Block Advance Causes Substantial Error in Stereology Estimates of Cavalieri Volume and Disector-Based Cell Counts

Background: Design-based (unbiased) stereology is considered the best practice for quantifying morphometric parameters of stained biological structures in tissue sections. These methods use systematic random sampling and unbiased geometric probes to ensure that estimates of stereology parameters converge on the expected (true) values as tissue sampling proceeds. A critical caveat is that the practical applications of these theoretically unbiased methods to biological tissue can confound the accuracy of stereology estimates. New Method: Here, we show that calculations based on block advance, i.e., the instrument setting, on sectioning partially processed tissue can introduce substantial nonrandom error into stereology estimates of total region volume ( \( \hat{V_{\mathrm{Ref}}} \) ) by Cavalieri point-counting and total cell number ( \( \hat{N_{\mathrm{Cell}}} \) ) by disector-based methods. Comparison with Existing Method: We provide proof that the use of block advance produces a similar error for both \( \hat{N_{\mathrm{Cell}}} \) calculated by the volume-based two-stage (NV · \( \hat{V_{\mathrm{Ref}}} \) ) approach and fraction-based methods. Finally, a formula is given for removing this error from \( \hat{V_{\mathrm{Ref}}} \) and \( \hat{N_{\mathrm{Cell}}} \) using the final post-processing section thickness.