How To Get A Cell Print

Further characterization of CTCs through CRS Services • FISH • Multiple protein markers (Cheshire) Extraction and characterization of DNA, RNA, and proteins • Gene Expression • Mutation Detection • Sequencing Rare cell capture

• Enumeration • Single protein marker

Cell capture for molecular characterization • CELLSEARCH® _Profile Kit (RUO) • CELLTRACKS® _Circulating Endothelial Cell Profile Kit (RUO)

QUICK CELL

CAPTURE AND

CHARACTERIZATION

GUIDE FOR

CELLSEARCH

®

CUSTOMERS

COMMERCIALLY AVAILABLE RARE CELL KITS

Cellular Kits for Rare Cell Capture and Staining

Circulating Tumor Cells

CELLSEARCH® _{Circulating Tumor Cell Kit (IVD) product code: 7900001}

Capture antigen: EpCAM CTC-identification marker: Cytokeratin (PE) White blood cell marker: CD45 (APC) Nuclear Stain: DAPI

Free channel: FITC

http://www.cellsearchruo.com/ruo-products/kits

Please refer to the Instructions for Use at https://www.cellsearchctc.com/support-resources.

CELLSEARCH® _{CXC Kit (RUO) product code: 7900017}

Capture antigen: EpCAM CTC-identification marker: Cytokeratin (FITC) White blood cell marker: CD45 (APC) Nuclear Stain: DAPI

Additional moderately expressed marker: PE

http://www.cellsearchruo.com/ruo-products/kits

CELLSEARCH® _{Epithelial Cell Kit (RUO) product code: 7900000}

Capture antigen: EpCAM CTC-identification marker: Cytokeratin (PE) White blood cell marker: CD45 (APC) Nuclear Stain: DAPI

Additional highly expressed marker: FITC

http://www.cellsearchruo.com/ruo-products/kits

Circulating Endothelial Cells

CELLSEARCH® _{Circulating Endothelial Cell Kit (RUO) product code: 7900009}

Capture antigen: CD146 CEC-identification marker: CD105 (PE) White blood cell marker: CD45 (APC) Nuclear Stain: DAPI

Additional highly expressed marker: FITC

http://www.cellsearchruo.com/ruo-products/kits

Circulating Melanoma Cells

CELLTRACKS® _{Circulating Melanoma Cell Kit (RUO) product code: 9594V}

Capture antigen: CD146 CMC-identification marker: HMW-MAA (PE) Nuclear Stain: DAPI Additional highly expressed marker: FITC White blood cell and endothelial cell markers: CD45 (APC), CD34 (APC)

http://www.cellsearchruo.com/ruo-products/kits

Please note that all of the above kits are commercially available and can be run on any installed CELLSEARCH® _instrument.

COMMERCIALLY AVAILABLE RARE CELL KITS

Cellular Kits for Rare Cell Capture and Staining

Control Cell Kits

CELLSEARCH® _{CTC Control Kit (IVD) product code: 7900003}

Each kit contains 24 single use bottles of fixed cells from a breast carcinoma

cell line (SK-BR-3) Each bottle contains both high & low control

http://www.cellsearchruo.com/ruo-products/kits

Please refer to the Instructions for Use at https://www.cellsearchctc.com/support-resources.

CELLSEARCH® _{Circulating Epithelial Cell Control Kit (RUO) product code: 7900002}

Each kit contains 24 single use bottles of fixed cells from a breast carcinoma

cell line (SK-BR-3) Each bottle contains both high & low control

http://www.cellsearchruo.com/ruo-products/kits

CELLSEARCH® _{CXC Control Kit (RUO) product code: 7900018}

Each kit contains 24 single use bottles of fixed cells from a breast carcinoma

cell line (SK-BR-3) Each bottle contains a high control

http://www.cellsearchruo.com/ruo-products/kits

CELLSEARCH® _{CEC/CMC Control Kit (RUO) product code: 9572V}

Each kit contains 24 single use bottles of fixed cells from SK-Mel28 Each bottle contains a high & low control

http://www.cellsearchruo.com/ruo-products/kits

Tumor Phenotyping Reagents (TPRs)

CELLSEARCH® _{Tumor Phenotyping Reagent HER-2/neu (RUO) product code: 7900006}

Fluorescein-conjugated monoclonal antibody that is to be used in conjunction with the CELLSEARCH® _{Epithelial Cell Kit to phenotype}

circulating epithelial cells for the presence of HER-2/neu

http://www.cellsearchruo.com/ruo-products/phenotyping-reagents

CELLSEARCH® _{Tumor Phenotyping Reagent EGFr (RUO) product code: 79000011}

Fluorescein-conjugated monoclonal antibody that is to be used in conjunction with the CELLSEARCH® _{Epithelial Cell Kit to phenotype}

circulating epithelial cells for the presence of EGFr

http://www.cellsearchruo.com/ruo-products/phenotyping-reagents

CELLSEARCH® _{Tumor Phenotyping Reagent IGF-1R (RUO) product code: 9597}

Allows for the qualitative determination of the presence of the IGF-1R antigen on circulating epithelial cells in the blood, and is to be used in conjunction with the CELLSEARCH® _{CXC Kit}

RNA DNA Proteins RT-PCR Microarray RNA Seq Next Gen Sequencing Mutation Detection Proteomics Taqman (ABI)

BioMark (Fluidigm) HiSeq/Miseq (Illumina)Cast PCR (ABI) —

COMMERCIALLY AVAILABLE RARE CELL KITS

Molecular Characterization of Rare Cells

Overview Using the profile kits, CTCs and CECs are captured and presented in a volume of 900 microliters. No fixation, staining or permeabilization is performed during capture. Cells are captured alive and can be maintained and potentially propagated under appropriate tissue culture conditions. The enriched sample also contains ferrofluid and a few thousands leukocytes which were non-specifically retained during the enrichment procedure. Samples should be processed as soon as possible to maximize nucleic acid integrity and recovery. Blood samples may be stored or transported in EDTA tubes at room temperature for up to 36 hours prior to processing. Note however that immediate processing is preferred since a loss of mRNA from epithelial cells may result if processing is delayed. Captured cells can be lysed and stored in Trizol or RLT buffers for future extraction of total RNA (including miRNA), DNA and proteins. Simultaneous extraction of RNA, DNA and proteins can be performed from each sample using: • Trizol protocol from Invitrogen (http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf) • AllPrep kits from Qiagen (http://www.qiagen.com/literature/render.aspx?id=2067) • All-in-One Purification kit from Norgen (http://www.norgenbiotek.com/display-product.php?ID=101) Numerous commercial kits can be used for separate isolates of RNA, miRNA, DNA and proteins. We recommend utilizing kits that are designed for small samples since CTC-enriched samples rarely contain more than 10,000 cells.

Molecular Kits for Rare Cell Capture

CELLSEARCH® _{Profile Kit (RUO) product code: 7900004}

CTCs are captured using EPCAM http://www.cellsearchruo.com/ruo-products/kits

CELLSEARCH® _{CEC Profile Kit (RUO) product code: 9564V}

CECs are captured using CD146 http://www.cellsearchruo.com/ruo-products/kits

Molecular Characterization Methods

Analyte

Methods

Platforms

Rare Cell Enumeration and

Single Marker Protein Expression

• Using any of our commercially available kits

• Other rare cell testing available (e.g. CMMC)

• A variety of Protein Markers (e.g. AR, ER, Ki-67 and more)

Custom Assay Development

Ferrofluid and Antibody Development

• For the development of other Rare Cell Capture and Protein Expression assays not currently commercially available

• Assay verification/pilot studies

Fluorescent in situ hybridization (FISH) — Custom

FISH assay development

• Unique probes prepared for each study • Utilizes special instrumentation for rare cell fixation in CELLSEARCH® _cartridges If desired, FISH can be performed following enumeration kits that were described above. Rare cell FISH allows interrogation of CTCs at the genetic level. Janssen Diagnostics was the first company to create REPEAT-FREE™ probes, which provide a clearer signal and are easier to score. By removing the repeat sequences, Janssen Diagnostics made it possible to deliver a level of performance key to understanding complex diseases. Numerous FISH assays have been developed for CTCs. Examples include Her-2/SE17, cMET/SE7, EGFR/SE7, AR/SEX, ERG break, AURK-A/Chr20, AURK-B/SE17, P53/SE17, IGF1-R/Chr15, cMYC/SE8 and PTEN/SE10. Additional assays can be developed upon request. Please note that subsequent FISH analysis requires special equipment and fixation of captured stained cells in cartridges. Contact CRS for additional information: http://www.cellsearchruo.com/contact-us.

Multiple Protein Markers (Cheshire)

Fluorescent signals from an initial set of markers can be “erased” using a proprietary Cheshire procedure and re-stained with a second set of biomarkers. For details about this protocol please contact CRS:

http://www.cellsearchruo.com/contact-us.

Molecular Characterization of Rare Cells

• Profile Kit Enrichment followed by lysis and storage of samples

• DNA & RNA Extraction

• Method development to enable rare cell characterization via downstream molecular platforms

CLINICAL RESEARCH SOLUTIONS (CRS) SERVICES

Assay Development

Process

Feasibility

3. Final antibody selection 4. Antibody conjugation

1. Create prototype assay 2. Test prototype

assay to specific agreed-upon criteria 3. Identify assay controls

1. Development report 2. Train clinical lab

scientists 3. Protocols 1. Test with clinical samples 2. Final report

Assay Verification

Assay Transfer

Assay Validation