It can be hard to measure the quality of cleaning. All flow core facilities have a posted cleaning protocol for users to follow once they finish their runs. Typically they involve some combination of bleach and water for 1-5 minutes each at a high flow rate. Cytometry cleaning protocols are designed to briefly decontaminate the system (OSHA recommends 10 minutes for equipment with a rinse to remove the residual bleach). The problem is it’s often difficult to get your users follow these protocols correctly.
Bleach is a chemical and can cause chemical reactions. I had an investigator come into my office the other day, puzzled over a problem with a cytometer. “My APC signal is gone. PE is fine, but APC is gone. Is it possible that the instrument is broken?” After talking with the investigator for a few minutes, I learned the he was setting the voltages using peak 6 beads. When comparing the PE and APC signals, there was a small shift in the PE signal, but the APC single was dramatically reduced. The investigator turned up the voltage to get the signal to where it should be, but when he put on his compensation control it was off-scale. I had seen this particular problem before and knew it was caused by residual bleach in the system.
The instrument that produced the below data was washed for 5 minutes with 10% bleach, followed by a water tube being placed on the sample injection port (SIP). Peak 6 beads were run and median fluorescent intensity was measured (shown in red). A second experiment was performed where the bleach was run for 5 minutes, followed by a 5 minute water wash (shown in blue). Look at the difference:
Mathematically, the data show:
|
PE (MFI) |
APC (MFI) |
|
|
Bleach |
9563 |
14948 |
|
+5 minute water |
9806 |
22585 |
|
% difference |
2.5% |
51.1% |
This simple experiment illustrates two things that many people forget, and is often not covered in basic instrument training
1. Always do the full wash after using bleach
2. APC is exquisitely sensitive to bleach
I recommend your users put on a fresh water tube, running it on high while they get their templates imported and setup.
