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I only have a minute or 2, so this has to be a short message, but I get essentially the same number (ie, to within a percent or better) for both images that you sent me:

From the JPG: 225,350 non-transparent pixels

From the NEF: 225,632 non-transparent pixels

All I did was control-click on the "Pen" layer in both.  That made a selection out of the non-transparent pixels, and then I read out the number of pixels at the bottom of the histogram window.

I think you are saying that you get the same results as me, but that when your PSD file includes two layers, (not just a single layer like in the ones you sent), then you get different numbers.  If this is correct, then the only way I can check this is (and hopefully, tell you what's going on) is if you send me a file with two layers.

Gotta run.

Tom M