CLINICAL HANDS ON PRESENTATION 14/09/2014 Marina Chatziioannou RnD - PowerPoint PPT Presentation

CLINICAL HANDS ON PRESENTATION 14/09/2014 Marina Chatziioannou RnD department R.G.C.C. Ltd

Points of interest 1. CTCs isolation − Manual or automated cell isolation − Magnetic bead epitope selection 2. Clinical flow cytometry − What for? − Epitope selection − Sample handling − Data analysis

CTCs isolation Topics 1. Sample arrival and handling 2. Manually or automated cell isolation 3. Magnetic bead epitope selection 4. Culture treatment

CTCs isolation Manually  Ficoll layering  Washing  Beads incubation  Magnetic CTCs isolation  Culture in FL25 Automated  Robosep handling  Culture in FL25

Epitopes (bead selection) Categorization 1. Epithelial origin 2. Hematological origin 3. Sarcomas 4. Non epithelial, non hematological, non sarcoma

Epithelial origin EpCAM i. Expressed exclusively in epithelia and epithelial-derived neoplasms. ii. Can be used as diagnostic marker for various cancers. iii. Plays a role in tumorigenesis and metastasis of carcinomas, so it can also act as a potential prognostic marker.

Hematological origin CD45 i. Leukocyte Common Antigen (LCA). ii. Its various forms are present on all differentiated hematopoietic cells except erythrocytes and plasma cells. iii. It is expressed in lymphomas, B-cell chronic lymphocytic leukemia, hairy cell leukemia, and acute nonlymphocytic leukemia. iv. A monoclonal antibody to CD45 is used in routine immunohistochemistry to differentiate between histological sections from lymphomas and carcinomas.

Sarcomas CD99 i. Ewing’s marker ii. Present to all sarcomas

Non epithelial, non hematological, non sarcomas CD45 negative selection i. For types of cancer that are not included in the previous categorizations. ii. For example: • Renal cancer • SCLC • NK lymphomas • Neuroblastomas • Adrenal cancer

Flow cytometry for clinical samples Topics • What for? • Epitopes selected • Sample handling • Data analysis

What for? • Easy, quick and reliable technique. • Gives accurate results. • Quick view of what to expect in the isolated culture from the same patient.

Flow cytometry clinical epitopes i. CD45:LCA ii. CD31: Endothelial cell marker iii. cMet: Metastatic cell marker iv. PanCK: Marker of cell of origin of various tumors (depends on cytokeratis pattern) v. CD227: Breast, Lung cancer marker vi. CD63: Melanoma cancer cell marker vii. PSMA: Prostate cancer cell marker

Sample handling Steps 1. Extracellular staining 2. Washing 3. Fixation 4. Washing 5. Permeabilization 6. Intracellular staining

Data analysis • Flow cytometry data file is analyzed and percentages of epitopes are produced. • Results are represented as below:

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Thank you for your time and patience!