METHOD FOR THE PREPARATION OF BIOLOGICAL SAMPLES AND COMPOSITION FOR MOUNTING MICROSCOPE SLIDES

Abstract

Subject-matter of the present invention is a method for the preparation of biological, cytological, histological and autopsical samples and a composition for mounting microscope slides. Subject-matter of the invention is also the samples mounted with the compositions described herein.

Claims

1. A method for mounting microscope slides for the analysis of biological, cytological, histological and autopsical samples, comprising dipping said microscope slides into at least one fluid composition comprising: a) at least one liquid component selected from xylene, ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and isoparaffin-based solvents; and b) one or more polymers or copolymers selected from polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polymethyl acrylate and polyethyl acrylate, in an amount of 15%-80%.

2. The method according to claim 1, for mounting microscope slides for the analysis of biological, cytological, histological and autopsical samples, comprising: (i) dipping said microscope slides into at least one fluid composition comprising: a) at least one liquid component selected from xylene, ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and isoparaffin-based solvents; and b) one or more polymers or copolymers selected from polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polymethyl acrylate and polyethyl acrylate, in an amount of 15%-80%; (ii) extracting said microscope slides from said composition; (iii) letting them drip; (iv) optionally repeating the steps from (i) to (iii); and (v) letting them air-dry or drying them with a suitable means.

3. The method according to claim 1, wherein, said one or more polymers (b) are present in an amount of 10-50%.

4. The method according to claim 1, wherein said at least one of said one or more polymers (b) is a methyl methacrylate/butyl methacrylate copolymer.

5. The method according to claim 1, wherein said at least one liquid component (a) is selected from ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and mixtures thereof.

6. A composition for mounting a microscope slide for the analysis of biological, cytological, histological and autopsical samples, comprising: a) a liquid component selected from ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and mixtures thereof; and b) one or more polymers or copolymers selected from polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polymethyl acrylate and polyethyl acrylate, in an amount of 15%-80%.

7. The composition according to claim 6, wherein said one or more polymers (b) are present in an amount of 10-50%.

8. The composition according to claim 7, wherein said one or more polymers (b) are present in an amount of 25-45%.

9. The composition according to claim 6, wherein said at least one of said one or more polymers (b) is a methyl methacrylate/butyl methacrylate copolymer and in that said at least one liquid component is selected from ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and mixtures thereof.

10. (canceled)

11. The method according to claim 1, wherein said composition comprises: a) a liquid component selected from ethyl acetate, butyl acetate, 1-methyl-2-methoxyethyl acetate and mixtures thereof; and b) one or more polymers or copolymers selected from polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polymethyl acrylate and polyethyl acrylate, in an amount of 15%-80%.

12. The method according to claim 1, wherein at least two steps (i) are carried out.

13. The method according to claim 12, wherein the compositions used in said at least two steps (i) have the same concentration.

14. The method according to claim 13, wherein said at least two steps (i) are carried out and in that said compositions have a concentration of about 20%.

15. The method according to claim 12, wherein the compositions used in said at least two steps (i) have different concentrations.

16. The method according to claim 15, wherein said at least two steps (i) are carried out and in that the first composition used has a concentration of about 30% and the second of about 20%.

17. Biological, cytological, histological or autopsical samples mounted with the composition according to claim 6.

18. Biological, cytological, histological or autopsical samples according to claim 17, wherein said samples do not comprise a coverslip.

19-20. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] FIGS. 1 and 2 depict the compositions described herein after drying, at the end of the mounting process.

[0131] FIG. 3 shows the ultra-transparency of the compositions described herein.

[0132] FIG. 4 shows a microscope image representing a biological sample mounted with the compositions described herein.

[0133] FIGS. 5 and 6 show the basket useful in the method of the invention.

EXPERIMENTAL SECTION

EXAMPLE 1

[0134] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0135] An opaque container is filled with 800 ml butyl acetate. 1.05 g n-octadecyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate (antioxidant agent) are added and left under stirring until complete dissolution, i.e. about 2 minutes. At this point, 200 g polymethyl methacrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear homogeneous mixture. Then 10.45 g/ml -methaacryloxypropyltrimethoxysilane (bond enhancer) and 10 g 2-ethylhexyl adipate are added (plasticizer).

[0136] The process is completely carried out at room temperature, the mechanical stirring is stopped only after obtaining a clear homogeneous mixture (about 20 minutes). The final formulation thus appears viscous and particle-free.

EXAMPLE 2

[0137] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0138] For the production of 1 liter of solution, an opaque container is filled with 700 ml xylene. 1.7 g butylhydroxytoluene (BHT) (antioxidant agent) are added and left under stirring until obtaining a clear homogeneous mixture, i.e. about 2 minutes. At this point, 300 g polymethyl methacrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear homogeneous mixture. Then, still at room temperature, 2 g liquid paraffin (anti-scratch agent) and 2.1 g 2-(5-tert-butyl-hydroxyphenyl)benzotriazole (UV absorber) are added. After about 30 minutes under stirring, a clear homogeneous mixture is obtained. The final formulation thus appears viscous and particle-free.

EXAMPLE 3

[0139] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0140] For the production of 1 liter of solution, an opaque container is filled with 600 ml xylene. 2 g n-octadecyl-3-(3,5-di-t-butyl-4-hydroxyphenyl)propionate (antioxidant agent) are added and left under stirring until obtaining a clear homogeneous mixture, i.e. about 2 minutes. At this point, 400 g polybutyl methacrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear homogeneous mixture. Then, still at room temperature, 15 g dibutyl phthalate (plasticizer) are added. After about 30 minutes under stirring a clear homogeneous mixture is obtained, which appears viscous and particle-free.

EXAMPLE 4

[0141] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0142] An opaque container is filled with 1000 ml xylene and 250 ml Diamount. 400 g polybutyl methacrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear, homogeneous, viscous and particle-free mixture.

EXAMPLE 5

[0143] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0144] An opaque container is filled with 800 ml xylene and 240 ml Micromount. 250 g polyethyl methacrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear, homogeneous, viscous and particle-free mixture.

EXAMPLE 6

[0145] Process for the Preparation of an Embodiment of a Composition for the Method According to the Invention

[0146] An opaque container is filled with 800 ml xylene and 100 ml OpticMount. 200 g polyethyl acrylate are added and left under stirring for about 2 hours at room temperature until obtaining a clear, homogeneous, viscous and particle-free mixture.

EXAMPLE 7

[0147] Process for the Preparation of an Embodiment of a Composition of the Invention

[0148] An opaque container is filled with 400 ml ethyl acetate and 400 ml 1-methyl-2-methoxyethyl acetate. 200 g Paraloid B-64 and 1 g BYK-320 (polyether-modified polymethylalkylsiloxane) are added and left under stirring for about 2 hours at room temperature until obtaining a clear, homogeneous, viscous and particle-free mixture.

EXAMPLE 8

[0149] Process for the Preparation of an Embodiment of a Composition of the Invention

[0150] An opaque container is filled with 350 ml ethyl acetate and 350 ml 1-methyl-2-methoxyethyl acetate. 300 g Elvacite 4021 (methyl methacrylate/n-butyl methacrylate copolymer) and 1.5 g BYK-320 (polyether-modified polymethylalkylsiloxane) are added and left under stirring for about 2 hours at room temperature until obtaining a clear, homogeneous, viscous and particle-free mixture.

EXAMPLE 9

[0151] Mounting Process

EXAMPLE 9.1 (COMPARATIVE)

[0152] Conventional Manual Mounting Process

[0153] At the end of the common process of manual staining, ending with alcohol washing, each of the 30 microscope slides underwent the following steps: (I) placing the microscope slide horizontally on the worktop; (II) delivering the proper amount of mounting medium on the microscope slide (about 2 drops); (III) placing the coverslip; and (IV) adjusting the coverslip. Overall time required to mount each microscope slide: about 12 seconds; multiplied for each microscope slide: about 360 seconds (i.e., about 6 minutes).

[0154] To the overall time spent for the conventional mounting of all the microscope slides, it should be added the drying time of the mounting medium, which takes about 20 minutes to be dried acceptably, for a total mounting time of about 26 minutes and 30 seconds.

[0155] Contrary to what has been described above, with the process according to the invention and considering two dippings, it is possible to save time, as it will be clear from the following examples.

[0156] Moreover, as it is clear from the just described example, the four steps reported above required for the conventional mounting, in addition to take a significant amount of time, are absolutely not free from errors or inaccuracy, these being particularly significant features. For this reason, the overall time of the procedure described above is further increased when: (1) the mounting medium is added in excess on the microscope slide and it would be required to remove such an excess; and (2) the mounting medium leaks from the microscope slide and it would be required to wait for the full and complete drying of the same for practical management of the workflow.

[0157] Finally, it is required to wait at least one day after the mounting process for the effective storage of the so-mounted microscope slides, since it is required to wait until they are complete dried.

EXAMPLE 9.2

[0158] Manual Mounting Process by Using the Method of the Invention

[0159] Following the common method of manual staining, ending with the xylene washing (or similar solvents), a basket containing 25 microscope slides is manually dipped into a tank containing the composition according to example 7 for about 1.5 minutes, after which the excess of the composition is removed by dripping for 1 minute and a second dipping into the same composition is carried out for about 1.5 minutes, followed by dripping for about 1 minute. Finally the mounted microscope slides are dried at 50 C. for 15 minutes, for an overall working-time of 20 minutes. Alternatively, the 25 mounted microscope slides can be placed in an oven at 65 C. for about 5 minutes, thus providing a mounting process having an overall time of about 10 minutes.

[0160] Microscope slides mounted in this way are shown in FIGS. 5 and 6.

EXAMPLE 9.3

[0161] Automatic Mounting Process by Using the Method of the Invention

[0162] Following the common staining process by means of an automatic stainer, ending with a xylene washing, 25 microscope slides (just stained) are taken by the automatic arm and dipped for 1.5 minutes into a tank still of the automatic stainer, containing the composition of the example 8. Then, the automatic arm of the automatic stainer transfers the microscope slides into an empty tank, in order to remove the excess of the composition for 1 minute and then they are dipped into a tank containing the composition of the example 7 for 10 seconds and then transferred into an empty tank, in order to remove the excess of the composition for 1 minute and to be completely air-dried, otherwise they are placed in an oven for a shorter drying.

[0163] The so-simplified mounting process of the microscope slides starts immediately after the staining and is completed within few minutes, without involving in any way the user.

[0164] It is clear that the mounting processes contemplating the use of the compositions described herein, i.e. Examples 9.2 and 9.3, provide an important time-saving with respect to the time needed for the conventional mounting, as described in Example 9.1, thus reducing the overall time for the preparation of the biological samples and optimizing the workflow and also getting rid of the problems related to air bubbles, flattening and possible inaccuracy due to the work by the user.