An unbiased correction factor for cell counts in histological sections

Abstract

It is common procedure to correct raw counts of cells (or nuclei or other such particles) in histological sections by multiplying them by a ‘correction factor’, to allow for sectioned particles being counted more than once. However, the derivation of the standard formulae assumes that the particles to be counted are of uniform size. Therefore, these formulae may be biased in practical situations. Here, a more general correction factor (C) not depending on this restrictive assumption is derived: C= ∫ m i=1 T/(T−h i/ ∫ m i=1 (T−R−S+h i/(T−h i) where T is section thickness, h i is the height of particle i measured perpendicular to the section plane, R and S are the heights, assumed constant, of the upper and lower ‘lost caps’, and the summation is performed over m (about 20) particles sampled randomly from among those lying wholly within individual sections. It is proposed that h i be measured by a differential focusing method; the formula will then be valid for particles of variable shape as well as size.