What happened to my red laser fluorophore signals!?

From: Allen, Chris
Sent: Wednesday, March 30, 2011 10:47 AM
To: Baumjohann, Dirk
Subject: RE: LSR II

Hi Dirk,
The problem you are describing is likely caused by a pressure/laser delay issue, not bubbles. In my experience, about 90% of the time, losing just the red laser signal is due to a pressure/laser delay problem, which is usually caused by priming itself (although many people seem to think priming is a good idea for all kinds of problems, most of the time it can actually cause more problems than it solves). Allow me to explain and then I'll give some suggestions. The way the system works is that is uses pressure to push everything along. Air is pumped into the sheath tank, pushing down on the fluid, which makes the fluid travel into the tubing. Air is also pumped into your sample tube and this pushes the fluid down in your sample, making it go up the probe. Because of this pressure-induced fluid flow, cells pass by the blue laser first, and then after a delay in time (laser delay), the cells hit the red laser. This laser delay time depends on two main factors, the distance between the lasers and the speed the cells are moving past them. The speed is of course primarily controlled by the pressure. Each time you run the CST beads, you are calibrating the laser delay.

When you prime the system, it drains all of the fluid out of the flow cell and fills it with air, and then fills it back up with fluid. It is analogous to trying to get rid of a bubble in a syringe by taking up a lot of air and then pushing it back out. When you do this, you will always see air in the waste line. So that is normal and does not indicate there was a bubble in the flow cell. However, after priming the system, it takes as much as 5 minutes for normal pressure to be restored. During that time, the pressure will fluctuate. Thus, let's say you prime the system and then immediately run the CST beads. This will likely set the wrong laser delay time, because the pressure has not reached equilibrium. Then later when the pressure does stabilize, the laser delay will be wrong, and the red laser signals will not be visible.

Conversely, if the CST laser delay is correct, but you prime the system and then run your sample, the pressure will also then be variable and the red laser signal will appear and disappear.

Most likely then, I think the issue is being caused by excessive priming. A related issue is that the air introduced into the waste line by priming can itself build up and create a "back-pressure" that can reduce the flow of the sample.
Thus, what I would try to is put the system on a water tube on RUN/HI and flick the orange waste line to try to get all the bubbles moving along (sometimes they get trapped at any bends in the tubing). Then let the system run for a full 5 minutes on water for the pressure to stabilize, and then run the CST beads. If you look at the CST report, the laser delay value should be near 60 or 61 for the red laser (you have to scroll down to the bottom). If it is, you should now be able to run your samples without a problem (just don't prime again!). If the laser delay has a different value, or the CST fails, then something else is wrong and you could indeed still have an air bubble. However, I think that is much less likely.

One other thing to check is to make sure the sample tubes being used do not have any cracks in them, as that can also affect the pressure in the tube if some air can leak out.

Usually air bubbles in the flow cell makes the signals get very low, particularly side scatter almost flattens out, or the signals appear noisy. In the unlikely event that there is a persistent air bubble, you can try an "aggressive debubble" procedure. Basically, you OPEN the sheath tank, and then hit prime, wait about 5 sec, hit run, and then repeat about 20 times. This will basically completely fill the system with air. Now, you seal the sheath tank, and drain the air out of the tubing on the side (there is a filter connected to the sheath tank, if you follow the tubing above that there is a little piece of tubing connected to a roller that you can use...roll out the clamp and drain the air and some fluid into a beaker until the air is gone). Now you have to prime the system many times and let it run the full cycle (about 20 sec) until it goes to standby to refill the system with fluid. Then you have to let the pressure stabilize as I mentioned above, try to flick the air bubbles in the waste line to move them along, hrun the system for 5 or 10 min, and then try running CST.

I will at Mt. Zion for much of the day, but let me know by email if you are able to get things working by flicking the waste line, letting the system re-run for a few minutes, and re-running the CST beads.