Is Perls stain specific for hemosiderin?

The paper by Thulborn et al. [1] on the distribution of ferritin and hemosiderin in cerebral hemorrhage implies that Perls Prussian blue stains hemosiderin and does not stain ferritin. This was most surprising to us, as we had just obtained excellent Perls stains of ferritincontaining regions of the brain [2] (Fig . 1 ). We think that given the current interest in iron in the brain and its effect on MR, clarification of the specificity of Perls stain is essential before this misconception is propagated further. The Perls method was developed in 1867 and, as described in standard histochemistry texts , is a stain for ferric iron , not just hemosiderin. It is based on the formation of ferric ferrocyanide (Prussian blue) that occurs when ferric ions, released by hydrochloric acid from iron-containing compounds, react with potassium ferrocyanide. The sensitivity can be intensified by using diaminobenzidine (DAB), as the ferrocyanide of the Perls reaction catalyzes the oxidation of DAB, which forms an insoluble brown precipitate [3]. The assumption by Thulborn et al. may have stemmed from earlier reports [4] that hemosiderin is more sensitive to the Perls reaction than ferritin is . This difference in earlier histochemical methods was due to leaching of iron in water-soluble compounds (ferritin is water soluble; hemosiderin is not). This problem nowadays is overcome by using the Perls-DAB method and cryostat-cut sections [3] . For example, in our study [2], we not only stained ferritin-containing brain nuclei with Perls-DAB but also correlated the optical density of the stain with the magnitude of MR hypointensity. Nevertheless, we do not dispute the conclusion of Thulborn et al. that ferritin contributes to hypointensity on MR images of late hemorrhage. After all , ferritin and hemosiderin have similar magnetic properties , and the effect of ferritin on MR has long been noted [5 , 6].