Critical Steps in Tissue Processing in Histopathology

Abstract

Histopathological diagnosis using Formalin-Fixed Paraffin Embedded (FFPE) tissues is essential for the prognostic and therapeutic management of cancer patients. Pathologists are being confronted with increasing demands, from both clinicians and patients, to provide immunophenotypic and gene expression data from FFPE tissues to allow the planning of personalized therapeutic regimens. Recent improvements in the protocols for pre-analysis processing of pathological tissues aim to better preserve cellular details and to conserve antigens and nucleic acid sequences. These developments have been recently patented. The international protocol for the transporting of surgical specimens from the surgical theatre to the pathology department is to immerse the specimen in formalin. The alternative method of sealing the specimens into bags under a vacuum and then cooling is a well-accepted and environmentally safe procedure that overcomes the many drawbacks linked to transfer in formalin. Importantly, RNA is notoriously poorly preserved in FFPE tissue. Due to this, successful procedures for the extraction of genetic information from archival tissues have been the object of several studies and patents. Novel molecular approaches for RT-qPCR and gene array analysis on FFPE tissues are presented here. Moreover, a major advance is reported in this study, the observation that tissue fixation in cold conditions allows a much better preservation of nucleic acid sequences.

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

“…Since the adoption at our institution of an innovative method for tissue collection, transport and storage of surgical specimens based on VPAC technology [5], [7], [8], we have demonstrated in these specimens optimal tissue morphology as well as integrity of antigens for ICC and excellent preservation of nucleic acids to perform molecular analyses [6], [9]. We now show a further implementation of VPAC technology.…”

“…Our results suggest the avoidance of insulating air around tissues with the VPAC system allows faster cooling at 4°C. From our experience on nucleic acid preservation [7], [9] it seems that it is the prompt cooling that principally influences preservation. This has also been confirmed by independent observations [27] that show that storage at 4°C preserved tissues to a higher degree than storage at room temperature, independently of whether the tissue was subjected to vacuum sealing or not.…”

“…Such a procedure allows for having fresh (i.e. not fixed) tissues and it has been demonstrated not to affect morphology and to best preserve nucleic acids (DNA and RNA) and proteins [6], [7], [9].…”

A Review of Preanalytical Factors Affecting Molecular, Protein, and Morphological Analysis of Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue: How Well Do You Know Your FFPE Specimen?

“…24,54,64,67 However, there is some disagreement as to whether a shorter fixation duration of 6 hours produced inferior 61,66 or comparable 57,62 ISH and RT-PCR results, and transcript-specific differences in RT-PCR amplicon levels were reported by 2 studies 68,69 for biospecimens fixed for 24 or 48 hours. While fixation at 378C resulted in poor RNA quality compared with ambient fixation, 25 reports conflict as to whether fixation at 48C generated superior 70,71 or equivalent 35 RNA quality and levels of amplifiable RNA compared with fixation at ambient temperatures. The performance of accelerated fixation techniques is method-specific for RNA analyses investigated to date, although reductions in processing time continue to be a shared benefit.…”

Current Challenges for HER2 Testing in Diagnostic Pathology: State of the Art and Controversial Issues

“…However, controlling the time of fixation is a difficult matter, because immersion in formalin of a large surgical specimen does not mean initiation of fixation of a tumor. Our group has successfully explored the under vacuum sealing of large specimens and cooling at 4°C for transport from the surgical theater to the pathology lab as a method that allows monitoring exactly the time of ischemia and of initiation of fixation and guarantees an optimal preservation of antigens (Bussolati et al, 2011; Comanescu et al, 2012). …”