in my first reaction to @mauricetoet’s question in this post I explained everything I think. As everyone could see, Maurice was able to perfectly calibrate his f/2.8 DSLR data with flat & light frames which had different iso values. Off course, his data is much more sensitive to a possible bad calibration with his fast focal ratio.
I offer you to demonstrate it on your data, it’s no trouble. I can even make a movie of it if you wish?
I would only need 2 bias frames of iso 1600 & iso 200, 2 flat frames, and 1 light frame to show you how it’s correctly done.
It really isn’t very complicated and it is done in a couple of minutes time.