Comparison of total lymphatic vessel density and hot spot analysis. Hot spot analysis and total LD were calculated. Hot spot analysis assessed lymphatic number by counting the number of immunohistochemically stained lymphatic vessels, in three regions with the highest LD, as assessed by eye, in a 100 μm border around the tumour. A) Hot spots (p < 0.05, unpaired t test) in 12 patients that later developed metastases (metastatic) and 12 patients which after 9 years follow-up did not. Error bars represent standard error of the mean. B) Total epi-tumoural LD and hot spot analysis were significantly correlated (p < 0.0001, Pearson, r
2 = 0.50). C) The Shields Index and hot spot analysis significantly correlated together (p < 0.01, Pearson, r
2 = 0.30) D) Time taken to calculate a Shields Index value compared to the time taken to calculate hot spot analysis in the same subset of patients (p < 0.0001, unpaired t test). E) Shields Index calculated by LD or hotspot are not different from each other.