Composition for fixing / preserving biological samples for analysis using a histological technique

Abstract

The present invention relates to a composition comprising a propolis, an essential oil of Origanum vulgare, an essential oil of Thymus vulgaris, an alcohol and a penetrating agent, said composition being particularly useful for the preservation of tissues and organs, especially with regard to biological samples for analysis using a histological technique.

Description

[0001] Composition for the fixation / preservation of biological samples intended for analysis by a histological technique

[0002] [1] This international application claims priority from European patent application EP24180552.2 filed on 06 / 06 / 2024, which is hereby incorporated by reference.

[0003] Scope of the invention

[0004] [2] The invention relates to the field of preservation and concerns a composition which can in particular be used in connection with histological techniques and also with body preservation techniques.

[0005] Previous art

[0006] [3] Formaldehyde, or formalin, is an organic compound belonging to the aldehyde family, of which it is the simplest member. At room temperature, it is a flammable gas. Ubiquitous in its occurrence, it is widely found in the products of incomplete combustion of carbon-containing substances. As such, it is present in the smoke of forest fires, emissions from thermal power plants, incinerators, refineries, and industrial boilers, as well as in the exhaust fumes of motor vehicles and tobacco smoke. Formaldehyde is also very prevalent inside buildings (much more so than in outdoor air). This high concentration results from the use of adhesives in construction, many types of which release formaldehyde after application.

[0007] [4] More generally, formaldehyde is found in a very large number of industries.

[0008] [5] In the chemical industry, formaldehyde is used as a precursor in the manufacture of numerous resins (urea-formaldehyde resin, melamine, and phenol-formaldehyde resin), as well as in the manufacture of paints and explosives. [6] In the pharmaceutical industry, it is used as a preservative (in some vaccines) and also as a fixative (related to its preservative function) for biological samples and for embalming corpses.

[0009] [7] Finally, and in connection with the food industry, formaldehyde is listed as a preservative under the code E240 and can therefore be present in small doses in human food (e.g., fruits and vegetables at concentrations ranging from 3 to 60 mg / kg). It should be noted that formaldehyde is also used in this industry for disinfection (livestock buildings and facilities), most often by nebulization or fumigation.

[0010] [8] Despite all its applications, formaldehyde has many harmful effects on human and animal health. Thus, it appears that formaldehyde has, in addition to irritant properties (above a concentration of 0.1 mg / kg in the air), neurotoxic, genotoxic, carcinogenic and reprotoxic properties.

[0011] [9] Within the European Union, formaldehyde is classified as follows:

[0012]

[0010] - carcinogenicity, category IB;

[0013]

[0011] - germ cell mutagenicity, category 2;

[0014]

[0012] - acute toxicity by skin contact or ingestion, category 3;

[0015]

[0013] - skin corrosion, category IB;

[0016]

[0014] - skin sensitization, category 1.

[0017]

[0015] Also, even though formaldehyde-based compositions remain very present today, there is still a real need to identify and develop healthier and more ecological compositions that can overcome the aforementioned disadvantages for one or more of its applications.

[0018]

[0016] While numerous attempts at substitution have been made, these have most often targeted compounds at least as toxic as formaldehyde. For example, Chinese patent application CN 104 094 923 seeks to replace, as far as possible, high concentrations of formaldehyde with other fixative and antimicrobial molecules, while striving to preserve tissue elasticity. To achieve this, the described composition uses glutaraldehyde (0.5% to 2%) in a solution of ethanol (40% to 65%) and glycerin (5% to 15%), with various additives, including sugar alcohols (e.g., sorbitol, mannitol) and bactericidal compounds (thymol, organic acids, etc.). Summary of the invention

[0019]

[0017] It is in this context that the inventor obtained, after a long development process, a composition which proves capable of fixing and preserving biological samples for several weeks, without altering either their appearance (and in particular their color), or more generally their structure.

[0020]

[0018] Consequently, the inventor has been able to demonstrate that this composition is particularly advantageous as a substitute for formaldehyde-based compositions for the fixation and preservation of biological samples, especially those intended for analysis by histological techniques such as immunohistochemistry, immunofluorescence, and in situ hybridization. It should be noted that even after evaporation of the solution, the biological samples show no degradation.

[0021]

[0019] Furthermore, the inventor has shown that this composition is particularly advantageous for the preparation of the deceased's body for viewing by the family prior to burial. Thus, the composition according to the invention also allows, in this context, the embalmer to avoid the toxicity inherent in the use of formaldehyde-based compositions.

[0022]

[0020] Consequently, a first object of the invention relates to such a composition, which comprises:

[0023]

[0021] - a propolis, the content of which is less than 1% (by dry weight of propolis relative to the total weight of the composition)

[0024]

[0022] - an essential oil of Origanum vulgare, the content of which is less than or equal to 1% (by weight relative to the total weight of the composition)

[0025]

[0023] - an essential oil of Thymus vulgaris, the content of which is less than or equal to 1% (by weight relative to the total weight of the composition)

[0026]

[0024] - an alcohol, with a content of at least 20% (by weight of alcohol relative to the total weight of the composition); and

[0027]

[0025] - a penetrating agent, with a content of at least 20% (by weight relative to the total weight of the composition.

[0028]

[0026] In patent FR 3 073 125, the inventor described a first composition intended for preserving tissues or for body preservation treatments, which comprised propolis and essential oils of Origanum vulgare and Thymus vulgaris. The composition in question now includes propolis in the form of a hydro-alcoholic extract, which represents approximately 30% to 60% of the total weight of the composition, given that propolis itself constitutes 30% of this extract. This composition then exhibited a brownish color that affected the color of the tissues it helped to preserve.

[0029]

[0027] Advantageously, the composition according to the invention does not include aldehyde in general, preferably not formaldehyde.

[0030]

[0028] A second object of the invention relates to the use of such a composition for the preservation of tissues or organs.

[0031]

[0029] According to a preferred embodiment, this use is aimed at fixing and preserving biological samples that are intended for analysis by a histological technique.

[0032]

[0030] According to another preferred embodiment, this use is aimed at the performance of thanatopraxy care; possibly such a use can be carried out with a composition diluted to 1 / 3, or even up to 1 / 5.

[0033]

[0031] A third object of the invention relates to a method of preserving and fixing a biological sample comprising a step of bringing said biological sample into contact with a composition as described above.

[0034] Detailed description of the invention

[0035]

[0032] The word propolis comes from ancient Greek which meant "entrance of a city", in reference to the reduction of the entrance of the hive with propolis to defend the colony.

[0036]

[0033] For the purposes of this invention, "propolis" refers to "bee propolis," a complex material produced by the European honeybee (Apis mellifera) from plant resin and wax. The worker bee carries the translucent, viscous resin in the pollen baskets on its hind legs (in the same way as pollen). These pellets range in color from light yellow to greenish-brown. They are not stored in the honeycomb cells but are used immediately by the builder bees. These bees modify the propolis by adding their own secretions (mainly wax and salivary secretions) and apply it as needed. The warmer the insect's environment, the higher the percentage of wax. Generally, propolis is viscous and sticky at around 20°C and becomes hard and brittle in cold weather, as well as with age.Ultimately, we will find a more concentrated propolis at the entrance hole and on the top of the hive frames.

[0037]

[0034] Advantageously, the propolis content in the composition is between 0.1% and 1% (by dry weight of propolis relative to the total weight of the composition), preferably between 0.1% and 0.5% and, particularly preferably, between 0.15% and 0.25%.

[0038]

[0035] The term "essential oil" as used in this document refers to its common use, namely an extract obtained by distillation according to conventional processes known to those skilled in the art, and classically by steam extraction.

[0039]

[0036] Origanum vulgare, or oregano, is a perennial herbaceous plant species in the mint family (Lamiaceae). Its plants generally reach a height of 30 to 80 cm, with red, square-sectioned, hairy stems bearing rounded, green, slightly toothed leaves. The flowers are pink or purple and are grouped in small panicles. Used as a culinary herb, oregano is harvested in July, including the flowers, stems, and leaves.

[0040]

[0037] Preferably, the essential oil of Origanum vulgare is an essential oil of flowers, stems and / or leaves of Origanum vulgare, preferably it is an essential oil of flowers of Origanum vulgare.

[0041]

[0038] Advantageously, the essential oil of Origanum vulgare is obtained by steam distillation of flowers, stems and / or leaves of Origanum vulgare, preferably by steam distillation of Origanum vulgare flowers.

[0042]

[0039] Advantageously, the content of Origanum vulgare essential oil in the composition is between 0.1% and 1% (by weight relative to the total weight of the composition), preferably between 0.3% and 0.7% and, particularly preferably, between 0.4% and 0.6%.

[0043]

[0040] Thymus vulgaris, or common thyme, also belongs to the Lamiaceae family and is the plant from which the culinary herb "thyme" is derived. This plant is a perennial, bushy, and highly aromatic subshrub (7 to 30 cm tall) widely found around the Mediterranean, from the sunny garrigue scrublands of southern Europe to North Africa. Its stems, woody at the base and herbaceous above, are almost cylindrical. It flowers from April to July with very small, oval flowers measuring 4 to 6 mm in length. Thymus vulgaris plants are further subdivided according to their level of expression of certain aromatic compounds (terpenes); these are referred to as "chemotypes." For example, we find chemotypes such as thymol, carvacrol, linalool, thuyanol, geraniol or paracymene.

[0044]

[0041] Preferably, Thymus vulgaris, the extract of which is used, belongs to the thymol chemotype.

[0045]

[0042] Advantageously, the essential oil of Thymus vulgaris is obtained by steam distillation of Thymus vulgaris,

[0046]

[0043] Advantageously, the content of Thymus vulgaris essential oil in the composition is between 0.1% and 1% (by weight relative to the total weight of the composition), preferably between 0.3% and 0.7% and, particularly preferably, between 0.4% and 0.6%.

[0047]

[0044] For the purposes of the present invention, "alcohol" means an organic compound in which one of the carbon atoms is bonded to a hydroxyl group (-OH). Examples of usable alcohols include methanol, ethanol, and isopropanol, with a preference for ethanol and isopropanol, and, more preferably, ethanol.

[0048]

[0045] Advantageously, the alcohol content in the composition is between 20% and 50% (by weight relative to the total weight of the composition), preferably between 25% and 40% and, particularly preferably, between 30% and 35%.

[0049]

[0046] The term "penetrating agent" refers to a compound capable of transporting liquids by drainage and capillary action. Penetrating agents that can be used in the composition according to the invention include sorbitol, ethylene glycol, propylene glycol, glycerol, and glycerin.

[0047] Advantageously, the content of the penetrating agent in the composition is between 20% and 50% (by weight relative to the total weight of the composition), preferably between 30% and 40%.

[0050]

[0048] According to a particular embodiment, the penetrating agent is a mixture of propylene glycol and glycerin.

[0051]

[0049] Advantageously, the propylene glycol content in the composition is between 25% and 40% (by weight relative to the total weight of the composition), preferably between 25% and 35% and, particularly preferably, about 30%.

[0052]

[0050] Advantageously, the glycerin content in the composition is between 1% and 10% (by weight relative to the total weight of the composition), preferably between 3% and 7% and, particularly preferably, about 5%.

[0053]

[0051] According to another preferred embodiment, the composition according to the invention further comprises boron salt, with a content less than or equal to 0.1% (by weight relative to the total weight of the composition).

[0054]

[0052] Boron salt is a borax-based chemical product that is well known to those skilled in the art and is used in the pharmaceutical field as an antiseptic, for food preservation in some countries, but also in the construction field to give better fire resistance to the materials that contain it.

[0055]

[0053] Advantageously, the content of boron salt extract in the composition is between 0.01% and 0.1% (by weight relative to the total weight of the composition), preferably between 0.03% and 0.07% and, particularly preferably, between 0.04% and 0.06%.

[0056]

[0054] Another object of the invention relates to the use of a composition of the invention for the preservation of tissues or organs.

[0057]

[0055] By “preservation” we mean the stabilization of the state of the tissue or organ over time.

[0058]

[0056] According to a preferred embodiment, the use according to the invention is aimed at fixing and preserving biological samples which are intended for analysis by a histological technique, such as immunohistochemistry, immunofluorescence, in situ hybridization.

[0059]

[0057] The solution of the invention thus makes it possible to avoid the use of a formalin-based solution while allowing the obtaining of biological samples perfectly usable by a histological technique (maintenance of the structure and appearance of the biological sample).

[0060]

[0058] Preferably, the use according to the invention is aimed at obtaining biological samples intended for paraffin coating.

[0061]

[0059] Such samples are subsequently prepared as sections (made with the aid of a microtome) a few microns thick, which are deposited on slides. The slides are then used in various histological techniques, most commonly in immunohistochemistry or immunofluorescence.

[0062]

[0060] According to another preferred embodiment, the use according to the invention is aimed at carrying out thanatopraxy care.

[0063]

[0061] The term "thanatopraxy" refers to all the means and techniques used to ensure the treatment and preservation of the bodies of deceased persons. Thanatopraxy aims to erase the visible effects of thanatomorphosis (stages of death), by delaying post-mortem decomposition and thus allowing the temporary preservation of the body under optimal hygienic and aesthetic conditions.

[0064]

[0062] In this context, the use of the composition of the invention can be done by injection (arterial or into the cardiac and / or abdominal and / or pulmonary cavities).

[0065]

[0063] Another object of the invention relates finally to a method of preserving and fixing a biological sample comprising a step of bringing said biological sample into contact with the composition according to the invention.

[0066]

[0064] By "biological sample" is meant a fragment of tissue or organ, which may correspond to a biopsy of a patient for example.

[0067]

[0065] Typically, this contact step is carried out by immersing the sample in a bath of the composition according to the invention, which immersion is carried out for a sufficient time to ensure the preservation and fixation of the biological sample.

[0066] As such, this contact step must be carried out for a period of at least 1 hour, typically between 1 and 48 hours, preferably between 2 and 24 hours.

[0068]

[0067] Generally, this step is carried out at room temperature, but it can be carried out at a lower temperature (above 0°C) and, in this case, will require a longer time.

[0069]

[0068] The following examples are given solely as an illustration of the object of the present invention and in no way constitute a limitation.

[0070] Examples

[0071]

[0069] Preparation of the ANTOLIS

[0072]

[0070] The manufacture of the composition according to the invention, with the name ANTOLIS, is described below for 10kg of composition (approximately 10 liters).

[0073]

[0071] 3.5 grams of boron salt are dissolved in 3,176.5 grams of demineralized water, which water is then mixed with 3,200 grams of 96% ethanol.

[0074]

[0072] To the mixture obtained are then successively added 20 grams of propolis powder, 50 grams of Thyme essential oil, 50 grams of oregano essential oil, 550 grams of glycerin and finally 2,950 grams of propylene glycol under vigorous stirring.

[0075]

[0073] At the end of the stirring, the solution presents some residues which are eliminated by filtration (typically at 150 microns) before packaging in a can.

[0076]

[0074] The hydroalcoholic solution obtained is translucent, light yellow in color, and can be stored at room temperature protected from light.

[0077]

[0075] A control composition was prepared according to the protocol described in the examples of French patent FR 3073125 B 1 which has a very high concentration of propolis (greater than 15%).

[0078]

[0076] Tissue preservation

[0079]

[0077] A 4cm cube of beef 2 is immersed in the ANTOLIS composition or in the control composition, then maintained at room temperature for 4 years. This test was also carried out over 6 months in an Anatomic Pathology laboratory by comparison with formalin-fixed surgical specimens, which were also found to show no alteration or decomposition, thus indicating that ANTOLIS has fixation properties similar to those of formalin.

[0080]

[0078] The results show that the ANTOLIS composition, although containing a much lower amount of propolis, ensures perfect preservation of the tissue over time. Simultaneously, no discoloration of the meat piece is revealed over time, unlike that resulting from prolonged immersion in the control composition.

[0081]

[0079] Use in histology

[0082]

[0080] A biological sample in the form of a cube with edges less than one centimeter in size is immersed in a volume at least 10 to 15 times greater of ANTOLIS composition and, as a control, in 10% formalin at neutral pH buffered with phosphate. This immersion is maintained for 24 hours before proceeding with tissue dehydration.

[0083]

[0081] To achieve this, the fabrics fixed in ANTOLIS or in formalin are subjected to successive passages in baths of 50%, 70%, 95%, and then 100% alcohol. The fabrics are then immersed in xylene baths before being embedded in paraffin.

[0084]

[0082] After coating, the tissues are placed in a microtome to make sections of thickness of about 5 microns (after roughing of the tissue included, to 20 microns) and spread them on glass slides.

[0085]

[0083] The glass slides, thus obtained from the samples, are then warmed with a semi-liquid glue to avoid any detachment, and then stained with HES (Hematoxylin-Eosin-Saffron) so that they can be interpreted in histology using a biological microscope.

[0086]

[0084] The results show that the ANTOLIS composition allows for the production of sections similar to those obtained with formalin and that it does not cause any staining or alteration in general that could compromise the analysis of the histological results.

[0087]

[0085] In conclusion, it appears that the ANTOLIS composition constitutes an alternative to formaldehyde-based compositions for the preservation and fixation of biological samples for use in histological techniques, particularly with regard to samples intended for paraffin embedding.

[0086] Preservation of bodies

[0088]

[0087] The ANTOLIS composition is used undiluted at a rate of 1 to 3 litres of composition for a body of average or standard build.

[0089]

[0088] In the case of arterial injection, this can be performed in the femoral, common carotid, or axillary arteries. In this case, a slow and steady injection is recommended. Cardiac drainage may also be necessary.

[0090]

[0089] In conclusion, the examples show that the ANTOLIS composition has good efficacy in terms of preserving / fixing tissues, without altering either their appearance (especially color), or their structure in general, which makes it particularly interesting both in the context of preserving / fixing biological samples intended for analysis by a histological technique and also in the context of thanatopraxy care.

Claims

DEMANDS 1. A composition that includes: - a propolis whose content is less than 1% (by dry weight of propolis in relation to the total weight of the composition); - an essential oil of Origanum vulgare, the content of which is less than or equal to 1% (by weight relative to the total weight of the composition); - an essential oil of Thymus vulgaris, the content of which is less than or equal to 1% (by weight relative to the total weight of the composition); - an alcohol, with a content of at least 20% (by weight of alcohol relative to the total weight of the composition); and - a penetrating agent, with a content of at least 20% (by weight relative to the total weight of the composition.

2. The composition according to claim 1, characterized in that it does not comprise formaldehyde.

3. The composition according to any one of claims 1 or 2, characterized in that the propolis content in the composition is between 0.1% and 1% (by dry weight of propolis relative to the total weight of the composition), preferably between 0.1% and 0.5% and, particularly preferably, between 0.15% and 0.25%.

4. The composition according to any one of claims 1 to 3, characterized in that the content of: Origanum vulgare essential oil in the composition is between 0.1% and 1% (by weight relative to the total weight of the composition), preferably between 0.3% and 0.7% and, particularly preferably, between 0.4% and 0.6%; and / or Thymus vulgaris essential oil in the composition is between 0.1% and 1% (by weight relative to the total weight of the composition), of preference between 0.3% and 0.7% and, in a particularly preferred manner between 0.4% and 0.6%.

5. The composition according to any one of claims 1 to 4, characterized in that the alcohol is ethanol with, preferably, an alcohol content in the composition of between 20% and 50%.

6. The composition according to any one of claims 1 to 5, characterized in that the penetrating agent is selected from the group comprising sorbitol, ethylene glycol, propylene glycol, glycerol and glycerin, 7. The composition according to any one of claims 1 to 6, characterized in that the content of penetrating agent in the composition is between 20% and 50% (by weight relative to the total weight of the composition), preferably between 30% and 40%.

8. The composition according to any one of claims 1 to 7, characterized in that the penetrating agent is a mixture of propylene glycol and glycerin and in that: the propylene glycol content in the composition is between 25% and 40% (by weight relative to the total weight of the composition), and the glycerin content in the composition is between 1% and 10% (by weight relative to the total weight of the composition).

9. The composition according to any one of claims 1 to 8, characterized in that it further comprises boron salt, with a content less than or equal to 0.1% (by weight relative to the total weight of the composition).

10. The composition according to the preceding claim, characterized in that the content of boron salt extract in the composition is between 0.01% and 0.1% (by weight relative to the total weight of the composition).

11. The use of a composition as defined in any one of claims 1 to 10 for the preservation of tissues or organs.

12. The use according to the preceding claim, characterized in that it relates to the fixation and preservation of biological samples intended for analysis by a histological technique.

13. The use according to the preceding claim, characterized in that it aims at obtaining biological samples intended for paraffin coating.

14. The use according to claim 11, characterized in that it is for the performance of thanatopraxy care.

15. A method for preserving and fixing a biological sample comprising a step of contacting said biological sample with a composition as defined in any one of claims 1 to 10.