Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

This experiment aims for sensitive detection, enumeration and characterization of circulating tumor cells from both human and mouse xenograft blood samples. First, collect the blood samples from humans and or mouse models of cancer and using a positive selection for epca positive cells enrich for CTCs in a background of contaminating leukocytes. Then with differential staining of CTCs and leukocytes, identify and enumerate the CTCs.

Perform the desired molecular characterization of these rare circulating tumor cells in the laboratory to aid in the selection of targeted therapies for cancer patients in the clinic. Results obtained using the cell search system demonstrate that CTC analysis can be effectively performed in both human and mouse xenograft blood samples. The development of additional user-defined CTC characterization protocols on the cell search system may ultimately aid in the selection of targeted therapies for patients.

In addition, comparable CTC assays adapted for utility in mouse xenografts will improve our understanding of the basic biology of these cells. Collect 7.5 milliliters of the blood sample from A CTC preservative tube and slowly dispense it into a processing tube. Add 6.5 milliliters of dilution buffer to each blood sample.

Invert five times to mix and centrifuge at 800 times G for 10 minutes. With the break in the off position for the control preparation, gently vortex the vial for five seconds and invert five times to mix. Then remove the cap top with an inverted processing tube and with one swift motion, transfer the sample.

Gently flick the sides of the control vial for maximum sample transfer. Carefully remove the inverted control vial ensuring that no liquid is lost. Collect the remaining contents from the vial and lid and gently add to the processing tube.

Follow the onscreen instructions on the preparation instrument to load all control and patient samples onto the system for processing. Follow the onscreen instructions on the preparation instrument to unload all the samples from the system. Now loosely cap each magnetic device cartridge and tap to release bubbles from the edges.

Then firmly cap the cartridge, lay the magnetic device flat and incubate in the dark for at least 20 minutes. Turn on the analysis instrument and initialize the lamp. Load the system verification cartridge onto the analysis instrument.

Select the QC test tab and follow the onscreen instructions to perform necessary quality control measures. Next, load a sample onto the analysis instrument and select the patient test tab. Click start to initialize the sample scanning.

Adjust all the edges as necessary using the directional keys. Then select accept To prepare user-defined markers, dilute the antibody of interest with bond primary antibody diluent. Place the marker reagent cup into position one in the reagent cartridge and load the cartridge onto the cell search system to enable custom marker addition.

Select user-defined assay when prompted by the preparation instrument. Input the marker name and select save. Once sample processing is complete, load a sample onto the analysis instrument and select the setup tab.

To initialize the ZI channel, select cell search CTC as the kit ID under the test protocol section. From this menu, select CTC research. Click edit button and set the exposure time as desired.

Collect a minimum of 50 microliters of mouse blood into a one milliliter EDTA microt retainer tube and mix by inversion to prevent clotting. For CTC enrichment, transfer the equivalent of 50 microliters of whole blood into a flow cytometry tube. Add 500 microliters of dilution buffer to each sample.

Washing down blood off the sides of the tube gently vortex the anti chem thorough fluid and add 25 microliters to each sample. Also, add 25 microliters of capture enhancement reagent. Vortex the samples gently and incubate at room temperature for 15 minutes.

Next, place the sample tubes into the magnet for 10 minutes. Carefully aspirate the residual liquid without touching the wall of the tube next to the magnet and discard, remove the sample tubes from the magnet and add 50 microliters of nucleic acid dye. 50 microliters of staining reagent 1.5 microliters of anti-US CD 45 A PC 5.0 microliters of anti-human HLA LOR 4 88 and 100 microliters of permeable reagent vortex gently and incubate in the dark for 20 minutes.

At room temperature. Now add 500 microliters of dilution buffer and vortex. Gently place the sample tubes into the magnet for 10 minutes.

Carefully aspirate the residual liquid and discard. Then resuspend the pellets in 350 microliters of dilution buffer vortex gently to mix. Aspirate the sample into a gel loading tip and transfer to the magnetic device by starting at the bottom of the cartridge and slowly withdrawing the tip as the sample is dispensed.

After removing air bubbles, using a sterile 22 gauge needle, firmly cap the cartridge, lay the magnetic device flat and incubate in the dark for at least 10 minutes. Load the sample onto the analysis instrument and select the setup tab. To clear existing data, click the format sample button.

Select cell search CTC as the kit ID and CTC research as the test protocol. From this menu, ensure that the appropriate exposure time has been selected. Select the patient test tab and select edit.

To input the sample information, input the remaining necessary information as indicated. Then save the sample information and click start to validate. Circulating tumor cell recovery, spiked human prostate cancer cells and uns spiked human blood samples from healthy volunteer donors were processed on the cell search system using the standard cell search CTC protocol.

As expected uns spiked samples were free of circulating tumor cells. The CTC recovery for spiked samples was 86.9%These cell search gallery images obtained from spiked samples are of optimal quality and the CTCs are easy to distinguish from non CTCs. The identification of CTCs is slightly more challenging from cancer patients with many cells appearing smaller in size and being less easily distinguishable from non CTCs.

In addition, when reviewing patient samples, six categories of events posed commonly discrepant items between viewers. These are small events, ZI positive events, dim cytokeratin or dappy staining, pixelated images, cytokeratin bleed through, and large or non-overlapping dpi. CTC kit selection must be considered when developing user-defined markers using a cell line known to demonstrate 98 4%positivity for the cancer stem cell markers CD 44.

The FCI based CTC kit only classified 69.3%of CTCs as CD 44 positive. However, using the PE based CX C kit, 98.8%of CTCs were classified as CD 44 Positive appropriate optimization of any user-defined marker requires validation using high antigen density, low antigen density, and negative cell lines for the marker of interest to ensure assay sensitivity and specificity. This experiment evaluates the sensitivity and specificity of the adapted mouse cell search protocol.

As expected. Uns spiked samples were free of CTCs using both assays and the CTC recovery. Using the adapted mouse kit and the standard automated system were comparable images obtained using the manual mouse adapted protocol did not differ from those observed using the standard automated technique.

The addition of HLA fits E to this assay ensures that human tumor cells can be easily distinguished from mouse squamous epithelial cells, serial dilution of spiked mouse blood samples, and the correlation of expected number of cells versus recovered number of cells. Confirm that this technique is as sensitive as the standard CSS protocol for the isolation of low number of CTCs. After watching this video, you should have a good understanding of how to perform automated and manual CTC enrichment from both human and me xenograph blood samples.

Additionally, you should have a greater understanding of the steps necessary for designing and optimizing CTC characterization protocols for use in combination with the cell search system.