JPH0331388B2 - - Google Patents
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- Publication number
- JPH0331388B2 JPH0331388B2 JP15593583A JP15593583A JPH0331388B2 JP H0331388 B2 JPH0331388 B2 JP H0331388B2 JP 15593583 A JP15593583 A JP 15593583A JP 15593583 A JP15593583 A JP 15593583A JP H0331388 B2 JPH0331388 B2 JP H0331388B2
- Authority
- JP
- Japan
- Prior art keywords
- staining
- staining solution
- acid
- ethanol
- solution
- 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.)
- Expired
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- 239000012192 staining solution Substances 0.000 claims description 79
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 26
- 238000010186 staining Methods 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 20
- 239000000975 dye Substances 0.000 claims description 19
- 238000007447 staining method Methods 0.000 claims description 18
- 150000007524 organic acids Chemical class 0.000 claims description 16
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 claims description 15
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 claims description 11
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 claims description 10
- 235000005985 organic acids Nutrition 0.000 claims description 10
- 150000003839 salts Chemical class 0.000 claims description 10
- UJMBCXLDXJUMFB-GLCFPVLVSA-K tartrazine Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)C1=NN(C=2C=CC(=CC=2)S([O-])(=O)=O)C(=O)C1\N=N\C1=CC=C(S([O-])(=O)=O)C=C1 UJMBCXLDXJUMFB-GLCFPVLVSA-K 0.000 claims description 10
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 claims description 9
- 235000012756 tartrazine Nutrition 0.000 claims description 9
- 239000004149 tartrazine Substances 0.000 claims description 9
- 229960000943 tartrazine Drugs 0.000 claims description 9
- 239000004214 Fast Green FCF Substances 0.000 claims description 8
- 235000019240 fast green FCF Nutrition 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- -1 organic acid salts Chemical class 0.000 claims description 6
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 5
- 229930195729 fatty acid Natural products 0.000 claims description 5
- 239000000194 fatty acid Substances 0.000 claims description 5
- 150000004665 fatty acids Chemical class 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 210000005128 keratinized epithelium Anatomy 0.000 claims description 4
- 210000003855 cell nucleus Anatomy 0.000 claims description 2
- HSXUHWZMNJHFRV-UHFFFAOYSA-L disodium;6-oxido-5-phenyldiazenyl-4-sulfonaphthalene-2-sulfonate Chemical compound [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1N=NC1=CC=CC=C1 HSXUHWZMNJHFRV-UHFFFAOYSA-L 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 134
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 28
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 14
- 239000001384 succinic acid Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000005711 Benzoic acid Substances 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 235000010233 benzoic acid Nutrition 0.000 description 7
- 238000007654 immersion Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 206010036790 Productive cough Diseases 0.000 description 4
- WLKAMFOFXYCYDK-UHFFFAOYSA-N [5-amino-4-[[3-[(2-amino-4-azaniumyl-5-methylphenyl)diazenyl]-4-methylphenyl]diazenyl]-2-methylphenyl]azanium;dichloride Chemical compound [Cl-].[Cl-].CC1=CC=C(N=NC=2C(=CC([NH3+])=C(C)C=2)N)C=C1N=NC1=CC(C)=C([NH3+])C=C1N WLKAMFOFXYCYDK-UHFFFAOYSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- QQVIHTHCMHWDBS-UHFFFAOYSA-N isophthalic acid Chemical compound OC(=O)C1=CC=CC(C(O)=O)=C1 QQVIHTHCMHWDBS-UHFFFAOYSA-N 0.000 description 4
- 239000012120 mounting media Substances 0.000 description 4
- 210000003802 sputum Anatomy 0.000 description 4
- 208000024794 sputum Diseases 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- LNETULKMXZVUST-UHFFFAOYSA-N 1-naphthoic acid Chemical compound C1=CC=C2C(C(=O)O)=CC=CC2=C1 LNETULKMXZVUST-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 159000000032 aromatic acids Chemical class 0.000 description 2
- 239000000981 basic dye Substances 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000003679 cervix uteri Anatomy 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 238000012137 double-staining Methods 0.000 description 2
- 238000005562 fading Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- DIZBQMTZXOUFTD-UHFFFAOYSA-N 2-(furan-2-yl)-3h-benzimidazole-5-carboxylic acid Chemical compound N1C2=CC(C(=O)O)=CC=C2N=C1C1=CC=CO1 DIZBQMTZXOUFTD-UHFFFAOYSA-N 0.000 description 1
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 1
- ATYSUIFFUCAXHH-UHFFFAOYSA-N 8-(dimethylamino)naphthalene-2-sulfonic acid Chemical compound C1=C(S(O)(=O)=O)C=C2C(N(C)C)=CC=CC2=C1 ATYSUIFFUCAXHH-UHFFFAOYSA-N 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- SJEYSFABYSGQBG-UHFFFAOYSA-M Patent blue Chemical compound [Na+].C1=CC(N(CC)CC)=CC=C1C(C=1C(=CC(=CC=1)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=CC(=[N+](CC)CC)C=C1 SJEYSFABYSGQBG-UHFFFAOYSA-M 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-N benzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-N 0.000 description 1
- 229940092714 benzenesulfonic acid Drugs 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- CVOQYKPWIVSMDC-UHFFFAOYSA-L dipotassium;butanedioate Chemical compound [K+].[K+].[O-]C(=O)CCC([O-])=O CVOQYKPWIVSMDC-UHFFFAOYSA-L 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 description 1
- 229910052808 lithium carbonate Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000000983 mordant dye Substances 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 238000001139 pH measurement Methods 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
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000011505 plaster Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 125000000542 sulfonic acid group Chemical group 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Description
本発明は、細胞の染色方法に関する。すなわち
臨床検査で細胞の異常を検出し、疾病の診断を行
なうための細胞診において、多色素−重染色を特
徴とする細胞の染色方法に関するものである。
細胞診は、細胞の異常を知る上で極めて重要な
もので、古くは、患者の各臓器からの滲出物、滲
出液あるいは擦過物などの中に含まれる細胞を、
顕微鏡下で観察し、正常細胞に混入している異型
細胞を選別し、さらに、この異型細胞が悪性なも
のかどうかを判別していた。その後、この際の細
胞の構造を見分けやすくするために種々の色素で
細胞を染めることにより、正常細胞と異型細胞の
形態を比較しやすくし、さらに染色性の差により
構成成分の相違を検出しやすくして、正常細胞と
異型細胞を容易に識別する方法が広く普及するよ
うになつた。この染色に用いられる色素は、酸性
染料、塩基性染料をはじめ、油溶染料、媒染染料
など多種多様であり、目的対象物によつてそれぞ
れ使い分けられている。
例えば、婦人科の膣脂膏、膣頚部及び膣内膜や
喀痰、尿、胃洗浄液の沈渣などの染色に用いられ
ているパパニコロ染色法は、ヘマトキシリン染色
液で細胞核を染色し、次にOG−６染色液（オレ
ンジＧ含有）で角化上皮を、EA染色液（エオシ
ンＹとライトグリーンSFY含有）でその他の部
分を染め分ける方法であり、多色素−重染色法の
代表的な方法として、ガン検診に広く普及してい
るものである。
パパニコロ染色法は、これまで種々の組成の染
色液とその染色操作法が発表され、使用されてお
り、例えば「細胞診教本−その基礎と実際−」
（宇宙堂八木書店）によれば、パパニコロ染色法
としてNo.268、No.267、No.267変法、パパニコロ
1963年版Atlas中のメモ、Walter Reed Army
Hospital（1968年）変法、以前のWalter Reed
Army Hospital（1960年）変法の６種の方法が記
載されている。
パパニコロ染色法は、カラフルでバランスのと
れた染色法であるが、以下のような欠点があるた
め新規改良が要望されるようになつた。すなわ
ち、EA染色液は、エオシンＹとライトグリーン
SFYの二種の色素を含有し、且つ塩基性色素の
ビスマルクブラウンを添加してエオシンＹとライ
トグリーンSFYの染色性を調整させ、３種の色
素の競合染色となつているため、染色液の液性
や、３種の色素の濃度及び比率が染色に大きく影
響を及ぼしており、再現性の良い結果を得るには
染色液の調製及び染色操作に高度の技術を必要と
した。しかも、エオシンＹ、ライトグリーン
SFYは光により分解されやすいため、染色後の
標本の褪色が早く、その保存安定性が非常に悪い
という問題点があつた。従つて、最近のガン検診
制度の進歩に伴い、パパニコロ染色法による細胞
診の自動化がなされるにつれ、以上述べてきたよ
うな問題点や欠点を解決又は軽減した新たな染色
操作法が切望されるようになつた。
本発明者らは、かかる問題点を解決すべく鋭意
研究の結果、パパニコロ染色液のOG−６の染色
液及びEA染色液による細胞染色の際、細胞組織
をより効果的に染め分けるには染色液のPHが影響
をもつのではないかとの着想から、その“見掛け
上の最適PH”が3.5〜6.5にあることを見出し、且
つそのPH調整のために有機酸又は／及びその塩類
の添加が非常に有効であることを見出し、更に、
OG−６染色液にオレンジＧとタートラジンの二
種の色素を使用することにより角化上皮の染色が
鮮明に行われ、EA染色液として、エオシンＹ、
ライトグリーンSFYの二種の色素に加えて、ア
シツドレツド、フアーストグリーンFCFの二種
の色素の内のいずれか一方又は両方を使用するこ
とによつて、染色性の向上と染色標本の保存安定
性の向上が達成できることを見出し、本発明を完
成するに到つた。
本発明に使用される有機酸は、好ましくは脂肪
酸類、例えば、酢酸、クエン酸、コハク酸、マレ
イン酸、ソルビン酸、酒石酸、乳酸、カプリル酸
ラウリン酸などが単独で又は二種以上の混合物と
して用いられるが、又芳香族酸類も問題なく使用
でき、ベンゼン環又はナフタレン環にスルホン酸
基又はカルボン酸基が直結した化合物、例えば、
ｐ−トルエンスルホン酸、ベンゼンスルホン酸、
ｐ−フエノールスルホン酸、１−ジメチルアミノ
ナフタレン−７−スルホン酸、安息香酸、１−ナ
フトエ酸、サリチル酸、スルホサリチル酸などが
単独で又は二種以上の混合物として用いられ、ま
た、脂肪酸類と芳香族酸類の任意の混合物として
も使用できる。又、これら有機酸の塩類として
は、アルカリ金属塩類、アンモニウム塩類、アミ
ン塩類などが使用でき、酸類と塩類の任意の組合
せによる混合物も差支えなく使用できる。なお、
これら有機酸又は有機酸の塩類は例示した化合物
に限定されるものではないことはいうまでもな
い。
以上述べたような有機酸又は／及び有機酸の塩
類を用いることにより、染色液の液性を、PH＝
3.5〜6.5に調整することが、本発明の染色法の重
要なポイントとなつている。ここでいう液性と
は、通常のガラス電極PHメーターによつて測定さ
れた“見掛けのPH”を意味し、この染色液のよう
に溶媒として60〜90v／ｖ％エタノールを用いた
場合、電極自体のアルコール誤差が大きいため、
正確なPH測定は困難であり、真のPH値とは区別さ
れた“見掛けのPH”として、その液性を示してあ
る。本発明者らは、OG−６染色液及びEA染色
液による細胞染色の際、細胞組織を最もよく染め
分けられるための“見掛け上の最適PH”が3.5〜
6.5にあることを見出し、且つそのPH調整のため
に有機酸又は／及びその塩の混合物を添加するこ
とが非常に有効であることも見出したのである。
用いる有機酸又は有機酸の塩類の濃度は、“見掛
けのPH”3.5〜6.5が得られる濃度であれば特に制
限はないが、通常用いられる濃度は0.05〜1w／
ｖ％である。しかし、この濃度範囲をはずれた溶
液でも何ら差支えはない。
例えば、脂肪酸類及び芳香族酸類を添加したと
きのEA染色液（色素としてエオシンＹ 0.45％、
アシツドレツド 0.005％、ライトグリーンSFY
0.02％、フアーストグリーンFCF 0.02％を含む）
の“見掛けのPH”を第１表、第２表に例示する。
酸濃度は、0.2w／ｖ％、溶倍は、80v／ｖ％エタ
ノールを用いた。
The present invention relates to a method for staining cells. That is, the present invention relates to a cell staining method characterized by multiple dye-double staining in cytodiagnosis for detecting cell abnormalities and diagnosing diseases in clinical examinations. Cytology is extremely important for detecting abnormalities in cells.
The cells were observed under a microscope to sort out atypical cells that were mixed in with normal cells, and then determine whether these atypical cells were malignant or not. Afterwards, cells are dyed with various dyes to make it easier to distinguish the structure of the cells, making it easier to compare the morphology of normal cells and atypical cells, and further detecting differences in their constituent components based on differences in staining properties. A method for easily distinguishing between normal cells and atypical cells has become widely used. There are a wide variety of dyes used for this dyeing, including acid dyes, basic dyes, oil-soluble dyes, and mordant dyes, and each is used depending on the intended object. For example, the Papanicolaou staining method, which is used in gynecology to stain vaginal plaster, vaginal cervix, vaginal lining, sputum, urine, and gastric lavage sediment, stains cell nuclei with hematoxylin staining solution, then OG-6 A staining solution (containing orange G) is used to stain the keratinized epithelium, and an EA staining solution (containing eosin Y and light green SFY) is used to stain the other areas.As a representative method of multi-dye double staining, it is used to treat cancer. It is widely used for medical examinations. Regarding the Papanicolaou staining method, staining liquids with various compositions and their staining procedures have been published and used, for example, in "Cytology Textbook - Its Basics and Practice".
According to (Uchudo Yagi Shoten), the Papanicolaou staining methods include No.268, No.267, No.267 modified method, and Papanicolaou staining method.
Notes in the 1963 Atlas, Walter Reed Army
Hospital (1968) variant, formerly Walter Reed
Army Hospital (1960) describes six modified methods. The Papanicolaou staining method is a colorful and well-balanced staining method, but it has the following drawbacks, and new improvements have been desired. That is, the EA staining solution consists of eosin Y and light green.
Contains two types of dyes, SFY, and the basic dye Bismarck Brown is added to adjust the stainability of eosin Y and light green SFY, making it a competitive stain of three types of dyes, so the staining solution The liquid properties and the concentration and ratio of the three types of dyes have a large effect on staining, and in order to obtain reproducible results, advanced techniques are required for preparing the staining liquid and performing the staining operation. Moreover, Eosin Y, light green
Since SFY is easily decomposed by light, the color of the specimen after staining fades quickly, and its storage stability is extremely poor. Therefore, with recent advances in the cancer screening system and the automation of cytodiagnosis using the Papanicolaou staining method, there is a strong need for a new staining operation method that solves or alleviates the problems and shortcomings mentioned above. It became like that. As a result of intensive research to solve such problems, the present inventors have discovered that when staining cells with Papanicolaou staining solution OG-6 staining solution and EA staining solution, it is possible to more effectively stain cell tissues. Based on the idea that the PH of the liquid may have an effect, we found that the "apparent optimum PH" is between 3.5 and 6.5, and that it is possible to add organic acids and/or their salts to adjust the PH. We found that it is very effective, and furthermore,
By using two types of dyes, Orange G and Tartrazine, in the OG-6 staining solution, the keratinized epithelium can be clearly stained, and as the EA staining solution, Eosin Y,
In addition to light green SFY, the use of either one or both of the two dyes, assisted red and fast green FCF, improves stainability and improves storage stability of stained specimens. The present inventors have discovered that the present invention can be improved. The organic acids used in the present invention are preferably fatty acids such as acetic acid, citric acid, succinic acid, maleic acid, sorbic acid, tartaric acid, lactic acid, caprylic acid, lauric acid, etc. alone or as a mixture of two or more. However, aromatic acids can also be used without problems, and compounds in which a sulfonic acid group or a carboxylic acid group is directly bonded to a benzene ring or a naphthalene ring, for example,
p-toluenesulfonic acid, benzenesulfonic acid,
p-phenolsulfonic acid, 1-dimethylaminonaphthalene-7-sulfonic acid, benzoic acid, 1-naphthoic acid, salicylic acid, sulfosalicylic acid, etc. are used alone or as a mixture of two or more, and fatty acids and aromatic Any mixture of acids can also be used. Further, as the salts of these organic acids, alkali metal salts, ammonium salts, amine salts, etc. can be used, and mixtures of acids and salts in any combination can also be used without any problem. In addition,
It goes without saying that these organic acids or salts of organic acids are not limited to the exemplified compounds. By using the organic acids and/or salts of organic acids as described above, the liquid properties of the staining solution can be adjusted to PH=
Adjustment to 3.5 to 6.5 is an important point in the staining method of the present invention. The term "liquid" here refers to the "apparent PH" measured with an ordinary glass electrode PH meter. Because the alcohol error itself is large,
Accurate PH measurement is difficult, and the liquid properties are indicated as "apparent PH", which is distinguished from the true PH value. The present inventors found that when staining cells with OG-6 staining solution and EA staining solution, the "apparent optimal pH" for the best staining of cell tissues is 3.5 to 3.5.
6.5, and also found that it is very effective to add a mixture of organic acids and/or their salts to adjust the pH.
The concentration of the organic acid or organic acid salt used is not particularly limited as long as it provides an "apparent pH" of 3.5 to 6.5, but the concentration usually used is 0.05 to 1w/w/
v%. However, there is no problem with solutions outside this concentration range. For example, EA staining solution when fatty acids and aromatic acids are added (eosin Y 0.45% as dye,
Ascended 0.005%, light green SFY
0.02%, First Green FCF 0.02%)
Tables 1 and 2 illustrate the “apparent PH” of
The acid concentration was 0.2 w/v%, and the solubility was 80 v/v% ethanol.
【表】【table】
【表】
また、有機酸と有機酸塩の混合物を添加したと
きのEA染色液（色素としてエオシンＹ 0.45％、
アシツドレツド 0.005％、ライトグリーンSFY
0.02％、フアーストグリーンFCF 0.02％を含む）
の“見掛けのPH”を第３表に例示する。酸及び塩
の濃度は0.2w／ｖ％、溶倍は、70v／ｖ％、エタ
ノールを用いた。[Table] Also, EA staining solution (0.45% eosin Y as dye,
Ascended 0.005%, light green SFY
0.02%, First Green FCF 0.02%)
Table 3 illustrates the “apparent PH” of The concentration of acid and salt was 0.2 w/v%, the solubility was 70 v/v%, and ethanol was used.
【表】
また、従来法に比べ、本発明は、染色の際の色
の鮮明さ及び染色後の標本の保存安定性が著しく
改善されている。すなわち、OG−６染色液にオ
レンジＧとタートラジンの２種の色素を用いるこ
とにより、角化上皮の染色が、従来のOG−６染
色に比べより鮮明に行なえるようになり、また、
EA染色液として、エオジンＹ、ライトグリーン
SFYの二種の色素に加えて、アシツドレツド、
フアーストグリーンFCFの二種の色素の内のい
ずれか一方又は両方を用いることにより、この保
存安定性がより一層改善され、従つて、従来のパ
パニコロ法におけるEA染色液に含まれていた塩
基性色素のビスマルクブラウンは全く用いる必要
がなくなつた。
本発明の細胞診染色方法をより詳細に下記に述
べる。OG−６染色液として、80v／ｖ％エタノ
ール中に、オレンジＧ 0.47w／ｖ％、タートラ
ジン0.01w／ｖ％、コハク酸 0.2w／ｖ％を含有
するもの、EA染色液として80v／ｖ％エタノー
ル中にエオシンＹ 0.35w／ｖ％、アシツドレツ
ド0.005w／ｖ％、ライトグリーンSFY 0.023w／
ｖ％、フアーストグリーンFCF 0.012w／ｖ％、
コハク酸0.2w／ｖ％を含有するものを調製する。
ヘマトキシリン液は、通常パパニコロ染色法で用
いられている、ヘマトキシリン染色液を用いる。
試料としては、例えば婦人科の膣頚部から擦過法
によりサンプリングし、スライドグラスに塗抹し
固定液で固定化した物を用いた。この固定化した
試料を、ヘマトキシリン染色液で６分間刻染色
し、蒸留水で洗浄し、0.25％塩酸中に６回前後出
入させてから流水で水洗する。次に、蒸留水→
50v／ｖ％エタノール→70v／ｖ％エタノール→
80v／ｖ％エタノールの順に濯いでからOG−６
染色液で３分間染色する。80v／ｖ％エタノール
洗浄後、１％リンタングステン酸を含む80v／ｖ
％エタノールに１分間浸漬し、次いで80v／ｖ％
エタノールで洗浄後、EA染色液で３分間染色す
る。次に、80v／ｖ％エタノール→エタノールの
順で濯いでから、キシレンに浸漬、透徹後、適当
な封入剤、例えば、エンテランニユー（メルク
社登録商標）、パーマウント（フイツシヤー・
サイエンテイフイツクカンパニー登録商標）、ビ
オライト（広研商事(株)商品名）などの封入剤で封
入する。この際、初めからEA染色液に0.2〜１％
のリンタングステン酸を加えたものを使用すれ
ば、上記のリンタングステン酸エタノール溶液に
浸漬する操作は省略しても差支えない。このEA
染色液は、従来のEA染色液に含まれているビス
マルクブラウン、炭酸リチウムは含んでおらず、
染色色素として従来のEA染色液に含まれている
エオシンＹ、ライトグリーンSFYの他に、アシ
ツドレツド、フアーストグリーンFCFのいずれ
か一方又は両方が含まれているので、好酸性細胞
質や好塩基性細胞質が従来のEA染色に比べて更
に多彩な色調に染色される。また、OG−６染色
液に、従来のOG−６染色液に含まれているオレ
ンジＧの他、タートラジンも含有させた場合、角
化表層細胞が従来のものに比べて鮮黄色に染色さ
れる。その結果、悪性細胞の検出が容易になつ
た。
上記例示した方法で、アシツドレツド、フアー
ストグリーンFCF、タートラジンのいずれをも
含有しない染色液を用いて染色を行なつた場合
は、従来のパパニコロ染色液を用いた場合と同様
のやや不鮮明な色調に染色された。
また、これまで従来のパパニコロ染色法で問題
となつていた染色標本の光照射による褪色、変色
問題も、本発明により改善され保存安定性に優れ
た染色標本を得ることが可能となつた。
以下に実施例を述べる。
実施例 １
染色試液
(1) ヘマトキシリン染色液
(2) OG−６染色液
オレンジG0.5ｇ及びコハク酸0.2ｇを80v／ｖ
％エタノールに溶解し全量100mlとする。
(3) EA染色液
エオシンY0.35ｇ、アシツドレツド0.01ｇ、
ライトグリーンSFY0.06ｇ、フアーストグリー
ンFCF0.02ｇ及びコハク酸0.25ｇを80v／ｖ％
エタノールに溶解し全量100mlとする。
染色方法
採取した喀痰を常法によりスライドグラスに塗
抹し、直ちにエーテル・アルコール固定液につけ
て固定する。固定した試料をヘマトキシリン染色
液で２分間核染色を行ない、その後蒸留水で洗浄
し、0.25％塩酸中に６回位出入させてから流水で
水洗する。次に、蒸留水→50v／ｖ％エタノール
→70v／ｖ％エタノール→80v／ｖ％エタノール
の順で濯いでから、OG−６染色液で１分間染色
する。80v／ｖ％エタノール洗浄後、１％リンタ
ングステン酸を含む80v／ｖ％エタノールに１分
間浸漬し、次いで80v／ｖ％エタノールで洗浄
後、EA染色液で２分間染色する。次に、80v／
ｖ％エタノール→エタノールの順で濯いでからキ
シレンに浸漬、透徹後、封入剤ビオライト（広研
商事(株)商品名）で封入する。
本法で染色した標本は、黄、赤、緑の染色が保
存中に褪色し難く、且つ従来法による染色標本に
比べて色調が鮮明であつた。
また、実施例１のコハク酸の代わりに安息香酸
２ｇ又はイソフタル酸0.3ｇを用いても、あるい
は、安息香酸１ｇ及びイソフタル酸0.1ｇを用い
ても、実施例１と同様の効果が得られた。更に、
実施例１のコハク酸の代わりにコハク酸0.16ｇ及
びコハク酸カリウム0.04ｇを用いても、実施例１
と同様の効果が得られた。
実施例 ２
染色液
(1) ヘマトキシリン染色液
(2) OG−６染色液
オレンジG0.47ｇ、タートラジン0.01ｇ及び
安息香酸0.5ｇ、コハク酸２ｇを80v／ｖ％エタ
ノールに溶解し全量100mlとする。
(3) EA染色液
エオシンY0.35ｇ、アシツドレツド0.005ｇ、
ライトグリーンSFY0.023ｇ、フアーストグリ
ーンFCF0.012ｇ及び安息香酸0.5ｇ、コハク酸
２ｇを80v／ｖ％エタノールに溶解し全量100
mlとする。
染色方法
実施例１に同じ。
本法で染色した標本は、角化表層細胞が、従来
法及び実施例１に比べ鮮黄色に染色された。その
他は実施例１に同じ。
また、実施例２の安息香酸0.5ｇ及びコハク酸
２ｇの代りに、安息香酸１ｇを単独で用いても、
あるいは、コハク酸１ｇを単独で用いても、実施
例２と同様の効果が得られた。
実施例 ３
染色液
(1) ヘマトキシリン染色液
(2) OG−６染色液
オレンジG0.44ｇ、タートラジン0.009ｇ、コ
ハク酸0.28ｇを82v／ｖ％エタノールに溶解し
全量100mlとする。
(3) EA染色液
エオシンY0.40ｇ、アシツドレツド0.005ｇ、
ライトグリーンSFY0.028ｇ、フアーストグリ
ーンFCF0.028ｇ、コハク酸0.25ｇ、リンタン
グステン酸0.5ｇを82v／ｖ％エタノールに溶解
し全量100mlとする。
染色方法
実施例１に同じ。但し、OG−６染色液による
染色後の「１％リンタングステン酸を含む80v／
ｖ％エタノールによる浸漬」は省略する。また、
OG−６染色液浸漬前後及びEA染色液浸漬前後
の浸漬工程に用いるエタノールは、82v／ｖ％の
ものを用いる。
本法で染色した標本は、角化表層細胞が、従来
法及び実施例１に比べ鮮黄色に染色される。その
他は実施例１に同じ。
実施例 ４
染色液
(1) ヘマトキシリン染色液
(2) OG−６染色液
オレンジG0.44ｇ、タートラジン0.0045ｇ、
コハク酸0.28ｇ、リンタングステン酸0.015ｇ
を80v／ｖ％エタノールに溶解し全量100mlと
する。
(3) EA染色液
実施例３に同じ。
染色方法
採取した喀痰をスライドグラスに塗抹し、
95v／ｖ％エタノールで固定した後、70v／ｖ％
エタノールに10回浸漬、次に50v／ｖ％エタノー
ルに10回浸漬、次に流水で軽く洗い、更に蒸留水
に10回浸漬した後、ヘマトキシリン染色液で３分
間染色後、流水で軽く洗う。次に、0.5％塩酸に
６回浸漬、次に流水で５分洗つた後、50v／ｖ％
エタノールで10回、70v／ｖ％エタノールで10
回、80v／ｖ％エタノールで10回、95v／ｖ％エ
タノールで10回浸漬後、OG−６染色液で１分間
染色する。次いで、95v／ｖ％エタノールで20回
浸漬し、EA染色液で１分間染色後、95v／ｖ％
エタノールで20回、エタノールで20回浸漬、次い
でキシレンに30回浸漬、透徹後、封入剤ビオライ
ト（広研商事(株)商品名）で封入する。
比較例 １
染色液
(1) ヘマトキシリン染色液
(2) OG−６染色液
オレンジG0.5ｇ、リンタングステン酸0.015
ｇを90v／ｖ％エタノールに溶解し、全量100
mlとする。
(3) EA染色液
エオシンY0.23ｇ、ライトグリーンSFY0.045
ｇ、ビスマルクブラウン0.05ｇ、リンタングス
テン酸0.6ｇを90v／ｖ％エタノールに溶解し、
全量100mlとする。
染色方法
採取した喀痰をスライドグラスに塗抹し、
95v／ｖ％エタノールで固定した後、実施例４と
同様に操作してヘマトキシリン液で染色後、OG
−６染色液、次いでEA染色液で染色を行なう。
但し、OG染色及びEA染色の染色時間は、それ
ぞれ１分30秒とする。
実施例４と比較例１により得られた標本を直射
日光下に保存した時の、各色調の変化を第４表に
示した。
比較例１では30日以後の赤及び緑の褪色が著し
く、60日経過後では赤は全く消失し、染色標本と
しての価値を失うが、実施例４の標本では、60日
経過後も僅かな褪色しか認められず、染色標本と
して使用可能である。[Table] Furthermore, compared to the conventional method, the present invention significantly improves the clarity of color during staining and the storage stability of the specimen after staining. In other words, by using two types of dyes, orange G and tartrazine, in the OG-6 staining solution, it is possible to stain the keratinized epithelium more clearly than with conventional OG-6 staining, and
Eosin Y, light green as EA staining solution
In addition to SFY's two types of dyes, Assidred,
By using one or both of the two types of dyes in Fast Green FCF, this storage stability is further improved, and the basicity contained in the EA staining solution in the conventional Papanicolaou method is improved. The dye Bismarck Brown no longer needs to be used at all. The cytodiagnostic staining method of the present invention will be described in more detail below. OG-6 staining solution contains 80v/v% ethanol containing Orange G 0.47w/v%, tartrazine 0.01w/v%, succinic acid 0.2w/v%, EA staining solution 80v/v%. Eosin Y 0.35w/v%, acid red 0.005w/v%, light green SFY 0.023w/v% in ethanol
v%, First Green FCF 0.012w/v%,
A sample containing 0.2% w/v of succinic acid is prepared.
As the hematoxylin solution, a hematoxylin staining solution that is normally used in the Papanicolaou staining method is used.
The sample used was, for example, sampled from the vaginal cervix of a gynecologist by the scraping method, smeared on a slide glass, and fixed with a fixative. This fixed sample is stained with hematoxylin staining solution for 6 minutes, washed with distilled water, placed in and out of 0.25% hydrochloric acid 6 times, and then washed with running water. Next, distilled water →
50v/v% ethanol→70v/v% ethanol→
After rinsing with 80v/v% ethanol, OG-6
Stain with staining solution for 3 minutes. 80v/v containing 1% phosphotungstic acid after washing with 80v/v% ethanol
% ethanol for 1 minute, then 80v/v%
After washing with ethanol, stain with EA staining solution for 3 minutes. Next, after rinsing in the order of 80v/v% ethanol → ethanol, immersion in xylene and clearing, use a suitable mounting medium such as Entellinyu (registered trademark of Merck & Co., Ltd.), Permount (Fitscher, etc.).
Mount with a mounting medium such as Scientific Photograph Company (registered trademark) or Biolite (Koken Shoji Co., Ltd. trade name). At this time, add 0.2 to 1% to the EA staining solution from the beginning.
If phosphotungstic acid added thereto is used, the above-mentioned immersion in the phosphotungstic acid ethanol solution can be omitted. This EA
The staining solution does not contain Bismarck Brown and lithium carbonate, which are contained in conventional EA staining solutions.
In addition to eosin Y and light green SFY, which are contained in conventional EA staining solutions, it contains one or both of acid red and fast green FCF as staining dyes. is dyed in a more diverse range of colors than conventional EA dyeing. In addition, when the OG-6 staining solution contains tartrazine in addition to Orange G, which is included in the conventional OG-6 staining solution, the keratinized superficial cells are stained bright yellow compared to the conventional OG-6 staining solution. . As a result, detection of malignant cells has become easier. When dyeing is carried out using a staining solution that does not contain Acid Red, Fast Green FCF, or Tartrazine using the method exemplified above, the color tone will be slightly unclear, similar to when using conventional Papanicolaou staining solution. stained. In addition, the present invention has improved the problem of fading and discoloration of stained specimens due to light irradiation, which had been a problem with conventional Papanicolaou staining methods, and it has become possible to obtain stained specimens with excellent storage stability. Examples will be described below. Example 1 Staining solution (1) Hematoxylin staining solution (2) OG-6 staining solution Orange G0.5g and succinic acid 0.2g at 80v/v
% ethanol to make a total volume of 100ml. (3) EA staining solution Eosin Y 0.35g, Assidred 0.01g,
Light Green SFY 0.06g, Fast Green FCF 0.02g and Succinic Acid 0.25g at 80v/v%
Dissolve in ethanol to make a total volume of 100ml. Staining method: Smear the collected sputum onto a slide glass using the standard method and immediately immerse it in ether/alcohol fixative to fix it. The fixed sample is nuclear stained with hematoxylin staining solution for 2 minutes, then washed with distilled water, placed in and out of 0.25% hydrochloric acid about 6 times, and then washed with running water. Next, rinse with distilled water → 50v/v% ethanol → 70v/v% ethanol → 80v/v% ethanol in this order, and then stain with OG-6 staining solution for 1 minute. After washing with 80v/v% ethanol, it is immersed in 80v/v% ethanol containing 1% phosphotungstic acid for 1 minute, then washed with 80v/v% ethanol, and then stained with EA staining solution for 2 minutes. Next, 80v/
After rinsing in the order of v% ethanol→ethanol, immersing it in xylene and clearing it, it is mounted with the mounting medium Biolite (trade name of Koken Shoji Co., Ltd.). In the specimens stained by this method, the yellow, red, and green staining did not easily fade during storage, and the color tone was clearer than that of specimens stained by the conventional method. Furthermore, the same effect as in Example 1 was obtained even if 2 g of benzoic acid or 0.3 g of isophthalic acid was used in place of succinic acid in Example 1, or even if 1 g of benzoic acid and 0.1 g of isophthalic acid were used. . Furthermore,
Even if 0.16 g of succinic acid and 0.04 g of potassium succinate were used instead of succinic acid in Example 1, Example 1
A similar effect was obtained. Example 2 Staining solution (1) Hematoxylin staining solution (2) OG-6 staining solution 0.47 g of orange G, 0.01 g of tartrazine, 0.5 g of benzoic acid, and 2 g of succinic acid are dissolved in 80 v/v% ethanol to make a total volume of 100 ml. (3) EA staining solution Eosin Y 0.35g, Assidred 0.005g,
Dissolve 0.023g of Light Green SFY, 0.012g of Fast Green FCF, 0.5g of benzoic acid, and 2g of succinic acid in 80v/v% ethanol, total volume 100.
ml. Staining method Same as Example 1. In the specimen stained by this method, the keratinized surface cells were stained brighter yellow than in the conventional method and Example 1. Other details are the same as in Example 1. Moreover, even if 1 g of benzoic acid is used alone in place of 0.5 g of benzoic acid and 2 g of succinic acid in Example 2,
Alternatively, even when 1 g of succinic acid was used alone, the same effect as in Example 2 was obtained. Example 3 Staining solution (1) Hematoxylin staining solution (2) OG-6 staining solution 0.44 g of orange G, 0.009 g of tartrazine, and 0.28 g of succinic acid were dissolved in 82 v/v% ethanol to make a total volume of 100 ml. (3) EA staining solution Eosin Y 0.40g, Assidred 0.005g,
Dissolve 0.028 g of Light Green SFY, 0.028 g of Fast Green FCF, 0.25 g of succinic acid, and 0.5 g of phosphotungstic acid in 82 v/v% ethanol to make a total volume of 100 ml. Staining method Same as Example 1. However, after staining with OG-6 staining solution, "80v/containing 1% phosphotungstic acid"
% ethanol immersion” is omitted. Also,
The ethanol used in the immersion steps before and after immersion in the OG-6 staining solution and before and after immersion in the EA staining solution is 82v/v%. In the specimen stained by this method, the keratinized surface cells are stained brighter yellow than in the conventional method and Example 1. Other details are the same as in Example 1. Example 4 Staining solution (1) Hematoxylin staining solution (2) OG-6 staining solution Orange G 0.44g, Tartrazine 0.0045g,
Succinic acid 0.28g, phosphotungstic acid 0.015g
Dissolve in 80v/v% ethanol to make a total volume of 100ml. (3) EA staining solution Same as Example 3. Staining method: Smear the collected sputum onto a slide glass.
After fixation with 95v/v% ethanol, 70v/v%
Immerse in ethanol 10 times, then immerse in 50v/v% ethanol 10 times, then rinse lightly with running water, further immerse in distilled water 10 times, stain with hematoxylin staining solution for 3 minutes, and rinse lightly with running water. Next, immerse in 0.5% hydrochloric acid 6 times, then wash with running water for 5 minutes, then 50v/v%
10 times with ethanol, 10 times with 70v/v% ethanol
After immersion in 80v/v% ethanol 10 times and 95v/v% ethanol 10 times, stain with OG-6 staining solution for 1 minute. Next, it was immersed in 95v/v% ethanol 20 times, stained with EA staining solution for 1 minute, and then stained with 95v/v% ethanol.
After being immersed in ethanol 20 times, 20 times in ethanol, and then 30 times in xylene for clearing, it is mounted with the mounting medium Biolite (trade name, Koken Shoji Co., Ltd.). Comparative example 1 Staining solution (1) Hematoxylin staining solution (2) OG-6 staining solution Orange G 0.5g, phosphotungstic acid 0.015
Dissolve g in 90v/v% ethanol, total volume 100
ml. (3) EA staining solution Eosin Y0.23g, Light Green SFY0.045
g, Bismarck Brown 0.05g, and phosphotungstic acid 0.6g dissolved in 90v/v% ethanol,
The total volume should be 100ml. Staining method: Smear the collected sputum onto a slide glass.
After fixing with 95v/v% ethanol, the same procedure as in Example 4 was carried out, and after staining with hematoxylin solution, OG
-6 staining solution and then EA staining solution.
However, the staining time for OG staining and EA staining is 1 minute and 30 seconds each. Table 4 shows the changes in each color tone when the samples obtained in Example 4 and Comparative Example 1 were stored under direct sunlight. In Comparative Example 1, the red and green colors significantly faded after 30 days, and after 60 days, the red color completely disappeared and lost its value as a stained specimen, but in the specimen of Example 4, there was only slight fading even after 60 days. It is not recognized and can be used as a stained specimen.
【表】【table】
【表】
判定方法：暗所に保存した同一染色標本の色調を
100として顕微鏡下、肉眼で評価。
評価点０は、全く脱色。[Table] Judgment method: Evaluate the color tone of the same stained specimen stored in the dark.
Evaluated under a microscope and with the naked eye as 100. An evaluation score of 0 means complete bleaching.
Claims (1)
いでOG−６染色液を用いて角化上皮を染色し、
更に、EA染色液を用いてその他の部分を染め分
ける細胞診染色法に於て、OG−６染色液として
有機酸又は／及び有機酸の塩類を加えた染色液を
用いるか、EA染色液として有機酸又は／及び有
機酸の塩類を加えた染色液を用いるか、或はOG
−６染色液、EA染色液共に有機酸又は／及び有
機酸の塩類を加えた染色液を用いることを特徴と
する、細胞の染色方法。 ２ 有機酸又は／及び有機酸の塩類が脂肪酸又
は／及び脂肪酸の塩類である、特許請求の範囲第
１項記載の染色方法。 ３ OG−６染色液としてオレンジＧ（CI 16230）
及びタートラジン（CI 19140）の二種の色素又
はこれにリンタングステン酸を加えたものを含ん
で成る染色液を用いる特許請求の範囲第１項又は
第２項に記載の染色方法。 ４ EA染色液としてエオジンＹ（CI 45380）、ラ
イトグリンーSFY（CI 42095）の二種の色素に加
えて、アシツドレツド（CI 45100）、フアースト
グリーンFCF（CI 42053）の二種の色素のいずれ
か一方又は両方を加えたもの又はこれにリンタン
グステン酸を加えたものを含んで成る染色液を用
いる特許請求の範囲第１項から第３項のいずれか
に記載の染色方法。[Claims] 1. Staining the cell nucleus with a hematoxylin staining solution, then staining the keratinized epithelium with an OG-6 staining solution,
Furthermore, in the cytodiagnosis staining method that uses EA staining solution to stain other areas, a staining solution to which an organic acid or/and organic acid salts have been added is used as OG-6 staining solution, or a staining solution containing an organic acid or/and organic acid salts is used as OG-6 staining solution, or as EA staining solution. Use a staining solution containing organic acids and/or organic acid salts, or use OG
-6 A method for staining cells, characterized by using a staining solution containing an organic acid or/and a salt of an organic acid in both the EA staining solution and the EA staining solution. 2. The dyeing method according to claim 1, wherein the organic acid and/or salts of organic acids are fatty acids and/or salts of fatty acids. 3 Orange G (CI 16230) as OG-6 staining solution
3. The dyeing method according to claim 1 or 2, which uses a staining solution comprising two types of dyes: and tartrazine (CI 19140), or a mixture thereof with phosphotungstic acid. 4 As an EA staining solution, in addition to two types of dyes: Eosin Y (CI 45380) and Light Green-SFY (CI 42095), one of two types of dyes: Assured Red (CI 45100) and Fast Green FCF (CI 42053). 4. The dyeing method according to any one of claims 1 to 3, which uses a dyeing solution containing the above-mentioned or both, or phosphotungstic acid added thereto.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15593583A JPS6047960A (en) | 1983-08-26 | 1983-08-26 | Staining method and staining test solution of cell in cytology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15593583A JPS6047960A (en) | 1983-08-26 | 1983-08-26 | Staining method and staining test solution of cell in cytology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6047960A JPS6047960A (en) | 1985-03-15 |
| JPH0331388B2 true JPH0331388B2 (en) | 1991-05-02 |
Family
ID=15616725
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15593583A Granted JPS6047960A (en) | 1983-08-26 | 1983-08-26 | Staining method and staining test solution of cell in cytology |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6047960A (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0711520B2 (en) * | 1986-09-25 | 1995-02-08 | 三菱化成株式会社 | How to make a permanent sample of cells |
| JPH0778502B2 (en) * | 1990-04-04 | 1995-08-23 | 株式会社京都医科学研究所 | Method for preserving cells in urine |
| KR20020006883A (en) * | 2000-07-14 | 2002-01-26 | 이수빈 | Cyto-Quik stain solution |
| GB0103605D0 (en) * | 2001-02-14 | 2001-03-28 | Cairns Hector | Stain |
| KR100458148B1 (en) | 2001-10-29 | 2004-11-26 | 포라 가세이 고교 가부시키가이샤 | A skin analysis system |
| JPWO2007029437A1 (en) * | 2005-09-06 | 2009-03-12 | ポーラ化成工業株式会社 | Staining solution for stratum corneum cells and method for staining stratum corneum cells using the same |
| JP2007114076A (en) * | 2005-10-21 | 2007-05-10 | Pola Chem Ind Inc | Regulation method for image of corneocyte |
| JP5491228B2 (en) * | 2010-02-16 | 2014-05-14 | 御木本製薬株式会社 | Staining solution and staining method |
-
1983
- 1983-08-26 JP JP15593583A patent/JPS6047960A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6047960A (en) | 1985-03-15 |
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