How thick is your section? The influence of section thickness on DNA-cytometry on histological sections

The exact determination of the thickness of paraffin sections used for DNA cytometry on histological slides is an inevitable prerequisite to obtain reliable and reproducible results. Although section thickness can be measured with great precision and accuracy using highly specialized equipment (e.g. confocal laser microscope, microinterferometer), their cost restricts availability for many laboratories. This paper describes a simple method to determine the actual thickness of routinely processed paraffin sections used for DNA cytometry employing standard laboratory equipment. To achieve this goal the given section is divided into two parts. One part is used for DNA quantitation whereas the other is re-embedded and resectioned orthogonally through its original thickness. The thickness of the re-embedded section can now easily be determined with any standard length measurement device available in light microscopy. Recalculation of the genuine DNA-content out of the measured fractioned nuclei with commercial software based on the section thickness determined by this method generated histograms with a 2c equivalent peak, whereas correction based on the microtome settings resulted in significant deviations as demonstrated on rat liver tissue.