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)	CF	D (μm)
3	12.3	3.6	8.7	0.29	7.29
4	13.3	4.8	8.5	0.36	7.12
5	14.7	6.0	8.7	0.41	7.32
6	15.7	7.2	8.6	0.46	7.17
9	19.4	10.7	8.6	0.55	7.24
Mean			8.6		7.23
1 SD			0.1		0.08

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.