Methodological aspects of 3D and automated 2D analyses of white matter neuronal density in temporal lobe epilepsy

White matter neuronal density has been correlated with clinical outcome after temporal lobectomy for refractory epilepsy. Both morphometric 2D (two‐dimensional) and stereological 3D (three‐dimensional) analyses of neuronal density have been performed. 3D analyses are thought to be more accurate than 2D counts, but more time‐consuming. We compared 3D and automated 2D measurements in the same specimens. Adjacent 20‐µm (for 3D analyses) and 5‐µm (for 2D analyses) sections from 10 temporal lobectomies were stained for NeuN immunohistochemistry. Analysis of 100% of a region of interest (ROI) in deep white matter was performed using an image analysis system (Histometrix, Kinetic Imaging, UK). 3D analyses were undertaken using × 63 magnification (6 h/case). Automated 2D analyses were undertaken using automatic neuronal identification at × 10 magnification with three to four repeats (1.5 h/case). The range of neuronal densities for 3D measurements was 2120–4910 neurones/mm3, and for automated 2D measurements 17.4–47.1 neurones/mm2. There was a linear correlation between the two methods with an r2 of 0.58. The limits of agreement for the two methods were 1718 to −2234 neurones/mm3. Count–recount variability was 1.4–9.9% for the 3D and 5.1–36.6% for the automated 2D measurements. We found a wide range of white matter neuronal densities using either analysis. The low agreement between methods, and the high count–recount variability for the automated 2D analyses, indicate that despite being more time‐consuming, rigorous 3D stereological analyses have to be performed to obtain reliable results. These findings have implications for studies requiring neuronal counts in normal and disease states.