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

University of Hohenheim, 70593 Stuttgart, Germany.
Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin (Impact Factor: 2.65). 07/2012; 461(3):259-69. DOI: 10.1007/s00428-012-1248-5
Source: PubMed


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 material
The online version of this article (doi:10.1007/s00428-012-1248-5) contains supplementary material, which is available to authorized users.

Download full-text


Available from: Thorben Nietner, Mar 30, 2015
30 Reads
  • Source
    • "Although not related to storage per se, it should be mentioned that inappropriate and prolonged fixation may damage antigenicity or cause diffusion artefacts, and that these effects may differ from one antigen to another [13]. Of course, the type of fixative also has an important impact on antigenicity [14]. Summarizing, the following factors may have a considerable impact on the quality of immunohistochemistry: "
    [Show abstract] [Hide abstract]
    ABSTRACT: The use of paraffin slides and tissue microarrays (TMA) is indispensable for translational research. However, storage of paraffin slides over time has a substantial detrimental effect on the quality and reliability of immunohistochemistry stains. Particularly affected by this issue may be any collaborative efforts where paraffin slides or TMAs are shipped to central laboratories and then 'biobanked' for some time until use. This article summarizes some of the key issues affecting loss of antigenicity on paraffin slides and some simple storage solutions to help maintain high quality immunohistochemistry results when paraffin slides must be stored for a certain time prior to use.
    Clinical and Translational Medicine 03/2014; 3(1):4. DOI:10.1186/2001-1326-3-4
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Identifying targets for personalized targeted therapy is the pathologist's domain and a treasure. For decades, pathologists have had to learn, understand, adopt and implement many new laboratory techniques as they arrived on the scene. Pathologists successfully integrate the results of those tests into final pathology reports that were, and still are, the basis of clinical therapeutic decisions. The molecular methods are different but no more difficult to comprehend in the era of "kit procedures". In recent years, the development of targeted therapies has influenced routine practices in pathology laboratories because the use of molecular techniques is required to include clinically useful predictive information in the pathology report. Pathologists have the knowledge and expertise to identify particular gene mutations using the appropriate molecular tests currently available. This review focuses on the most important recent developments in KRAS mutation testing in metastatic colorectal cancer (CRC), and shows that a pathologist is involved in 10 stages of this procedure. Recent studies have shown that highly sensitive, simple, reliable and rapid assays may significantly improve the identification of CRC patients resistant to anti-EGFR therapy. Thus, direct sequencing does not seem to be an optimal procedure of KRAS testing for clinical purposes. Twelve currently available high-sensitivity diagnostic assays (with the CE-IVD mark) for KRAS mutation testing are briefly described and compared. The suggested pathology report content for somatic mutation tests is described. In conclusion, evidence is presented that sending away paraffin blocks with tumor tissue for KRAS mutation testing may not be in the best interest of patients. Instead, an evidence-based approach indicates that KRAS mutation testing should be performed in pathology departments, only with the use of CE-IVD/FDA-approved KRAS tests, and with the obligatory, periodic participation in the KRAS EQA scheme organized by the European Society of Pathology as an independent international body.
    Polish journal of pathology: official journal of the Polish Society of Pathologists 11/2012; 63(3):145-64. DOI:10.5114/pjp.2012.31499 · 1.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recent advances in molecular biology and pathology have opened new opportunities for refining our knowledge of pathophysiologic events and biomarkers. Particular interest in applying these novel methods to current and archived tissues collected in experimental and epidemiological/clinical studies is evident. Until now, it has not always been possible to use archived alcohol-fixed paraffin-embedded (AFPE) tissues for immunohistochemisty (IHC), because AFPE slices and blocks were not often amenable to standard IHC methods. In order to solve this problem, we developed a simple method of post-fixation, which allows to use, on AFPE slices, standard IHC protocols already used for formalin-fixed paraffin-embedded (FFPE) samples. For the assessment of post-fixation processing and to test the feasibility of IHC, we selected the spleen from Sprague-Dawley rats as a demonstrative tissue. Antibodies to PAX5, CD3, CD68 and Ki-67, were tested on FFPE, AFPE and AFPE post-fixed spleen samples. The specificity of antibodies to bind different epitopes expressed in spleen tissue was maintained in FFPE and AFPE post-fixed sections, according to anatomical localization. Post-fixation of AFPE samples did not affect tissue morphology and IHC results were comparable to the FFPE sections in terms of sensitivity, specificity and intensity of staining. In addition to providing an opportunity to use archived tissues, this new post-fixation method would dramatically reduce the use of formaldehyde during histopathology procedures, thus minimizing worker exposure to this dangerous carcinogenic substance.
Show more