WO2006081999A1

WO2006081999A1 - Feeding funnel for rod-shaped biological samples

Info

Publication number: WO2006081999A1
Application number: PCT/EP2006/000755
Authority: WO
Inventors: Ulrich Vogel
Original assignee: Eberhard-Karls-Universität Tübingen
Priority date: 2005-02-01
Filing date: 2006-01-28
Publication date: 2006-08-10
Also published as: DE102005005518A1

Abstract

The invention relates to a device for introducing rod-shaped biological samples into a receptacle, an associated insertion rod, a kit encompassing the inventive device and said insertion rod, and a method for introducing a rod-shaped biological sample into a receptacle.

Description

INTRODUCER FOR HEAVY BIOLOGICAL SAMPLES

The present invention relates to a device for introducing rod-shaped biological samples into a container, an associated push-rod, a kit comprising the device and the push-in rod, and a method for introducing a rod-shaped biological sample into a container. Biological samples, such as biological tissue, can be stored in a paraffinized state for decades without loss of antigenicity and without damaging DNA and RNA. In such preserved biological samples of human, animal or plant tissue, from microorganisms, cell cultures or macromolecules, histological, immunohistochemical and molecular biological investigations can be carried out by methods known in the art.

Conserved or paraffinized tissues are often analyzed using a so-called Tissue MicroArray (TMA). Such a TMA was first reported by Kononen et al. (1998), Tissue microarrays for high-throughput molecular profiling of tumor specimens, Nat. Med. 4, pages 844-847. A TMA consists of a geometric arrangement of tissue samples, usually in a plurality of columns or rows in a carrier, such as. a support block made of paraffin, in which the tissue samples are in the individual wells or holes.

The tissue samples are usually prepared prior to assembly of the TMA by means of a cannulated tissue punch in the form of small tissue cylinders z .B. punched out of paraffin tissue blocks in which the tissues are cast. Such tissue cylinders then usually have a diameter of 0, 6 to 1, 5 mm and a length of up to 5 mm. After punching, the tissue cylinders are then inserted directly into the wells of the support block to form the actual TMA. This is done in such a way that the tissue punch, in the top of which the tissue cylinder is located, directly over the corresponding depression is brought and then in an upper opening of the tissue punch a push rod, such as a stylet, is introduced, with which the tissue cylinder is pushed out of the punch and thereby transferred into the recess of the support block. This will be repeated, if necessary. with paraffin blocks of tissues of various kinds, as long as repeated until all the required wells are filled with tissue cylinders and the actual TMA is ready for use.

The most common tissue punching and the most commonly used method for creating TMAs using the so-called conon method are described by Beecher Instruments, Sun Prairie, WI, and Zymed Laboratories Inc. , South San Francisco, CA, both United States of America.

A summary of TMAs can be found in the publication Mengel et al. (2003), Rapid and Large-Scale Transition of New Tumor Biomarkers to Clinical Biopsy Material by Innovative Tissue Microarray Systems, Applied Immunohistochemistry & Molecular Morphology 11, pp. 261-268; the content of this publication is incorporated herein by reference.

Wan et al. (1987), A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously, Journal of Immunological Methods 103, pages 121-129, have described for the first time the phenomenon mentioned at the outset, namely that paraffinized tissue, for example tumor tissue in the form of tissue cylinders for further use in small vessels, such as e.g. so-called . Eppendorf hat or tube, can be stored. In this manner In addition, archives of defined tissue cylinders can be created, which can then be sent without any problems and thus analyzed by specialized research teams, for example.

A disadvantage in this context, however, that such archived tissue cylinder can be introduced into the wells of the support blocks or comparable containers extremely difficult and thus hardly available for analysis in TMA with the method distributed by Beecher Instruments and world wide used. It is well known that the small tissue cylinders are extremely difficult to handle, for example by means of tweezers. When attempting to introduce the tissue cylinders into the depressions of the carrier plate, both the depressions and the tissue cylinders can be destroyed or at least severely damaged. This is due to the high fragility and sensitivity of the paraffinated biomaterial and the i.d.R. made of paraffin Trägerbloσkes. For example, Mengel et al. (supra) that archived and stored tissue cylinders can not be analyzed in TMA at all because of these difficulties.

Von Wasielewski et al. (2002), Tissue Array Technology for Testing Interlaboratory and interobserver Reproducibility of Immunohistochemical Estrogen Receptor Analysis in a Large Therapeutic Trial. At the. J. Clin. Pathol. 118, pages 675-682, therefore describe a TMA technology, in which - as described above - the tissue cylinders are transferred after their punching directly into the wells of the microtiter plate. Prefabricated archived tissue cylinders can be added not be used in this known method. An identical procedure with corresponding problems is known from DE 100 01 136 A1 as well as from US 2003/0038401 A1.

Meanwhile, the companies Zymed, United States of America, and Zytomed, Berlin, Germany, offer as a service to introduce prefabricated tissue cylinders into the wells of the carrier blocks which are part of the so-called Max Array System of Zymed. To do this, the tissue cylinders must be shipped to the appropriate two companies. There, the tissue cylinders are introduced by means of a method unknown in the wells. As a result, the investigating scientist or pathologist can not immediately begin the TMA analysis, rather he has to wait for the return of the loaded carrier blocks and incur associated high costs.

It is therefore an object of the present invention to provide a device with which the above-described disadvantages of the prior art are avoided. In particular, a device is to be provided by means of which the scientist, pathologist or even a simple assistant can easily handle locally fabricated tissue cylinders and insert them into the wells of the carrier blocks or corresponding vessels belonging to the TMAs without these being damaged or destroyed. Such a device should also be inexpensive to produce as a mass product.

This object is achieved by the provision of a device for introducing rod-shaped biological samples into a container which has a body with a through-channel wherein the through channel has an inlet opening for introducing the sample and an outlet opening, via which the sample from the body is insertable into the container, wherein the inlet opening has an expansion.

The object underlying the invention is thereby completely solved.

The inventors have realized that by the simple measure of providing a widening at the inlet opening of the through-channel, the gentle introduction of the rod-shaped biological samples, for example paraffinized tissue cylinders, is particularly well possible. It was particularly surprising that the extremely sensitive and fragile biological samples are not damaged or only to a very limited extent when hitting the widening forming walls. The sample is then deflected in the direction of the passageway and so gently introduced into the latter. Via the passageway, the biological sample then passes through the outlet opening into the depression of the support block of the TMA located immediately below it. The introduction of the biological sample into the depression can take place with the aid of a push rod or a stylet, with which pressure is exerted carefully on the biological sample, so that a passage of the latter through the passage is facilitated.

The present invention also has the advantage that it is particularly cost-effective to implement. Thus, commercially available tissue punches, such as those offered for example by Beecher Instruments, USA, can be made by attaching a speaking expansion can be easily modified according to the invention. Such a widening can be realized, for example, by a conical boring or a counterbore in the inlet opening of the commercially available tissue punch.

With this device, prefabricated and optionally archived tissue cylinders can be introduced as biological samples into a container in a particularly advantageous manner. The use of such tissue cylinders has the advantage that they are clearly visible against directly punched-out tissue cylinders and are not covered by the tube of the tissue punch. Thus, by appropriate selection of the biological samples TMAs can be constructed with tissue cylinders of the same length, which is not possible when using immediately before punched tissue cylinders because of the widely varying thickness of routinely produced paraffin tissue blocks. Furthermore, the use of archived tissue cylinders allows a time-saving and thus very efficient work, since the identification of the tissue of interest in the paraffin tissue block must be done only once and not every time in reassembling the TMA.

The term "rod-shaped biological sample" means according to the invention such a sample, which may consist of any biological material, such as human, animal or plant tissue, as well as microorganisms, cell cultures or macromolecules contained in a matrix which gives the sample an external shape , Such a matrix is provided, for example, by paraffin or any other suitable substance into which the biomaterial is embedded. Rod-shaped in this context means that the biological Sample has an elongated shape, such as in a cuboid, a cylinder, a conical or egg-shaped structure, a sausage-shaped structure, etc.

However, according to the invention, the cross-section of the biological sample can have any shape, for example circular, oval, star-shaped, square, etc. be. This depends on the one hand on the way in which the biological sample was obtained, for example the shape of the tissue punch with which the sample was punched out of a paraffin tissue block. In addition, the cross section of the biological sample may depend on the nature of the latter, i. depending on the tissue used, or on the matrix forming agent, for example, the particular paraffin used.

The container may have any configuration, for example a depression or a receiving hole of a support block or a similar device, which is provided as part of a TMA, as long as the container is suitable for receiving a corresponding biological sample.

According to the invention, expansion means any broadening of the through-channel in the region of the inlet opening, so that the average cross-sectional area of the expansion is greater than the average cross-sectional area of the adjoining through-channel.

It is preferred if the expansion as an insertion funnel, preferably frusto-conical, more preferably as a counterbore is formed. This measure has the advantage that hereby the design conditions for a safe and particularly gentle introduction of the biological sample are created in the passageway. These can be realized in a particularly simple manner, for example using conventional drilling or milling tools to provide a counterbore. Further special technical precautions or the use of special devices or special materials are not required.

It has been found to be particularly advantageous if the sides of the insertion funnel with the longitudinal axis of the passage channel form an angle which is at 85 ° to 5 °, preferably at 55 ° to 45 °, more preferably at about 35 °.

As the inventors have found, the biological sample is deflected particularly well and gently in the direction of the passage channel at such an inclination of the sides of the insertion funnel. Also, commercially available drills or cutters that are available in appropriate dimensions can be used to implement this embodiment.

According to a variant of the invention, the insertion funnel is formed spherical disc-shaped.

As the inventors were able to ascertain, even with this variant of the inlet opening, a gentle introduction of the biological sample is made possible by a corresponding orientation of the latter in the direction of the main duct. Also are in the state the art drill or cutter available with which such a design is easy to implement.

Another particularly suitable variant is that the insertion funnel is formed trumpet-shaped.

This configuration, in which the slope of the expansion in the direction of the passageway increases, ensures that the biological sample is targeted and gently introduced into the passageway.

In the device according to the invention, it is preferred if the edges which are formed by the widening on the body are rounded.

This measure has the advantage that edges, which are formed, for example, when mounting the counterbore in the region of the inlet opening, are removed and the biological sample can no longer be damaged by these during insertion. The introduction of the biological sample into the receiving bores of the support block is thereby possible in a more gentle manner.

According to a preferred development, the cross-section of the through-channel is adapted to the cross-section of the biological sample.

This measure has the advantage that the biological sample is guided in the through-channel and supported laterally. The sample is thereby aligned in the axis of the through-channel, whereby a tilting and possibly. Tilting and damaging the The latter is prevented in the channel. The sample is thus gently introduced into the wells of the support block. The cross-section of the through-channel can be substantially circular, for example, whereby the device is adapted to archived or stored tissue cylinders, which likewise have a substantially circular cross-section.

It is preferred if the passage is formed substantially cylindrical.

This measure has the advantage that such a passage can be easily manufactured, for example, by a corresponding hole, and further the biological sample can be gently guided into the recess of the support block, to the aid of a push rod or a stylet is made possible.

The passageway preferably has a diameter of 0, 3 to 2, 5 mm, preferably from 1, 0 mm, on.

This measure has the advantage that herewith in the device according to the invention the structural requirements for the use of the customary used in connection with TMAs punched fabric cylinders are created. Namely, these usually have diameters of 0, 6 to 1, 5 mm, care must be taken that the passageway has a diameter which, depending on the punch diameter is approximately equal to or slightly larger than the diameter of the biological sample to a Hindurσhtreten the The latter through the passage to ensure without getting stuck. According to a development of the invention, the device further comprises a hollow body, which extends from the outlet opening as an extension of the passage channel in the direction opposite to the inlet opening direction.

This measure has the advantage that the biological sample can be introduced particularly easily into the depression or the vessel. Thus, the hollow body is usually over, if necessary. on or in the recess of the support block (on) brought. As a result, the entire device can be aligned more accurately over the container and the biological sample is thus introduced safely and there is no risk of it missing the depression.

In this case, the hollow body is preferably formed by a needle whose end opposite the inlet opening is designed such that biological preparations, preferably paraffin tissue, can be punched out.

This measure has the advantage that the device can be used not only for introducing the biological sample into a container, but rather also as a tissue punch, without the need for expensive constructional measures are required. Thus, such a needle can be pressed into a prefabricated Paraffingewebeblock, wherein in the tip of the needle, a tissue cylinder is formed, which can then be transferred into the recess of a support block for TMA analysis or in a closable vessel for the purpose of archiving. The end of the needle is sharp-edged, for which a corresponding grinding can be provided. It is preferred if the hollow body is formed by a cannula.

This measure has the particular advantage that suitable hollow bodies already present in the prior art are used, which can serve both for the purposeful introduction of the biological sample into the container and for the punching out of biological samples from, for example, paraffin blocks.

According to the invention, the body and the hollow body may be integrally formed.

This measure has the advantage that such a device can be produced inexpensively in one operation, for example as an injection-molded part, without having to connect both parts by design measures. It is ensured at the same time that the body and the hollow body are tightly connected and a smooth-walled configuration of the through-channel without any projections is simplified possible.

Alternatively, the body and the hollow body are formed in two pieces.

This measure has the advantage that in the prior art already existing components, such as a hollow needle or cannula, can be used as a hollow body and, if necessary. only the body is to be manufactured separately. Body and hollow body are then connected together. For this purpose, for example, a needle is inserted into the body and fixed by means of a fixing screw. Both parts can for example Metal are made and are therefore particularly robust, easy to clean and therefore reusable.

It is preferred if the body of the device is cylindrical.

The cylindrical design allows a particularly simple production and handling of the device.

Another object of the present invention relates to a push rod, whose cross-section is designed such that it can be inserted into the passage of the device described above.

Such a push rod, which may be made of any material, such as metal or plastic of any kind, is a valuable aid to the operator of the device. Thus, by means of the insertion rod, which can be realized by a stylet, the biological sample is carefully pressed through the passageway and thus gently into the recess of the container or the support block are introduced. In this case, the cross section of the insertion rod is substantially formed as the cross section of the passage channel, it being ensured that the insertion rod in cross-section is slightly smaller than the cross section of the through-channel.

Another object of the present invention relates to a kit having the device described above and the insertion rod described above. Thus, the operator is provided with a set which contains at least two objects which are particularly suitable for the advantageous establishment of a TMA from archived tissue cylinders. It is understood that such a kit may contain other items, such as a user manual for using the device or the insertion rod, the production of a paraffinized tissue block, a TMA, etc.

Against this background, the present invention is also a method for introducing a rod-shaped biological sample into a container, comprising the steps of: (a) positioning an outlet opening of an introducer above the container, (b) introducing the sample into an inlet opening of the introducer, and (c) allowing the sample to pass through a passageway of the introducer into the container, using as introducer the device of the invention described above, and step (c) is preferably performed with the aid of the above-described inserter rod.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

The present invention will now be explained in more detail with reference to exemplary embodiments, which are purely illustrative and in no way restrict the scope of the present invention. Reference is made to the accompanying figures, in which: Fig. 1 shows a schematic representation of the device according to the invention;

Fig. 2 shows various embodiments of the expansion;

Fig. Figure 3 shows the provision of rounded edges in the area of the expansion;

Fig. 4 shows a particular embodiment of the device according to the invention, and

Fig. 5 shows the individual steps of the method according to the invention.

embodiments

Einführvorrichtunq

In Fig. 1, the insertion device according to the invention is designated as a whole by 10. This device can be made of any suitable material, for example a metal, such as stainless steel, but also any suitable plastic. The device 10 has a body 12 with a passage 14. The body 10 further has at its upper end an inlet opening 16, via which the biological sample can be introduced, and at its lower end an outlet opening 18, via which the sample from the body can be introduced into a container. The inlet opening 16 has a widening 20, which facilitates the introduction of the sample into the through-channel. In Fig. 2, various embodiments of the expansion are shown. Part of Figure A shows the expansion in the form of a truncated cone-shaped insertion funnel 22, which can be realized by means of a counterbore in the inlet opening 16. The sides of the insertion funnel 22 form with the longitudinal axis of the passage channel 14 an angle α, which is preferably 35 °. The through-passage has a diameter d of 0, 3 to 2, 5 mm, preferably of 1, 0 mm.

In partial illustration B, the widening 20 is shown as a spherical disk-shaped insertion funnel 24.

Part Figure C shows the expansion 20 as a trumpet-shaped insertion funnel 26.

In Fig. 3, an embodiment of the device according to the invention in the region of the expansion 20 in the form of a frusto-conical insertion funnel 22 is shown in which the edges 28 which are formed by the widening of the body, are rounded.

Fig. 4 shows the cross section of an embodiment of the device according to the invention, wherein the body 12 is formed in the form of a holding part 30, which may have a cylindrical shape. Through this holding part 30, the passage channel 14 extends through. Furthermore, the holding part 30 has the inlet opening 16 and the widening 20 at its upper end. At the lower end of the holding member 30 connects as an extension of the passage channel 14 to a cannula 32, which forms a hollow body. This cannula has at its lower end a sharp-edged end 34, which can be used as a punch, with which logical preparations, such as paraffin tissue from a prefabricated block, can be punched out.

Holding part 30 and cannula or hollow body 32 may be integrally formed, for example in the form of an injection molded part. Holding part 30 and cannula or hollow body 32 may equally be formed in two pieces. For this purpose, a cannula 32 can be connected directly to the lower end of the through-channel 14 of the holding part 30, wherein holding part 30 and cannula 32 are glued or welded together for this purpose, for example. In the two-piece embodiment, it is also possible that the cannula 32 is inserted into the passage 14 of the holding part 30 and secured there accordingly. This can also be done by gluing or welding the cannula 32 to the walls of the passageway 14 inside the holding part 30. Other design measures are conceivable, such as the provision of a stop, the provision of a fixing screw, which is screwed laterally into the holding part and is pressed onto the outside of the inserted cannula 32, etc.

The device of the invention may alternatively be realized by modifying the tissue punch of the Manual Tissue Arrayer MTA-I from Beecher Instruments, United States of America, or another commercially available tissue punch. For this purpose, a counterbore is drilled in the upper opening of the tissue punch of the MTA-I or another commercially available tissue punch with the aid of a countersink with the desired dimensions. The punch of the MTA-I and other suitable for the modification according to the invention tissue punching are described at http: //www.beecherinstruments .com /; the content of this Pages is incorporated by reference into the present application.

Inventive method

In Fig. 5 is a rod-shaped biological sample or tissue cylinder, generally designated 36. This can be brought by means of tweezers in the cone-shaped insertion funnel 22 of Einführvorriσhtung 10; Teilabbildung A. After the impact of the tissue cylinder 36 on the sides of the Einführtriσhters 22 this is directed by the truncated cone shape or conical bore in the direction of the passage channel 14; Partial picture B. The tissue cylinder 36 can then pass through the passageway 14, sub-picture C, for which purpose a push-in rod 38, such as a pestle or stylet, can be used; Partial picture D. In this case, the tissue cylinder 36 can be carefully pushed through the passageway 14 until it is inserted into a receptacle 40, such as the bore in a support block from which the TMA is formed.

In this way, for the first time, a prefabricated rod-shaped biological sample, such as a paraffinized tissue cylinder 36, can be gently introduced into the receiving opening of a tissue punch and from there into a bore of a TMA.

Claims

claims

A device (10) for introducing rod-shaped biological samples (36) into a container (40) having a body (12) with a passageway (14), said passageway having an inlet opening (16) for introducing the sample and a Outlet opening (18) through which the sample from the body into the container is insertable, characterized in that the inlet opening has a widening (20; 22, 24, 26).

2. Apparatus according to claim 1, characterized in that the widening (20) is designed as an insertion funnel.

3. Apparatus according to claim 2, characterized in that the insertion funnel is frusto-conical (22), preferably designed as a counterbore.

4. Apparatus according to claim 3, characterized in that the sides of the insertion funnel with the longitudinal axis of the passage channel (14) form an angle α, at 85 ° to 5 °, preferably at 55 ° to 45 °, more preferably at about 35 ° lies .

5. Apparatus according to claim 2, characterized in that the insertion funnel is formed spherical disc-shaped (24).

6. Apparatus according to claim 2, characterized in that the insertion funnel-shaped (26) is formed.

7. Device according to one of the preceding claims, characterized in that the edges which are formed by the widening (20) on the body (12), rounded (28).

8. Device according to one of the preceding claims, characterized in that the cross section of the through-channel (14) is adapted to the cross-section of the biological sample (36).

9. Device according to one of the preceding claims, characterized in that the passage channel (14) is formed substantially cylindrical.

10. Device according to one of the preceding claims, characterized in that the passage channel (14) has a diameter d of 0, 3 to 2, 5 mm, preferably of 1, 0 mm.

11. Device according to one of the preceding claims, characterized in that it further comprises a hollow body (32) extending from the outlet opening (18) as an extension of the passage channel (14) in the inlet opening (16) opposite direction.

12. The device according to claim 11, characterized in that the hollow body (32) is formed by a needle whose inlet opening (16) opposite end (34) is designed such that biological preparations, preferably paraffin tissue, are punched out.

13. The apparatus according to claim 12, characterized in that the hollow body (32) is formed by a cannula.

14. Device according to one of claims 11 to 13, characterized in that the body (12) and the hollow body (32) are integrally formed.

15. Device according to one of claims 11 to 13, characterized in that the body (12) and the hollow body (32) are formed in two pieces.

16. Device according to one of the preceding claims, characterized in that the body (12) is cylindrical.

17. insertion rod (38) whose cross-section is formed such that it in the passageway (14) of the device (10) according to one of claims 1 to 16 is einσhiebbar.

18. Kit comprising the device (10) according to any one of claims 1 to 16 and the insertion rod (38) according to claim 17.

19. A method for introducing a rod-shaped biological sample (36) into a container (40), comprising the steps of:

(a) positioning an outlet opening (18) of an insertion device (10) above the container, (B) introducing the sample into an inlet opening (16) of the insertion device, and

(c) passing the sample through a passageway (14) of the introducer into the container,

characterized in that the device according to one of claims 1 to 16 is used as insertion device.

20. The method according to claim 19, characterized in that step (c) takes place with the aid of the insertion rod (38) according to claim 17.