CN102791124A - 用于生物细胞的固定溶液 - Google Patents
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Abstract
该固定溶液用于在体外保存包含具核细胞和红细胞的细胞学样品,并且包含醇以用于固定所述细胞。它包含生理盐水以便避免在细胞壁处的渗透压休克,福尔马林和聚乙二醇以用于保持在所述溶液中进行固定的具核细胞和红细胞的大小和完整性。该溶液不包含丙酮或酮类化合物,也不包含乙酸。
Description
本发明涉及用于生物细胞的固定溶液,其用于在体外保存细胞学样品,该固定溶液的类型为包含醇以用于固定所述细胞。更特别地,本发明适用于细胞学领域。
固定溶液,或固定剂,用于保存或保持包含细胞的细胞学取样物或样品,以便由细胞学家以后对其进行分析。这些细胞样品可以例如通过在患者中进行针穿刺、清洗、刷擦或小便采集来获得。所述细胞可以来自任何器官,例如子宫、肝脏、胃、乳房等。细胞样品可以例如为液体取样物,例如尿、腹水、胸膜液或心包液;涂片,例如宫颈涂片、阴道涂片...;器官(例如,浅表腺体例如乳腺、甲状腺,或者深层腺体例如胰腺或肝脏,等等)的穿刺物。
“细胞学固定剂”或“细胞学固定溶液”是指通常在细胞学分析中使用以实现细胞固定的溶液。
固定是这样的操作,所述操作旨在使细胞的形态尽可能地保持于在其取样之前它们所处的状态。最好的固定剂是那些固定剂,所述固定剂通过快速作用,产生尽可能少的可能妨碍细胞内部形态的分析的二次修饰或人为现象。
乙酸福尔马林醇(alcool formoléacétique)或AFA是已知的并且很久以来尤其用于农业科学和兽医学环境。AFA包含甲醇或乙醇、福尔马林和冰醋酸。福尔马林由甲酸、甲醛、低聚甲醛和甲醇组成。一个实例为Locquin的AFA,其组成如下:
-80°乙醇 100cc,
-38%的实验室福尔马林 10cc,
-冰醋酸 5cc,
-蔗糖 10g,
-蒸馏水 20cc。
然而,冰醋酸破坏红细胞并且裂解其内容物,其中涉及血红蛋白沉积,这在标准染色例如帕帕尼古拉乌(Papanicolaou)染色或梅-格-吉(May-Grünwald Giemsa)染色(MGG)之后的分析中是非常有妨碍的,或者对于免疫细胞化学研究来说是非常有妨碍的,等等。此外,在该浓度下,乙醇从规定角度来看被认为是易燃的,并且福尔马林被认为是有毒的和致癌的。
本发明的目标是提供用于生物细胞的固定溶液,其使得能够良好地保持具核细胞和红细胞的完整性以为了其分析,但对于使用者来说危险性更小。
为此,本发明的目标在于上述类型的用于生物细胞的固定溶液,其包含生理盐水以便避免在细胞壁处的渗透压休克,福尔马林和聚乙二醇以用于保持在所述溶液中进行固定的具核细胞和红细胞的大小和完整性。
根据本发明的其他方面,所述用于生物细胞的固定溶液包含一个或多个下述特征:
-所述固定溶液不包含丙酮或酮类化合物,也不包含乙酸,以便保护红细胞的完整性;
-所述醇为乙醇或异丙醇;
-所述醇为乙醇和异丙醇的混合物;
-醇的量大致小于该固定溶液的45体积%;
-福尔马林的量大致为该固定溶液的大约0.2-1体积%;
-所述固定溶液包含保证该固定溶液的pH大致为6.4-7.4的缓冲液;和
-所述固定溶液包含80体积%至95体积%的:
-590ml的生理盐水,
-203ml的异丙醇,
-193ml的纯乙醇,
-0.01体积%的叠氮化钠,
以及20体积%至5体积%的经缓冲的4%福尔马林。
因此,该溶液使得能够更好地保持细胞的比例,特别是相对于细胞尺寸而言的细胞核尺寸,并且尤其是极好地保持红细胞。
此外,该固定溶液是不太易燃的、非毒性的和非致癌的。
经由阅读仅作为实例而给出并通过参考附图来进行的下面的描述,将会更好地理解本发明,在所述附图中:
-图1为取自乳房的细胞样品的照片,所述样品保持在根据本发明的固定溶液中,
-图2为取自甲状腺的细胞样品的照片,所述样品保持在根据本发明的固定溶液中。
本发明涉及用于在体外保存细胞学样品的固定溶液,所述细胞学样品包含准备供由细胞学家进行分析之用的生物细胞。
所述固定溶液包含等渗的氯化钠水溶液或生理盐水,以代替习惯上作为通常使用的固定溶液的基质而使用的蒸馏水或去离子水。
生理盐水很久以来就是已知的,并且包含按照9克氯化钠/1升水稀释在蒸馏水中的氯化钠。生理盐水是等渗的,这使细胞维持在合适的渗透性状态下,以避免对于其造成的在细胞壁处的任何渗透压休克并因此避免由于细胞质膜和核膜破裂而引起的细胞破坏。生理盐水的渗透性等于308毫渗透压摩尔/升。
渗透性是介质的浓度。这借助了渗透的概念,渗透是溶剂穿越分隔两种具有不同浓度的溶液(例如固定溶液和细胞的细胞质液体)的半透膜而进行的扩散。
此外,所述固定溶液包含醇以用于通过在该含醇介质中固定细胞来使所述细胞保持在其状态。
优选地,醇的量大致小于该固定溶液的45体积%,以便是不太易燃的。因此,该低的醇含量使得更容易运输和储存,因为该固定溶液的危险性更小。“不太易燃的”特别是指,根据本发明的溶液不需要在该溶液的包装上使用标明产品易燃性的安全象形符号。
所述醇为例如乙醇或异丙醇。
根据一个变化形式,所述醇为乙醇和异丙醇的混合物。
然而,单独的醇是脱水性还原剂,因此通过细胞核和细胞质的收缩而改变细胞的大小。
为了克服该缺点,所述固定溶液还包含福尔马林以用于保持细胞的大小。以已知的和已经由Saccomanno等人发表的方式,所述固定溶液还可以包含或聚乙二醇。以已知的和已经发表的方式,所述福尔马林可以与脱钙剂或抗聚集剂例如乙二胺四乙酸(EDTA)及其盐相联合。以也是已知的和已经发表的方式,可以向包含粘液的取样物添加粘液溶解试剂例如二硫苏糖醇(DTT)或乙酰半胱氨酸。
福尔马林使得能够保持红细胞和具核细胞而不裂解它们,这保证了在大小方面的参考,从而允许细胞学家作更确切的诊断。
特别地,正如可以在根据本发明的固定溶液中保持的细胞取样物的图1和2中看到的,在所述图中标注为1的红细胞被完好地保持并且是完整的,这使得能够进行这些取样物的确切分析。
优选地,福尔马林的量大致为该固定溶液的大约0.2-1体积%。在该浓度下,根据由国家研究和安全研究所(Institut National deRecherche et de Sécurité,INRS)所编订的毒理学记录卡,福尔马林或甲醛被认为仅是刺激性的,这与1-25%的通常所使用的浓度(在该浓度下,福尔马林是腐蚀性的和致癌的)不同。因此,根据本发明的溶液的包装也不需要使用标明腐蚀性产品的安全象形符号。
因此,在低于1%的浓度下,可以毫无危险地操作该溶液,其中关于操作它的预防措施水平是不太高的。
以已知的方式,所述固定溶液可以包含抗微生物剂。
所述固定溶液包含保证该固定溶液的pH大致为6.4-7.4的缓冲液。该pH范围相应于保持人体细胞和血液的最佳条件。
下面的实施例举例说明了本发明而不限制其范围。
实施例1:
由下列组分形成的溶液:
80体积%的:
-590ml的生理盐水,
-203ml的异丙醇,
-193ml的纯乙醇,
-0.01体积%的叠氮化钠,
以及20体积%的经缓冲的4%福尔马林。
备选地,所述溶液可以由90体积%的上述混合物和10体积%的经缓冲的4%福尔马林形成,或者由95体积%的上述混合物和5体积%的经缓冲的4%福尔马林形成。
进一步地,将会注意到,根据本发明的固定溶液不包含丙酮或酮类化合物,也不包含乙酸。因为这些产品裂解红细胞,其通过破裂而释放结合在所述细胞上的血红蛋白。某些类型的染色,例如帕帕尼古拉乌染色,由于联合了数种核染料的染色试剂的复杂性和血红蛋白,因而致使细胞学分析(尤其是细胞核分析)非常困难甚至不可能。同样地,免疫细胞化学研究经常受到血红蛋白沉积妨碍。
根据本发明,通过帕帕尼古拉乌染色来进行的样品的以后分析,或者在本文所描述的并且不包含丙酮或酮类化合物也不包含乙酸的固定溶液中进行固定的取样物的免疫细胞化学研究,得到了改善。
因此,上面所描述的固定溶液使得能够极好地保持具核细胞和红细胞的完整性以为了其分析。
Claims (7)
1.用于在体外保存包含具核细胞和红细胞的细胞学样品的固定溶液,其包含醇以用于固定所述细胞,生理盐水以便避免在细胞壁处的渗透压休克,福尔马林和聚乙二醇以用于保持在所述溶液中进行固定的具核细胞和红细胞的大小和完整性;所述固定溶液的特征在于,不包含丙酮或酮类化合物,也不包含乙酸,以便保护红细胞的完整性。
2.根据权利要求1的固定溶液,其特征在于,所述醇为乙醇或异丙醇。
3.根据权利要求1的固定溶液,其特征在于,所述醇为乙醇和异丙醇的混合物。
4.根据权利要求1至3中任一项的固定溶液,其特征在于,醇的量大致小于该固定溶液的45体积%。
5.根据权利要求1至4中任一项的固定溶液,其特征在于,福尔马林的量大致为该固定溶液的大约0.2-1体积%。
6.根据权利要求1至5中任一项的固定溶液,其特征在于,它包含保证该固定溶液的pH大致为6.4-7.4的缓冲液。
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FR1050536A FR2955458B1 (fr) | 2010-01-27 | 2010-01-27 | Solution de fixation pour des cellules biologiques |
PCT/FR2011/050101 WO2011092414A1 (fr) | 2010-01-27 | 2011-01-20 | Solution de fixation pour des cellules biologiques |
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EP (1) | EP2528433A1 (zh) |
JP (1) | JP2013518087A (zh) |
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Cited By (2)
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CN106706395A (zh) * | 2016-12-08 | 2017-05-24 | 武汉宏兹生物技术有限公司 | 一种新型环保固定液 |
CN112504793A (zh) * | 2020-10-14 | 2021-03-16 | 核工业总医院 | 用于渗透和固定血细胞的试剂及分析方法 |
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WO2014151437A1 (en) * | 2013-03-15 | 2014-09-25 | Creatv Microtech, Inc. | Urine preservative reagent for microfiltration |
CN111972396A (zh) * | 2019-05-21 | 2020-11-24 | 威海威高医用材料有限公司 | 宫颈脱落细胞保存液及制备方法和细胞保存方法 |
CN113068683B (zh) * | 2021-03-03 | 2022-04-01 | 北京诚智光辉科技有限公司 | 一种液基细胞检查用细胞保存液及其制备方法 |
CN114208812A (zh) * | 2021-12-22 | 2022-03-22 | 桂林优利特医疗电子有限公司 | 一种液基细胞保存液 |
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JP2005211055A (ja) * | 2004-02-02 | 2005-08-11 | Osaka Prefecture | 固定液 |
UA76372C2 (en) * | 2005-02-01 | 2006-07-17 | Inst Oncology Acad Med Sci Ua | Method for producing cytological preparation of dendritic cells |
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2011
- 2011-01-20 KR KR1020127021833A patent/KR20120128131A/ko not_active Application Discontinuation
- 2011-01-20 EP EP11704659A patent/EP2528433A1/fr not_active Withdrawn
- 2011-01-20 US US13/575,325 patent/US20130059330A1/en not_active Abandoned
- 2011-01-20 RU RU2012136482/13A patent/RU2551570C2/ru not_active IP Right Cessation
- 2011-01-20 WO PCT/FR2011/050101 patent/WO2011092414A1/fr active Application Filing
- 2011-01-20 CN CN201180013370.3A patent/CN102791124B/zh not_active Expired - Fee Related
- 2011-01-20 JP JP2012550492A patent/JP2013518087A/ja active Pending
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US4857300A (en) * | 1987-07-27 | 1989-08-15 | Cytocorp, Inc. | Cytological and histological fixative formulation and methods for using same |
US20020094577A1 (en) * | 1998-06-30 | 2002-07-18 | Guirguis Raouf A. | Cytological and histological fixative composition and methods of use |
WO2000002031A1 (en) * | 1998-07-07 | 2000-01-13 | Lamina, Inc. | Improved method for mixing and processing specimen samples |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106706395A (zh) * | 2016-12-08 | 2017-05-24 | 武汉宏兹生物技术有限公司 | 一种新型环保固定液 |
CN106706395B (zh) * | 2016-12-08 | 2021-03-02 | 横琴宏恩医疗科技有限公司 | 一种环保固定液 |
CN112504793A (zh) * | 2020-10-14 | 2021-03-16 | 核工业总医院 | 用于渗透和固定血细胞的试剂及分析方法 |
CN112504793B (zh) * | 2020-10-14 | 2021-10-01 | 核工业总医院 | 用于渗透和固定血细胞的试剂及分析方法 |
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KR20120128131A (ko) | 2012-11-26 |
RU2551570C2 (ru) | 2015-05-27 |
US20130059330A1 (en) | 2013-03-07 |
RU2012136482A (ru) | 2014-03-10 |
FR2955458A1 (fr) | 2011-07-29 |
WO2011092414A1 (fr) | 2011-08-04 |
JP2013518087A (ja) | 2013-05-20 |
EP2528433A1 (fr) | 2012-12-05 |
FR2955458B1 (fr) | 2014-09-05 |
CN102791124B (zh) | 2014-12-03 |
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