DNA extraction and molecular analysis of non-tumoral liver, spleen, and brain from autopsy samples: the effect of formalin fixation and paraffin embedding.

The use of molecular biology in combination with morphological analysis is increasing because of the treatments by target therapies. However, to improve the methods for obtaining DNA for molecular analyses from formalin-fixed, paraffin-embedded (FFPE) tissue is a challenge. The aim of this study was to evaluate the DNA extracted from FFPE tissue blocks (non-tumoral liver, spleen, and brain), obtained from autopsy, 8-24 h post mortem, using three methods of DNA extraction. PCR of the ß-actin (136 pb) and human amelogenin (AMEL 212-218 bp/106-112 bp) genes, as well as short tandem repeat (STR) (100-400 bp fragments), reported in forensic scientific analysis, was performed to evaluate the effectiveness of the methods of DNA extraction. We used 28 archived (1 and 5 years) and 12 recent autopsy cases. The commercial kit showed reproducible and consistent results in the PCR amplification of the ß-actin and AMEL genes and in analysis by STR used in forensic analysis. This is the first report using non-tumoral samples from FFPE autopsy tissues, comparing the three most common methods of DNA extraction and using the STR previously described in forensics. Our study has clarified the challenges for pathologists in applying the molecular biology approach in combination with methods suited for morphology, which must be improved. The data provided here should be used in other molecular studies in FFPE samples.

Morphometric analysis of the fate of secretory epithelial cells in the ventral lobe of the rat prostate during massive apoptosis

Orchiectomy causes marked, rapid involution of the prostatic secretory epithelium. Concurrently, macrophages, which in normal glands are small and rarely occur at the base of the secretory epithelium, increase in size and number. Apoptotic cells are engulfed by companion epithelial cells and also by macrophages. In secretory cells and macrophages, dense bodies progressively increase in number and store membranes derived from dead cells of the secretory epithelium. In this work, we examined the contributions of the various routes of disposal of demised secretory epithelial cells of the rat prostate, induced to enter in apoptosis by retrieval of androgen. Specifi cally, we sought to determine how much membrane surface area derived from apoptotic cells of the secretory epithelium could be stored in dense bodies, and how these data compared with the disposal of dead cells via the glandular lumen. Glands from unoperated controls (day 0) and from rats examined 12 h and 1, 2, 3, 4, 5, 6, 7, 8, and 9 days after orchiectomy were studied morphometrically. The total membrane surface area of rough and smooth endoplasmic reticulum, Golgi apparatus, mitochondria and vesicles declined from 6.75 x 103 ìm2 in non-castrated rats to 1.12 x 103 ìm2 nine days after castration. Similarly, the total surface area of the secretory epithelium decreased from 10.6 x 1011 ìm2 in non-castrated rats to 0.204 x 1011 ìm2 nine days after castration. Geometrical models revealed that 1 ìm3 of dense body accommodated at least 142 ìm2 of myelin-like membrane surface area. Three to four days after castration, the total volume of intramacrophage dense bodies peaked (~5 x 106 ìm3) and represented 1-2% of the volume of intraepithelial dense bodies (~4 x 108 ìm3). The minimum membrane surface area that could be stored in dense bodies of the secretory epithelium on post-castration days 0, 1, 2, 3, 4 and 9 was 1.4%, 9%, 16%, 23%, 28% and 44%, respectively, of the total membrane surface area of the...