Solution for preliminary processing for immunohistochemical staining and condensed solution

FIELD: chemistry.

SUBSTANCE: described are: solution for preliminary processing for immunohistochemical staining, which elutes paraffin-containing mounting medium from microscope slide with tissue sample, embedded into medium, and extracts antigenicity of tissue sample, and which can be used three or more times, and solution concentrate for preliminary processing for immunohistochemical staining, which provides possibility of easy obtaining of solution for preliminary processing. Solution for preliminary processing for immunohistochemical staining contains antigen-extracting agent, certain non-ionic surface-active substances in specified range and cyclodextrin or its derivative in certain amount, with not less than 80% of water constituting the remaining part. Composition of antigen-extracting agent is such that pH of solution for preliminary processing is in specified range, and content of cyclodextrin or its derivative represents specified amount.

EFFECT: obtaining solutions for preliminary processing for immunohistochemical staining.

21 cl, 5 tbl, 11 ex

 

AREA of TECHNOLOGY

The present invention relates to a solution for pre-treatment and concentrate for immunohistochemical staining, the solution ensures the possibility of performing immunohistochemical staining for a short time and with less labor, and immunohistochemical staining includes: elution containing paraffin wax potting medium with the slide with the tissue sample as the antigen, encapsulated in a medium; antigen retrieval; flushing; interaction with the antibody; and staining. In particular, the present invention relates to a solution for pre-treatment and concentrate for immunohistochemical staining, and the solution functions as for elution with the slide with the tissue sample, bathed in a medium containing paraffin wax potting medium for immunohistochemical staining, and to retrieve antigenicity, allowing sufficient intensity of staining for subsequent immunohistochemical staining, and that supports-eluted priming Wednesday, dispergirovannoyj in it, even after its first use, and can be used three or more times.

The LEVEL of TECHNOLOGY

When pathomorphological study pretreatment d�I dewaxing and antigen retrieval for immunohistochemical staining, the slides with the tissue sample, usually includes, as described in non-patent publication 1: the first stage dewaxing by passing fixed with formalin, embedded in paraffin, cut tissue through the layer of organic solvent, such as xylene, benzene or toluene, 3 times for 3 minutes each; the second stage of rehydration the coverslip and tissue sample by passing through an amphiphilic solution, such as ethanol, 4 times for 3 minutes each; and the third stage of extraction of antigen from registrationpage sample of tissue by heat treatment registrationpage tissue sample immersed in the solution for extraction of the antigen such as citrate buffer or a solution of Tris-EDTA.

The organic solvents used in the first stage, pre-processing, such as xylene, benzene or toluene, are highly toxic and volatile that requires the use of equipment for exhaust ventilation such as a fume cupboard, and has adverse effects on the environment, which employs technical staff.

As a substitute adverse organic solvents, commercially available high security agents for dewaxing, such as Hemo-De (trade name, produced by FALMA), an organic solvent, extracted and purified from citrus peels, Clear-Pls and Hemo-Clear (both trade names, manufactured by FALMA) using aliphatic hydrocarbons (alkanes) and Tissue Clear (trade name, manufactured by SACURA FINETEK JAPAN CO., LTD).

Instead of using high deparaffinizing agents, more desirable convenient methods, where the three stages of pre-processing are performed simultaneously in the same solution, and some suggestions were made.

For example, in patent document 1, a method of pre-treatment, where the dewaxing and antigen retrieval are performed simultaneously by heating to a temperature above the melting point of paraffin, embedded in paraffin tissue sample in a solution containing paraffin solvent organic solvent selected from aromatic hydrocarbon, terpene, or isoparaffinic hydrocarbon, a polar organic solvent to extract the antigen component and a surfactant. In patent publication 1 also stated that the removal of residual surfactants in washing stage following the pre-processing method, the washing solution may contain a cyclodextrin that binds to a surface-active substance.

On the other hand, in patent publication 1 in example 7 shows that when the above solution, the use�creating pre-processing, used consistently, the residual paraffin-eluted in the solution after pre-treatment, coupled with the next slide, so this solution is not suitable for further use.

In addition, when the solution composition described in patent publication 1, prepared to obtain alkaline pH, immunohistochemical staining after pretreatment, the glass slide with coupled to the tissue sample has a tendency to repel staining reagent in parts, free from the tissue sample, which leads to uneven dyeing.

In patent publications 2 and 3 indicate that the dewaxing and antigen retrieval can be performed simultaneously using a solution containing various buffers, surfactants, ethylene glycol, and the like, as well as in the pre-processing method described in patent publication 1.

However, in these publications, the consistent use of a solution for pre-treatment also is not provided, similarly to the solution of the patent publication 1, and there is no description of this technology, which is used for dispersing paraffin-eluted in the solution, or the like. Thus, it is likely that solutions for pretreatment, description�record in these publications, the sequential application also have the same problems as those mentioned in patent publication 1.

Incidentally, some solutions for pretreatment for immunohistochemical staining, which simultaneously perform dewaxing and retrieval of antigens, are already on sale, such as the solutions disclosed in the above patent publications, including the Trilogy solution for a single application (trade name, manufactured by a company Cell Marque Corporation), or a solution of Target Retrieval Solution pH9 (3-in-1) (trade name, manufactured by DAKO) for three consecutive application within one week.

The composition of these commercially available products are unknown, but they do not contain cyclodextrin. Furthermore, in all such products, hydrophobic paraffin-eluted in the solution for pre-treatment during the application, often floats on the surface of the solution.

Such insoluble paraffin on the surface of the solution adheres to the slide with the tissue sample when it is removed from the solution, so it requires more washing stages before subsequent staining, which complicates the operation. Otherwise, insoluble paraffin adversely affect subsequent immunohistochemical staining. In addition,when the dyeing is performed in the automatic device for immunohistochemical staining, insoluble paraffin wax can have an adverse impact on the device for staining.

The publication of the level of technology

Patent publication

Patent publication 1: JP-2001-505297-A.

Patent publication 2: JP-2003-526086-A.

Patent publication 3: U.S. patent No. 6649368-B1.

Non-patent publication

Non-patent document 1: “Koso Koutai Hou” (Imminoenzymatic technique), Revised 4thEdition, Watanabe and Nakane, Kakusai Kikaku (2002).

A BRIEF SUMMARY of the INVENTION

Issues to be addressed by the invention

The aim of the present invention to provide a solution for pretreatment for immunohistochemical staining, which operates immunohistochemical staining for elution containing paraffin wax potting medium with the slide with the tissue sample in the form of a tissue section, coupled to the slide, and filled into the environment, and to extract the antigens from the tissue sample, which provides a sufficient intensity of staining for subsequent immunohistochemical staining, which supports-eluted priming Wednesday dispersed in it after its first use, which can be used three or more times consecutively and which is characterized by high safety.

Another purpose of�of bretania is the obtaining of a solution for pretreatment for immunohistochemical staining, which solves the problems described above, which, after staining followed by treatment with a solution containing paraffin wax potting medium with the slide with the tissue sample in the form of a tissue section, coupled to the slide and filled Wednesday, and extraction of antigens from the tissue sample, inhibits repulsion of coloring reagent on the part of the slide without tissue sample to prevent through this uneven staining.

Another objective of the present invention is the preparation of a concentrate of solution for pretreatment for immunohistochemical staining, from which we can easily obtain the solution for pretreatment for immunohistochemical staining, achieving the above objectives.

Means for solving the problems

The applicants have conducted intensive studies to achieve the above mentioned objectives. First, researchers have focused on obtaining solutions for pretreatment for immunohistochemical staining, which for security reasons does not contain conventional organic solvents, such as xylene, benzene or toluene, and which is mainly composed of water based on the environment and other factors. In this regard, the researchers studied various surfactants as comp�component solution for pre-treatment, providing and effects sufficient elution containing paraffin wax potting medium with the slide with the tissue sample embedded in the environment, and inhibition of swimming-eluted potting medium on the surface of the solution for the dispersion medium in the solution in order to enable subsequent use of the solution for pre-processing.

As a result, it was revealed that the two above-mentioned effect cannot be obtained simultaneously by only one surface-active substance that these two effects can be obtained in some degree by the use of two specific surfactants, but if the resulting alkaline solution for pre-treatment, and it is used consistently, three times or more, it is likely the repulsion of the coloring agent on the part of the slide without a sample, which leads to uneven dyeing of the stage of painting after using consistently used solution for pre-processing.

Then, a search was undertaken of the components, which was thought to solve such problems, by combining two specific surfactants with various polymer compounds.

In addition, to confirm the ability of a single solution is simultaneously eluted priming with�food and extract antigens and exert little effect on the intensity of staining at the stage of staining, to solve the above problems, we investigated various combinations of components with conventional agents for the extraction of antigens.

In addition, researchers have focused on the use of cyclodextrin and its derivatives, which were not used in the solution for pretreatment for immunohistochemical staining, but, as described in patent publication 1, is able to remove residual surfactants in their content in the washing solution used solution after pre-processing.

Eventually, through experimentation it was found that when the cyclodextrin or its derivative is added in an amount of 2 wt%. in the solution for pre-treatment along with the two above defined surfactant agent and to retrieve the antigen, the action themselves surfactants ingibirovalos, probably due to binding of surface-active substances, as described in patent publication 1, the dispersibility is markedly deteriorated, and the problem is insoluble paraffin arose again, and should have been addressed. In addition, the intensity of staining at the stage of staining was significantly decreased, and the expected effect cannot be achieved. Unexpectedly, however, it was confirmed that cyclo�extrin or its derivative in a specific content can prevent the inhibition of the action of surface-active substances, can inhibit the deterioration of staining intensity and can solve the above various problems.

In addition, it was found that when the amphiphilic solution was added to the above composition, the colour intensity is further increased, and it was possible to improve the inhibition effect of repulsion of coloring reagent, which otherwise occurs when the solution for pre-treatment is alkaline.

In accordance with the present invention the resulting solution for pretreatment for immunohistochemical staining for elution containing paraffin priming solution with the slide with the tissue sample, filled in the specified environment, and to retrieve the antigenicity of the specified tissue sample, wherein said solution for pre-treatment can be used three or more times, wherein said solution for pre-treatment agent contains to retrieve antigens, non-ionic surfactant polyoxyethylene-alkylphenolate and cyclodextrin or its derivative, and the remaining amount is not less than 80% of the mass. water, where the contents of the specified agent for the extraction of antigens is such that the specified pH of the solution for pre-treatment ranging from 5.0 to 10.0, and contains�R specified cyclodextrin or its derivative is from 0.01 to 1.0 wt.%, and where the specified solution for pre-treatment does not necessarily contain amphiphilic solution.

In accordance with the present invention is also the resulting concentrate of solution for pretreatment for immunohistochemical staining, containing less water than the above solution for pretreatment for immunohistochemical staining, where the specified solution for pretreatment is designed for use as a solution for pretreatment for immunohistochemical staining at a given pH by adding thereto a predetermined amount of water when used.

In accordance with the present invention, in addition, received the above concentrate of solution for pretreatment for immunohistochemical staining, where the specified specified pH ranges from 5.0 to 7.0, and where the specified concentrate of solution for pre-treatment is delivered in one part.

In accordance with the present invention is also obtained from the above concentrate of solution for pretreatment for immunohistochemical staining, where the specified concentrate of solution for pre-treatment comes in two parts and consists of a first concentrate solution containing the agent to retrieve the antigens, and the WTO�CSOs concentrate solution containing non-ionic surfactant polyoxyethylene-alkylphenolate, nonionic surfactant, polyoxyethylenesorbitan, cyclodextrin or its derivative and, optionally, an amphiphilic solution, and where the specified concentrate of solution for pre-treatment is intended for use by mixing the specified first concentrate solution, the specified second concentrate solution and a predetermined amount of water to install a specified predetermined pH from 8.0 to 10.0.

Solution for pretreatment for immunohistochemical staining according to the present invention contains the agent to retrieve antigens, two specific surfactants and certain content of the cyclodextrin or its derivative as the rest of not less than 80% of the mass. water. Thus, the solution is functioning and for elution containing paraffin wax potting medium used in the immunohistochemical staining, the slides with the tissue sample embedded in the environment, and to retrieve the antigenicity of the tissue sample; provides sufficient opportunity of staining intensity for subsequent immunohistochemical staining; supports dispergirovannoyj in it-eluted priming environment even after the first use for p�of edatrexate the insoluble wax on the surface of the solution after use; and can be used three or more times consecutively. In addition, the present solution for pre-treatment, not containing organic solvents, such as xylene, has high safety and environmental advantages. In addition, even when received in the form of an alkaline solution, solution for pretreatment for immunohistochemical staining according to the present invention still inhibits repulsion of coloring reagent.

Therefore, by use of a solution for pretreatment for immunohistochemical staining according to the present invention, immunohistochemical staining can be performed in a short period of time and with lower labor costs.

Solution for pretreatment for immunohistochemical staining according to the present invention, when it further comprises an amphiphilic solution, provides the possibility of an even stronger intensity of staining and ensures the best effect of inhibition of repulsion staining reagent.

Concentrate of solution for pretreatment for immunohistochemical staining according to the present invention containing less water than the solution for pretreatment for immunogen�chimicheskogo staining in accordance with the present invention, can easily be used as a solution for pretreatment for immunohistochemical staining at a given pH by adding a predetermined amount of water when used.

Embodiments of the INVENTION

Now the present invention will be explained in detail.

Solution for pretreatment for immunohistochemical staining and concentrate of this solution in accordance with the present invention are intended for so-called immunohistochemical staining, in which a specific substance (antigen) present in normal or neoplastic tissues or cells of living organisms, is detected based on the specific reactions of antigen-antibody for pathological diagnosis.

Tissue samples subject to immunohistochemical staining, usually fixed with formalin, alcohol and similar substances, and poured in the priming medium containing paraffin for long term storage. Get a slice of this fixed with formalin, embedded in paraffin block and interlock them with the slide for immunohistochemical staining, dewaxing and retrieval of antigens, which should be carried out depending on the type of primary antigen that is to be used.

General procedures for deparaffinization� and retrieve antigens include three stages, i.e. dewaxing, for example, fixed with formalin, embedded in paraffin tissue sample, as described above, an organic solution, such as xylene, benzene or toluene, rehydrated deparaffinizing tissue sample amphiphilic solution, such as ethanol, and extraction of antigens present in negidrirovannah deparaffinizing the tissue sample. After these stages is carried out immunohistochemical staining, including washing and staining.

The present invention provides the ability to simultaneously conduct a three-stage pre-treatment in the same solution, i.e. the solution for pretreatment for immunohistochemical staining. Solutions for pre-treatment used so that previously were offered and there was a sale, but most of them were intended for a single use, and few of them can be used three or more times consecutively, for example, consistently about three to four times. The present invention relates to the preparation of such a solution for pre-treatment and concentrate that can be used three times or more, preferably, three to five times.

Solution for pre-treatment in accordance with the present invention contains not less than 80 wt.%, before�occhialino, not less than 85 wt.%, more preferably, not less than 90% of the mass. water does not contain commonly used organic solvents, such as xylene, because of concerns about security, and contains the extraction of the antigens of the agent, two specific non-ionic surfactant, and a cyclodextrin or its derivative. The water preferably is a deionized water. In this case, the water content includes any moisture content of the other components.

Agent to retrieve antigens not specifically limited, as long as it is conventional and provides the ability to retrieve antigenicity by heat treatment, and can be suitably selected agents capable of regulating the pH, depending on the tissue to be treated. Examples of the agent to retrieve the antigens may include various buffers such as citrate buffers, Tris buffers, SSC (saline-sodium-citrate) buffers and these compounds are mixed with a chelating agent such as EDTA.

The content of the extraction of the antigens of the agent can be appropriately selected depending on be treated tissue so that the pH of the solution for pre-treatment of the present invention is within the range from 5.0 to 10.0, preferably from 6.0 to 9.0.

Two specific surface-AK�active substances are non-ionic surface-active substance polyoxyethylene (hereinafter abbreviated as POE)-alkylphenolate and nonionic surfactant POE-sorbitan.

In the solution for pre-treatment of the present invention, the nonionic surfactant POE-alkylphenolate mainly involved in dewaxing, and may preferably be, for example, at least one of NP-40, Triton X-100, Triton X-104 or IGEPAL CA-630.

The content of nonionic surfactant POE alkylphenolate can be correspondingly selected based on the above functions, and is usually from 0.01 to 1.0 wt.%, preferably, from 0.01 to 0.5 wt.%, more preferably, from 0.05 to 0.3 wt%. At too low concentration of the nonionic surfactant POE alkylphenolate, the effect of dewaxing may be small, while when too high content may be difficult to maintain the set temperature when heated to extract the antigens that will be discussed below.

In the solution for pre-treatment of the present invention non-ionic surfactant POE-sorbitan mainly performs the function of maintaining the dispersion in it-eluted paraffin for three or more times of use, through interaction with cyclodextrin or its derivatives, which will be discussed below. Nonionic surfactant POE-sorbitan can represent at least one of T�ina 20, Tween 40 or Tween 80.

The content of nonionic surfactant POE-sorbitan can be correspondingly selected based on the above basic functions, and is usually from 0.3 to 3.0 wt.%, preferably, from 0.5 to 1.5 wt.%, more preferably, from 0.5 to 1.0 wt%. At too low concentration of the nonionic surfactant POE-sorbitan can be broken dispersibility of the wax, if used consistently, whereas a too high content may be difficult to maintain the set temperature when heated to extract the antigens that will be discussed below.

In the solution for pre-treatment of the present invention the cyclodextrin or its derivative is mainly involved in the inhibition of repulsion staining reagent, the specimen slide on the stage of staining after pre-treatment, as well as in maintenance-eluted wax dispersed in the solution for pre-treatment. The cyclodextrin or its derivative may be at least one of α-, β - and γ-cyclodextrin, which are natural products, in particular, with improved water solubility by adding a hydroxyl group, etc., Preferred examples may include hydroxyacylglutathione, so�e as hydroxyalkyl-α-cyclodextrin, hydroxyalkyl-β-cyclodextrin and hydroxyalkyl-γ-cyclodextrin, and particularly preferred hydroxyalkyl-β-cyclodextrin. Alkyl may be, for example, ethyl or propyl.

The content of the cyclodextrin or its derivative is from 0.01 to 1.0 wt.%, preferably, from 0.05 to 0.5 wt%, more preferably, from 0.05 to 0.3 wt%. When the content of the cyclodextrin or its derivative is less than 0.01 wt%. it is impossible to achieve the effect of inhibition of repulsion staining reagent object glass. On the other hand, when the content of more than 1.0 wt%. occurs adverse effects on the above effect of maintaining-eluted wax dispersed in the solution for pre-treatment, and inhibited the effect of surface-active substances. Also inhibited the retrieval of antigens and it is impossible to achieve a sufficient intensity of staining. Thus, it is necessary to closely monitor the upper limit of the content of the cyclodextrin or its derivative, as can also be inhibited desirable effect of the present invention.

Solution for pre-treatment of the present invention may optionally contain other components, in addition to the above main components, with the aim of improving the desired effects or the existence of other RMS�Chow without inhibiting the desired effects of the present invention.

As such other components, amphiphilic solution may preferably be contained to ensure improved intensity of staining staining after pre-processing and to further improve the effect of inhibition of repulsion staining reagent from the slides combination with cyclodextrin or its derivatives.

Amphiphilic solution is not specifically limited, can be expected to improve the above effects, and may preferably be, for example, at least one of ethylene glycol, propylene glycol or 1,3-butyleneglycol. Among them, ethylene glycol is of particular advantage to use three or more consecutive times, because, in addition to improving the above effects, it has the effect of inhibiting the re-coupling with the slide of small-eluted particles of wax dispersed in the solution for pre-processing.

The content of the amphiphilic solution, if it is part of the solution for pre-treatment in accordance with the present invention may accordingly be selected according to its effect and can usually be from 1.0 to 5.0 wt.%, preferably, from 2.0 to 4.0 wt%. At too low concentration of the amphiphilic solution expected Ulu�tion effects may not be achieved, whereas at too high content may adversely suppressed the effect of inhibition of insoluble paraffin, which is one of the effects of solution for pre-treatment of the present invention.

Other components may optionally include, in addition to the amphiphilic solution, at least one of preservatives and disinfectants such as sodium azide, thimerosal, gentamicin, ProClin (trade name, manufactured by SUPELCO), p-hydroxybenzoate (paraben), benzalkonium chloride and ticlopidine chloride. The content of such other components may be suitably selected based on their functions until inhibited the effects of the present invention.

Concentrate of solution for pretreatment for immunohistochemical staining according to the present invention is suitable for obtaining the above solution for pre-treatment of the present invention, contains less water than the solution for pre-treatment of the present invention, and is intended for use at a given pH by adding thereto a predetermined amount of water when used.

The level of concentration can be suitably selected and can usually be 5-20-fold, preferably, 5-to 10-fold.

Concentrate of solution for pre �of processing for immunohistochemical staining according to the present invention, when a given pH ranging from 5.0 to 7.0, preferably is supplied in one piece for improved stability during storage.

On the other hand, when the specified pH is from 8.0 to 10.0, a concentrate of solution for pre-treatment can be delivered in two parts and is composed of a first concentrate solution containing the extracting agent antigens and, optionally, a preservative, and a second concentrate solution containing non-ionic surfactant polyoxyethylene-alkylphenolate, non-ionic surfactant polyoxyethylene-sorbitan, cyclodextrin or its derivative and optionally amphiphilic solution and the buffer.

Concentrate of solution for pretreatment for immunohistochemical staining according to the present invention, supplied or in one or in two parts, can be turned into a solution for pre-treatment of the present invention by mixing with a predetermined amount of water, such as deionized water, for installation at a given pH.

Solution for pre-treatment of the present invention can be used either in manual mode or in an automated device. In particular, it is convenient and preferable to use the automated device with adjustable temperature and or�automatic device for immunohistochemical staining.

Pretreatment for immunohistochemical staining using a solution for pre-treatment of the present invention can be carried out, for example, the introduction of the sample, such as a glass slide with a tissue sample, filled in containing the wax potting medium, in the solution for pre-treatment of the present invention, the temperature of which is adjusted typically from 20 to 70°C, preferably to a level not lower than the melting point of the filling medium, such as paraffin, and by shaking the system many times to facilitate dewaxing. At this point, usually to confirm the removal of paraffin.

Then, the temperature of the slurry for pretreatment raise to stimulate the extraction of antigens in the optional conditions of increasing pressure to shorten the reaction time. The temperature is usually from 80 to 130°C, preferably from 90 to 120°C, and retention time at this temperature is usually from 1 to 70 minutes, preferably about 5 to 60 minutes.

After treatment at elevated temperature the temperature of the slurry for pretreatment decreases again to approximately 20-70°C, and the system is shaken to complete through this pre-processing.

Solution for pre-treatment according to the present invent�Oia can use three or more usually, three to five, preferably three to four consecutive times, so that pre-processing of the next tissue sample could be similarly used with a solution for pre-treatment, and if the solution is designed for five-time use, similar to the staining results can be obtained from the first and fifth pre-processing.

Pre-treated microscope slides can be subjected to immunohistochemical staining by performing a subsequent step of rinsing stage and immunohistochemical staining by ordinary methods.

EXAMPLES

The present invention will be explained in more detail with reference to examples, reference examples and comparative examples which do not limit the present invention.

In the following examples, evaluation was conducted as follows.

The subject of evaluation (1): Possible or impossible to carry out a simultaneous dewaxing and retrieval of antigens in the same solution for pre-processing.

Assessments were designated as "Y" values and "N" for impossible estimates.

The subject of evaluation (2): Visual observation of insoluble paraffin on the surface of the solution after dewaxing and antigen retrieval solution for pre�kiteley processing.

Assessments were designated as 5 points for solutions with a touch on the surface, 4 points for solutions with a slight touch on the surface, 3 points for solutions with small masses of plaque on the surface, 2 points for solutions with mixed small and large masses of plaque on the surface and 1 point for solutions with large masses of plaque on the surface (results presented as average of three times).

The subject of evaluation (3): Visual observation of repulsion reagent for immunohistochemical staining on a slide.

Assessments were designated as 5 points for observations without repulsion, 4 points for observations with several small pieces of repulsion, 3 points for observations with multiple small parts of repulsion, 2 points for observations with mixed small and large parts repulsion and 1 point for observations with large parts of repulsion or serious problems.

The subject of evaluation (4): the Intensity of staining, observed under the optical microscope

The evaluation was denoted in the form of two 2 points for observations with higher intensity, compared to control examples 1 to 3, 1 point for observations with slightly higher intensity compared to the control examples 1-3, 0 points for observations with intensity, compared�the ima with the control examples 1-3, -1 point for observations with a somewhat lower intensity compared to control examples 1 to 3 and -2 points for observations with lower intensity compared with the control examples 1-3.

The subject of evaluation (5): the thickness of the fabric after dewaxing and retrieval of antigens with a solution for pre-processing (the thinner the fabric, the more paraffin and similar materials present within the tissue sample was enough-eluted).

The evaluation was denoted as 5 points for observations, comparable with the control examples 1-3, 4 points for observation, where tissue samples were slightly thicker compared to the control examples 1-3, 3 points for observation, where tissue samples were slightly thicker compared to the control examples 1-3, 2 points for observation, where tissue samples were thicker compared to control examples 1 to 3 and 1 points for observation, where tissue samples were significantly thicker compared with the control examples 1-3.

The subject of evaluation (6): Determining whether or not three consecutive times using the solution for pre-processing from the point of view of the valuation items(1)-(5).

The evaluation was denoted as "Y" for possible evaluation and "N" for cases impossible to evaluate.

Control examples 1-3(dewaxing, rehydration and the extraction�to the amount of antigens was carried out separately in accordance with the usual practice)

(A) Pre-processing

Dewaxing and rehydration

Got a slice thickness of 3 μm formalin fixed, embedded in paraffin tissue of the tonsils, was affixed to a subject covered glasses (cover MAS (magnesium silicate of aluminum) manufactured by MATSUNAMI GLASS IND., LTD.) and dried at 37°C for 18 hours. A glass slide was deparaffinization three-minute sedimentation in the layer of xylene three times, and then rehydratable three-minute settlement in a layer of ethanol four times. After final settling in the layer of ethanol a glass slide was washed in phosphate buffer at pH 7.6 three times for three minutes each.

Extraction of antigens

Negidrirovannogo and washed a glass slide was placed in a heat resistant container filled with a citrate buffer with pH 6.0 (control example 1), citrate buffer with pH 7.0 (control example 2) or a buffer of Tris-HCl with a pH of 9.0, containing EDTA (control example 3), and none of the buffers did not contain a surfactant. Each heat-resistant container was placed in a tabletop autoclave (manufactured by ALP CO., LTD) and autecological at 121°C for 20 minutes. After autoclaving, each heat-resistant container was removed and left to sediment at room temperature for 20 min to retrieve antigenicity.

<> (B) Irrigation

After completing the 20-minute sedimentation, predmestie glass was transferred into a phosphate buffer with a pH of 7.6 and washed with five defending three times.

(C) Immunohistochemical staining

(1) Manual immunohistochemical staining of

A glass slide, subjected to a washing stage (B), was placed in a 3% solution of hydrogen peroxide/methanol, slightly shaken and left to sediment for 10 minutes. Then the slide was transferred into a phosphate buffer with a pH of 7.6 and washed with five defending three times.

Moisture was removed from the resulting glass slides and marker PAP PEN (manufactured by DAIDO SANGYO Co., LTD.) draw a circle around the tissue sample. Then, as the primary antibody on a glass slide was added dropwise CD3 rabbit monoclonal antibody (SP7) (trade name, manufactured by NICHIREI BIOSCIENCES INC.), and the reaction was performed at 25°C for 60 minutes. After completion of the reaction, the slide was transferred into a phosphate buffer with a pH of 7.6 and washed with five defending three times.

Then the moisture was removed from the washed glass slides and placed on a glass slide was added dropwise dye Histofine Simple Stain MAX-PO (MULTI) (trade name, manufactured by NICHIREI BIOSCIENCE INC.) as secondary antibody, and the reaction was performed at 25°C within 30 minutes. After completion of the reaction, the slide was transferred into a phosphate buffer with a pH of 7.6 and washed with five defending three times.

After washing, the slide, devoid of moisture, was added a solution of DAB (dimethylaminoazobenzene), obtained using the set of DAB substrate (trade name, manufactured by NICHEREI BIOSCIENCES INC.) as chromogenic substrate, and the reaction was carried out at room temperature for 5 minutes. The slide was washed in running water for 5 minutes, remove moisture, the reaction was carried out with Mayer hematoxylin for 30 seconds to bring out the colours and washed in running water for 5 minutes.

Then the moisture was removed, and slides were subjected to dehydration and purification by passing through a layer of ethanol three times, a single three-minute defending in the layer of ethanol, the single transmittance through the layer of xylene and two-time defending five-minute in xylene layer and then placed in an aqueous environment for histological slice (manufactured by NICHEREI BIOSCIENCES INC.).

(2)Immunohistochemical staining in the automatic device for immunohistochemical staining

A glass slide, subjected to a washing stage (B), installed in the rack slides automatic device for immunohistochemical OCD�shivani, HISTOSTAINER (trade name, manufactured by NICHEREI BIOSCIENCES INC.) (hereinafter referred to as HISTOSTAINER). The fabric was covered with a phosphate buffer with a pH of 7.6 to prevent drying. A glass slide, mounted in the device, washed in PBS for HISTOSTAINER and air dried. The three percent solution of hydrogen peroxide (manufactured by NICHEREI BIOSCIENCES INC.) dropwise added to the slide in the device and was subjected to reaction for 5 minutes.

After completion of the reaction, the slide was washed with PBS for HISTOSTAINER and air dried. CD3 rabbit monoclonal antibody (SP7) (trade name, manufactured by NICHIREI BIOSCIENCES INC.) as the primary antibodies was added dropwise on a glass slide, and the reaction was carried out at room temperature for 30 minutes. After completion of the reaction, the slide was washed twice PBS for HISTOSTAINER. After air drying on a glass slide was added dropwise dye HISTOFINE SIMPLE STAIN MAX-PO (MULTI) for HISTOSTAINER (trade name, manufactured by NICHIREI BIOSCIENCES INC.) as a secondary antibody and the reaction was carried out at room temperature for 30 minutes. After completion of the reaction, the slide was washed twice PBS for HISTOSTAINER.

Then on the slide as a chromogenic substrate, obtained using the set of DAB substrate (trade name, release�ICDO company NICHEREI BIOSCIENCES INC.), added a solution of DAB, and the reaction was carried out at room temperature for 10 minutes. After completion of the reaction microscope slides were washed once in PBS for HISTOSTAINER and repeatedly rinsed in water. Painted the glass slide was removed and washed in running water for 5 minutes. Thereafter, moisture was removed and placed on a glass slide, were influenced by Mayer hematoxylin for 30 seconds followed by rinsing in running water for 5 minutes.

Then the moisture was removed, and slides were subjected to dehydration and purification by passing through a layer of ethanol three times, a single three-minute defending in the layer of ethanol, the single transmittance through the layer of xylene and two-time defending five-minute in xylene layer and then placed in an aqueous environment for histological slice (manufactured by NICHEREI BIOSCIENCES INC.).

Example 1: get solution for pre-treatment at pH 9,0 without amphiphilic solution

Received a tenfold solution concentrate of buffer Tris-HCl containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To the thus obtained tenfold�th concentrate solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

(A) Pre-processing

Dewaxing and antigen retrieval

The above solution for pre-treatment was placed in an automatic device with adjustable temperature PTModule (trade name, manufactured by THERMO FISHER SCIENTIFIC K. K.) (hereinafter abbreviated referred to as PTModule) and heated to 65°C.

On the other hand, got a slice thickness of 3 μm formalin fixed, embedded in paraffin tissue of the tonsils, was affixed to a subject covered glasses (cover MAS, manufactured by MATSUNAMI GLASS IND., LTD.) and dried at 37°C for 18 hours. A glass slide was installed in the rack slides automatic device for immunohistochemical staining HISTOSTAINER.

When the temperature of the solution in PTModule reached 65°C, the lid opened, and the rack slides, in which was set a glass slide was placed in a solution and in�trachial several times. The tripod was recovered from the solution to confirm that the slide did not remain paraffin, and then installed in PTModule and closed the lid. A glass slide was subjected to heat treatment in PTModule in accordance with a pre-installed program (the temperature of the solution was raised from 65°C to 100°C, kept at 100°C for 40 minutes and was reduced to 65°C).

(B) Irrigation

After heat treatment, the glass slide when installed in the rack slides, slightly shook, then transferred to phosphate buffer pH 7.6 containing 0.05% of the mass. Tween 20, and left for sedimentation for 5 minutes. Conducted visual observation to detect any residual wax on the tissue sample or slide (was there any residual paraffin, which could not be removed by washing) and any insoluble paraffin on the surface of the solution after the solution for pre-treatment was left to cool to room temperature after pre-processing.

(C) Immunohistochemical staining in the automatic device for immunohistochemical staining

A glass slide, mounted in a tripod for HISTOSTAINER and subjected to a washing stage (B), installed in HISTOSTAINER without additional pressures, p�washed in PBS for HISTOSTAINER and air dried. The three percent solution of hydrogen peroxide (manufactured by NICHEREI BIOSCIENCES INC.) dropwise added to a glass slide and subjected to reaction for 5 minutes.

After completion of the reaction, the slide was washed with PBS for HISTOSTAINER and air dried. CD3 rabbit monoclonal antibody (SP7) (HISTOSTAINER) (trade name, manufactured by NICHIREI BIOSCIENCES INC.) as the primary antibodies was added dropwise on a glass slide, and the reaction was carried out at room temperature for 30 minutes. After completion of the reaction, the slide was washed twice PBS for HISTOSTAINER. After air drying on a glass slide was added dropwise dye HISTOFINE SIMPLE STAIN MAX-PO (MULTI) (HISTOSTAINER) (trade name, manufactured by NICHIREI BIOSCIENCES INC.) as secondary antibody, and the reaction was carried out at room temperature for 30 minutes. After completion of the reaction, the slide was washed twice PBS for HISTOSTAINER.

Then on the slide as a chromogenic substrate, obtained using the set of DAB substrate (for HISTOSTAINER) (trade name, manufactured by NICHEREI BIOSCIENCES INC.), added a solution of DAB, and the reaction was carried out at room temperature for 10 minutes. After completion of the reaction microscope slides were washed once in PBS for HISTOSTAINER and repeatedly rinsed in water. Krashen�e a glass slide was removed and washed in running water for 5 minutes. Thereafter, moisture was removed and placed on a glass slide, were influenced by Mayer hematoxylin for 30 seconds followed by rinsing in running water for 5 minutes.

Moisture was removed, and slides were subjected to dehydration and purification by passing through a layer of ethanol three times, a single three-minute defending in the layer of ethanol, the single transmittance through the layer of xylene and two-time defending five-minute in xylene layer and then placed in an aqueous environment for histological slice (manufactured by NICHEREI BIOSCIENCES INC.).

The above stage (A) to(C) were repeated three times without changing the solution for pre-treatment. The results of the above assessments for the third time shown in table 1 together with the results of control examples 1-3, which were the results of one treatment.

Example 2: fetching solution for pre-treatment at a pH of 9.0, containing amphiphilic solution

Received a tenfold solution concentrate of buffer Tris-HCl containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To thus obtained a tenfold concentrate restoratively Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. and ethylene glycol at a final concentration of 3.0 wt%. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results are shown in table 1.

In addition, the evaluation also conducted in the same manner as in example 2 with α-, β - or γ-cyclodextrin is hydroxypropyl-β-cyclodextrin, to achieve the same results. It was found that hydroxypropyl-β-cyclodextrin was the most preferred from the standpoint of ease of manipulation, since α - and β-cyclodextrin were not soluble at room temperature, and their effects are not manifested until dissolved after heat treatment.

Example 3: preparation of solution for pre-treatment with pH 6.0 without amphiphilic solution

Received a ten-fold concentrate of citrate buffer and filters�whether through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could constitute 6.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. and ethylene glycol at a final concentration of 3.0 wt%. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results are shown in table 1.

Example 4: Obtain solution for pre-treatment with pH 6.0 containing amphiphilic solution

Received a ten-fold concentrate of citrate buffer and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received no�e, was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could constitute 6.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. and ethylene glycol at a final concentration of 3.0 wt%. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results are shown in table 1.

Example 5: obtain the solution for pre-treatment with pH 7.0 without amphiphilic solution

Received a ten-fold concentrate of citrate buffer and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment�and could amount to 7.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. and ethylene glycol at a final concentration of 3.0 wt%. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results are shown in table 1.

Example 6: obtain the solution for pre-treatment with pH 7.0 containing amphiphilic solution

Received a ten-fold concentrate of citrate buffer and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 7.0. To thus obtained a ten-fold concentrated solution was added Triton X100 to a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and hydroxypropyl-β-cyclodextrin at a final concentration of 0.1% wt. and ethylene glycol at a final concentration of 3.0 wt%. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results are shown in table 1.

Comparative example 1: get solution for pre-treatment with pH 9,0 without amphiphilic solution and telodendria

Received a ten-fold concentrate of Tris-HCl buffer containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both �Einie surfactant, for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. In this case, the solution for pre-treatment of comparative example 1 could not be used consistently, so that the results of the second use is shown in table 1.

Comparative example 2: fetching solution for pre-treatment with pH 9,0 without telodendria

Received a ten-fold concentrate of Tris-HCl buffer containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration of 1.0% wt., both non-ionic surfactants, and polyethylene glycol in a horse�concentrations of 30% wt. for getting through this concentrate of solution for pre-processing.

Thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then there was a stage with (A) through (C), and evaluation was performed in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above.

Comparative examples 3 and 4: Obtain solution for pre-treatment with pH 9,0 without cyclodextrin

Solutions for pre-treatment was obtained in the same manner as in comparative example 2, except that ethylene glycol was replaced by propylene glycol (comparative example 3) or 1,3-butyleneglycol (comparative example 4), and stage (A) to (C), and evaluation was performed in the same manner as in example 1.

Table 1
The subject of evaluation (1)The subject of evaluation (2)The subject of evaluation (3)The subject of evaluation (4)The subject of evaluation (5)The subject of evaluation (6)
To�control example 1 --5 points0 points5 points-

tr>
Test case 2--5 points0 points5 points-
Test case 3--5 points0 points5 points-
Example 1Y5 points4 points0 points4 pointsY
Example 2Y4.5 points4 points2 points4 pointsY
Example 3Y4.5 points 5 points0 points4 pointsY
Example 4Y4.5 points5 points2 points4 pointsY
Example 5Y4.5 points5 points0 points4 pointsY
Example 6Y4.5 points5 points2 points4 pointsY
Comparative example 1Y5 points1 point0 points4 pointsN
Comparative example 2Y4.5 points2 points2 points4 pointsN
Comparative example 3Y4 points2 score0 points4 pointsN
Comparative example 4Y4 points2 points1 point4 pointsN

The results shown in table 1, show that the solutions for pre-treatment of the comparative examples 1-4 without cyclodextrin cannot be used consistently due to the repulsion coloring agent subject glasses (the subject of evaluation (3)). When using the solutions for pre-treatment of the comparative examples 2-4, containing amphiphilic solution, the repulsion some were eliminated, compared with comparative example 1, but the solutions for pre-treatment did not have sufficient properties to withstand the use of three or more consecutive times. In contrast, solutions of examples can be used three times consecutively. In particular, it is seen that with amphiphilic solution, the intensity of staining (the subject of evaluation (4)) had a trend to greater improvement

In addition, it was confirmed that in example 2, when Triton X has been replaced by NP-40, Triton X-114 or IGEPAL CA-630, tween 20 was replaced by Tween 40 or Tween 80 were achieved similar effects.

The control comparative example 1: get solution for pre-treatment with a pH of 9.0, a study with one nonionic surface-active substance

Received a tenfold solution concentrate of buffer Tris-HCl containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To thus obtained a ten-fold concentrated solution was added each of nonionic surfactants as shown in table 2, at a final concentration of 0.1% wt. for getting through this concentrate of solution for pre-processing.

Each of the thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then once held the stage with (A) through (C), and evaluations were carried out in respect of the valuation items (1) to(4) in the same manner as in example 1, except that a solution of DL� pretreatment was replaced by solutions, obtained above. The results are shown in table 2.

4 points
Table 2
View surfactantsThe subject of evaluation (1)The subject of evaluation (2)The subject of evaluation (3)The subject of evaluation (4)
Triton X-100Y1 point2 points0 points
NP-40Y1 point2 points0 points
Triton X-114Y1 point2 points0 points
Tween 20N4 points1 point-1 point
Twin 40N4 points1 point-1 point
Tween 80N1 point-1 point

The results shown in table 2, show that with only one surface-active substance consistent use may not be exercised.

The control comparative example 2: fetching solution for pre-treatment with a pH of 9.0, a study with two non-ionic surfactants

Received a tenfold solution concentrate of buffer Tris-HCl containing EDTA, and filtered through a filter with a pore size of 0.22 μm, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0. To thus obtained a ten-fold concentrated solution was added Triton X-100 at a final concentration of 0.1% wt. and tween 20 at a final concentration as shown in table 2, both non-ionic surfactant, to obtain through this concentrates solutions for pre-processing.

Each of the thus obtained concentrate of solution for pre-treatment was diluted 10 times with deionized water to obtain a solution for pre-processing.

Then we hosted two stages with (A) through (C), and evaluate p�bodily in respect of the valuation items (1) to(5) in the same manner as in example 1, except that the solution for pretreatment was replaced by a solution obtained above. The results of the second times of use are shown in table 3.

Table 3
The final concentration of Tween 20The subject of evaluation (1)The subject of evaluation (2)The subject of evaluation (3)The subject of evaluation (4)The subject of evaluation (5)
0,1% of the mass.Y2 points1 point0 points3 points
0.3% of the mass.Y3 points1 point0 points3 points
0.5% mass.Y4 points1 point0 points4 points
1,0% of the mass.Y5 points1 point 0 points4 points
2,0% of the mass.Y5 points1 point0 points4 points
3.0% of the mass.Y5 points1 point0 points4 points

The results shown in table 3, show that even with two specific surfactants can be implemented consistent use.

Examples 7-10 and comparative examples 5 and 6: to study cyclodextrin

Received solutions for pre-processing and evaluation were performed in the same manner as in example 2, except that a final concentration of hydroxypropyl-β-cyclodextrin and 0.1% of the mass. changed to a final concentration of 0.01% wt. (example 7), of 0.05% of the mass. (example 8), 0.5% by weight. (example 9), 1,0% of the mass. (example 10), 2.0% by mass. (comparative example 5) or 15.0% of the mass. (comparative example 6). The results are shown in table 4 together with results of example 2.

The final concentration of hydroxypropyl-β-cyclodextrin
Table 4
The subject of evaluation (1)The subject of evaluation (2)The subject of evaluation (3)The subject of evaluation (4)The subject of evaluation (5)The subject of evaluation (6)
Example 20,1% of the mass.Y4.5 points4 points2 points4 pointsY
Example 70.01% of the mass.Y4 points3 points2 points4 pointsY
Example 80,005% wt.Y4 points3.5 points2 points4 pointsY
Example 90.5% mass.Y4 points4 points 1 point4 pointsY
Example 101,0% of the mass.Y3.5 points4.5 points0 points4 pointsY
Comparative example 52,0% of the mass.Y2.5 points4.8 points-1 point4 pointsN
Comparative example 615.0% of the mass.Y2 points5 points-1 point4 pointsN

The results shown in table 4, show that if the content of the cyclodextrin, even if it is a part, is outside the defined range, the effect of output consistent use cannot be obtained.

Example 11: Example of a concentrate of solution for pre-treatment, supplied in two parts

Got A solution composed of �eraticator solution concentrate of 500 mm buffer Tris-HCl, containing 100 mm EDTA, and when the concentrate of solution for pre-treatment, to be received below was diluted ten times with this buffer, the pH of the resulting solution for pre-treatment could amount to 9.0, and 0.01% of the mass. sodium azide as preservative.

On the other hand, obtained the solution B composed of 1 mm citrate buffer, 1% of the mass. Triton X-100 and 10% of the mass. Tween 20, both non-ionic surfactants, 1% of the mass. hydroxypropyl-β-cyclodextrin, and 30% of the mass. of ethylene glycol.

Solutions A and B were mixed with deionized water in the ratio 1:1:8: to a solution for pre-treatment with a pH of 9.0.

Evaluation of solution for pre-treatment was performed in the same manner as in example 2. It was revealed that in respect of all items of the assessment were obtained results similar to example 2.

The control comparative example 3: Study of polysaccharides, hydrocarbon polymers or higher polymers instead cyclodextrin

Obtained the solution for pre-treatment in the same manner as in example 1, except that hydroxypropyl-β-cyclodextrin was replaced the various polysaccharides, hydrocarbon polymers or higher polymers, shown in table 5.

The thus obtained solutions for pre - � treatment was evaluated in respect to the valuation items (1) to(3) and (6) in the same manner as in example 1. The results are shown in table 5.

Table 5
The polymer is hydroxypropyl-β-cyclodextrinThe subject of evaluation (1)The subject of evaluation (2)The subject of evaluation (3)The subject of evaluation (6)
Dextran (Mm (molecular mass) 35000-45000) (1% wt.)N1 point1 pointN
Dextran (Mm 400000-500000) (1% wt.)N1 point1 pointN
Dextran (2000000 Mm) (1% wt.)Y2 points3 pointsN
Dextran (Mm 5000000-20000000) (1% wt.)Y1 point3 pointsN
D-sorbitol (1% wt.)N1 point2 pointsN
Trehalose (1% wt.)N1 point2 pointsN
PVP (Mm 58000) (1% wt.)Y4 points2 pointsN
PVP (Mm 58000) (5% wt.)Y4 points2 pointsN
PVP (Mm 34000) (1% wt.)Y4 points2 pointsN
PVP (Mm 130000) (1% wt.)Y3 points1 pointN
PVP/VA (Mm 58000) (1% wt.)Y4 points2 pointsN
PVP/VA (Mm 58000) (3% mass.)Y4 points2 pointsN
PVP/VA (Mm 58000) (5% wt.)Y 4 points2 pointsN
PVP: polyvinylpyrrolidone;
PVP/VA copolymer of polyvinylpyrrolidone and vinyl acetate

The results shown in table 5, demonstrate that when in solution for pre-treatment of dekstranov having a linear sugar, contains dekstrana having a relatively low molecular weight, monosaccharide, such as sorbitol, or a disaccharide, such as trehalose, dewaxing, which in other cases is achieved, can not be implemented. It is assumed that the effects of Tween 20 or Triton X-100 ingibirovalis these components. On the other hand, it was found that dekstrana having a high molecular weight, when heated, emit that smell substances.

PVP and its copolymers are used as excipients medicines for dispersing water-insoluble substances. In anticipation of this effect is explored some PVP, and the results, as shown above in table 5, indicate that there has been good progress in relation to insoluble paraffin (the subject of evaluation (2)), but the results are sufficient for consistent use, have not been achieved in relation to the repulsion of coloring reagent on glass slides (est subject�and (3)). It was also found that PVP with higher molecular weight had a higher viscosity and, thus, was difficult to use.

1. Solution for pretreatment for immunohistochemical staining for elution containing paraffin wax potting medium with the slide with the tissue sample, filled in the specified environment, and to retrieve the antigenicity of the specified tissue sample, wherein said solution for pre-treatment can be used three times or more,
moreover this solution for pre-processing contains the agent for the extraction of antigens selected from citrate buffer, citrate buffer, mixed with a chelating agent, SSC buffer, SSC buffer, mixed with a chelating agent, or Tris-buffer, mixed with a chelating agent, a nonionic surface-active agent on the basis of polyoxyethylene-alkylphenolate, nonionic surfactant based on polyoxyethylene-sorbitan and cyclodextrin or its derivative, and the remaining amount is not less than 80 wt.% water, where the contents of the specified agent for the extraction of antigens is such that the specified pH of the solution for pre-treatment ranging from 5.0 to 10.0, and the contents of the specified cyclodextrin or its derivative is from 0.01 to 1.0 wt.%

2. Solution d�I pretreatment for immunohistochemical staining according to claim 1, additionally containing amphiphilic solution.

3. Solution for pretreatment for immunohistochemical staining according to claim 1 or 2, where the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-alkylphenolate is from 0.01 to 1.0 wt.%, and the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-sorbitane is from 0.3 to 3.0 wt.%.

4. Solution for pretreatment for immunohistochemical staining according to claim 1 or 2, where the specified nonionic surfactant on the basis of polyoxyethylene-alkylphenolate represents at least one of NP-40, Triton X-100, Triton X-114 or IGEPAL CA-630, and the specified non-ionic surfactant based on polyoxyethylene-sorbitan represents at least one of Tween 20, Tween 40 or Tween 80.

5. Solution for pretreatment for immunohistochemical staining according to claim 1 or 2, where the specified cyclodextrin or its derivative is at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkyl-α-cyclodextrin, hydroxyalkyl-β-cyclodextrin and hydroxyalkyl-γ-cyclodextrin.

6. Solution for pretreatment for immunohistochemical staining according to claim 2, where you provide during account creation�th amphiphilic solution is a, at least one of ethylene glycol, propylene glycol or 1,3-butyleneglycol.

7. Solution for pretreatment for immunohistochemical staining according to claim 1 or 2, further comprising a preservative and/or disinfectant.

8. Concentrate of solution for pretreatment for immunohistochemical staining for elution containing paraffin wax potting medium with the slide with the tissue sample, filled in the specified environment, and to retrieve the antigenicity of a specified sample of tissue, wherein the specified concentrate of solution for pre-processing contains the agent for the extraction of antigens selected from citrate buffer, citrate buffer, mixed with a chelating agent, SSC buffer, SSC buffer, mixed with a chelating agent, or Tris-buffer, mixed with a chelating agent, a nonionic surface-active agent on the basis of polyoxyethylene-alkylphenolate, non-ionic surface-active agent on the basis of polyoxyethylene-sorbitan and cyclodextrin or its derivative, and the remainder is water, but its content is less than 80 wt.%, where the specified concentrate of solution for pre-treatment designed for use as a solution for pretreatment for immunohistochemical staining with satandard by adding thereto a predetermined amount of water when using, over and above the specified pH ranges from 5.0 to 7.0.

9. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 8, further comprising the amphiphilic solution.

10. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 8 or 9, where the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-alkylphenolate is from 0.1 to 10.0 wt.%, and the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-sorbitane is from 3.0 to 30.0% by weight, and the content of the cyclodextrin or its derivative is 0.1 to 10.0 wt.%.

11. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 9 where the above-mentioned amphiphilic solution represents at least one of ethylene glycol, propylene glycol or 1,3-butyleneglycol.

12. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 8 or 9, where the specified nonionic surfactant on the basis of polyoxyethylene-alkylphenolate represents at least one of NP-40, Triton X-100, Triton X-114 or IGEPAL CA-630, and the specified non-ionic surfactant based on polyoxyethylene-sorbitan performance�possessing a, at least one of Tween 20, Tween 40 or Tween 80.

13. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 8 or 9, where the specified cyclodextrin or its derivative is at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkyl-α-cyclodextrin, hydroxyalkyl-β-cyclodextrin and hydroxyalkyl-γ-cyclodextrin.

14. Concentrate of solution for pretreatment for immunohistochemical staining according to claim 8 or 9, further comprising a preservative and/or disinfectant.

15. Set to obtain the solution for pretreatment for immunohistochemical staining for elution containing paraffin wax potting medium with the slide with the tissue sample, filled in the specified environment, and to retrieve the antigenicity of a specified sample of tissue, wherein the specified set consists of a first concentrate solution and a second concentrate solution where the above-mentioned first concentrate solution contains an agent for the extraction of antigens selected from citrate buffer, citrate buffer, mixed with a chelating agent, SSC buffer, SSC buffer, mixed with a chelating agent, or Tris-buffer, mixed with a chelating agent, where this second concentrate solution contains a nonionic �poverhnosti-active substance on the basis of polyoxyethylene-alkylphenolate, non-ionic surface-active agent on the basis of polyoxyethylenesorbitan and cyclodextrin or its derivative, where mixing of the specified first concentrate solution, the specified second concentrate solution and a predetermined quantity of water into said solution for pre-treatment, having a pH of from 8.0 to 10.0 to its application.

16. The kit according to claim 15, where the specified second concentrate solution further comprises an amphiphilic solution.

17. The kit according to claim 15 or 16, where the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-alkylphenolate is from 0.1 to 10.0 wt.%, the contents of the specified non-ionic surfactants on the basis of polyoxyethylene-sorbitane is from 3.0 to 30.0% by weight, and the contents of the specified cyclodextrin or its derivative is 0.1 to 10.0 wt.%.

18. The kit according to claim 16, where the specified amphiphilic solution represents at least one of ethylene glycol, propylene glycol or 1,3-butyleneglycol.

19. The kit according to claim 15 or 16, where the specified nonionic surfactant on the basis of polyoxyethylene-alkylphenolate represents at least one of NP-40, Triton X-100, Triton X-114 or IGEPAL CA-630, and the specified non-ionic surfactant based on polyoxyethylene-sorbitan represents, in m�Nisha least one of the Tween 20, Tween 40 or Tween 80.

20. The kit according to claim 15 or 16, where the specified cyclodextrin or its derivative is at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyalkyl-α-cyclodextrin, hydroxyalkyl-β-cyclodextrin and hydroxyalkyl-γ-cyclodextrin.

21. The kit according to claim 15 or 16, further comprising a preservative and/or disinfectant.



 

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18 cl, 6 ex, 5 tbl

FIELD: medicine.

SUBSTANCE: invention relates to immunology and medical diagnostics and represents a method of the immunochromatographic determination of specific antibodies. The method is based on the contact of a membrane test-strip with an analysed liquid sample and initiated by the said contact movement of reagents, contained in the sample or applied on the membrane and forming immune complexes in the course of interactions in the membrane pores or on its surface, on the test-strip membranes. The intensity of colouration of a mark, which bound in the analytical zone, is detected visually. Characteristic peculiarity of the claimed method of the antibody determination consists in the fact that a conjugate of colloid gold with an antigen is used in the test, and the reagent for binding common antibodies is immobilised in the analytical zone of the test-strip, whereas in a standard scheme of immunochromatography for the determination of specific antibodies the reagent for binding antibodies is conjugated with colloid gold and the antigen is immobilised in the analytical zone of the test-strip.

EFFECT: application of the claimed method for analysis makes it possible to increase the analysis sensitivity and/or reduce the antigen consumption.

2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: implementing the method of treating is ensured by sampling a mammal in need of reducing circulating galectin-3 levels, and performing a blood plasmapheresis in the above mammal. The plasmapheresis is performed to reduce circulating active gal-3 levels so as to remove at least ten percent of circulating gal-2 in individual's serum by means of the plasmapheresis. Using the invention provides binding and blocking gal-3 activity in the circulation or removing bulks of gal-3 from the circulation.

EFFECT: improving the existing conservative management, suppressing or reducing inflammation and fibrosis caused by the other ones, and enabling intervention into various painful conditions, which are not treated otherwise.

23 cl

FIELD: medicine.

SUBSTANCE: invention represents a differential diagnostic technique for types of choroidal neovascularisation (CNV) accompanying age-related wet macular dystrophy characterised by measuring the concentration of vascular endothelial growth factor that is followed by evaluating the results by the measured values. If the concentration is more than 650 pg/ml, classical CNV is diagnosed; the value of 300-400 pg/ml enables diagnosing combined CNV; and the value of less than 160 pg/ml shows active fibrovascular CNV.

EFFECT: higher accuracy and simplifying the differential diagnostic technique of types of choroidal neovascularisation accompanying age-related wet macular dystrophy.

3 ex

FIELD: biotechnology.

SUBSTANCE: purpose of present invention is to provide a method of simultaneous detection of immunoglobulins of class G to specific antigens of agents of TORCH-complex infectious. The solid phase for immunosorbent is used as polystyrene microplate of low luminescent grade of plastic. The discrete microregions are applied to the bottom of a microplate well in the form of at least two microzones. The first immunospecific component is used as the mixtures of recombinant antigens. The test sample for analysis may be used as blood serum or whole blood dry spots. The second immunospecific component is used as monoclonal murine antibodies.

EFFECT: increase in accuracy, sensitivity and specificity of detection of IgG-antibodies as markers of TORCH-infections.

8 cl, 5 dwg, 7 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: group of inventions represents a method of obtaining a test-system for the diagnostics of the cytomegalovirus infection, including obtaining a purified viral lysate, preparation of an immunosorbent, containing specific CMV proteins pp 150, pp 130, gp 75, pp 65, gp 55, pp52, p 38, pp 28, preparation of solutions for electrophoresis, preparation of a solution for dilution of serums, preparation of a 5-fold concentrate of a washing solution, preparation of a conjugate, packing of the test-system, as well as the test-system, obtained by the said method. Application of a natural lysate highly-purified antigen of the cytomegalovirus strain "CMV AD 169" in obtaining the immunosorbent and the presence in it of a complete spectrum of the CMV specific proteins (in total 8 proteins), has the following advantages: the analysis sensitivity increases considerably, the setting time decreases, the necessity of preliminary dilution of the samples is absent, an initial stage of immunosorbent blocking is absent, more convenient packing considerably reduces the analysis time.

EFFECT: method is referent, maximum highly sensitive, highly specific, confirming the diagnosis in case of suspicion of the infection if positive or doubtful results are obtained.

4 cl, 6 tbl

FIELD: medicine.

SUBSTANCE: suspension of the cells Tetrahymena pyriformis, R3 agent and an analysed sample are inserted into measuring cells of a device for automated counting of living infusoria, and a living cell count is measured every minute. If a dynamic pattern of living cell count with time for the analysed sample and a reference representing a pool of 10 healthy donors' serums appears to coincide, the activities of complement component C3 in these samples seem to be equal.

EFFECT: effective method for calculating the activity of complement applicable in diagnosing a number of diseases.

2 cl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to traumatology, and can be used for prediction of delayed fracture union. Described is predicting the delayed fracture union by measuring relative growth factor (TGFβ1), lymphocytic-thrombocytic adhesion (LTA), deoxypyridinoline (DPYD), recording a microcirculation value (MV) in the patient's limbs on the 10th post-traumatic day and calculating a coefficient according to the presented formula; the derived value is used to predict the delayed fracture union.

EFFECT: higher prediction accuracy.

1 tbl, 3 ex

FIELD: test equipment.

SUBSTANCE: invention relates to laboratory test equipment, namely to the device for forming and testing of samples of thin coatings in loading devices, for example, for testing of thin ceramic heat-shielding coatings for tensile strength. The device is a two-piece unit intended for placement in the load device comprising two cylindrical and circular details the external surface of which is intended for application, at least, of one layer of thin coating and forming of a sample. One of cylindrical details has on the axis a cylindrical cavity, and another one a companion cylindrical ledge placed through a ring hole in a cavity and connecting the details. The external surface of cylindrical details has adhesion, and a ring surface has applied coatings without adhesion, and serve, respectively, for forming of a sample as a connecting layer and/or non- adhesion thin coating.

EFFECT: improvement of reliability of study of strength properties of thin coatings by forming of non-adhesion longitudinal superficial sample on the two-piece unit suitable for loading by longitudinal and temperature loads.

5 cl, 3 dwg

FIELD: chemistry.

SUBSTANCE: group of inventions relates to making preparations of adhering or non-adhering cells and/or particles, contained in liquid. Compartment (10) for making said preparations contains accumulation chamber (20) for storing liquid in accumulation chamber in suspended state against force of gravity, acting on liquid, only due to forces of adhesion and/or superficial tension. Accumulation chamber is made with possibility of storing liquid, which contains cells and/or particles, and discharge of stored liquid, containing said cells and/or particles, through output opening (22) by application of specified external force, in particular centrifugal force. Compartment contains channel (30), located adjacently to output opening (22) of accumulation chamber (20), with output opening (22) of accumulation chamber (20) leading to said channel. Channel (30) has section, larger than section of output opening (22), and wall in the place of transition from output opening (22) into channel (30) forms edge (32). Compartment also includes subject section (50) for reception of output liquid, containing said cells and/or particles, and absorbing means (40), located adjacently to subject section (50) between channel (30) and subject section (50). Absorbing means (40) has opening (42) , making it possible for liquid, containing said cells and/or particles, move through opening (42) onto subject section (50), and additionally removes liquid from liquid, containing said cells and/or particles, on subject section (50) in such a way as to leave said cells and/or particles on subject section (50) for analysis.

EFFECT: realisation of more efficient, reliable and high-quality making of preparations of cells and/or particles, contained in liquid.

25 cl, 14 dwg

FIELD: measurement equipment.

SUBSTANCE: invention relates to measurement of total gas content in non-traditional container rocks, such as gas-bearing container beds, which may be found in sedimentary rocks, volcanic or metamorphic rocks. The method includes stages of well drilling in the measurement range in a container bed to create a volume of drilling mud in annular space, which contains fragments of drilled rock and gas. At the same time the volume of annular space has the front edge and the rear edge, diversion of the front edge of the annular space volume so that entire volume of annular space is trapped in a degassing system for storage without its exposure to atmosphere, interruption of diversion of annular space volume after trapping of the front edge of annular space volume in the degassing system for storage in order to determine quantity of gas in terms of annular volume; and also in-situ calculation of the total gas volume in the container bed with account of gas and fragments of drilled rock in terms of fragments of drilled rock and gas, contained in the annular space volume.

EFFECT: increased reliability and accuracy of the method and the device for measurement of total gas content in non-traditional container rock.

25 cl, 2 dwg

FIELD: biotechnology.

SUBSTANCE: invention refers to a method for identifying living and dead mesozooplankton in seawater samples, which involves taking samples, staining the organisms with suitable colouring material, giving a visual estimation of the colour intensity of the units under the microscope, which is combined with microphotographying the units with an adjustable camera without changing the settings keeping throughout a photographic session of at least one sample; thereafter colour and brightness specifications average for each unit are measured in the formed images with using a painting program, e.g. Adobe Photoshop package, and the units are referred to living or dead by a discriminative analysis of the varied digital values.

EFFECT: improving the method.

FIELD: veterinary medicine.

SUBSTANCE: method comprises collection of urine after natural urination of animal into a sterile container. At that frozen urine samples are taken with the snow in winter, with the outdoor temperature is 10-50°C below zero.

EFFECT: use of the proposed method enables to extend the range of the animals tested to carrying the pathogenic leptospira, to provide the most long-term storage of the biological material selected, and to improve the accuracy of determining the source of leptospirosis.

FIELD: machine building.

SUBSTANCE: pads with dimensions and shape identical to the sample which are made from the material providing for total rigidity of both pads that is less or equal to the rigidity of the sample being tested, are glued to two opposite surfaces of the sample thus a laboratory assembly is produced and then set in collet clamps of a testing machine. Each clamp is located between the edge of the end face and the beginning of fillet arc of the assembly. An extensometer is installed on the assembly surface. Load is applied to the assembly and basing on the extensometer readings the curve "deformation - stress" of the laboratory assembly is drawn up and used to restore the diagram of the sample deformation. Stress in the sample σs is expressed via the stress of the laboratory assembly σla and the pad σp, provided with deformation equality, according to the formula σs=3·σla-2·σp.

EFFECT: possibility to implement Saint-Venant principle and provision for homogeneous stress in the working part of a sample made from brittle material, provision for uniaxial tension in the working part of the sample from tested material, prevention of bending, provision for more force measurement points on the equal deformation base.

2 cl, 4 dwg

FIELD: measurement equipment.

SUBSTANCE: invention relates to a method for acquisition and processing of geochemical survey data, which represents a gradient method of geochemical survey. The method involves acquisition at each sampling point of a set of samples by alternating sampling of soil samples and gas samples at the interval of 0.5-1 m downwards from ground surface. Then, analysis of soil and gas samples for their geochemical indicator or indicators is performed, and charts of geochemical indicator(s) and charts of its gradient depending on depth are built as per analysis results for each sampling point. Formation of profiles of geochemical indicator(s) and profiles of its gradient is performed for each depth; with that, the profile is built along the survey line. As per the obtained charts, isolines of geochemical indicator(s) and isolines of its gradient for the profile are built, as per which three-dimensional viewing diagram of the collected data of the area is formed. After that, determination as per characteristics of variations of geochemical indicator(s) is performed depending on depth and abnormalities of its gradients in the three-dimensional viewing diagram of the area rich in metal ores or deposits.

EFFECT: acquisition of large amount of information, namely information on longitudinal variations, other than common geochemical survey.

5 cl, 5 dwg

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a method for retrieval of an antigen in a formaldehyde-fixed tissue sample, and to a kit used in said method. The method includes incubating the formaldehyde-fixed tissue sample in a first antigen retrieval solution at a temperature higher than 90°C; transferring the formaldehyde-fixed tissue sample to a second antigen retrieval solution; and incubating the formaldehyde-fixed tissue sample in the second antigen retrieval solution at a temperature higher than 90°C. The first antigen retrieval solution comprises a buffer solution having a pH range of between about 5 and about 7 and the second antigen retrieval solution comprises a buffer solution having a pH range of between about 7.5 and about 11. Alternatively, the first antigen retrieval solution comprises a buffer solution having a pH range of between about 7.5 and about 11 and the second antigen retrieval solution comprises a buffer solution having a pH range of between about 5 and about 7. The kit comprises a first antigen retrieval solution which retrieves at least a portion of unretrieved antigens in the sample, and a second antigen retrieval solution which retrieves at least some of another portion of unretrieved antigens in the sample. The first solution comprises citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine-N,N′-bis(2-ethanesulphonic acid), dimethylarsinic acid, 2-(N-morpholino)ethanesulphonic acid, or a combination thereof, and the second solution comprises tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulphonic acid (TAPS), N,N′-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulphonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulphonic acid (TES), or a combination thereof.

EFFECT: said steps of using the first and second antigen retrieval solutions improve antigen retrieval in tissue compared to use of the first solution without the second solution and vice versa.

17 cl, 5 dwg

FIELD: machine building.

SUBSTANCE: manufacturing method of part samples from composite materials involves markup and cut-out of samples from part allowance. Part allowance is used to cut-out a ring which longitudinal section corresponds to transverse section of the sample workpiece. Then a flat template corresponding to longitudinal section of the sample workpiece is manufactured. In its central part there performed is an area of lower width with transverse matchmark applied on it in the middle. Location of sample workpieces is marked by the template along the perimeter of ring butt end by means of alignment of the template matchmark with material folds. Then sample workpieces are cut-out and machined to manufacture samples with thinned working area in the central part.

EFFECT: improving efficiency of manufacturing quality control of large parts from composite materials.

4 dwg

FIELD: metallurgy.

SUBSTANCE: invention refers to a device for surface etching for metallographic analysis of samples. The device includes a cell for etching and facilities insulating an etched zone from surrounding areas of the surface. At that the cell involves facilities for attachment to an etched object, and the specified insulating facilities are made as an elastic packing. Also the cell has an attached reservoir with an etching solution, a reservoir with a washing solution and an inlet pipe for collection of a waste solution.

EFFECT: device structure ensures an improvement of the surface preparation cleanliness for analysis and results reproducibility, also it ensures the possibility of operation not only on horizontal, but on inclined and vertical surfaces of structural elements of operating equipment in the field as well.

6 cl, 2 dwg

FIELD: automatical aids for sampling liquids.

SUBSTANCE: system for sampling and delivering filtrate has filter submerged into tested medium and connected with collecting tank and vacuum pressure source which is connected with top hole of collecting tank by means of pneumatic pipe. System has sample receiving tank connected with collecting tank and control unit which has first output to be connected with vacuum pressure source. Collecting tank has two separated chambers - washing chamber and dispatching chamber. Lower hole of washing chamber has to be lower hole of collecting tank and side hole of dispatching chamber has to be side hole of collecting tank. Floating valve is installed inside washing chamber to shut off lower and top holes. Filter is connected with lower hole of collecting tank through sampling pipe. Side hole of collecting tank is connected with lower hole of tank for receiving samples through sampling pipe. Flow-type sensor and check valve are installed inside transportation pipe. Output of flow-type sensor is connected with input of control unit; second output of control unit is connected with control input of analyzer.

EFFECT: improved precision of measurement of sample ion composition; prolonged service life of filter.

1 cl, 1 dwg

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