HK1165553B - Use of a bis-maleic anhydride cross-linking agent for fixation of a cell or tissue sample - Google Patents

Description

Use of a bismaleic anhydride cross-linking agent for fixing a cell or tissue sample

Technical Field

The present application relates to novel bis-maleic anhydrides. The present application relates in particular to the following findings: the bismaleic anhydride cross-linking agent may be used to preserve/fix a cell sample or a tissue sample. Due to its great advantage, the bismaleic anhydride cross-linker can be used in methods that require fixation of a cell or tissue sample and at the same time require a fixative with little effect on the detection of proteins or nucleic acids in subsequent procedures, such as immunohistochemistry, fluorescence in situ hybridization or RT-PCR. The present application also demonstrates that the use of a bismaleic anhydride cross-linking agent as a fixative greatly facilitates subsequent detection of an analyte of interest in a previously fixed cell or tissue sample.

Background

To date, there is no generally applicable, "ideal" way to prepare a cell sample or tissue sample, e.g., for immunohistochemistry or detection of a nucleic acid of interest, respectively. The reversibility of the immobilization and of the negative effects introduced by the immobilization has a major influence on the detectability of the polypeptide antigen and the nucleic acid, respectively, and on the reproducibility of the results obtained therefrom.

For successful immunostaining of antigens in a cell sample or tissue sample, at least three criteria are met: a) retention of the antigen in its place (retention), b) accessibility of the antigen (accessibility) and c) correct construction/preservation of the antigen/epitope of interest. It appears that none of the fixation and/or detection operations fully satisfy all three criteria. For operations known in the art, the best performance for one or both of these criteria results in a cost of reduced performance for at least one other criterion.

Some fixatives are available and used in the routine of clinical pathology laboratories, such as glutaraldehyde, formaldehyde and acetone, or other organic solvents. However, the vast majority of fixing operations are based on the use of crosslinking agents, such as formaldehyde. The fixative solution is typically an aqueous formaldehyde solution containing sodium phosphate, which is formulated to provide a buffer to a pH of 7.2-7.6 (minimal pH change upon addition of a small amount of strong acid or base) and an approximately isotonic solution (a solution with the same osmotic pressure as the mammalian extracellular fluid, typically based on physiological saline).

According to the state of the art, it is necessary that the fixing operation is exactly correct.

If the fixing time is too short, only coagulation of the protein by the alcohol used to dehydrate the sample occurs, and no fixing occurs. This may, for example, negatively affect the preservation of the tissue morphology or impair the long-term preservation stability.

With prolonged formaldehyde fixation, the cross-linked protein molecules form a dense network that can impair the penetration of paraffin or/and the entry of antibody molecules. Thus, the antigen of interest may be reversibly or even irreversibly masked. Further, epitopes may be chemically modified ("destroyed") (e.g., by reaction with formaldehyde).

Furthermore, it is known that most of the enzyme activities are impaired after formaldehyde fixation.

As mentioned above, the fixation in formaldehyde is most widely used in clinical pathology. The main reason for this is most probably that by fixation with formaldehyde, the antigen of interest is captured in the position it occupied in the living body. By virtue of the methylene bridge introduced after formaldehyde fixation, the morphology of the cell sample or tissue sample is also well preserved. However, these positive effects entail a cost in terms of sample permeability and result in a fixation that causes changes in accessibility and/or conformation of the antigen/epitope of interest, damage to the nucleic acid and inactivation of the enzymatic activity.

Cross-linking resulting from formaldehyde fixation may mask or damage the epitope, resulting in false negative immunostaining. This failure is even more likely to occur when the primary immunizing agent is a monoclonal antibody than when polyclonal antisera is used. This is why many attempts have been made and found in the literature in connection with treatments to reverse the formaldehyde fixation effect.

For long term storage, fixed cell or tissue samples typically need to be dehydrated and embedded in a suitable embedding medium. Paraffin embedding is generally preferred over plastic embedding or cutting of non-embedded samples with a vibrating microtome (vibrating microtome) or in a cryostat.

As mentioned above, to some extent, all fixed operations represent various compromise schemes. Best morphological preservation often comes at the cost of accessibility to the antibody or destruction of the antigen or epitope thereon.

However, and equally important to the present invention, not only is there a high variability introduced during the preparation of the sample (e.g. fixation of the sample or further processing such as embedding with paraffin), but there may also be an even greater variability, which is caused by various means and pathways to restore immunoreactivity or accessibility in nucleic acid detection, i.e. in a procedure known as antigen retrieval.

Despite the widespread and important use of, for example, immunohistochemical methods or methods for detecting nucleic acids of interest in cell or tissue samples, there is still a great need for further improvements. Such improvements may for example relate to a gentler fixation of the cell sample or tissue sample, an improvement in antigen retrieval or/and a better comparability and reproducibility of the results, and may even relate to the possibility of using antibodies in which the corresponding antigen or epitope is destroyed in standard procedures such as formaldehyde fixation.

The inventors of the present invention have surprisingly found that the use of bismaleic anhydride as a cross-linking agent in the preparation/fixation of a cell or tissue sample has great advantages and may and will lead to significant improvements with respect to at least one or even several of the problems known in the art.

Disclosure of Invention

The present invention relates to a method for in vitro fixation of a cell sample or a tissue sample, wherein said cell sample or said tissue sample is incubated with a bismaleic anhydride cross-linker, thereby fixing said cell sample or said tissue sample.

The present application further discloses a method of preserving a cell sample or tissue sample, the method comprising the steps of fixing the tissue sample with a bismaleic anhydride cross-linking agent and embedding the fixed sample in paraffin.

The present invention also discloses a method for performing immunohistochemistry on a cell sample or a tissue sample, the method comprising the steps of: fixing a cell or tissue sample with a bismaleimide cross-linker, embedding the fixed sample in paraffin, deparaffinizing the sample, removing the bismaleimide cross-linking and immunodetecting the epitope of interest.

The present application also describes a method for the in vitro detection of a nucleic acid of interest by in situ hybridization on a cell sample or a tissue sample, said method comprising the steps of: fixing a cell or tissue sample with a bismaleimide cross-linker, embedding the fixed sample in paraffin, deparaffinizing the sample, removing the bismaleimide cross-linking and detecting the nucleic acid of interest by in situ hybridization.

The present application further presents a method for the in vitro detection of a nucleic acid of interest by RT-PCR in a cell sample or a tissue sample, said method comprising the steps of: fixing a cell or tissue sample with a bismaleimide cross-linker, embedding the fixed sample in paraffin, deparaffinizing the sample, removing the bismaleimide cross-linking and detecting the nucleic acid of interest by performing RT-PCR.

The present application also shows that both the polypeptide of interest and the nucleic acid of interest can be detected in the same sample prepared from a cell sample or a tissue sample based on the method of the present invention. The present invention relates to a method for the in vitro detection of at least one polypeptide of interest and the detection of at least one nucleic acid of interest by immunohistochemistry in a test sample comprising a cell sample or a tissue sample, said method comprising the steps of: fixing a cell or tissue sample with a bismaleic anhydride cross-linker, embedding said fixed sample in paraffin, deparaffinizing said sample, removing the bismaleic amide cross-linking and immunodetecting at least one polypeptide of interest and detecting at least one nucleic acid of interest by performing RT-PCR or fluorescence in situ hybridization.

The invention further relates to the use of a bismaleic anhydride cross-linking agent for fixing a cell or tissue sample and to the use of a bismaleic anhydride cross-linking agent for the manufacture of a fixing agent for fixing a cell or tissue sample.

Detailed Description

In a first embodiment, the invention relates to a method for in vitro fixation of a cell sample or a tissue sample, wherein said cell sample or said tissue sample is incubated with bismaleic anhydride of formula I,

wherein R1 and R2 are independently selected from: hydrogen, methyl, ethyl, propyl, isopropyl and butyl, wherein X is a linker between 1 and 30 atoms in length, and thereby immobilizing the cell sample or the tissue sample.

Sometimes the compound of formula I will be referred to simply as a "bismaleic anhydride crosslinker" or "bismaleic anhydride".

It is to be understood that "method for immobilization" in the present invention means "treatment method", "immobilization" equals "crosslinking", "immobilized" will be alternatively expressed as "crosslinked", and "immobilization" by and in the method of the present invention relates to "crosslinking comprising (comprising) bismaleamides" or "crosslinking comprising (s)) bismaleamides". From a convenience point of view, and in view of the fact that a person skilled in the art is fully aware of the meanings attached to terms such as immobilisation, fixative or fixing, and for convenience these terms will generally only be used throughout the specification.

It is also to be understood that in a scientifically correct sense, it is not the cell sample or tissue sample that is fixed or cross-linked, but the biomolecules contained in such a sample are cross-linked or immobilized in the immobilization method as disclosed in the present invention. The cross-links in these biomolecules may be intramolecular cross-links as well as intermolecular cross-links.

If at least two maleic anhydrides, which are linked to each other by a linking group X (at least bismaleic anhydride), are reacted with at least two primary amines, at least two amide bonds are formed and the at least two primary amines are crosslinked by the at least two amide bonds and by the linking group X. For convenience purposes, this type of crosslinking will be referred to hereinafter as "crosslinking of bismaleimides".

The articles "a" and "an" as used in this application are intended to mean one or more than one (i.e., at least one) of the grammatical object of the article.

The expression "one or more" or "at least one" means 1 to 20, preferably 1 to 15, further preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

The present invention is based on the surprising and surprising discovery that bismaleic anhydride can be used to gently and reversibly fix cell or tissue samples. Without wishing to be bound by theory, it is believed that the great advantage is due to the fact that a) the bismaleic anhydride crosslinker forms bismaleimide crosslinks quickly and efficiently, b) the crosslinker, bismaleic acid, can be easily removed whenever desired and/or c) the possibility of negative effects such as distortion or destruction of epitopes is reduced by using a linker X of suitable length compared to the relatively short crosslinker formaldehyde.

To fully understand the great advantages of the methods of the present application, it would be useful to discuss in more detail those most frequently used tissue fixation procedures, i.e., procedures based on the use of formaldehyde as a fixative.

Formaldehyde-based fixatives are typically obtained from formalin, which is a solution containing 37% w/w (═ 40% w/v) formaldehyde in water. The working fixative was formalin at ten fold dilution (4 grams per 100 ml). Paraformaldehyde can be used as a starting material to prepare nearly identical composition solutions. Paraformaldehyde is a solid polymer that changes to formaldehyde when heated (in slightly basic water) to 60 ℃. Although the phrase "fixed in 4% paraformaldehyde" is commonly used in the literature, it is not entirely true. Most of the formaldehyde in dilute aqueous solutions exists in the form of methylene glycol, which is formed by the addition of molecular water to a formaldehyde molecule:

the concentration of free formaldehyde in the fixative solution is very low. However, it is the free formaldehyde that enters the fixed chemical reaction, not the methylene glycol.

The fixation by formaldehyde mainly chemically reacts with primary amines, such as primary amines present in polypeptides.

Formaldehyde fixation is not shown as a two-step process. In the first step, formaldehyde is rapidly bound. By this rapid binding, formaldehyde may stop autolysis, but it does not stabilize the fine structure of the tissue and does not provide effective protection against the destructive effects of subsequent treatments such as paraffin embedding.

In the first stage (hours), the formaldehyde molecule binds to various parts of the protein molecule, in particular the lysine side chain amino groups and the nitrogen atom of the peptide bond:

in the second stage, the bound hydroxymethyl groups react with other nitrogen atoms of the same or nearby protein molecule, thereby creating methylene crosslinks or bridges. These methylene groups (-CH)₂-) the bridge is stable and explains the insolubility and rigidity of protein-containing tissues that have been fixed by formaldehyde. One possible reaction is:

protein-NH-CH₂OH+NH₂Protein → protein-NH-CH₂-NH-protein + H₂O

In addition to the reaction with proteins, formaldehyde can also bind to some basic lipids.

The chemical reaction discussed above, which requires "complete" formaldehyde fixation for both steps, may potentially explain some of the difficulties faced when working with formaldehyde-fixed materials. Brief exposure to formaldehyde does not result in sufficient cross-linking to immobilize small proteins or other small analytes. Too long a fixation can lead to irreversible damage, especially due to the formation of excessive methylene bridges. For reasonable preservation of the structure, it is generally accepted that the sample should be preserved in formaldehyde solution at least overnight or even for about 24 hours.

In immunohistochemistry, the antigenic epitope of interest must be accessible to the primary antibody. An epitope is a small portion of a macromolecule that specifically binds to the binding site of an antibody molecule, such as an amino acid sequence of about 5 to about 10 amino acids. Monoclonal antibodies recognize only one epitope. On the other hand, polyclonal antisera can recognize several different epitopes.

Reversing undesired negative side effects such as formaldehyde fixation is known in literature under the heading of e.g. antigen repair or epitope repair.

At least three different pathways of antigen repair are widely used, alone or in combination: partial enzymatic digestion, heating and/or different chemicals that are supposed to reverse the effect of formaldehyde.

For partial proteolytic digestion, for example, inexpensive grade porcine trypsin (containing some chymotrypsin) is used. The principle of using proteolytic enzymes is that breaking some peptide bonds will form gaps in the matrix (matrix) of the cross-linked protein, allowing access to e.g. antibody molecules. However, proteolytic enzymes often attack all proteins, including the antigen of interest. Wire walking (light-rope walk) refers to digesting just enough length and one can easily understand that the conditions of digestion will vary from tissue to tissue and/or from antigen to antigen.

Most of the formaldehyde bound to the fixed tissue can be removed by heat-induced antigen retrieval. However, heating may also lead to irreversible destruction of the target analyte. Heating is known to damage, for example, thermolabile epitopes or thermolabile enzymes.

Heat-induced antigen retrieval is often combined with the use of specific "retrieval" or "extraction" buffers. However, the success of these procedures is unpredictable and the unpredictable consequences of these procedures are one of the major puzzles in the field of immunohistochemistry, for example.

Many different buffers and pH-values (e.g. citrate; glycine/HCl-mainly acidic pH-values in the range of about 2.5 to about 6, and Tris-based alkaline buffers with pH ranges of about 9 to 10) have been used, alone or in combination with chemicals which are believed to at least partially reverse the negative effects of methylene cross-linking introduced by formaldehyde. The chemical substance is used by dissolving it in water alone or in a buffer such as EDTA, citraconic acid, lead thiocyanate, aluminum chloride or zinc sulfate.

The great diversity of components used in solutions for high temperature antigen retrieval suggests that more than one mechanism may be involved. Most antigens can be repaired at a near neutral pH, but some require more basic media and others require acidic conditions. In some cases, binding of tissue-bound calcium ions can mask the epitope, necessitating removal of the metal ion by chelation. Other components of the repair solution include heavy metal ions (which can expose epitopes by coagulation-like action on the protein), and chaotropic substances (which can modify the shape of the protein by changing the structure of hydrone clusters).

For the analysis of nucleic acids, for example from formaldehyde-fixed paraffin-embedded (FFPE) tissues, methods for the repair of various antigens (analytes), which are generally very different from the methods required for the immunological detection of the epitope of interest, are recommended and used in the art. However, it is also accepted that formaldehyde fixation can have a negative effect on nucleic acids. For example, messenger RNAs (m-RNAs) in FFPE tissue are known to be at least partially disrupted, making detection of m-RNAs greater than 100 nucleotides in length a particularly challenging task.

Reference is now made to the properties and advantages of the bismaleic anhydride crosslinker shown in the present application: the reaction of maleic anhydride with a primary amine can be described by the following reaction scheme:

it is important that on the one hand the amide bond formed is more stable during (long term) storage of a sample immobilized on the basis of such a reagent and on the other hand the amide bond can be easily cleaved and the primary amine can be easily and under mild conditions recovered.

The formation of amide bonds between primary amino groups and maleic anhydride groups occurs rapidly at neutral and basic pH. Preferably, the incubation of the cell sample or tissue sample with the bismaleic anhydride of formula I is performed at a pH of 7.0 or higher. Also preferred is a pH below pH 12. It is further preferred that the pH value for fixation is in the range of pH 7.0 to pH 11.0, inclusive. It is also preferred that the incubation is performed at a pH value of 7.5 to 10.0, inclusive. For obvious reasons, buffer substances containing primary amines should be avoided.

Preferably, the fixative will not only be buffered to stabilize the pH during fixation, but the buffer will also have a physiological salt concentration. It is further advantageous and preferred if the bismaleic anhydride cross-linker is provided in a buffer also comprising a water-miscible organic solvent. Preferred organic solvents in this context are ethanol, N' -Dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

The amide bond formed by the reaction of maleic anhydride with a primary amine is stable at basic pH. If stored, the sample immobilized with bismaleic anhydride of formula I is preferably stored at neutral or basic pH (using the same preferred ranges given above for the immobilization or incubation steps).

Samples of cells contained in suspension or 1 μm³Or smaller, small tissue samples may be fixed within a short period of time, such as, for example, 10 to 60 minutes. However, in clinical routines, tissue samples will typically be greater than 1 μm³Much larger. Therefore, for routine purposes, it is preferred to incubate the cell or tissue sample with the bismaleimide crosslinker for 1-72 hours. As the skilled person will consider, cell samples or tissue samples are fixed for 1-72 hours. Also preferred in the method of the invention is an incubation of the cell sample or tissue sample in a fixative comprising bismaleic anhydride of formula I for 2 to 48 hours.

The bismaleic anhydride crosslinker may, for example, be dissolved in an organic solvent at high concentrations and may be diluted to give the appropriate working or final concentration. The final concentration of the bismaleic anhydride cross-linker used in the incubation/immobilization step may vary to some extent. Preferred final concentrations are between 0.1 and 20%. Further preferred final concentrations will be between 0.25 and 10% (by weight per volume).

The amide bond formed between the primary amino group and the maleic anhydride group in the immobilization process disclosed in the present application can be rapidly cleaved under acidic buffer conditions. After incubation in acidic pH-buffer, the amide bonds are cleaved, the bismaleimide cross-links are removed and the bismaleic anhydride can be washed away. At the same time, the primary amine, which was previously part of the amide bond with the crosslinker, is recovered and re-presented. Preferably, the cross-linking of the bismaleimide is removed by incubating the immobilized sample (i.e. the sample immobilized by using the bismaleic anhydride of formula I) at an acidic pH. Preferably, the cross-linking of the bismaleimide is removed by incubation in a buffer having a pH from 2.0 to 6.5. It is also preferred that the cross-linking buffer used to remove the bismaleimide will have a pH of from 2.5 to 6.5 or from 3.0 to 6.0, inclusive. Removal occurs rapidly, the faster the reaction rate, the more acidic the buffer. Preferably, the fixed sample is incubated for 2 minutes to 2 hours, also preferably 5 minutes to 1 hour to remove cross-linking of the bismaleimide.

It has been found that the residues R1 and R2 of the bismaleic anhydride crosslinker used in the process disclosed in the present application need to be selected from hydrogen, methyl, ethyl, propyl, isopropyl or butyl.

Further preferred, the residue of formula I R1 and/or R2 is selected from hydrogen, methyl or ethyl. Also preferred for use in the process of the present invention are bismaleic anhydrides of formula I wherein R1 is hydrogen or methyl and R2 is hydrogen or methyl.

If R1 and R2 are identical, the amide bond formed during fixation of the cell sample or tissue sample can be reversed under the same conditions. This would be advantageous whenever as much crosslinker as possible should be removed and at the same time as much primary amine as possible should be recovered. Thus, in a preferred embodiment, the bismaleic anhydride used in the process of the invention is a bismaleic anhydride of formula I, wherein R1 and R2 are the same.

As will be appreciated by the skilled artisan, since bismaleic anhydrides have been found to be useful in the fixation of cell or tissue samples, a wide variety of compounds comprising at least two maleic anhydrides linked by a linker can be designed and used.

The linking group X used in the bismaleic anhydrides used in the process of the present invention has a length of between 1 and 30 atoms. The term length must be understood to include the number of atoms given. A linker of 30 atoms in length has a backbone consisting of 30 atoms.

It will be apparent that the linker X may be designed in many ways, and that no undue limitation or restriction would be appropriate, as may be required by this particular application. However, some preferred examples of such linkers will be given.

In a preferred embodiment, the bismaleic anhydride of formula I for use in the process of the present invention will have a linking group X comprising a backbone consisting of carbon atoms and optionally one or more heteroatoms selected from O, N and S. It is also preferred that the heteroatoms included in the backbone of the linking group X will be O or N or both.

The linking group X may carry a side chain. In a preferred embodiment, the bismaleic anhydride of formula I for use in the process of the present invention will have a linker X containing one or more side chains designed to carry one or more additional maleic anhydride groups, respectively. Preferably, the bismaleic anhydride of formula I for use in the process of the present invention will have a linker X containing 1-3 maleic acid groups attached to one or more side chains, resulting in a compound of formula 1 having 3-5 maleic anhydride groups.

Preferably, the linker X used in the compounds of formula I used in the methods of the invention will have a molecular weight of 10kD or less. It is also preferred that the linker X will have a molecular weight of 5kD or less, 3kD or less, 2kD or less or 1kD or less. In a preferred embodiment, the bismaleimide crosslinker for use in the methods of the present application will have a molecular weight of 1kD or less.

Although it is possible to design and use maleic anhydride crosslinkers comprising three, four, five or even more maleic anhydride groups, it is preferred to use bismaleic anhydride crosslinkers having exactly two maleic anhydride groups linked by a linker X.

However, in some applications, side chains in the linker X will be applicable in other applications, a linker X with a main chain without side chains will be preferred. A pendant-free linker is a linker having only a backbone atom and an atom directly attached to the backbone atom.

In a preferred embodiment, the bismaleic anhydride of formula I used in the process of the present invention will have a linker X of 1-20 atoms in length.

In a preferred embodiment, the linker X in the bismaleic anhydride of formula I used in the process of the present invention is selected from the following linkers: a linker of 1 to 30 atoms in length, the backbone consisting of carbon atoms and optionally one or more heteroatoms selected from O, N and S; 1-20 methylene (-CH)₂-) a linker of the unit; a 3 to 30 atom linker group consisting of methylene (-CH)₂-) ethylene (-C), ethylene (-C)₂H₄-) and/or propylene (-C)₃H₆-) units and oxygen, wherein the number of ether linkages having oxygen is from 1 to 8; the main chain is a connecting group of 5-30 atoms, comprising a plurality of methylene groups and 1-4 carbonyl units bonded through ester bonds or amide bonds, or a connecting group of 11-30 atoms, comprising 6-25 methylene groups, 2 carbonyl units bonded through ester bonds or amide bonds, and further comprising 1-6 oxygen atoms bonded through ether bonds.

In a preferred embodiment, the bismaleimides of formula I for use in the process of the inventionThe linking group X of the anhydride is selected from the following linking groups: a linker of 1 to 30 atoms in length, the backbone consisting of carbon atoms and optionally one or more heteroatoms selected from O, N and S; 1-6 methylene (-CH)₂-) a linker of the unit; at a 3 to 30 atom linkage with a methylene group (-CH)₂-) ethylene (-C), ethylene (-C)₂H₄-) and/or propylene (-C)₃H₆-) units and oxygen, wherein the number of ether linkages having oxygen is from 1 to 8; the main chain is a connecting group of 5-30 atoms, comprising a plurality of methylene groups and 1-4 carbonyl units bonded through ester bonds or amide bonds, or a connecting group of 11-30 atoms, comprising 6-25 methylene groups, 2 carbonyl units bonded through ester bonds or amide bonds, and further comprising 1-6 oxygen atoms bonded through ether bonds.

In a preferred embodiment, the bismaleic anhydride of formula I used in the process of the present invention will have from 1 to 20 methylene groups (-CH)₂-) linker X of the unit. Preferably, such a linker will have from 1 to 8 methylene units and also preferably from 2 to 6 methylene units.

In a preferred embodiment, the present application relates to bismaleic anhydrides of formula I wherein the linking group X consists of five methylene units.

In a preferred embodiment, the bismaleic anhydride of formula I used in the process of the present invention will have a 3 to 30 atom linkage X consisting of a methylene group (-CH)₂-) ethylene (-C), ethylene (-C)₂H₄-) and/or propylene (-C)₃H₆-) units and oxygen, wherein the number of ether linkages having oxygen is from 1 to 6.

Examples of such linkers are given in formula II below.

Preferably, such a linker would include 4-8 methylene, ethylene (-C)₂H₄-) and/or propylene (-C)₃H₆-) units and 1-8 ether linkages. It is also preferred that the linking group will have 4 to 6 methylene units and 1 or 2 ether linkages.

In a preferred embodiment, the bismaleic anhydride of formula I used in the process of the present invention will have a backbone X containing 5 to 30 atoms, comprising a plurality of methylene groups and 1 to 4 carbonyl units bonded by ester or amide linkages. It is also preferred that the linker X will have a length of 8-20 atoms, including 4-16 methylene groups and 2-4 carbonyl units bonded to the backbone of the linker X by ester or amide linkages. It is also preferred that the linker X will comprise 4 to 12 methylene groups and 2-4 carbonyl units bonded to the backbone of the linker via ester or amide linkages. In another preferred embodiment, the linker X will comprise 4-8 methylene groups and 2 carbonyl units bound to the linker backbone via ester or amide bonds. Examples of such bismaleic anhydrides are given in formula III and formula IV below.

In a preferred embodiment, the bismaleic anhydrides of formula I used in the process of the present invention will have 11 to 30 atoms; a linker group X comprising 6 to 25 methylene groups, 2 carbonyl units in the main chain bound to the linker group X through ester or amide bonds, and 1 to 6 oxygen atoms connected through ether linkages. Preferred examples are described in formulas V to VII below.

Formula V

Cell or tissue samples may include cells obtained from in vitro cell or tissue culture or may represent samples that may be obtained in clinical routines. In a preferred embodiment, the cell sample or tissue sample will comprise cells of interest that are studied in a clinical routine. In the field of oncology, such cell or tissue samples may for example comprise circulating tumor cells or be tissues suspected or known to contain tumor cells. Preferred samples are whole blood and tissue samples, such as samples obtained by surgery or biopsy.

As will be appreciated by those skilled in the art, any of the methods of the present invention are performed in vitro. After analysis, the patient's sample is either stored or discarded. The sample is not returned to the patient.

For example, cells contained in blood are typically at least partially separated from plasma and may be fixed in suspension or embedded into agar. Tissue samples (e.g., obtained by resection or biopsy) are typically washed briefly in physiological buffer or transferred directly to a solution containing a suitable fixative.

As mentioned further above, formaldehyde is fixed as a two-step process and thus it is not easy to control it. The bismaleic anhydrides used in the process of the present invention have the great advantage that they only require one type of reaction to occur, i.e. the formation of an amide bond. Crosslinking has thus already taken place once at least two amide bonds which are linked to one another via the linker X have formed between at least two primary amino groups and the maleic anhydride group. The formation of methylene bridges in the second type of chemical reaction is not required.

Although perhaps the most critical step, the fixation of a cell sample or tissue sample is in most cases only one of several potentially critical steps in a clinical routine.

Typically, cell or tissue samples are studied using a microscope. For this purpose, it is necessary to prepare samples with a suitable thickness for staining and microscopic investigation. In the case of tissue samples, frozen samples or so-called paraffin blocks (see below) are usually sliced into so-called thin sections with a microtome. The thin slices are typically 2-10 μm thick. In the case where the sample is a tissue sample, the analytical method used in the study of such a sample is preferably performed on a thin slice of tissue.

In clinical pathology, it is routine to take means that allow long-term storage of cell or tissue samples. Although tissue preservation, for example, may also be achieved by cryogenic storage (e.g., at about-70 ℃), routine storage conditions are storage at 4-8 ℃, or even at ambient temperatures.

After fixation of the cell sample or tissue sample, the direct use of such a sample for the removal of the bismaleic anhydride cross-linker and further analysis is possible and represents a preferred embodiment in the methods disclosed above in the present application. The sample can be analyzed using any of the methods for FFPE materials set forth in more detail below.

Although embedding the fixed sample in paraffin is only one of several options for the most widely used procedure in clinical routines in the methods disclosed above in this application after fixation of the cell or tissue sample. Paraffin embedding represents an intermediate step between fixation and analysis.

For long term storage at e.g. ambient temperature, it is standard practice to dehydrate and embed cell or tissue samples in a suitable medium. The skilled artisan is fully aware of the operational details and need not be given here. The methods disclosed in this application employing machines for automated tissue processing such as embedding, deparaffinization, and/or staining are also preferably practiced.

In clinical routines, paraffin is most widely used for embedding and preserving samples for e.g. subsequent histopathology, immunohistochemistry, etc. The method of the invention is suitable for routine methods for embedding in paraffin. Thus in a preferred embodiment, the present invention relates to a method for preserving a cell sample or a tissue sample, said method comprising the steps of

a) Fixing the tissue sample with a bismaleimide cross-linker, and

b) embedding the fixed sample of step (a) in paraffin.

By this procedure, a paraffin block that can be easily cut into thin sections is obtained.

Once embedded, the cell or tissue sample may be stored until the assay is expired. The analysis may be performed within hours or days or, as the case may be, after several years. Before subsequent analysis can be performed, it is necessary to remove the paraffin and rehydrate the sample of interest on, for example, paraffin-embedded tissue sections. Various methods for removing paraffin wax can be used, and the skilled artisan will remove paraffin wax in a suitable manner without difficulty.

As mentioned above, various types of analysis may be performed on a cell sample or a tissue sample. Morphology, enzymatic activity, immunoreactivity, and/or nucleic acid are typically assessed. As will be appreciated, each of these kinds of evaluations will depend in large part on the extent to which the structure and function of the sample to be studied is preserved.

Because of the negative effects of formaldehyde on enzyme activity that are commonly observed, studies on enzyme properties are not generally at risk if formaldehyde is used as a fixative. Due to the gentle immobilization in the method disclosed herein, it is more likely that the enzyme activity is less affected or may even not be affected as the case may be. In a preferred embodiment, the present invention relates to a method for analyzing an enzymatic activity in a cell sample or a tissue sample, said method comprising the steps of: the samples were fixed with bismaleimide cross-linker, the bismaleimide cross-linking removed and assayed for enzyme activity.

The method of immobilization with the bismaleic cross-linker of the present application, which has significant advantages, can be used in routine immunohistochemistry. In a preferred embodiment, the present invention therefore relates to a method for immunohistochemistry on a cell sample or a tissue sample, said method comprising the steps of

a) Fixing a cell sample or tissue sample with a bismaleimide cross-linking agent,

b) embedding the fixed sample of step (a) in paraffin,

c) the sample is deparaffinized by subjecting the sample to deparaffinization,

d) removing the bismaleimide cross-linking, and

e) immunodetection of the epitope of interest.

In one embodiment, the present invention relates to a method comprising fixing a cell sample or tissue sample as disclosed in the present invention, said method further comprising the steps of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-linking, and

e) immunodetection of the epitope of interest.

If the cell sample or tissue sample is fixed with a bismaleimide crosslinker and the crosslinking of the bismaleimide is released prior to analysis, the primary amine initially present in the sample will become available again. It is expected that this would represent a major advantage over other fixatives such as formaldehyde, which are known to generally have deleterious and irreversible effects on many epitopes. This negative effect may be particularly severe if a single epitope recognized by a monoclonal antibody is attacked or destroyed. Many of these potentially extremely valuable monoclonal antibodies have not gained attention and market penetration because they do not function in standard immunohistochemistry based on formalin-fixed paraffin-embedded tissue (FFPET). It is likely that many monoclonal antibodies not useful for FFPET will function on samples immobilized with the bismaleic anhydride crosslinker. In a preferred embodiment, the invention therefore relates to an immunohistochemical method, substantially as described in the preceding paragraph, with the following additional technical features: the epitope of interest is an epitope that is masked or destroyed when the cell sample or tissue sample comprising the epitope is fixed with a formaldehyde fixative. In other words, the method is performed using an antibody that does not act on FFPET.

As mentioned above, the methods disclosed herein work well with polypeptides having, for example, enzymatic or antigenic properties. Surprisingly, the methods disclosed herein are also advantageous in the detection of nucleic acids of interest.

In a preferred embodiment, the nucleic acid is deoxyribonucleic acid (DNA), such as occurs in the nucleus of a eukaryotic cell. In a preferred embodiment, the DNA is analyzed by in situ hybridization methods. Methods for In Situ Hybridization (ISH) are well known to the skilled artisan. Gene amplification can be measured, for example, using in situ hybridization methods such as fluorescence in situ hybridization technique (FISH), chromogenic in situ hybridization technique (CISH), or silver in situ hybridization technique (SISH). In a preferred embodiment, the present invention relates to a method for the in vitro detection of a nucleic acid of interest by in situ hybridization on a cell sample or a tissue sample, said method comprising the following steps

a) Fixing a cell sample or tissue sample with a bismaleimide cross-linking agent,

b) embedding the fixed sample of step (a) in paraffin,

c) the sample is deparaffinized by subjecting the sample to deparaffinization,

d) removing the bismaleimide cross-linking, and

e) the nucleic acid of interest is detected by performing in situ hybridization.

In one embodiment, the present invention relates to a method comprising fixing a cell sample or tissue sample as disclosed in the present invention, said method further comprising the steps of

a) Embedding the fixed sample of step (a) in paraffin,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-linking, and

d) the nucleic acid of interest is detected by performing in situ hybridization.

Surprisingly, the immobilization method described in the present invention is also advantageous in the detection of m-RNA in cell or tissue samples prepared with a bismaleic anhydride cross-linker. The expression level of the m-RNA of interest can be determined by suitable techniques, such as Northern Blot (Northern Blot), real-time polymerase chain reaction (RT-PCR), and the like. All these detection techniques are well known in the art and can be deduced from standard textbooks such as Lottspeich (Bioanalytik, Spektrum Akademischer Verlag, 1998) or Sambrook and Russell (2001, Molecular Cloning: A Laboratory Manual, CSHPress, Cold Spring Harbor, NY, USA). Preferably, m-RNA is detected using real-time polymerase chain reaction (RT-PCR).

In a preferred embodiment, the invention therefore relates to a method for the in vitro detection of a nucleic acid of interest by RT-PCR in a cell sample or a tissue sample, said method comprising the following steps

a) Fixing a cell or tissue sample with a bismaleic anhydride cross-linker by the methods described herein above,

b) embedding the fixed sample of step (a) in paraffin,

c) the sample is deparaffinized by subjecting the sample to deparaffinization,

d) removing the bismaleimide cross-linking, and

e) the nucleic acid of interest is detected by performing RT-PCR.

In one embodiment, the present invention relates to a method comprising fixing a cell sample or tissue sample as disclosed in the present invention, said method further comprising the steps of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-linking, and

d) the nucleic acid of interest is detected by performing RT-PCR.

In a further preferred embodiment, the nucleic acid is isolated from a cell sample or tissue sample that has been fixed with a bismaleic anhydride cross-linking agent and the isolated nucleic acid is further analyzed. Further preferred are such isolated nucleic acids for mutation analysis. In a preferred embodiment, the present invention therefore relates to a method for performing an in vitro mutation analysis on a nucleic acid sample isolated from a cell sample or a tissue sample, said method comprising the steps of

a) Fixing a cell sample or tissue sample with a bismaleimide cross-linking agent,

b) embedding the fixed sample of step (a) in paraffin,

c) the sample is deparaffinized by subjecting the sample to deparaffinization,

d) the cross-linking of the bismaleimide is removed,

e) isolating the nucleic acid, and

f) performing mutation analysis using the nucleic acid isolated in step (e).

In one embodiment, the present invention relates to a method comprising fixing a cell sample or tissue sample as disclosed in the present invention, said method further comprising the steps of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) the cross-linking of the bismaleimide is removed,

d) isolating the nucleic acid, and

e) performing mutation analysis using the nucleic acid isolated in step (d).

Determining the presence or absence of a particular mutation can be performed in a variety of ways. Such methods include, but are not limited to, PCR, hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry, or DNA sequencing, including microsequence sequencing. In particular embodiments, hybridization with allele-specific probes can be performed in two ways: (1) allele-specific oligonucleotides bound to a solid phase (glass, silicon, nylon membrane) and labeled samples in solution, as in many DNA chip applications, or (2) bound samples (typically cloned DNA or PCR amplified DNA) and labeled oligonucleotides in solution (allele-specific or short to allow sequencing by hybridization). Preferably, the determination of the presence or absence of a mutation comprises determining an appropriate nucleotide sequence comprising the site of the mutation by a method such as Polymerase Chain Reaction (PCR), DNA sequencing, oligonucleotide probe hybridization analysis, or mass spectrometry.

With the methods currently available through the disclosures provided in this application, it is even possible to detect the nucleic acid of interest and the polypeptide of interest in the same sample. In a further preferred embodiment, the present invention relates to a method for the in vitro detection of at least one polypeptide of interest and the detection of at least one nucleic acid of interest by immunohistochemistry in a test sample comprising a cell sample or a tissue sample, said method comprising the steps of

a) Fixing a cell sample or tissue sample with a bismaleimide cross-linking agent,

b) embedding the fixed sample of step (a) in paraffin,

c) the sample is deparaffinized by subjecting the sample to deparaffinization,

d) removing the bismaleimide cross-linking, and

e) immunodetection of at least one polypeptide of interest and detection of at least one nucleic acid of interest by performing RT-PCR or fluorescent in situ hybridization.

In one embodiment, the present invention relates to a method comprising fixing a cell sample or tissue sample as disclosed in the present invention, said method further comprising the steps of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-linking, and

d) immunodetection of at least one polypeptide of interest and detection of at least one nucleic acid of interest by performing RT-PCR or fluorescent in situ hybridization.

As discussed above and as will be further exemplified in the examples given, the use of a bismaleic anhydride cross-linker has significant advantages in many respects over other fixatives and procedures used in current pathology laboratory routines. In a very preferred embodiment, the present invention therefore relates to the use of a bismaleic anhydride cross-linking agent in the fixation of a cell sample or a tissue sample.

In a still further preferred embodiment, the bismaleic anhydride crosslinker is used in the preparation of ready-to-use fixatives. Preferably, the present invention therefore relates to the use of bismaleic anhydride for the preparation of a fixative for the fixation of a cell or tissue sample.

Preferably, the bismaleic anhydride cross-linking agent used for fixing the cell sample or the tissue sample or used in preparing a fixative for fixing the cell sample or the tissue sample will be a bismaleic cross-linking agent as defined in formula I. Preferably, when carrying out the immobilization method disclosed in the present invention, the linker X of formula I will be selected from the linkers disclosed as preferred. In a further embodiment, the cross-linking agent will be selected from the compounds as described in formulas II, III, IV, V, VI, VII, VIII and IX.

Since the use of bismaleic anhydride crosslinkers has great advantages, in particular the fact that reversibility is known to be easy to achieve, it can be easily envisaged that such agents may be combined with other fixatives, for example fixatives leading to permanent fixation. In this way it is possible to further improve the long-term preservation of the tissue, however with the possibility of easily restoring at least the relevant part of the nucleic acid or antigen of interest.

In another preferred embodiment, the present application relates to a method for fixing a cell or tissue sample, wherein a mixture comprising a bismaleic anhydride cross-linking agent and a second cross-linking agent selected from formaldehyde and/or glutaraldehyde is used. Preferably, such a mixture is one described hereinafter. In yet another preferred embodiment, the present application relates to a fixative comprising a bismaleic anhydride cross-linker and a fixative selected from the group consisting of formaldehyde and/or glutaraldehyde. The other components of such fixatives will be selected from the preferred embodiments given for the bismaleic anhydride fixatives described above. Preferably, the volume-based ratio between the bismaleic anhydride crosslinker used and either formaldehyde or glutaraldehyde or the sum of both (if a mixture is used) will be from 1: 10 to 10: 1 (weight/weight). The total final concentration of fixative in such a mixture will be as set forth above for fixatives containing only bismaleic anhydride crosslinker. Preferably, such a fixative comprising a bismaleic anhydride crosslinker and formaldehyde or glutaraldehyde or both will have a pH in the range of 8.0 to 11.0.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

Drawings

Figure 1 shows a stained 4 μm thin section obtained from H322M xenograft-bearing SCID beige mice stained with monoclonal antibody 5G11 that specifically binds to IGF-1R. Tissue fixation has been performed with different fixatives (using 4% formaldehyde ("formalin"), 4% or 8% biscitraconic acid), respectively. Immunohistochemical localization of IGF-1R showed comparable staining intensity or quality between the fixative formalin (FIG. 1; A) or the biscitraconic acid used at 4% (FIG. 1: B) and 8% (FIG. 1: C), respectively, after heat-induced antigen repair of formaldehyde-fixed tissue and removal of the biscitraconic acid from the tissue fixed with biscitraconic acid by using an acidic buffer.

Figure 2 shows a stained 4 μm thin section obtained from H322M xenograft tumor-bearing SCID beige mice stained with monoclonal antibody 3C6 that specifically binds to EGFR. Tissue fixation has been performed with different fixatives (using 4% formaldehyde ("formalin"), 4% or 8% biscitraconic acid), respectively. Immunohistochemical localization of EGFR showed comparable staining intensity or quality between the fixative formalin (fig. 2; a) or the biscitraconic acid used at 4% (fig. 2: B) and 8% (fig. 2: C), respectively, after protease-assisted antigen retrieval of formaldehyde-fixed tissues and removal of the biscitraconic acid from the tissues fixed with biscitraconic acid by using an acidic buffer.

Figure 3 shows PCR amplification of EGFR gene using mRNA isolated from MDA468 cells that have been subjected to different pretreatment/immobilization protocols. Shown is the amplification of EGFR-mRNA from MDA-MB468 cells (fresh and non-fixed, both stored in RPMI for 4 hours, fresh MDA-MB468 cells treated/fixed in 10% DMSO, 80% DMSO, 10% DMSO with 1% biscitraconic acid, 80% DMSO with 1% biscitraconic acid, and water "Wasser" (as a negative control)). As is evident from the amplification curves and the inserted tables, mRNA from all samples was amplified in a rather similar manner.

Figure 4 shows PCR amplification of HER2 gene using mRNA isolated from MDA468 cells that have been subjected to different pretreatment/immobilization protocols. Shown is the amplification of HER2-mRNA from MDA-MB468 cells (fresh and non-fixed, both stored at RPM for 4 hours, fresh MDA-MB468 cells treated/fixed in 10% DMSO, 80% DMSO, 10% DMSO and 1% biscitraconic acid, 80% DMSO and 1% biscitraconic acid, respectively, and water "Wasser" (as negative control)). As is evident from the amplification curves and the inserted tables, mRNA from all samples was amplified in a rather similar manner.

Example 1:

synthesis of "Dicitraconic acid" of formula VIII

Synthesis of methyl-3-tolylcarbamoyl-acrylic acid

To a solution of 3.2ml of 3-methyl-furan-2, 5-dione (citraconic anhydride) in 25ml of diethyl ether, a solution of 3.74g of p-tolylamine in 25ml of diethyl ether was added dropwise over 15 minutes. The yellow suspension was stirred for 1 hour and filtered. The residue was washed with diethyl ether and dried in vacuo.

Yield: 7.22g, 94%

Synthesis of 3-methyl-1-p-tolyl-pyrrole-2, 5-dione

7.22g of methyl-3-tolylcarbamoyl-acrylic acid are suspended in 60ml of acetic anhydride. The suspension was heated at reflux for 3 hours. After cooling to room temperature, the solvent was removed in vacuo. The residue was recrystallized from ethanol.

Yield: 4.32g, 65%

Synthesis of 3- (5- (4-methyl-2, 5-dione-1-p-tolyl-2, 5-dihydro-1H-pyrrol-3-yl) -pentyl-) -4-methyl-1-p-tolyl-pyrrole-2, 5-dione

12g of 3-methyl-1-p-tolyl-pyrrole-2, 5-dione and 15.6g of triphenylphosphine were dissolved in 155ml of acetic acid and 2.15ml of glutaraldehyde were added. The reaction mixture was refluxed for 20 hours. Acetic acid was removed by distillation and the residue was heated to 150-160 ℃ and held for 6 hours.

The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate 7: 3). The product was further purified by digestion in methanol, filtered and dried.

Yield: 2g, 35%

Synthesis of 3- (5- (4-methyl-2, 5-dioxo-2, 5-dihydro-furan-3-yl) -pentyl) -4-methyl-furan-2, 5-dione ("biscitraconic acid")

1.07g of 3- (5- (4-methyl-2, 5-dione-1-p-tolyl-2, 5-dihydro-1H-pyrrol-3-yl) -pentyl-) -4-methyl-1-p-tolyl-pyrrole-2, 5-dione are dissolved in 30ml of a 1: 1 mixture of tetrahydrofuran and methanol. After addition of 3.48g of potassium hydroxide dissolved in water, the mixture was heated at reflux for 3 hours. The solvent was removed by distillation and the residue was purified by column chromatography on silica gel (petroleum ether: ethyl acetate 7: 3). The product was dried in vacuo.

Yield: 402mg, 60%

Example 2:

synthesis of 3- (5- (4-methyl-2, 5-dioxo-2, 5-dihydro-furan-3-yl) -3-oxa-pentyl) -4-methyl-furan-2, 5-dione (formula IX):

the crosslinker 3- (5- (4-methyl-2, 5-dioxo-2, 5-dihydro-furan-3-yl) -3-oxa-pentyl) -4-methyl-furan-2, 5-dione (formula IX) was synthesized in analogy to the procedure described in example 1, using 3-oxa-1, 5-glutaraldehyde instead of 1, 5-glutaraldehyde. The starting material 3-oxa-1, 5-glutaraldehyde is described by Bowers, S. et al, Bioorganic & Medicinal Chemistry Letters 19(2009) 6952-.

Example 3:

staining with IGF-1R antibody

H322M xenograft tumor-bearing SCID beige mice were sacrificed. The tumors were removed and cut into 3 pieces of approximately the same size. The tissue samples were then transferred to individual fixative solutions. For immobilization with biscitraconic acid, the material was dissolved in DMSO to a final concentration of 80% by repeated pipetting at room temperature. After completion of dissolutionThe solution was used as is, or diluted 1: 1 in DMSO and further diluted in 1x PBS pH 7.4, eventually forming PBS with final concentrations of 10% DMSO and 8% or 4% biscitraconic acid, respectively. To test the effect of different concentrations of biscitraconic acid on the fixation effect, solutions containing 4% or 8% biscitraconic acid (w/v) were prepared. For the preparation of the formaldehyde fixative formalin (40% (w/v) paraformaldehyde in H₂Solution in O) was dissolved 1: 10 in 1 XPBS pH 7.4.

All tumor samples were fixed overnight at room temperature for 12 hours. The following day, the tissue samples were treated with H₂O wash for 1 hour. The fixed tumor tissue was then embedded in paraffin.

Sections of paraffin-embedded tissue samples fixed with different fixatives (4% formalin, 4% or 8% biscitraconic acid) were cut at 4 μm using a conventional rotary microtome. For immunohistochemical localization of IGF-1R, the cut tissue sections were loaded on glass slides. Deparaffinization of tissue samples was performed on a Ventana Benchmark XT automated IHC stainer (Ventana, Tucson). For localization of IGF-1R in FFPE tissue by immunohistochemistry with < IGF-1R >5G11 monoclonal antibody (Ventana, Tucson), heat-induced antigen retrieval was required prior to staining of formalin-fixed samples. Heat-induced antigen retrieval for immunohistochemical detection of IGF-1R was performed by incubating tissue sections on Ventana Benchmark XT at 95 ℃ in buffer CC1(Ventana, Tucson) for 1 hour. Antigen retrieval in thin sections previously fixed with biscitraconic acid and then embedded with paraffin can be performed by simply incubating the tissue sections in a buffer with an acidic pH. Thin sections were incubated in buffer at pH 5.8 for 2 hours. After antigen retrieval, all slides were placed on a Ventana Benchmark and stained for IGF-1R (16 min for one antibody incubation time). Bound primary antibodies were detected with the Ventana iview DAB detection kit. Examination of the stained sections showed that tissue fixation with biscitraconic acid retained the morphology of the tissue (fig. 1B and C). In addition, biscitraconic acid can be recovered by simple incubation in an acidic buffer solution. Immunohistochemical localization of IGF-1R did not show significant differences in staining intensity or morphological quality between formalin (FIG. 1; A) or biscitraconic acid-fixed tissues (FIG. 1; B and C).

The results obtained in this example demonstrate that fixation with biscitraconic acid enables preservation and easy and gentle repair of epitopes that become accessible in formalin-fixed tissue only after the tissue has been treated with a method known as heat-induced antigen repair.

Example 4:

staining with EGFR antibody

By a procedure similar to example 2, formalin or biscitraconic acid fixed tissues were prepared for EGFR staining using the antibody 3C6(< EGFR > mAB 3C 6; Ventana, Tucson). This antibody is known to rely on protease pre-treatment of tissue sections derived from FFPE in order to regain access to its epitopes in such FFPE samples. As shown in figure 2, no immunohistochemical localization of EGFR was found to distinguish between formalin fixation and protease-assisted epitope repair or biscitraconic fixation and repair (by incubation in acidic buffer).

The results obtained in this example demonstrate that fixation with biscitraconic acid enables preservation and easy and gentle repair of epitopes that become accessible in formalin-fixed tissue only after the tissue has been treated with protease.

As demonstrated in the examples 2 and 3 in their entirety, biscitraconic acid fixation and repair is a method for the detection of different epitopes that heretofore have required repair in formalin-fixed tissue by one or more different repair methods (heat or protease induced). As a prototype of other bismaleic anhydrides, biscitraconic acid, when used, does not require a stringent repair procedure for different antibodies (otherwise requiring a very different repair procedure). Conversely, normalization and reproducibility of heat-induced or protease-assisted repair is not easy, and it would be possible to obtain more reproducible antigen/epitope accessibility/reactivity by using a gentle and easy method of removing the fixative based on the bis-maleic anhydride crosslinker as shown above.

Example 5:

DNA isolation and qPCR for gene amplification analysis

MDA-MB468 cells were first fixed in different fixative reagents for 10 min and then neutralized in citrate buffer (pH 4.4). The different samples presented in FIG. 3 are MDA-MB468 cells (fresh MDA-MB468 cells, both fresh and left unfixed at RPM for 4 hours, treated/fixed in 10% DMSO, 80% DMSO, 10% DMSO with 1% biscitraconic acid, and 80% DMSO with 1% biscitraconic acid, respectively).

After different fixing operations, use 1x 10⁷DNA isolation was performed on individual MDA-MB468 cells. For isolation of DNA, a high purity template preparation kit (Roche Diagnostics GmbH, catalog No.: 11796828) was used according to the manufacturer's instructions. The isolated DNA was stored at-20 ℃.

The amplification status of EGFR and HER2 was measured in MDA-MB468 cells. Therefore, quantitative PCR based on the use of hydrolysis probes (Taqman probes) was performed using gene-specific primers and probes (see table 1). The probe for the target gene was labeled with Fam at 5 'and BHQ-2 at 3'.

TABLE 1: primers and probes for target genes HER2 and EGFR

For each gene, a separate, validated PCR-mix was used (see table 2).

TABLE 2: PCR-mixture composition for qPCR assay

Each mixture consisted of 5. mu.M forward and reverse primers and 2. mu.M probe. The Z05 polymerase used was included in a COBASS Taqman RNA reaction mixture (LUO M/N58004938) purchased from Roche Molecular Diagnostics (Branchburg, USA) and magnesium acetate [25mM ] (Fluka, Cat. No.: 63049) was added at different concentrations according to different oligonucleotide (oligo) mixtures. Mu.l of DNA template was used and topped up to 5. mu.l with nuclease-free water. Three measurements were made for each sample. The measurements were carried out using a LightCycler 480(Roche Diagnostics GmbH) and suitable 96-well plates and sealing foils. The following thermal cycling parameters (table 3) were used in the cycler (cycler).

Table 3:LightCycler thermal cycle parameters

Name of program	Number of cycles	Analysis mode
			Decontamination	1	Quantification of
Amplification of	47	Quantification of
			Cooling down	1	Is free of

Claims (15)

1. A method for in vitro fixation of a cell sample or a tissue sample, wherein said cell sample or said tissue sample is incubated with bismaleic anhydride of formula I,

formula I

Wherein R1 and R2 are independently selected from: hydrogen, methyl, ethyl, propyl, isopropyl and butyl,

wherein X is a linker between 1 and 30 atoms in length,

thereby fixing the cell sample or the tissue sample.

2. The process of claim 1, wherein in the bismaleic anhydride, R1 is hydrogen or methyl and R2 is hydrogen or methyl.

3. The process of claim 1 or 2, wherein in the bismaleic anhydride, R1 and R2 are the same.

4. The process of claim 1 wherein in the bismaleic anhydride the linking group X is 1 to 20 atoms in length.

5. The process of claim 1 wherein in said bismaleic anhydride the backbone of the linking group X consists of carbon atoms and optionally one or more heteroatoms selected from O, N and S.

6. The method of claim 1, wherein the sample is incubated with bismaleic anhydride for 1-72 hours.

7. The method of claim 1, wherein the method further comprises the step of embedding the fixed sample in paraffin.

8. The method of claim 1, further comprising the step of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-links formed in the process of claim 1, and

e) immunodetection of the epitope of interest.

9. The method of claim 8, wherein the epitope of interest is an epitope that is masked or destroyed when the cell sample or tissue sample comprising the epitope is fixed with a formaldehyde fixative.

10. The method of claim 1, further comprising the step of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-links formed in the process of claim 1, and

d) the nucleic acid of interest is detected by performing in situ hybridization.

11. The method of claim 1, further comprising the step of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-links formed in the process of claim 1, and

d) the nucleic acid of interest is detected by performing RT-PCR.

12. The method of claim 1, further comprising the step of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the bismaleimide cross-links formed in the process of claim 1, and

d) immunodetection of at least one polypeptide of interest and detection of at least one nucleic acid of interest by performing RT-PCR or fluorescent in situ hybridization.

13. The method of claim 1, further comprising the step of

a) The fixed sample was embedded in paraffin wax,

b) the sample is deparaffinized by subjecting the sample to deparaffinization,

c) removing the cross-links of the bismaleimide formed in the process of claim 1,

d) isolating the nucleic acid, and

e) performing mutation analysis using the nucleic acid isolated in step (d).

14. Use of bismaleic anhydride of formula I according to claim 1 for fixing a cell sample or a tissue sample.

15. Use of bismaleic anhydride of formula I according to claim 1 for the preparation of a fixative for fixation of a cell or tissue sample.