Effects of fixation and demineralization on immunohistochemical assessment of canine bone marrow

The objective of this study was to evaluate the effect of different fixatives and demineralizers on immunohistochemical (IHC) detection of antigens in bone marrow (BM). Sternal BM samples were collected within 24 hours of death from dogs with spontaneous disease, fixed with acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) for 24 hours, and decalcified with formic acid, hydrochloric acid, or ethylenediaminetetraacetic acid (EDTA) for 1, 12, or 24 hours, respectively. Immunohistochemical sections for CD3, CD20, CD34, CD204, FLT3, and factor VIII-related antigen (FVIII-rAG) were scored for background, signal intensity, and percent positive cells by 4 independent raters. Some endothelial but not hematopoietic cells were CD34 immunopositive, precluding further assessment. Immunopositive BM cells ranged from 1.3% to 18.3% (CD3), 1.3% to 22.3% (CD20), 0.5% to 22.5% (CD204), and 0% to 17.6% (FLT3). Background scores were similar for all antigens and fixatives except for CD3, for which NBF yielded lower scores than AZF. The signal intensity for all IHC assays was similar for AZF and NBF. The percentage of FLT3- and FVIII-rAG-immunopositive cells was slightly higher in AZF- than NBF-fixed samples. Different demineralizers yielded similar results for all IHC assays except higher background for CD20/EDTA. Signal intensity was higher for CD204 in samples demineralized with EDTA rather than acids. Higher percentages of CD204 and FLT3 immunopositive cells were noted with EDTA relative to acid demineralization. Overall, with the use of stringently standardized pre-analytic and processing protocols, all IHC assays yielded acceptable results, and differences between protocols were considered minor.