FIXATIVE COMPOSITION FOR SAMPLES OF BIOLOGICAL MATERIAL

Abstract

Object of the invention is a novel fixative composition for samples of biological material that is non-toxic and that can also easily and safely be used in big quantities.

Claims

1. Fixative composition for samples of biological material comprising, as essential components, formaldehyde and paraffin.

2. The fixative composition according to claim 1, wherein said paraffin is in oily liquid form and insoluble in water.

3. The fixative composition according to claim 1, further comprising an aqueous solution, wherein said formaldehyde is present in said aqueous solution.

4. The fixative composition according to claim 3, wherein said aqueous solution is buffered.

5. The fixative composition according to claim 1, further comprising a buffered aqueous solution that includes said formaldehyde and wherein said fixative composition comprises said buffered aqueous solution that includes said formaldehyde, paraffin in liquid form, and at least one of agar and agarose.

6. The fixative composition according to claim 1, further comprising a buffered aqueous solution that includes said formaldehyde and wherein said fixative composition comprises said buffered aqueous solution that includes said formaldehyde, paraffin in liquid form, and cetyl alcohol.

7. The fixative composition according to claim 1, further comprising a buffered aqueous solution that includes said formaldehyde and wherein said fixative composition comprises said buffered aqueous solution that includes said formaldehyde, paraffin in liquid form, and cetyl palmitate.

8. The fixative composition according to claim 3, characterized in that said formaldehyde in said aqueous solution and said paraffin in liquid form are present in a ratio ranging from 70:30 to 95:5.

9. The fixative composition according to claim 8, wherein said ratio is 90:10.

10. The fixative composition according to claim 5, characterized in that said at least one of agar and agarose is present in amounts between 0.1 and 0.2% with respect to the total weight of the fixative composition.

11. A container for the collection of samples of biological material prefilled with the fixative composition of claim 3.

12. A container for the collection of samples of biological material prefilled with the fixative composition of claim 5.

13. (canceled)

14. A method for preserving samples of biological material, said method comprising placing a sample of biological material in the fixative composition of claim 1.

15. The method as in claim 14, wherein said formaldehyde is present in a buffered aqueous solution.

16. The method as in claim 14, wherein said formaldehyde is present in an aqueous solution and wherein said fixative composition comprises said buffered aqueous solution of said formaldehyde, paraffin in liquid form, and at least one of agar and agarose.

17. The method as in claim 14, wherein said formaldehyde is present in an aqueous solution and wherein said fixative composition comprises said buffered aqueous solution of said formaldehyde, paraffin in liquid form, and cetyl alcohol.

18. The method as in claim 14, wherein said formaldehyde is present in an aqueous solution

Description

[0039] The present invention will be now described in more detail based on the experimental part reported here below, given by way of example only and non-limiting the invention.

[0040] A comparative study has been made between the composition according to the known art, provided as a filler of the containers for the collection of samples of biological material according to the present invention, and a base of formaldehyde only in water (hereinafter referred to as F) and the composition according to the invention, comprising formaldehyde, water and paraffin oil (referred to as FS).

[0041] The comparative study has been made in order to assess possible fixative variables of the two compositions according to defined time slots, in particular to assess the fixative properties of the composition according to the known art compared to those of the composition according to the invention.

[0042] COMPOSITION ACCORDING TO THE KNOWN ART (F): buffered neutral formalin

[0043] COMPOSITION ACCORDING TO THE PRESENT INVENTION (FS): buffered neutral formalin plus paraffin oil

[0044] This study has been made in order to assess the fixative properties of the composition according to the invention (FS) in comparison with the fixation with traditional method (buffered neutral formalin F).

Comparative Tests of Type 1

[0045] The test provided for a comparative study between the fixation characteristics of the two compositions listed below: [0046] fixation with buffered neutral formalin (composition) (F); [0047] fixation with the composition according to the invention (buffered neutral formalin+paraffin oil) (FS);

Characteristics to be Tested:

[0048] Dispersion of formalin into the environment; [0049] Slowing down of the fixation time; [0050] Possible interferences due to the addition of paraffin oil;

Materials and Methods:

[0051] Sampling and fixation of hepatic bovine tissue; [0052] Fixation steps defined in: 6 hours; 24 hours; 48 hours; 72 hours; [0053] Subsequent processing, prior to fixation in the above listed steps, of the hepatic withdrawals with Donatello processor in LaboPath; [0054] Sample inclusion in the control unit Canova at LaboPath; [0055] Cut of sections in paraffin with the Galileo Auto microtome in LaboPath; [0056] Microscope examination.

20 Apr. 2015

Container Configuration

[0057] Eight 250 ml containers (pre-filled at 150 ml) containing buffered neutral formalin, pH 7.2-7.4 (composition according to the known art, called composition F) (FIG. 1), have been used.

[0058] It was decided to add 20 ml paraffin oil in each of four of the aforementioned eight containers, so obtaining the composition according to the present invention (composition FS) (FIG. 2)

Samples Used

[0059] Hepatic bovine tissue.

[0060] 8 withdrawals (one per each container), so to be able to work in double to assess possible differences, have been carried out.

Withdrawal Size(FIG. 3)

[0061] length: 3 cm

[0062] width: 2 cm

[0063] thickness: 4 mm

Timing

[0064] The 8 hepatic withdrawals have been simultaneously dipped in the containers containing neutral formalin pH 7.2-7.4 (FIG. 4) (F1, F2, F3, F4 compositions) and neutral formalin pH 7.2-7.4+paraffin oil (FIG. 5) (FS1, FS2, FS3, FS4 compositions) at 10:15 of 20 Apr. 2015.

[0065] Ist processing: 6 h after dipping;

[0066] IInd processing: 24 h after dipping;

[0067] IIIrd processing: 48 h after dipping;

[0068] IVth processing: 72 h after dipping.

Description of the Phenomenon

[0069] The withdrawal remains in suspension for a few seconds in dipping the sample in the can containing the composition FS according to the invention. During this permanence it becomes wet in oil and, subsequently, spontaneously precipitates towards the bottom of the container. In this step it can be observed how the oil is detaching from the tissue and coming back to the surface.

[0070] The tissue is rinsed out of the oil in the dipping step in formalin. In this step, the fixation times listed above have been respected. The tissue sample is again oily following the contact with the paraffin oil occurring during the extraction of the same from the container.

Processing Protocol

[0071] Keeping in mind the different fixation times inside the containers, processing has been started from water.

[0072] The reagent OTTIX PLUS is currently marketed by Diapath S.p.A.

[0073] OTTIX PLUS is a reagent ready for use, non-toxic, free from aromatic solvents.

[0074] It is able to simultaneously act as a dehydrating and clearing agent in all of the pathological anatomy protocols and is a substitute both of alcohols and solvents, such as xylene and/or toluene.

[0075] It allows carrying out the staining with guaranteed results and equivalent to the traditional method in a completely safe way. It is classified as an in vitro diagnostic device.

[0076] Ottix Plus contains: [0077] EXXSOL DSP 100/140 IN BULK [0078] Ethanol [0079] Propan-2-ol

[0080] WATER10 min

[0081] 70% ALCOHOL45 min

[0082] 95% ALCOHOL1 h

[0083] 99% ALCOHOL3 h

[0084] OTTIX PLUS1 h

[0085] OTTIX PLUS1 h

[0086] OTTIX PLUS1 h

[0087] PARAFFIN1 h

[0088] PARAFFIN1 h and 30 min

[0089] PARAFFIN1 h and 30 min

F1+FS1

[0090] The processing has been started at 16:15 and has duration of 11 h and 55 min stationing in the last paraffin.

21 Apr. 2015

[0091] After processing, the samples have been included. (FIGS. 6-10)

[0092] 2 and 3 sections which, placed on microscope slides, have been dried in oven at 70 C. for 30 min and stained according to Hematoxylin-Eosin staining protocol in use, have been cut.

Hematoxylin-Eosin Staining Protocol

[0093] Ottix Plus 10 min

[0094] Ottix Plus 10 min

[0095] Ottix Plus 5 min

[0096] 99% Alcohol 10 min

[0097] 95% Alcohol 5 min

[0098] 70% Alcohol 3 min

[0099] 50% Alcohol 3 min

[0100] Distilled water 3 min

Mayer Hematoxylin 5 Minutes

[0101] Tap water 5 minutes

Eosin 2 Minutes

[0102] Tap water 3 minutes

[0103] Distilled water 1 min

[0104] 95% Alcohol 3 min

[0105] 95% Alcohol 3 min

[0106] 99% Alcohol 5 min

[0107] Ottix Plus 3 minutes

[0108] Ottix Plus 5 min

[0109] Ottix Plus 5 min

[0110] On the tissue section present on the microscope slide, after having carried out the aforementioned staining protocol, the strut is dispensed and the microscope slide applied.

[0111] The observation with an optical microscope follows.

F2+FS2

[0112] After the fixation (24 h), the F2 and FS2 samples have been processed following the same protocol.

22 Apr. 2015

[0113] After processing, the samples have been included. (FIGS. 11-15)

[0114] 2 and 3 sections which, placed on microscope slides, have been dried in oven at 70 C. for 30 min and stained according to Hematoxylin-Eosin staining protocol in use, have been cut. (FIG. 16)

Results

[0115] Adequate fixation in both withdrawals.

F3+FS3

[0116] After the fixation (48 h), the F3 and FS3 samples have been processed.

23 Apr. 2015

[0117] After processing, the samples have been included in paraffin. (FIGS. 17-21)

[0118] 2 and 3 sections which, placed on microscope slides, have been dried in oven at 70 C. for 30 min and stained according to Hematoxylin-Eosin staining protocol, have been cut. (FIG. 22)

Results

[0119] Adequate fixation in both withdrawals.

F4+FS4

[0120] After the fixation (72 h), the F4 and FS4 samples have been processed following the same protocol.

24 Apr. 2015

[0121] After processing, the samples have been included. (FIGS. 23-27)

[0122] 2 and 3 sections which, placed on microscope slides, have been dried in oven at 70 C. for 30 min and stained according to Hematoxylin-Eosin staining protocol, have been cut. (FIG. 28)

Results

[0123] Adequate fixation in both withdrawals.

Conclusions

[0124] The fixation of the hepatic tissues, carried out with the two compositions (composition F according to the known art and composition FS according to the present invention) object of the comparative tests, has been tested according to different fixation times: 6 h, 24 h, 48 h, 72 h.

[0125] On the tissue samples treated with the two compositions, macroscopic differences are appreciable in 6 hours: the FS1 tissue is less fixed in the center (phenomenon of delayed fixation).

[0126] On the tissue samples treated with the two compositions in 24-48-72 hours, neither macroscopic nor microscopic differences are appreciable.

[0127] The hypothesis that the paraffin oil, for its intrinsic hydrophobicity characteristics, could delay the standard fixation being of about 1 mm/hour for the first three hours, occurred in the samples (FS1) fixed in the first 6 hours.

[0128] The above described phenomenon of the spontaneous rinsing of the post-dipping surgical sample, after briefly staying in the oily phase, de facto clears the film that was thought to be able to permanently isolate the sample from the fixative means. The different weight of the oil compared to the aqueous phase makes the oil droplets to gradually come back to the surface, thus freeing the tissue in the fixative means which it has been dipped in.

[0129] This influences the penetration of the formalin in the first 6 hours, whereas the phenomenon of delayed fixation in the subsequent tests (24-48-72 hours) looks less evident.

[0130] The carried out tests demonstrated that the composition according to the present invention is inert, doesn't create problems to the biological samples, has no Risk Phrases different or in addition to those of the buffered neutral formalin according to the known art.

Comparative Tests of Type 2

[0131] Comparative Study Among Buffered Neutral Formalin (Composition F) and Formalin with the Addition of Paraffin Oil-Agar Mixture (Composition FS-A).

Assessment of Fixation Variables of the Two Compositions in Defined Time Slots.

[0132] Polymerization of the paraffin oil.

[0133] Gelling of the paraffin oil by using Agar 0.1 and 0.2%.

[0134] The purpose of these further comparative tests is to assess the following aspects: [0135] Possibility to give to the oil layer a structure, so that to reduce the contact with the biological sample dipped in the container, in order to avoid phenomena of delayed fixation (consequent hypo-fixation if the sample was dipped for 6 h only) [0136] Possibility to further minimize the evaporation and the perception of the typical pungent odor of formalin.

[0137] This study is carried out in order to assess the fixative properties of the formalin, with the addition of a Paraffin oil-Agar mixture (composition according to the present invention), in comparison with the fixation through traditional method (buffered neutral formalin, composition according to the known art).

Comparative Tests of Type 1

[0138] The test provided for a comparative study between the fixation characteristics of the two compositions and precisely: [0139] fixation with buffered neutral formalin (composition F according to the known art); [0140] alternative fixative composition (buffered neutral formalin+Paraffin oil-Agar mixture, FS-A composition according to the present invention).

Characteristics to be Tested:

[0141] Dispersion of formalin into the environment; [0142] Slowing down of the fixation time; [0143] Possible interferences due to the addition of the Paraffin oil-Agar mixture.

Materials and Methods:

[0144] Sampling and fixation of hepatic porcine tissue; [0145] Fixation steps defined in: 6 hours; [0146] Subsequent processing, prior to fixation, of the hepatic withdrawals with Donatello processor; [0147] Sample inclusion in the control unit Canova; [0148] Cut of sections in paraffin with the Galileo Auto microtome; [0149] Microscope examination.

Container Configuration:

[0150] Three 150 ml containers (pre-filled at 90 ml) containing buffered neutral formalin (according to the known art), pH 7.2-7.4, have been used. (FIGS. 29-31)

[0151] It was decided to add the Paraffin oil-Agar mixture in two containers in different amounts, by varying the ratio with the formalin and keeping the same final filling volume (90 ml). [0152] 40 ml formalin+50 ml Oil-Agar mixture (FIG. 30); [0153] 50 ml formalin+40 ml Oil-Agar mixture (FIG. 31).

Samples Used

[0154] Hepatic porcine tissue.

[0155] 3 withdrawals (one per each container), so to be able to work in triple to assess possible differences, have been carried out.

Withdrawal Size: (FIG. 32)

[0156] Length: 2 cm;

[0157] Width: 1 cm;

[0158] Thickness: 4 mm.

Timing

[0159] The 3 hepatic withdrawals have been simultaneously dipped in the containers containing 90 ml neutral formalin pH 7.2-7.4, 50 ml neutral formalin pH 7.2-7.4+40 ml Paraffin oil-Agar mixture and 40 ml neutral formalin pH 7.2-7.4+50 ml Paraffin oil-Agar mixture (FIG. 33).

[0160] Processing: after 6 h from dipping

Description of the Phenomenon

[0161] The withdrawal remains in suspension in dipping the sample in the can with the Oil-Agar mixture and it is necessary, with the aid of the pliers, to push the withdrawal towards the bottom of the container. In this step, the withdrawal becomes wet in Paraffin oil-Agar but, once on the bottom of the container, it can be observed how the composition is detaching from the tissue and comes back to the surface.

[0162] The tissue is rinsed out of the Oil-Agar mixture in the dipping step in formalin. In this step, the fixation times listed above have been respected. The tissue sample is again oily following the contact with the paraffin oil during the extraction of the same from the container.

N.B:

[0163] The hepatic tissue sample (FIG. 37, containers 2 and 3 from the left) is oily following the contact with the paraffin oil occurred during the extraction of the same from the container.

Processing Protocol:

[0164] Keeping in mind the fixation times inside the containers, the processing step has been started from water.

[0165] WATER10 min

[0166] OTTIX SHAPER1 h

[0167] OTTIX PLUS1 h

[0168] OTTIX PLUS1 h and 30 min

[0169] OTTIX PLUS1 h and 30 min

[0170] OTTIX PLUS2 h

[0171] PARAFFIN1 h and 30 min

[0172] PARAFFIN2 h

[0173] PARAFFIN2 h

[0174] The processing has duration of 12 h and 40 min stationing in the last paraffin.

17 Jul. 2015

[0175] After processing, the samples have been included. (FIGS. 38-45)

[0176] 2 sections which, placed on microscope slides, have been dried in oven at 70 C. for 30 min and stained according to Hematoxylin-Eosin staining protocol, have been cut. (FIGS. 46-47)

Conclusions

[0177] The fixation of the hepatic tissues carried out with the two compositions object of the comparative tests, formalin (according to the known art) and formalin+Paraffin oil-Agar mixture (according to the present invention), has been tested with fixation times of 6 h (critical time highlighted in the previous tests with paraffin oil-formalin only).

[0178] In this case, in the tissue samples fixed with the two mixtures in 6 hours, macroscopic and microscopic differences are not appreciable.

[0179] Consequently, the composition according to the present invention of formalin, paraffin oil and Agar was found particularly suitable to be used in the fixation processes of samples of biological material requiring short fixation times, around about 6 hours.