Study highlights how processing choices shape canine marrow IHC

CURRENT FULL VERSION: A new study in Veterinary Pathology takes aim at a familiar but underappreciated problem in diagnostic pathology: how much the preanalytic steps, specifically fixation and bone demineralization, can change what pathologists see on immunohistochemistry in canine bone marrow. The paper evaluated marrow samples from dogs with spontaneous disease after fixation in acetic acid-zinc-formalin or 10% neutral-buffered formalin and decalcification with hydrochloric acid, formic acid, or EDTA, with the goal of identifying which combinations best preserve antigen detection for IHC. That question matters because bone marrow is one of the few tissues where decalcification is unavoidable, yet clinicians may still need immunophenotyping or molecular follow-up from the same sample. (acvp.org)

The work builds on earlier research from the same University of Guelph group. In a 2024 Veterinary Pathology paper, the investigators reported that for canine bone marrow, acid-zinc-formalin and neutral-buffered formalin produced similar histomorphology, but EDTA decalcification outperformed hydrochloric acid and formic acid for both morphology and PCR-based DNA amplification. In that study, only 3% of acid-decalcified samples generated any amplification, versus 81% of EDTA-decalcified samples, underscoring how strongly decalcification chemistry can shape downstream test performance. The authors explicitly noted then that the effects on IHC still needed to be evaluated, making the new paper a logical next step rather than a standalone finding. (pmc.ncbi.nlm.nih.gov)

That broader trajectory also fits with a growing push toward standardization in veterinary pathology. A 2025 Veterinary Pathology paper from the Japanese College of Veterinary Pathologists’ tissue processing committee found that unbuffered formalin and prolonged fixation can attenuate immunolabeling and reduce PCR performance, and recommended 10% neutral-buffered formalin within 72 hours for high-quality formalin-fixed, paraffin-embedded samples used in histology and molecular diagnostics. Taken together, the newer veterinary literature is pointing to the same conclusion: tissue handling decisions made before a slide ever reaches a pathologist can materially affect diagnostic quality. (pubmed.ncbi.nlm.nih.gov)

Although the full text of the IHC-focused canine marrow paper was not readily accessible in the search results, the study design in the supplied source is clear. The investigators used sternal bone marrow collected within 24 hours of death from dogs with spontaneous disease, fixed samples for 24 hours in either acetic acid-zinc-formalin or neutral-buffered formalin, and decalcified them using formic acid, hydrochloric acid, or EDTA for different durations before immunohistochemistry. Based on the team’s earlier marrow work and the comparative pathology literature, the most clinically relevant issue is whether gentler decalcification preserves diagnostically important antigens better than strong acids do. (pmc.ncbi.nlm.nih.gov)

Outside veterinary medicine, that pattern is already well established. A 2019 study on bone samples found hydrochloric acid and prolonged formic acid decalcification could produce false-negative immunohistochemistry and molecular results, while EDTA and short-course formic acid better preserved antigenicity. Another bone marrow biopsy study reported that EDTA maintained intact nuclear protein staining, while hydrochloric acid produced poor-quality IHC images. More recently, a 2025 pilot study comparing bone marrow biopsy IHC protocols found the fewest inadequate stains with a B5-based fixative plus an EDTA-based decalcifier. Those are human data, but they offer useful context because the underlying chemistry of antigen preservation is the same. Similar principles are showing up in other calcified-tissue workflows too: an Animals study describing DNA extraction from deer antlers and prepared trophy skulls used an EDTA-based digestion buffer with Proteinase K, mechanical homogenization, organic solvent purification, and centrifugal filtration, and successfully generated complete microsatellite genotypes from all 60 tested samples. That was a forensic and conservation application rather than diagnostic histopathology, but it adds another example of EDTA-supported processing helping preserve usable nucleic acids in mineralized tissues. (pubmed.ncbi.nlm.nih.gov)

Expert-style guidance in pathology references has long leaned the same way. Veterinary pathology teaching resources and histology protocols generally recommend full fixation before decalcification and note that EDTA best preserves tissue antigens, RNA, and morphology, even though it is slower than acid-based approaches. Formic acid is often treated as a compromise option when turnaround time matters, while hydrochloric acid is faster but more likely to degrade morphology and immunoreactivity. That speed-versus-quality tradeoff is exactly what makes this study relevant to real-world diagnostic labs. (veteriankey.com)

Why it matters: For veterinarians and pathologists, marrow biopsy interpretation is often highest stakes when the differential list is narrow and treatment decisions hinge on lineage, clonality, or disease classification. If a decalcifier suppresses antigen staining, the result may be a weaker panel, a more equivocal diagnosis, or the need for additional testing that adds cost and time for the pet parent. The practical implication is that labs may need to think of marrow processing as part of diagnostic test validation, not just specimen prep. Standardizing around neutral-buffered formalin and EDTA, especially for cases likely to need IHC or molecular testing, could improve consistency, but it may also require workflow changes and acceptance of longer decalcification times. The same preservation-first logic is relevant when hard tissues are later used for PCR or genotyping, as shown by successful EDTA-based DNA recovery from processed deer antlers and trophy skulls. (acvp.org)

What to watch: The next step is whether veterinary diagnostic labs translate these findings into protocol changes, and whether future studies identify marker-by-marker effects for common canine marrow IHC panels, along with the turnaround-time tradeoffs of EDTA-based workflows. Comparative pathology suggests the field is moving toward more standardized, assay-conscious tissue handling, but adoption will likely depend on how strongly the new canine data show clinically meaningful differences in stain quality and interpretability. It will also be worth watching whether lessons from other mineralized tissues, including forensic DNA recovery protocols that avoid harsher processing, further influence how veterinary labs balance speed against analyte preservation. (pubmed.ncbi.nlm.nih.gov)