Perls' Prussian blue staining and chemistry of Prussian blue and Turnbull blue

Perls’ Prussian blue staining or reaction is used to detect haemosiderin, which is stored or sequestrated non-haeme iron. Methodologically, the iron in haemosiderin is released as Fe³⁺ (Fe^III) by hydrochloric acid (HCl), and Fe^III reacts with potassium ferrocyanide (K₄[Fe^II(CN)₆]) to form Prussian blue. Iron released from ferritin, another stored non-haeme iron, is too scarce to be detected. Haeme iron, including haemoglobin and cytochromes, is not released by HCl. Thus, haemosiderin can easily be detected under the microscope as distinct blue deposits with minimal background staining. The chemistry of cyanide-bridged iron complexes, including Prussian blue and Turnbull blue, is the basis for understanding Perls' staining. Prussian blue is a cubic lattice with Fe^II or Fe^III ions alternately aligned at the corners to give Fe^II–CN–Fe^III formations at the edges. Physicochemically, Prussian blue is soluble in water (dispersible as a colloid) or insoluble depending on how it is formed. As with Perls' staining, Prussian blue is expected to take a soluble form because of excess K₄[Fe^II(CN)₆] compared to Fe³⁺ released from tissues. Notably, Prussian blue used in Perls' staining does not in fact dissipate into the staining solution but remains on the tissue, rendering this a practical method for histological detection of haemosiderin and also exogenous iron of forensic significance. However, further examinations of its mechanisms are needed to evaluate the applicability of this method on various forensics cases.