The one-step nucleic acid amplification (OSNA) is an intraoperative technique which detects and quantifies the presence of cytokeratin 19 (CK-19) mRNA in a lymph node by polymerase chain reaction (PCR) .
We adopted the standard CK-19 mRNA copy count to stratify the size of metastasis where mRNA copy count of <250 copies/[micro]L, 250-5000 copies/[micro]L, and >5000 copies/[micro]L is considered as negative, micrometastases, and macrometastases, respectively .
Upregulated expression of cytokeratin-19 (CK-19
), the parent protein of CYFRA 21-1, has proven to be an independent risk factor of intraliver and lymph node metastasis in hepatocellular carcinoma [20, 21].
hAMSC expression of CK-19 and vimentin was detected by immunofluorescence.
hAMSCs in the 3rd passage barely expressed CK-19. hAMSCs were positive for vimentin expression.
We developed a multiplexed cell staining protocol to characterize the cryopreserved differentiated HepaRG cells used in this study, combining Hoechst 33342 dye to stain all cell nuclei within the cultures, an antibody against Cytokeratin 19 (CK-19
), a marker of epithelial cells, and an antibody against CYP3A4.
This system is based on a combination of immunocytochemistry and immunofluorescence techniques, in which the immunomagnetic selection of CTCs with antibodies specific for the epithelial marker EpCAM is followed by staining of EpCAM-positive cells for CKs (CK-8, CK-18, and CK-19
Real-time quantification of CK-19
mRNA-positive cells in peripheral blood of breast cancer patients using the light-cycler system.
The subsequent approaches used to detect CTCs are (i) immunocytochemistry with anticytokeratin (anti-CK) antibodies; (ii) RT-PCR targeting various epithelial mRNAs, including CK-19
mRNA; and (iii) epithelial immunospot (EPISPOT) assays detecting tumor-specific proteins released by CTCs [e.g., prostate-specific antigen (PSA)].
Immunocytochemically, the tumor cells showed positive staining for wide-spectrum cytokeratin, CK-19
, and epithelial membrane antigen, but negative staining for vimentin, S100 protein, desmin, smooth muscle actin, human melanoma antigen (HMB-45), and [[alpha].sub.1]-antichymotrypsin.
Imaging approaches are based on the characterization of isolated cells as CTCs through fluorescently labeled antibodies mainly against epithelial antigens such as CK-19
. A limited number of other protein markers have also been used for this reason.
(6) Antibody against cytokeratin 19 (CK-19
) (mouse monoclonal; 1:40; Dako Corporation, Carpinteria, Calif) was used.