The present patent application claims priority of the patent application RO A/00075 filed on 28/01/2014 and of the patent application RO A/00078 filed on 29/01/2014. The present patent application discloses various novel and inventive technical solutions improving the technical solutions already disclosed in the patent application no. PCT/RO2012/000017, published under no. WO2013/002661; the disclosure of all the aforementioned applications, RO A/00075, RO A/00078 and PCT/RO2012/000017 is herein incorporated by reference in its entirety.

Field of the invention

This invention addresses generally to the field of histopathology and refers to a biomimetic gellable colloid with customizable / tunable composition that can be gelled and stabilized on demand. The biomimetic gellable colloid of the present invention is intended for use, inter alia as a sectionable support for processing and for encoding histological samples.

Biomimetic colloid within the meaning of the present invention refers to a man-made colloid that imitate a biological material, for example the histological sample to be processed and/or microtome sectioned for diagnostic purposes, without being limited to this. It may also imitate any biological composition (human, animal, plant, microbiological and the like).

The present invention also relates to processes for the preparation of said colloid, to sectionable matrices for receiving the histological samples to be processed and sectioned for diagnostic purposes made from said colloid and to methods for preparing said matrices, as well as to various other uses of the biomimetic gellable colloid of the invention.

The invention also relates to a sectionable code intended for use as a means of unambiguous and irreversible identification of the histological preparations as well as to various methods for encoding histological preparations with the aid of the gellable colloid.

The biomimetic gellable colloid of the present invention will be hereinafter also referred to as gellable colloid or simply colloid.

Background of the invention

There is a continuous need for the use of aids in the histological processing of explants, biopsies, cell suspensions or sediments resulting from various interventions such as puncture aspiration, centrifugation of liquids (as urine, cerebrospinal fluid, pleural effusions, ascites and the like), etc.

There is a continuous need to develop safer methods of writing unique identification codes for the histological preparations. These codes are used in cytology and pathology laboratories as aids in the histological processing of explants, biopsies or suspensions.

In 2013, over 7.5 billion paraffin blocks were prepared in the US only and used for the preparation of over 15 billion sections for microscope examination.

Traditionally, the only solution which is still functional in pathology laboratories for simultaneous embedding of cell suspensions/sediments, hystoids, spheroids, or of several small or loose fragments, with the purpose of obtaining histological preparations, is the use of either matrices of natural origin (ovalbumin, fibrin, agarose, or mixtures of agarose with molten gelatine, etc.) or of synthetic matrices, such as proprietary compounds primarily based on mixtures of carbohydrate hydrocolloids (for example Histogel™, Richard Allan Scientific).

Occasionally, direct processing into centrifuge tubes or placement of the cells after centrifugation in pouches of collodion (nitrocellulose) or porous paper (tea bags), or in synthetic sponges is used, but these methods involve cumbersome techniques and give inconsistent results.

For the preparations on ice, a mixture of hydrocolloids such as OCT (optimal cutting temperature, Tissue-Tek) is used, with or without a surrounding matrix of fibrin, agarose or Histogel. Occasionally, the OCT mixture is also used as a matrix for paraffin preparations, but the results are still unsatisfactory.

Unfortunately, all the current techniques except for the Cellient method described herein below, are laborious and have a series of drawbacks which limit their implementation. Moreover, these techniques may be used only in laboratories provided with adequate pathology or cytology equipment and not in operating rooms or biological sampling rooms. Considerable delays resulting from the handling and transport of biological samples adversely affect the quality of the histological preparations.

Most of the currently used materials (OCT, agarose, hydroxyethyl cellulose and the like, all characterized by positive electric charge, the same as the microscope slides) lead to the obtaining of preparations which require a relatively slow processing, are difficult to section and result in sections with reduced adhesion to the microscope slides which increases the risk of detachment during staining. Another disadvantage lies in the use of both agarose and hydroxyethyl cellulose at high temperatures (i.e. at melting temperatures of over 50 - 60°C), which may induce serious artefacts upon the contact with the biological material to be examined. The whole procedure is relatively long (15 - 30 minutes) and comprises subjecting the cells to a heating cycle (50 - 60°C) followed by a cooling cycle (4 - 10°C or even -20°C in some methodologies). This may lead to undesirable artefacts.

Another disadvantage lies in the fact that both the agar and the Histogel require special precautions with respect to the protocols used for dehydration and clarification. Some reagents (aliphatic solvents, limonenes, etc.) are not recommended for processing agar or Histogel blocks. Accelerated methods of processing (high temperature, with or without microwaves) are also unsuitable for the agar blocks.

Methods using ovalbumin or fibrin clot are also relatively laborious. The fibrin clot method uses expensive ingredients such as fibrin, which is also considered a biohazard ingredient since it is derived from blood plasma, usually of human origin. Moreover, the preparations resulting from the use of such a method are unsuitable for immunohistochemical staining of immunoglobulins, kappa and lambda light chains, albumin, etc. This method is also not compatible with cells that have been pre-treated with aldehyde fixatives. The ovalbumin method requires the use of concentrated ethanol (95%) for clotting which may lead to significant artefacts and irreversible distortion of some antigenic determinants.

The method used by Hologic (Cellient™ Automated Cell Block System) is the closest prior art technology. Although it is a relatively simple and fast method, it results into the obtaining of extremely thin blocks which are difficult to cut and it allows only individual processing of the preparations; it also requires dedicated and expensive equipment which translates into an extremely low efficiency at a high price.

Given that the separation of cellular elements is achieved by filtration, there is a risk of loss of some components during processing (i.e. anything which is able to pass through the pores of the filter membranes which are under 8 μηι in size), thus the diagnostic accuracy is affected. Moreover, the filtration process may also result in compression artefacts.

All the above methods have the disadvantage of not allowing an accurate calibration of the concentration of cells present in the final preparation and of not providing a sufficient number of histological sections. In the absence of a sufficient number of sections from the same block, modern molecular techniques which require multiple markers and a large number of control slides may not contribute to the accuracy of the cytological diagnosis {J. Histochem. Cytochem. May 2000, vol. 48. no. 5, 709 - 718).

Another way of processing multiple parts is the use of a sectionable "platform" (Tissue- Tek® Paraform® Sectionable Cassette System, Sakura Finetek U.S.A., Inc.] which in theory allows the correct orientation of small fragments before starting the processing and which may virtually facilitate the automation of the next steps (i.e. shaping and sectioning of the paraffin block). Unfortunately, this system is not applicable to multiple biopsies (> 4), especially when they do not have identical diameters and shapes (i.e. in 99% of cases). It is also well known that such sectionable platforms increase the difficulty of sectioning the preparation.

The international patent application no. PCT/RO2012/000017 published under no. WO2013/002661 entitled "Matrix for receiving a tissue sample and use thereof and the US patent application no. 60/420,027 entitled "The HistoBest method for preparing multiuse tissue arrays" disclose the use of plant or animal tissue which is processed into matrices with preformed receptacles for tandem processing of biopsies/explants. The plant or animal tissue is used either as such or as a starting material for preparing composite materials to be further processed into said matrices.

All the techniques presently in use are cumbersome, expensive, capricious and do not allow for quality sections, unless used by experienced professionals. Therefore, many laboratories do not use them and prefer conventional and more expensive methods.

As far as the unambiguous identification of histological preparations is concerned, none of the prior art methods enable the labelling with unique codes. Thus conventional methods are used, such as handwriting, use of specialized printers or laser etching. Alphanumeric codes or ID / 2D barcodes are used. Given the error rate of all these methods (approximately 0.5 - 3.5%) and the disastrous consequences the errors may have in establishing a correct diagnosis, the methodological rules currently in force require the simultaneous use of at least two different methods of identification for biological specimens and the use of molecular biology techniques for the identification of the patients in all the cases where there is a suspicion of malignancy or of labelling inconsistency. The risk of error is thus significantly reduced, but not entirely eliminated. Material costs and the time involved by these procedures are substantial.

All the drawbacks of the above-mentioned methods are overcome by the use of the biomimetic gellable colloid and of the sectionable code disclosed in the present patent application. Summary of the invention

The present invention thus refers to a biomimetic gellable colloid for coding and processing histological specimens. The gellable colloid of this invention is characterized by a uniformly dispersed phase which comprises a hydrophilic component and may be gelled/solidified upon demand. Preferably, the colloid is a nano-/microcolloid [a nano-/microemulsion). The colloid may optionally further comprise a hydrophobic component.

According to one specific embodiment, the gellable colloid comprises both a hydrophilic and a hydrophobic component. According to a preferred embodiment, the colloid displays vesicles of which over 50% have diameters below 10 microns.

The colloid of the invention is customizable / tunable, i.e. it has chemical and mechanical properties that can be tailored depending upon the intended use / application.

The present invention also provides for a process for preparing the gellable colloid of the invention. The process includes the step of preparing a water dispersion which comprises the hydrophilic component, optionally in admixture with the hydrophobic component when such a component is used, followed by emulsification.

Another object of this invention is a sectionable matrix for receiving a histological specimen obtained from the gellable colloid of the invention. A sectionable matrix within the meaning of the present invention also comprises the meaning of sectionable support for the processing of histological preparations.

The present invention further relates to a process for the preparation of such a sectionable matrix intended for receiving a histological specimen, wherein the shaping of the gellable colloid is carried out in a preformed mould, followed by the gelation / solidification of the colloid.

In addition, the invention refers to a sectionable code prepared with the aid of the inventive gellable colloid which is used for the orientation and unambiguous identification of a histological preparation.

Brief description of the drawings

Figure 1 shows a microscopic section through a pre-formed matrix prepared from the tissue surrogate as per the technique described in PCT/RO2012/000017 - magnification x 100; the sectionable matrix was paraffin processed via conventional methods, sectioned at 4 - 5 μπι, stained with hematoxylin-eosin and photographed at x 100 magnification; an amorphous conglomerate of protein may be observed together with irregular carbohydrate containing particles and empty spaces of various dimensions wherefrom lipids were extracted by the organic solvents employed during processing.

Figure 2 shows a microscopic section through a pre-formed matrix prepared from one embodiment of the gellable colloid described in the present invention comprising both a hydrophilic and a hydrophobic component - magnification x 100; the sectionable matrix was loaded with renal pig biopsies as shown in the lower part of the figure, paraffin processed via conventional methods, sectioned at 4-5 μπι, stained with hematoxylin-eosin and photographed at x 100 magnification; the sectionable matrix is displaying an arrangement of small vesicles, comparable in size (1-2 μιη in diameter] and devoid of content as the lipids were extracted by the organic solvents employed during processing; the vesicles are uniformly dispersed within an amorphous mass of proteins.

Figure 3 shows one embodiment of creating a unique identifier for the histological preparation; a pre-formed sectionable matrix provided with grooves of 1 mm width and 1 mm depth and loaded with renal pig biopsies is shown; the pre-formed matrix is provided with small cavities pre-punched into the projections formed between the grooves, said cavities being pre-filled with the biomimetic gellable colloid of the invention previously marked in contrasting colors and with a specific disposition which creates a unique code.

Figure 4 shows a transverse section through a surgical specimen of human prostate (adenocarcinoma] previously immersed into a fluid biomimetic gellable colloid which was subsequently ionically (CaCb) and chemically (formaldehyde] polymerized; the specimen was sliced at 5 mm and paraffin processed via conventional methods; such an outer layer of biomimetic material may be used for marking structures of interest during grossing or important anatomical landmarks; during microscopic examination, the previously dyed biomimetic material facilitates for example, the identification of malignant extra capsular invasion.

Detailed description of the invention

The present invention discloses a biomimetic gellable colloid for coding and processing histological specimens for microtome sectioning and diagnostic examination. The dispersed phase of the colloid is uniform / homogeneous and the colloid may be solidified / gelled upon demand. Its chemical and mechanical properties may be tailored by varying its composition (ingredients, proportions, etc.] depending upon the intended application: for example, a fluid gellable colloid may be prepared to incorporate a histological specimen, a gellable colloid may be used in the preparation of a preformed matrix for receiving histological samples, a gellable colloid may be used for the embedding of a sectionable code in the histological preparations or according to other applications as further set forth in the description.

The term "sectionable" as used in expressions such as "sectionable matrix", "sectionable code", "sectionable preparation" refers to the operation of microtome sectioning.

For the purposes of this application, the colloid may be defined as a mixture of two phases, a dispersed phase and a continuous phase. These phases may be immiscible or not.

The emulsion is a type of colloid in which both the dispersed phase and the continuous phase are liquid. The gel is a type of colloid in which the dispersed phase is liquid and the continuous phase is a solid. The sol is a type of colloid in which the dispersed phase is solid and the continuous phase is liquid. The solid sol is a type of colloid in which both phases are solid.

The gellable colloid of this invention may exist in any of these states (emulsion, gel, sol, solid sol), depending upon the preparative phase and upon the intended application.

The gellable colloid of this invention comprises a hydrophilic component and may be gelled/solidified upon demand. Preferably, the colloid is a nano-/microcolloid (a nano- /microemulsion).

The colloid may optionally further comprise a hydrophobic component. According to one specific embodiment, the gellable colloid comprises both a hydrophilic and a hydrophobic component. Optionally, it may contain one or more enzymes and/or other aids for the desired modification of some physical and chemical properties.

An illustrative example of the colloid in the sol phase is a gellable colloid according to the invention wherein the continuous phase has a lower transition temperature and the dispersed phase comprises solid hydrophobic vesicles. In one embodiment, the dispersed phase comprises stearic acid containing vesicles and vesicles of hydrogenated oils with a melting point which has been previously adjusted such as they become solid at the working temperature.

The hydrophilic component may comprise a protein component and/or a polymerizable carbohydrate and/or a pre-polymerized carbohydrate, e.g. a micro- or nanofibrillar insoluble carbohydrate, in an aqueous phase such as water or an organic or inoganic buffer.

Optionally, the hydrophilic component may comprise other compounds such as mineral or organic salts, cation sequestrants/chelating agents (to prevent self-oxidation of lipids and/or the polymerization of the carbohydrates which is catalysed by several metals), surfactants, chaotropic agents, kosmotropic/osmolyte agents, pigments, polymerisation and/or stabilization agents, plasticizers, preservatives, or mixtures thereof.

The protein component may comprise for example, proteins, peptides, polypeptides (e.g. gelatine) or mixtures thereof; purified, semi-purified or non-purified; natural, of animal or plant origin, or recombinant.

The purified proteins of animal origin may be derived from mammals, birds, fish, etc., or may be synthetic, such as recombinant.

One may prefer the use of protein isolates of animal origin, potentially combined with gelatine in different proportions, depending upon the desired mechanical properties of the end product. Non-limitative examples of gelatine which may be used according to the invention are gelatine type A or B, Bloom 20-350 and the like. More preferably, the protein component comprises a mixture of gelatines with different molecular mass (between 5,000 - 1,000,000).

The pre-polymerized carbohydrates serve to increase viscosity, emulsificability and stability of the emulsion, and in the same time help to create an additional network within the protein polymer (co-polymerization). Exemplary non-limiting pre-polymerized carbohydrates are micro- or nanofibrillar insoluble carbohydrates, e.g. microfibrillar plant cellulose, microbial cellulose (nanocellulose), etc.

A polymerizable carbohydrate means one or more polymerizable carbohydrates, linear or branched, of natural or synthetic origin, or mixtures thereof in various proportions. Polymerizable carbohydrates may be optionally functionalized by chemical methods well known to the person skilled in the art. Polymerizable carbohydrates are preferably polyanionic, for example sodium alginate or alginate esters, gum arabic, polyvinylpyrrolidone, guar gum, xanthan gum, konjac gum, carboxymethylcellulose, etc.

The polymerizable carbohydrates have the same functions as the above mentioned pre- polymerized carbohydrates, i.e. to increase viscosity, emulsificability and stability of the emulsion, also helping the creation of an additional network within the protein polymer (co- polymerization), in this case however after the polymerization step which occurs on demand. While the polymerizable carbohydrates are water soluble, the pre-polymerized carbohydrates are insoluble. The polymerizable carbohydrates can be used separately or in various combinations. Some combinations exhibit synergism. Polymerization of carbohydrates may be temporary (thermally or chemically reversible] or irreversible, depending on the needs.

The protein component and/or the polymerizable carbohydrate may be gelled either separately within the hydrophilic component, or preferably together.

Non-limiting examples of organic or mineral salts are any organic or mineral salts commonly used for the preparation of pre-determined pH buffers (for example for a pH between 4 and 9, preferably between 6 and 7). It is required that the pH is chosen such as to be different from the isoelectric point of the protein.

Non-limiting examples of cation sequestrants/chelating agents which may be mentioned are ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), sodium hexametaphosphate (Calgon™), etc.

Non-limiting examples of surfactants which may be mentioned are anionic agents, such as alkylbenzene sulfonates, cationic agents, for example quaternary ammonium salts, non-ionic surfactants, for example polyoxyethylene from the potassium sorbate range, Tween, Triton or Brij, non-ionic agents such as glycoside from the octyl-tioglycoside range, maltosides, detergents from the HEGA or MEGA range, or zwitterionic agents from the CHAPS range. Alternatively, fatty alcohols may be used such as undecanol (11 carbon atoms], dodecanol (12 carbon atoms), tridecanol (13 carbon atoms), 1-tetradecanol (14 carbon atoms), pentadecanol (15 carbon atoms), cetyl alcohol (16 carbon atoms), 1-octadecanol (18 carbon atoms), etc.

Non-limiting examples of chaotropic agents which may be mentioned are, inter alia, urea, thiourea, guanidine chloride.

Non-limiting examples of kosmotropic / osmolytic agents are non-ionic kosmotropic agents such as proline, lysine, trehalose, or ionic kosmotropic agents such as trimethylamine, N-oxide (TMAO) or sodium glutamate.

Non-limiting examples of pigments are soluble pigments (hydrophilic or lipophilic) or insoluble, preferably insoluble pigments, for example those in the Davidson® Tissue Marking Dye range or drawing ink.

Non-limiting examples of polymerization and/or stabilization agents are chemical agents such as carbodiimides, poly L-glutamic acid, acrylic acid, alkylenes, citric acid, polyphenols (e.g. caffeic, chlorogenic, caftaric, tannic acids, etc.), flavonoids (e.g. quercetin, rutin, _, etc.), oxidized mono- or disaccharides, oxo-lactose, dialdehydes based on a carbohydrate group (for example galacto-hexodialdose, etc.), genipin and other iridoid glycosides, secoiridoids, preferably oleuropein, thiol-reactive groups (for example, polyethylene glycol), dextran, oxidized dextran, dialdehyde dextran, aldehydes of glyoxal type, formaldehyde or glutaraldehyde; enzymatic agents such as oxidases which are catalysed by copper ions (laccase, bilirubin oxidase, etc.), enzymes from the transglutaminase group (calcium-dependent and calcium-independent, of natural origin or recombinant, etc.), tyrosinase, etc.; physical agents, such as UV radiation, gamma radiation, etc.

Non-limiting examples of plasticizers are glycerol, ethylene glycol, diethylene glycol, butylene glycol, etc. Plasticizers facilitate the emulsification and serve as wetting agents in the mixture; the mixture becomes more fluid and is more easily injected / poured into the mould, while the thus obtained end product is less brittle.

Non-limiting examples of preservatives are benzyl benzoate, sodium benzoate, thimerosal, etc.

The hydrophobic component comprises one or more compounds of the group consisting of saturated fatty acids, such as butyric acid, an acid with 4 carbon atoms which is found, for example in butter; lauric acid, an acid with 12 carbon atoms which is found, for example in coconut oil, palm kernel oil, breast milk; myristic acid, an acid with 14 carbon atoms which is found, for example in the cow's milk; palmitic acid, an acid with 16 carbon atoms which is found, for example in palm oil and meat; stearic acid, an acid with 18 carbon atoms which is found, for example in meat and cocoa butter; unsaturated fatty acids, for example linolenic acid, stearidonic acid, linoleic acid, arachidonic acid, palmitoleic acid, oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, etc., which are found in various combinations, for example in plants or animals, such as in olive oil, jojoba oil, avocado oil, walnut, palm, coconut, peanut, soybean, flax, lard, tallow, etc.; alkyl amides of fatty acids, condensation products of fatty acids with proteins, simple or complex lipids, sulphated, ethoxylated or propoxylated fatty alcohols, sterols, hydrocarbons, such as, without any limitation, paraffin wax or oil, hydrophobic solvents and the like, alone or in various mixtures thereof, selected such as to adjust the point of solidification / crystallisation to a desirable value according to the specific application / use.

Non-limiting examples of fats are triglycerides, mixtures of triglycerides, e.g. Miglyol®, Softisan®, etc.; glycolipids, lipoproteins, oils, such as olive oil, castor oil, coconut, palm, peanut, soybean, flax, jojoba, avocado and hydrogenated oils, such as peanut, castor oil, etc.; synthetic oils such as silicone oil or paraffin wax, natural, synthetic or semi-synthetic waxes, such as beeswax, oleic, myristic, cetylic or palmitic acid esters, waxes derived from petroleum, phospholipids.

The hydrophobic component serves as a porogen in the composition of the gellable colloid. It may be progressively removed by solvent extraction during the histological processing and it also assists in the prevention of the collapsing of the hydrophilic component of the colloid.

If it is necessary to subject the mixture of components of the hydrophobic phase to repeat cycles of solidification/re-melting, it is preferred that the hydrophobic phase acts as an eutectic mixture. When the gellable colloid is used for the production of pre-polymerized receptacles, hypo- or hypereutectic hydrophobic mixtures may be advantageous, depending upon the targeted tissue and upon the specific physical and chemical properties intended to achieve /optimize.

According to an embodiment of the present invention, the continuous phase is hydrophilic and the dispersed phase is hydrophobic (for example oil-in-water emulsion). This embodiment is preferable for example, for paraffin preparations. By the judicious selection of the composition and parameters of the preparations, homogeneous hydrophobic vesicles with controlled sizes are obtained. By controlling the size of the vesicles in the dispersed hydrophobic component and by a suitable choice of the compounds that it comprises (compounds of suitable extractability, for example, oils), its rate of dissolution and elimination from the mixture during the processing of the histological sectionable preparation can be controlled (for example, ethanol, solvents, molten wax, etc.).

According to one embodiment, the continuous phase is hydrophobic and the dispersed phase is hydrophilic (for example water-in-oil emulsion).

According to yet another embodiment, both phases are hydrophilic, i.e. a water-in-water emulsion, e.g. when micro- /nanocellulose is employed.

The latest two mentioned embodiments (the water-in-oil and water-in-water emulsions) provide a higher resistance to the dehydration which normally occurs in the course of long- term archiving of extemporaneous preparations which are sectioned on ice.

It is apparent to the person skilled in the art that any combination of these types of emulsions can be prepared (e.g., oil-in-water-in-oil or water-in-oil-in-water, etc.), as well as micellar emulsions showing complex lamellar structures. According to a preferred embodiment, more than 50% of the vesicles of the dispersed phase of the gellable colloid have a diameter below 10 microns (micro- / nanocolloid, micro- / nanoemulsion].

The colloid of the present invention helps the pathologist to correctly orient and place the histological sample(s) before processing, sectioning and diagnostic examination.

Preformed sectionable matrices may be manufactured upon the solidification of the gellable colloid of the invention, in different shapes and sizes, suitable for receiving and processing various histological samples.

Alternatively, histological samples may be incorporated into the biomimetic gellable colloid of the invention while in a fluid state, followed by its gelation upon demand in view of the processing of the thus obtained solidified preparation for diagnostic examination.

Furthermore, any suitable combination of these methods may be used, e.g. firstly positioning the histological samples in the receptacles of various shapes of a preformed sectionable matrix obtained by the gelation and prior stabilization of the colloid of the invention, followed by the "sealing" of the histological samples in said receptacles with said gellable colloid in a fluid phase and its solidification upon demand.

Thus, the gellable colloid may be used as a sectionable mechanical support in the histological processing of any diagnostic specimens, of either plant, animal or human origin.

The same gellable colloid may be used as a means for writing sectionable unique codes / identifiers which may be for example embedded simultaneously with the histological preparations, thus also being firmly attached thereto. The embedding of sectionable codes in the histological preparations with the aid of the gellable colloid of the invention eliminates any risk of misidentification of the preparation and of the microscopic sections deriving therefrom.

The gellable colloid of the invention makes all the areas of interest in the histological samples accessible to analysis, in such a manner that the histological sections become as informative as possible, allowing for a quick and accurate diagnosis at a reasonable price. The sectionable code in some specific embodiments facilitates the three-dimensional identification of the location of biological structures processed for diagnostic, similarly to a holographic map.

Moreover, the gellable colloid of the invention significantly facilitates the microtome sectioning, especially of small, heterogeneous, friable and/or multiple fragments. Tissue fragments embedded in the gellable colloid enjoy a greater protection during processing. As the gellable colloid mechanically protects the tissue sample, it is possible to increase the stirring of the solutions used during processing, which automatically leads to a significant shortening of the time elapsed from the specimen sampling to the obtaining of the examination result (TAT - turnaround time).

The gellable colloid may be used for the marking of certain areas of interest by applying it in a thin layer during the preliminary examination of large surgical parts for the purpose of subsequent identification of topographic landmarks that would otherwise be impossible to find after the "grossing" of the parts to be sent for microscopic analysis. The "grossing" means the dissection of representative fragments small enough to be processed histologically (e.g. of maximum 0.5 x 2.4 x 3.5 cm).

Currently, this is done with the aid of marking inks (an insoluble pigment applied on the outer layer of the grossed fragments). Marking inks have the disadvantage of providing a very superficial mark which is quite hard to find even when using high-magnification microscope lenses.

The gellable colloid of the invention can be solidified/coagulated/gelled upon demand, after the application on the fresh or fixed surgical piece. Gelation may be chemical (e.g. by the use of calcium salts) or physical (e.g. by the application of the colloid to the surgical piece in a fluid state, more precisely in a state of sol at 22-35°C, followed by gelation of the colloid occurring within seconds or tens of seconds at a temperature lower by 5-10°C than the working temperature at which the colloid is in the sol phase). Alternatively both methods may be used sequentially (for example gelling by cooling, followed by chemical stabilization). The person skilled in the art may consider other methods as well.

The gellable colloid of the invention is a semi-synthetic material, a man made material which may simulate practically all the various types and subtypes of animal/human/plant tissues. The composition of the colloid may be optimized to incorporate cellular suspensions/sediments or from microorganisms, hystoids, spheroids and/or multiple biopsies/explants of any tissue origin, by varying the proportions of the compounds in any of its components (hydrophilic, hydrophobic if used, more specifically, peptides, proteins, lipids, carbohydrates, water, etc.) in such a way that the physical properties (e.g. phase-shift temperature, the degree of shrinkage during dehydration, sectionability) are as close as possible to the properties of the tissue of interest, for example the specimen embedded within the gellable colloid. By simultaneously processing multiple biopsies/explants and by the precise orientation now performed by the pathologist during the preliminary examination, significant savings are obtained and an improved accuracy of the diagnosis.

The use of the gellable colloid of the invention for permanent and unambiguous identification purposes of the diagnostic preparations permanently removes the risk of inadvertent mixing of samples/diagnoses and at the same time significantly lowers the costs.

According to one more specific embodiment, the invention relates to a gellable colloid comprising a protein component in a proportion of 3-20% by weight, preferably in a proportion of 6-16% by weight, a hydrophobic component in a proportion of 5-50% by weight, preferably in a proportion of 7-25% by weight, a polymerizable carbohydrate in a proportion of 0.1-5% by weight, preferably in a proportion of 0.5-3.5% by weight, the aqueous phase and, optionally, a plasticizer in a proportion of 1-25% by weight, preferably in a proportion of 5- 15% by weight, optionally, a pigment, possibly insoluble or conjugated with one of the already mentioned components of the mixture, in a proportion of 0.01-10% by weight, preferably in a proportion of 0.1-2% and, optionally, a delayed-polymerization agent, either ionic which releases for example the calcium from some water-insoluble salts thereof, or enzymatic, or of another type.

According to a more specific embodiment, the protein component is a gelatine or a mixture of fractionated gelatines with molecular weight ranging from 5,000 to 1,000,000.

According to another specific embodiment, purified or non-purified protein isolates are used, possibly of animal origin, possibly combined in various proportions with gelatine, for example with type A or B, Bloom 20-350 (depending upon the intended hardness of the end product], with or without thickening additives.

According to another embodiment, the hydrophobic component has a melting point ranging between -20°C and +80°C (e.g. -20°C when castor oil is used, +70°C when stearic acid is used]. By the judicious selection of the hydrophobic component, a melting/solidification point in the area of interest may be obtained.

According to another embodiment, the plasticizer is glycerine.

According to yet another embodiment, the polymerizable carbohydrate may be alginic acid, one or more salts of the alginic acid or mixtures thereof, preferably sodium alginate. Sodium alginate acts as a thickening and emulsifying agent. In combination with salts of calcium, barium, strontium, lead, aluminium or other cations, for example with sodium tetraborate, it contributes to the solidification of the colloid upon demand.

The invention also relates to a process for preparing a gellable colloid according to the invention, comprising the following steps: preparation of a mixture comprising the hydrophilic component and optionally the hydrophobic component when also used, possibly together with any other optional ingredient from those already listed hereinbefore, as well as with any other agent that the person skilled in the art may consider, for example, plasticizer, pigment, delayed- polymerization agent, a preservative, etc., followed by emulsification of the mixture thus obtained by conventional methods.

Preferably, the emulsification of the mixture is aimed to preparing an emulsion in which more than 50% of the vesicles have a diameter below 10 microns or around this value, more preferably below 5 microns, and even better below 2 microns.

The process may comprise a step of "erasing" the thermal memory of the gellable protein while the emulsification step can be repeated as necessary.

A fluid gellable colloid is thus obtained and this colloid may be used as such or may be stored for a long term by refrigeration or freezing, to be later thawed before use.

The gelation point of the protein component may be decreased below the room temperature, either by the use of certain varieties of gelatine, for example the gelatine extracted from cold water fish, or by the controlled hydrolysis of gelatines of types A and B (either enzymatic or chemical or both], or by the addition of chaotropic agents (urea, thiourea, guanidine chloride, etc.), or by a combination of these methods.

The protein component may be hydrated and solubilized in the buffer solution (e.g. phosphate buffer, Tris, acetate, borate, etc.), at a pH value which is different from the isoelectric point of the protein, preferably from 6 to 8. It is preferred that the protein solution is heated up to 60- 70°C and maintained at this temperature for 1-2 hours prior to emulsification in order to erase the "thermal history" of the gellable protein which may be for example the gelatine.

The invention also relates to a process for preparing a preformed sectionable matrix for the receipt of a histological preparation, obtainable from the fluid gellable colloid according to the invention, said process comprising the following steps: - shaping/forming the fluid gellable colloid into a preformed mould of a size suitable for the intended application (for example by injection, casting, manufacturing of a fabric with preformed filaments, extrusion, etc.), and

- gelation/solidification of the colloid (for example, by one or more methods selected from physical methods, e.g. cooling, irradiation, etc.; chemical methods, e.g. enzyme treatment, treatment with chemicals, ionic solutions, pH alteration, etc.; combined in any sequence).

The sectionable preformed matrix thus obtained may be immediately used or may be sequentially stabilized/reinforced.

Non-limiting methods of stabilizing/reinforcing said matrix are physical methods (e.g. freezing, freeze-drying, radiation polymerization, etc.), chemical methods (e.g. polymerization, stabilization with the previously specified polymerization/stabilization agents, with mineral salts, e.g. cations such as those mentioned above, and the like), enzymatic methods, etc.

The person skilled in the art may use the methods of stabilization/ reinforcing /consolidation of the matrix in any sequence and in any combination, taking into account the specific enzyme or chemical incompatibilities. Moreover, the method of stabilization will be chosen according to the final application. For example, if a sectionable matrix - histological sample assembly is intended for freeze-sectioning, the enzyme stabilization may not be necessary. If the preparation is fixed in a calcium containing fixative, the stabilization phase with cationic solutions may be reduced or eliminated, etc.

The method may further comprise a phase of long-term preservation of the preformed sectionable matrices obtained. Non-limiting examples of methods for preservation are freezing, keeping in preservative solutions, for example in formaldehyde, freeze-drying, etc.

The preparation of the mixture for obtaining the gellable colloid of the invention consists of mixing the ingredients in any order. It is preferred to mix the powder into the liquid phase (containing also, for example, the hydrophobic component and glycerol, when used) less the aqueous phase, whereupon the pre-warmed aqueous phase is added with constant stirring.

Depending upon the composition, the working temperature, the rate of mixing of the components and the cooling rate of the mixture, water-in-water, water-in-oil, oil-in-water, oil- in-water-in-oil, water-in-oil-in-water emulsions may be obtained, as well as complex micellar structures or reticulated structures by stopping the phase separation (for example by "pinning" in the "spinodal decomposition phase"). Each type of emulsion has different properties, the differences being sometimes significant in terms of behaviour of the gellable colloid during the histological processing, some types being more suitable for preparations obtained by dehydration and inclusion in paraffin, and others for extemporaneous preparations that are sectioned on ice.

After many trials, it has been observed that the oil-in-water emulsions are preferable for paraffin preparations. For the preparations on ice, both types of emulsions may be used, but the water-in-oil and the water-in-water types display a greater resistance to the dehydration which occurs during long-term archiving.

By vigorously mixing with: i) a static microfluidic mixer or ii) a rotor/stator mixer, possibly "inline", or in) a propeller/paddle mixer that does not introduce air into the solution, preferably a mixer working under vacuum; followed by the judicious selection of the mixing speed [fast enough to obtain emulsification, but without reaching levels that could favour the thermal denaturation of proteins), very homogeneous emulsions will be obtained, with small or very small vesicles [microemulsions or nanoemulsions).

Viscosity-enhancing agents may be added to these emulsions and make them stable, without the appearance of separation, coalescence or Ostwald ripening phenomena for long periods of time (hours, days, months, years). Gelation can be performed sequentially or simultaneously by controlled cooling of the mixture or by enzymatic and/or ionic means.

By the selection of any of the following in any combination: the mixing method, the surfactants, the speed of gelation (e.g. the cooling temperature and the cooling rate, or the concentration of the enzyme and/or the temperature of the enzymatic reaction), the size of the vesicles incorporated / stabilized in the end product (gel) may be controlled, being preferred the vesicles with diameters between a few tens of nanometres up to 10 microns.

In addition, hydrophobic vesicles with lamellar or reticulate arrangement may be made if needed; a structure may be obtained where the hydrophobic vesicles partially communicate similar to a crystallized labyrinth structure, e.g. with an aspect of open cell sponge, but without the complete coalescence that would generate large hydrophobic "lakes" which might hinder the extractability during the histological processing.

This reticulate arrangement facilitates the mandatory dehydration and clarification phases prior to paraffin infiltration. Breaking of the hydrophobic vesicles may be achieved either during the preparation of the emulsions (for example by rapid cooling which leads to the crystallization of the fat or of the solid fatty acids at room temperature), or after the gelation of the mixture, for example by slow freezing or by repeated freeze/thaw cycles or by repeated freeze-drying/rehydration cycles of the matrices, carried out in order to obtain large ice crystals which will break the proteic walls of the vesicles.

According to an embodiment, the hydrophobic component may be added to the solution in which the other components have been previously dissolved, pre-heated to a temperature slightly higher than the melting point of the hydrophobic component. Alternatively, all the ingredients except for the aqueous phase are dry-mixed and suspended into the hydrophobic phase, while the aqueous solution pre-heated to the desired temperature is then added to this mixture.

The final properties of the gellable colloid may be tuned by the incorporation of plasticizers, surfactants, kosmotropic and/or osmolytic agents, or by the incorporation of chaotropic agents.

Kosmotrops and/or osmolytes are used for the increasing of the organization of the water molecules in the solvation layer of solubilized proteins, in order to stabilize the macromolecules and therefore, to increase the intermolecular interactions and aggregation. Other examples of kosmotropic agents include non-ionic kosmotropic agents such as proline, lysine, trehalose, or ionic kosmotropic agents such as trimethylamine N-oxide (TMAO) and sodium glutamate.

The chaotropic agents have the opposite action of the kosmotropic agents. Extensive testing of the urea concentration in the range of from 1/10 to 2/1 relative to the total concentration of the protein has shown that the transition sol - gel temperature range, the flexibility and other mechanical properties of the resulting gellable colloid can be predictably modified.

Other excipients which may be used in the composition of the gellable colloid according to the invention are surfactants (emulsifiers). They may be used either separately or in various combinations, selected according to the HLB value (Hydrophile-Lipophile Balance).

Other excipients which still may be used in the composition of the gellable colloid according to the invention are insoluble pigments which increase the optical contrast [within the visible light, ultraviolet or infrared spectra). Their use facilitates the sectioning and examination of the microscopic preparations. In the manufacturing process, successive layers in different colours may be applied by moulding, spraying, etc., significantly facilitating the quick orientation of the blocks in the correct plane of sectioning.

In addition, substances having specific optical properties (e.g. specific colours, fluorescence at specific wavelengths in the infrared or ultraviolet spectra) may be incorporated in discrete locations, for example with tattoo needles, to prevent forgery or for the purpose of creating landmarks that will facilitate the microscopic analysis of the resulted preparations or unique sectionable codes as barcodes, bi- or tri-dimensional matrices, alphanumeric codes, etc.

A standard version of the invention process includes the preparation of a mixture comprising a protein component in a proportion of 3-20% by weight, a hydrophobic component in a proportion of 5-25% by weight, a polymerizable carbohydrate in a proportion of 0.2 - 3% by weight, the aqueous phase (for example 50 mM acetate buffer, pH 7.0, 10 raM EDTA, 1% sodium benzoate], optionally a plasticizer in a proportion of 5-25% by weight, optionally an insoluble pigment in a proportion of 0.1 to 10% by weight and optionally a polymerization agent.

The emulsification of the obtained mixture is made preferably under vacuum, at 5,000-25,000 rpm, preferably at 7,000-17,000 rpm, and more preferably at about 10,000 rpm, at a temperature between 20°C - 60°C, preferably between 22°C - 40°C and the emulsion is homogenized until the desired size of vesicles is obtained. The mixture is left for 60 minutes at 60°C for erasing the thermal memory of the gellable protein, cooled to the injection temperature (for example, by 5-10°C above than the sol-gel transition temperature], the emulsification step is repeated if necessary, the homogeneous mixture thus obtained is then injected into the desired shapes (preformed moulds of suitable size for the intended applications) and optionally allowed to cool as required (e.g. for 2 to 18 hours at 4-12°C].

Patent application no. PCT/RO2012/000017 published under no. WO2013/002661 discloses a multitude of appropriate matrices for the purpose of the present invention. However, the person skilled in the art may also consider any other shapes.

The resulting matrices of the desired shapes, e.g. matrices with channels/grooves or slots/wells, are carefully removed from the moulds and optionally stabilized, e.g. by immersion in 1-5% aqueous solution of CaCh or other cationic solution, at room temperature or below, for a period of time from 5 minutes to 3 hours or longer, preferably from 5 minutes to 1 hour.

The resulting shaped material may be optionally stabilized by spraying with an aqueous solution of 1-5% CaCh or with other cationic solution.

The resulting shaped material may be optionally subjected to enzymatic polymerization, for example in a bacterial transglutaminase solution (0.5 - 10 u/g protein] at room temperature for 24 hours or at 40 - 50°C for 4-10 hours. The resulting shaped material may be optionally polymerized by physical means: by repeated freeze/thaw cycles or repeated [cryojdrying/rehydration cycles. If delayed-polymerization agents have been included in the mixture, these steps can be shortened or omitted.

The resulting shaped material may be washed with distilled water and may be further stabilized with 0.1-5% glutaraldehyde at pH 7-8 for 1-3 hours. Free aldehyde groups can be neutralized using 1-10% potassium metabisulfite. Optionally, additional stabilization may be performed with 0.1-5% tannic acid for 5-60 minutes. Optionally, the residual tannic acid is neutralized with 1-5% sodium bicarbonate for 1.5 hours.

Optionally, the resulting shaped material is sectioned to the appropriate sizes for various applications desired.

Optionally, the end product is packed in airtight packages containing formaldehyde or other preservatives. Optionally, the end product is freeze-dried and stored dry. Optionally, the end product is rehydrated at the time of use. Optionally, the end product is chemically sterilized or by irradiation with gamma rays. Optionally, the end product is frozen for storage until needed.

Optionally, the resulting shaped material can be histologically processed (e.g. by dehydration, clearing, infiltration] to serve as a sectionable matrix for histological preparations which are in the same preparative stage as the matrix, thus generating multiplex preparations, e.g. a Tissue MicroArray as disclosed in the US patent application no. 60/420,027 entitled "The HistoBest method for preparing multiuse tissue arrays".

Optionally, the resulting dehydrated sectionable matrix may be used as a "filter" for the capture and concentration of sediments, cells, cultures of microorganisms, spheroids and hystoids isolated from various effusions, lavages, aspirates or secretions taken for diagnostic purposes. The matrix may be for example thin or may be optionally preformed in the form of a receptacle or funnel.

The matrices loaded with sediment, cells, cultures of microorganisms, spheroids, hystoids are further covered with a protective coating of the gellable colloid in sol state, followed by the quick stabilization of this layer, for example by cooling or by ionic polymerization, fixation and conventional histological processing.

A sectionable matrix thus obtained from the colloid of the invention simulates / mimics a biological material, e.g. the animal tissue if desired. The various properties of this material are tunable, for example by varying its chemical composition and/or by selecting a certain method of preparation. The gellable colloid of the invention in any selected composition and with any method used for its preparation is easily microtome sectionable, resulting in sections with chemical properties ensuring satisfactory adhesion to microscope slides. This is accomplished for example by the protein component displaying negative electric charges which complement the positive charges of the microscope slides and not only. Robust preparations are obtained which are very stable in the course of staining. The tinctorial affinities of the sections are therefore predictable and of low-intensity, without hampering the microscopic examination.

The nature of the emulsion depends inter alia upon the mixing speed, the agents used and their proportions. The obtained gellable colloid is sectionable both in paraffin preparations and in extemporaneous preparations, e.g. on ice, enabling the embedment of multiple biopsies/fragments which can be oriented as desired.

The gellable colloid in its fluid state can also be easily integrated into any procedure which automates the collection and the orientation of biopsies, for example in the SmartBx system (UC-CARE Medical Systems, Israel) to significantly improve the performance. Up to date, this system does not allow the simultaneous placement of more than 2-3 biopsies and the resulting blocks are thin making difficult the sectioning of the paraffin blocks.

The gellable colloid can also serve as a 3D ink for the marking of the topographic landmarks which allows for the embedding of unique identification codes along the whole thickness of the preparation. The codes are sectionable and are visible on each resulting microscopic slide. The specimens are thus permanently embedded in the histological preparation together with the unique identifier which prevents any inadvertent migration during transport or processing.

The codes also enable the precise dating of the preparations, e.g. with tracers having predetermined physicochemical properties, like radioactive isotopes, fluorophores with controlled degradation, etc. They also enable the identification of the area/thickness wherefrom the microscopic section was sliced. Calibrated balls made out of latex or of the inventive gellable colloid previously coloured with various pigments may be incorporated into the preparations, to facilitate morphometric analyses.

Therefore the invention also refers to the use of the gellable colloid in a fluid state as a marking ink of topographic landmarks of histological specimens.

It is another object of the invention to use the gellable colloid in a fluid state for the filtration/concentration, encasement and orientation of cellular suspensions/sediments, hystoids, spheroids and histological specimens in view of processing for examination. It is another object of the invention to inject the gellable colloid in a fluid state into the vasculature or hollow organs for the purpose of avoiding their collapse during histological processing.

It is another object of the invention to use the gellable colloid in a fluid state for a precise / correct incorporation and orientation of multiple specimens in a single histological preparation, thus enabling the simultaneous processing, sectioning, staining and histochemical and/or molecular, analysis thereof, and of course the simultaneous examination of all the specimens.

It is another object of the invention to use the gellable colloid in a fluid state for the purpose of writing / printing unique identification codes for histological specimens.

The fluid gellable colloid may be formed into moulds and hardened in various shapes to obtain sectionable matrices, e.g. with grooves or wells of different sizes, or it may be frozen for long- term storage and subsequently thawed before use or it may be also kept in a semifluid state for long periods of time.

The gellable colloid already shaped in matrices can be chemically or physically sterilized, e.g. with formaldehyde, glutaraldehyde, ethylene oxide or gamma irradiation and/or it can be stabilized by the addition of preservation agents, such as benzyl benzoate, sodium benzoate, thimerosal, anti-oxidants, e.g. ascorbic acid, butylated hydroxytoluene, tocopherols, and the like.

Dedicated injection moulds may be used to increase the histological processing speed of the end product, e.g. moulds which allow the production of sectionable matrices with very thin walls or with a specific geometry.

According to another embodiment, moulds that form microgrooves on the "sole" of the sectionable matrix may be used, e.g. with specific patterns like diagonal hatching, etc., increasing thereby the contact and exchange surface with the dehydration and clarification solutions. A specific arrangement and sizing of these grooves enable an accurate adjustment and control of the contractions that will occur during dehydration, both in amplitude and spatial orientation.

Polyphenols such as tannic acid, gallic acid and the like, may be used to increase the mechanical strength of the sectionable matrices as well as to increase the adhesion of the histological samples to the matrix. To this end, the sectionable matrices may be immersed for different periods of time (varying largely for example from about 5 to about 180 minutes) in solutions of different concentrations (e.g. 0.1 - 10%). Alternatively this chemical treatment may be applied only on one side of the matrix to create an asymmetric matrix. This obtained reinforced surface is advantageous because it confers to the sectionable matrix a certain mechanical strength during the various manipulations the person skilled in the art might envisage. Although this "reinforcement" may affect the sectioning properties of the matrix to some extent, the auxiliary reinforced layer may be removed prior to sectioning, leaving the remainder of the matrix surrounding the histological samples unaffected.

The gelation of the colloid may be for example accomplished by the polymerization of gelatine or of other components that make up the protein component. Polymerization may be performed by:

• physical means, e.g. by cooling at a temperature below the gelation point of the specific mixture, heat denaturation, high pressure, etc.;

• chemical means, e.g. with carbodiimides; poly L-glutamic acid; polyacrylic acid; alkylene; citric acid; polyphenols like caffeic, chlorogenic, caftaric, tannic acids, etc.; flavonoids like quercetin, rutin, etc.; oxidized mono- or disaccharides; oxo-lactose; dialdehydes based on a carbohydrate group, like galacto-hexodialdose, etc.; genipin and other iridoid glycosides; secoiridoids, preferably oleuropein; thiol-reactive groups, e.g. polyethylene glycol; dextran; oxidized dextran; dialdehyde dextran; glyoxal aldehydes; formaldehyde or glutaraldehyde;

• enzymatic polymerization, e.g. using copper catalyzed oxidases like laccase, bilirubin oxidase, etc.; enzymes from the group of transglutaminases which may be calcium- dependent or calcium-independent, naturally occurring or recombinant; tyrosinase, etc.; or

• UV- or gamma-irradiation,

• repeated freezing/thawing cycles,

• repeated (cryo)desiccation/rehydration cycles.

These methods of gelation/polymerisation are illustrative only and may be combined in any order to obtain gels with specific properties. Other methods are readily available to the skilled person.

To manufacture sectionable matrices with high stability, greater mechanical strength and extremely accurate geometrical shapes, for example as those designed for simultaneous processing of multiple biopsies, the fluid colloid of the invention is preferably gelled in sequential manner using several methods.

According to a preferred method of the invention, cooling and slow maturation of the hydrolysed collagen network is performed in a first step (partial reconstitution of the triple helix], followed by optional additional polymerization of the matrices in a second phase, and/or thermo stabilization thereof with ionic methods, e.g. with calcium, barium, strontium, lead or aluminium ions when alginic acid was used as a polymerizable carbohydrate, or with a potassium salt when carrageenan was used.

The use of ionic gelation of the alginic acid or sodium alginate leads to the obtaining of oriented porous structures: a polymerization front is formed on the surface that comes into contact with the ionic solution and tubular/duct-shaped structures are generated perpendicularly oriented to the contact surface. Under certain conditions this ionic polymerization is reversible, e.g. when performing a treatment with acids such as citric acid, hydrochloric acid, etc., or with salts like phosphate, polyphosphate, or with chelating agents, like EDTA, Calgon™, etc., the dissolution of the gel occurs, unless additional methods of stabilization were previously used, e.g. controlled freezing.

Polymerization may also be enzymatically supplemented and/or augmented, either by immersing for longer periods of time the resulted matrices in enzyme solutions, e.g. transglutaminase, tyrosinase, etc., or by prior incorporation of the enzymes into the gellable colloid. In the latter case, the enzymes will not significantly contribute to the stabilization of the collagen network until the end product is brought to the working temperature; e.g. the bacterial transglutaminase is practically inactive at temperatures below 20°C, achieving a peak of the enzymatic activity only at 40-50°C.

Additional stabilization of the matrix may also be performed by chemical polymerization with formaldehyde, glutaraldehyde, genipin, etc.

A surface-alteration treatment may also be used to optimize the sectionable matrix. An increased adhesion is desirable when histological samples are placed in preformed grooves / cavities and this may be achieved e.g. by chemical treatment, e.g. with tannic acid or with some polyphenols which create extremely abrasive nanostructures, or by the application of adhesives such as vegetable gum, dextrin, etc.

The gellable colloid of the invention may also be used for the preparation of paraffin blocks containing sediments, isolated cells from primary or cultured cell lines, cultures of microorganisms, hystoids cultured for the creation of control standards for immunohistochemical analysis or molecular biology, cells and spheroids separated from various effusions, lavages, aspirates or secretions harvested for diagnostic purposes (sputum, saliva, urine, pleural, pericardial, peritoneal or cerebrospinal fluid, milk, etc.).

The gellable colloid of the invention may also be used for the sampling and orientation of explants and tissue fragments for microscopic examination.

The above mentioned applications require the use of the gellable colloid in a fluid state. After the incorporation and thorough mixing (e.g. using static mixers) of the cells, cultures of microorganisms, sediments, spheroids, isolated hystoids and the like, or after the orientation of the harvested fragments, in an appropriate amount of fluid gellable colloid, the gelation can be achieved very quickly and upon demand, by various methods, e.g. by:

• cooling by 10-15°C when the mixture was previously heated to a temperature slightly higher than its transition temperature to the gel state;

• optimizing the mixture of gelatines with different molecular weights and/or the combinations of gelatine with chaotropic agents - e.g. when gelatines and/or chaotropic agents are used in the mixture - to obtain different types of gellable colloid in a fluid state; the resulted colloid is characterized by a gel - sol transition range in the region of the ambient temperature; for example when the colloid is heated to a temperature above 25°C it will melt and remain in a fluid state, gelation being achieved only if it is cooled to a temperature below 20°C, while after the gelation occurs at the temperature below 20°C, the colloid will further remain in a gel state at room temperature (laboratory standard: 22.5 ± 1°C);

• pre-mixing with an enzyme or with low-solubility calcium salts, depending upon the selected gelation/polymerisation method; the working time can be adjusted as desired, e.g. to \, 2, 3 or more minutes.

After completing the gelation stage, the whole gelled colloid - histological preparation assembly can be frozen for freeze sectioning or can be chemically fixed for paraffin processing.

Alternatively, a colloid may be prepared which remains fluid or semi-fluid even at low temperatures, e.g. during refrigeration, using for example chaotropic agents or low-molecular weight fractionated gelatines (< 5000 D), while gelation/polymerization may be triggered upon demand, e.g. after the histological samples were properly oriented, e.g. by prior mixing or by spraying with an enzyme,.or with bi- or trivalent metal salts, for example calcium or barium salts.

Alternatively, CaC loaded liposomes may be used, e.g. made from 90% dipalmitoylphosphatidylcholine and 10% dimyristoylphosphatidylcholine, which can be broken upon demand, e.g. thermally, with gamma-rays or ultrasounds, by pH variations, or with "photo-caged" calcium, etc., to activate the polymerisation of the sodium alginate and/or of the calcium-activated transglutaminase for example.

Incorporation into the gellable colloid of the invention of compounds which remain fluid at low temperatures and are gelled by simple heating is also envisaged. An illustrative such compound is poloxamer 407.

It is another object of the invention to provide a sectionable code which uniquely identifies the final histological preparation. The unique identifier may be created in a sectionable receptacle made out of the gelled colloid of the invention in which biological specimens are placed and sealed using the gellable colloid in a fluid state. The whole assembly consisting of the biological specimen, the polymerised receptacle containing the unique identifier and the fluid colloid sealing it, will be irreversibly polymerized to ensure the permanence of the preparation.

The sectionable codes may be created by etching the codes into the end product or by including the codes at the time of injection using special moulds to this end, similar to those used for printing. Alternatively the codes may be injected into the gelled colloid using techniques similar to the tattooing techniques, using inks, fluorophores, radioactive tracers, etc., or fluid gellable colloid coloured with inks or incorporating fluorophores, radioactive tracers, etc.

Another method which has been successfully implemented allows for a very quick creation of unique codes in the sectionable colloid of the invention, during or after its gelation, by using high resolution embroidery machines and a microtome sectionable yarn which does not degrade during histological processing, e.g. natural or semi-synthetic silk or a resorbable yarn such as a polyglycolic acid, a polylactic acid, a polydioxanone or a caprolactone yarn. The method may also be applied without any kind of yarn, the needle of the embroidery machine creating the inscription itself by controlled punctual removal of small amounts of the gelled colloid. The resulted gaps may be left as such or may be subsequently filled in with colloid in a fluid state, preferably coloured in a colour which provides a contrast to the colour of the background matrix.

Other methods that can be used for writing unique sectionable codes in the gelled colloid of the invention are, inter alia: photolithography, photolithogravure, laser or electro vaporization, high-frequency ultrasound. The colloid in a fluid state may be injected into moulds with a variable geometry to create symbols similar to the Braille system, or in disposable moulds (sacrificial) created from wax, sugar, salt, shellac, rosin, etc., for example similar to the additive 3D printing methods.

Another method which has been successfully implemented allows for the very quick creation of unique codes in the sectionable gelled colloid, stabilized either by freezing or by dehydration and infiltration with paraffin or polyvinyl alcohols with molecular weights over 4000 D, i.e. solid at ambient temperature. Said method comprises the step wherein the stabilized matrices are engraved with unique codes either mechanically, e.g by milling, ultrasound, water jet, or by vaporization, e.g. with laser, plasma jet, electric discharge, etc. After engraving, the stabilized gelled colloid is reverse processed to bring it in a state which makes it suitable for diagnostic purposes, i.e. by thawing, rehydration, etc.

Unique coding of a histological specimen may also be achieved by combinations of characters and/or tracers, e.g. light in the visible, UV, X-rays and/or IR spectra, by activation with alpha, beta, gamma rays and/or with electron beams. Low penetrating radioactive isotopes with alpha emission may also be used, e.g. Americium 241, being harmless to the users.

The composition of the mixture of tracers and their rate of degradation may encode even more information. It may also allow the analysis of the age of preparation, similar to the radiocarbon dating.

By exploiting the specific properties of certain fluorescent substances, "non-invasive" marking may be achieved, with analogue codes and/or with barcodes, e.g. ID, 2D or 3D (hologram).

By simple laser irradiation at a given wavelength, some fluorophores can be selectively inactivated and the code is printed as a negative image (e.g. fluorescent background and non- fluorescent characters). Such a technology is well known to the skilled person in the fluorescence microscopy who is familiar with the "moth-eaten" images appearing in the areas previously exposed to UV or laser, especially when pictures are taken and the exposure is more intense.

As far as the fluorescent markers are concerned, a large variety of markers may be used. Non- limiting examples of such markers are chemicals such as fluorescein and its derivatives, rhodamine and its derivatives, quinine, fluorescent proteins extracted from cephalopods or obtained by genetic engineering, as well as very inexpensive and absolutely non-toxic fluorescent proteins as porphyrin derivatives, bilirubin, chlorophyll, erythrosine, etc. Nanoparticles, e.g. quantum dots and others may also be used. Other possible markers to be used for the development of unique sectionable codes with the gellable colloid of the present invention are inter alia, electro / mechano / chemo responsive elastomers, either natural or synthetic.

Additionally, radio frequency identification tags [RFID] or near field chips [NFC] may be incorporated in the end product. Although non-sectionable, they have both successfully withstood the rigors of histological processing.

The person skilled in the art may now envisage many means of "encryption/encoding" for the histological preparations with the aid of the colloid of the invention. What is surprising and unexpected is the incorporation of these means in a sectionable matrix which now becomes tamper-proof and error proof.

The number of possible combinations is extremely high and in the same time sufficient for the intended purpose. Very simple analogue codes may be imagined for an easy and inexpensive industrial production with existing confortable technologies, easy to be read/decoded and adequate to carry enough information to make them unique, i.e. bearing a unique spectral signature.

EXAMPLES

Example 1

Sectionable colloid for obtaining pre-formed matrices for explants, endoscopic or percutaneous biopsies, prepared with protein isolates and additives and use of said matrices in obtaining paraffin preparations

The following components are mixed in a pre-heated vessel:

- total protein isolate with additives (for example Scanpro EC 40, from BHJ, USA] 100 g

- castor-oil (pharmaceutical grade] 100 mL

- glycerine 100 mL

- potassium sorbate 10 g

- sodium benzoate 10 g

- drawing ink 10 mL

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM) acetate buffer solution (100 mM], pH 7.0, preheated to 60°C. The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA] at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled gradually to 40°C; drawing ink is optionally added and then the mixture is emulsified again at 17,000 rpm.

The mixture is injected into silicone rubber moulds designed for percutaneous prostate biopsies (harvested with a 1 mm diameter needle, max. 22-24 mm biopsy length). The moulds filled with gellable colloid are cooled for 18 hours at 4-10°C. The cold-cured material is extracted from the injection moulds and incubated with stirring, at room temperature, in a solution of 1% CaCI ₂ in distilled water for one hour. The material is then washed with distilled water for 5 minutes and then incubated with continuous stirring in a solution of microbial transglutaminase (2 units per gram of protein in the composition, Activa, Ajinamoto, Japan) dissolved in an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, 10 mL per gram of enzyme, for 24 hours, at room temperature. The material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of glutaraldehyde, 0.25% in distilled water, pH 7.0, for one hour. The resulting material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of potassium metabisulfite 1% in distilled water, for 60 minutes.

The material is subsequently washed with distilled water for 5 minutes, partitioned into segments with 10 grooves and 25 mm width and the resulting matrices are stored in a formaldehyde solution 4% in 50mM calcium acetate buffer solution, at room temperature.

The matrices are loaded by placing biopsies harvested from a surgical specimen into the preformed grooves and the entire assembly is placed in a standard histological cassette to be histologically processed by conventional methods: fixation in formaldehyde, 4% in 50 mM calcium acetate buffer solution at room temperature for 24 hours, dehydration in multiple changes of 45 minutes each, as follows: ethanol 70%, 80%, 90%, 95%, 100% (three changes), xylene (three changes), melted paraffin at 60°C (three changes); the obtained paraffin blocks are semi-serially sectioned to 4-5 micrometres; the sections are applied on microscope slides and stained with haematoxylin-eosin.

Results: the paraffin blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; the matrix shows however an intense stainability and an extremely high heterogeneity; it may require that the pathologist be more concentrated during the microscopic examination, but it does not interfere with the accuracy of the histopathological diagnosis. Example 2

Sectionable colloid for obtaining pre-formed matrices for explants, endoscopic or percutaneous biopsies, prepared with purified protein isolates and use of said matrices in obtaining paraffin preparations

The following components are mixed in a pre-heated vessel:

- total protein isolate (e.g. Scanpro T95 from BHJ, USA) 100 g

- castor oil (pharmaceutical grade) 100 mL

- glycerine 100 mL

- sodium alginate 10 g

- potassium sorbate 10 g

- sodium benzoate 10 g

- drawing ink 10 mL

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, preheated to 60°C.

The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA) at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled slowly to 40°C, followed by the addition of the drawing ink; the mixture is emulsified again at 17,000 rpm.

The mixture is injected into silicone rubber moulds designed for percutaneous prostate biopsies (harvested with a 1 mm diameter needle, max. 22-24 mm biopsy length). The moulds filled with gellable colloid are cooled for 18 hours at 4-10°C. The cold-cured material is extracted from the injection moulds and incubated with stirring, at room temperature, in a solution of 1% CaCh in distilled water for one hour. The material is then washed with distilled water for 5 minutes and then incubated with continuous stirring in a solution of microbial transglutaminase (2 units per gram of protein in the composition, Activa, Ajinamoto, Japan) dissolved in an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, 10 mL per gram of enzyme, for 24 hours, at room temperature. The material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of glutaraldehyde, 0.25% in distilled water, pH 7.0, for one hour. The resulting material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of potassium metabisulfite 1% in distilled water, for 60 minutes. The obtained material is stable, but slippery. To facilitate the placement of the cylindrical biopsies, a short treatment with 1% tannic acid is performed for 15 minutes resulting into the formation of a thin layer (less than 1 μηι) of rough nanocomposite on the surface of the material having a well-known structure of hexagonal lattice when examined with a scanning electrone microscope. The excess of tannic acid is neutralized with 1% sodium bicarbonate for one hour and then washing with distilled water is carried out for another hour. The material is further washed with distilled water for 5 minutes, partitioned into segments with 10 grooves and 25 mm width and the resulting matrices are stored in a formaldehyde solution 4% in 50 mM calcium acetate buffer, at room temperature.

The matrices are loaded by placing biopsies harvested from a surgical specimen into the preformed grooves and the entire assembly is placed in a standard histological cassette to be histologically processed by conventional methods: fixation in formaldehyde, 4% in 50 mM calcium acetate buffer at room temperature for 24 hours, dehydration in multiple changes of 45 minutes each, as follows: ethanol 70%, 80%, 90%, 95%, 100% (three changes], xylene (three changes), melted paraffin at 60°C (three changes); the obtained paraffin blocks are semi-serially sectioned to 4-5 micrometres; the sections are applied on microscope slides and stained with haematoxylin-eosin.

Results: the paraffin blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; the matrix shows a moderate stainability, but it is homogenous; here and there foreign bodies can be seen: spores, microorganisms, cell debris - all from the protein component; also visible as gaps are the vesicles from which the hydrophobic material (oil) was extracted during the histological processing; they are of various sizes, some exceeding 100 micrometres; the sectionable matrix does not create significant difficulties during microscopic examination and does not interfere with the accuracy of the histopathological diagnosis.

Example 3

Sectionable colloid for obtaining pre-formed matrices for explants, endoscopic or percutaneous biopsies, prepared with gelatine and chaotropic agents, and use of said matrices in obtaining paraffin preparations

The following components are mixed in a pre-heated vessel:

- gelatine type A, 250 Bloom (for example, PB Gelatins, UK) 100 g

- urea 50 g

- castor oil (pharmaceutical grade) 100 mL - glycerine 100 mL

- sodium alginate 15 g

- potassium sorbate 10 g

- sodium benzoate 10 g

- drawing ink 10 mL

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, preheated to 60°C.

The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA) at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled slowly to 40°C, followed by the addition of the drawing ink; the mixture is emulsified again at 17,000 rpm.

The mixture is injected into silicone rubber moulds sized for percutaneous prostate biopsies (harvested with a 1 mm diameter needle, max. 22-24 mm biopsy length). The moulds filled with gellable colloid are cooled for 18 hours at 4-10°C. The cold-cured material is extracted from the injection moulds and incubated with stirring, at room temperature, in a solution of 1% CaCl ₂ in distilled water for one hour. The material is then washed with distilled water for 5 minutes and then incubated with continuous stirring in a solution of microbial transglutaminase (2 units per gram of protein in the composition, Activa, Ajinamoto, Japan) dissolved in an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, 10 mL per gram of enzyme, for 24 hours, at room temperature. The material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of glutaraldehyde, 0.25% in distilled water, pH 7.0, for one hour. The resulting material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of potassium metabisulfite 1% in distilled water, for 60 minutes.

The obtained material is stable, but slippery. To facilitate the placement of the cylindrical biopsies, a short treatment with 1% tannic acid is performed for 15 minutes resulting into the formation of a thin layer (less than 1 μηι) of rough nanocomposite on the surface of the material having a well-known structure of hexagonal lattice when examined with a scanning electrone microscope. The excess of tannic acid is neutralized with 1% sodium bicarbonate for one hour and then washing with distilled water is carried out for another hour. The material is further washed with distilled water for 5 minutes, partitioned into segments with 10 grooves and 25 mm width and the resulting matrices are stored in a formaldehyde solution 4% in 50 mM calcium acetate buffer, at room temperature. The matrices are loaded by placing biopsies harvested from a surgical specimen into the preformed grooves and the entire assembly is placed in a standard histological cassette to be histologically processed by conventional methods: fixation in formaldehyde, 4% in 50 mM calcium acetate buffer at room temperature for 24 hours, dehydration in multiple changes of 45 minutes each, as follows: ethanol 70%, 80%, 90%, 95%, 100% (three changes], xylene (three changes], melted paraffin at 60°C (three changes]; the obtained paraffin blocks are semi-serially sectioned to 4-5 micrometres; the sections are applied on microscope slides and stained with haematoxylin-eosin.

Results: the paraffin blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; the matrix shows a low stainability and it is highly homogenous; gaps having uniform sizes of 1-2 micrometres are visible; these are the vesicles from which the hydrophobic material (oil] was extracted during the histological processing; the sectionable matrix does not create difficulties during microscopic examination and does not interfere with the accuracy of the histopathological diagnosis.

Example 4

Sectionable colloid for obtaining pre-formed matrices for explants, endoscopic or percutaneous biopsies, prepared with gelatine and chaotropic agents, and use of said matrices in obtaining preparations on ice

The following components are mixed in a pre-heated vessel:

- gelatine type A, 250 Bloom (for example, PB Gelatins, UK] 100 g

- urea 50 g

- castor oil (pharmaceutical grade] 100 mL

- glycerine 100 mL

- sodium alginate 15 g

- potassium sorbate 10 g

- sodium benzoate 10 g

- drawing ink 10 mL

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM] acetate buffer solution (100 mM], pH 7.0, preheated to 60°C.

The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA] at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled slowly to 40°C, followed by the addition of the drawing ink; the mixture is emulsified again at 17,000 rpm.

The mixture is injected into silicone rubber moulds sized for percutaneous breast biopsies (harvested with a 1.6 mm diameter needle, max. 22-24 mm biopsy length). The moulds filled with gellable colloid are cooled for 18 hours at 4-10°C. The cold-cured material is extracted from the injection moulds and incubated with stirring, at room temperature, in a solution of 1% CaC in distilled water for one hour. The material is then washed with distilled water for 5 minutes and then incubated with continuous stirring in a solution of microbial transglutaminase (2 units per gram of protein in the composition, Activa, Ajinamoto, Japan) dissolved in an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, 10 mL per gram of enzyme, for 24 hours, at room temperature. The material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of glutaraldehyde, 0.25% in distilled water, pH 7.0, for one hour. The resulting material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of potassium metabisulfite 1% in distilled water, for 60 minutes.

The obtained material is stable, but slippery. To facilitate the placement of the cylindrical biopsies, a short treatment with 1% tannic acid is performed for 15 minutes resulting into the formation of a thin layer (less than 1 μιη) of rough nanocomposite on the surface of the material having a well-known structure of hexagonal lattice when examined with a scanning electrone microscope. The excess of tannic acid is neutralized with 1% sodium bicarbonate for one hour and then washing with distilled water is carried out for another hour. The material is further washed with distilled water for 5 minutes, partitioned into segments with 10 grooves and 25 mm width and the resulting matrices are stored in a formaldehyde solution 4% in 50 mM calcium acetate buffer, at room temperature.

The matrices are loaded by placing the biopsies harvested from a surgical piece into the preformed grooves and the entire assembly is frozen in isopentane cooled with dry ice (-80°C). After applying a thin layer of mounting medium (OCT, Sakura Finetek, USA), the preparation is mounted in a cryostat and sectioned to 7-8 micrometres at -20°C. The sections are applied on microscope slides and stained with haematoxylin-eosin.

Results: the frozen blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; the matrix shows a low stainability and it is highly homogenous; the sectionable matrix does not create difficulties during microscopic examination and does not interfere with the accuracy of the histopathological diagnosis. Example 5

Fluid sectionable gellable colloid for marking large histological specimens or for incorporation of sediments, cells, cultures of micro-organisms, hystoids and spheroids isolated from various effusions, lavages, aspirates or secretions harvested for diagnostic purposes, and its use in obtaining paraffin preparations

The following components are mixed in a pre-heated vessel:

- gelatine type A, 250 Bloom (for example, PB Gelatins, UK) 100 g

- urea 120 g

- castor oil (pharmaceutical grade) 100 mL

- glycerine 100 mL

- sodium alginate 15 g

-potassium sorbate 10 g

- sodium benzoate 10 g

- drawing ink 10 mL

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, preheated to 60°C.

The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA) at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled slowly to 40°C, followed by the addition of the drawing ink; the mixture is emulsified again at 17,000 rpm.

The mixture is partitioned in vials, sealed and stored refrigerated (4-8°C). Prior to use, the sectionable colloid is returned to a fluid state, either by quick heating on a water bath at 30°C, or by slow heating for 60 minutes at room temperature. Large specimens are either submerged into the colloid or marked by the application of the sectionable fluid colloid with a brush or spatula. The tissue fragments previously taken from the area of interest by the pathologist are properly oriented and placed together with a few drops of fluid sectionable colloid, either in preformed matrices with wells or in moulds cooled with ice. The matrices with wells or the moulds are completely filled with the sectionable fluid colloid and cooled on ice for 1-2 minutes. The same is done also in the case of the large specimens marked with the sectionable colloid on their surface. The gelled sectionable colloid is stabilized quasi-instantaneously by spraying or by immersion with CaC solution (1%) or by any calcium or barium salt containing histological fixative (for example, Baker's Formalin, containing 1% CaCl ₂ , or formalin buffered with calcium acetate). The resulting specimens comprising the biological material embedded in the gellable colloid are histologically processed using conventional methods.

Results: the paraffin blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; even loose tissue fragments remain intact and the preparations do not disintegrate during sectioning; the risk of contamination of water from the flotation bath is eliminated; common artefacts arising from the sudden distension of the sections when floating on hot water in the flotation bath are virtually eliminated; the surface marking of large specimens is clearly visible to the naked eye and microtome sectioning shows no difficulty; the sectionable matrix does not create difficulties during microscopic examination and does not interfere with the accuracy of the histopathological diagnosis.

Example 6

Sectionable colloid for the obtaining of unique sectionable codes to unambiguously identify histological preparations

The following components are mixed in a pre-heated vessel:

- gelatine type A, 250 Bloom (for example, PB Gelatins, UK) 100 g

- urea 50 g

- castor oil (pharmaceutical grade) 100 mL

- glycerine 100 mL

- sodium alginate 15 g

- potassium sorbate 10 g

- sodium benzoate 10 g

The dry powders are completely incorporated in the castor oil and glycerine, followed by the gradual addition of 1000 mL of an EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, preheated to 60°C.

The mixture is vacuum-emulsified with a high-speed homogenizer (Dupont Sorvall Omni- Mixer, USA) at 5000 rpm for 2 minutes. The resulting mixture is kept at 60°C for 60 minutes. The mixture is then emulsified at 10,000 rpm for 2 minutes, cooled slowly to 40°and emulsified again at 17,000 rpm.

The mixture is injected into silicone rubber moulds sized for groups of 6 percutaneous prostate biopsies (harvested with a 1 mm diameter needle, max. 22-24 mm biopsy length). A 4 mm wide strip is provided for each group of 6 grooves. The moulds filled with gellable colloid are cooled for 18 hours at 4-10°C. The cold-cured material is extracted from the injection moulds and incubated with stirring at room temperature in a solution of 1% CaCh in distilled water, for one hour. The material is then washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of microbial transglutaminase (2 units per gram of protein in the composition, Activa, Ajinamoto, japan], dissolved in EDTA containing (1 mM) acetate buffer solution (100 mM), pH 7.0, 10 mL per gram of enzyme, for 24 hours at room temperature. The material is subsequently washed with distilled water for 5 minutes and then incubated with continuous stirring in a solution of glutaraldehyde, 0.25% in distilled water, pH 7.0, for one hour. The material is further washed with distilled water for 5 minutes and incubated with continuous stirring in a solution of potassium metabisulfite, 1% in distilled water, for 60 minutes. · ^■

The obtained material is washed with distilled water for 5 minutes and histologically processed using conventional methods: fixation in formaldehyde, 4% in 50 mM calcium acetate buffer, at room temperature, for 24 hours, dehydration in multiple changes of 45 minutes each as follows: ethylic alcohol 70%, 80%, 90%, 95%, 100% (three changes], xylene (three changes), melted paraffin at 60°C (three changes). The paraffin infiltrated material is engraved using alphanumeric codes with a C0 ₂ laser (Epilog Mini 24, Epilog Laser, USA). The engraved material is histologically rehydrated in several changes of 45 minutes each as follows: xylene at 60°C (three changes), ethanol 100% (three changes), 95%, 90%, 80%, 70%, distilled water (three changes). The gaps vaporized by laser engraving are filled with fluid colloid prepared according to Example 5, but coloured with a drawing ink in a contrasting colour. The resulted marked material is stabilized by incubation with stirring at room temperature in a solution of 1% CaCb, followed by incubation for 24 hours in a formaldehyde solution 4% in 50mM calcium acetate buffer at room temperature. The material is partitioned into segments with 6 grooves and an engraved strip, having a 25 mm width and the resulted matrices are stored in a formaldehyde solution, 4% in 50mM calcium acetate buffer, at room temperature.

The matrices are loaded by placing biopsies harvested from a surgical specimen into the preformed grooves and the entire assembly is placed in a standard histological cassette to be histologically processed by conventional methods: fixation in formaldehyde, 4% in 50 mM calcium acetate buffer at room temperature for 24 hours, dehydration in multiple changes of 45 minutes each, as follows: ethanol 70%, 80%, 90%, 95%, 100% (three changes), xylene (three changes), melted paraffin at 60°C (three changes); the obtained paraffin blocks are semi-serially sectioned to 4-5 micrometres; the sections are applied on microscope slides and stained with haematoxylin-eosin. Results: the paraffin blocks are relatively easy sectioned and the obtained sections show the perfectly aligned biopsies with great accuracy, without any fragment being lost during processing; the sectionable code is visible on all the sections obtained, even to the naked eye; the sectionable matrix and the sectionable code do not interfere with the accuracy of the histopathological diagnosis; the sectionable code facilitate rapid and unambiguous identification of the paraffin blocks, sections and stained microscopic preparations.

The invention has been illustrated by the description of some embodiments only. Although some examples have been described in a greater detail, they are not intended to limit in any way the scope of protection. The possibility of combination of various features herein disclosed, within the scope of the present invention, is not limited to the explicitly disclosed combinations. The features may be combined in any other manner according to the needs of the user, all these combinations being implicitly disclosed and protected by the present application. The person skilled in the art may easily notice other advantages of the disclosed technical solutions and envisage other modifications which are also within the scope of this invention. The invention is not limited to any specific details, to standard or preferred embodiments or to any disclosed examples.