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

Abstract

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.

Fig 1. Five wet preparations after biopsy sampling, newly stored in Jores II solution for further investigations.

Fig 2. Electropherogram shows mutations of KRAS in Da236 (G12D) and BRAF in Ht13 (V600E).

Arrows indicate the mutations.

Fig 3. Histological and immunohistological staining of wet preparations.

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).