Method of preparing bone tissue preparation and set for its realisation


G01N1/28 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

FIELD: medicine.

SUBSTANCE: invention relates to medicine, veterinary and biology. Sample fixation, decalcification, washing with water, dehydration in alcohol solutions and pouring with paraffin are performed. Sample fixation is performed for 24 hours in alcohol-based molecular fixing agent FineFix, which contains FineFix and 96° alcohol in ratio 1:2.5. Decalcification is carried out for 2-5 days in 5-8% buffered solution of formic acid with daily replacement of decalcifying solution and control of decalcification completeness. Ratio sample:decalcifying solution constitutes 1:20. When decalcification is finished, sample washing with water is performed and sample is re-submerged into alcohol solution of molecular fixing agent FineFix for 6-12 hours before dehydration stage. Set for preparation of bone tissue preparation contains alcohol-based molecular fixing agent FineFix, concentrated solution of decalcifying agent, prepared with ratio 40 g of sodium citrate, 100 ml of 90% solution of formic acid, 300 ml of distilled water, and working solutions for control of decalcification completeness, containing saturated ammonium oxalate solution and 25% water solution of ammonia.

EFFECT: invention makes it possible to obtain high-quality preparations suitable for further histological and immunohistological analysis without application of highly toxic components.

2 cl, 2 dwg

 

The present invention relates to the field of medicine, veterinary medicine and biology.

Histological examination of bone tissue is a required component diagnosis of many diseases and conditions and is used in clinical and experimental studies. The quality of the product plays a crucial role in diagnosis and in establishing the fabric of the relationship.

There is a method of preparing bone, including bone cutting and grinding (Microscopic techniques / Ed. by Sarkisov D.S. Petrov JUL - M.: Medicine, 1996. - S).

The disadvantages of this method include the fact that the resulting preparations are unsuitable for histological studies, as in thick slices obscured the structure of the bone, and when grinding also destroyed cellular elements.

Also known a method of preparation of fragments of the bone tissue to the research, including fixation in buffered 10%solution of neutral formalin for 24-48 hours, rinse in water for 24-48 hours, declinatio 5-7 .5%solution of nitric acid to the full decalcomania, subsequent processing of the preparation of bone tissue in 5%solution of sodium sulfate or lithium within 1 day, washing in water for 24-48 hours, dehydrated in alcohols and fill in paraffin (ICRI is scopacasa technique / edited Sarkisov D.S. Petrov JUL - M.: Medicine, 1996. - S, 457, 467).

However, this method does not allow to achieve good preservation of cellular structures, especially in the surrounding soft tissues (muscle, periosteum, bone marrow), significantly affects the quality standard histologische colors, and does not allow subsequent immunohistochemical and immunofluorescence studies due to the damage of the secondary structure of protein epitopes.

The closest in technical essence to the present invention is a method of preparation of bone samples for morphological diagnosis, including the fixation of the sample, its declinatio, washing with water, dehydration in ethanol solutions with increasing concentration and fill in paraffin (Medvedev N.N., Nikolaev V.G. and other Method of preparation of bone samples for morphological diagnosis. Pat. EN 2241994, IPC7G01N 33/48, G01N 1/28, Application: 2003129253/15, 30.09.2003, Published: 10.12.2004).

The known method is as follows. After fixation of bone samples in formalin solution are declinatio within 7-8 days in a 5%solution of nitric acid on the basis of a 5%formalin solution, after which bone samples for 2 hours, washed under running tap water and 12 hours immersed in a 5%solution of sulfuric acid lithium, then for 20-30 mine is again washed with running water and subjected to dehydration in alcohols of increasing concentration.

The disadvantages of this method include the duration of the procedure of decalcomania, use as a fixative formalin, and as calcinator solution of nitric acid, which prevents the subsequent conduct immunohistochemical and immunofluorescence studies of samples due to the significant damage to the surrounding tissue and cellular structures when conducting decalcomania.

The task of the invention is to develop a method of preparing bone, suitable for histological and immunomorphological research, and set for its implementation.

The technical result of the proposed method is the preservation of the histological structures of the bone and surrounding tissues, as well as reducing the complexity and toxic effects in the manufacture of the drug.

The technical result is achieved in that the method of preparation of preparation of bone tissue includes the fixation of the sample, its declinatio, washing with water, dehydration in ethanol solutions and fill in paraffin.

The difference of the proposed method lies in the fact that the fixation of the sample bone should be performed within 24 hours in molecular clamp, which does not include aldehydes, for example, raster FineFix alcohol-based, containing FineFix is 96° ethyl alcohol in the ratio of 1:2,5.

FineFix - Besfamilnov the latch, which includes the following components: water, 1,2-PROPANEDIOL, polyvinyl alcohol and from 0.05 to 2.00 wt.-% Monomeric carbohydrate containing at least 3 carbon atoms and the second means for storing ethanol (patent EP 145 5174 B1 Fixative, the application Number EP20030005010, publication date: December 15, 2004, priority date: March 5, 2003. Other numbers of patent: US 20050074422. Author: Francesco Visinoni, The Applicant Is A Milestone S.r.L).

Distinctive technique proposed method is that declinatio carried out in 2-5 days 5-8% buffered solution of formic acid at a daily changing decalcifying solution and verify the completeness decalcomanie ratio sample:decalcifying solution is 1:20.

The differences between the proposed method also includes receiving repeated premises of the sample bone in alcohol solution molecular retainer for 6-12 hours, which is carried out after decalcomania and washing the sample with water.

Comparative analysis of the proposed technical solutions to the prototype allows to make a conclusion on the conformity of the proposed technical solution the criteria of the invention of "novelty."

Distinctive techniques of the proposed method ensures the safety of histological structures of the bone and surrounding tissues, preservation of antigenic epitopes,and reduce labor intensity and reduce the time of manufacture of the drug. So, the timing of decalcomanie compact bone small laboratory animals (mice, rats) are 2-4 days, rabbits 4-5 days, fragments of human bone tissue - 4-5 days. This eliminates the toxic effects of formalin on the researcher.

The inventive method achieves perceived by the applicant of the technical result, namely, the preservation of the histological structures of the bone and surrounding tissue, reducing the complexity and faster manufacturing of drugs.

Therefore, the inventive method allows to obtain preparations of high quality, suitable for routine histological examination and for carrying out immunohistological studies. In addition eliminates the use of highly toxic formalin.

The above can conclude that the technical solutions according to the criterion of "inventive step".

The method constituting the invention, intended for use in medicine, veterinary medicine and biology. The possibility of its fulfillment is confirmed as described in the application techniques and equipment.

The above gives grounds to believe that the proposed solution meets the criteria of the invention "industrial applicability".

Implementation of the proposed method is illustrated by a specific example is on the run. The following example serves to illustrate, but not limit the invention.

Samples of bone tissue fixed in alcohol solution molecular latch FineFix (280 ml FineFix+720 ml of 96° alcohol) for 24 hours. Then without washing the sample is placed in an individual well-closed container in a 5%buffered solution of formic acid. A concentrated solution of calcinator, the so-called matrix solution, at the rate of 40 g sodium citrate, 100 ml of 90% formic acid, 300 ml of distilled water; before use the solution was adjusted with distilled water to obtain a 5-8% solution of formic acid. For each sample the ratio of the sample:decalcifying solution is 1:20.

Daily assessment of the completeness of decalcomanie calcium-oxalate method (3 ml used decalcifying fluid, to bring the pH to 7.0 with concentrated (25%) ammonia solution, then add 5 ml of saturated ammonium oxalate solution, mix well, leave on for 30 minutes while the liquid remains clear - decalcomania completed). In the presence of calcium used in decalcifying solution, the sample is placed in a fresh portion of calcinator.

After decalcomanie carry out washing of the samples with water for 5 minutes and then placed in alcohol solution FneFix 12 hours.

This was followed by dehydration in alcohols of increasing concentration ranging from 70° solution and ending at 100° alcohol. Fill in paraffin carried out according to the standard technique (Microscopic techniques / Ed. by Sarkisov D.S. Petrov JUL - M.: Medicine, 1996. - S).

The proposed method produced 162 block of samples of bone tissue. Obtained high-quality histological preparations as the application of routine methods of staining (staining with hematoxylin-eosin)and when applying immunomorphological methods of dyeing.

When the preparation of bone tissue using a set containing molecular latch FineFix alcohol-based, containing FineFix and 96° ethyl alcohol in the ratio of 1:2.5, the concentrated solution of calcinating and working solutions to monitor the completeness of decalcomanie: 25% ammonia solution and a saturated solution of ammonium oxalate.

The essence of the proposed method is illustrated in figures histological preparations of bone tissue, obtained by the proposed method.

Thus, figure 1 shows the histological preparation of the bone, stained hematoxylin-eosin. Shows the security structures of the bone and surrounding tissues And muscles, periosteum, bone, D - bone marrow, E - cartilage, 40x magnification.

Figure 2 shows the staining of bone marrow cells in detalizirovan the th bone immunohistochemical method on carbonic anhydrase. Used primary antibody CA2, Epitomics® catalog No. 5204-1. Demonstrated good quality staining. F - brightly coloured osteoclasts, D - bone, magnification 400x.

Thus, the proposed method allows to obtain preparations of bone tissue, suitable for histological and immunomorphological research. When this bone and the surrounding tissue to maintain its structure, are preserved antigenic epitopes, there is no use of highly toxic components.

1. The method of preparation of preparation of bone tissue, including the fixation of the sample, decalcomania, washing with water, dehydration in ethanol solutions and fill in paraffin, characterized in that the fixation of the sample should be performed within 24 h in the molecular latch FineFix alcohol-based, containing FineFix and 96%ethanol in the ratio 1:2,5, declinatio carried out in 2-5 days 5-8% buffered solution of formic acid at a daily changing decalcifying solution and verify the completeness decalcomanie ratio sample:decalcifying solution is 1:20, after decalcomanie spend washing the sample with water and to the stage of dehydration re-place the sample in alcohol solution molecular latch FineFix 6-12 hours

2. Kit for the preparation of the preparation of bone tissue, characterized the decomposing those it contains a molecular clamp FineFix alcohol-based, concentrated solution of calcinator made at the rate of 40 g sodium citrate, 100 ml of 90%formic acid, 300 ml of distilled water and working solutions to monitor the completeness of decalcomania: a saturated solution of ammonium oxalate and 25%aqueous ammonia solution.



 

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