Systematic comparison of tissue fixation with alternative fixatives to conventional tissue fixation with buffered formalin in a xenograft-based model

Abstract

In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene®, HOPE®, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene® was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE® fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE®, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-012-1248-5) contains supplementary material, which is available to authorized users.

Carbonic Anhydrase IX (CAIX) Does Not Differentiate Between Benign and Malignant Mesothelium

“…We hypothesize that this could represent an artifact of cell block preparation and the use of alcohol in the CytoLyt (Cytyc, Boxborough, MA), which could potentially have a deleterious effect on certain immunomarkers, although the rest of the immunohistochemical stains in our study were not affected. 12,13 We also speculate that atypical/malignant mesothelial cells have more rigid cell walls than do benign ones, which make them more resistant to centrifugation and cell block preparation, resulting in better membranous immunostaining for CAIX. We believe that this is a limitation of this study and can be adequately addressed by use of alternative fixatives or cell block preparation methods.…”

A Comparative Study of Fixatives for Axolotl Blastema Tissue

“…AFA is a hybrid fixative and has the favorable features of consisting alcohol such as exceptional antigen conservation for post fixation processes. Moreover, on top of high grade protein conservation, preservation quality of nucleic acids in AFA makes it a commonly used fixative in wide range of histological examinations (Nietner et al, 2012). Our data reveal that AFA is the most suitable fixative for blastema tissue to perform histological and immune-histological staining.…”

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories

“…According to College of American Pathologists (CAP) guidelines, changes in biospecimen processing that can impact testing must be validated. To switch to a new formalin-free fixative, immunohistochemistry procedures for each antibody in a laboratory’s repertoire and diverse molecular assays would need to be revalidated [58]. Furthermore, formalin-free fixatives and lyophilization face the challenge of producing histology equal or superior to that of FFPE in the subjective eyes of demanding pathologists.…”