Paraffin Treasures: Do They Last Forever?

Introduction

Tissue samples are routinely fixed with buffered formaldehyde and embedded in paraffin to preserve tissue architecture and cell morphology. Formalin fixation and paraffin embedding have been the clinical standard for preserving these samples. Once the diagnostic process is over, formalin-fixed paraffin-embedded tissues (FFPE) can be stored for a long period of time. The large amount of paraffin blocks stored in Pathology Departments have become valuable sources of material for both diagnostic and research purposes, and are currently being integrated into tissue and tumor banks. Obtaining nucleic acids from FFPE tissues is challenging, as formaldehyde (the main constituent of formalin) creates cross-linkage between nucleic acids and proteins, which could be limiting for further use of the nucleic acids. Moreover, the integrity of nucleic acids can be damaged by pre-fixation procedures, fixation related factors, post-fixation parameters, and the subsequent storage.^1–4

To evaluate nucleic acid degradation over time, we have performed a comparison between the quality of DNA obtained from FFPE samples shortly after the processing (less than 1 month) and after 8 years of storage in standard conditions (room temperature). DNA extraction from FFPE tissue requires special protocols because the material is often scarce and degraded. However, FFPE tissues have several advantages over fresh or frozen tissue samples, i.e., they are easy to handle and long-term storage is relatively inexpensive.

Material and Methods

Results and Discussion

In 38 out of 43 cases, assessment of DNA quality showed that DNA extracted in 2002, that had been kept frozen at −20°C, was of higher integrity than the DNA extracted from the same samples in 2010 (8 years later) (Table 1 and Fig. 1), and only in five cases was the same amplifiable length of nuclear DNA obtained (400 bp). The difference between the quality of the DNA from both groups of samples was statistically significant (p<0.001). Similar results have been reported comparing recently prepared versus 5 year-old FFPE breast cancer cells. ⁵

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FIG. 1.

(A) Analysis of DNA quality in four representative cases extracted in 2002 (lanes 1, 3, 5, and 7) and in 2010 (lanes 2, 4, 6, and 8). Lane 9 corresponds to a negative control with no DNA (C-), lane 10 corresponds to a positive control showing amplification of all segments (C+) and lane 10 is the molecular weight marker (M). (B) Analysis of DNA quality in three cases performed immediately after sample acquisition in 2005 (upper panel), and reassessed at present with the remaining DNA that had been stored frozen (middle panel), and with new DNA extractions (lower panel).

We further compared DNA quality variation by analyzing three additional cases, in which DNA quality assessment had been performed immediately after sample acquisition (2005). We reassessed at present (2014) the quality of the remaining DNA that had been stored frozen and compared it also with a new DNA extraction. As shown in Figure 1B, no significant differences were observed when comparing the amplifiable length of nuclear DNA in 2005 and at present, indicating that although some degradation may have occurred during these 9 years, it had not affected significantly the suitability of this DNA to undergo molecular studies.

Analysis of molecular biomarkers or genetic alterations by PCR with DNA from FFPE samples, such as T-cell receptor or immunoglobulin heavy or light chain rearrangements in cases with a suspicion of lymphomas, or gene mutations in several types of solid tumors or hematologic neoplasms, is usually optimized by analyzing amplicons not larger than 300–400 bp. Nevertheless, it is important to have the best possible quality of DNA. New markers are being rapidly developed, and the analysis of old samples may be important in daily practice in making clinical decisions. In these cases, we sometimes face problems in amplifying and obtaining an informative result due to low DNA quality from stored FFPE samples. Similar observations have been made regarding RNA extracted from FFPE tissues, which is usually heavily degraded due to its higher instability, regardless of the extraction protocols used. Results suggest that RNA should be isolated preferably within 1 year after tissue embedding (i.e., before fragmentation during storage of FFPE samples becomes limiting for performance of the RNA in enzymatic assays).^6,7

In summary, although it has been demonstrated that DNA degrades over time, a comparison of DNA extracted from human biopsies at two different time points had not been reported. We have shown that DNA obtained after several years from FFPE tissues is, in the majority of cases, of lower quality in terms of amplifiable length of nuclear DNA by PCR, and only a minority of these samples are still equally amplifiable. Frozen tissue still remains the best source of high-quality nucleic acids and should be maintained as possible at the institutional biorepository. However, when no frozen tissue is available and it is suspected that a molecular analysis may be necessary in the future, it would be important to obtain DNA shortly after the diagnostic process is over and preserve it in the frozen state, to assure that its quality and usefulness can be preserved over time.