Abstract

Objective. Standardization of the pre-analytical phases of bone marrow trephine biopsy (BM) has yet to be achieved. In particular, several fixative and decalcifying reagents with specific benefits and drawbacks are described, but only a few direct comparisons are available. This study aims to test the most used fixation and decalcification protocols and evaluate their effect on tissue antigenicity via immunohistochemistry (IHC).
Methods. To avoid damaging and exhausting diagnostic BMs, we used “surrogate” BMs obtained from dedicated grossing of a non-pathologic spleen. Eleven fixation and decalcification protocols were tested, and their performances were evaluated via IHC protein expression of 25 biomarkers.
Results. The IHC yield varied based on the fixative and decalcifying reagents, but the overall quality is mainly related to the fixative rather than the decalcifying phases. The protocol with the lowest number of inadequate IHC stains (5 out of 25) combined commercially available B5-based fixative and EDTA-based decalcifying reagents. The worst metrics (8 inadequate IHC stains out of 25) were obtained with a protocol based on “inhouse” B5-based and EDTA-based reagents.
Conclusions. We compared different protocols and found the best combination of fixative and decalcifying reagents for accurate IHC staining. These findings can improve bone marrow sample handling and standardization in pathology laboratories.

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Authors

Michele Paudice - IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy

Alessandro Gambella - Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

Simona Pigozzi - IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy; Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

Michela Campora - Department of Pathology, Santa Chiara Hospital, APSS Trento, Trento, Italy

Francesca Pitto - IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy; Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

Silvia Bozzano - Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

Luca Mastracci - IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy; Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

Federica Grillo - IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy; 2 Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy

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Copyright (c) 2025 Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology

How to Cite
Paudice, M., Gambella, A., Pigozzi, S., Campora, M., Pitto, F., Bozzano, S., Mastracci, L., & Grillo, F. (2025). One protocol to rule them all: a pilot study to identify the best fixation and decalcification approach for bone marrow biopsy immunohistochemistry. Pathologica - Journal of the Italian Society of Anatomic Pathology and Diagnostic Cytopathology, 117(2). Retrieved from https://www.pathologica.it/article/view/1350
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