Validation of Fixation and Decalcification Protocols for Optimizing Immunohistochemical Staining for Bone Marrow Trephine Biopsy

Background: Processing of bone marrow trephine biopsies involves the use of various fixatives and decalcifying agents, which can impact immunohistochemistry (IHC) results. However, a simultaneous analysis of both simultaneous fixatives and decalcifying agents has not been conducted. Objective: To determine the optimal protocol that would yield superior IHC staining results. Materials and Methods: Twenty reactive tonsillectomy specimens were collected. Forty-two tissue pieces of size 2×2×2 mm, from each tonsil were subjected to different fixation and decalcification protocols. The tested fixatives included 10% buffered formalin and aceto-zinc formalin solution (AZF), with fixation durations of 2, 4, 8, and 24 hours. Decalcification was performed using 5% nitric acid, “Decal II® (Surgipath)”, 10% formic acid, 20% ethylene diamine tetraacetic acid (EDTA) (pH 7.1), and 10% EDTA (pH 7.4). The effect of each protocol on staining quality was assessed using the tissue microarray (TMA) approach. The tested IHC staining panel included CD3, CD20, PAX5, CD30, CD5, cyclin D1, CD10, BCL6, and Ki67. Results: Ten percent buffered formalin demonstrated significantly better IHC staining results compared to AZF, particularly for nuclear-stained antibodies. The duration of fixation of 2, 4, 8, or 24 hours, did not significantly affect the staining outcomes. Among the decalcifying agents, both 10% formic acid and 10% EDTA provided superior IHC staining results compared to the others. Both agents yielded similar staining outcomes without a significant difference. Conclusion: For optimized IHC staining in bone marrow trephine biopsy, 10% buffered formalin is recommended as the preferred fixative, while either 10% formic acid or 10% EDTA can be used for decalcification.