Title of Invention


Abstract The invention relates to the processing of a biological sample for histological analysis. In particular, it relates to a rapid automated processing system that can be operated with continuous throughput and that eliminates the use of toxic solvents such as xylene. Provided is a method for processing a biological sample for histological analysis, comprising contacting the sample with a composition comprising a supercritical or near supercritical fluid followed by impregnating the sample under a pressure of more than 1 bar with an embedding medium, preferably paraffin. Also provided is a processor (1) for preparing at least one sample (10) for histological analysis,. Fig. 1
Full Text

The present invention relates to the processing of a biological sample firom fixation to impregnation for histological analysis. In particular, it relates to a rapid and safe automated processing system that can be operated with continuous throughput and that eliminates the use of toxic and (in)flammable solvents such as xylene .
Conventional methods for preparing a sample (e.g. tissue) for histology involve incubation in separate solutions of phosphate-buffered 10% formaldehyde for fixation, incubation in a series of increasing concentrations of alcohol for dehydration, and incubation in xylene for clearing tissue of dehydration agent, prior to impregnation. Because of the time required for this process, usually 8 hours or longer, it is customary to complete these separate steps - fixation, dehydration, clearing, and impregnation-overnight in automated mechanical instruments designed for those tasks (see, for example, US3,892,197; US4,141,312; and US5,049,510).
The ultimate goal of tissue processing is to provide the specimen with intemsd and external support from a medium of like hardness so that the specimen can withstand microtomy without damage. The most common embedding or support medium is paraffin but many other substances are also used. Microtomy is the process of cutting or sectioning an embedded sample or specimen into thin slices of approximately 2 - 8 microns in thickness with a sharp steel knife in a microtome. Slices are then picked up on sUdes, usually microscope slides.
Standard paraffin processing procedures include e3q)osure to chemical dehydration through graded alcohol solutions, then immersion in a transition solution (commonly referred to as a clearant) followed by impregnation with paraffin. Dehydration means the removal of water. During processing procedures, dehydration is used to remove the free water molecules

and, if performed correctly, also the molecularly boimd water. Dehydration is normally accomplished using alcohol solutions; most commonly ethanol, isopropylalcohol (isopropanol); occasionally methanol; or butanol for plant sm.d tissue. If specimens are improperly dehydrated and water is left in the the clearant and impregnation agent (for example penetrate the tissue and it will be soft and mushy. Excessive dehydration will remove the bound water, causing shrunken, hard, brittle specimens that require excessive rehydration before sectioning.
Fat in a tissue sample is removed with a solvent because fat impairs clearing and impregnation. Inadequate fat removal (defatting) can result in spreading artefacts of tissue sections, wrinkling of tissue sections, and poor staining. Fat may be removed from the tissue specimen with various solvents such as, for example, acetone, chloroform or xylene.
Following dehydration of a sample, a "clearing" agent is used to remove alcohol used for dehydration from the sample and to prepare the sample or specimen for the impregnation medium. Clearing agents, also referred to as "dealcohohzation" agents, must be miscible with both the dehydrating agent and the impregnation/embedding medixun. Inadequate clearing, which can be caused by water remaining in the specimen or by inadequate exposure times, causes poor paraffin infiltration which will result in soft, mushy specimens. On the other hand, excessive exposure to clearing agents wiU produce hard, brittle specimens caused by the denaturation of the tissue proteins that is very similar to the effect of excessive dehydration.
Xylene (dimethylbenzehe) has been the most widely used clearant for many years. It is an aromatic hydrocarbon that rapidly replaces alcohol and has a refractive index capable of rendering the tissue transparent. A major drawback of xylene is that it is veiy cimibersome to use, because it is highly volatile, flammable and a suspected carcinogen. Xylene should therefore only be used with adequate ventilation, and skin contact should be avoided. In addition, xylene is e3q)ensive.

Effective replacements for xylene have been actively sought for. A first substitute that was presented in 1981 was limonene. Unfortunately, that chemical has cast a shadow over the subject because of several issues. Limonene is oily and cannot be repyded reliably (the recycled solution is different from the original product). Its odour is overpowering and quickly pervades neighbouring rooms and halls. Most troublesome is the fact that it causes serious sensitization reactions in e^osed workers. Other xylene substitutes axe short chain aliphatic hydrocarbons (alkanes). Bssentdal oils can also be used as xylene substitute but they are not as common. However, none of these xylene substitutes has proven as useful and cost-effective as xylene .
Conventional procedures for tissue processing can be performed both m£Lnually or in an automated fsishion. Most histopathology labs now use automated tissue processing machines which use multiple containers and reqiiire 6-20 hours for processing. Automated tissue processors implementing such conventional processes are manu&ictured and sold by, for example, Shandon (HYPERCENTER and PATHCENTRE models), Miles-Sakura (TISSUE-TEK models), and Mopec-Medite (TPC15 model).
A disadvantage of the systems of the prior art is that such automated systems have not been capable of continuous throughput. Given the time required to complete tissue processing, cassettes containing tissues are loaded into the system during the day and tissue processing is completed in an overnight cycle. Thus, operation of the prior art systems did not allow tissue-containing cassettes to be processed to completion during the work day.
Typically such conventional methodology demands sending tissue specimens from the operating room, medical ofGLce or other sites, to a pathology laboratory sometime during the working day, followed by overnight batch processing of the specimens, so that a tissue specimen suitable for blocking and sectioning is at the earliest only available on the morning of the next day; and rendering a diagnosis by a pathologist based on microscopic examination of sections prepared from a blocked and sectioned specimen is only possible

later on that next day. This minimallb^ requires almost 24 hours between receipt of the specimen and dehvery of the pathologist's report.
In addition to the minimiim one-day delay in giving a medical practitioner (e.g. a surgeon) the benefit of a report irom. ihe pathologist, there are also problems associated with impeded work flow in the pathology laboratoty necessitated by the requisite batch processing of specimens, the safety concerns that attend having instruments operating overnight, the risk of possible instrument fJEulures and the need to monitor the instruments, and the waste of using large volmnes of reagents for such processing when automated. Moreover, expensive measures are required to prevent exposure of laboratory personnel to noxious fumes and toxic substances associated with the reagents (such as xylene) used in this process. Also, the large volumes of solvent waste and paraffin debris produced by the conventional methodology will pollute the environment if not properly disposed.
There is an ever present interest in expediting tissue processing and analysis for diagnostic purposes. Furthermore, recent healthcare focus has been directed to lessening the cost of various procedures including tissue processmg. The costs of tissue processing are related to the time for processing and analysis of the specimens, the space required for the personnel and equipment in the laboratory, the volume of reagents (both the purchase price of the pure chemicals and the charges for discarding waste), and the nxmiber of personnel required. More importantly, patients and their physicians depend on evaluation and diagnosis by the pathologist to guide treatment. Reducing the amount of time needed to complete tissue processing woidd lessen the anxiety experienced during the period between obtaining the specimen and delivering the pathologist's report to the physician. Thus, a significant reduction in the time required for processing of a histological sample is very desirable. Others have also recognized the need to shorten the time required for tissue processing, but they have made only modest improvements in the conventional methods. To accelerate tissue processing, U.S. Pat. Nos. 4,656,047, 4,839,194,

and 5,244,787 use microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No. 5,023,187 uses infrared energy. U.S. Pat. No. 5,104,640 disclosed a non-aqueous composition of a fixative, a stabilizing agent, and a solubilizing agent that adheres a blood smear to a slide.
The inventors now describe a method for tissue processing in a manner distinct from any of the procedures currently used. Provided is a method for processing a biological sample for histological (or pathological) analysis, comprising contacting the sample with a composition comprising a supercritical or a near supercritical fluid followed by impregnating the sample with an embedding medium under a pressure of more than 1 bar.
A method in accordance with the present invention is faster than any of the reported methods, causes minimal damage to the processed tissue, avoids the use of organic solvents including xylene, uses minimal amounts of reagents, and, surprisingly, results in a superior specimen for subsequent cytological, histological or anatomical analyBis. In addition, with a method according to the invention it is no longer necessary to use large (plastic) containers with an inflammable liquid such as ethanol or xylene.
A supercritical fluid, sometimes called a supercritical gas fluid or a fliddum, is any substance above its supercritical temperature (Tc) and its supercritical pressure (Pc). For every substance, there is a temperature above which it can no longer exist as a Hquid, no matter how much pressiire is applied. Likewise, there is a pressure above which the substance can no longer exist as a gas no matter how high the temperature is raised. This point is called the supercritical point; the critical temperature and critical pressure are the defining boxmdaries on a phase diagram for a pure substance.
In the supercritical area there is only one state-of-the-fluid. A supercritical fluid exhibits physicochemical properties intermediate between those of liquids and gases. Supercritical fluids (also known as highly condensed gases) are able to spread out along a surface more easily than a true hquid

because they have lower siirface tensions than liquids. At the same time, a supercritical fluid maintains a liquid's abHily to dissolve substances that are soluble in the compounds, which a gas cannot do.
According to the invention, a (tissue) sample is contacted wibh or surrounded by a supercritical fbnd , comprising pressurizing the sample with the composition comprising a (near) supercritical fluid to above the critical pressure ^c) of the supercritical fluid and heating the sample with the supercriticfd fluid to above the critical temperature (Te) of the supercritical fluid. The supercritical fluid penetrates a sample as it passes by a sample in a h^h-pressure vessel. It may not alwasrs be necessaiy to use a substance that is at or above its supercritical point (i.e. above its Tc and Pc), as long as the properties (especially the solubilizing capacity) of the stibstance are of use in a method for histoprocessing. For example, a near supercriticcd fluid, whssh is at a pressure and temperature in the vicinity of the supercritical point can also be advantageously used in a method provided herein. According to the invention, the term 'near supercritical fluid" is defined as a fluid at a temperature in the range of about 0.7 to about 1.4 times its Tc and at a pressure in the range of about U.a to about 7 times its Pc.
In a preferred embodiment, a sample is contacted with a composition comprising (near) supercritical carbon dioxide (CO2). The supercritical pressure of CO2 is about 7,3 MPa (73 bar) and the supercritical temperature is approximately 31 "Celsius. Biological tissues contain proteins which denatiire by temperature above approximately 60 *C. The relatively low supercritical temperature of CO2 allows to contact a sample with a supercritical fluid at a temperature that has essentially no detrimental effects on the biological sample. However, other (near) supercritical fluids with a relatively low supercritical temperature (preferably lower than 60 "C) are also suitable for use in a method according to the invention, for example xenon, nitrous oxide, ethane, HFC-H6, chlorotrifluormethane, ethylene, sulfur hexafluoride and trifluormethane. CO2 is extremely attractive in industrial appUcations becaiise

it is the second most abimdant and the second least expensive solvent on earth. It is non-flammable, non-toxic, readily available in hi^ purity.
An embedding or supporting meditun provides the sample with mechanical support such that sections can be prepared, e.g. for microscopical examination. A preferred embedding medium according to the invention is a liquid embedding medium, for example hquid paraffin. Paraffin has been chosen as embedding medium in the examples herein because it is soluble in supercritical CO2 (or, vice versa, CO2 is soluble in paraffin) inea^ensive, ea^ to handle, and ribbon sectioning is &icihtated by the coherence of structures provided by this material. Other suitable embedding or impregnation materials are commercial wax formulae, mixtures of waxes of different melting points (e. g., Hquid mineral oil and solid paraffin), paraplast, bioloid, embedol, plastics, and the like.
According to the invention, a sample is typically first immersed into ethanol or other tjrpe of dehydrating agent in order to replace water in the sample. The sample is then pressurized with a (near) supercritical fluid to remove the dehydrating agent such as ethanol. At (near) supercritical conditions, the dehydrating agent and the fluid are misdble i.e. they dissolve completely in each other whatever the proportions of the components is. Subsequently, the (near) supercritical flvdd is replaced by infiltrating an embedding medium while maintaining an elevated pressure i.e. a pressure above the pressure of I bar (1 atmosphere, 1 kg/cm^). The (near) supercritical fluid within the sample is dissolved in the embedding medium while at the same time it is being gradually replaced by the paraffin. The elevated pressure during the infiltration step ensures that no gas is trapped in the tissue and that the cellular structures remain preserved. Preferably, the pressure applied is at least 50 bar, more preferably at least 100 bar, such as 120 or 150 bar or even higher, such as axound 200 bar. Generally speaking, the higher the pressiire apphed, the higher the solubihty of the (near) supercritical fluid in the embedding medium and the more efficient the fluid will be replaced by the

embedding medium. For example, it has been observed that the solubility of CO2 in liquid paraffin is approximately 15 % by weight at 80 bar at 55°C, approximately 30% (w/w) at 120 bar and around 50% at 180 bar. The temperature at which impregnation according to the invention can be periformed can vary and depends among others on the embedding medium used. Typically, a temperature above the melting point of the embedding medium is chosen. In case of paraffin, this is a temperature of 56-58°C. However, under elevated pressure the melting point is usually reduced. For example, at a pressure of 110 bar paraffin starts to melt at 51°C and is completely melted at 6TC. When increasing the temperature of a reactor comprising a sample (see Fig. 1) above the melting temperature, the pressure in the reactor is increased. In one embodiment of the invention, a sample is contacted with CO2 at a pressure of 150 bar at around 40°C during 0.5-1 hour to remove ethanol from the sample. Subsequently, the sample is heated to eS^G while maintaining the density of CO2 such that the pressure increases to around 220-250 bar. liquid paraffin is allowed to enter the reactor, while CO2 is allowed to leave the reactor while trying to medntain a constant pressure. The reactor is completely filled with parafEbi over a time period of about 30 minutes to remove and/or dissolve CO2 from the sample. Duriag this stage of the process, the pressure may drop, e.g. to 100-140 bar. Once the sample is fully immersed in the paraffin, the pressxire can be gradually decreased to allow difEusion of CO2 from the tissue into the paraffin and to avoid that CO2 bubbles become trapped in the tissue.
After the sample is depressurized, it may be advantageous to leave the samples for some period of time (e.g. 10 -60 minutes) in the warm paraffin.
The phenomena of enhanced solubilities in supercritical fluids has been known since the late 1800s. For decades they have been used in food processing industries to extract flavouriag compounds such as caffeine and hop oil. The solubilizing power of supercritical fluids is sensitive to small changes in the operating conditions, and it is possible to fine-ttme the pressure and the

temperature to tailor the solvent capacity of a supercritical fluid for a particular process. The desirable and unique properties of supercritical fluids have provided the impetus for applying supercritical fluid technology to various other problems, e.g. the cleaning of fabrics or the sanitization of contaminated soil.
Supercritical fluid extraction has been used in methods for preparing sterilized tissue for incorporation into xenografts and bioprostethic devices. US Pat. AppL No. US2003/0072677 describes the use of supercritical fluids to renoiove infectious materials from tissues and to treat the tissue with a chemical agent. Unlike the present iavention, US2003/0072677 does not relate to processing samples for further examination, let alone to the apphcation of supercritical fluids in histological (embedding) procedures. US Patent No. 6,493,964 discloses a supercritical poiat drying apparatus for sample preparation in electron microscopy and. semiconductor wafer production. It utilizes the technique of substituting a supercritical 'transitional' fluid for the dehydrating fluid in the ceU structure and then removing the transitional fluid. However, US 6,493,964 does not relate to sample impregnation and doe not teach or suggest that supercritical fluids are advantageously used as intermediate between dehydration agents and an embedding medium while maintaining an elevated pressure, that such use results in a superior quality of the specimen (see below) compared to conventional procedures. EP0822403 relates to processing of organic tissues to prepare them for further investigation using an inert gas at increased pressure, such that the sample can be treated at higher temperatures. The pressure may be built up by introducing an inert gas in the container comprising the tissue sample, for example CO2. It is mentioned that a dehydration/clearing step is preferably carried out simultaneously at a pressure up to 10 bar and at a temperature from room temperature to up to 90°C. Under these conditions, CO2 is not in a supercritical or near supercritical state. Thus, the process of EP0822403 entails an entirely different concept compared to a method of the invention and

does not iavolve a (near) supercritical fluid. Moreover, EP0822403 mentions that impregnation is preferably carried out under vacuimi.
In a method for histological processing according to the invention, a sample is treated with a (near) supercritical fluid and subsequently impregnated with an embedding medium xmder elevated pressure. In one embodiment, a method of the invention further comprises dehydration, defattii^ and/or decalcification of said sample prior to impregnation. Said additional processing step(s) may be performed using the conventional procedures mentioned above. Preferably however, they are performed making use of a supercritical fluid. In one embodiment, a method is provided for processing a sample wherein s£iid processing comprises dehydration of said sample using a supercritical fluid. For example, a sample is contacted with a supercritical fluid to solubilize and remove water flrom said sample prior to impregnating the sample with an embedding medium. A supercritical fltdd can be mixed with an other solvent, to aid in extracting certain substances (e.g. water) from a sample. In one embodiment, a sample is dehydrated with a composition comprising a supercritical fluid and a dehydrating agent, preferably an alcohol, such as ethylalcohol (ethanol; EtOH) or a detergent, such as Tween. Dehydration using a supercritical fluid as provided herein is typically accomplished fast, sometimes even within minutes. Thus, the invention combines the improved embedding procedure with an attractive alternative for the time-consuming traditional step-wise dehydration using graded alcohol solutions. Advantageously, the supercritical fluid dissolves and extracts other substances from a sample, such as fats and lipids, thereby facilitating cutting a sample into thin slices. In a specific embodiment, processing of a sample, in particular a calcified tissue like a bone specimen, comprises removing calcium from a sample. Decalcification of a sample is important for cutting thin shces firom bone tissues and other calcified particle in tissues, because calcified structures are in general difficult to cut. Traditional decalcifying protocols require the additional incubation of a fixed

sample during one to five nights in an acidic decalcifying solution (typically formic acid, acetic acid, hydrochloric add or nitric add). Decalcification is also performed using a calciiun-chelator such as EDTA (ethylenediaminetetra acetic add). According to the invention, decalcification of a sample is accomplished simpler and faster when compared to existing decalcification methods. Hereto, a biological sample is contacted with a composition comprising a (near) supercritical fluid, wherein said composition additionally comprises a decalcifying agent. Suitable decalcifying agents include adds such as carboxyUc acids, for instance formic add or acetic add, and other chemicals capedble of binding or sequestering caldum.
When a composition comprising a supercritical flxdd and additionally comprising a co-solvent (e.g. for water and/or caldum) is used, the supercritical fluid and the co-solvent (e.g. an alcohol and/or an add) can be supplied as a mixture in a cylinder. Another method of supplying the additional co-solvent can be achieved by using an additional pump systems onsite to iniy the required co-solvent to the supercritical fluid.
Histological analysis as used herein refers to any type of analysis that may be performed to study the appearance, properties and behaviour of a tissue, a cell, an organ or an organism. It can be carried out by inspecting a processed sample under a microscope. A (component of a) processed sample can be contacted with one or more reagents, such as a dye, a reagent or a probe specifically reactive with one or more components (such as proteins, nucleic acids, carbohydrates) present in the sample to identify or mark a certain cell type or tissue. A tissue refers to a group or layer of cells which are essentially alike and work together to perform a specific function. Typical probes indude antibodies (e.g. for immunohistochemistry), nucleic acid (RNA; DNA) probes (e.g. for in situ hybridisation or PCR techniques), substrates for use in enzyme histochemistry (e.g. NADH for detecting acetyl cholinesterase or ATP-ase activity) and conventional staining chemicals such as hematoxylin and eosin (H&E); Aldan Blue for sulfated mucosubstances; Brown-Brenn Gram stain for

Gram-positive and Gram - negative bacteria; Congo Red for amyloid; Giemsa for H. pyhri] and Bone Marrow; Gomori's Modified Iron stain; and many other probes known to a person skilled in the art that are of use to identify or mark a certain cell type or tissue, be it normal or diseased.
Pathological analysis refers to histological analysis that is related to pathology. Typically, pathological analysis is performed by a pathologist who diagnoses disease by studying a sample comprising cells and tissues under a microscope. In a preferred embodiment, a pathological analysis comprises analysis of a himian sample to stage or grade a disease.
According to the invention, a sample oompnses a biological sample, such as a tissue specimen. In the context of the invention, a "tissue specimen" is any piece of tissue that may be processed by a method disclosed herein. It may also refer to single cells &om any biological fluid (e. g., ascites, blood, pleiural exudate), or a cell suspension obtained from aspiration of sohd organs or lavage of body cavities. Single cells may be pelleted by sedimentation or buoyant centrifugation prior to processing. It may also refer to an intact organ, or even an intact organism, or a part thereof. Oi^^anisms iaclude imicellular and multicellular organisms, and range firom bacteria, fimgi, insects and plemts to mammals. Sohd pieces (1 e., tissue sUces or needle biopsies) from a human subject are commonly processed for histology and pathology. Where conventional methods for the fixation and embedding of an organ (e.g. brain) require up to 6-8 weeks, a method of the invention now allows impregnation of an (intact) organ or a part thereof with a solid embedding medium within a day and without the use of toxic (clearant) solvents.
With a method of the invention, it is possible to reduce total proqessiag time (firom fixation to impregnation) from the conventional 8-12 hours to less than 2 hours, preferably less than 1.5 hours, more preferred less than one hour. Nowhere in the prior art it has been taught or suggested that the entire process of preparing diagnostic tissue slides could be accomplished in less than 1.5-2 hoiu*s, starting from the preparation of a specimen firom a

fixed or non-fixed tissue and ending with impregnation, with continuous processing of specimens, circumventing the use of toxic, possibly carcinogenic clearants and a superior quality of the sample. WO 01/44783 discloses a tissue processor system including an improved microwave unit that allows rapid processing under two hours and, optionally, without the use of xylene clearants. However, the protocol of WO 01/44783 can only process a tissue specimen with a thickness of less than about three millimetres. In contrast, in a method of the present invention, samples vip with a thickness to 5 millimeters, such as 8 mm or even more than 1 centimeter may be rapidly processed using a supercritical fluid. As mentioned above, and imlike WO 01/44783, a method of the invention is not limited to tissue samples or small tissue sections. A method according to the invention allows to process a sample with a voliime rangiag firom about 0,001 cm' ( e.g. a biopsy) or 1 cm' (e.g. a skin specimen) up to 10 cm^ (e.g. a small tiunor) or even up to larger samples such as those with a volume of 2000 cm' (e.g. cui organ such as complete brains). In general, according to a method of the invention, the larger the sample, the more time it will take to process the sample. However, ia comparison with the prior art the time gain adueved with the present invention also increases with the size of the specimen. For example, a tissue specimen of 20 x 15 x 5 mm is rapidly dehydrated and impregnated according to a method of the invention. This offers a considerable advantage, in that a sample can be processed of such a voltmie or size that it is possible to obtain multiple (microtome) slices fi-om said sample. For instance, following histological inspection of a sample, a pathologist may want to inspect the same sample that has been stained with a specific reagent, such as an antibody, to aid in the histological analysis. Processing a sample according to the invention aEows to simply provide a second, third or even higher number of (parallel) slices firom the same sample. If the specimen is already less than 3 millimeter before processing, as is the case in WO 01/44783, this is obviously not possible. Instead, multiple samples need to be taken at the outset and their relative

orientation needs to be carefully registered to reconstruct their connection in situ.
Histological analysis of various types of himian tissue specimens that were processed using a supercritical fluid according to the invention surprisingly revealed a superior quality of the specimen compared to specimens from the same tissue sample that were processed according to conventional procedures. For example, the keratin staining pattern of a human colon specimen following processing using carbon dioxide shown in Figure 3 is more intense compared to the keratin staining pattern of Figure 2, showing a specimen from the same colon sample which was processed using conventional methods. likewise, improved histological analysis could be performed on a vitnentin-stained human gall bladder specimen processed according to a method of the invention (compare Figures 3 and 4) and on a human nerve specimen stained for S-100 protein (compare Figures 6 and 7).
In one embodiment of the invention, a sample is fixed according to conventional methods prior to being processed accordii^ to a method according to the invention, for instance using a formaldehyde solution (also known as formalin). Formaldehyde (CH2O) reacts with terminal free -NH2 groups of proteins and forms covalent methylene bridges between two components of a protein or between two different proteins. However, a major drawback of conventional fixation and tissue processing (i.e. into parafBn blocks) lies in the fact that it can cause irreversible damage (e.g., hydrolysis of a phosphodiester bond and/or deamidation) to the structure of nucleic adds (e.g., DNA, and especially RNA). Accordingly, fixing and processing of a (tissue) sample into a paraffin blocks hmits the application of genetic techniques for diagnosis and research.
In one embodiment of the present iavention, a sample that has been previously frozen is processed according to a method of the invention. It is known in the field that most DNA and certainly RNA analyses require special precautions with handling of sample material, such as immediate ("snap")

freezing of fresh tissues into hquid nitrogen to prevent nucleic acid degradation. A method of the present invention can be used for impregnating a (snap) frozen sample and the invention thiis provides a method for obtaining a processed biological sample that can subsequently be analysed for various types of histological analysis, including nucleic acid (DNA, RNA) analysis. On the other hand however, histological diagnosis of a frozen section may suffer from disadvantages in comparison to sections prepared from paraffin blocks. For instance, frozen sample are prone to dehydration. Storage of frozen samples therefore requires measures to prevent dehydration. Importantly, frozen tissues often show many artefacts that are caused by the presence of ice crystals in the sample. Thus, it may sometimes be dif&cult to adequately weigh the advantages and disadvantages that are associated with either a fixed or frozen samples against each other.
A method in accordance with the invention for processing a sample now provides an elegant solution to these problems, because in addition to processiag a fixed or frozen sample, it also allows for rapid processing a fresh sample that has not been fixed or firozen prior to being contacted with a supercritical fLxdd. Instead of freezing or using a chemical fixative, the high pressure encountered by the sample during contacting with a supercritical fluid and the rapid impregnation with an embedding medium (e.g. paraffin wax) imder elevated pressure ensures an optimal preservation of the structure and architecture. Surprisingly, no damage to the tissue sample occurs if pressure is increased and subsequently gradually decreased. Pressurizing the tissue can be performed relatively fast. The ceUs within the tissue are filled with liqxdd which will withstand a rapid pressure increase. However, the sample should be gradually depressxurised to avoid rapid expansion of the fluid and rupture of the cells. Thus, the invention provides an attractive alternative for snap freezing a sample and allows for preparing a sample that is compatible with mvdtiple types of (pathological) analysis including histological, biochemical and nucleic acid analysis, without the use of formalin.

Importantly, in addition to the reduction in time reqxiired for tissue processing, the rapid tissue preparation using a supercritical fluid allows for preserving tissue structures and morphology that are bst with conventional methodology. Glycogen, which is an important compound giving strength to biological staructures, is almost always lost using the conventional methodology. Lymphatic vessels, particularly of the myometriimi, collapse during conventional processing while they remain essentially intact when a method of the present invention is used. Moreover, studies with tissues processed in accordance with the invention indicate better preservation of DNA and RNA extraction as compared to conventional processing methods. Tissues obtained in hospitals and other siugical settings can be processed for both histological and genetic studies soon after delivery to the laboratory. In addition, because a sample processed in accordance with the invention is typically well preserved, archival sample material may be made available for future research and other appUcations.
The present invention does not prohibit preparation of nucleic adds, DNA or RNA, from processed samples. Thus, genetic study is possible for specimens collected routinely in the clinical pathology laboratory. The combiaed power of these technologies will be great. Histological observations may be correlated with results from genetic studies by analy^dng one histochemical section by staining or immunohistochemistry, and analyzing nucleic acids from an adjacent section £)r genetic euialysis (using for example PCR techniques). For example, diseased and normal regions of the same section may be compared to detect genetic differences (e. g., mutations, levels of transcription), disease progression may be characterized by comparing genetic differences in samples taken at several time points, and tumor evolution may be assessed by following the accumulation of genetic diEferences from primary cancer to metastasis.
A further advantage of a method according to the invention relates to sample or specimen orientation. Specimen orientation is key to reach the end

result - the correct diagnosis. In existing procedures, a processed (embedded) specimen is placed in a specimen holder or mold to enable sectiDning of the tissue in a microtome. Fixation and immobilization of a sample, frequently small and delicate, in the correct orientation into a holder is ofben troublesome. Because most glues are not compatible with the organic solvents used in histoprocessing, molds with a roughened or "sticky" sur&ce are typically used to attach a specimen to the bottom of a mold or holder. However, these holders often do not suf&dently fix a specimen such that it can be sectioned. Other specimen holders are provided with snap-on Hds to immobilize a sample by simply clamping a sample in between the bottom and the lid. Still, these holders are not suitable for deUcate specimens such as skin or epithelial tissues because the pressure required to clamp a specimen easily disrupts the integrity of such tissues, e.g. evidenced by coUapsed blood vessels. The invention now provides a solution to these problems. Since a method of the invention does no longer reqidre organic solvents (xylenes) which previously disqualified the use of glues, a specimen can now simply be glued in a desired orientation to the bottom of a holder. Moreover, as mentioned earlier, a method of the invention is suitably used to impregnate samples of considerably larger size than could be used thus far. As a consequence, it is now even possible to first orient an intact tissue, organ or even organism, prior to processing. For example, a tumour is removed in the operating theatre. Instead of cutting the tumour into multiple small size specimens, thereby carefully keeping track of their relative orientation, the invention now permits to orient and embed the whole, iatact tumour.
We have developed a simple, safe, low cost, e^editious, and rehable process that permits preparation of impregnated tissue blocks suitable for microtome sectioning in less than 1.5 hoiurs from the moment tissue is received in the pathology laboratory. The invention allows continuous processing and flow of specimens, either fresh, fixed or frozen, is adaptable to automation, precludes the need for formalin and xylene with their noxious fumes, allows

standardizsation of tissue processing, and requires considerably smaller volumes of reagents than conventional methods. By "continuous" processing, we mean accessing the system of the invention with additional tissue specimens at intervals determined by the time required to complete an individual step of the process (i.e., a few minutes) instead of the time required to complete the process (i.e., an hour to several hours). At any given time, there can be samples at different stages of processing. In other words, a continuous throughput and flow of specimens along the various stages of tissue processing is made possible by the invention. Continuous processing may be accomplished manually or by an automated instrument, sudi as a tissue processor.
In one aspect of the invention, a processor is provided for use in a method of the invention. An embodiment of such a processor according to the present invention wiU now be described by way of example with reference to Figure 1 in the accompanying drawing.
Figure 1 schematically shows an example of a processor according to the invention.
The processor 1 of Figure 1 comprises a storage tank 3 and a conduit system 4. The conduit system 4 has an inlet 2 for supplying to the processor a substance, in this example assumed to be liquid carbon dioxide. The carbon dioxide is transported through a conduit of the conduit system 4 through a cooler 13 into the storage tank 3. The processor 1 comprises downstream of the storage tank 3 presstiriadng means 5 and heating means 6, both for bringing the carbon dioxide at the required conditions. The carbon dioxide is suppUed to a process reactor 9. This reactor 9 may contain samples 10 to be prepared by the processor 1 for histological analysis. The reactor 9 comprises heating and/or cooling means 14 for maintaining the carbon dioxide in the reactor 9 at the required conditions. With these conditioning means 14 of the reactor 9 the different steps of the different methods of the invention as described above can

be performed in the process reactor 9. Downstream of the process reactor 9 the processor 1 further comprises a pressiire control valve 16, subsequently followed by separation means 11 for separating different substances from the mixture of substances leaving the reactor 9. The extracted substances sudi as alcohol, parafBn, water, etcetera, can leave tiie extraction means throuih outlets 12. Some of these substances, for example paraffin, can be re-used. The carbon dioxide left over from the nuxture can be recycled. To that end, it is fed from the separation means 11 via conduit 4 into a recyding means 13, for example a carbon dioxide gas cooler 13 comprised in the conduit ^stem 4 of the processor 1, after which the liquefied carbon dioxide is fed to the storage tank 3 again. When the reactor 9 is sufficiently flushed with carbon dioxide, piunp 5 is stopped and the pressure vessel 7 containing the embedding medium (e.g. paraffin) is emptied into reactor 9 by opening valve 17, such that the embedding agent flows on gravity through conduit 8 and flows into reactor 9. Pressure vessel 7 is kept under pressure because it is connected to the conduit 4. Here the samples 10 are impregnated with the embeddiag medium, where after the reactor 9 and pressxure vessel 9 can subsequently depressurised through conduit 4A into separation vessel 11 and emptied through outlet 15.
Having described an example of a processor 1 according to the invention, many modifications thereto will become apparent to those skilled in the art without deviation from the invention as defined by the scope of the appended claims. For example, it is possible to apply in a single processor a larger number of reactors, pressure vessels, storage tanks, pumps, control means, valves, etcetera. The pumps for instance may be used to add co-solvents or dehydration agents (e.g. ethanol, acetone, formaldehyde, etcetera).

Human gall bladder, colon, and nerve specimens impregnated with ethanol were placed in a 1 VLtre reactor. The reactor was maintained at a temperature of 40°C. The reactor was closed and pressurized to 150 bar with carbon dioxide at 40°C. While maintainiag the pressure at 160 bar applying a control valve, the reactor was flushed with firesh carbon dioxide at a rate of 10 kg/h applying a pump. After 45 minutes, the pump was stopped and the temperature was raised to GCC. Raising the pressure to approximately 230 bar. Molten parafBn of GO'C was slowfy pimiped into the reactor at a rate of 2 htre/h, impregnating the specimen, while maintaining a pressure of 210 bar. After the reactor was nearly completely filled with parafGn, the reactor was depressurized in 10 minutes. Excess paraffin was drained from the reactor and the reactor was cooled to 40*0, after which the embedded specimens were collected irora. the reactor. The specimens were histologically analysed according to conventional procedures.

Figure 1
Example of a processor according to the invention.
Figure 2
Hximan colon specimen (lOOx; insert 400x) processed according to the
conventional high throughput Sakura VIP-300 bench-top automated tissue
processor. Keratine was stained using Keratine 20 (monoclonal Mouse Anti
Human Cytokeratin 20 Clone Ks 20.8 done no M7019 lot 067 from Dako). A
Ventana NexES"™ automated immunostainer was used.
Specificity: epithelial ceUs of the mucosa of the large intestine are stained, no
backgroimd staining visible.
Intensity: diffuse staining pattern along the entire crsnpt.
Human Colon specimen (lOOx; insert 400x) from the same sample as shown in
Fig.2 but processed using super critical carbon dioxide. Staining was
performed as described for Figure 2.
Specificity: epithelial cells in the mucosa of the large intestine are stained, no
background staining visible.
Intensity: diffuse staining along the entire crypt, staining is enhanced
compared with the conventional method (VIP).
Figure 4
Human gall bladder specimen (lOOx; insert 400x) processed by standard
procedure VIP300.
Vimentin was stained using an anti-vimeutin antibody (clone vim 3B4) cat nr
112457 Boehringer Mannheim) and the Ventana staining according to

Specificity: mesenchjnmal cells in the lamina propria and the deeper layers of
the gallbladder are stained. Epithelial cells are negative.
Intensity: diffuse intracytoplasmatic staining of the mesenchymal cells.
Figure 5
Human gall bladder specimen (lOOx; insert 400x) of the same sample as shown
in Fig. 4 but processed using supercritical carbon dioxide.
Staining was performed as described for Fig. 4.
Specificity: mesenchymal cells are stained in the lamina propria and deeper
layers of the gall bladder.
Intensity: enhanced compared with the conventional procedure (VIP).
Fifirure 6
Human nerve specimen (400x) processed using the standard procedure
VTP300. Staining was performed using antibody: S-100 Code no.Z 311 lotno.
026 Dako. Ventana staining according to protocol
Specificity: nei^es are stained and other structures are negative.
Intensity: diffiise intracytoplasmatic staining of the Schwann cells and the
neurons. No background staining visible.
Human nerve specimen (400x) of the same sample as shown in Fig. 6 but
processed using supercritical carbon dioxide.
Staining was performed as described for Fig. 6.
Specificity : nerves are stained and other structures are negative.
Intensity: strong staining of the Schwann cells and the neurons. No
background staining visible.

1. A method for processing a biological sample for histological analysis,
comprising the steps of:
a) contacting the sample with a dehydrating agent;
b) removing the dehydrating agent with a composition and
c) replacing the supercritical fluid by infiltrating an embedding medium, preferably paraffin, at a pressure of at least 1 bar
characterized in that said composition comprises a supercritical or a near supercritical fluid at a temperature in the range of 0.7 to 1.4 times its critical temperature and at a pressure in the range of 0.3 to 7 times its critical pressure.
2. The method as claimed in claim 1, wherein said supercritical or near
supercritical fluid is carbon dioxide.
3. The method as claimed in claim 1 or 2, wherein said biological sample is a fresh, frozen or fixed tissue sample, preferably a fresh, non-fixed sample.
4. The method as claimed in any one of claims 1 to 3, wherein said biological sample comprises an organ or a part thereof
5. The method as claimed in any one of claims 1 to 4, wherein said sample is dehydrated, defatted and/or decalcified prior to impregnation by using a composition comprising a supercritical fluid.

6. The method as claimed in claim 5, wherein said composition additionally
comprises a dehydrating agent, preferably an alcohol.
7. The method as claimed in claim 5 or 6, wherein said composition additionally
comprises a decalcifying agent, preferably an acid.
8. A processor (1) for preparing at least one sample (10) for histological analysis,
comprising at least one process reactor (9) for the at least one sample (10),
wherein the processor (1) comprises supplying means (4) for supplying to the
reactor (9) at least one substance and at least one supplying means (7) for adding
the embedding medium to the reactor (9) through conduit (8) the processor being
characterized in that one of the substance supplied is in supercritical phase or near
supercritical phase and the processor further comprises pressurizing and/or heating
means (5, 6) for bringing said substance in supercritical phase or near supercritical
phase at the required pressure in the range of 0,3 to 7 times its critical pressure
and/or temperature in the range of 0.7 to 1.4 times its critical temperature.
9. The processor (1) as claimed in claim 8, further comprising separating means
(11) for separating substances from a mixture of substances leaving the reactor (9).
10. The processor (1) as claimed in claim 8 or 9, further comprising recycling
means (13) for recycling substances discharged from the reactor (9).


0364-chenp-2006 complete specification as granted.pdf






0364-chenp-2006-form 1.pdf

0364-chenp-2006-form 18.pdf

0364-chenp-2006-form 3.pdf

0364-chenp-2006-form 5.pdf




364-CHENP-2006 PETITIONS.pdf


364-chenp-2006 complete specification as granted.pdf

364-chenp-2006 drawiings.pdf

Patent Number 234403
Indian Patent Application Number 364/CHENP/2006
PG Journal Number 29/2009
Publication Date 17-Jul-2009
Grant Date 27-May-2009
Date of Filing 30-Jan-2006
Applicant Address OOSTERSINGEL 59 NL-9713 EZ GRONINGEN
# Inventor's Name Inventor's Address
PCT International Classification Number G01N1/30
PCT International Application Number PCT/NL04/00462
PCT International Filing date 2004-06-30
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 03077047.3 2003-06-30 EUROPEAN UNION