IsoFlux Cytation Imager – CTC Enumeration

Automated Imager – Instructions for the Enumeration of IsoFlux Samples

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INTENDED USE

The intended use for the IsoFlux™ Cytation Imager is as an instrument for the enumeration of IsoFlux enriched circulating tumor cell (CTCs) samples. This instrument is used with the IsoFlux System, a bench-top instrument for semi-automated cell isolation. The IsoFlux Cytation Imager is for Research Use Only.

SUMMARY AND EXPLANATION

Circulating tumor cells (CTCs) are cancer cells that shed from a primary or metastatic tumor and enter the peripheral circulation. Carcinomas are cancers of epithelial origin and include breast, prostate, lung, and colorectal cancers. These tumors shed CTCs that are of epithelial origin. CTCs are distinct from other blood cells since cells of epithelial origin are not normally found in the circulation.

The IsoFlux Cytation Imager is designed to standardize and automate the enumeration of CTCs isolated from biological samples using the IsoFlux System. CTCs are enriched from the sample using an immunomagnetic capture reagent while the sample flows through a microfluidic cartridge designed for cellular isolation. The IsoFlux produces an enriched cell pellet that can subsequently be used for further testing, in this case staining, mounting, imaging, and semi-automated enumeration.

PRINCIPLES OF THE PROCEDURE

The IsoFlux workflows use CTCs isolated from the blood of cancer patients to perform a variety of assays. For CTC enumeration and other assays where fluorescent signal is the readout, microscopy of the cell samples post enrichment is required. The IsoFlux Cytation Imager is an automated microscope that is uniquely configured to work with CTC samples, from the original image acquisition to the final analysis of identified CTCs.

This procedure outlines the steps needed to perform image acquisition for one or more IsoFlux samples using the Cytation Imager, as well as protocols for cell identification and enumeration using the associated Gen 5 software.

MATERIALS PROVIDED

  • IsoFlux Cytation Imager
  • Protocol files and instructions for sample image acquisition  Protocol files and instructions for sample image counting

MATERIALS REQUIRED, NOT PROVIDED

  • Permanent magnets (accessory parts included with IsoFlux instrument: large round and small cylindrical magnets)
  • SensoPlate Glass bottom 24-well plates
  • Glass Cover slips (12mm circular)
  • Test tube racks
  • Calibrated micro-pipettors and tips
  • Serological pipettes and pipettor

WARNINGS AND PRECAUTIONS

  • For Research Use Only
  • Please read the entire contents of the Instructions for Use before processing samples.
  • Caution: Care should be taken to collect and transfer blood samples before processing. Cells are fragile and can be damaged or lost if not handled properly.
  • Caution: All personnel should follow universal precautions for biological sample handling and use personal protective equipment (i.e., safety glasses, laboratory coat, gloves, etc.).
  • Caution: Microbial contamination of reagents can cause erroneous results and should be avoided.
  • Warning: All biological specimens, cartridges and other materials coming into contact with the specimen(s) are considered bio-hazardous. Handle as if capable of transmitting infection. Treat and dispose of waste using proper precautions and in accordance with local, state, and federal regulations. Never pipette by mouth.
  • Operator training is required to perform the test procedure.

SAMPLE MOUNTING PROCEDURE

This is an example of mounting sample onto a glass surface; in this case, we recommend using the Greiner glass bottom plate. Mounting on slide is also possible. However, make sure to invert the slide when placing the slide in the Cytation such that the thin cover slip faces the objective.    

  1. After sample is enriched and stained according to IsoFlux protocol (630-0126, IsoFlux Enumeration IFU), mount the cells as shown below.
  2. Transfer the stained cells/beads into a SensoPlate™ well, centered on a small round magnet.
  3. Remove the buffer while keeping the stained cells/beads at the center of the well over the magnet.
  4. Remove the plate from the magnet. Dispense 2.5μL of Mounting Media over the cells/beads spot. Place the cells/beads over the large round magnet.
  5. Place the glass coverslip over the cells/beads spot while keeping the magnet in place. Center the coverslip if necessary. Remove the plate from the magnet.
     
  6. (Optional) Dab the edges of the coverslip with clear nail polish – perform this step if the sample is to wait over 6 hours before
  7. Cover the SensoPlate™ with plate sealer and lid. The sample is now ready for imaging and can be stored at 4oC for at least 2 weeks if kept moist and away from light.

SAMPLE IMAGING USING THE ISOFLUX CYTATION

These instructions are for use with the Cytation with laser auto-focus option. For systems without this option, please refer to set-up in the Cytation user manual under "Understanding Auto Focus" (page 57-64).


  1. Start the Cytation software (Gen5 application)
  2. BioTek Initial Settings:
    System → Plate type → Greiner 24-well
  3. Create new experiment from existing protocol:
  4. Select BIOTEK FLUXION ENUMERATION PROTOCOL*
  5. Double click on PROTOCOL
  6. Double click on PROCEDURE
  7. Make sure under PLATE TYPE, Greiner 24-well is selected and USE LID box is ticked.
  8. Double click on READ: GFP 469,525, DAPI 377,447, RFP 531, 593.
  9. The tray will slide out and GEN5 software will prompt to place the plate into the reader
  10. Plate the SensoPlate™ into the plate holder. Click OK
  11. Image Montage should be checked and a 9x7 grid defined. Check that Auto for Stitching is also ticked (see above)
  12. Select channel 1 for GFP. Click OPTIONS
  13. Check that Autofocus is selected.
  14. Click the microscope icon on right hand side (CAPTURE REFERENCE SCAN).
  15. The instrument always aligns at center of well A1. Choose the appropriate well as reference. For example, if the sample is in well B6, then select this well.
  16. Make sure the focus current position is at 1126. The GFP image should be relatively sharp at this focus. If multiple wells are to be scanned on the same plate, only one reference image* is needed.
    *Re-do the reference scan whenever placing a plate into the instrument, regardless of using a new or a previously used/scanned plate. A small difference can affect the focus and therefore the analysis of the images. 
  17. Click SAVE SETTINGS.
  18. The Cytation3 will now capture the reference image. Select OK to exit.
  19. Under FILE, save experiment (all related images will be stored under this experiment name). Name the experiment accordingly.
  20. System is now ready for scanning samples
  21. Click on GREEN PLAY ARROW
  22. The tray will slide out and GEN5 software will prompt to place the plate into the reader
  23. The SensoPlate™ should already sit the plate holder. Click OK
  24. Select the appropriate well(s) to scan. Click OK
  25. Sample now are being scanned. Gen5 will automatically count after finishing the last scan. A warning “Failed to calculate …” may pop up. Click OK and proceed to the analysis and cell scoring as described below.

ANALYSIS AND CELL SCORING

  1. After the scan is completed, Gen5 will automatically run the initial analysis and the total number of possible cells will be given in the spreadsheet format.
  2. Select the appropriate well. From Image pull-down menu, choose Stitched GFP.
  3. Double click on Data Reduction on the left panel
  4. Tick “I want to edit an existing Image Analysis step”
  5. Select “Cellular Analysis Cell Count”
  6. Gen5 now re-run the analysis. Upon completion the results will be displayed in a panel on the right.
  7. From Object details pull-down menu, select DAPI+ AND RFP- Stitch
  8. The right panel now will display all possible objects meeting the criteria GFP+ DAPI+ and RFP- (corresponding to Cyto-keratin+, DAPI+, CD45-); it is normal to identify 1.5 to 2x more objects than there are cells at this point. However, if a very large number of objects are identified as possible CTCs there is probably an issue with automated recognition thresholds and they need to be adjusted.
  9. Move the mouse cursor over to the image on the left. Right click and select zoom. Repeat zoom as necessary until the display object is between 20 to 50µm
  10. Move the mouse cursor over to the right panel and click on any of the possible CTCs listed (in the example, these are object numbers 6, 9, 13, 92 etc...). The image on the left now displays the selected object somewhat at the center. Object being selected is highlighted red; all possible CTCs are highlighted pink; objects do not meet the criteria are highlighted yellow.
  11. In order to review all objects, start with the first object at the top of the list and determine if this is a CTC or not; continue by either clicking on the next row in the worksheet or by using the down arrow.
  12. Uncheck highlight objects. This will allow faster rendering of image to find the possible object for scoring. (Gen5 will re-highlight objects whenever the selection is moved to a different region of the image. This will take time and thus will slow down the image review process. For this reason, we suggest unhighlight normally and only re-highlight when needed.)
  13. In the above example, all objects automatically identified are CTCs
  14. In edge cases, turning off one or two of the 3 color channels captured may clarify the provenance of the object. This can be done by checking or un-checking the boxes pertaining to each channel on the far left of the panel.
  15. Continue arrow key to move down the list to confirm the selected objects are CTC and not debris.
  16. The list of possible objects (basically the CTC count without visual confirmation) can be export into an excel file.