Using micro-synchrotron radiation x-ray fluorescence (µ-SRXRF) for trace metal imaging in the development of MRI contrast agents for prostate cancer imaging.

BACKGROUND: Contrast agents (CA) are administered in magnetic resonance imaging (MRI) clinical exams to measure tissue perfusion, enhance image contrast between adjacent tissues, or provide additional biochemical information in molecular MRI. The efficacy of a CA is determined by the tissue distribution of the agent and its concentration in the extracellular space of all tissues. METHODS: In this work, micro-synchrotron radiation x-ray fluorescence (µ-SRXRF) was used to examine and characterize a gadolinium-based zinc-sensitive agent (GdL2) currently under development for detection of prostate cancer (PCa) by MRI. Prostate tissue samples were collected from control mice and mice with known PCa after an MRI exam that included injection of GdL2. The samples were raster scanned to investigate trends in Zn, Gd, Cu, Fe, S, P, and Ca. RESULTS: Significant Zn and Gd co-localization was observed in both healthy and malignant tissues. In addition, a marked decrease in Zn was found in the lateral lobe of the prostate obtained from mice with PCa. CONCLUSION: We demonstrate here that µ-SRXRF is a useful tool for monitoring the distribution of several elements including Zn and Gd in animal models of cancer. The optimized procedures for tissue preparation, processing, data collection, and analysis are described.

Influence of zinc and zinc chelator on HT-29 colorectal cell line.

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

Zinc presence in invasive ductal carcinoma of the breast and its correlation with oestrogen receptor status.

Zinc is known to play an important role in many cellular processes, and the levels of zinc are controlled by specific transporters from the ZIP (SLC39A) influx transporter group and the ZnT (SLC30A) efflux transporter group. The distribution of zinc was measured in 59 samples of invasive ductal carcinoma of breast using synchrotron radiation micro probe x-ray fluorescence facilities. The samples were formalin fixed paraffin embedded tissue micro arrays (TMAs) enabling a high throughput of samples and allowing us to correlate the distribution of trace metals with tumour cell distribution and, for the first time, important biological variables. The samples were divided into two classes, 34 oestrogen receptor positive (ER+ve) and 25 oestrogen receptor negative (ER-ve) based on quantitative immunohistochemistry assessment. The overall levels of zinc (i.e. in tumour and surrounding tissue) in the ER+ve samples were on average 60% higher than those in the ER-ve samples. The zinc levels were higher in the ER+ve tumour areas compared to the ER-ve tumour areas with the mean levels in the ER+ve samples being approximately 80% higher than the mean ER-ve levels. However, the non-tumour tissue regions of the samples contained on average the same levels of zinc in both types of breast cancers. The relative levels of zinc in tumour areas of the tissue were compared with levels in areas of non-tumour surrounding tissue. There was a significant increase in zinc in the tumour regions of the ER+ve samples compared to the surrounding regions (P < 0.001) and a non-significant increase in the ER-ve samples. When comparing the increase in zinc in the tumour regions expressed as a percentage of the surrounding non-tumour tissue zinc level in the same sample, a significant difference between the ER+ve and ER-ve samples was found (P < 0.01).

Human soft tissue analysis using x-ray or gamma-ray techniques.

This topical review is intended to describe the x-ray techniques used for human soft tissue analysis. X-ray techniques have been applied to human soft tissue characterization and interesting results have been presented over the last few decades. The motivation behind such studies is to provide improved patient outcome by using the data obtained to better understand a disease process and improve diagnosis. An overview of theoretical background as well as a complete set of references is presented. For each study, a brief summary of the methodology and results is given. The x-ray techniques include x-ray diffraction, x-ray fluorescence, Compton scattering, Compton to coherent scattering ratio and attenuation measurements. The soft tissues that have been classified using x-rays or gamma rays include brain, breast, colon, fat, kidney, liver, lung, muscle, prostate, skin, thyroid and uterus.

The distribution of trace elements Ca, Fe, Cu and Zn and the determination of copper oxidation state in breast tumour tissue using muSRXRF and muXANES.

A micro beam synchrotron x-ray fluorescence (muSRXRF) technique has been used to determine the localization of metals in primary invasive ductal carcinoma of breast. Nine samples were examined, all of which were formalin fixed tissues arranged as micro arrays of 1.0 mm diameter and 10 microm thickness. Cu was the particular interest in this study although 2D maps of the elements Ca, Fe and Zn, which are also of physiological importance, are presented. The distribution of these metals was obtained at approximately 18 microm spatial resolution and compared with light transmission images of adjacent sections that were H and E stained to reveal the location of the cancer cell clusters. Correlations were found between these reference images and the elemental distributions indicating an increase in all element concentrations in the tumour regions of all samples, with the exception of Fe, which in some cases showed a reverse of this trend. On average over all samples the percentage difference from the normal tissue elemental concentrations are Ca approximately 67%, Cu approximately 64% and Zn approximately 145%. Micro x-ray absorption near edge spectroscopy (muXANES) was used to estimate the oxidation state of Cu in 19 normal and 17 tumour regions spread over five samples. The shape and the position of both normal and tumour regions suggest that they contain mixtures of copper ions with a significant fraction of Cu2+. However, the shape of the spectra does not exclude the presence of Cu+. Tumour regions were found to have a higher fraction of Cu+ compared to the normal samples.

Feasibility of the use of a handheld XRF analyzer to measure skin iron to monitor iron levels in critical organs.

There exists a need for accurate, non-invasive point-of-care tests to detect body iron burden. This study investigated the use of x-ray fluorescence (XRF) measurements of skin iron as a marker for organ iron content in rats. This study also evaluated a novel application of a commercial XRF device, commonly used in mining and construction, as a rapid, portable, and non-invasive measurement tool. Rats (n = 32) were loaded with iron dextran and the iron signal of each animal's skin, liver, and kidney was measured using a conventional XRF system. A quadratic correlation was observed between liver and skin iron signal (R2 = 0.92) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.65). As such, it is concluded that skin iron content can act as a marker for both liver and kidney iron content. The same skin samples were measured using the portable XRF device and compared to the liver and kidney samples measured in the conventional XRF system. Again, a quadratic correlation was observed between liver and skin iron signal (R2 = 0.91) and a linear correlation was observed between kidney and skin iron signal (R2 = 0.83). Thus, the portable XRF device can provide rapid non-invasive, skin XRF measurements. Dosimetry was performed using the portable XRF device to assess the radiological hazard associated with its use. The average skin equivalent dose from this device is 30 ± 10 mSv/min, when the device is collimated and operated at 40 kV. In conclusion, skin iron XRF measurements can act as a surrogate marker for liver iron content, and can be measured using a commercial XRF device for a portable, fast, and non-invasive measurement.

The localisation and micro-mapping of copper and other trace elements in breast tumours using a synchrotron micro-XRF system.

Trace elements have critical roles in cancer biology. The quantity and distribution of the elements Cl, Ca, K, P, S, Ti, Fe, Cu and Zn in samples of primary breast cancer have been assessed. The samples were formalin fixed tissue specimens formatted as microarrays of cores 1.0 mm diameter and 10 microm thick each. The data were obtained using a synchrotron X-ray fluorescence microprobe system. The spatial resolution of elemental maps was approximately 20 microm. Maps were compared with light transmission images of the samples and then the images were stained for cancer. The synchrotron system proved successful in producing data that could be mapped into high-resolution images where clear structure could be identified. Correlation of these distributions with the concentrations of cancer cells was achieved in some samples.

Aberrant expression of HLA-DR antigens by insulin-containing beta-cells in recent-onset type I diabetes mellitus.

The pancreases of 14 children who died of type I diabetes were studied immunohistochemically for aberrant expression of HLA-DR antigens on islet endocrine cells. Two cases in which no residual insulin-secreting beta-cells were present had no evidence of HLA-DR expression on endocrine cells. Insulin-containing islets were present in the remainder, and HLA-DR-positive endocrine cells were demonstrable in all of them. Endocrine cells expressing HLA-DR were present in 171 of 630 insulin-containing islets from all the cases. However, HLA-DR-positive endocrine cells were not seen in 2060 insulin-deficient islets, providing evidence that of the four hormone-producing cells in the pancreas only the beta-cells expressed HLA-DR. Sections double stained for HLA-DR and the pancreatic hormones confirmed this view. Most islets in which HLA-DR-positive endocrine cells were seen had no evidence of insulitis, suggesting that within an individual islet, aberrant expression of HLA-DR on beta-cells may precede the inflammatory infiltrate.

Effect of sample preparation techniques on the concentrations and distributions of elements in biological tissues using µSRXRF: a comparative study.

Routine tissue sample preparation using chemical fixatives is known to preserve the morphology of the tissue being studied. A competitive method, cryofixation followed by freeze drying, involves no chemical agents and maintains the biological function of the tissue. The possible effects of both sample preparation techniques in terms of the distribution of bio-metals (calcium (Ca), copper (Cu) zinc (Zn), and iron (Fe) specifically) in human skin tissue samples was investigated. Micro synchrotron radiation x-ray fluorescence (µSRXRF) was used to map bio-metal distribution in epidermal and dermal layers of human skin samples from various locations of the body that have been prepared using both techniques. For Ca, Cu and Zn, there were statistically significant differences between the epidermis and dermis using the freeze drying technique (p = 0.02, p < 0.01, and p < 0.01, respectively). Also using the formalin fixed, paraffin embedded technique the levels of Ca, Cu and Zn, were significantly different between the epidermis and dermis layers (p = 0.03, p < 0.01, and p < 0.01, respectively). However, the difference in levels of Fe between the epidermis and dermis was unclear and further analysis was required. The epidermis was further divided into two sub-layers, one mainly composed of the stratum corneum and the other deeper layer, the stratum basale. It was found that the difference between the distribution of Fe in the two epidermal layers using the freeze drying technique resulted in a statistically significant difference (p = 0.012). This same region also showed a difference in Fe using the formalin fixed, paraffin embedded technique (p < 0.01). The formalin fixed, paraffin embedded technique also showed a difference between the deeper epidermal layer and the dermis (p < 0.01). It can be concluded that studies involving Ca, Cu and Zn might show similar results using both sample preparation techniques, however studies involving Fe would need more special attention.

Localization of alpha-interferon in the human feto-placental unit.

The distribution of alpha-interferon in human placental tissue was investigated by immunocytochemical study of paraffin wax-embedded tissue sections using a sheep alpha-interferon antiserum. Fifty-eight placentas of gestational ages from 8 to 40 weeks were examined. alpha-Interferon was present in the syncytiotrophoblast of the chorionic villi of all placentas and was also in macrophages in 28 cases. The appearances suggest production of interferon in human placental trophoblast and, in view of its diverse biological effects, support the concept of a role for alpha-interferon in the complex series of events required for successful gestation.

Detection of messenger RNA using a digoxigenin end labelled oligodeoxynucleotide probe.

A synthetic oligodeoxynucleotide sequence complementary to the mRNA for the adrenocorticotrophin (ACTH) precursor pro-opiomelanocortin (POMC) was end labelled using digoxigenin. The probe was used to detect POMC mRNA both on nitrocellulose filters and by non-isotopic in situ hybridisation (NISH) in tissue sections. Digoxigenin was identified using anti-digoxigenin alkaline phosphatase. The model system examined was the rat pituitary gland. Removal of both adrenal glands and dexamethasone administration were used to change the concentrations of POMC mRNA in the rat anterior lobe. The labelled probe reacted with a single band of appropriate molecular weight in Northern blot analysis. The distribution of signal in tissue sections and the changes induced by experimental manipulation were as predicted. The results indicate that this method of NISH will prove useful in the detection of specific messenger RNAs in tissue sections of buffered, formalin fixed, paraffin wax embedded material.

Liver membrane antibodies in alcoholic liver disease. II. Antibodies to ethanol-altered hepatocytes.

Using an indirect immunofluorescence technique, circulating liver membrane antibodies against normal rabbit hepatocytes and ethanol-altered rabbit hepatocytes have been sought in a series of patients with histologically confirmed alcoholic liver disease. Liver membrane antibodies against normal hepatocytes were found in 18 (28%) of the 65 sera examined, but with ethanol-altered hepatocytes as substrate liver membrane antibodies were found in 48 (74%) of the sera. Isolation of F(ab')2 fragments confirmed that the positive results were due to antibody binding. Liver membrane antibodies against ethanol-altered hepatocytes are peculiar to alcoholic liver disease, and there is a similar incidence in the various histological types of alcoholic liver disease. Absorption studies suggest that the liver membrane antibodies are directed against new or altered antigens which are not present in normal hepatocytes. These new or altered antigens may also appear after pretreatment with other primary alcohols and seem likely to be induced by a haptenic effect of the alcohol or a metabolic break-down product. These studies represent a novel approach to the further investigation of the possible role of immunological mechanisms in alcohol-induced liver injury.

p53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

AIMS: To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. METHODS: Immunocytochemistry for p53 was performed in 65 specimens of formalin fixed, paraffin wax embedded cervical tissue using a polyclonal antibody against recombinant p53. Microwave oven heating was used for antigen retrieval. Eight normal biopsy specimens, eight cases with histological features of HPV infection, and 49 cases of cervical intraepithelial neoplasia (CIN) were examined. Thirty one cases of CIN were examined. Thirty one cases of CIN were examined for evidence of HPV infection using in situ hybridisation with probes directed against wide spectrum HPV, HPV 16 and HPV 18. RESULTS: p53 immunoreactivity was seen in seven of eight (87%) of specimens with histological features of HPV infection, five of eight (62%) normal specimens, 13 of 22 (59%) CIN III, three of 14 (21%) CIN II and five of 13 (38%) CIN I specimens. The numbers of positive nuclei were small in cases of CIN and the location of positive nuclei within the epithelium paralleled the degree of dysplasia. Eleven of 15 (73%) CIN specimens which were immunoreactive for p53 yielded a positive signal for HPV by in situ hybridisation. A positive signal for HPV was also seen in 10 of 16 (63%) of CIN specimens in which p53 staining was absent. CONCLUSIONS: p53 immunoreactivity can be demonstrated in a small proportion of cells in the cervical squamous epithelium in a significant proportion of cases of CIN. This immunoreactivity seems to be independent of the presence of HPV, as assessed by in situ hybridisation. p53 immunoreactivity also occurs in non-neoplastic cervical squamous epithelium with a pattern of distribution within the epithelium which differs from that seen in CIN. Antigen retrieval by microwave oven heating enhances p53 immunostaining and may result in visualisation of cellular p53 in the absence of mutation.

Detection of mRNA by in situ hybridisation and in northern blot analysis using oligodeoxynucleotide probes labelled with alkaline phosphatase.

AIMS: To assess whether a reduction in intensity of signal observed using an alkaline phosphatase labelled oligodeoxynucleotide probe could be explained on the basis of procedural steps rather than reduced sensitivity. METHOD: Signal intensity was assessed on in situ hybridisation for pro-opiomelanocortin (POMC) mRNA in rat pituitary and for somatostatin mRNA in human pancreas and in northern blot analysis for POMC mRNA in the presence and absence of formamide. The direct effects of formamide on the alkaline phosphatase detection step were assessed using histochemical enzyme detection in rat kidney. RESULTS: All signals were reduced in systems containing formamide. CONCLUSIONS: In the absence of formamide clear, strong signals for specific mRNAs can be obtained by in situ hybridisation and northern blot analysis using oligodeoxynucleotide probes directly labelled with alkaline phosphatase. Formamide seems to inhibit the activity of alkaline phosphatase.

Sequential malt lymphomas of the stomach, small intestine, and gall bladder.

Low grade lymphomas of mucosa associated lymphoid tissue (MALT) are indolent neoplasms that, although tending to remain localised for many years, may spread to other mucosal sites. A 53 year old woman treated by total gastrectomy for low grade MALT lymphoma of the stomach developed a recurrence in the small bowel 18 years later, and a further recurrence involving the gall bladder after three years in complete clinical remission after chemotherapy. In situ hybridisation showed that the small intestine and gall bladder recurrences had the same pattern of light chain restriction. Tumour from all three sites was shown to be derived from a single clone by the demonstration of an identical immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction. The case illustrates the propensity of MALT lymphomas to "home" to mucosal sites and gives an insight into their behavior over an extended follow up.

Fine needle aspiration cytology diagnosis of malignant lymphoma and reactive lymphoid hyperplasia.

AIMS: To assess the diagnostic accuracy of lymph node fine needle aspiration (FNA) cytology to distinguish reactive lymphoid hyperplasia from malignant lymphoma, and to evaluate the contribution of ancillary techniques applied to cytological material. METHODS: Two hundred and seventy seven consecutive lymph node FNA specimens reported to be consistent with reactive lymphoid hyperplasia (n = 213) or suggestive/diagnostic of malignant lymphoma (n = 64) were reviewed. Follow up data were obtained by case record review or by histological correlation. The value of immunocytochemistry, in situ hybridisation for immunoglobulin light chain mRNA, and polymerase chain reaction (PCR) towards the final clinicopathological diagnosis was assessed in 92, 61, and 45 cases, respectively. RESULTS: Sixty one of 67 lymphomas and 207 of 209 reactive lymph nodes were accurately diagnosed by FNA cytology. There were six false negative aspirates including three cases of follicular lymphoma, two cases of Hodgkin's disease, and one chronic lymphocytic leukaemia. Two FNA specimens considered suspicious of lymphoma proved reactive on histology or clinical follow up. One metastatic small cell carcinoma was wrongly diagnosed as lymphoma. Ancillary studies contributed to the correct diagnosis in most cases although occasional misleading results were obtained, particularly with PCR. CONCLUSIONS: FNA cytology accurately distinguished reactive lymphoid hyperplasia from malignant lymphoma in 97% of cases. However, occasional wrong diagnoses occurred owing to sampling error or misinterpretation. Ancillary studies can be applied to cytological samples and contribute to the diagnosis in most cases.

Immunoglobulin light chain mRNA detected by in situ hybridisation in diagnostic fine needle aspiration cytology specimens.

AIMS: To demonstrate expression of immunoglobulin light chain mRNA in diagnostic fine needle aspiration (FNA) cytology specimens using an in situ hybridisation (ISH) technique; and to evaluate ISH in a series of reactive lymphoid proliferations and malignant lymphomas. METHODS: Forty diagnostic FNA specimens showing a lymphoid cell population were examined for immunoglobulin light chain mRNA expression using ISH. Aspirates were obtained from lymph node (n = 34), salivary gland (n = 3), subcutaneous tissue, thyroid and breast (n = 1 each). The cases included 20 B cell lymphomas, five cases of Hodgkin's disease and 15 reactive lymphoid proliferations. Comparison with light chain immunoreactivity was made in 36 cases and histological correlation from biopsy material was available in 24. RESULTS: Immunoglobulin light chain restriction was demonstrated in 14 of 20 B cell lymphomas using ISH and in six of 17 B cell lymphomas using immunocytochemistry. A polytypic pattern of light chain expression was observed in four of five cases of Hodgkin's disease with both techniques, and in 12 of 15 and 11 of 14 reactive lymphoid proliferations using ISH and immunocytochemistry, respectively. CONCLUSIONS: The assessment of immunoglobulin light chain expression is a useful adjunct to morphology in the diagnosis of reactive and malignant lymphoid proliferations in FNA specimens. Light chain restriction can be shown using either immunocytochemistry or ISH, but the latter is more sensitive in the diagnosis of B cell lymphoma.

Detection of cytomegalovirus in upper gastrointestinal biopsies from heart transplant recipients: comparison of light microscopy, immunocytochemistry, in situ hybridisation, and nested PCR.

AIM: To establish the diagnostic value of in situ hybridisation and the nested polymerase chain reaction (PCR) in detecting clinically relevant cytomegalovirus (CMV) infection in upper gastrointestinal biopsies from heart transplant patients. METHODS: Test sensitivity and specificity for detection of CMV early gene RNA by in situ hybridisation and CMV intermediate early gene by PCR were established and then compared with haematoxylin and eosin (H&E) and immunocytochemical detection of CMV in order to establish the best pathological diagnostic approach. All investigations were carried out on formalin fixed, paraffin embedded tissue. RESULTS: Nested PCR had the highest test sensitivity, followed by in situ hybridisation and immunocytochemistry with the same sensitivity; H&E had the lowest. H&E and immunocytochemistry were the most specific but both had a significant false negative rate which was less of a problem with PCR. However, PCR gave no other diagnostic information, and in situ hybridisation was no better than immunocytochemistry. Both in situ hybridisation and PCR were technically complex and more expensive. CONCLUSIONS: H&E and immunocytochemistry represent the best initial screen for CMV and other diseases in upper gastrointestinal biopsies from heart transplant patients. If H&E and immunocytochemistry were negative, nested PCR could significantly increase the diagnostic yield of clinically relevant CMV infection. In situ hybridisation appeared to have no advantages and some drawbacks compared with immunocytochemistry and PCR.

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction (PCR) in the diagnosis of B cell lymphoma. METHODS: Analyses were applied to formalin fixed, paraffin wax embedded, routine diagnostic specimens from cases with a provisional diagnosis of reactive lymph node (n = 23), B cell lymphoma (n = 21), and T cell lymphoma (n = 4). Nonisotopic ISH for kappa and lambda immunoglobulin light chain mRNA was performed using both fluorescein and digoxigenin labelled oligodeoxynucleotide probe cocktails. PCR was carried out on DNA extracted from sections using primers to framework 3 (Fr3) of the V segments and to conserved sequences from the J regions of the immunoglobulin heavy chain genes. RESULTS: All reactive lymph nodes showed a polyclonal pattern of light chain mRNA by ISH, although one showed an excess of kappa positive cells. Nineteen of 21 (90%) cases of B cell lymphoma showed light chain restriction, and a further case showed a vast excess of kappa positive cells. By PCR, 20 of 23 reactive nodes (87%) showed a polyclonal pattern. In 13 of 21 B cell lymphomas (62%) a clonal band was detected. CONCLUSION: In the diagnosis of B cell lymphoma in routinely processed diagnostic material ISH for light chain mRNA was more sensitive (90%) than PCR for heavy chain gene rearrangement using Fr3 and J region primers (62%).

Immunolocalization of alpha interferon in liver disease.

The expression of immunoreactive alpha interferon was examined in 78 liver biopsy specimens using an indirect immunoperoxidase technique. Biopsy specimens included cases of acute viral hepatitis, chronic active hepatitis, primary biliary cirrhosis, alcoholic hepatitis, large bile duct obstruction and normal liver. Kupffer cells were positive for alpha interferon in all cases. Hepatocytes were negative for alpha interferon in normal liver but in acute viral hepatitis were positive in perivenular and necrotic areas. Hepatocytes were positive in periportal areas, associated with piecemeal necrosis, in chronic active hepatitis and primary biliary cirrhosis, and were positive in perivenular areas in alcoholic hepatitis and large bile duct obstruction. The unexpected finding of alpha interferon in hepatocytes in non-viral liver disease indicates that the presence of this substance in liver cells cannot be taken as a specific marker of viral infection.