Journal of Clinical Pathology, 1978, 31, 784-790 Histological demonstration of copper and copper-associated protein in chronic liver diseases S. JAIN, P. J. SCHEUER, BARBARA ARCHER, S. P. NEWMAN, AND SHEILA SHERLOCK From the Departments of Medicine, Histopathology, and Physics, Royal Free Hospital, Pond Street, London NW3 2QG, UK SUMMARY Liver copper concentrations in percutaneous biopsy specimens were measured by neutron activation analysis and compared with histological staining for copper by rubeanic acid and rhodanine, and with copper-associated protein stained by orcein. Liver copper concentrations were elevated in 31 of 35 biopsies from patients with primary biliary cirrhosis (PBC), and discrimination between normal and elevated liver copper was correct in 32 of the 35 biopsies by staining with rubeanic acid, and in 31 of the 35 by staining with rhodanine. Orcein staining of copper-associated protein was positive in 33 of the 35 biopsies. All 17 biopsy specimens from patients with Wilson's disease had high liver copper concentrations, but only nine had positive staining for copper, and six were orcein positive. Similarly, histological stains gave little indication of the liver copper con- centrations in tissue from 16 patients with chronic active hepatitis. Staining of liver sections can be useful in detecting elevation of liver copper in PBC, but not in Wilson's disease, where the absolute concentration must be measured. Excess copper appears to accumulate in the liver in different chemical forms in PBC and Wilson's disease. Hepatic copper concentration is high in patients This suggests that copper is retained in copper- with Wilson's disease (Cumings, 1948) and is protein complexes in the cytoplasm (Salaspuro and responsible for the liver damage (Sternlieb, 1972). Sipponen, 1976; Sipponen, 1976) as a result of Patients with the untreated disease usually have cholestasis of any cause. liver copper concentrations above 4 0 mmol/kg of In order to evaluate different methods of demon- dry liver, normal range 0-25-0 90 mmol/kg (Sternlieb strating excess copper in patients with chronic and Scheinberg, 1968). Approximately 80 % of liver disease, we have compared the results of liver absorbed copper is normally excreted in bile and copper measurement by neutron activation analysis lost in the faces (Cartwright and Wintrobe, 1964), with copper staining by rubeanic acid and rhodanine, and the cholestasis of primary biliary cirrhosis and with staining of copper-associated protein by (PBC) and long-standing extrahepatic biliary ob- the orcein method. struction can cause elevation of liver copper concen- trations above 40 mmol/kg (Hunt et al., 1963; Material and methods Smallwood et al., 1968). Some patients with chronic active hepatitis (CAH) and alcoholic cirrhosis also NEUTRON ACTIVATION ANALYSIS (NAA) have raised liver copper concentrations, but not to Percutaneous liver biopsies were performed with the levels found in Wilson's disease and PBC Menghini needles, which were prepared by washing (Smallwood et al., 1968; Fleming et al., 1974). in a 1 % solution of ethylene-diamine-tetra-acetic Cytoplasmic material, which stains with orcein, acid (EDTA) to remove surface copper. A sample of has been found in the same cellular and subcellular each biopsy was separated, freeze-dried, weighed, locations as stainable copper in liver biopsy speci- and submitted for NAA by the technique of Todd mens from patients with prolonged cholestasis. et al. (1967). HISTOLOGICAL STAINING METHODS Received for publication 12 January 1978 Paraffin sections of liver biopsy specimens, 4-5 784 Histological demonstration of copper and copper-associated protein in chronic liver diseases 785 microns, were fixed in neutral buffered formol- The sampling error of copper measurement in saline. The sections were stained with rubeanic cirrhotic livers was assessed by taking multiple acid (Cook, 1974), with reagent supplied by Raymond cores with a Menghini needle from a cirrhotic liver A. Lamb, London, using the staining solution at obtained at necropsy. The coefficients of variation room temperature for 24 hours, and with rhodanine of the liver copper concentrations ranged from (BDH Chemicals Ltd) by the method of Lindquist 361 %, when portions of less than 1 mg dry weight (1969). The sections were also stained by Shikata's were compared, to 4.0 % on samples weighing orcein method (BDH Chemicals Ltd), using a 3-5 mg. These results show that, provided more modified oxidising solution (Deodhar et al., 1975). than 3 mg of liver (10 mg wet weight) is used, This stains sulphonic acid residues formed from sampling error is unlikely to be important as the sulphydryl groups during oxidation, including variation is within that of the measuring technique. hepatitis B surface antigen (HB5Ag) (Shikata et al., These results are similar to those of Fleming et al. 1974), a copper-associated protein (Salaspuro and (1974), using atomic absorption spectrophoto- Sipponen, 1976), and elastic fibres. metry. Each slide was examined by one observer (PJS) without knowledge of the liver copper concentration HISTOLOGICAL STAINING METHODS of the biopsy specimen, and the degree of staining by In order to evaluate the three methods used for each of the three methods was scored 0 to 3. where copper and copper-associated protein, the effect of O was negative, 1 minimal, 2 positive, and 3 heavy prior removal of copper from the sections was staining (Fig. 1). Both extent and density of staining investigated. Sodium diethyldithiocarbamate (DTC) were taken into account. (Hopkin and Williams Ltd), which forms a coloured complex with copper (Howell, 1959) and which can Evaluation of methods be dissolved in alcohol, was used to remove copper from sections (Scheuer and Barka, 1963). Because NEUTRON ACTIVATION ANALYSIS removal of the coloured compound can be checked The reproducibility of the technique was tested by optically, this method was preferred to non-specific measuring the copper concentration of differing copper-removing procedures such as the per- weights of a powder made up of glucose and copper manganate-oxalic acid step in the orcein method sulphate, with a copper concentration of approxi- (Deodhar et al., 1975). Mallory's haematoxylin mately 3 mmol/kg. The weights of the samples method for copper (Mallory and Parker, 1939) was submitted for analysis ranged from 1 to 10 mg. The also used because this method is thought to rely on coefficient of variation in 15 samples was 12.4%. the presence of metallic copper to mordant the r .t. ys -4ik .. ..tt # ... KBl;i x<Ub X t '** wox :e .e . ~ ~ W O. .pA lf .-f': .,, e:- Fie-S1,. A1 GradeVtf ""CJ3 *x xAs r copper staining in periportal liver cells ,,> s in primary biliary *!>g ov §§S,.P cirrhosis. Rhodanine b: # , 1 t x 380 lp ^ { 1.1'I* -4. - . 4ni r9 l,s * & h k:'iM....g: U ;S !B jr-,'?.t 4.. ;} 14 .R JI A.,* ' r ~~~-# YE ZX' ..::3w.~~~A 786 S. Jain, P. J. Scheuer, Barbara Archer, S. P. Newman, and Sheila Sherlock haematoxylin, and is thus considered very specific There was no difficulty in distinguishing orcein- for the method. positive granules of copper-associated protein As shown in Table 1, staining by Mallory's (Fig. 2) from hepatitis B surface antigen in liver-cell haematoxylin, rhodanine, and rubeanic acid was cytoplasm or elastic tissue which orcein also stained. prevented by prior removal of copper by means of Lipofuscin pigment was also easily distinguished DTC and alcohol. Shikata's orcein staining was from copper-associated protein, for it did not stain not affected. This is in agreement with the conclusion with orcein and was usually in a different location, of Salaspuro and Sipponen (1976), that orcein being found mainly around centrilobular veins. stains a copper-binding protein rather than copper Copper-associated protein was mainly periportal, itself. Problems of technique and interpretation of and was often seen in areas of piecemeal necrosis the different staining methods were relatively minor. within the cytoplasm of swollen liver cells. In some There was gradual fading of rhodanine staining biopsy specimens, particularly those with abundant over a period of several days, particularly in biopsy orcein-positive granules, the presence of copper- specimens from patients with PBC and when copper associated protein could be predicted in sections was scanty. A similar problem was noted by Lindquist stained by other methods; the material often (1969), using Permount as a mounting medium for appeared as grey pigment granules, somewhat the sections. In our experiments, the degree of resembling intracellular deposits of bile, and was fading differed according to the mounting medium usually PAS positive after diastase digestion. used. This was greatest with Eukitt, less with DPX and Diatex, and least with Ralmount (all products Patients of Raymond A. Lamb). Liver copper concentration and staining for copper and copper-associated protein was performed on Table 1 Effect ofremoval ofcopper from sections biopsy specimens from six patients who were found by DTC and alcohol on copper staining methods to have no liver disease, 35 patients with PBC, and Stain 17 with Wilson's disease (six were on D-penicillamine treatment). Sixteen patients with CAH were studied; Mallory's Rhodanine Rubeanic acid Shikata eight were HBsAg positive, and eight were HB8Ag haematoxylin negative women with clinical and serological Untreated + + + 4 features of 'lupoid' hepatitis receiving oral cortico- Copper removed - - - + steroid therapy. .. 4 I. 4! i',ie *.::Ad.-