Editorial
Published: 2022-09-22
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Fixation in histopathology: the mandate to renew

Department of Medical Sciences, University of Turin, Italy
Fixation Formalin Glyoxal GAF Histopathology

 

One and a half century ago Edwin Klebs and Wilhelm His sr. invented the paraffin embedding procedures which pathologists are still using today 1. A few years later, Ferdinand Bloom proposed formaldehyde as the fixative of choice 2.

The need to renew these ancient procedures is dictated by pressing requests for reproducibility, standardization, and respect for the environment to prevent obnoxious exposure of workers. Interest on these topics is enhanced by the fact that in recent times the requirements for tissue preservation are concerning not only the fine structural integrity, but molecular components as well (nucleic acids and protein antigens) to ensure the validity of tests predicting and conditioning the planning of personalized therapies 3.

The fixation step is the single most critical factor in all the histopathological procedure. Its accomplishment (as the name implies) is to realize the block (“fix”) of the structural and molecular components as they were in their living conditions. In histopathology practice, tissues are immersed in formalin (the 1:10 solution of commercial formaldehyde in water, corresponding to a 4% concentration of the reagent), which is known to penetrate at 1 mm/hour, a rate which however varies according to several factors (type of tissue, temperature, pressure, time progression). This implies that small specimens are rapidly and uniformly fixed, while in large tissue blocks the central areas fail to be rapidly reached by the fixative and undergo progressive autolysis.

To prevent this deleterious process, we proposed the adoption of the Under-Vacuum Sealing and Cooling procedure 4 and several reports have confirmed the merits of this procedure in terms of compliance by the personnel and safety for the tissues 5. In addition, the procedure is environmentally sound by preventing exposure to formalin vapors and allows sampling of specimens for tissue banking 6 up to hours after removal from the body.

The mechanism of fixation by formaldehyde has been deeply investigated and can basically be described as “fast penetration, slow fixation”. The chemical mechanism is conditioned by the fact that the 4% formaldehyde solution in phosphate buffer pH 7.2-7.4 (so called “phosphate buffered formalin”, PBF), the formulation now universally employed, has a high osmolarity, but in water solution the active aldehyde reagent is only a fraction (approximately 1/10) and is in equilibrium with methyl glycol, which is inactive. Complete tissue fixation is known to occur after 24-48 h at room temperature, although in small specimens such as core biopsies, a 6 h fixation is regarded as satisfactory.

Caution in the use of formalin has extensively been recommended. The reagent can be allergenic and cause eczemas when in solution, but the main risk is linked to vapors which can be inhaled. The reagent is toxic and carcinogenic and should be abolished, although in EU its use for histological fixation has been temporally permitted (Directive (EU) 2019/983 of the European Parliament and of the Council of 5 June 2019 amending Directive 2004/37/EC on the protection of workers from the risks related to exposure to carcinogens or mutagens at work) provided that the air concentration is kept under 0,5 ppm. This permission will end in July 2024, when the limit will be brought to 0. ppm. Recently, concern was raised over the mental effects of occupational exposure to formaldehyde vapors 7.

Substitution of formalin (PBF) as the fixative of choice in histopathology could only be considered with a reagent sharing the same merits in guaranteeing reliable diagnoses. A fixative substituting PBF should therefore be nontoxic, but with performance characteristics similar (non inferior) in terms of fixation speed, modalities of fixation (no over-fixation) and preservation of structural and molecular features.

We have been reasoning that the ideal candidate would have been glyoxal, the smallest aldehyde next to formaldehyde. The reagent is practically non-volatile, and this prevents exposure by inhalation. However, acidic glyoxal-containing fixative are detrimental for nucleic acid preservation 8,9 since commercial glyoxal contains strong acids (mainly glyoxylic and glycolic acids). We found that the removal of strong acids, using an ion exchange resin, allowed to produce a mildly basic solution (pH 7.2-7.4) and the proposed reagent, glyoxal acid-free (GAF) fixative proved to provide microscopical patterns and molecular preservation (immune-histochemical reactivity and nucleic acid sequence integrity) like those obtained, on parallel samples, by PBF fixation 10.

In EU, chances to substitute formalin could only be afforded using the criteria and rules set for medical devices. In fact, a histological fixative is not considered a medical product, it’s a Medical-Diagnostic in Vitro Device (IVDR) Class A, rule 5C which must follow the Regulation (EU) 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices, a Regulation which is going to become effective in May 26h, 2022. To ensure a high level of safety and performance, demonstration of compliance with the general safety and performance requirements laid down in Regulation 2017/746 should be controlled by a CRO (Contract Research Organization), based on clinical evidence to be sourced from performance studies to be carried out under the responsibility of a sponsor. Data sheet of IVDR should be implemented with information on the clinical performance and clinical evidence, presenting an Evaluation Report of the performance.

To validate GAF as a fixative alternative to formalin, we decided to fund a Start Up (ADDAX Biosciences srl., Turin, Italy) and proceed by selecting a CRO (1MED, Manno, Switzerland) which produced a Project finalized to a validation trial. The Project was focused on a relatively homogeneous group of “small” specimens (core and endoscopic biopsies from breast, colon, lung, endometrium, and prostate). The Project started in 2019 and ended in Feb. 2022. In 3 European centers (in Candiolo-Turin, Barcelona, and Manchester), punch biopsies were collected from fresh surgical specimens and fixed in alternative in PBF and in GAF fixative, then embedded in paraffin. Sections were stained with haematoxylin-eosin and, in immune-histochemistry, for 4 markers of interest for each type of tissue. A total of 100 cases were processed in parallel, which amounts to a total of 1000 slides (100 H&E+ 400 IHC x 2) which were scanned and prepared for a blind reviewing using a protocol prepared by CRS4 (Pula, Italy). The results were statistically analyzed and validated by the CRO. The final Report of the ADDAX-GAF Validation Trial issued on Feb. 2022, contains all the data (Protocol, sequences, conduction and organization, reviewers’ responses, statistical analysis, and results). The full data of the Validation Trial can be accessed by linking at:

The conclusions of the study report: Confirming the non-inferiority of GAF respect to PBF, the data of the trial highlight the capability of the investigational device to ensure the structural preservation of the: tissue, nuclei, cytoplasm, and diagnostic value of the preparations (for the sections of all organs tested).

The histological preparations collected in the ADDAX-GAF Validation Trial (amounting to a total of 1000 scanned images) can be viewed and analysed by linking to:

Separate datasets for the different types of biopsies (from breast, colon, lung, endometrium, and prostate) are also available.

The positive results of the study outline the possibility of substituting formalin as the histological fixative of choice and represent a critical step towards introduction of novel, reliable and environmentally safe procedures, in respect of EU directories.

References

  1. van der Lem T, de Bakker M, Keuck G. Wilhelm His Sr. and the development of paraffin embedding. Pathologe. 2021; 42:55-61. DOI
  2. Fox CH, Johnson FB, Whiting J. Formaldehyde fixation. J Histochem Cytochem. 1985; 33:845-853. DOI
  3. Vendrell JA, Grand D, Rouquette I. High-throughput detection of clinically targetable alterations using next-generation sequencing. Oncotarget. 2017; 8:40345-40358. DOI
  4. Bussolati G, Chiusa L, Cimino A. Tissue transfer to pathology labs: under vacuum is the safe alternative to formalin. Virchows Arch. 2008; 452:229-231. DOI
  5. Di Novi C, Minniti D, Barbaro S. Vacuum-based preservation of surgical specimens: an environmentally safe step towards a formalin-free hospital. Sci Total Environ. 2010; 408:3092-3095. DOI
  6. Veneroni S, Dugo M, Daidone MG. Applicability of under vacuum fresh tissue sealing and cooling to omics analysis of tumor tissues. Biopreserv Biobank. 2016; 14:480-490. DOI
  7. Letellier N, Gutierrez LA, Pilorget C. Association Between Occupational Exposure to Formaldehyde and Cognitive Impairment. Neurology. 2022; 98:e633-e640. DOI
  8. Willmore-Payne C, Metzger K, Layfield LJ. Effects of fixative and fixation protocols on assessment of Her-2/neu oncogene amplification status by fluorescence in situ hybridization. Appl Immunohistochem Mol Morphol. 2007; 15:84-87. DOI
  9. Marcon N, Bressenot A, Montagne K. Le glyoxal: un possible substitut polyvalent du formaldéhyde en anatomie pathologique? [Glyoxal: a possible polyvalent substitute for formaldehyde in pathology?]. Ann Pathol. 2009; 29:460-467. DOI
  10. Bussolati G, Annaratone L, Berrino E. Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples. PLoS One. 2017; 12:e0182965. DOI

Affiliations

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Gianni Bussolati

Department of Medical Sciences, University of Turin, Italy
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Copyright

© Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology , 2022

How to Cite

[1]
Bussolati, G. 2022. Fixation in histopathology: the mandate to renew. Pathologica - Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology. 114, 4 (Sep. 2022), 275-277. DOI:https://doi.org/10.32074/1591-951X-782.
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