CTC represent an important biological marker, and provide insight into the development and progression of the disease. At the Ljubljana Oncology Institute, we have established a method for isolating CTCs from a blood sample. After isolation, we prepared cytological preparations, which we then identified with cytopathological analysis. We worked on the samples of 10 patients, median age 53 taken in the GALIA clinical survey. Immunofluorescence (IF) staining allows us to mark and identify several different cell markers at once on the same sample. For this purpose, we have developed an in-house protocol in which we label 4 cell markers. We used methanol-fixed preparations for IF staining. The established protocol on CTC samples obtained from the blood of breast cancer hospitals has not always proven to be optimal. In particular, we noticed pale nuclei and uneven labeling of CD45. It also showed in some cases the absence of blood markers, nor did it show thrombocytopenia. For this reason, we compared the established in-house IF protocol with the Portrait®+ protocol (Portrait®+ CTC Staining Kit, CelLBxHealth plc.), which was developed and validated for CTC labeling. The Portrait®+ protocol contains a larger set of cell markers, which is a trade secret. For Portrait®+, we used two types of control preparations: one fixed in methanol, the other in acetone. Finally, we performed the imaging with a Zeiss Axio Observer fluorescence microscope and created images using the Imaris software and GraphPad Prism software version 10.6.1. For statistical analysis and graph drawing. With methanol, we observed a greater degeneration of cells and a greater impact on morphology. Portrait®+ had more blood markers, but more red staining was present, which could be mistaken for erythrocytes, but there were no antibodies for erythrocytes. In the samples stained with the Portrait®+ protocol, we determined a higher number of single CTCs in the pool of CTC clusters than when stained with the in-house protocol.
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