Bone appositional rate and percentage of doubly and singly labeled surfaces: comparison of data from 5 and 20 microns...

On theoretical grounds, it seemed plausible that the apparent distance between two fluorescent labels can surpass the actual distance between them by a percentage that is related to the true distance: section thickness ratio. For similar reasons, the percentage of double labels discernible as such would be influenced by the same ratio. These assumptions were tested in a series of doubly labeled biopsies in which a large variation of appositional rates had been found. The apparent mean distance between labels and, hence, the appositional rate as measured in 20 microns sections was significantly lower than in the 5 microns sections from the same biopsies. Yet, 20 microns sections cannot be used to counteract distortion by oblique sectioning because of the bias obscuring the lower range of true interlabel distances. The percentage of doubly labeled surfaces was from 13 to over 50% lower in the 20 microns sections. Thus, overlap of double labels by geometrical projection in 20 microns sections causes a considerable reduction of formation rates derived from the parameters investigated. The problem is resolved, to a large extent, by using sections as thin as possible.