Electron microscopic detection of copper in the liver of two patients with morbus Wilson by EELS and EDX.

A 20-year-old male patient with morbus Wilson was liver transplanted because of terminal failure of liver function. The explanted liver showed a strong macronodular cirrhosis as typically seen in Wilson disease. There were visible granular accumulations in the hepatocytes after the rubeanic acid or rhodanine method for histochemical detection of copper. The electron microscopic studies on ultrathin sections revealed numerous electron-dense lysosomes and residual bodies. The elemental analysis in transmission electron microscope (TEM) with electron energy loss spectroscopy (EELS) and in scanning electron microscope (SEM) with energy dispersive x-ray analysis (EDX) showed copper-specific signals of electron-dense accumulations inside these dark lysosomes and residual bodies. In a second case, Wilson disease was diagnosed after autopsy of a 31-year-old patient by liver electron microscopy and EELS; strong electron-dense lysosomes and residual bodies with positive copper signals were found inside hepatocytes. For negative control, hepatocytes with iron accumulation after idiopathic hemochromatosis and liver cirrhosis were also analyzed by EELS in TEM, which showed strong iron, but only a few or no copper signals. Atomic absorption spectroscopy (AAS) in 16 liver samples of healthy and cirrhotic liver revealed only in both cases of Wilson disease a strong increased copper concentration higher than 100 microg Cu/g. The electron microscopic detection of copper-containing hepatocytic lysosomes is helpful for the diagnosis of early stages of Wilson disease in addition to the quantification of hepatic copper by AAS.