WO2014122058A1 - Procédé de coloration d'échantillons cellulaires dans le cadre de la miscroscopie - Google Patents

Procédé de coloration d'échantillons cellulaires dans le cadre de la miscroscopie Download PDF

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Publication number
WO2014122058A1
WO2014122058A1 PCT/EP2014/051761 EP2014051761W WO2014122058A1 WO 2014122058 A1 WO2014122058 A1 WO 2014122058A1 EP 2014051761 W EP2014051761 W EP 2014051761W WO 2014122058 A1 WO2014122058 A1 WO 2014122058A1
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WO
WIPO (PCT)
Prior art keywords
staining
solution
cell sample
weight
cell
Prior art date
Application number
PCT/EP2014/051761
Other languages
German (de)
English (en)
Inventor
Gabriela Altmann
Oliver Lade
Lance Anthony Ladic
Saskia Rausch
Original Assignee
Siemens Aktiengesellschaft
Siemens Healthcare Diagnostics Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Siemens Aktiengesellschaft, Siemens Healthcare Diagnostics Inc. filed Critical Siemens Aktiengesellschaft
Publication of WO2014122058A1 publication Critical patent/WO2014122058A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • the present invention relates to a method of staining cell samples for microscopy, an aqueous staining solution for use in the method, and the use of the staining aqueous solution for staining cell samples.
  • Staining methods for the staining of cell preparations have long been used in microscopy to visualize animal or plant cell and tissue constituents and to allow identification and differentiation of such tissues. Staining methods are widely used, among others, in histology, cytology and microbiology.
  • Hematoxylin-eosin staining is a standard staining technique for staining cell preparations for microscopy. It serves to differentiate different tissue structures in the microscopic picture by means of two different single stains and is one of the most common routine staining methods for morphological examinations. With the aid of HE staining, pathological changes in biopsies and surgical specimens can be examined.
  • the HE stain plays u.a. in the diagnosis of early detection of cancer, for example, for the early detection of oral carcinoma or cervical cancer.
  • HE staining the cell samples are first stained with hematoxylin after fixation on a slide (Ii staining). Hematoxylin stains all of the acidic or ba ⁇ sophilen structures blue, particular nuclei with the DAR deoxyribonucleic acid contained in (DNA) and the somen enriched ribo- rough endoplasmic reticulum (RER). After H staining, the nuclei initially appear reddish brown due to the low pH of the staining solution. By increasing the pH, the hue turns into the typical blue violet (blueing); this is effected by means of flushing in freshwater ⁇ ser with neutral pH, for example, tap water.
  • E staining eosin staining
  • Eosin stains all acidophilic or basic (eosinophilic) structures red, which mainly includes the cytoplasmic proteins.
  • Eosin G also called Eosin Y
  • Eosin B is used.
  • a standard protocol for performing HE staining involves the following steps:
  • hematoxylin staining for example, acid hematoxylin according to Ehrlich or acidic hemalaun according to Mayer is used.
  • the staining solution contains: hematoxylin, Natriumj odat, potash alum ⁇ , chloral hydrate, hydrochloric acid, 0.1% strength ammonia solution, sodium bicarbonate and citric acid.
  • the staining solution contains: hematoxylin, isopropanol 96%, glycerol, potassium alum, acetic acid, sodium iodate, sodium bicarbonate and ammonia solution
  • the eosin solution is prepared by dissolving Eosin G or B in distilled or demineralized water.
  • the object of the present invention is therefore to provide a method for staining cell samples, by which cell samples can be stained quickly and without the use of hazardous or health-endangering substances.
  • the object is achieved by a method for staining cell samples according to claim 1 and the use of a dyeing solution according to claim 12.
  • the method according to the invention for staining cell samples for microscopy comprises the following steps:
  • the staining solution invention comprises hematoxylin, sodium umjodat, potassium aluminum sulfate dodecahydrate and Zitronen Text ⁇ re.
  • the staining solution contains no chloral hydrate.
  • the inventors of the present invention were able to show that using the method according to the invention, the stained ⁇ cell samples surprisingly even without additional dewatering and fixation steps, after the second Entfär ⁇ tion step, over a longer period
  • a dye solution could be provided that does not require the addition of health-hazardous chloral hydrate and, surprisingly, a dyeing entspre ⁇ accordingly comprises the processes known from the prior art, staining solutions, and is stable over a longer period, and as can be ⁇ derver Listet.
  • the method according to the invention minimizes expenditure, time and risk potential as well as the need for technical and personal protective equipment.
  • the process poses no disposal problems because no hazardous ingredients are used. It is therefore also applicable to less well trained medical personnel.
  • the method according to the invention comprises a plurality of method steps whose advantageous embodiments are described below with reference to the attached FIGURE.
  • step Sl (a) applying the cell sample to a substrate
  • a carrier material ⁇ As support material any comes for microscopy suitable carrier material in Be ⁇ costume.
  • a transparent carrier material is ver ⁇ turns.
  • a slide is used as the carrier material.
  • any slide as it is familiar to those skilled in microscopy can be used.
  • Slide can be made of any material, preferably it consists of a plastic or glass, more preferably glass.
  • a cell sample any cell sample can be used. It may be a bacterial, plant, animal or human cell sample, preferably a human cell sample. Preferably, it is a cell sample of the oral mucus ⁇ skin, the mucous membrane of the cervix uteri, or the mucosa of vulva, more preferably a sample of the oral mucus ⁇ skin, in particular squamous the oral mucosa.
  • the cell sample contains carcinoma cells.
  • the cell sample can be obtained by any method known to those skilled in the art, preferably by brush biopsy.
  • the cell sample is applied to the substrate by, for example, painting the sample on the substrate.
  • the fixation of the cell sample on the carrier material is carried out by drying the cell sample.
  • the duration of the drying is preferably 0.5 to 10 minutes, particularly preferably 1 to 5 minutes, very particularly preferably 1.5 to 2.5 minutes, in particular 2 minutes.
  • the drying is preferably carried out at a temperature of 10 ° C - 30 ° C, more preferably 15 ° C - 25 ° C, most preferably 22 ° C.
  • An additional fixation of the cells toomme ⁇ sponding preferably takes place on the slide using commercially available fixing solutions.
  • An alcoholic Fixierlö ⁇ solution is preferably used, particularly preferably a fixing solution contained ⁇ th ethanol.
  • the fixation can be done using 1-2 spray sizes of Merckofix® fixation spray from Merck, Darmstadt, Germany.
  • step (b) staining the cell sample by means of a first staining solution;
  • step (Sl) to the carrier material applied ⁇ cell sample is then stained in step 2 by means of a first staining solution.
  • any commercial dyeing solution known to those skilled in the art may be used to stain cells.
  • An aqueous solution is preferably used containing toxylin hematoma, as commonly used by those skilled in the HE staining and is ⁇ be written in the art, more preferably the hematoxylin solution contains no chloral hydrate, is especially preferred according to the invention an aqueous Dyeing solution as disclosed in the present specification.
  • the dyeing is preferably carried out by dipping the carrier into the object ⁇ staining solution.
  • the slide is 0.2 - 5 min, particularly ⁇ vorzugt be 0.5 - 2.5 min, most preferably 0.75 - 2 minutes, in particular 1 min dipped into the first dye solution.
  • step S3 After staining the cell sample by means of a first staining solution, in step S3, a first decolorization of the im
  • Step S2 stained cells.
  • the decolorization takes place by means of water, preferably not by rinsing with running water, more preferably by immersing the support material in a vessel with water.
  • any fresh water can be used with a pH in the neutral range, preferably water is ⁇ used. If the deinking by means of water is not sufficient, can be decolored with an aqueous sodium hydrogen carbonate bicarbonate solution ⁇ optional.
  • aqueous solution of sodium hydrogencarbonate a solution of sodium hydrogen carbonate in water is used, preferably a solution with 0.01 - 1% by weight sodium bicarbonate, preferably 0.05 Sonders be ⁇ -.. 0.5% by weight, very particularly before ⁇ Trains t 0.075 - 0.25% by weight, in particular with 0.1% by weight
  • the decolorization preferably takes place over a period of 1 second (sec) - 5 min, more preferably 10 sec - 2.5 min, very particularly preferably 20 sec - 1 min, in particular 30 sec.
  • step S3 cell sample is stained at step S4 with ⁇ means of a second staining solution.
  • ⁇ means of a second staining solution.
  • any commercial dyeing solution known to the person skilled in the art can be used for staining cells.
  • a ⁇ membered aq solution preferably containing eosin used as commonly used by those skilled in the HE staining, particularly forthcoming yakt an eosin solution containing 0.05 - 1 g eosin, very particularly ⁇ preferred 0.1 - 0.5 g eosin, in particular 0.3 g eosin in 100 ml of water.
  • Eosin G also called Eosin Y
  • Eosin B is used, preferably Eosin G.
  • distilled or demineralized water is used for the eosin solution.
  • Glycerol may preferably be added to the eosin solution, more preferably 1 to 25 ml of water per 100 ml of water are replaced by glycerol, very particularly preferably 5 to 20 ml, in particular 10 to 15 ml, particularly preferably 13 ml.
  • the eosin solution is preferably added per 100 ml total volume 0.01-1 g of sodium chloride, more preferably 0.05-0.5 g, most preferably 0.1 g.
  • the dyeing is preferably carried out by dipping the carrier in the second object ⁇ staining solution.
  • the slide is 0.2 - 5 min, particularly ⁇ vorzugt be 0.5 - 2.5 min, most preferably 0.75 - 2 minutes, in particular 1 min immersed in the second dye solution.
  • step S5 After staining the cell sample in step (S4) by means of a second staining solution, a second decolorization of the cell sample takes place in step S5.
  • the decolorization takes place by means of water, preferably not by rinsing with running water, more preferably by immersing the support material in a vessel with water.
  • any fresh water having a pH in the neutral range may be used, but tap water is preferably used.
  • the decolorization is preferably carried out over a period of 1 second to 5 minutes, particularly preferably 10 seconds to 2.5 minutes, very particularly preferably 20 seconds to 1 minute, in particular 30 seconds.
  • decolorizing 100 - 2000 ml of water verwen- det particularly preferably 200 - 1000 ml, especially before Trains t ⁇ 250-750 ml, in particular 500 ml.
  • step S3 water For decolorizing the ver ⁇ applied already in step S3 water can be used again.
  • the inventive method comprises, after Ver ⁇ method step S5 no fixation and dehydration steps with alcoholic solutions, particularly preferably, the he ⁇ invention process according to the process step S5 no fixation and dehydration steps, especially before ⁇ Trains t, the method according to the invention by the procedural ⁇ rens intimid on S5 no further steps more and ends.
  • the drive by the inventive Ver ⁇ stained cell samples are rungs- without additional drainage and fixing steps, stable over a longer period.
  • the cells remain stable even at higher temperatures around 40 ° C over a period of over two weeks.
  • the staining solution invention comprises hematoxylin, sodium umjodat, potassium aluminum sulfate dodecahydrate and Zitronen Text ⁇ re.
  • the staining solution according to the invention contains no chloral hydrate.
  • the staining solution preferably contains no further components than hematoxylin, sodium iodate, potassium aluminum sulfate dodecahydrate and citric acid.
  • the dyeing solution according to the invention is preferably prepared by dissolving the components in water, more preferably in distilled or demineralized water.
  • Hematoxylin is preferred in an amount of 0.01-10
  • % More preferably 0.01 - 5 wt.%, In particular 0.02 -. 2% by weight, particularly preferably 0.03 -. 1% by weight, particularly preferably 0.05 insbesonde ⁇ re -. 0.5 wt. %, in particular 0.1% by weight, in each case based on the total weight of the dyeing solution used.
  • Sodium iodate is preferably used in an amount of 0.001-2% by weight, more preferably 0.005-1.5% by weight, in particular 0.01-1% by weight, particularly preferably 0.015-0.5% by weight, particularly particularly preferably 0.018 0.1% by weight, in particular 0.02% by weight, based in each case on the total weight of the dyeing solution.
  • Potassium aluminum sulfate dodecahydrate is preferably used in an amount of 0.1-20% by weight, more preferably 1-10% by weight, in particular 2-8% by weight, particularly preferably 3-7% by weight, in particular particularly 4-6 % By weight, in particular 5% by weight, in each case based on the total weight of the dyeing solution.
  • Citric acid is preferably used in an amount of 0.01-5% by weight, more preferably 0.05-2.5% by weight, in particular 0.075-1% by weight, particularly preferably 0.08-0.5% by weight, in particular, particularly preferably 0.1% by weight, based in each case on the total weight of the dyeing solution.
  • the staining solution according to the invention can be used for staining of cell samples, for example for the diagnosis of carcinoma cells Kar ⁇ .
  • the staining solution according to the invention is preferably used in the process OF INVENTION ⁇ to the invention, particularly preferred as the first dye solution.
  • the coloring solution according to the invention surprisingly achieved even without the addition of chloral hydrate a coloring power according to the known prior art solutions Rheinlö ⁇ and is stable over a long period of time and can be reused.
  • Squamous cell epithelial cells obtained by brush biopsy were applied to a glass slide.
  • the cells were dried by drying the cells on the slide for 2 min at room temperature (20 ° C).
  • a hematoxylin (H) staining was Immersing the slide for 1 minute in 300 ml of the use solution ⁇ carried out according to Example. 1
  • the staining solution was placed in a dyeing trough (PMP) from Th. Geyer Gmbh, Renningen, Germany (measuring 80 mm ⁇ 100 mm ⁇ 70 mm (width ⁇ length ⁇ height).)
  • the slide was stained by means of a staining rack made by Th. Geyer Gmbh, with horizontal bottom for 20 slides, immersed in the solution.
  • an eosin (E) staining by immersing the slide for 1 minute in 300 ml of the eosin solution was carried out ge ⁇ Telss Example 2.
  • the staining solution was placed in a staining dish (PMP) the company Th. Geyer Gmbh, Germany (with dimensions of 80 mm x 100 mm x 70 mm (width x length x height) placed in front ⁇ .
  • the slide was by means of an accessory staining of Th. Geyer Gmbh, with horizontal bottom for 20 slides, immersed in the solution.
  • the comparison of the staining solution according to the invention according to Example 1 with hematoxylin-staining solutions of the prior art shows that the staining solution according to the invention comprises a dyeing ⁇ power according to the processes known from the prior art dyeing solutions and is stable over a long period of time and can be reused.
  • the comparison of the cell sample stained in Example 3 by means of the method according to the invention with a cell sample stained by a method according to the prior art shows that the method according to the invention significantly reduces the time required and the number of method steps, and nevertheless leads to stained cell samples. which are stable over a longer period.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
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  • Analytical Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Immunology (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention concerne un procédé permettant de colorer des échantillons cellulaires dans le cadre de la microscopie, qui comprend les étapes suivantes, en particulier mise en place (S1) de l'échantillon cellulaire sur un matériau support; coloration (S2) de l'échantillon cellulaire au moyen d'une première solution colorante; première décoloration (S3) de l'échantillon cellulaire; coloration (S4) de l'échantillon cellulaire au moyen d'une seconde solution colorante et seconde décoloration (S5) de l'échantillon cellulaire.
PCT/EP2014/051761 2013-02-11 2014-01-30 Procédé de coloration d'échantillons cellulaires dans le cadre de la miscroscopie WO2014122058A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE201310202183 DE102013202183A1 (de) 2013-02-11 2013-02-11 Verfahren zum Anfärben von Zellproben für die Mikroskopie
DE102013202183.6 2013-02-11

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WO2014122058A1 true WO2014122058A1 (fr) 2014-08-14

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040064A2 (fr) * 2001-11-07 2003-05-15 Bioview Ltd. Kits et methodes de preparation d'echantillons cellulaires optimises pour une coloration duale

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69031682T2 (de) * 1990-03-07 1998-03-05 Cytyc Corp Thionin Färbungstechnik
DE19709348C2 (de) * 1996-05-29 1999-07-01 Schubert Walter Dr Md Automatisches Multi-Epitop-Ligand-Kartierungsverfahren
DE10128552B4 (de) * 2001-06-13 2015-01-22 Jens Stockhausen Verfahren zur Zellanalyse und Zellanalyseeinrichtung
DE102004037860A1 (de) * 2004-08-04 2006-03-16 Friedrich-Alexander-Universität Erlangen-Nürnberg Turmormarker zur Diagnose von Karzinomen und/oder davon abstammender Metastasen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040064A2 (fr) * 2001-11-07 2003-05-15 Bioview Ltd. Kits et methodes de preparation d'echantillons cellulaires optimises pour une coloration duale

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BLASIAK B ET AL: "Detection of T2 changes in an early mouse brain tumor", MAGNETIC RESONANCE IMAGING, ELSEVIER SCIENCE, TARRYTOWN, NY, US, vol. 28, no. 6, 1 July 2010 (2010-07-01), pages 784 - 789, XP027093166, ISSN: 0730-725X, [retrieved on 20100415] *
GODWIN AVWIORO: "Histochemical uses of haematoxylin - a review", JPCS, vol. 1, 2011, XP055108696
GODWIN AVWIORO: "HISTOCHEMICAL USES OF HAEMATOXYLIN -A REVIEW", JPCS, vol. 1, 1 April 2011 (2011-04-01), pages 24 - 34, XP055108696 *
GULYAS B ET AL: "The norepinephrine transporter (NET) radioligand (S,S)-[<18>F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study", NEUROCHEMISTRY INTERNATIONAL, PERGAMON PRESS, OXFORD, GB, vol. 56, no. 6-7, 1 May 2010 (2010-05-01), pages 789 - 798, XP027020050, ISSN: 0197-0186, [retrieved on 20100306] *
RÜEGG; MEINEN: "Histopathology in Hematoxylin & Eosin stained muscle sections", TREAT-NMD / CURE CMD VERÖFFENTLICHT

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