Table 2.

Validation of the CF using the multiple section thickness method

Section Thickness (T) (μm) Podocytes per Cross-Section (No) (n)Podocytes per Nuclear Tuft (Nt) (n)Extra Podocyte Nuclei (Ne) (n)CFD (μm)
312.33.68.70.297.29
413.34.88.50.367.12
514.76.08.70.417.32
615.77.28.60.467.17
919.410.78.60.557.24
Mean8.67.23
1 SD0.10.08
• As the section thickness increases, the observed (apparent) number of podocytes per tuft cross-section (No) increases as shown in Figure 5, C and D. Figure 5C shows that the true podocyte number is given by y=mx, where x is the section thickness. From the slope of the line in Figure 5D, the value for m is 1.19. Therefore, the true number of podocyte nuclei per tuft (Nt) can be calculated for each section thickness as shown in Table 1 (Nt=1.19×T). The number of extra podocyte nuclei (Ne) counted in any glomerular cross-section due to nuclei bisected at the edge of the section (as illustrated in Figure 5A) is derived from No−Nt and, as expected, is a constant value whatever the section thickness (8.6±0.1 μm). The CF is given by Nt/No for any section thickness as shown in Table 1. The value for the mean caliper diameter of podocyte nuclei can be derived from the formula D=(1/CF−1)×T by substituting known values for the CF and section thickness T. The value for D derived for rat podocyte nuclei (7.2±0.1 μm) is not significantly different from the value directly measured in thick sections of 7.1±0.1 μm.