Special Stains vs. Immunohistochemistry: What They Are and When Your Pathologist Uses Each
Tips for Submission · Canine, Feline
Most histopathology diagnoses are made on a routine hematoxylin and eosin (H&E) stained section. But some cases require an additional step to answer a question the H&E alone cannot resolve. Special stains and immunohistochemistry (IHC) are the two main categories of ancillary testing available on fixed tissue. They work differently, have different strengths and limitations, and are selected based on what the H&E findings and clinical context suggest. Understanding what each does — and why one might be chosen over the other — makes the pathology report easier to read and the diagnostic process more transparent.
Special stains: chemistry applied to tissue
Special stains use chemical reactions to selectively color specific substances in tissue based on their composition. They do not use antibodies. They work because certain dyes or reagents have a predictable chemical affinity for specific targets — fungal cell walls, collagen, iron deposits, bacterial cell walls, or the waxy coat of mycobacteria.
Strengths
Special stains are fast, relatively inexpensive, and highly specific for the substances they target. When the diagnostic question is chemical in nature — is fungus present, is this collagen or edema, are there acid-fast organisms here — they provide a direct, reliable answer. They do not require antibody validation or specialized platforms beyond basic laboratory equipment, and results are typically available within one to two days.
Limitations
Special stains can only identify substances with characteristic chemical properties that a dye or reagent can target. They cannot identify cell lineage, determine whether a tumor is malignant or benign, or distinguish between different types of cells that look similar on H&E. They are the right tool for a specific category of question — and the wrong tool for anything outside it.
Common special stains and their uses
| Stain | Target | Common use in veterinary pathology |
|---|---|---|
| PAS | Fungal cell walls, glycoproteins | Suspected fungal infection: Aspergillus, Cryptococcus, Histoplasma, Blastomyces |
| GMS | Fungal organisms | Same as PAS; often used together for confirmation |
| Masson's trichrome | Collagen (blue), muscle (red) | Hepatic fibrosis grading; stromal characterization in tumors |
| Prussian blue (Perl's) | Iron / hemosiderin | Hepatic siderosis; pulmonary hemorrhage |
| Gram stain | Bacteria (G+ purple, G− pink) | Bacterial infection in tissue; septic lesions |
| Ziehl-Neelsen / Fite's | Acid-fast organisms | Mycobacterial infection: leproid granuloma, feline leprosy, disseminated mycobacteriosis |
Immunohistochemistry: antibodies applied to tissue
IHC uses antibodies to detect specific proteins within tissue sections. The antibody binds to its target protein and a detection system produces a visible color reaction — typically brown — at the site of binding. What the pathologist sees under the microscope is which cells in the section are expressing the target protein.
IHC answers a fundamentally different question than special stains. Where special stains identify substances based on chemistry, IHC identifies cells based on what proteins they produce. This makes IHC the appropriate tool for determining cell lineage, confirming a tumor type, assessing prognostic markers, or detecting infectious agents that have no reliable chemical staining target.
Strengths
IHC is highly specific for the proteins it targets and can resolve diagnostic questions that H&E and special stains cannot. It can distinguish between tumor types that look identical on routine staining, confirm the origin of an undifferentiated neoplasm, identify infectious agents at the protein level, and provide prognostic information through markers like Ki-67. For many of the most diagnostically challenging cases in veterinary oncology — amelanotic melanomas, poorly differentiated round cell tumors, undifferentiated carcinomas — IHC is the definitive next step.
Limitations
IHC requires validated antibodies that perform reliably on formalin-fixed tissue in the species being tested. Not all antibodies validated for human tissue perform equivalently in dogs and cats, and species-specific validation is an important quality consideration. Interpretation requires familiarity with expected staining patterns in different species, tissues, and tumor types — a positive result is only meaningful in the context of the specific antibody, the staining pattern, and the clinical and morphologic picture.
Sarcomas deserve a specific note: soft tissue sarcomas are among the most challenging tumors to subtype by IHC. Unlike carcinomas and round cell tumors, which have well-defined lineage markers, sarcomas frequently share overlapping protein expression profiles that do not cleanly distinguish one subtype from another. A poorly differentiated sarcoma may yield a report confirming mesenchymal origin without being able to assign a specific histogenetic subtype. This is not a failure — it reflects the biology of these tumors. The clinically relevant management decision — surgical excision with adequate margins — is the same regardless.
Common IHC markers and their uses
| Marker | What it identifies | Common use |
|---|---|---|
| Pancytokeratin (AE1/AE3) | Epithelial cells | Confirm carcinoma; distinguish from sarcoma |
| Vimentin | Mesenchymal cells (broad) | Support sarcoma diagnosis; not subtype-specific |
| Melan-A / PNL2 | Melanocytic cells | Confirm melanoma, including amelanotic tumors |
| S100 | Neural, melanocytic, others | Broad panel marker; nonspecific alone |
| CD3 | T-lymphocytes | T-cell lymphoma immunophenotyping |
| CD79a / CD20 | B-lymphocytes | B-cell lymphoma immunophenotyping |
| c-Kit (CD117) | Mast cells; KIT protein | Poorly granulated MCT; mutation pattern assessment |
| Ki-67 | Proliferating cells | Proliferation index; prognostic adjunct in MCT, STS, mammary, oral tumors |
| Synaptophysin / Chromogranin A | Neuroendocrine cells | Pheochromocytoma, islet cell tumor, medullary thyroid carcinoma |
| SMA / Desmin | Smooth / striated muscle | Suggest myogenic differentiation in sarcomas (not specific) |
| Feline coronavirus antigen | FIP virus | Confirm FIP in pyogranulomatous lesions |
| CDV antigen | Canine distemper virus | Confirm distemper in neurologic or systemic disease |
Why IHC costs more — briefly
IHC requires validated antibodies, positive and negative controls on every run, automated staining platforms, and more technician time than special stains. A panel of multiple markers multiplies those inputs. Because IHC carries real cost, it is never added to a case without first contacting the submitting veterinarian — the pathologist will explain what the additional testing is expected to answer and obtain approval before proceeding.
The most useful thing you can do
The pathologist selects ancillary tests based on what the H&E shows and what the clinical context suggests. The single most useful contribution from the submitting clinician is a complete clinical history: signalment, lesion characteristics, duration, prior treatments, and the clinical differential list. That context allows the pathologist to frame the diagnostic question precisely — and to select the most efficient path to answering it.
Eric Snook, DVM, PhD, DACVP — Vetopathy. Questions about what a pathology report's ancillary test recommendations mean? Direct pathologist contact is available before and after every report.