I've found an interesting issue with AGS 3.2.1. If you compile a game in 32-bit color, and you have a GUI that has no background picture set, then any 16-bit button pics on the GUI will not be displayed. The workaround is to set a 16-bit background picture that is blank, and it is also averted by using winsetup to select "downgrade 32-bit to 16-bit".
http://www.adventuregamestudio.co.uk/forums/index.php?issue=498.0
In related news, if you have a 32-bit game that contains a 16-bit room... if you do RawDrawImage within this room, that works fine. However, if you save the game in this room, quit, then restart the game and restore, only half of the background will have been saved in the save file, and the rest is garbled or blank.
Also, if I understand correctly, certain functions like Label.TextColor still expect a 16-bit color value even in 32-bit mode, whereas others like game.read_dialog_option_color expect a value to match the color mode, and yet others like SetFadeColor expect a 32-bit color value even while in 16-bit mode. Probably worth mentioning in the manual, at least.
I also ran into this. I'd like to mention, that you also can't raw draw 16bit sprites on a 32bit GUI, even if it has the blank backgroundsprite. On room surfaces this is perfectly possible.
--edit: oops sorry: my dynamic sprite had an alpha channel. Without the alpha channel, it works for the GUI and 16bit sprites.