Application of alternative fixatives to formalin in diagnostic pathology

Abstract

Fixation is a critical step in the preparation of tissues for histopathology. The aim of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine stainings), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved to be the best fixative for morphology. The morphology obtained with Bouin was comparable to the one with formalin. Hollande was the best fixative for histochemistry. Bouin proved to be equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved the possibility to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

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“…The MKI67 and p53 labeling indices were established as the percentage of tumoral cells with positive nuclei. For the grading, a mitotic count O2 was considered positive and the threshold for MKI67 index positivity set at O1% rather than O3%, taking into account the use of Bouin-Hollande fixative solution that results in a lower sensitivity for the MKI67 antibodies than formalin (16,17). For p53, detection was considered as positive if there were more than ten strongly positive nuclei per 10 HPF (HPF of 0.30 mm 2 ; 400!…”

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“…However, Bouin's solution has very low pH, contains picric acid, which can be explosive, and is sensitive to friction and shock when dry. Further, a recent study showed that fixation in Bouin's solution did not provide IHC results comparable to those obtained with formalin . Fixatives containing mercuric chloride like Zenker's fixative and B5 were also widely shown to be suitable for IHC studies , but are toxic; their disposal as hazardous waste is costly, and these reagents are not allowed in many countries at this time.…”

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“…Some of the frequently encountered issues include the techniques having a high cost, being labor‐intensive, and yielding less than adequate cellular material for diagnosis or ancillary testing. Some techniques use fixatives other than formalin, which may interfere with immunohistochemical (IHC) studies . The automated method currently available is expensive and time‐consuming, produces limited numbers of slides, and uses alcohol as the fixative.…”

“…Some techniques use fixatives other than formalin, which may interfere with immunohistochemical (IHC) studies. 8 The automated method currently available is expensive and time-consuming, produces limited numbers of slides, and uses alcohol as the fixative. To resolve these issues, we developed a formalin-fixed, paraffin-embedded cytology cell block technique, AFFECT, to make cell blocks.…”