (2) The sample prepared in (1) Is measured by a flow cytometer to measure at least two scattered lights. (3) a step of obtaining a two-dimensional distribution using two different scattered lights from the scattered light signals of the individual cells measured in (2); 4) a step of classifying and counting normal leukocytes into at least four from the two-dimensional distribution; (5) setting an area of damaged lymphocytes from the two-dimensional distribution;
Step of Classifying and Counting Damaged Lymphocytes (6) Setting the Area of Damaged Monocytes from the Two-Dimensional Distribution and Classifying and Counting Damaged Monocytes (7) Setting the Area of Damaged Neutrophils from the Two-Dimensional Distribution (8) setting the area of damaged eosinophils from the two-dimensional distribution and classifying and counting damaged eosinophils (9) the number of lymphocytes measured in (4) Step of correcting the number of lymphocytes using the number of damaged lymphocytes measured in (5) (10) Monocytes using the number of monocytes measured in (4) and the number of damaged monocytes measured in (6) Step of correcting the number (11) Step of correcting the number of neutrophils using the number of neutrophils measured in (4) and the number of damaged neutrophils measured in (7) (12) Measured in (4) Correcting the number of eosinophils using the obtained number of eosinophils and the number of damaged eosinophils measured in (8).
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
載の損傷細胞分類計数方法。2. A hemolytic agent that 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 is composed of at least one nonionic surfactant and at least one cation. The method for classification and counting of damaged cells according to claim 1, wherein the surfactant is a surfactant and has a pH of 4.5 to 11.0.
する有機酸もしくはその塩を含有する請求項１〜２に記
載の損傷細胞分類計数方法。3. The method according to claim 1, 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記載の損傷細胞分類計数方法。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 classifying and counting damaged cells according to any one of claims 1 to 3.
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