Compositions for processing histological and cytological preparations comprising reagents and antioxidant agents that counteract the effect of molecular damage and DNA and RNA fragmentation

Abstract

The addition of hydrophilic and / or lipophilic antioxidants to aldehyde histological fixatives and / or to the materials for embedding the preparations allows to improve the preservation of antigenic characteristics of tissue markers and the preservation of the integrity of DNA and RNA sequences.

Description

[0001] “COMPOSITIONS FOR PROCESSING HISTOLOGICAL AND CYTOLOGICAL PREPARATIONS COMPRISING REAGENTS AND ANTIOXIDANT AGENTS THAT COUNTERACT THE EFFECT OF MOLECULAR DAMAGE AND DNA AND RNA FRAGMENTATION”

[0002] The present invention relates to compositions for the fixation, dehydration and clarification of histological and cytological preparations comprising reagents and antioxidant agents embedded in paraffin.

[0003] Prior art

[0004] The process of cellular fixation and treatment of histological tissues leading to embedding in paraffin underlies the examination of biopsy material and is widely practised worldwide, giving rise to the production of millions of preparations and blocks used for diagnostic purposes. On the material embedded in paraffin and then stored in archives, sections used for morphological analysis, immunohistochemical reactions and in situ hybridization (called FISH) and extraction of nucleic acids used for genetic and molecular biology examinations are practised. The cytological examination involves the fixation of isolated cells, followed by staining, any immunohistochemical examinations and DNA and / or RNA extraction, while for the study of the tissues, after the initial fixation, dehydration follows, by means of a series of baths in alcoholic liquids (generally ethyl alcohol) of the fixated tissues, subsequent clarification, a passage in paraffin solvent liquids (generally xylol) and finally embedding in liquid paraffin (at 65 °C). Once the paraffin solidifies (at room temperature) the biopsy samples are then packaged in the form of blocks, suitably labelled, which are used to carry out histological sections of a thickness of 4-5 microns that are stained with Hematoxylin- Eosin for histopathological diagnosis and for subsequent molecular analyses. The process of embedding histological preparations in paraffin was originally introduced by Wilhelm His Sr. in 1868 (van der Lem et al., 2021) and has continued without significant modifications for about 150 years, as it allows thin sections to be made and the material to be preserved for a long time, since it is believed that, paraffin being inert, the material thus preserved is stable.

[0005] Further sections can be obtained from the paraffin blocks which are used for immunohistochemical reactions in order to recognise and localise different antigens (see Dabbs, 2021) for diagnostic and therapy setting purposes.

[0006] Sections can also be obtained from the same blocks, from which it is possible to extract nucleic acids, both DNA and RNA, for gene analysis. This type of investigation is currently widely used to characterise the molecular and mutational characteristics of tumours, as gene sequencing makes it possible to determine the presence of alterations (hyper-expressions, deletions, mutations) that not only have diagnostic significance, but also for setting specific therapies for the various tumours.

[0007] From the above not only does the enormous popularity of the use of the process of paraffin embedding of tissues emerge, a process that is currently implemented with equipment in which the various steps are implemented in automatic and standardized sequencing (called processors, for example HistoCore PEGASUS Plus Tissue Processor of the company Leica Biosystems). The use of this procedure, considered central and indispensable in histopathological practice, is related to two factors: 1) it allows to make thin and uniform sections of the tissues and 2) it blocks and keeps constant, in an inert material such as paraffin, the morphological, antigenic and molecular characteristics of the tissues.

[0008] Before being processed for paraffin-embedding, the isolated cells and biopsy fragments are fixed, mainly using aldehyde fixatives, such as formaldehyde or glyoxal, or alcohol-based fixatives, such as ethyl alcohol. The cells are generally fixed with alcoholic fixatives, while tissue fixation is currently carried out with aqueous fixatives containing aldehydes, in particular Neutral Buffered Formalin (NBF). Due to the toxicity and carcinogenicity of this reagent, it was proposed to replace formaldehyde with another aldehyde, glyoxal. The fixation of histological cells and tissues with acid-free glyoxal (Glyoxal acid-free = GAF) guarantees optimal structural and molecular preservation (Bussolati et al., 2017), allowing a replacement of a toxic and carcinogenic reagent such as formaldehyde with a non-toxic and environmentally friendly fixative. Comparative studies have shown that the fixative GAF guarantees results similar ("not inferior") to buffered formalin (Neutral Buffered Formalin) from a morphological and immunohistochemical point of view (Ryska et al., 2023). A better preservation of nucleic acids has also been detected (Berrino et al., 2024). These data make it possible to envisage the replacement of the routine use of NBF in histological practice with the fixative GAF, as it is non-toxic and non-carcinogenic and able to provide similar or even better results with respect to formalin.

[0009] The process of fixation and embedding in paraffin alters the molecular characteristics and it is well known that in tissues fixed in formalin and embedded in paraffin (Formalin-fixed Paraffin- embedded = FFPE tissue blocks) the antigenic characteristics, however altered, can be "returned" by means of Antigen Retrieval procedures (See Dabbs, 2021). Nucleic acids are also altered in tissues fixed in Formalin and embedded in Paraffin, especially due to nucleotide chain fragmentation phenomena (see Berrino et al, 2024). This phenomenon has considerable relevance in the correct execution of gene sequencing analyses and is a cause of impediment in carrying out NGS (Next Generation Sequencing) analyses in a fraction of cases.

[0010] The above antigenic and gene analysis difficulties increase in FFPE blocks with the passage of time. In fact, it is common practice to store paraffin blocks after diagnosis, as new investigations may become necessary over time, both for the emergence of new therapeutic possibilities that require specific antigenic or gene characterisation, and for retrospective studies. In Italy, as well as in many other countries, the law therefore requires the storage of FFPE blocks for a long time, at least 10 years.

[0011] The reasons why years-old archival blocks encounter difficulties in gene / antigen analysis over time are not known (Yi et al, 2020; Zimmermann, 2008). Some authors (Xie et al. 2011) have suggested that the rate of residual moisture has a deleterious effect on the degradation of archival tissues. Various works (Manjunath et al., 2022; Masuda et al., 1999; Kojima et al., 2014) suggest that the factors responsible for degradation of RNA and antigenic components in archival tissues are chemical alterations induced by formaldehyde fixation.

[0012] From the above, the problem of optimal preservation of cellular antigens and nucleic acids during and at the end of the necessary processes of fixation, dehydration, clarification and embedding in paraffin emerges, in order to allow molecular analyses useful for diagnostic and therapeutic purposes.

[0013] Description of the Invention

[0014] It has been found that the prevention or blocking of oxidation, obtainable by means of the addition of lipophilic antioxidants, allows to obtain advantageous effects in the processes of cell fixation and embedding of histological tissues, improving the preservation of antigens and preserving nucleic acid sequences.

[0015] The invention therefore relates to compositions necessary in the various subsequent steps of the histological process, therefore: for the fixation of cytological or histological preparations comprising a solution of an aldehyde fixative, preferably formaldehyde or acid-free glyoxal, and lipophilic antioxidant agents that counteract the effect of molecular damage and DNA fragmentation caused by oxidation.

[0016] Preferred aldehyde fixatives are neutral buffered formalin (NBF) and acid-free glyoxal (GAF).

[0017] Acid-free glyoxal solutions typically have weight concentrations comprised between 0.1 and 10%.

[0018] The fat-soluble antioxidants are preferably chosen from sterically hindered phenolic antioxidants, thiodipropionic acid dialkyl esters, hindered amines (HALS), propyl gallate, hydroxyphenyl benzotriazoles, 2-hydroxybenzophenones, stable phosphites.

[0019] Examples of sterically hindered phenolic antioxidants comprise: tetrakis[methylene(3,5-di-tert-butyl-4-hydroxyhydrocinnamate)]methane (Irganox 1010®); tetrakis[methylene(3,5-di-tert-butyl-4-hydroxyphenyl)propionate methane (Irganox L101®); octadecyl 3,5-di-tert-butyl-4-hydroxyhydrocinnamate (Irganox 1076®);

[0020] N,N ’ -hexamethylenebis(3 , 5 -di-tert-butyl-4-hydroxyhydrocinnamamide (Irganox 1098®); hexamethylenebis[3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate] (Irganox L109®);

[0021] 1.3.5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-l,3,5-triazine-2,4,6(lH,3H,5H)-trione (Irganox 245®);

[0022] 2,2'-thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (Irganox LI 15®);

[0023] 1.3.5 -Tris(3 , 5 -di- tert-butyl-4-hydroxybenzyl)- s-triazine ;

[0024] 2-(2H-benzotriazol-2-yl)-4,6-di-tert-butylphenol (Chimassorb 994®).

[0025] Examples of hydroxyphenyl benzotriazoles comprise phenol, 2-(5-chloro-2H-benzotriazol-2- yl)-6-(l,l-dimethylethyl)-4-methyl (Tinuvin 326®); branched and linear phenol, 2-(2H- benzotriazol-2-yl)-4-methyl-6-dodecyl-, (Tinuvin 571®).

[0026] An example of an antioxidant from the 2-hydroxybenzophenone class is methanone, 2- hydroxy-4-(octyloxy)-phenyl (Chimassorb 81®).

[0027] Examples of hindered light stabilising amines (HALS) comprise N,N'-bis(2,2,6,6-tetramethyl- 4-piperidinyl) sebacate (Tinuvin 770®); mixture of bis(l,2,2,6,6-pentamethyl-4-piperidyl) sebacate and methyl l,2,2,6,6-pentamethyl-4-piperidyl sebacate (Tinuvin 765®).

[0028] An example of an antioxidant from the class of stable phosphites is tris(2,4-di-tert-butylphenyl) phosphite (Irgafos 168®).

[0029] Other lipophilic antioxidants advantageously employable according to the invention comprise 4-( 1 -methyl- 1 -phenylethyl)N - [4-( 1 -methyl- 1 -phenylethyl)phenyl] amine (N augard 445®) , octylated / butylated diphenyl amine (Irganox L57®); alkylated phenyl alpha-naphthylamine (Irganox L06®). A preferred antioxidant mixture consists of Irganox 1010, Irganox 1076 and Irgafos 168.

[0030] The antioxidant agents or mixtures thereof can be used in a concentration by weight comprised between 0.01 and 5%.

[0031] The invention further relates to a method for fixing and preserving a sample of human, animal and plant cells and tissues for the purpose of histological or cytological evaluation, immunohistochemistry and gene analysis comprising:

[0032] • a) fixation of the sample by contact with the compositions of the invention;

[0033] • b) dehydration, clarification and embedding of the sample fixed in step a) in paraffin, wherein all or part of the reagents are added with fat- soluble antioxidants in single form or as a synergistic mixture thereof.

[0034] The dehydration can be performed by treatment with ethanol or isopropanol while the clarification is performed by treatment with xylol and paraffin solvents.

[0035] Another object of the invention relates to a kit for the fixation of histological and cytological preparations comprising a container containing the compositions of the invention, reagents for the dehydration and clarification of the fixed histological preparation and paraffin and any other reagents commonly used in histology, all or in part added with lipophilic antioxidants.

[0036] The antioxidants comprised in the kit, alone or in a synergistic mixture, are added to the histological reagents by fixation and / or dehydration, clarification and embedding in paraffin.

[0037] Detailed description of the invention

[0038] Antioxidant agents are a heterogeneous group of compounds that, added at low concentration, act by delaying or preventing the oxidation of other chemical compounds, neutralising free radicals and the harmful action of oxygen (Cadet et al., 2017; Azat Aziz et al, 2019; Santos-Sanchez et al, 2019: Zehiroglu et al, 2019; Flieger et al, 2021).

[0039] In addition to organic antioxidants, inorganic antioxidants have also been described, in particular selenium, zinc and manganese salts (Zago and Oteiza, 2001; Jarosz et al, 2017; Maywald and Rink, 2022). In order to exert an antioxidant action during the cell and tissue fixation process and throughout the various histological treatment steps for embedding in paraffin, different agents (with different solubility) must be employed in the various steps.

[0040] For the histological steps of dehydration, clarification and paraffin embedding performed in organic solvents, fat-soluble antioxidants soluble in mineral oils such as BHT, BHA, Chimassorb 81, Tinuvin 326, Tinuvin 765, Tinuvin 571, Irqanox L57, Irganox L109, Irganox L101, Irganox LI 15, Irganox L06, Irgafos 168, Naugard 445, BHT, BHD (Uyar and Meydan, 2023) are used according to the invention. These reagents were dissolved, in a concentration varying between 0.001 and 10% (preferably 0.1%) in the reagents used for paraffin embedding: 95% ethyl alcohol, absolute ethyl alcohol, xylol, paraffin for embedding dissolved at 65-70°C. These reagents are used for the paraffin embedding of previously fixed histological tissues. Subsequently, the tissues were embedded in blocks, using antioxidant- added paraffin (A.O. paraffin)

[0041] It is known that the use of multiple antioxidants instead of a single antioxidant can offer several advantages, for the same total amount. This strategy, often referred to as “antioxidant synergy,” exploits the different properties and mechanisms of action of various antioxidants to provide more complete protection against oxidative stress (Seeram et al., 2005; Kurin et al., 2012; Betts JW, Wareham, 2014; Commisso et al., 2017;).

[0042] The antioxidants can be used either individually, or preferably in synergistic mixtures in which each component has relative percentages ranging between 1 and 90%. The mixture, in percentage ranging between 0.01 and 3%, preferably in final percentage equal to about 0.1%, was added to the reagents used in the paraffin embedding process: ethyl alcohol, xylol, paraffin for embedding.

[0043] The antioxidants, in synergistic combination, were used on mouse or human tissues and on human cells in culture. After fixation (in NBF or GAF), the cells were treated by nucleic acid extraction. In parallel, the tissues were embedded in paraffin using reagents (alcohol, xylol and paraffin) added or not with single fat- soluble antioxidants or in synergistic association. Sections used for morphological, immunohistochemical or molecular biology studies were obtained from the paraffin blocks.

[0044] Similar studies were carried out on tissues included in paraffin, added or not with antioxidants, 6 months after embedding, so as to check whether the addition of antioxidants to the paraffin used for embedding had an effect on the degree of preservation of antigenic characteristics of tissue markers and on the preservation of the integrity of DNA and RNA sequences.

[0045] Human cells cultured in vitro (HEK 293 cells and HeLa cells) were fixed in 95% ethanol (Sigma Aldrich) added or not with fat-soluble antioxidants (propyl gallate, BHT or BHA in 0.1% proportion), or in NBF (Neutral Buffered Formalin, Diapath, Martinengo, BG) or in GAF (Glyoxal Acid-free, produced by ADD AX Biosciences, srl, Turin) added with zinc or selenium salts, ascorbic acid and / or propyl gallate, in amounts varying between 0.001 and 5%, preferably 0.1%. It was checked that the pH and the characteristics of the reagent did not vary with respect to the original NBF or GAF. After fixation, RNA preservation analysis was performed (see below). Cells fixed in ethanol, NBF, or antioxidant-free GAF were used as a control.

[0046] Similarly, fragments of mouse tissues or human tissues, both normal and pathological, were immersed, immediately after sampling, in 95% ethanol, added or not with antioxidants or fixed in NBF or fixed in GAF. After a period of 20-24 h of tissue fixation at room temperature, the tissues were processed for paraffin embedding (see below).

[0047] In parallel and for comparison, similar tissues were fixed in GAF (free of antioxidants), in commercial formalin or in 95% ethyl alcohol (see below).

[0048] Mouse tissues or human tissues, both normal and pathological, were immersed, immediately after sampling, in 95% ethyl alcohol added or not with fat-soluble antioxidant. Histological tissues fixed in GAF, (added or not with antioxidant), in 95% alcohol (added or not with antioxidant) or in NBF (added or not with antioxidant), were processed by paraffin embedding using the HistoCore PEGASUS Plus Tissue Processor from Leica Biosystems or the HistoPro 300 processor from Histolyne Laboratories, Pantigliate (MI) Italy.

[0049] The reagents present in the processors (95% ethyl alcohol, absolute ethyl alcohol, xylol, paraffin) were added or not with fat-soluble antioxidant in isolated form (propyl gallate, BHT or BHA or Irganox 1010 (Sigma) or preferably in the form of a synergistic mixture (in concentrations ranging between 0.001 and 3%, preferably 0.1%).

[0050] The samples were finally embedded in paraffin blocks, using embedding paraffin (Sigma) added or not with antioxidant.

[0051] Histological sections of tissues fixed in ethanol + antioxidant or in NBF + antioxidant or in GAF+ antioxidant and embedded in paraffin using antioxidant- added reagents were compared with sections of the same tissues, fixed in NBF or GAF or ethanol and embedded in paraffin according to the routine method, with antioxidant- free reagents. The sections were stained in hematoxylin-eosin. Both immunohistochemistry reactions were performed for the search of various antigens (in particular Ki67, Cytokeratin, ER, PgR, HER2, PDL1, CD3, CD45).

[0052] Nucleic acids (DNA and RNA) were extracted from histological tissues fixed in NBF or GAF added or not with antioxidant or in ethanol added or not with antioxidant and embedded in paraffin, alternatively processed with reagents added with antioxidants or according to the traditional method. The amount of extracted nucleic acid, the length (in bp) of the chain and the integrity of the nucleotide sequence (DIN = DNA Integrity Number or RIN = RNA Integrity Number) were checked. These studies were performed both immediately after the embedding process and in blocks preserved for a long time (6 months).

[0053] As preservation criteria of the nucleic acid sequences (DNA and RNA), the percentage evaluation of fragments of different length (in base pairs: bp) and Integrity numbers (respectively DIN = DNA Integrity Number and RIN = RNA Integrity Number) were used, analysed by the AGILENT equipment following dedicated algorithms.

[0054] The data obtained indicate that the addition of antioxidants to the fixative GAF, formalin NBF or ethanol during fixation and the addition of fat-soluble antioxidants to the reagents used for dehydration, clarification and embedding in paraffin and to paraffin itself allow better antigenic conservation and preservation even over time of the nucleotide sequences of histological tissues. The addition of antioxidants limited to a single step of the histological process (alternatively, during fixation or during paraffin-embedding) produces a marked improvement in the degree of DNA preservation. Optimal results are however obtained when all the steps of the histological process (fixation, dehydration, clarification, embedding in paraffin) are carried out using reagents with added antioxidants. The following examples illustrate the invention in greater detail.

[0055] Example 1. Effect of antioxidant addition to reagents by fixation in alcohol and / or embedding in paraffin on DNA preservation

[0056] In tissues fixed in 95% alcohol free of antioxidants, and subsequently embedded in paraffin using 95% alcohol, absolute alcohol, xylol and paraffin to which a mixture of BHT, BHA and Irganox 1010® 0.1% had been added, the extracted DNA shows better characteristics (less fragmentation; longer fragment length) with respect to tissues fixed in 95% alcohol and processed according to the traditional method (antioxidant- free reagents). However, an optimal result is obtained by using a process in which all the reagents (95% alcohol for fixation, 95% and absolute alcohol for dehydration, xylol for clarification and paraffin for embedding) were added with the antioxidants BHT, BHA and Irganox 1010.

[0057] AO 57; Mouse kidney, fixation in 95% ethyl alcohol, free of antioxidants. Subsequent embedding in paraffin according to the routine procedure. Sections from which DNA was extracted were obtained from the paraffin blocks.

[0058] AO6: Mouse kidney, fixation in 95% ethyl alcohol, free of antioxidants. Subsequently, Processed by paraffin embedding using reagents added with BHT, BHA and Irganox 1010.

[0059] AO 16, Mouse kidney, fixation in 95% ethyl alcohol, with antioxidant added with BHT, BHA and Irganox 0.1%. Subsequently, processed by paraffin embedding, in which all the reagents (dehydration + clarification + paraffin embedding) had been added with a mixture of BHT and Irganox 1010 0.1%.

[0060] For DNA extraction, quantification and quality evaluation, nine sections (5 pm thick) were obtained from paraffin embedded tissue blocks. The paraffin was removed from the sections with 1 ml of xylene. .After overnight incubation at 56°C with proteinase K, the DNA was isolated from five sections using the MagCore Genomic DNA FFPE kit on the MagCore Automatic Extraction Tool (RBC Bioscience, Taiwan) according to the manufacturer's protocol.

[0061] DNA integrity was assessed using the high sensitivity DNA analysis kit (Agilent Technologies, Santa Clara, CA) on HS DNA chips. The samples were diluted to 2 ng / pL and DNA length analysis was performed according to manufacturer's instructions.

[0062] As highlighted in Figure 1: ( AO57) in tissues fixed in 95% ethyl alcohol and embedded in paraffin in the absence of antioxidants (AO), the sizes of the obtainable DNA fragments (in analogy to what is described in the literature) are largely between KMX) and 5000 bp, indicating intense fragmentation. (DIN - 3.5).

[0063] The use of antioxidants added in the reagents for dehydration, clarification and paraffin embedding, on the contrary', (Figure 1 AO 6) allows to obtain much better preserved fragments up to 20,000 bp, (DIN = 5.9).

[0064] Optimal results (Figure 1: AO 16) were obtained in tissues where all the processes (fixation, dehydration, clarification, paraffin embedding) were carried out in the presence of antioxidant (AO). The majority of the DNA fragments have a length greater than 5000 bp, as indicated by Figure 1.

[0065] In Figure 1 the black arrow indicates DNA sequences less than 5000 bp in length and the grey arrow indicates DNA sequences greater than 5000 bp in length.

[0066] From the results shown in Figure 1, it is evident that the use of antioxidants conditions the length of the nucleotide fragments. Optimal genomic DNA preservation results are obtained by adding the antioxidant mixture to all the steps before and during paraffin-embedding.

[0067] Example 2. Effect of antioxidants added to GAF for DNA preservation

[0068] Fresh mouse and human tissues (kidney, liver, colon, colon and breast carcinoma) destined for disposal as redundant with respect to diagnostic needs, were used for fixation. Adjacent sections of tissue fragments were fixed respectively in GAF fixative (Addax Biosciences s.r.l., Turin, Italy) and in GAF fixative added with antioxidants (synergistic mixture comprising ascorbic acid, zinc chloride and propyl gallate) in 0.1% proportion. The tissues remained in the respective fixatives for 20 hours, at room temperature, then processed for embedding in paraffin (Leica inclusion apparatus: Leica ASP 300 S). The sections were collected on slides.

[0069] For DNA extraction, quantification and quality evaluation, nine sections (5 pm thick) were obtained from paraffin embedded tissue blocks. The paraffin w'as removed from the sections with 1 ml of xylene. After overnight incubation at 56°C with proteinase K, the DNA was isolated from five sections using the MagCore Genomic DNA FFPE kit on the MagCore Automatic Extraction Tool (RBC Bioscience, Taiwan) according to the manufacturer's protocol.

[0070] DNA integrity was assessed using the high sensitivity DNA analysis kit (Agilent Technologies, Santa Clara, CA) on HS DNA chips. The samples were diluted to 2 ng / pl. and DNA length analysis was performed according to manufacturer’s instructions.

[0071] As shown in Figure 2a in tissues fixed in GAF, the dimensions of the obtainable DNA fragments (in analogy with what is described in the literature) are largely between 1000 and 5000 bp, indicating intense fragmentation and DIN of 3.8. On the contrary, fixing in GAF added with a synergistic mixture of antioxidants (Figure 2 b) allows to obtain much better preserved fragments up to 20,000 bp and with DIN of 5.6.

[0072] Example 3. Effect of antioxidant addition to paraffin inclusion reagents in tissues fixed in NBF on DNA preservation

[0073] Mouse tissues (kidney, liver) were fixed in NBF for 24 hours at room temperature. Subsequently, the tissues were embedded in paraffin using the automatic processor HistoCore PEGASUS Plus Tissue Processor from Leica Biosystems), wherein alternatively embedding reagents (95% ethanol, absolute ethanol, xylol, paraffin for embedding) were added or not with BHT, BHA and Irganox 1010 antioxidant mixture in 0.1% percentage.

[0074] Nucleic acids (DNA and RNA) were extracted from histological tissues fixed in NBF and embedded in paraffin, alternatively processed with reagents added with antioxidants or according to the traditional method. The amount of extracted nucleic acid, the length (in bp) of the chain and the integrity of the nucleotide sequence (DIN = DNA Integrity Number or RIN = RNA Integrity Number) were checked.

[0075] In Figure 3, AO20 refers to tissues fixed in neutral buffered formalin (NBF) free of antioxidants and processed according to the current procedure (antioxidant-free reagents), the DNA is strongly altered and fragmented (majority of fragments < 5000 bp; DIN (DNA Integrity Number) = 4.2.

[0076] AO 19 refers to tissues fixed in formalin (NBF) processed with reagents (alcohol, xylol, paraffin containing a mixture of BHT, BHA, and Irganox 1010. The DNA is less fragmented (majority of fragments > 5000 bp; DIN (DNA Integrity Number) = 6.1.

[0077] Example 4. Preservation of HER2 antigens and DNA over time

[0078] Tissue fragments (wall of large intestine and colon carcinomas; breast tissues and breast carcinoma) removed for operative interventions were used. The fragments were in excess with respect to diagnostic requirements.

[0079] Fragments freshly assembled at the dissection table were about 3-4 mm thick and about 2 cm wide, thus being similar to those currently used for diagnostic purposes. The fragments, introduced in special boxes for identification purposes, were immersed in fixative liquid.

[0080] The fragments were immersed in GAF fixative or, alternatively, in antioxidant- added GAF fixative (synergistic mixture comprising ascorbic acid, zinc chloride and propyl gallate) in 0.1% proportion. The fixation at room temperature was continued for 24 hours (as used in routine fixation), then the fragments in the respective identification boxes were processed as usual with dehydration and embedding in paraffin (Leica Processor, Milan) or, alternatively in a processor in which reagents (95% alcohol, absolute alcohol, xylol, paraffin) were added with a mixture of BHT, BHA and Irganox 1010 in 0.1% proportion. Histological sections were obtained from the relative paraffin blocks, which were stained in Hematoxylin-Eosin, as per routine, in parallel.

[0081] Immunohistochemical stains for the following antigens were performed on histological sections of the fragments fixed with GAF (prep.l) and with GAF added with antioxidants and embedded in paraffin using reagents added with BHT and Irganox 1010: Broad spectrum cytokeratin; Cytokeratin CK19, CDX2, Ki67, HER2. FISH (Fluorescent in situ Hybridisation) reactions were also carried out on these sections for the following genes: AEK and HER2. On the above material (fixed in parallel in GAF, as per routine, and in GAF + ascorbic acid), gene analyses were also carried out by sequencing for the evaluation of K-RAS and its mutations.

[0082] Results

[0083] Upon microscopic examination, tissues fixed in GAF and those fixed in GAF + antioxidants exhibited optimal fixation.

[0084] The immunohistochemical reactions for CK 19, CDX2 and Ki67 and HER2 performed in parallel on GAF and GAF + antioxidant fixed tissues gave overlapping results.

[0085] Similarly, analyses performed in FISH and with sequencing to assess the AEK, HER2 and K-RAS genes showed similar results.

[0086] Six months after the tissues were embedded, the paraffin blocks (which had been conserved at room temperature) were sectioned again.

[0087] The results indicate that for some antigens (CK 19, CDX2) similar results were obtained in tissues fixed in GAF or in tissues fixed in GAF + antioxidants (and embedded in paraffin + antioxidants), and similar to those obtained on the same tissues 6 months earlier. In contrast, other antigens such as Ki67 and HER2 showed decreased antigenic reactivity in GAF-fixed tissues. This effect was particularly noticeable after the sections had been left in air, unstained, for a week. In tissues fixed in GAF + antioxidants (and processed in paraffin +BHT, BHA and Irganox 1010), however, this loss of antigenic reactivity was not observed and the results were comparable to those obtained 6 months earlier. DNA integrity extraction and analyses were carried out on the same sections.

[0088] For DNA extraction, quantification and quality evaluation, sections (5 pm thick) were obtained from paraffin embedded tissue blocks. The paraffin was removed from the sections with 1 ml of xylene. After overnight incubation at 56°C with proteinase K, the DNA was isolated from five sections using the MagCore Genomic DNA FFPE kit on the MagCore Automatic Extraction Tool (RBC Bioscience, Taiwan) according to the manufacturer's protocol.

[0089] DNA integrity was assessed using the high sensitivity DNA analysis kit (Agilent Technologies, Santa Clara, CA) on HS DN A chips. The samples were diluted to 2 ng / pL and DNA length analysis was performed according to manufacturer’s instructions.

[0090] In tissues fixed in GAF, the dimensions of the obtainable DNA fragments (in analogy with what is described in the literature) are largely between 1000 and 5000 bp, indicating intense fragmentation, On the contrary, GAF + antioxidant fixation and processed and preserved for 6 months in paraffin added with antioxidant resulted in much better preserved fragments (up to 20,000 bp), similar to those observed in the same tissues 6 months earlier.

[0091] In conclusion, the data obtained indicate that the fixation in fixative added with antioxidants, processed and preserved for a long time in paraffin added to a mixture of fatsoluble antioxidants allows to preserve the antigenic characteristics of tissue markers over time and to avoid the degradation of nucleic acid chains, in particular DNA.

[0092] Example 5. Effect of the addition of antioxidants (AO) to the fixative GAF, in order to obtain a better conservation of RNA in cultured cells.

[0093] Hek293T cells, cultured in RPMI+10% foetal bovine serum (Sigma- Aldrich), were centrifuged at 300-400 x g for 3 minutes to remove the medium, and then fixed by adding 1 mL of GAF plus 0.1% AO (synergistic mixture of water-soluble, inorganic and organic antioxidants) (a) or only GAF (b)" at room temperature for 10 minutes. After centrifugation (400 x g for 3 minutes) RNA was extracted using TRIzol (Invitrogen) following the manufacturer's instructions. The total isolated RNA was eluted with 20 uL nuclease-free water (Sigma-Aldrich) and subjected to automated electrophoresis by means of TapeStation (Agilent) for sample RNA quality control.

[0094] Figures 4a and 4b demonstrate at TapeStation electrophoresis analysis the presence of high quality RNA (RIN 10) in Hek239 cells fixed in GAF + antioxidants 0.1%, compared to cells fixed in GAF alone, wherein the presence of RNA degraded with RIN 2 is highlighted.

[0095] Bibliography van der Lem T, de Bakker M, Keuck G, Richardson MK. Wilhelm His Sr. and the development of paraffin embedding. Pathologe. 2021 Nov;42(Suppl 1):55~61. doi: 10.1007 / s()0292-021-00947-4. Epub 2021 Jul 8. PMID: 34236458; PMCID: PMC8571243.

[0096] Manjunath HS, Al Khulaifi M, Sidahmed H, Ammar A, Vadakekolathu J, Rutella S, Al- Mohannadi MJ, Elawad M, Mifsud W, Charles A, Maccalli C, Tomei S. Gene Expression Profiling of FFPE Samples: A Titration Test. Technol Cancer Res Treat. 2022 Jan- Dec;21: 1533033822U29710. doi: 10.1177 / 15330338221129710. PMID: 36415121.

[0097] Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res. 1999;27:4436-4443.

[0098] Kojima K, April C, Canasto-Chibuque C, et al. Transcriptome profiling of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissue for disease classification. PLoS One. 2014;9: e86961.

[0099] Xie R, Chung JY, Ylaya K, Williams RL, Guerrero N, Nakatsuka. N, Badie C, Hewitt SM. Factors influencing the degradation of archival formalin-fixed paraffin-embedded tissue sections. J Histochem Cytochem. 2011 Apr;59(4):356-65. doi: 10.1369 / 0022155411398488. Epub 2011 Feb 10. PMID: 21411807: PMCID: PMC320H47.

[0100] Yi QQ, Yang R, Shi JF, Zeng NY, Liang DY, Sha S, Chang Q. Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity. J Int Med Res. 2020 Jun;48(6):300060520931259. doi: 10.1177 / 0300060520931259. PMID: 32567435; PMCID: PMC7309401.

[0101] Zimmermann J, Hajibabaei M, Blackburn DC, Hanken J, Cantin E, Posfai J, Evans TC Jr. DNA damage in preserved specimens and tissue samples: a molecular assessment. Front Zool. 2008 Oct 23; 5: 18. doi: 10.1186 / 1742-9994-5-18. PMID: 18947416; PMCID: PMC2579423.

[0102] Dabbs D.J., Diagnostic Immunohistochemistry. Theranostic and Genomic Applications. 6th Edition, 2021. Elsevier.).

[0103] Berrino E., Bellomo SE, Chesta A, Detillo P, Bragoni A, Gagliardi A, Naccarati A, Cereda M, Witel G, Sapino A, Bussolati B, Bussolati G, Marchio C., Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive. Genomic Profiling. Lab Invest 104 (2024) 100280.

[0104] Cadet J, Davies KJ A, Medeiros MH, Di Mascio P, Wagner J R. Formation and repair of oxidatively generated damage in cellular DNA. Free Radic Biol Med. 201 / Jun; 107: 13 34.doi: 10.1016 / j.freeradbionied.2016.12.049. Epub 2017 Jan 2. PMID: 28057600; PMCID:

[0105] PMC5457722.

[0106] Cadet J, Davies KJA. Oxidative DNA damage & repair: An introduction. Free Radic Biol Med. 2017 Jun: 107 :2-12. doi: 10.1016 / j.freeradbiomed.2017.03.030.

[0107] Uyar, B. and Meydan, I. (2023), "Performance of commercial antioxidants in white mineral oils”, Industrial Lubrication and Tribology, Vol. 75 No. 2, pp. 221- 229. https: / / doi.org / 10.! 108 / ILT-09-2022-208:

[0108] Bussolati G, Annaratone L, Berrino E, Miglio U, Panero M, Cupo M, Gugliotta P, Venesio T, Sapino A, Marchib C. Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples. PLoS One. 2017 Aug 10;12(8): e0182965. doi: 10.1371.

[0109] Ryska A, Sapino A, Landolfi S, Valero IS, Cajal SRY, Oliveira P, Detillo P, Lianas L, Frexia F, Nicolosi PA, Monti T, Bussolati B, Marchid C, Bussolati G. Glyoxal acid-free (GAF) histological fixative is a suitable alternative to formalin: results from an open-label comparative non-mferiority study. Virchows Arch. 2023 Nov 24. doi: 10.1007 / s00428-023-03692-6.

[0110] Flieger J, Flieger W, Baj J, Maciejewski R. Antioxidants: Classification, Natural Sources, Activity / Capacity Measurements, and Usefulness for the Synthesis of Nanoparticles. Materials (Basel). 2021 Jul 25;14(15):4135. doi: 10.3390 / mal4154135.

[0111] Zehiroglu C, Ozturk Sarikaya SB. The importance of antioxidants and place in today's scientific and technological studies. J Food Sci Technol. 2019 Nov;56(ll ):4757-4774. doi: 10.1007 / sl3197-019-03952-x.

[0112] Azat Aziz M, Shehab Diab A and Abdulrazak Mohammed A., Antioxidant Categories and Mode of Action. Chapter in “Antioxidants” ed. by Eniad Shalaby. IntechOpen, 2019 DOI: 10.5772 / in techopen.83544.

[0113] Santos -Sanchez N.F., Salas-Coronado R., Villanueva-Canongo C. and Hernandez- Carlos B. Antioxidant Compounds and Their Antioxidant Mechanism. Chapter in “Antioxidants” ed. by Emad Shalaby. IntechOpen, 2019 DOI: 10.5772 / intechopen.85270.

[0114] Maywald M, Rink L. Zinc in Human Health and Infectious Diseases. Biomolecules. 2022 Nov 24; 12( 12 ): 1748. doi: 10.3390 / bioml2121748.

[0115] Jarosz M, Olbert M, Wyszogrodzka G, Mlyniec K, Librowski T. Antioxidant and antiinflammatory effects of zinc. Zinc-dependent NF-KB signaling. Inflammopharmacology. 2017 Feb;25(l): 11-24. doi: 10.1007 / sl0787-017-0309-4.

[0116] Zago .MP, Oteiza Pl. The antioxidant properties of zinc: interactions with iron and antioxidants. Free Radio Biol Med. 2001 Jul 15;31(2):266-74. doi: 10.1016 / s0891 - 5849(01)00583-4.

[0117] Kurin E, Mucaji P, Nagy M. In vitro antioxidant activities of three red wine polyfenolos and their mixtures: an interaction study. Molecules. 2012 Dec 3;17(12): 14336-48. doi: 10.3390 / molecules 171214336.

[0118] Betts JW, Wareham DW. In vitro activity of curcumin in combination with epigalloca techin gallate (EGCG) versus multidrug-resistant Acinetobacter baumannii. BMC Microbiol. 2014 Jun 27;14: 172. doi: 10.1186 / 1471-2180- 14-172.

[0119] Commisso M, Bianconi M, Di Carlo F, Poletti S, Bulgarini A, Munari F, Negri S. Stocchero M, Ceoldo S, Avesani L, Assfalg M, Zoccatelii G, Guzzo F. Multi-approach metabolomics analysis and artificial simplified phytocomplexes reveal cultivar-dependent synergy between polyfenolos and ascorbic acid in fruits of the sweet cherry (Prunus avium L.). PLoS One. 2017 Jul 21:12(7): eO18O889. doi:10.1371 / joumal.pone.0180889.

[0120] Seeram NP, Adams LS, Henning SM, Niu Y, Zhang Y, Nair MG, Heber D. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyfenolos as found in pomegranate juice. J Nutr Biochem. 2005 Jun;16(6):360-7. doi: 10.1016 / j.j nutbio.2005.01.006.

Claims

CLAIMS1. Compositions for the fixation of histological and cytological preparations comprising a solution of an aldehyde fixative, preferably formaldehyde or acid-free glyoxal, and antioxidant agents that counteract the effect of molecular damage and DNA fragmentation caused by oxidation.

2. Compositions according to claim 1 wherein the fixative solution is a buffered aqueous solution having a pH between 7.0 and 8.0 (preferably 7.2-7.4) and the antioxidant agents are single or a synergistic mixture of antioxidants selected from water-soluble inorganic (such as zinc or selenium salts) and / or organic antioxidants, preferably ascorbic acid or propyl gallate.

3. Compositions according to claim 1 wherein the solution is an alcoholic solution, preferably ethanol or methanol and the antioxidant is lipophilic or water-soluble.

4. Compositions according to any one of claims 1 to 2 wherein the fixing agent is Neutral Buffered Formalin (NBF).

5. Compositions according to any one of claims 1 to 2 wherein the fixing agent is Glyoxal Acid Free (Glyoxal Acid Free = GAF).

6. Compositions according to claim 5 wherein the glyoxal solution has a weight concentration of between 0.1 and 10%.

7. Compositions according to claim 1-6 wherein the antioxidant agent has a weight concentration of between 0.01 and 5%.

8. A method for fixing and preserving a sample of human, animal and plant cells and tissues for the purpose of histological or cytological evaluation, immunohistochemistry and gene analysis comprising:• (a) fixation of the sample by contact with the compositions of claims 1-7;• (b) dehydration, clarification and embedding of the sample fixed in step (a) in paraffin, wherein all or part of the reagents are fortified with fat- soluble antioxidants in single form or as a synergistic mixture thereof.

9. A method according to claim 8 wherein the fat-soluble antioxidants are chosen from dialkyl ester of thiodipropionic acid), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (2[3] - t-butyl-4-hydroxyanisole), propyl gallate or are combined in a synergistic mixture thereof.

10. A method according to claim 9 wherein the dehydration is carried out by. treatment with ethanol or isopropanol.

11. A method according to claim 9 wherein the clarifying is carried out by treatment with xylol and paraffin solvents.

12. A kit for the fixation of histological and cytological preparations comprising a container containing the composition of claims 1-7, and reagents for dehydration and clarification of the fixed histological preparation and paraffin and any other reagents commonly used in histology, all or in part additivated with lipophilic antioxidants.

13. Kits according to claim 12, containing single antioxidants or in synergistic mixture, to be added to histological reagents for fixation and / or dehydration, clarification and inclusion in paraffin.

Images