A question on ImageQuantTL
I would definitely go for option A. If you are assuming that the staining in band 6 is all due to whatever you are measuring, there is no reason to think that the signal at the top of lane 1 is not meaningful.

That said, this type of measures is only semi-quantitative, so being too "picky" in how you choose your region probably won't really increase the precision of your result. If you want to get a quantitative measure I am afraid you will have to turn to some other technique.

Finally note that the area obviously means a lot if you are looking at total intensity of the bands, rather than average intensity.