DE102013202183A1 - Method for staining cell samples for microscopy - Google Patents

Abstract

Method for staining cell samples for microscopy with several steps, namely applying (S1) the cell sample to a carrier material; Staining (S2) the cell sample using a first staining solution; First destaining (S3) of the cell sample; (Staining (S4) the cell sample using a second staining solution; and second destaining (S5) the cell sample.

Description

The present invention relates to a method for 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 (HE staining) is a standard staining technique for staining cell preparations for microscopy. It is used to distinguish different tissue structures in the microscopic picture from two different single stains and is one of the most widely used routine staining methods for morphological studies. With the help 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.

In the case of HE staining, the cell samples are first stained with hematoxylin after fixation on a microscope slide (H staining). Hematoxylin stains all acidic or basophilic structures blue, in particular cell nuclei with the deoxyribonucleic acid (DNA) contained therein and the ribosome-enriched 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 done by rinsing in fresh water of neutral pH, e.g. Tap water causes.

This is followed by eosin staining (E staining) with an eosin solution. Eosin stains all acidophilic or basic (eosinophilic) structures red, which mainly includes the cytoplasmic proteins. Eosin G (also called Eosin Y) or Eosin B is used.

By further rinsing over alcohol solutions in ascending concentration up to absolute alcohol, the water is displaced from the tissue section. Finally, the dewatered cut is clarified in an organic solvent such as xylene.

• (1) Fixierung der zu untersuchenden Zellen auf dem Objektträger mittels 1–2 Spraystöße eines alkoholischen Fixiersprays
• (2) Hämatoxylin-Färbung (H-Färbung) 1–2 min
• (3) Entfärbung 3 min, unter laufenden warmen Wasser
• (4) Entfärbung 2 min, unter laufenden kalten Wasser
• (5) Entfärbung 1 min, mit destilliertem oder demineralisiertem Wasser
• (6) Eosin-Färbung (E-Färbung) 2–3 min
• (7) Entwässerung / Fixierung mit 70 % Ethanol
• (8) Entwässerung / Fixierung 3 min mit 90 % Ethanol
• (9) Entwässerung / Fixierung 3 min mit 96 % Ethanol
• (10) Entwässerung / Fixierung 2 mal 5 min mit 100 % Ethanol
• (11) Entwässerung / Fixierung 2 mal 10 min mit 100% Xylol

A standard protocol for performing HE staining involves the following steps:

• (1) Fixation of the cells to be examined on the slide by means of 1-2 sprays of an alcoholic fixative spray
• (2) Hematoxylin staining (H staining) 1-2 min
• (3) discoloring for 3 min, under running warm water
• (4) decolorization 2 min, under running cold water
• (5) decolorization for 1 min, with distilled or demineralized water
• (6) Eosin staining (E staining) 2-3 min
• (7) drainage / fixation with 70% ethanol
• (8) Drainage / fixation for 3 minutes with 90% ethanol
• (9) Drainage / fixation for 3 minutes with 96% ethanol
• (10) drainage / fixation 2 times 5 min with 100% ethanol
• (11) drainage / fixation 2 times 10 min. With 100% xylene

The total duration of the process is over 45 minutes and in the decolorization under running water in step (3) and (4) 3 to 12 liters of water are used.

The prior art discloses various staining solutions for use in HE staining.

So are off Godwin Avwioro, "Histochemical uses of haematoxylin - a review", JPCS Vol (1), 2011 ; and standard method (SOP) Rüegg and Mine, "Histopathology in Hematoxylin & Eosin stained muscle sections", SOP_ID: MDC1A_M.1.2.004, published in TREAT-NMD / CURE CMD, HE staining solutions and methods ,

Hematoxylin staining

For hematoxylin staining, for example, acid hematoxylin according to Ehrlich or acidic hemalaun according to Mayer is used.

Hämalaun to Mayer:

The staining solution contains: hematoxylin, sodium iodate, potassium alum, chloral hydrate, hydrochloric acid 0.1%, ammonia solution, sodium bicarbonate and citric acid.

Hematoxylin according to Ehrlich:

The staining solution contains: hematoxylin, isopropanol 96%, glycerol, potassium alum, acetic acid, sodium iodate, sodium bicarbonate and ammonia solution

eosin staining

eosin:

The eosin solution is prepared by dissolving Eosin G or B in distilled or demineralized water.

The staining protocols known from the prior art have the disadvantage that they use staining solutions which contain toxic / hazardous substances and have several fixing and dehydration steps with toxic and combustible alcohols. Due to the many different steps to be performed, these protocols are time-consuming and complex. In addition, when dyeing is carried out, there is a) a health risk for medical personnel due to the health-endangering solutions used and b) a need for appropriate technical and personal precautions (e.g., fume hood, chemical storage, protective clothing, gloves, goggles).

To facilitate the use of microscopic diagnostic procedures, e.g. For cancer screening, even for less well-trained medical staff in a poorly equipped environment, for example, in remote areas that have a poor medical infrastructure, harnessing simple protocols are necessary, which have a low hazard potential for users by avoiding hazardous chemicals , do not cause any disposal problems and are also less time-consuming and complex. 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 staining solution according to claim 12.

• (a) Aufbringen der Zellprobe auf ein Trägermaterial
• (b) Färben der Zellprobe mittels einer ersten Färbelösung;
• (c) Entfärben der Zellprobe;
• (d) Färben der Zellprobe mittels einer zweiten Färbelösung; und
• (e) Entfärben der Zellprobe;

wobei das Verfahren nach dem Verfahrensschritt (e) keine Fixier- und Entwässerungsschritte mit alkoholischen Lösungen aufweist. The method according to the invention for staining cell samples for microscopy comprises the following steps:

• (a) applying the cell sample to a substrate
• (b) staining the cell sample by means of a first staining solution;
• (c) decolorizing the cell sample;
• (d) staining the cell sample by means of a second staining solution; and
• (e) decolorizing the cell sample;

the process after process step (e) having no fixing and dewatering steps with alcoholic solutions.

The staining solution of the present invention contains hematoxylin, sodium iodate, potassium aluminum sulfate dodecahydrate and citric acid. 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 remain stable over an extended period even without additional dewatering and fixation steps, after the second decolorization step.

In addition, a dyeing solution could be provided which manages without the addition of harmful chloral hydrate and surprisingly has a dyeing capacity according to the known from the prior art staining solutions and is stable over a longer period and can be reused.

The inventive method minimizes effort, time and risk potential and the need for technical and personal protective equipment. In addition, the process poses no disposal problems because no hazardous ingredients are used. It is therefore also applicable to less well trained medical personnel.

Inventive method

The inventive method comprises a plurality of method steps whose advantageous embodiments are described below with reference to the accompanying figure.

(a) applying the cell sample to a substrate

First, the cell sample to be examined is applied to a carrier material in step S1. Suitable carrier material is any carrier material suitable for microscopy. The person skilled in such carrier materials are familiar. Preferably, a transparent carrier material is used.

Preferably, a slide is used as the carrier material. As a slide any slide as it is familiar to those skilled in microscopy can be used. The slide can consist of any material, preferably it consists of a plastic or glass, more preferably of glass.

As 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. It is preferably a cell sample of the oral mucosa, the mucous membrane of the cervix uteri or the mucous membrane of the vulva, particularly preferably a sample of the oral mucosa, in particular the squamous epithelial cells of the oral mucosa.

Preferably, 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 0.5-10 minutes, more preferably 1-5 minutes, most preferably 1.5-2.5 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.

Preferably, an additional fixation of the cells to be examined on the slide by means of commercially available fixing solutions. Preferably, an alcoholic fixing solution is used, particularly preferably a fixing solution containing ethanol. For example, the fixation with 1-2 spray bursts of Merckofix ^® fixation sprays can be done from Merck, Darmstadt, Germany.

(b) staining the cell sample by means of a first staining solution;

The cell sample applied to the carrier material in step (S1) is then stained in step 2 by means of a first staining solution. For dyeing, any commercial dyeing solution known to those skilled in the art may be used to stain cells. Preferably, an aqueous solution containing hematoxylin is used, as is commonly used by the person skilled in the art in HE staining, and it is described in the prior art, more preferably the hematoxylin solution contains no chloral hydrate, most preferably an aqueous staining solution according to the invention is used, such as it is disclosed in the present specification.

The dyeing is preferably carried out by immersing the slide in the dyeing solution.

Preferably, the slide is immersed in the first staining solution for 0.2-5 minutes, more preferably 0.5-2.5 minutes, most preferably 0.75-2 minutes, especially 1 minute.

(c) First decolorization of the cell sample

After staining the cell sample by means of a first staining solution, in step S3, a first decolorization of the stained in step S2 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.

For decolorizing any fresh water with a pH in the neutral range can be used, preferably tap water is used.

If discoloring by means of water is not sufficient, it is optionally also possible to decolorize with an aqueous sodium bicarbonate solution.

The aqueous sodium bicarbonate solution used is a solution of sodium bicarbonate in water, preferably a solution containing 0.01-1% by weight of sodium bicarbonate, particularly preferably 0.05-0.5% by weight, very particularly preferably 0.075-0.25% by weight .%, In particular with 0.1 wt.% Sodium bicarbonate, based on the total weight of the sodium bicarbonate solution.

The decolorization preferably takes place over a period of 1 second (sec) - 5 min, particularly preferably 10 sec-2.5 min, very particularly preferably 20 sec-1 min, in particular 30 sec.

For decolorizing, 100-2000 ml of water are preferably used, particularly preferably 200-1000 ml, very particularly preferably 250-750 ml, in particular 500 ml.

(D) staining the cell sample by means of a second staining solution

The cell sample decolorized in step S3 is stained by means of a second staining solution in step S4. For dyeing, any commercial dyeing solution known to those skilled in the art may be used to stain cells. Preferred is a aqueous solution containing eosin used, as commonly used in the HE staining, more preferably an eosin solution containing 0.05-1 g eosin, most preferably 0.1-0.5 g eosin, especially 0.3 g eosin in 100 ml of water.

Eosin G (also called Eosin Y) or Eosin B is used, preferably Eosin G.

Preferably, distilled or demineralized water is used for the eosin solution.

Glycerol may preferably be added to the eosin solution, particularly preferably 1-25 ml of water per 100 ml of water are replaced by glycerol, very particularly preferably 5-20 ml, in particular 10-15 ml, particularly preferably 13 ml.

The eosin solution is preferably added per 100 ml total volume 0.01-1 g sodium chloride, more preferably 0.05-0.5 g, most preferably 0.1 g.

The dyeing is preferably carried out by immersing the slide in the second dyeing solution.

Preferably, the slide is immersed in the second staining solution for 0.2-5 minutes, more preferably 0.5-2.5 minutes, most preferably 0.75-2 minutes, especially 1 minute.

(e) second decolorization of the cell sample

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.

For decolorizing, any fresh water having a pH in the neutral range may be used, but tap water is preferably used.

Preferably, the decolorization takes place over a period of 1 sec-5 min, more preferably 10 sec-2.5 min, very particularly preferably 20 sec-1 min, in particular 30 sec.

For decolorizing, 100-2000 ml of water are preferably used, particularly preferably 200-1000 ml, very particularly preferably 250-750 ml, in particular 500 ml.

For destaining, the water already used in step S3 can be used again.

Preferably, the method according to the invention does not have any fixing and dewatering steps with alcoholic solutions after process step S5. The process according to the invention particularly preferably does not have any fixing and dewatering steps after process step S5. The process according to the invention after process step S5 most preferably has no further process steps more on and is finished.

Surprisingly, the cell samples stained by the method according to the invention are stable over an extended period of time even without additional dewatering and fixation steps. Thus, the cells remain stable even at higher temperatures around 40 ° C over a period of over two weeks.

Dyeing solution according to the invention

The staining solution of the present invention contains hematoxylin, sodium iodate, potassium aluminum sulfate dodecahydrate and citric acid. The staining solution according to the invention contains no chloral hydrate.

Preferably, the staining solution contains no other 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 preferably used in an amount of 0.01-10% by weight, particularly preferably 0.01-5% by weight, in particular 0.02-2% by weight, particularly preferably 0.03-1% by weight, especially especially preferably 0.05-0.5% by weight, in particular 0.1% by weight, in each case based on the total weight of the dyeing solution.

Sodium iodate is preferably used in an amount of 0.001-2% by weight, particularly 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 wt.%, In particular 0.02 wt.%, Each based on the total weight of the dyeing solution used.

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, particularly particularly preferably 4-6% by weight, in particular 5% by weight, in each case based on the total weight of the dyeing solution used.

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 cell samples, for example for the diagnosis of carcinoma cells.

The dyeing solution according to the invention is preferably used in the process according to the invention, particularly preferably as a first dyeing solution.

The staining solution according to the invention surprisingly reaches a staining capacity according to the staining solutions known from the prior art even without the addition of chloral hydrate and is stable over a longer period of time and can be reused.

Examples

example 1

Hematoxylin solution

Required chemicals:

Hematoxylin, sodium iodate, potassium aluminum sulfate dodecahydrate, citric acid, eosin

• Stock solution:

1 g of hematoxylin is dissolved in 1 liter of distilled water and 0.2 g of sodium iodate and 50 g of sodium aluminum sulfate dodecahydrate are added.

• working solution:

In 100 ml of the stock solution, 0.1 g of citric acid are dissolved.

Example 2

• eosin solution:

0.3 g of eosin are dissolved in 87 ml of distilled water and then 13 ml of glycerol and 0.1 g of NaCl are added.

Example 3

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). Subsequently, hematoxylin (H) staining was performed by immersing the slide for 1 minute in 300 ml of the use solution 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). Geyer Gmbh, with horizontal bottom for 20 slides, immersed in the solution.

This was followed by decolorization by immersing the slide in a glass jar with 500 ml of tap water for 30 seconds.

Subsequently, eosin (E) staining was performed by immersing the slide for 1 minute in 300 ml of the eosin solution according to Example 2. The dyeing solution was placed in a dyeing trough (PMP) from Th. Geyer Gmbh, Germany (measuring 80 mm.times.100 mm.times.70 mm (width.times.length.times.height).) The slide was prepared by means of a dyeing pad from Th. Geyer Gmbh , with horizontal bottom for 20 slides, immersed in the solution.

This was followed by a second decolorization by immersing the slide for 30 seconds in the glass vessel with 500 ml of tap water, which was already used for the first decolorization.

The comparison of the staining solution according to the invention with hematoxylin staining solutions of the prior art shows that the staining solution according to the invention has a staining capacity according to the staining solutions known from the prior art and is stable over a longer period 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.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Godwin Avwioro, "Histochemical uses of haematoxylin - a review", JPCS Vol (1), 2011 [0012]

Claims (15)

A method of staining cell samples (biopsy samples) for microscopy comprising the steps of: (a) applying (S1) the cell sample to a support material; (b) staining (S2) the cell sample by means of a first staining solution; (c) first decolorization (S3) of the cell sample; (d) staining (S4) the cell sample by means of a second staining solution; and (e) second decolorization (S5) of the cell sample; characterized in that the method after the process step S5 has no fixing and dewatering steps with alcoholic solutions. A method according to claim 1, characterized in that the method after the fifth process step (S5) has no fixing and dewatering steps. A method according to claim 1 or 2, characterized in that the fixation of the cell samples is carried out on the carrier material by means of an alcoholic fixing solution. A method according to any one of claims 1 to 3, characterized in that the method has no decolorization steps under running water. A method according to any one of claims 1 to 4, characterized in that the cell samples are surface epithelial cells of the oral mucosa. Method according to one of claims 1 to 5, characterized in that the cell samples were obtained by means of brush biopsy. A method according to any one of claims 1 to 6, characterized in that a hematoxylin-containing staining solution is used as the first staining solution. A method according to any one of claims 1 to 7, characterized in that the first staining solution does not contain chloral hydrate. Method according to one of claims 1 to 8, characterized in that a solution according to one of claims 12 to 14 is used as the first coloring solution. A method according to any one of claims 1 to 9, characterized in that a eosin-containing staining solution is used as the second staining solution. Method according to one of claims 1 to 10, for the in vitro detection of carcinoma cells. An aqueous staining solution for use in a method according to any one of claims 1 to 11, comprising hematoxylin, sodium iodate, potassium aluminum sulfate dodecahydrate and citric acid, characterized in that the staining solution does not contain chloral hydrate. An aqueous staining solution according to claim 12, wherein the staining solution contains 0.01-10 wt% hematoxylin, 0.001-2 wt% sodium iodate, 0.1-20 wt% potassium aluminum sulfate dodecahydrate, and 0.01-5 wt% citric acid , An aqueous staining solution according to claim 12 or 13, wherein the staining solution is used as a first staining solution in a method according to any one of claims 1-11. Use of the staining solution according to one of claims 11 to 13 for the staining of cell samples, preferably of surface epithelial cells.

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