FIXATIVE

Description

[0001] The present invention relates to a fixative.

[0002] Various fixatives are known, for example, from DE 10 2012 101 896 A1. One of the disclosed fixatives comprises alcohol as a solvent in which a polyamine is dissolved as a denaturing agent. However, DE 10 2012 101 896 A1 also discloses that certain alcohols, such as ethanol, are disadvantageous as a solvent because they dehydrate the tissue to be preserved. Another of the disclosed fixatives comprises water as a solvent in which a polyamine is dissolved as a denaturing agent. However, a particular disadvantage of fixating tissue with polyamines dissolved in water as a denaturing agent is that the structural change in the organic tissue to be preserved, which is necessary for preservation, proceeds too slowly and, for example, undesired artifacts form, or the fixation process does not fully capture all structures of the cells and tissue.

[0003] EP 2 873 962 A1 discloses a fixative according to the preamble of claim 1.

[0004] The object of the invention is therefore to accelerate the effect of the denaturing agent without destroying the tissue to be preserved and thus to improve fixation.

[0005] According to one aspect of the invention, a fixative for preserving organic tissue and cell aggregates comprises a hydrophilic denaturant for structurally altering the molecules of the organic tissue, a lipophobic solvent in which the denaturant is dissolved, and an amphiphilic infiltration agent dissolved in the solvent for dissolving fats on the surface of the biological tissue to accelerate penetration of the denaturant into the biological tissue.

[0006] The specified fixative is based on the idea that a denaturing agent is used dissolved in a solvent and has a hydrophilic effect. The denaturing agent must be able to dissolve in the solvent; therefore, the solvent cannot be chosen arbitrarily. The solvent must not alter the organic tissue to be preserved and should therefore be lipophobic. On the other hand, fat on the cell walls of the organic tissue to be preserved slows the effect of the denaturing agent, which increases the likelihood of osmotic damage to the organic tissue to be preserved, which in turn leads to artifacts that should be avoided (examples: hemolytic effect, structural, biochemical, and morphological destruction of erythrocytes and other cells, cell structures, for example, also of protozoa, deterioration of immunological properties).The fixative according to the invention is defined in claim 1.

[0007] For the denaturant to penetrate the tissue to be preserved quickly, it would be desirable for the fat on the cell walls to be dissolved as quickly as possible. Therefore, with the specified fixative, it is proposed to additionally dissolve an amphiphilic, i.e., simultaneously lipophilic and hydrophilic, infiltration agent in the solvent. This infiltration agent, due to its hydrophilicity, dissolves in the solvent and, due to its lipophilicity, dissolves the fat on the cell walls of the biological tissue to be preserved. This accelerates the penetration of the denaturant into the biological tissue to be preserved, and improves the effect of the fixative, allowing it to reach all intercellular and extracellular areas or components of the tissue to be preserved.

[0008] With the specified fixative, satisfactory preservation results can be achieved even with polyamine, especially with urotropin as denaturing agent.

[0009] Water should preferably be used as a solvent because, on the one hand, it does not attack the tissue to be preserved and, on the other hand, it is very cost-effective.

[0010] The amphiphilic infiltration agent should preferably exhibit hygroscopic properties. This way, the infiltration agent itself acts as a fixative and supports the actual denaturant in its preservation without exhibiting adverse denaturing and hygroscopic properties.

[0011] The amphiphilic infiltration agent may contain at least one short-chain alkanol with a maximum of ten carbon units. Short-chain alcohols have both polar OH groups and non-polar CH groups and are therefore amphiphilic. The short-chain alkanol can be selected from methanol, ethanol, isopropanol, glycerin, and butanol. According to the invention, the specified fixative contains 80 to 99.8 wt.% of the solvent, 0.1 to 10 wt.% of the denaturant, and 0.1 to 10 wt.% of the infiltration agent. With these mixing ratios, active infiltration is achieved by dissolving lipophilic membrane structures of tissue cells. In the fixative mentioned above, the use of amphiphilic ethanol as a solvent merely provides a passive functional aid, which does not work or only works imperfectly with many tissue types.

[0012] According to the invention, the infiltration agent contains 0.1 to 10 wt.% methanol and / or 0.1 to 10 wt.% ethanol and / or 0.1 to 10 wt.% isopropanol and / or 0.1 to 10 wt.% glycerol and / or 0.1 to 10 wt.% butanol. In particular, the fixative according to the invention contains three or four of the aforementioned alcohols and can thus be a ternary or quaternary mixture.

[0013] The denaturant may contain a polyamine, particularly urotropine (also called hexamethylenetetramine or methenamine). The fixative may further comprise an acidifying agent capable of releasing protons that react with the denaturing agent to form an aldehyde, which in turn accelerates the preservation of the biological tissue. The acidifying agent can be used to adjust the pH of the fixative. For example, if urotropine is used alone in water as a solvent, the pH of this aqueous solution is alkaline and unstable. For an effective fixative, the pH should be adjusted to a stable value between 2.5 and 10, preferably below 8.

[0014] The acidifying agent for adjusting the pH value can be an organic acid, an inorganic acid, an acidic salt or a mixture thereof.

[0015] The organic acid can be selected from aliphatic saturated and unsaturated monocarboxylic acids, di- and tricarboxylic acids, aromatic carboxylic acids, for example salicylic acids, benzoic acids, heterocyclic carboxylic acids, or mixtures thereof. Examples of suitable organic acids are formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, succinic acid, malonic acid, glutaric acid, tartaric acid, malic acid, citric acid, sorbic acid, ascorbic acid, and mixtures thereof.

[0016] The inorganic acid can be selected from hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, nitric acid, nitrous acid and polythionic acids and their salts, as well as mixtures thereof.

[0017] The acidic salt may be selected from quaternary ammonium compounds, ammonium chloride and mixtures thereof.

[0018] Bactericidal substances (such as sodium azide and quaternary ammonium compounds) can also be added to the fixative. The addition of surfactants (anionic and cationic, non-ionic and amphoteric surfactants—including quaternary ammonium compounds with surfactant activity) improves the infiltration and penetration ability of the fixative mentioned in the invention in physiological and non-physiological environments. The concentration can be selected from 0.001 to 10.0%.

[0019] The solutions with the above-mentioned contents may also contain fragrances and dyes.

[0020] Compared to conventional tissue and cell staining with formalin and other previously used fixatives, which have many disadvantages, the specified fixative produces few or no artifacts. Many structures in the tissue, cells, and subcellular areas are better stained by the various subsequent staining procedures. The use of the newly developed fixative concept also benefits serological-immunological and molecular biological examinations, improving the possibilities for professional and diagnostic assessment and further processing.

[0021] The coloring of the fixative allows for visual differentiation from other aqueous diagnostic and chemical solutions. This can increase laboratory safety. The characteristic color, which is intended to be protected, is pink. The fragrances also serve to enhance laboratory safety.

[0022] The specified fixative does not cause any significant cross-linking of DNA, RNA, or proteins, as is the case with most known fixatives based on current technology. This has enormous advantages for DNA / RNA studies, molecular biological genetic engineering, and all other biotechnological work and investigations involving protein structures. Fixatives that alter primary, secondary, tertiary, or quaternary protein structures, often irreversibly, do not meet modern analytical and preparative working conditions and their quality management requirements. The severe, sometimes irreversible protein denaturation not only creates an artificial barrier, hinders, and delays the fixation process, but also produces artifacts that, for example, favor false positive or false negative results in diagnostics.Formalin-based fixatives and their analogues are a good example of these disadvantages. However, the disadvantages of purely alcohol-based fixatives must also be mentioned here. The fixative described combines the advantages of all previous state-of-the-art fixatives without exhibiting their disadvantages.

[0023] The above-described properties, features, and advantages of this invention, as well as the manner in which they are achieved, will become clearer and more readily understood in connection with the following description of the exemplary embodiments, which are explained in more detail in conjunction with the drawings. Fig. 1 a schematic representation of a tissue body to be preserved in a fixative.

[0024] In the figures, identical technical elements are provided with identical reference symbols and described only once. The figures are purely schematic and, above all, do not represent the actual geometric relationships.

[0025] It will be Fig. 1 Reference is made. Schematically shown is a beaker 2 containing a fixative solution 4 and a tissue 6 to be fixed with the fixative solution 4. The tissue 6 to be fixed can be a very fatty organ of a human, such as the central nervous system, the liver, the muscles, the heart, or the blood vessels. The rich fat content of the tissue 6 to be fixed is in Fig. 1 indicated by a dashed line 8 around the tissue 6 to be fixed.

[0026] In the present embodiment, the fixing solution 4 contains 98% of a solvent in the form of water, 1% of a denaturing agent in the form of urotropin and 1% of an infiltration agent to be described later.

[0027] For example, the preservative effect of urotropin is known from DE 10 2012 101 896 A1. However, the preservation process is very slow, and a high fat content 8 in the organic tissue 6 to be preserved significantly prolongs the preservation process.

[0028] For this reason, the infiltration agent is also dissolved in the solvent of the fixation solution 4. Its function is to dissolve the fatty portion 8 of the tissue to be fixed and thus accelerate the infiltration of the dentating agent. In order for the infiltration agent to dissolve in the fixation solution 4, it must also be hydrophilic, which is why only amphiphilic substances are suitable for the infiltration agent.

[0029] In the present embodiment, a quaternary mixture of methanol, ethanol, isopropanol, and glycerin, each in equal proportions, was chosen for the infiltration agent. By adding this infiltration agent to the fixing solution 4, the time required for the entire fixing and preservation process could be significantly reduced.

[0030] Although the use of an amphiphilic agent in the form of alcohol in the fixative solution is already known from DE 10 2012 101 896 A1, the alcohol is used there as the solvent itself. The alcohol specified there, when mixed with urotropin, an agent with strongly polar and hydrophilic properties, promotes passive penetration into biological tissue.

[0031] The property of the alcohols previously mentioned as infiltration agents dissolved in water, on the one hand, extends the penetration of the fixative urotropin in a significantly improved way, namely by softening / dissolving - and opening - the lipophilic membrane structures, on the other hand, these alcohols themselves act in the proposed concentrations and mixtures as ideal fixatives - without having the unfavorable denaturing and hygroscopic properties of the pure alcohol fixations when the alcohol itself is used as solvent.

[0032] In addition, the fixing properties of the alcohols dissolved in water together with urotropin in the specified concentrations are not only present individually, but also cumulatively, resulting in a significant increase in the fixative quality and potency in all tissue types, regardless of the fat content.

[0033] In summary, the specified fixative solution can be assessed as follows: Amphiphilic substances, such as the alcohols listed as examples, are not used as solvents carrying the denaturant, but rather as auxiliary agents. The substance groups specified for the specified fixative, at the specified concentrations and in the suggested mixing ratio, provide ideal fixative properties in physiological environments, as well as in fatty or fatty tissue.

[0034] The specified fixative eliminates the previous disadvantages of the individual fixative amphiphilic substances, because these disadvantages no longer occur due to dissolution in a hydrophobic solvent.

[0035] Infiltration of the denaturant is actively achieved by dissolving lipophilic membrane structures of tissue cells. The use of amphiphilic substances as solvents has only provided a passive functional aid, which is either ineffective or incomplete in many tissue types.

Claims

1. Fixative for the preservation of organic tissue and cell associations, comprising: - a denaturing agent containing a hydrophilic polyamine for structurally altering the molecules of the organic tissue, - a lipophobic solvent in which the denaturing agent is dissolved, and - an amphiphilic infiltration agent dissolved in the solvent for dissolving fats on the surface of the biological tissue to accelerate the penetration of the denaturing agent into the biological tissue, characterized by - 80 to 99.8 wt.% of the solvent, - 0.1 to 10 wt.% of the denaturing agent, and - 0.1 to 10 wt.% of the infiltration agent, wherein the amphiphilic infiltration agent contains methanol or ethanol, and the infiltration agent contains at least three, preferably four, of the following alcohols: - 0.1 to 10 wt.% methanol; and / or - 0.1 to 10 wt.% ethanol; and / or - 0.1 to 10 wt.% isopropanol; and / or - 0.1 to 10 wt.% glycerol; and / or - 0.1 to 10 wt.% butanol.

2. Fixative according to claim 1, wherein the solvent contains water.

3. Fixative according to claim 1 or 2, wherein the amphiphilic infiltration agent exhibits hygroscopic properties.

4. Fixative according to any of the preceding claims, wherein the polyamine is hexamethylenetetramine.

5. Fixative according to any of the preceding claims, further comprising an acidifying agent configured to donate protons that react with the denaturing agent.

6. Fixative according to claim 5, wherein the acidifying agent is an organic acid, an inorganic acid, an acid-reactive salt, or a mixture thereof.

7. Fixative according to any of the preceding claims, further comprising a bactericidal substance.