Dear Editor,
We read with interest the study by Creutzenberg et al. (Citation2012) and Schaudien et al. (Citation2012) on the presence of nanoparticles in vivo following lung exposure in rats. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultra-thin lung slices. A particular methodological challenge is the two dimensional presentation of larger particles in the ultrathin TEM section planes and the analytical approach to extract informative data thereof. The typical thickness of an ultrathin section is about 80 nm or smaller (CitationHayat, 1986). Therefore, a larger particle (e.g. with a diameter of 300 nm) is cut into a slice at a random section plane. The profiles of this slice visible and measured in the TEM sections predictably underestimate the true particle diameter: this is the TEM slicing bias. This slicing bias is amplified by the systematic oversampling of large particles (Boyce et al., Citation2010; Cruz-Orive, Citation1987; Hsia et al., Citation2010). We developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias and contrasted observed and expected numbers in 2 × 2 tables by odds ratios (Morfeld et al., Citation2012). Our results showed pronounced overestimates of the prevalence of small particles if the bias is not taken into account. Creutzenberg et al. (Citation2012) and Schaudien et al. (Citation2012) did not take care of the TEM slicing bias in their analyses. The results presented in Table 3, Figure 2 and Figure 3 in Creutzenberg et al. (Citation2012) are distorted suggesting erroneously the presence of too many small particles. This may irritate readers who are interested in the question whether nano-objects are present in the lung after exposure to nanostructured materials. As an example for such an irritation we note that the MAK committee appears to take such an observation for granted: “Ultrafine primary particles are measured according to their mobility-equivalent diameter (DM) <100 nm (corresponds to a diffusion-equivalent diameter (Dae) <100 nm). They can occur as single particles in the workplace air or more often as basic units of aggregates and agglomerates. In these forms they can be seen under an electron microscope”. (Deutsche Forschungsgemeinschaft, Citation2012). We would suggest correcting the particle size distributions given in Creutzenberg et al. (Citation2012) for the immanent TEM slicing bias to avoid confusion.
Declaration of interest
BASF SE and Evonik Industries produce TiO2 or produce products containing TiO2.
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- Boyce RW, Dorph-Petersen KA, Lyck L, Gundersen HJ. 2010. Design-based stereology: Introduction to basic concepts and practical approaches for estimation of cell number. Toxicol Pathol 38:1011–1025.
- Creutzenberg O, Bellmann B, Korolewitz R, Koch W, Mangelsdorf I, Tillmann T, Schaudien D. 2012. Change in agglomeration status and toxicokinetic fate of various nanoparticles in vivo following lung exposure in rats. Inhal Toxicol 24:821–830.
- Cruz-Orive LM. 1987. Particle number can be estimated using a disector of unknown thickness: The selector. J Microsc 145:121–142.
- Deutsche Forschungsgemeinschaft (DFG). 2012. List of MAK and BAT Values 2012: Maximum Concentrations and Biological Tolerance Values at the Workplace, Chapter 5 (Aerosols), p. 192. Available at: http://onlinelibrary.wiley.com/book/10.1002/9783527666034. Accessed on 29 October 2012.
- Hayat MA. (1986). Basic techniques for transmission electron microscopy. Orlando: Academic Press.
- Hsia CC, Hyde DM, Ochs M, Weibel ER. 2010. An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure. Am J Respir Crit Care Med 181:394–418.
- Morfeld P, Treumann S, Ma-Hock L, Bruch J, Landsiedel R. 2012. Deposition behaviour of inhaled nanostructured TiO2 in rats: Fractions of particle diameters below 100 nm (nanoscale) and the slicing bias of transmission electron microscopy. Inhal Toxicol 24:939–951.
- Schaudien D, Knebel JW, Mangesldorf I, Voss J-U, Koch W, Creutzenberg O. 2012. Dispersion and retention of dusts consisting of ultrafine primary particles in lungs. Research Project F 2133. Dortmund/Berlin/Dresden: Federal Institute for Occupational Safety and Health (BAuA). Available at: http://www.baua.de/en/Publications/Expert-Papers/F2133.html. Accessed on 29th October 2012.