Molecular and Immunohistochemical Characterization of Historical Long-Term Preserved Fixed Tissues from Different Human Organs.

University and museum collections are very important sources of biological samples that can be used to asses the past and present genetic diversity of many species. Modern genetic and immunohistochemical techniques can be used on long-term preserved fixed tissues from museum specimens to answer epidemiological questions. A proof of principle was established to apply modern molecular genetics and immunohistochemical methods to these old specimens and to verify the original diagnosis. We analysed 19 specimens from our university collection including human organs that had been in fixative for more than 80 years. The tissues originated from lung, colon, brain, heart, adrenal gland, uterus and skin. We isolated amplifiable DNA from these wet preparations and performed mutational analysis of BRAF, KRAS and EGFR. The tissues were also embedded in paraffin and used for modern histology and immunohistochemistry. Our data show that amplifiable DNA is extractable and ranged from 0.25 to 22.77 μg of total DNA. In three specimens BRAFV600E or KRASG12D mutations were found. Additionally, expression of different proteins like vimentin and GFAP was detected immunohistochemical in six investigated specimens. On the basis of our results the original diagnosis was altered in three specimens. Our work showed that it is possible to extract amplifiable DNA suitable for sequence analysis from long-term fixed tissue. Furthermore, histology and immunohistochemistry is feasible in specimens fixed long time ago. We conclude that these old preparations are suitable for further epidemiological research and that our methods open up new opportunities for future studies

Five wet preparations after biopsy sampling, newly stored in Jores II solution for further investigations.

<p>Five wet preparations after biopsy sampling, newly stored in Jores II solution for further investigations.</p

Comparison of total DNA amount and purity of analysed specimens.

<p>Comparison of total DNA amount and purity of analysed specimens.</p

Histological and immunohistological staining of wet preparations.

<p>H&E stain (A through D) and immunohistochemical staining (E through H). Weak vimentin expression in adrenal gland of Nn28 (E) and Lu176 (F). Absent TTF1 expression in heart of He146 (G). Strong GFAP expression in brain (H). F, H by 20x objective; A through E and G by 40x objective).</p

Five wet preparations after biopsy sampling, newly stored in Jores II solution for further investigations.

<p>Five wet preparations after biopsy sampling, newly stored in Jores II solution for further investigations.</p

DNA concentration and DNA purity of the different investigated organs.

<p>DNA concentration and DNA purity of the different investigated organs.</p

Immunohistochemical staining results and corresponding IRS^a.

<p>IRS represents the product of score resulting from percentage of positive cells and score resulting from intensity of staining.</p><p>^aIRS, immunoreactive score</p><p>Immunohistochemical staining results and corresponding IRS<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135297#t006fn002" target="_blank">^a</a>.</p

Overview of investigated genes of the different specimens, amplifiability of DNA and also results of mutation analysis.

<p>^a(+) indicates positive amplification.</p><p>^b(-) indicates negative amplification.</p><p>^cwt, wild type</p><p>Overview of investigated genes of the different specimens, amplifiability of DNA and also results of mutation analysis.</p

Overview of specimens, fixation time and their corresponding disease.

<p>Specimens marked in bold showed re-assessed diagnosis after new validation.</p><p>Overview of specimens, fixation time and their corresponding disease.</p