Paudice Michele, Gambella Alessandro, Pigozzi Simona, Campora Michela, Pitto Francesca, Bozzano Silvia, Mastracci Luca, Grillo Federica
IRCCS San Martino Policlinic Hospital of Genoa, Genoa, Italy.
Pathology Unit, Department of Surgical Sciences and Integrated Diagnostics (DISC), University of Genoa, Genoa, Italy.
Pathologica. 2025 Apr;117(2):145-152. doi: 10.32074/1591-951X-1065.
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).
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.
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.
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.
骨髓环钻活检（BM）分析前阶段的标准化尚未实现。特别是，有几种具有特定优缺点的固定剂和脱钙试剂被描述，但仅有少数直接比较。本研究旨在测试最常用的固定和脱钙方案，并通过免疫组织化学（IHC）评估它们对组织抗原性的影响。
为避免损坏和耗尽诊断性BM，我们使用从非病理性脾脏的专门大体检查中获得的“替代”BM。测试了11种固定和脱钙方案，并通过25种生物标志物的IHC蛋白表达评估其性能。
IHC产量因固定剂和脱钙试剂而异，但总体质量主要与固定剂有关，而非脱钙阶段。免疫组化染色不足数量最少（25种中有5种）的方案将市售的基于B5的固定剂和基于EDTA的脱钙试剂结合使用。基于“自制”的基于B5和基于EDTA的试剂的方案获得了最差的指标（25种中有8种免疫组化染色不足）。
我们比较了不同方案，发现了用于准确免疫组化染色的固定剂和脱钙试剂的最佳组合。这些发现可改善病理实验室中骨髓样本的处理和标准化。
