Ancillary Techniques: Panels, Controls, and Preanalytical Reality
Tests are tools with failure modes
Ancillary studies require pattern recognition plus respect for assay limits. For immunohistochemistry, understand antigen retrieval, clone selection, expected subcellular localization (nuclear, cytoplasmic, membranous), and the role of internal and external controls. Diagnostic work uses panels, not single antibodies. Learn anchoring patterns: p63/p40 for squamous and myoepithelial cells; TTF-1 and napsin A for lung adenocarcinoma; PAX8 for Müllerian, renal, and thyroid lesions; GATA3 for breast and urothelium; CDX2 for intestinal differentiation; and mismatch repair proteins with their lost-partner logic.
Electron microscopy still answers questions IHC cannot: basement membrane duplication, desmosomes, dense-core granules, melanosomes, and viral particles. Molecular pathology spans PCR, FISH, and next-generation sequencing. Know the preanalytical threats-fixation time, decalcification, tumor cellularity, and formalin-induced C:G to T:A transitions-and the vocabulary of variant allele fraction, limit of detection, and orthogonal confirmation.
Traps: treating one positive marker as proof; ignoring a failed internal control; calling a low-level variant real without confirmation; confusing germline with somatic findings; and forgetting that a negative result requires adequate tumor content.
Rule of thumb: no result without a control. If the internal control fails, the test is invalid-not negative.
Worked example: a poorly differentiated tumor of uncertain origin. Start broad (CK7, CK20, TTF-1, napsin A, p40, GATA3, PAX8, CDX2, S100, CD45), then narrow by pattern. Report discordant results honestly and consider molecular profiling rather than forcing a single site.