Table 3. . Immunohistochemical markers for evaluating differentiation in biphenotypic (hepatobiliary) primary liver carcinoma.

	Hepatocyte differentiation	Biliary differentiation	Stem cell differentiation with stem cell morphology	Stem cell differentiation without stem cell morphology
Common markers (use as panel or use in sequence until a positive result is obtained)	HepPar1, Arginase-1, canalicular staining with CD10, canalicular staining with pCEA, AFP	Keratin 7, Keratin 19, cytoplasmic staining for CEA (pCEA or mCEA), EMA (Muc-1)	Keratin 19, NCAM (CD56), EpCAM, EMA (Muc-1), c-kit (CD117)	No available common markers
Experimental markers (supplemental to ‘common markers’)	Nuclear staining for HNF4a	Nuclear staining for Sox-9	Nestin, Keratin 14, CD133, nuclear staining for Sox-9	Nuclear staining for Oct-4; Nuclear staining for Nanog; Nuclear staining for Sall-4

Bold text highlights the antibodies to be used.

For each differentiation state within a b(HB)-PLC – hepatocellular, biliary and stem cell – several markers are available; however, availability or cost may limit the utility of such large panels. On the other hand, lack of consistency between studies and/or centers can result in data that cannot be easily compared between different studies. To limit these difficulties, the panels in the upper row are recommended to be used either: as a panel with all antibodies applied to every case; or in a sequence, top to bottom, stopping with the first positive antibody. The markers in the bottom row are recognized as having utility for studying b(HB)-PLC, but are considered supplemental to the primary panels listed first. It is emphasized that morphology is primary in the assessment of subpopulations and immunophenotyping is secondary. In particular, stem cell components are assessed by routine light (H&E) microscopy before confirming by immunohistochemistry.