While these conditions are not optimal, there is nothing incorrect about them. Causes for this include 1.The “spillover” detector is set to a higher voltage, so that even if more photons are reaching the primary detector, the spillover detector gives a higher output, or 2.The primary detector has a very narrow bandpass filter, and/or the spillover channel has a very wide filter, so that more photons are actually reaching the spillover detector. This can still be valid, even though Diva gives an error message when that happens. Depending on your system setup, it is possible that you could have greater than 100% compensation, meaning your signal in a spillover channel is higher than in your primary channel. With the new digital systems today, the entire signal from each detector is quantified, and then compensation is applied mathematically. With small amounts of spillover this worked fine, but as you tried to compensate higher and higher values, it would be less accurate. From what I understand, this bias against high compensation is mostly a holdover from the early days of flow when compensation was done with pulse subtraction–a circuit would create a negative pulse proportional to the signal pulse in the primary channel to reduce the signal in the spillover channel and adjust for the spectral overlap. ISAC 2010 had a session about minimizing the compensation values for a panel, which some saw as a useless exercise. There is a stigma in flow against high compensation values, and their validity is called into question. ![]() ![]() Voltages can be set to give you almost any value for the compensation matrix, and spillover for a tandem can change over time as the reagent breaks down, resulting in different “correct” compensation values (one comparison of spillover from PE-Cy5 into PE was 45% for an old vial of reagent, vs 7% for an unopened one of the same lot at the same settings). The actual values in themselves don’t give enough information to make this determination. I often hear of people looking at a compensation matrix and deciding if it is “good” or “bad”.
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