Value of histochemical stains for copper in the diagnosis of Wilson's disease.

AIMS: The histochemical demonstration of hepatic copper is important in the diagnosis of Wilson's disease (WD). Conflicting results have been published with regard to the ability of different histochemical methods to demonstrate copper storage in the liver. Therefore, we evaluated the diagnostic value of three available histochemical methods in a large series of patients affected by WD. METHODS AND RESULTS: Seventy-nine consecutive liver needle biopsies, from 74 patients, 39 males and 35 females, aged 4-60 years (mean age 28.5 years) were stained with orcein, rhodanine and using Timm's method. On the basis of the histological picture, liver biopsies were subdivided into three groups: group A, steatosis; group B, interface hepatitis; group C, chronic hepatitis with bridging fibrosis and/or cirrhosis. In group A, 30.4% of the cases were positive using Timm's method, vs 13.2% using the rhodanine and 17.5% using the orcein method. In group B, Timm's method was positive in 40.1% while rhodanine and orcein showed positivity in 26.7%. In group C, the Timm's method stained 58.6%, rhodanine 36.6% and orcein 29.3% positively. CONCLUSIONS: Our data show that: (1) Timm's silver stain is the most sensitive method for the demonstration of copper in all cases of WD; (2) rhodanine and orcein have minor value in the diagnosis of WD, especially in the early stages of the disease; (3) to increase the diagnostic value of histochemistry for copper multiple histochemical stains in serial sections are required; and (4) although hepatic copper concentration is highest in the early stages of WD, the histochemical demonstration fails in a large number of cases.

Chronic caregiving stress alters peripheral blood immune parameters: the role of age and severity of stress.

BACKGROUND: To examine the impact of chronic psychological stress on the immune system, a series of cellular and humoral immunological parameters was compared in 18 female caregivers of handicapped people and 18 age- and sexmatched controls. METHODS: The immunological parameters included assessment of T cell number (T cells, T helper, and T suppressor/cytotoxic) and function (delayed-type cutaneous hypersensitivity), antibody titers for latent herpesviruses (cytomegalovirus and herpes simplex virus 1 and 2), and markers of inflammation (complement C3 and C4 factors and c-reactive protein). Serum immunoglobulins (IgG, IgM, IgA, IgE) and titers for the nonlatent virus roseola were used to control for nonspecific elevations in serum proteins. Results were associated with the age of the investigated subjects, the severity of stress (family burden) and the degree of disability of the handicapped people. RESULTS: Caregivers had a significantly lower percentage of T cells, a significantly higher percentage of T suppressor/cytotoxic cells and a significantly lower T helper:suppressor ratio. Subjects were also analyzed after division into two groups according to the median age (45 years). Compared to their matched controls, older caregivers (mean age = 50.3) also had lower numbers of T cells and T helper cells and higher antibody titers for cytomegalovirus. In addition, in the caregiver population severity of stress was significantly positively correlated with T suppressor/cytotoxic cells and negatively correlated with T helper:suppressor ratio. No other differneces in the immune parameters were found between caregivers and controls. CONCLUSIONS: The results indicate that psychological stress differentially affects various aspects of the immune system and confirm the relevant role of age and severity of stress in modulating these influences.

Zinc content and distribution in the newborn liver.

The newborn liver is a proven model for the study of liver storage of copper and iron. We analyzed zinc concentration and distribution in the livers of newborns and infants using a systematic tissue-sampling technique. We studied 14 newborns of 26-41 weeks of gestation (WG). One stillborn, and three infants (52-90 days old). At autopsy, a longitudinal liver slice extending from the right to the left lobe was subdivided into 10 samples that were analyzed for zinc concentration by atomic absorption spectroscopy. The mean zinc concentration in the newborn liver was 639 micrograms/g of dry tissue (dt). A striking interindividual variability in zinc liver stores was observed; the hepatic concentration of the metal ranged from 300 to 1,400 micrograms/g dt. We found a correlation between zinc liver content and gestational age. In newborns of 27-32 WG, the hepatic zinc concentration was significantly higher (p < 0.01) than in newborns of 34-41 WG. Zinc stores decreased in the postnatal period; in the infant group, the mean liver zinc concentration was 148 micrograms/g dt. The analysis of zinc concentration in 10 blocks from each liver revealed a regular distribution of the metal, without significant differences between liver lobes. Our data show that the newborn liver can be considered an interesting model for the study of zinc storage, which appears to correlate inversely with gestational age. From a practical point of view, the observed regular distribution of zinc implies that, at least in this model, zinc content determined in a small liver sample is representative of zinc content in the whole liver.

Iron concentration and distribution in the newborn liver.

Recent observations on a correlation between fetal serum ferritin and gestational age, consistent with an increase in fetal iron stores during pregnancy, led us to study liver iron content in 22 human stillborns, newborns and infants of different gestational and postnatal age. At autopsy, a longitudinal liver slice was subdivided into ten blocks. Each sample was analyzed for iron content by atomic absorption spectroscopy. The mean iron concentration in the studied livers was 21.6 microM/g dry tissue (d.t.). A striking interindividual variability in iron content was observed: the hepatic concentration of the metal ranged from 3.3 to 64.4 microM/g d.t. No correlation was found between the hepatic iron concentration and gestational age or other clinical parameters of the patients studied. Moreover, the total storage iron of the liver did not appear to be correlated with the gestational age. The analysis of iron concentration in ten blocks in each liver revealed an irregular distribution of the metal. Lobar differences were observed, with a tendency of the left lobe to accumulate more iron than the right one. Furthermore, striking differences in iron content were found between adjacent liver samples, ranging in one instance from 4.5 up to 109.0 microM/g of dry tissue. Perls' stain for iron was positive in 7 out of the 22 livers examined, showing an irregular acinar distribution, with preferential periportal localization. Our data show that the newborn liver can be considered an interesting model for the study of iron storage.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning electron microscopy of chronic hepatitis C. An OsO4 maceration study on human biopsies.

A study at the scanning electron microscope (SEM) on the liver changes in chronic hepatitis C was carried out in human needle biopsies from four patients. Intracellular structures were visualized by a novel modification of the OsO4 maceration method that allows to investigate human pathological specimens. At low magnification we observed both sinusoidal and hepatic cells alterations: sinusoids appeared occluded by lymphocytes, hypertrophic Kupffer cells, activated perisinusoidal cells, necrotic material and apoptotic bodies. Some hepatocytes showed ballooning, arrangement in rosettes, and structural changes related to apoptosis: cell rounding, detachment from neighbouring cells, clustering of cytoplasmic organelles and cell fragmentation. We also found periterminal, sinusoidal, and pericellular severe fibrosis, and bile duct damage of moderate degree. At higher magnification, after removing the intracellular matrix, all the intracellular structures appeared normal, except for focal dilatation of smooth endoplasmic reticulum. Our findings clearly demonstrate the usefulness of the OsO4 maceration method for the study of chronic hepatitis and of liver disease in general. Thank to this technique, in fact, SEM becomes a diagnostic tool complementary to light microscopy and transmission electron microscopy (TEM), for its unique ability to give both low magnification panoramic views and detailed high magnification 3D images of cell organelles.

Copper distribution within and between newborn livers.

Copper concentration was determined in different liver samples (blocks, needle biopsy and bile) obtained from 9 newborns and 3 infants at autopsy. Tissue blocks, sampled at ten equally spaced intervals between the left and the right lobe, revealed in newborns high copper concentrations, ranging from 93 to 335 mg/Kg of dry tissue with an upward trend from the right to the left lobe. The situation differed in infants where a lower and more evenly distributed copper content, ranging from 49 to 123 mg/Kg of dry tissue, was found, which seemed to approximate the situation found in adults. Copper concentration was generally high in bile, with an overall range from 99 to 413 mg/Kg of dry material. This fact was particularly significant in infants, considering their low hepatic copper content. Copper content determined in percutaneous biopts was significantly correlated to (P less than 0.01), though only moderately predictive of (r2 = 0.54), the average copper content calculated from the ten liver blocks.