PRESERVATION OF NUCLEIC ACID SEQUENCES BY FIXING TISSUES IN BUFFERED FORMALIN PREPARED WITH ACID-DEPRIVED FORMALDEHYDE

Abstract

The invention relates to a method of preservation of nucleic acid sequences in histological tissues which comprises: treating a concentrated formaldehyde solution in water with basic ion-exchange resins; diluting the resulting acid-deprived formaldehyde solution with phosphate buffer pH 7.2-7.4 up to a concentration ranging between 2 and 4%; contacting the resulting acid-deprived formaldehyde solution obtained with the tissue samples; optionally embedding the samples fixed in paraffin.

Claims

1. A method of preservation of nucleic acid sequences in histological tissues which comprises: a) treating a concentrated formaldehyde solution in water with basic ion-exchange resins; b) diluting the acid-deprived formaldehyde solution obtained from step a) with phosphate buffer pH 7.2-7.4 up to a concentration ranging between 2 and 4%; c) contacting the acid-deprived formaldehyde solution obtained from step b) with the tissue samples; d) optionally embedding the samples fixed in step c) in paraffin.

2. A method according to claim 1, wherein the concentrated formaldehyde solution has a concentration of 40% by weight.

3. A method according to claim 1, wherein the concentration of the acid-deprived formaldehyde solution is 4% by weight.

4. A method according to claim 1, wherein the ion-exchange resin is an Amberlyst A21 resin.

5. A method according to claim 1, wherein the samples are treated with the acid-deprived formaldehyde solution for a time ranging between 3 and 72 hours.

6. Method of fixing histological and cytological samples, said method comprising contacting a tissue sample with a 4% by weight acid-deprived formaldehyde solution in phosphate buffer pH 7.2, and fixing said tissue sample.

7. The method according to claim 6, wherein said tissue sample is DNA or RNA.

8. A method of analyzing DNA or RNA, said method comprising contacting said DNA or said RNA with a 4% by weight acid-deprived formaldehyde solution in phosphate buffer pH 7.2.

Description

DESCRIPTION OF THE INVENTION

[0009] The purpose of the invention is therefore to propose an approach designed to improve the genetic integrity of organic tissue samples fixed with formalin, since the fixation with PFB currently in use gives disappointing results, as described above.

[0010] It has now been discovered that when the commercial formaldehyde solution is deprived of acids using ion-exchange resins, thereby eliminating the formation of sodium formate, fixation in the resulting acid-free reagent (Acid-Deprived, Phosphate-Buffered Formalin=AD-PBF) gives rise to better preservation and lower fragmentation of nucleic acids, especially DNA, than is the case when commercial phosphate-buffered formalin-fixed tissues (PBF) are used. The improvement was markedly significant in AD-PBF-fixed paraffin-embedded tissues stored for a long time.

[0011] The subject of the invention is therefore a preservation method for nucleic acid sequences in histological tissues and cytological samples which comprises: [0012] a. treating a concentrated solution of formaldehyde in water with basic ion-exchange resins; [0013] b. diluting the acid-deprived formaldehyde solution obtained from step a) with phosphate buffer pH 7.2-7.4 to a concentration ranging between 2 and 4%, preferably to a concentration of 4%; [0014] c. placing the acid-deprived formaldehyde solution obtained from step b) in contact with the tissue samples; [0015] d. optionally embedding the fixed samples from step c) in paraffin. The concentrated formaldehyde solution used in step a) is available on the market, and has a concentration ranging between 30 and 40% by weight.

[0016] Any basic resin able to neutralise the acids present in the formaldehyde solution and prevent their formation can be used as ion-exchange resin. An example of a resin suitable for said purpose is Amberlyst A21® resin.

[0017] Histological and cytological samples are typically treated with the acid-deprived formaldehyde solution for a time ranging between 3 and 72 hours.

[0018] The following examples illustrate the invention in greater detail.

Example 1

[0019] 40% formaldehyde solutions were obtained on the market (Sigma-Aldrich, Milan; Carlo Erba, Milan). The pH of said solutions ranged between 2.6 and 2.9. Amberlyst resin A21 (Dow Chemicals, Milan), a basic ion-exchange resin, was washed with H2O, after which 10 g of said resin was added to 100 ml of 40% formaldehyde. Said mixture was stirred for 60 min. at room temp., and then filtered. The pH of the filtrate ranged between 6.8 and 7.3. The filtrate was mixed at the ratio of 1:10 in phosphate buffer pH 7.2, and an acid-deprived 4% formaldehyde solution in phosphate buffer (AD-PBF) was obtained.

[0020] Fresh human tissues (kidney, liver, colon, colon carcinoma and breast carcinoma), destined for disposal because they were superfluous to diagnostic requirements, were used for fixation. Adjacent sections of tissue fragments were fixed in AD-PBF (see above) and commercial buffered formalin (DiaPath, Bergamo). The tissues remained in their respective fixatives for 20 hours at room temp., and were then processed for embedding in paraffin (Leica embedding apparatus: Leica ASP 300 S).

[0021] The paraffin-embedded tissue blocks were cut to obtain sections stained with haematoxylin-eosin. For the extraction, quantitation and evaluation of DNA and RNA quality, nine sections (thickness 5 μm) were obtained from paraffin-embedded tissue blocks of 10 tissues (see above) fixed in parallel in AD-PBF and PBF. The sections were deparaffinised with 1 ml of xylene. After overnight incubation at 56° C. with proteinase K, the DNA was isolated from five sections using the MagCore Genomic DNA FFPE kit on the MagCore automatic extraction instrument (RBC Bioscience, Taiwan), according to the manufacturer's protocol. The RNA was obtained by using the remaining four sections with the RecoverAll total nucleic acid isolation kit for FFPE (ThermoFisher Scientific, USA), according to the manufacturer's protocols. Both DNA and RNA extracts were quantified by Qubit BR assay on a Qubit Fluorometer (Invitrogen, Carlsbad, Calif., USA) and NanoDrop Spectrophotometer (ThermoFisher Scientific). DNA and RNA integrity was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, USA).

[0022] DNA integrity was evaluated with the high-sensitivity DNA analysis kit (Agilent Technologies, Santa Clara, Calif.) on DNA HS chips. The samples were diluted to 2 ng/μL, and DNA length analysis was conducted according to the manufacturer's instructions. The average size of the DNA fragment of the AD-PBF and PBF samples was evaluated using 5000 nt as threshold for the longest DNA fragments (>5000 nt). Their distribution relative to said threshold was compared statistically with the Chi-square test.

[0023] RNA integrity was evaluated with the Agilent RNA 6000 nano kit. The size distribution of the DNA fragments was calculated from the readings of the Agilent 2100 Bioanalyzer, using smear analysis with a threshold of 200 nt; the percentage of DNA fragments with a size >200 nt (DV200 metric) was recorded.

[0024] FIG. 1 compares the DNA extracted from tissue fixed in PBF or AD-PBF. Biopsies obtained in parallel from the same colon (left) and breast (right) carcinoma sample were fixed in PBF or AD-PBF, and the DNA extracted was analysed with the Agilent Bioanalyzer. The image shows the size of the DNA fragments obtainable in decreasing order (colour intensity scale as shown in the sidebar). The presence of DNA of larger size, and therefore less fragmented, is evident in the biopsies fixed with AD-PBF.

[0025] As shown in FIG. 1, in tissues fixed in PBF, the size of the vast majority of the DNA fragments obtainable (by analogy with the findings described in the literature) ranges between 1000 and 5000 bp, indicating intense fragmentation. Conversely, fixation in AD-PBF, and therefore removal of acid radicals from the fixative, gives rise to fragments which are much better preserved up to 20,000 bp.

Example 2

[0026] Tissues fixed in AD-PBF and, in parallel, in PBF and embedded in paraffin, were stored at room temperature for 12 months, after which the analysis procedure of Example 1 was repeated.

[0027] The DNA extracted from the tissues was analysed with the Agilent Bioanalyzer apparatus.

[0028] FIG. 2 shows the DNA extracted from the same colon carcinoma sample fixed in PBF or AD-PBF, embedded in paraffin, and stored for a year. The extracted DNA was analysed with the Agilent Bioanalyzer. The curves show the size of the DNA fragments obtainable as the size increases. The fact that longer DNA fragments were present in the biopsies fixed with AD-PBF (B) than in those fixed with PBF (A) clearly appears.

Example 3

[0029] In order to check the preservation of nucleic acids, and specifically of DNA, in tissues fixed alternatively in Phosphate buffered Formalin and in AD Formalin, a study was conducted in 27 cases of human cancers (colon, breast and lung cancers). Specimens (approximate size: 1×2×0.3 cm) were collected fresh from the tissues and fixed in parallel in Acid-Deprived (A-D) Formalin, buffered at pH 7.2 with Phosphate Buffer 0.1 M and in a Phosphate buffered Formalin (PBF) from the commerce (Roti-Histofix 4.5 acid free (pH 7) phosphate-buffered formaldehyde solution, Prodotti Gianni, Milan, Italy). The specimens were immersed in the alternative fixatives for 24 h., at room temp., then processed routinely for paraffin embedding.

[0030] Section from the paraffin blocks (10 sections, 5 micron thick) were processed for DNA extraction, then analyzed for assessing the size of the fragments, matching in each case the size of base-pair fragments. The direct comparison was represented either in lines (matching size vs frequency) and using the Kolmogorv-smirnoff test to evaluate the lines tendency or, alternatively, Box plots (see FIGS. 3-5) featured in 3 different families of base-pair fragments (0-5000, 5000-20000, >20000). The data were statistically analyzed with paired tests.

[0031] The results clearly indicate that tissue fixation in PBF results in a higher fragmentation of DNA, since in tissues fixed in AD Formalin there is a higher number of fragments longer than 5000 bp. The data indicate that tissues fixed in AD Formalin are more fit for a successful DNA analysis of tumor tissues, permitting a more proper definition of the theragnostic features.

BIBLIOGRAPHY

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