Interpretation, Localization Patterns, and Reporting
Localization is the first question
Before calling anything positive, decide which compartment should stain. Nuclear markers (ER, PR, Ki67, p53, TTF-1, PAX8, SOX10) must show nuclear staining; cytoplasmic markers (cytokeratins, synaptophysin, CD68, most intermediate filaments) stain the cytoplasm; membranous markers (HER2, CD20, CD3, E-cadherin) require crisp membrane staining. Dot-like Golgi accentuation (CD30, chromogranin) and granular cytoplasmic patterns carry specific diagnostic meaning. Staining in the wrong compartment is usually background, not biology.
Semiquantitative scoring
ER and PR are reported as percentage of positive tumor nuclei plus intensity, often with Allred or H-score. HER2 uses a four-tier system: 0, 1+, 2+ (equivocal, reflex to ISH), and 3+ (strong complete membrane staining in more than 10% of tumor cells). Ki67 is a percentage of tumor nuclei, and its interpretation is notoriously subject to counting rules and preanalytic variation.
Reporting elements
A defensible report names the antibody clone, vendor, dilution, retrieval method, detection system and platform, and states that appropriate controls were adequate. It gives the result in the correct semiquantitative format, comments on background or necrosis when relevant, and notes any uninterpretable areas.
Mimics and traps
Melanin, hemosiderin, lipofuscin, and formalin pigment can mimic DAB. Necrotic tissue and crush artifact bind antibody nonspecifically. Edge artifact and retraction spaces produce false positivity at tissue borders. A faint blush in the wrong compartment should never be called positive, and cytoplasmic HER2 staining is not scored as membranous.
Rule of thumb
If the pattern does not match the expected subcellular localization, distrust the stain before you trust the finding.