All tests have limits, there is no 100% in any test. For instance, in order to even start the process, RGCC needs to identify and isolate the CTCs before proceeding to the next step of analysis.

There are a few cases that CTC may not be identified hence the process will not proceed and declared as 0 CTCs. Common reasons for this occurring are as follows:

1. The sample was not properly preserved during transportation and the CTCs were destroyed. There is a minimal chance that this was the case since the sample successfully passed the initial QC.
2. The sample was properly collected, passed QC check, and the test ran and detected the protein markers CD31 (presence of endothelial cells) and panCK (epithelial cells) but no CTCs. The presence of endothelial cells is considered a contaminant from the blood draw, which can mask or hide a low CTC count and can result in a 0 CTC count. This is an uncommon finding, but it does occur. The test does not necessarily call for an immediate new blood sample, but the patient should be informed that the test may be falsely negative and should be repeated in a few months. If a repeat sampling in 3-6 months reveals 0 CTC count and the biomarkers CD31 and panCK are both negative, there is a high confidence that the test result is correct in not detecting CTC, but it still does not eliminate the possibility of false negative if it is below the level of detection.
3. The patient received a systemic therapy (chemotherapy agents, steroidal agents, anti-hormonal therapies, immunotherapy, or targeted therapies), this will eliminate the CTCs from the peripheral circulation (and it will affect the solid tumor in a later stage). For this reason, the guidelines are specific that the blood sampling should take place 14 days after suspending those kinds of therapies.
4. There is a chance that CTCs will merge or fuse with monocytes and generate hybrid cells which are very difficult to isolate, and the approach of isolation is only on the size and content of the nucleus. This kind is rare, but the isolation approach should be different in these cases which means a more time-consuming approach is required. In these cases, we will need to redo the analysis utilizing different steps of isolation.

Also, we must keep in mind the following facts:

1. OncoTrace has specific values of sensitivity (86.2%) and specificity (83.9%). In this case the sensitivity is important, because at the level of 86.2% there is a 13.8% chance that a false negative may occur for the four reasons stated above.
2. We also recommend the OncoTrace as a follow up tool. It is not the ideal test for screening purposes because there is a very low limit of detection (1.3 CTC in 10 million WBC or 1 CTC in 25 mL of whole blood). This is exactly the reason why the new Onco-D-Clare test was developed.