One protocol to rule them all: a pilot study to identify the best fixation and decalcification approach for bone marrow biopsy immunohistochemistry.

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 "in-house" 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.