WO2014079927A1

WO2014079927A1 - Method and system for snap freezing tissues

Info

Publication number: WO2014079927A1
Application number: PCT/EP2013/074382
Authority: WO
Inventors: Theo VAN DER LEIJ
Original assignee: Pamgene B.V.
Priority date: 2012-11-23
Filing date: 2013-11-21
Publication date: 2014-05-30

Abstract

The present invention relates to a system and method for snap freezing a biopsy tissue, and, more particularly to a system and method that preserves the molecular activity in said biopsy tissue. The present invention also aims to provide a frozen biopsy tissue sample, wherein histological parameters are maintained. More in particular, the object of the present invention is to provide methods and devices where a biopsy sample is removed from a biopsy needle, collected, frozen down steeply and thermally isolated to control thawing and to allow for transportation from the location where the biopsy is taken to another location where the biopsy tissue can be examined and/o rarchived.

Description

METHOD AND SYSTEM FOR SNAP FREEZING TISSUES FIELD OF THE INVENTION

The present invention relates to a system and method for snap freezing a biopsy tissue, and, more particularly to a system and method to preserve molecular activity in said biopsy tissue. The present invention also aims to provide a frozen biopsy tissue sample, wherein histological parameters are maintained. More in particular, the object of the present invention is to provide methods and devices where a biopsy sample is removed from a biopsy needle, collected, frozen down steeply and thermally isolated to control thawing and to allow for transportation from the location where the biopsy is taken to another location where the biopsy tissue can be examined and/or archived. More specifically, the present invention provides methods where a biopsy sample is snap frozen without the use of hazardous coolants such as liquid nitrogen. BACKGROUND OF THE INVENTION

Access to frozen biopsies is of tremendous importance to improve the treatment choices for patients. In order to be able to perform contemporary molecular biology technologies, such as RNA testing or protein (kinase, protease, nucleic hormone receptor...) activity testing, fresh/frozen tissue samples are a prerequisite. The problem is that frozen patient material such as a biopsy is not regarded as a routine source for diagnostic testing in clinical practice, and the routine practice in a hospital does not include a simple and reliable solution for collecting, storing and preserving these samples.

When a disease such as cancer, a cardiovascular, an inflammatory or an infectious disease is suspected, the best hope for long-term survival for the patient is an accurate diagnosis in the earliest detectable stage of the disease. For this purpose tissue acquisition is required for which a variety of techniques can be applied such as a transdermal macro biopsy, a vacuum assisted biopsy, a laparoscopic tissue acquisition, tru-cut biopsy, or a fine needle aspiration.

Current practice in a hospital typically uses chemical fixation (formalin) directly after tissue acquisition to preserve tissue from degradation, and to maintain the structure of the cells. The later is needed to diagnose for instance cancer based on the histological examination of tissue samples. Normal formalin embedded tissue samples however, can not be used to apply contemporary molecular biology technologies resulting in the need for acquiring additional fresh frozen biopsies.

Currently, fresh frozen biopsies are acquired using snap freezing of the tissue by plunging the biopsy in a cryogenic coolant such as liquid nitrogen or cooled isopentane. Due its hazardous nature, special care has to be taken when using cryogenic coolants such as liquid nitrogen or liquid helium or a coolant such as dry ice, sometimes referred to as "Cardice". This type of sample collection is not routinely performed by physicians due to the lack of such cryogenic coolants at the location where the patient is treated. Accordingly, the fresh tissue is transported to the laboratory where the snap freezing of the tissue can take place. However, to ensure minimal degradation of the specimen and hence no limitation on the types of studies that can be conducted or any influence on the usefulness of the data obtained from the tissue analysis, the freezing must occur as soon as possible after excision of the biopsy.

Accordingly, there is a need for a system and method for acquiring fresh frozen biopsy samples at the location where the biopsy is taken, and in which the molecular activity is being preserved and the histological parameters are maintained, and, which features a means for transportation keeping the biopsy frozen and allowing for further examination and archiving at a laboratory at a different location then the location where the biopsy is taken.

SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to systems and methods for freezing biopsy specimen, and more particularly to a cryogenic system and method that aims to temporary preserve a biopsy specimen and in which the molecular activity is being preserved and the histological parameters are maintained. The system and method of the present invention are designed to collect, store and preserve samples featuring means for transportation while keeping the biopsy frozen and allowing further examination and archiving at a laboratory at a location different from the location where the biopsy is taken. The present invention aims to stabilize tissue characteristics based on rapid, deep freezing of the tissue without the use of cryo-protectants.

The present invention in particularly relates to methods for freezing, preferably snap- freezing, a biopsy specimen, the method comprising the steps of:

a) collecting a biopsy specimen into a specimen container b) positioning the container into a precooled thermal mass enabling controlled freezing and thawing of the tissue sample which is kept in said specimen container, wherein the thermal mass has been cooled to a temperature below the eutectic temperature of said biopsy specimen; and;

c) applying a thermal insulator around the thermal mass, thereby maintaining said specimen container comprising said biopsy specimen frozen below the eutectic temperature when the thermal mass is disconnected from the cooling system, thereby providing a frozen biopsy specimen having a preserved molecular activity and histological characteristics.

In particular, the method as provided herein is a method for freezing and transporting a biopsy specimen, the method comprising the steps of:

a) collecting a biopsy specimen into a specimen container;

b) positioning said specimen container into a precooled thermal mass enabling controlled freezing and thawing of said biopsy specimen which is kept in said specimen container, wherein said thermal mass is solid and has been cooled to a temperature below the eutectic temperature of said biopsy specimen;

c) applying a thermal insulator around said thermal mass, thereby maintaining said specimen container comprising said biopsy specimen frozen below the eutectic temperature when the thermal mass is disconnected from the cooling system, thereby providing a frozen biopsy specimen having a preserved molecular activity and histological characteristics; and;

d) disconnecting said thermal mass and said thermal insulator from the cooling system and transporting it to another location.

More particularly, said specimen container is shaped such that the maximal distance between the inner side of the specimen container and the outer side of the biopsy specimen is 2 mm or less. More particularly, said specimen container comprises a pressure sensitive adhesive located on the inside of said specimen container.

According to a particular embodiment the thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK. More particularly, the thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower.

According to a particular embodiment the thermal mass is precooled by means of an electrical powered cooling system. More particularly, said cooling system is a thermoelectric cooling, sorption cooling, Stirling cooling Joule Thomson cooling, Brayton cooling, Pulse-Tube cooling, Gifford-McMahon cooling or any vapour compression cooling system.

According to a particular embodiment, the method according to the present invention further comprises the step of sectioning the frozen biopsy specimen.

More particularly, said specimen container is constructed to enable removal of the tissue specimen from the biopsy needle by moving the needle along a release element restraining said sample wherein said release element is an integral part of the specimen container.

More particularly, said specimen container is constructed to enable removal of the tissue specimen from the biopsy needle by moving the needle along a release element shaped by a clasp to hold the sample wherein said clasp is an integral part of the specimen container.

In a particular embodiment, the present invention relates to a method for determining molecular activity and histological parameters for a biopsy sample, comprising the steps of

a) freezing, preferably snap-freezing, a biopsy specimen according to the methods disclosed herein; thereby obtaining a frozen biopsy specimen;

b) sectioning said frozen biopsy specimen; thereby keeping the specimen frozen; and;

c) examining the histological parameters and molecular activity of said biopsy specimen, thereby determining the molecular activity and histological parameters for said biopsy sample.

More particularly, said molecular activity is the activity of kinases, phosphotases or nucleic hormone receptors. More particularly, the examination of said biopsy specimen includes immunohistochemistry and/or RNA examination.

In a particular embodiment, the present invention relates to a device for performing a method as described herein, comprising

- a specimen container for holding a biopsy specimen, wherein the maximal distance between the inner side of the specimen container and the outer side of the biopsy specimen is less than 2 mm; a thermal mass adapted to receive and entirely cover said specimen container, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK;

a thermal insulator adapted to receive and at least partially cover said thermal mass, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower; and;

a cooling system for cooling said thermal mass to a temperature below the eutectic temperature of said biopsy specimen.

More particularly, said device for performing the method as described herein comprises a specimen container for holding a biopsy specimen, wherein said specimen container is positioned inside of a thermal mass adapted to receive and entirely cover said specimen container, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK, wherein said thermal mass is positioned at least partially inside of a thermal insulator adapted to receive and at least partially cover said thermal mass, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower, wherein said thermal insulator is positioned on top of a cooling system for cooling said thermal mass to a temperature below the eutectic temperature of said biopsy specimen.

More particularly said specimen container comprises a pressure sensitive adhesive. More particularly said specimen container further comprises a release element shaped as a clasp for removing said tissue specimen from the biopsy needle.

BRIEF DESCRIPTION OF FIGURES

Figure 1 provides, as summarised in the description of the invention, a graphical representation of the specimen container, the cooling system, the thermal mass and the thermal insulator.

Figure 2 provides a graphical representation of the specimen container, the thermal mass and the thermal insulator.

Figure 3 provides a graphical representation of a release mechanism for the transfer of the biopsy specimen from the biopsy needle into the specimen container.

1 - Biopsy needle; 2 - Biopsy sample; 3 - Specimen container; 4 - Cooling system; 5 - Thermal mass; 6 - Thermal insulator; 7 - Needle notch; 8 - Release element; 9 - Clasp. DETAILED DESCRIPTION OF THE INVENTION

Before the present method and devices used in the invention are described, it is to be understood that this invention is not limited to particular methods, components, or devices described, as such methods, components, and devices may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the preferred methods and materials are now described.

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +/-5% or less, more preferably or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Embodiments of the present invention are directed to systems and methods for freezing biopsy specimen, and more particularly to a cryogenic system and method that aims to temporary preserve a biopsy specimen and in which the molecular activity is being preserved and the histological parameters are maintained. The system and method of the present invention are designed to collect, store and preserve samples featuring means for transportation while keeping the biopsy frozen and allowing further examination and archiving at a laboratory at a location different from the location where the biopsy is taken. The present invention aims to stabilize tissue characteristics based on rapid, deep freezing of the tissue without the use of cryo-protectants. During tissue freezing, the most fundamental physical change taking place is the transport of water across a cell's semi-permeable membrane. The cell walls are generally considered freely permeable to water. As cooling proceeds, the intracellular as well as the extracellular fluid reaches the freezing temperature. It is normally assumed that ice forms in the extracellular fluid first. At the cellular level, the osmotic water migration is higher for a slow cooling process, leading to dehydration and a large change in the cell size. Fast cooling causes the least amount of migration and will leave the cell closer to its original shape and size. Additionally, also the rate at which frozen samples are re- warmed is of importance. Damage caused by rapid warming is due to osmotic stresses that may occur during rapid re-hydration of the cell, and is less likely to occur in rapidly cooled cells because these do not dehydrate significantly during cooling.

Techniques of cryo-preservation are commonly used to prevent degradation of e.g. tissue, cells, embryos and sperm. These techniques typically involve loading the sample with cryo-protective agents and then applying freezing procedures that prevent specimen damage due to ice crystal formation. Cryo-preservation to enable specimen inspection in a Transmission Electron Microscope requires vitrification procedures in an attempt to prevent ice formation throughout the entire sample. To that end the sample must be frozen extremely fast to a temperature of below e.g. 136 K (EP2381236).

To acquire fresh frozen biopsies enabling contemporary molecular biology technologies, current practices are to snap freeze the tissue by plunging the biopsy in a cryogenic coolant such as liquid nitrogen or a coolant such as dry ice. Various snap freezing protocols can be applied but universal efficacy depends on the tissue origin, the operator skills as well as the thawing of the samples. Further, freezing tissue in liquid nitrogen will create an insulating gas layer between the specimen and the liquid and will therefore delay the speed of freezing, i.e. the Leidenfrost effect. To prevent cell damage caused by said insulating effect a transition fluid such as isopentane is being used, a fluid considered to be a very good cryo-conductor. The exact mechanisms and temperature gradients needed to preserve the bio-molecular processes and histological parameters of tissue are not known, and the current usage of cryogenic coolants is mainly based on practical experiences.

As disclosed herein, the inventors have found a method for freezing, preferably snap- freezing, a biopsy specimen, the method comprising the steps of:

a) collecting a biopsy specimen into a specimen container;

b) placing the container into a precooled thermal mass enabling controlled freezing of said tissue sample held in said specimen container; wherein said thermal mass is precooled to a temperature below the eutectic temperature of said biopsy specimen; and;

c) applying a thermal insulator around said thermal mass, thereby maintaining said specimen container, comprising said biopsy specimen, at a temperature below the eutectic temperature of said biopsy specimen when the thermal mass is disconnected from the cooling system.

a) collecting a biopsy specimen into a specimen container;

It should be clear that the application of the thermal insulator around the thermal mass may occur at any time prior to the collection of the biopsy specimen into the specimen container, or the positioning of the specimen container in the precooled thermal mass. Alternatively, the order of the steps of the method of the invention is maintained. In particular, the method as provided herein is a method for freezing and transporting a biopsy specimen, the method comprising the steps of:

a) collecting a biopsy specimen into a specimen container;

b) positioning said specimen container into a precooled thermal mass enabling controlled freezing and thawing of said biopsy specimen which is kept in said specimen container, wherein said thermal mass is solid and has been cooled to a temperature below the eutectic temperature of said biopsy specimen; and wherein said thermal mass is at least partially insulated by a thermal insulator surrounding said thermal mass, thereby maintaining said specimen container comprising said biopsy specimen frozen below the eutectic temperature when the thermal mass is disconnected from the cooling system, thereby providing a frozen biopsy specimen having a preserved molecular activity and histological characteristics; and;

c) disconnecting said thermal mass and said thermal insulator from the cooling system and transporting it to another location.

In a particular embodiment, the thermal mass is fully or at least partially insulated by a thermal insulator as described herein.

By conducting the method according to the present invention it has been found that the frozen biopsy specimen is characterized by having a preserved molecular activity and histological characteristics.

In a particular embodiment, the method according to the present invention provides that said specimen container is shaped such that the maximal distance between the inner side of the specimen container and the outer side of the biopsy specimen is 2 mm or less, and more particularly, 1.8, 1 .6, 1.4, 1 .2 or 1 mm or less. Accordingly, said specimen container provides that when the biopsy specimen is loaded into the specimen container, the maximal distance between the inner surface of the container and the outer side of the specimen is less than 2 mm, thereby assuring optimal freezing conditions for the specimen. Typically, the specimen will make contact with at least part of the inner surface of the specimen container, and the free surface of the specimen will be at a maximal distance of 2mm from the nearest inner surface of the specimen container.

In a particular embodiment the method according to the present invention provides that said specimen container is constructed of a material having a heat conductivity of at least 0.1 W/mK, more particularly, at least 0.2, 0.3, 0.4, 0.5, 0.75 or 1 W/mK. In particular embodiments the material used for the specimen container is plastic (such as polypropene) or metal, more particularly a reinforced plastic, a cryoplastic or a metallic material.

A reinforced plastic material refers to a composite material made of a polymer matrix reinforced with fibers. The fibers are usually glass, carbon, or aramid. The polymer is usually an epoxy, vinylester or polyester thermosetting plastic.

In a particular embodiment the method according to the present invention provides that said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK. In order to ensure good heat transfer between the thermal mass and the specimen container, the distance between both should be kept as small as possible. Preferably, the thermal mass encloses the specimen container, thereby making contact with most of the outer surface of the specimen container.

In a particular embodiment the method according to the present invention provides that said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower. Typical insulating materials known in the art may be used for the thermal insulator. Preferably aerogels, vacuum or multi layer insulation materials (MLI). MLI refers to is thermal insulation composed of multiple layers of thin sheets made of very thin plastic sheets (about 6 micrometres), such as Mylar or Kapton, coated on one side with a thin layer of metal on both sides, typically silver or aluminium.

By applying a cooling method and system applying a thermal mass having a high thermal conductivity of 200 W/mK or higher, and/or a specimen container tightly holding the biopsy specimen, a frozen biopsy sample can be obtained, wherein the molecular activity and histological characteristics of the biopsy are preserved, and this without having to use without the use of cryogenic coolants or cryo-preservatives which are completely banned from places where surgical interventions for collecting biopsy samples take place.

Embodiments of the present invention are designed to control the freezing temperature and are based on sufficiently high cooling rates obtained through the usage of a thermal mass constructed of a material composition with a thermal conductivity of at least 200 W/mK. Typically, the thermal mass is a solid material. Materials having a thermal conductivity of at least 200 W/mK include but are not limited to aluminium, beryllium, copper, gold, silver and/or combination thereof. Preferably aluminium or copper are used as material for the thermal mass.

Further the specimen container has been designed to reduce the thermally insulating air gap between the major part of the removed biopsy and the specimen container to control the freezing and thawing rates and will be typically less than 2 mm and more particularly, 1 .8, 1.6, 1.4, 1 .2 or 1 mm or less. The cooling system of said embodiment will enable a thermal mass to be cooled to a temperature of -80°C or below. The thermal mass in combination with the thermal insulator will control thawing of the tissue specimen by keeping the temperature of the tissue specimen inside the specimen container to below the eutectic temperature of the tissue specimen when the thermal mass is disconnected from the cooling system enabling transportation of at least one hour.

The present invention provides methods and devices that enable freezing, preferably snap-freezing, and/or transporting a tissue specimen without the use of cryogenic coolants thereby facilitating freezing, preferably snap-freezing, at the location were the biopsy is taken. More in particular, freezing, preferably snap-freezing, in the room or its surroundings where a patient is treated and where the biopsy is acquired will reduce the lag time between the excision and the freezing of the biopsy tissue. A reduced lag time will ensure minimal degradation of the biopsy specimen. Moreover freezing, preferably snap-freezing, is only being performed at laboratories were cryogenic coolants are allowed.

Accordingly, the object of the present invention is to provide methods and devices that remove a biopsy from a biopsy needle, collect it, freeze it down steeply and thermally isolate it to allow controlled thawing and transportation of the frozen biopsy from one location to another location where the tissue can be examined and archived.

The method according to the present invention also provides in disconnecting the thermal mass and the thermal insulator from the cooling system and transporting it to another location. As such, this provides in a regular sized system which does not require any provision of energy in the form or for instance batteries during the transportation. By disconnecting and transporting the thermal mass comprising the specimen container and the thermal insulator, the disconnected system can be manipulated and transported easily to the designated location. As used in the present invention, the terms "biopsy", "biopsy specimen", "tissue specimen", "biopsy tissue" or "biopsy sample" refers to a biological sample obtained from an organism (patient) such as human or from components (e.g. tissue or cells) of such an organism. Said biological sample preferably comprises sampled cells or tissues used for medical examination. A biopsy is typically removed from a living subject to determine the presence or extent of a disease. Said biopsy may be an excisional biopsy or an incisional biopsy (also referred to as core biopsy). In particular embodiments said biopsy is a tissue biopsy, fine needle biopsy, fine needle aspiration biopsy, core needle biopsy, vacuum assisted biopsy, open surgical biopsy or material from a resected tissue.

Typical surgical interventions where biopsies are obtained include for instance, a tru- cut or core needle biopsy, which uses a large, fitted needle to extract a sample of tissue about the size of a piece of pencil lead. A core needle biopsy can take place in a clinic or hospital and it can be performed by physician e.g. an internist, interventional radiologist, or surgeon.

Biopsy samples are typically collected and used for the diagnoses of different diseases including, but not limited to precancerous conditions (suspicious lesions or masses), cancer, cardiovascular diseases, inflammatory diseases or infectious diseases. Some examples of different types of biopsies for specific conditions are:

• lung biopsy in a case of suspected lung cancer

• biopsy of the temporal arteries for suspected vasculitis

• bowel biopsy for conditions such as inflammatory bowel disease, Crohn's disease or ulcerative colitis

• kidney biopsy for renal conditions such as Crescentic glomerulonephritis.

• lymph node biopsy for a variety of infectious or autoimmune diseases

• gingival biopsy for amyloidosis

• a biopsy of a transplanted organ to determine rejection or that the disease that necessitated transplant has not recurred

• testicular biopsy for evaluating the fertility of men

Types of biopsy include bone marrow biopsy, gastrointestinal tract biopsy, needle core biopsies or aspirates of the pancreas, lung biopsy, liver biopsy, prostate biopsy, nervous system biopsy (brain biopsy, nerve biopsy, meningeal biopsy...), urogenital biopsies (renal biopsy, endometrial biopsy, cervical conisation...), breast biopsy, lymph node biopsy, muscle biopsy, skin biopsy...

In a particular embodiment, the method according to the present invention provides in the cooling of the thermal mass is by means of an electrical powered cooling system. Preferably, said electrical powered cooling system is a thermoelectric cooling, sorption cooling, Stirling cooling Joule Thomson cooling, Brayton cooling, Pulse-Tube cooling, Gifford-McMahon cooling or any vapour compression cooling system. Preferably said electrical powered cooling system is a thermoelectric cooling system. The main advantages of an electrical powered cooling system such as a Peltier cooler (thermoelectric cooling) is the lack of moving parts or circulating liquid, and its small size and flexible shape, allowing easy use in places where a patient is treated and where the biopsy is acquired. Accordingly, the thermal mass can be cooled by a cooling system without the use of hazardous cryogenic coolants such as liquid nitrogen. The cooling system will utilize any electricity powered cooling technology to enable system use at hospitals, academic institutes or any other institutes where biopsies are being taken.

In a particular embodiment, the thermal mass is cooled to a temperature below the eutectic temperature of the biopsy specimen prior to placing the specimen container into the thermal mass. In this manner the thermal mass can be provided in a pre- cooled manner to the place where the biopsy is taken. Once the biopsy is taken it is inserted into the specimen container which is subsequently introduced into the already pre-cooled thermal mass. Accordingly, once the specimen container is inserted into the thermal mass the specimen temperature is freezing is initiated, thereby drastically reducing the time between the removal of the biopsy and the freezing. In a particular embodiment the specimen container comprises an integrated release mechanism to remove the tissue specimen from the biopsy needle. Accordingly, the release element forms an integral part of the specimen container. The removal of the tissue specimen from the biopsy needle may occur using different types and forms of release mechanisms typically known in the art. For instance, the specimen container may comprise a release element shaped as a clasp. By moving the needle along the release element for instance shaped as a clasp, the biopsy sample will be withheld by the release mechanism, thereby removing the specimen from the biopsy needle and into the specimen container. The provision of a specimen container comprising an integrated system for placing the biopsy specimen from the biopsy needle into the specimen container will allow the user to safely transfer the biopsy specimen without having to risk contamination through external objects. Also, an integrated release mechanism will further assure that the biopsy specimen in placed onto the optimal position in the specimen container. Any external interference needed for the transfer of the biopsy specimen will only result in a suboptimal placement of the biopsy specimen in the specimen container leading to suboptimal freezing of the biopsy specimen.

The method according to the present invention therefore provides that said specimen container is constructed to enable removal of the tissue specimen from the biopsy needle by moving the needle along a release element for instance shaped by a clasp to hold the sample wherein said clasp is an integral part of the specimen container. This allows removal of tissue from a biopsy needle, resolving the cumbersome operation of the removal of fresh biopsy material from the notch of the biopsy needle. Additionally the specimen container is featured to match biopsy needles of various dimensions such as for instance needles to execute macro-biopsies with dimensions between 8 and 14 gauge or needles to execute micro-biopsies with dimensions between 14 and 20 gauge. Moreover the biopsy needles may be based on e.g. vacuum assisted needles, tru-cut needles or Menghini needles. Further, the gap between the major part of the removed biopsy and the specimen container will be typically less than 2 mm, and more particularly, less than 1 .8, 1.6, 1.4, 1.2 or 1 mm.

In a further particular embodiment said specimen container comprises an adhesive or bonding agent inside the specimen container. More particularly, said adhesive or bonding agent is a compound that maintains the adhesive characteristics at low temperatures and this without affecting the molecular (kinase) activity of the tissue sample. More particularly, said adhesive is a pressure sensitive adhesive. The provision of a (pressure sensitive) adhesive in the specimen container allows the fixation and attachment of the biopsy specimen in the specimen container. This may be important in view of later manipulations to which the biopsy specimen may be subjected. When for instance sectioning of the tissue sample is required, the biopsy specimen needs to be attached to the container in order to be able to perform the sectioning in an accurate manner. Typically, tissues are embedded in compounds such as OCT prior to sectioning. However, the use of OCT may not be considered in the methods according to the present invention as the use of OCT will actually damage or harm the molecular (kinase) activity of the tissue sample. Accordingly, it has been found that the use of a pressure sensitive adhesive such as adhesives made out of acrylate, silicones or rubber, and preferably acrylate in the specimen container may be used to attach the biopsy sample in the specimen container, the used pressure sensitive adhesive, being resistant to the low freezing temperatures, thereby allowing the sectioning of the biopsy sample in a frozen state, thereby avoiding the use of compounds such as OCT, and maintaining the actual molecular activity of the frozen tissue sample. In a particular embodiment, the method according to the present invention further comprises the step of sectioning the frozen biopsy specimen.

According to a particular embodiment the present invention pertains to a method for determining the molecular activity and histological parameters for a biopsy sample, comprising the steps of

a) freezing, preferably snap-freezing, a biopsy specimen according the methods of the present invention; thereby obtaining a frozen biopsy specimen;

More particularly, said molecular activity is the activity of kinases, phosphotases or nucleic hormone receptors.

According to another particular embodiment the examination of said biopsy specimen includes immunohistochemistry and/or RNA examination.

Accordingly, in a particular embodiment the method according to the present invention comprises the steps of:

(a) transferring a biopsy sample from the biopsy needle into a specimen container; wherein the removal of the tissue from the biopsy needle occurs using a release mechanism integrated into the specimen container, for instance by moving the biopsy needle along a release element shaped by a clasp to hold the sample, which clasp is an integral part of the specimen container;

(b) collecting the biopsy sample into the specimen container;

(c) applying a cooling system to cool down a thermal mass and keep its temperature down as long as the user requires so;

(d) positioning the specimen container into the pre-cooled thermal mass enabling steep freezing of the specimen container holding the biopsy sample; (e) applying a thermal insulator around the thermal mass holding the specimen container with the biopsy sample therein to control the temperature of the biopsy sample when the thermal mass is disconnected from the cooling system enabling transport of the biopsy sample in a deep frozen state; and,

(f) applying a transportable system which consist of the thermal insulator, the thermal mass and the specimen container as referred to in the previous steps, thereby keeping biopsy sample frozen during transport from the location where the biopsy is taken from a patient to the location where the biopsy will be examined and archived;

(g) applying sectioning of the (snap) frozen biopsy specimen; thereby obtaining multiple frozen biopsy specimen sections;

(h) performing a histological analysis of part of said frozen biopsy specimen sections;

(h) performing a molecular (protein) analysis of part of said frozen biopsy specimen sections.

It should be noted that the application of the thermal insulator around the thermal mass may occur prior to, during or after cooling the thermal mass.

In a particular embodiment, the present invention relates to a device for performing the method according to the present invention, wherein said device comprises

- a specimen container (3) for holding a biopsy specimen (2), wherein the distance between the inner side of the specimen container and the outer side of the biopsy specimen is less than 2 mm, and more particularly, less than 1 .8, 1 .6, 1.4, 1.2 or 1 mm.;

a thermal mass (5) adapted to receive and entirely cover said specimen container, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK;

a thermal insulator (6) adapted to receive and at least partially cover said thermal mass, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower; and;

- a cooling system (4) for cooling said thermal mass to a temperature below the eutectic temperature of said biopsy specimen.

More particularly, the device for performing a method according to the present invention comprises a specimen container for holding a biopsy specimen, wherein said specimen container is positioned inside of a thermal mass adapted to receive and entirely cover said specimen container, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK, wherein said thermal mass is positioned at least partially inside of a thermal insulator adapted to receive and at least partially cover said thermal mass, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower, wherein said thermal insulator is positioned on top of a cooling system for cooling said thermal mass to a temperature below the eutectic temperature of said biopsy specimen.

In a particular embodiment, the device according to the present invention provides in an additional insulating void space between the thermal mass and the thermal insulator, the void being filled with a gas such as air to further improve the insulation of the thermal mass.

More particularly, said specimen container further comprises a pressure sensitive adhesive located on the inside of said specimen container.

More particularly, said specimen container (3) further comprises an integrated release element (8) for instance shaped as a clasp (9) for removing said tissue specimen (2) from the biopsy needle (1 ).

In accordance with features in embodiments of the present invention, the steps of freezing and thawing of the tissue while utilizing the thermal mass are being done in a controlled way using the configuration of the thermal insulator, the thermal mass and the specimen container to provide temperature gradients (rate, duration etc) required to preserve molecular activity in a biopsy tissue and to maintain histological parameters. To prevent damage of the morphology of tissue samples ice crystallization has to be controlled. It has been postulated that as cooling rates increase the size of the intracellular and extracellular ice crystals decreases. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention. To obtain such cooling rates the thermal mass should preferably be constructed of a material composition with a thermal conductivity of at least 200 W/mK. Temperature control to below - 80°C has been shown to result in only small ice crystal formation so that cell damage may not harm histological examination. It should be understood that the desired result is to maintain the temperature below that at which ice begins to crystallize. Cooling temperatures down to about -123°C are therefore preferred, i.e. the vitrification temperature; the temperature it is thought that ice crystallization begins to occur at in untreated cells. In order to sufficiently preserve the biomolecular processes of the tissue, the tissue should be frozen and kept frozen to a temperature of around - 30°C which is around the eutectic point of the intracellular and extracellular fluids at which biomolecular processes will cease and stabilize. The specimen container, thermal mass and thermal insulator according to the present invention should in particular make it possible to reproducibly set defined freezing or thawing rates wherein temperature gradients or variations in the cooling or thawing rate inside a biopsy sample are being avoided.

The following examples are offered by way of illustration, and not by way of limitation. EXAMPLES

EXAMPLE 1 - Determining the effect of cooling on histological parameters of tissue.

The method of the present invention allows steep freezing of a biopsy. This experiment shows the effect of cooling rate and cooling depth on the histological parameters of biopsies. A well known and often used snap freezing protocol was applied to serve as a standard, and compared to the results of two different but alternative snap freezing protocols to be used in the present invention. The experiment was further intended to reveal the effect of the thermal conductivity due to the thermally insulating air layer which is apparent between tissue and specimen container. This experiment also shows the applicability of a thermal mass having a thermal conductivity of about 200 W/mK to allow for snap freezing.

At day 1 , snap freezing of the biopsies was executed. All specimens were acquired applying a 12 Gauge biopsy needle having a 15mm needle notch and using colon cancer resection tissue.

In the standard snap freezing procedure applied, directly after excision of the biopsy, the biopsy specimen was placed into an empty 2ml cryovial, the cryovial closed, and the cryovial was immediately submerged into liquid nitrogen. Specimen was frozen in 30-60 seconds. Immediately after the snap freezing procedure the cryovials holding the biopsy specimen were placed in a - 80°C freezer.

The alternative snap freezing procedure applied a thermal mass, consisting of an aluminium block having a thermal conductivity of approximately 200 W/mK and containing cavities in which 2 ml cryovials can be placed tightly so as to reduce the thermally insulating effect of the air gap between the outer-side of the cryovial and the inner-side of the cavity of the aluminium block. In this procedure the aluminium blocks in which the 2 ml cryovials were placed, were pre-cooled for one hour in a - 80°C freezer. The blocks holding the 2 ml cryovials were taken out of the freezer and, directly after excision of the biopsy, the biopsy specimen was placed into an empty 2ml cryovial, the cryovial closed and the aluminium block holding the cryovials and the biopsies was placed back in the - 80°C freezer.

The second alternative snap freezing procedure applied was equal to the first alternative snap freezing method but made use of 0.5 ml cryovials and a thermal mass having cavities to allow for 0.5 ml cryovial.

At day 2, standard hospital procedures were applied to cut 5 μηη tissues sections from each snap frozen biopsy for histological evaluation using a cryotome at an operating temperature of around -20°C. All biopsies were subjected to the same thawing times to prevent various sized ice crystal formations affecting the histological parameters. Also standard hospital procedures were used for HE staining to allow for histological examination. Examination of the tissue sections indicated that the histological characteristics are preserved when the alternative method according to an embodiment of the invention is performed.

Consequently, the present example shows that the snap freezing method according to particular embodiments of the present invention provides frozen tissue samples having histological parameters of at least equal quality compared to samples from conventional methods.

EXAMPLE 2 - Determining the effect of cooling on protein activity of tissue.

In a separate study the study in Experiment 1 is repeated and the protein activity of the tissue samples is analysed. It is observed that the protein activity (kinase activity) is preserved in the tissue samples treated according to the methods of the present invention.

Claims

1 . A method for freezing and transporting a biopsy specimen, the method comprising the steps of:

a) collecting a biopsy specimen into a specimen container;

2. Method according to claim 1 , wherein said specimen container is shaped such that the maximal distance between the inner side of the specimen container and the outer side of the biopsy specimen is 2 mm or less.

3. Method according to claim 1 or 2, wherein said specimen container comprises a pressure sensitive adhesive located on the inside of said specimen container.

4. Method according to any of claims 1 to 3, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK.

5. Method according to any of claims 1 to 4, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower.

6. Method according to any of claims 1 to 5, wherein said thermal mass is cooled by means of an electrical powered cooling system.

7. Method according to claim 6, wherein said cooling system is a thermoelectric cooling, sorption cooling, Stirling cooling Joule Thomson cooling, Brayton cooling, Pulse-Tube cooling, Gifford-McMahon cooling or any vapour compression cooling system.

8. Method according to any of claims 1 to 7, further comprising the step of sectioning said frozen biopsy specimen.

9. Method according to any of claims 1 to 8, wherein said specimen container is constructed to enable removal of said tissue specimen from the biopsy needle by moving said biopsy needle along a release element for restraining said sample said release element being an integral part of said specimen container.

10. Method for determining molecular activity and histological parameters for a biopsy sample, comprising the steps of

a) freezing a biopsy specimen according to any of claims 1 to 9; thereby obtaining a frozen biopsy specimen;

1 1 . Method according to claim 10, wherein said molecular activity is the activity of kinases, phosphotases or nucleic hormone receptors.

12. Method according to claim 10 or 1 1 , wherein the examination of said biopsy specimen includes immunohistochemistry and/or RNA examination.

13. A device for performing a method according to any of claims 1 to 9, comprising a specimen container for holding a biopsy specimen, wherein said specimen container is positioned inside of a thermal mass adapted to receive and entirely cover said specimen container, wherein said thermal mass is constructed of a material having a heat conductivity of at least 200 W/mK, wherein said thermal mass is positioned at least partially inside of a thermal insulator adapted to receive and at least partially cover said thermal mass, wherein said thermal insulator is constructed of a material having a heat conductivity of 0.1 W/mK or lower, wherein said thermal insulator is positioned on top of a cooling system for cooling said thermal mass to a temperature below the eutectic temperature of said biopsy specimen.

14. A device according to claim 13, wherein said specimen container comprises a pressure sensitive adhesive located on the inside of said specimen container.

15. A device according to claim 13 or 14, wherein said specimen container further comprises a release element for removing said tissue specimen from the biopsy needle.