Study highlights processing effects on canine bone marrow IHC

CURRENT FULL VERSION: A new study in Veterinary Pathology puts a spotlight on a familiar but consequential lab question: how much do fixation and demineralization choices shape immunohistochemical assessment of canine bone marrow? For veterinary teams managing suspected marrow infiltration, leukemia, lymphoma, myelodysplastic disease, or other hematopoietic disorders, the answer matters because the quality of the stain can depend as much on sample processing as on the biology of the case itself. The paper focuses on canine marrow specifically, using sternal samples from dogs with spontaneous disease and comparing two fixatives and three demineralization approaches. (acvp.org)

The study also fits into a broader research program from the same group at the University of Guelph and collaborators. In a related Veterinary Pathology paper published online June 6, 2024, and in print in November 2024, the authors reported that EDTA decalcification produced better marrow histomorphology scores than hydrochloric acid or formic acid, and dramatically improved the odds of successful DNA amplification. That earlier work concluded that EDTA should be used when PCR-based testing may be needed after histologic review. (pubmed.ncbi.nlm.nih.gov)

That background is important because marrow biopsies often need to do more than one job. A single sample may be used for routine morphology, immunophenotyping, clonality testing, or other molecular assays. Established pathology guidance has long warned that acidic decalcifiers are faster, but can damage fragile epitopes and nucleic acids, while EDTA is slower and generally better at preserving antigenicity. An ILAR Journal review on pathology methods recommends immediate fixation in 10% neutral-buffered formalin and gentle EDTA decalcification when histochemical and immunohistochemical staining may be required. Human pathology literature points in the same direction: a 2020 Modern Pathology study found that hydrochloric acid and prolonged formic acid decalcification could produce false-negative immunohistochemistry and molecular results, whereas EDTA and short-formic protocols preserved antigenicity more reliably. (academic.oup.com)

Support for EDTA-based processing also shows up outside bone marrow pathology. A recent Animals paper described a simplified, low-cost organic solvent protocol for DNA extraction from deer antlers and prepared trophy skulls that avoided commercial kits and cryogenic grinding. The method used bead-based mechanical homogenization, a 4-hour enzymatic digestion in EDTA buffer with N-lauryl sarcosine and proteinase K, followed by phenol-chloroform-isoamyl alcohol purification and centrifugal filtration. Across 60 samples—30 antlers and 30 pedicle portions from roe deer, fallow deer, and red deer—the authors obtained DNA suitable for downstream testing and successfully generated complete microsatellite genotypes from all 60 samples by multiplex PCR. That is not directly comparable to canine marrow IHC, but it reinforces the broader practical point that EDTA-based handling can preserve molecular utility even in heavily mineralized tissues.

In the canine marrow work summarized here, the authors collected sternal bone marrow 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 then demineralized them with formic acid, hydrochloric acid, or EDTA for different durations. Even without the full text of the new IHC paper available in the search results, the design closely mirrors the group’s earlier marrow-processing study and addresses a key unresolved question that paper explicitly flagged: whether the choice of fixative and demineralizer changes immunohistochemical performance in canine marrow. (acvp.org)

There doesn’t appear to be a separate institutional press release or broad industry announcement tied to this paper in the search results, and expert reaction specific to this publication was limited. Still, the wider technical literature is fairly consistent. Older immunopathology studies found that routine decalcification does not affect all antigens equally, which helps explain why labs often validate marker-specific protocols rather than relying on a one-size-fits-all assumption. More recent work, including veterinary and translational studies, has reinforced that EDTA tends to preserve immunoreactivity better than strong acids, even if it adds processing time. One PubMed-indexed study also suggested ultrasound-assisted EDTA decalcification may shorten turnaround without compromising histology or immunohistochemistry, which could be relevant for labs trying to balance quality and workflow. (pubmed.ncbi.nlm.nih.gov)

Why it matters: For veterinary professionals, this is really about diagnostic confidence. When a marrow core is being used to distinguish reactive from neoplastic infiltrates, classify lineage, or support staging in oncology cases, preanalytic artifacts can distort the result before the pathologist ever sees the slide. If a lab uses a rapid acid decalcifier to speed sectioning, that may help turnaround, but it can also reduce the reliability of IHC or molecular follow-up. In practical terms, this paper strengthens the case for thinking about marrow processing upstream, not after a weak stain or failed assay forces a repeat. That’s especially relevant for referral hospitals, oncology services, and commercial labs handling small or hard-to-repeat samples. Evidence from other mineralized tissues points the same way: EDTA-based workflows have been shown to recover DNA of sufficient quality for complete genotyping even from processed antlers and trophy skulls, suggesting that slower, gentler demineralization can preserve downstream options that harsher methods may compromise. (academic.oup.com)

It may also matter for standardization across institutions. Recent canine marrow research has emphasized the value of standardized marrow assessment in diseases such as myelodysplastic syndrome and acute myeloid leukemia, where interpretation can already be challenging. Better control of fixation and demineralization variables could reduce another source of inconsistency between cases and between laboratories. That won’t eliminate biologic complexity, but it can make the technical side of the work more dependable. (pubmed.ncbi.nlm.nih.gov)

What to watch: The next step is whether diagnostic veterinary pathology labs translate these findings into validated marrow protocols, including marker-specific IHC guidance and decisions about when EDTA-based processing should be the default despite slower turnaround. (academic.oup.com)