I invented the liquid nitrogen freeze method while working on a project for
EPA. I found that neither the pin prick nor Steve Tabor's - cut and
freeze in freezer- produced consistent results. The problem is that physical
damage (pricking, cutting out bits of comb) can induce a repair behavior.
Hygenic behavior is supposedly controlled by two genes, each with a bit
different behavior. Also, removal of paper is probably not a good test -
its just part of a two step process.
As per area of brood - the larger the area you kill, the more certain you
will be to see bees take action - that's again not simply hygienic
behavior, but a response to a damage 'crisis'.
What most have forgotten - our data showed that several small patches over
more than on brood frame provided the most reliable test.