Adenovirus and ileocecal intussusception.

Intranuclear inclusion bodies were found by light microscopy in epithelial cells in more than one-third of the specimens from children operated on for ileocecal intussusception. Electron microscopic examination done on hematoxylin and eosin-stained slides showed the intranuclear inclusion bodies to be composed of viral particles in large and small crystalline arrays. Adenovirus of serotypes 2, 3, and 5 were isolated from the five cases with inclusions in which isolation was attempted. These findings strongly suggest a pathogenetic role for adenovirus in those cases of intussusception in which intranuclear inclusion bodies are found in the epithelial cells of the appendix or the terminal ileum.     

Histological diagnosis of cytomegalovirus hepatitis in liver allografts.

AIMS--To determine the incidence of histologically documented cytomegalovirus (CMV) hepatitis following orthotopic liver transplantation (OLT) and to assess the effectiveness of immunohistochemistry and in situ hybridisation (ISH) in detecting CMV. To describe the histological pattern most frequently associated with CMV hepatitis in order to select the biopsy group in which these modern techniques are most effective. METHODS--A prospective histological study was carried out on 853 biopsy specimens, obtained from 191 liver allografts (160 patients). Specimens were stained with haematoxylin and eosin and immunohistochemically (avidin-biotin complex) using monoclonal antibodies directed against early and late CMV antigens. A retrospective selection was made of 23 specimens with viral inclusion bodies in cytomegalic cells (group A) to characterise the most frequently associated histological pattern, and of 34 other specimens without viral inclusion bodies (group B) but with the same microscopic features as group A. Re-cuts from both specimen groups were studied using immunohistochemistry and ISH with a CMV specific complementary DNA probe. RESULTS--CMV infection was confirmed in 35 specimens (29 by immunohistochemistry, 23 by presence of inclusion bodies in haematoxylin and eosin stained sections, 16 by ISH) from 27 patients (incidence 16.9%). CMV hepatitis was diagnosed within 46 +/- 19 (range 21-114) days posttransplant. Twenty on (91.3%) of the 23 biopsy specimens with inclusion bodies (group A) displayed heterogeneous inflammatory foci disseminated throughout the hepatic lobule. Nineteen specimens (82.6%) were positive by immunohistochemistry and 14 (60.9%) by ISH. In eight (23.5%) of the 34 group B specimens CMV infection was confirmed by immunohistochemistry (n = 6) or ISH (n = 2). Another 12 (35.3%) of the group B specimens negative on staining with haematoxylin and eosin, immunohistochemistry and ISH came from allografts in which previous or subsequent biopsy specimens were CMV positive. CONCLUSIONS--Demonstration of cytomegalic inclusion bodies in haematoxylin and eosin sections is sufficient for a diagnosis of CMV hepatitis. The routine use of immunohistochemistry in all allograft biopsy specimens in more sensitive than demonstration of inclusion bodies by staining with haematoxylin and eosin but may yield false negative results because of the focal distribution of positive cells. ISH was less sensitive than staining with haematoxylin and eosin and/or immunohistochemistry. A histological picture of "disseminated focal hepatitis" without viral inclusion bodies selects a group of allograft biopsy specimens in which immunohistochemistry and/or ISH may improve detection of CMV.     

Fatal adenovirus 32 infection in a bone marrow transplant recipient.

A case of disseminated adenovirus type 32 infection causing severe hepatitis, gastrointestinal ulceration and also with respiratory involvement is reported in a bone marrow transplant recipient. Typical viral inclusions were seen in the postmortem histological sections and adenovirus infection was confirmed using in situ hybridisation and isolation of adenovirus type 32 from separate organs at necropsy. This is the first case in which adenovirus 32 was the cause of fatal disseminated disease in a bone marrow transplant recipient.     

Inclusion bodies containing adenovirus-like particles in the kidneys of psittacine birds

Two types of intranuclear inclusion bodies were found in the epithelial cells of renal tubules or rami ureterici of 12 psittacine birds. One type was large and stained moderately with haematoxylin, poorly with eosin and contained a few granules which stained with haematoxylin. The other was medium in size, circular, almost filling the entire nucleus or irregularly shaped with a clear halo, and stained homogeneously with eosin. Ultrastructurally, the large inclusions consisted of virus particles and fine filamentous materials. The particles exhibited an oval or hexagonal configuration, ranging from 55 to 88 nm in diameter, morphologically characteristic of adenovirus particles. The medium-sized inclusions were composed of aggregates of fine granules. The adenovirus infections were regarded as opportunistic infections.     

Inclusion bodies and hepatopathies in psittacines

Intranuclear inclusion bodies in hepatic cells of seven Peach-faced Lovebirds (Agapornis roseicollis), two Black-masked Love birds (A. per sonata) and two galahs (Cacatua roseicapilla) were examined electron microscopically. Thin sections were prepared from formalin-fixed Epon-embedded tissue or from haematoxylin and eosin stained sections that were subsequently embedded in Epon. Discrete and diffuse eosino-philic inclusions in hepatocytes of eight birds were composed of filaments and virus particles were not demonstrated within them. Virus-like particles of at least three types were found in diffuse, lightly basophilic inclusions of similar appearance in hepatocytes and Kupffer cells or endothelial cells in four birds. Particles, 66 nm in diameter which were probably adenovirus were demonstrated in hepatocytes in one A. roseicollis. Particles 26 nm to 39 nm in diameter which resembled papovaviruses were seen in one A. personata and one C. roseicapilla. Particles of 40 nm to 50 nm in diameter were seen in one A. personata but because of the method of preparation it was not possible to state whether they were adenovirus or papovavirus.     

Down-regulation of CD95 (Fas/Apo-1) in the epithelia of adenovirus-infected appendices

 

Aims:

Adenoviral inclusions are commonly seen in appendices from infants with intussusception. They are associated with focal epithelial budding and less frequently with epithelial shedding. These morphological changes could depend on the opposing effects of adenoviral gene products on CD95-mediated apoptosis.  

Methods and results:

Appendices from intussusceptions with viral inclusions ( n  = 4) and normal appendices ( n  = 10) were studied by immunochemistry with anti-adenovirus, anti-CD95 and anti-HLA-DR antibodies. Apoptosis was studied by the TUNEL method. The mucosa of normal appendices contained no adenoviral protein. CD95 was present in all epithelial cells except Paneth cells. HLA-DR was absent in epithelial cells and apoptosis was seen only in germinal centres and in a few surface epithelial cells. The epithelium of appendices from intussusceptions contained nuclear inclusions labelled with anti-adenovirus antibody, always found in the epithelial buds. The epithelial CD95 pattern was drastically altered in adenovirus-infected appendices. CD95 was absent from the budding foci. In these foci, HLA-DR was overexpressed. There was also increased epithelial apoptosis in areas remote from those lacking CD95 antigen.  

Conclusions:

The appearance of epithelial budding or shedding in appendices from intussusception could be due to focal in situ differences in the expression of adenoviral genes.     

Appendicitis revisited: a comparative study of Malawian and English appendices

The incidence of appendicitis shows a marked variation between populations which has been attributed to dietary differences. Neural mechanisms and serotonin discharge from subepithelial neurosecretory cells have been previously implicated in pain referable to the appendix and appendicitis. Forty consecutive appendicectomy specimens from Malawi were studied by staining with haematoxylin and eosin, an alcian blue - PAS diastase sequence coupled with lead haematoxylin (PbH) and immunohistology for serotonin and NSE. The findings were compared with those in appendices removed at the Middlesex Hospital, London, to see if there were any differences between a population with a low risk of appendicitis (Malawi) and a high risk population (England). Acute transmural appendicitis was seen in fewer appendices from Malawi (27.5 per cent) than in English appendices (58 per cent). Subepithelial neurosecretory cells identified with PbH were present in 20 per cent of appendices from Malawi and 69 per cent of English appendices. These cells in both series showed immunohistochemical staining for serotonin. Nerve hyperplasia identified by staining for NSE in the appendix was present in 17.5 per cent and 81 per cent of non-inflamed appendices from Malawi and England respectively. Appearance of subepithelial neurosecretory cells and hyperplasia appear to be concomitants of an increased risk of appendicitis. Neural mechanisms may participate in adapting to a low residue diet and in some cases may generate appendicitis.     

The value of an elastic tissue stain in detecting venous invasion in colorectal cancer

BACKGROUND: Venous invasion by tumour is an independent prognostic indicator of both prognosis and risk of development of distant metastases in colorectal carcinoma. The use of special stains to aid its detection in pathology specimens is not currently universally recommended. AIMS: To determine whether an elastica stain significantly increases the incidence of detection of vascular invasion compared with routinely stained sections. METHODS: Serial sections from the 75 cases of colorectal carcinoma were stained by haematoxylin and eosin (H&E) only and elastica counterstained with H&E. The incidence of both intramural and extramural venous invasion was recorded and compared with that seen when the tumours were originally reported. RESULTS: Extramural venous invasion had been noted in 14 of the pathology reports and was seen in 18 cases when only the H&E sections were viewed in the study. It was present in 32 cases when elastica stained sections were analysed. Intramural venous invasion was seen in eight cases on H&E sections and 30 cases on elastica stained sections. CONCLUSION: The use of elastica stained serial sections to detect venous invasion in tumours should be recommended in guidelines for the reporting of colorectal carcinomas.     

Intraoperative examination in neuropathology: proceedings evaluations

INTRODUCTION: In the neuropathological intraoperative diagnostic is extremely important having guide lines to obtain a lot of information about the slide. In our study we have considered only the intraoperative the cases diagnosed within a month comparing the histological evaluation using the cryostat and the cytological examination of touch or smear preparation. MATERIALS AND METHODS: Our study includes 7 meningiomas, 2 metastatic carcinomas, 1 anaplastic astrocitoma, 1 mieloplaxis tumor, 1 anaplastic chordoma. For the cytological examination we have got ready 8 touch or smear preparations and we have executed 4 proceedings: stainings with Giemsa, toluidine blue and haematoxylin and eosin, after fixation with 95% alcohol and staining with haematoxylin and eosin after fixation with alcohol and a small quantity of acetic acid. For the histological examination we have got ready 8 preparations, using the cryostat. We have fixed the slides with 95% alcohol or 10% formalin and stained them with haematoxylin and eosin. In the 50% of the cases we have treated the sections with microwave at 400W. RESULTS: Two pathologists have examinated the 192 sections prepared, judging each slide considering the legibility: unsatisfactory, quite good, good and excellent. CONCLUSIONS: Considering the cytological examination, Giemsa and toluidine blue permit the best legibility of nuclear and cytoplasmatic morphological details. Legibility is worse in staining with haematoxylin and eosin. Addition of acetic acid makes variable results. In the histological slides the best result are obtained using 95% alcohol for fixing. Microwave use doesn't determine improvement.     

Adenovirus infection of the large bowel in HIV positive patients.

AIMS: To describe the microscopic appearance of adenovirus infection in the large bowel of human immunodeficiency virus (HIV) positive patients with diarrhoea. METHODS: Large bowel biopsy specimens from 10 HIV positive patients, eight of whom were also infected with other gastrointestinal pathogens, with diarrhoea were examined, together with six small bowel biopsy specimens from the same group of patients. Eight of the patients had AIDS. The biopsy specimens were examined by light microscopy performed on haematoxylin and eosin stained and immunoperoxidase preparations, the latter using a commercially available antibody (Serotec MCA 489). Confirmation was obtained with electron microscopy. RESULTS: The morphological appearance of cells infected with adenovirus showed characteristic nuclear and cellular changes, although the inflammatory reaction was non-specific. Immunoperoxidase staining for adenovirus was sensitive and specific, and the presence of viral inclusions consistent with adenovirus was confirmed by electron microscopy. CONCLUSIONS: The light microscopic features of adenovirus infection are distinctive and immunocytochemistry with a commercially available antibody is a sensitive and specific means of confirming the diagnosis. Further studies of the role of adenovirus in causing diarrhoea in these patients are indicated.     

Diagnosis of progressive multifocal leucoencephalopathy by hybridisation techniques.

In situ hybridisation with acetyl-aminofluorene (AAF) and 35S-labelled DNA probes for polyomaviruses, was used to detect JC virus DNA in brain necropsy material in a patient with progressive multifocal leucoencephalopathy (PML). In a second patient PML was diagnosed from a brain necropsy specimen using the same technique. The main infected cell type were oligodendrocytes; dot hybridisation was used to estimate the number of viral copies in each infected cell. Southern blot hybridisation for further analysis of the viral genome was also carried out. In situ hybridisation with non-radioactive labelled polyomavirus DNA provides a simple and specific means for studying viral DNA in formaldehyde fixed tissue sections from patients with suspected PML. Even in small biopsy samples hybridisation results can be correlated with standard histopathological, immunocytochemical, and electron microscopic findings.     

Comparison of in situ hybridization and immunohistochemistry for detection of cytomegalovirus and herpes simplex virus

In situ hybridization (ISH) and immunohistochemistry (IHC) were compared for detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) in routinely processed tissue. Fifty-four formalin-fixed paraffin-embedded tissue samples infected with CMV (36 tissues) or HSV (18 tissues) from 30 autopsies were studied. All tissues had either positive viral cultures (38 of 54) or characteristic viral inclusions on hematoxylin and eosin examination (39 of 54). The tissues examined included lung (28), liver (nine), kidney (five), heart (three), adrenal (two), spleen (two), and thymus, pancreas, appendix, esophagus, and duodenum (one each). Studies by ISH were performed with two detection systems, using biotinylated probes to CMV and HSV (Enzo Biochem, New York, NY). Using ISH with an alkaline phosphatase detection system, infected cells were detected in 33 of 54 tissues (CMV: 23 of 36, HSV: 10 of 18). Using ISH with a peroxidase detection system, infected cells were identified in 30 of 54 tissues (CMV: 22 of 36, HSV: eight of 18). With IHC, antibodies to CMV and HSV stained the infected cells in 34 of 54 tissues (CMV: 24 of 36, HSV: 10 of 18). All infections detected with ISH were also detected with IHC. We conclude that these techniques for ISH and IHC are equally effective for detecting CMV and HSV in paraffin sections. The results of both techniques correlate better with viral inclusions than with culture results. The ISH stains are more difficult to prepare and in some cases are more difficult to interpret. Therefore, IHC may be preferable to ISH for detecting CMV and HSV in routine diagnostic work.     

Diagnosis of psittacine beak and feather disease (PBFD) viral infection, avian polyomavirus infection, adenovirus infection and herpesvirus infection in psittacine tissues using DNA in situ hybridization

The evaluation of the usefulness of DNA probes in a diagnostic setting to identify nuclear inclusions in selected viral infections (psittacine beak and feather disease viral infection, avian polyomavirus infection, adenovirus infection and Pacheco's parrot disease) is reported. A DNA in situ hybridization method was used to detect viral nucleic acid in sections of paraffin-embedded tissues coming from birds naturally and/or experimentally infected. It is concluded that DNA probes used for polyomavirus (FN-19) and adenovirus (FN-23) are able to identify nucleic acid of each virus in the cells with nuclear inclusions, and when used for psittacine beak and feather disease virus (FN-8), and Pacheco's parrot disease virus (FN-49) are able to detect viral nucleic acid in cells with or without inclusions.     

Detection of Helicobacter pylori in gastric biopsy and resection specimens.

AIMS: To compare the sensitivity of detecting Helicobacter pylori in gastric biopsy and resection specimens using tinctorial and silver impregnation stains, immunohistochemistry and the polymerase chain reaction (PCR). METHODS: Formalin fixed, paraffin wax embedded tissue from 33 gastric biopsy specimens (26 showing chronic gastritis and seven showing low grade mucosa associated lymphoid tissue (MALT) lymphoma) together with blocks of uninvolved mucosa from gastrectomy specimens for MALT lymphoma (five cases) were studied. Consecutive sections were stained using haematoxylin and eosin, Giemsa, the Warthin-Starry silver stain, and a polyclonal antibody directed against H pylori using an immunoperoxidase technique following heat induced antigen retrieval. PCR analysis of DNA extracted from a further section was carried out using primers which amplified a 411 base pair fragment of the urease A gene. RESULTS: H pylori was detected in 14 (37%) sections stained with haematoxylin and eosin, 21 (55%) with Giemsa, 23 (61%) with Warthin-Starry, and 25 (66%) stained with the antibody. Seventeen (45%) cases were positive on PCR. Immunohistochemistry was positive in all cases in which H pylori was detected by other methods. CONCLUSION: Immunohistochemistry using an immunoperoxidase technique following heat induced antigen retrieval for detecting H pylori in gastric biopsy and resection specimens is highly sensitive and easy to use.     

An autofluorescence method for the diagnosis of early ischaemic myocardial lesions

Summary A systematic study of an autofluorescence method is described to improve the early histological diagnosis of myocardial ischaemia. Our results on 732 autopsy cases including 182 cases of sudden death show that the autofluorescence examination of haematoxylin and eosin stained sections of the myocardium is not only reliable in the identification of recent ischaemic lesions, but contributes to a better histological evaluation. In 24 cases undetected by white light examination it allowed recognition of ischaemic lesions.     

Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique

Rapid methods of specific viral diagnosis in formalin fixed, paraffin embedded tissues include identification of viral incusions in routinely stained histologic sections, immunologic staining of viral antigens, and in situ nucleic acid hybridization. To correlate in situ hybridization with immunologic detection methods, sequential two-color staining was used on tissues from 12 patients, each containing characteristic cytomegalovirus (CMV) inclusions, using a biotinylated CMV DNA probe in an avidin-alkaline phosphatase-linked reaction followed by avidin-biotin complex immunoperoxidase staining of CMV antigen. CMV genetic material was seen in all 17 tissues. CMV antigen was detected in 11 of 17 tissues (65%). The DNA hybridization technique provided more intense staining, detected greater numbers of inclusions, and had less background staining than the immunoperoxidase technique. The alkaline phosphatase reaction product was stable through subsequent immunostaining steps, and immunologic reactivity of CMV antigen was not significantly reduced by prior hybridization steps. CMV DNA probe was localized predominantly within cell nuclei, while CMV antigen immunostaining was predominantly cytoplasmic. It was concluded that sequential in situ hybridization and immunocytochemistry can be performed on standard histologic sections. Furthermore, it is likely that the majority of CMV nucleic acid detected by this tissue hybridization technique is unencapsidated, intranuclear viral DNA and not DNA contained within complete CMV nucleocapsids.     

Adenovirus pulmonary infections identified by PCR and in situ hybridisation in bone marrow transplant recipients.

AIMS--To investigate adenovirus pulmonary infections in bone marrow transplant (BMT) recipients. METHODS--Formalin fixed, paraffin wax embedded lung tissue was examined from 13 necropsy cases after BMT using PCR and in situ hybridisation to detect adenovirus DNA. The E1A region of the adenoviral genome was targeted for PCR. In situ hybridisation was performed only in the PCR positive cases. RESULTS--Of the 13 lung specimens analysed, nine cases were negative for adenoviral nucleic acid. Four (30%) PCR and two (15%) in situ hybridisation positive cases were found. In some of the patients there were clinical and pathological indications that some diseases might be associated with adenovirus infection--haemorrhagic cystitis (three cases); necrotising pneumonia (one case). In necrotising pneumonia in which no pathogenic agents had been shown by conventional histological study, the in situ hybridisation technique showed positive staining for adenovirus. In a patient who died of renal failure caused by adenovirus nephritis, both PCR and in situ hybridisation were positive in the lung as well as in the kidney, although no histological change was found. Two PCR positive cases lacked positive sites for adenovirus by in situ hybridisation. CONCLUSIONS--The combination of PCR and in situ hybridisation could be useful for diagnosing adenovirus infection of the lung in BMT recipients. These results provide a basis for exploring further the clinical use of PCR and in situ hybridisation to diagnose adenovirus infection.     

Cytokeratin immunostaining for detection of biliary epithelium: its use in counting bile ducts in cases of liver allograft rejection.

AIMS--To see how useful the application of a bile duct specific cytokeratin antibody (AE1) was in identifying and counting bile ducts in liver allograft biopsy specimens. METHODS--Eighteen liver biopsy specimens showing acute rejection and 17 biopsy specimens plus six hepatectomy specimens showing chronic rejection were studied. Serial sections were cut and stained with haematoxylin and eosin and AE1 antibody. Two pathologists (RFH and KP) examined the sections with respect to a range of histological features. RESULTS--Similar numbers of bile ducts were identified on haematoxylin and eosin sections as on corresponding sections stained by AE1 in cases of acute rejection and end stage chronic rejection. Greater numbers of bile ducts were identified by AE1 during the early stages of chronic rejection, especially when dense portal inflammatory infiltrates were present. These were often incomplete structures or individual cells within portal tracts, and bile ducts subsequently disappeared in all cases. Ductular proliferation was clearly shown by AE1 in acute rejection and the extent seemed to correlate with the severity of rejection present. By contrast, no ductular proliferation was observed in chronic rejection. CONCLUSIONS--Haematoxylin and eosin stained sections are adequate for counting bile ducts in most biopsy specimens from patients with suspected chronic rejection. Immunostaining for biliary cytokeratins using AE1 is of limited use in occasional cases where bile ducts are obscured by inflammatory cells.     

Histological diagnosis of intestinal microsporidiosis in patients with AIDS.

Fifty nine patients seropositive for human immunodeficiency virus (HIV) and diarrhoea and 20 with weight loss were investigated for microsporidiosis using light and electron microscopical examination of duodenal and jejunal biopsy specimens. Eight cases of microsporidiosis were found, in five of whom it was the sole pathogen. In all eight cases the organism was identified at light microscopy without prior knowledge of the electron microscopical findings. All stages of the life cycle are best seen in resin sections cut at 1 micron and stained with Giemsa, but spores could easily be identified in paraffin sections cut at 5 microns and stained with haematoxylin and eosin. In all cases the parasite was identified both in duodenal pinch and jejunal "Crosby" capsule biopsy specimens. All cases of microsporidiosis occurred in patients with diarrhoea. Both electron and light microscopical examination suggested that the pathogenic mechanism involves the shedding of infected enterocytes containing large numbers of spores. It is suggested that the optimal way to diagnose microsporidiosis is by light microscopical examination of duodenal pinch biopsy specimens.     

Preparation and light-microscopic examination of fixed hematopoietic cells in soft agar.

Hematopoietic cells from the blood or bone marrow (of leukemic and nonleukemic patients) grown in vitro using soft agar tissue-culture technics may be fixed in formalin, embedded in paraffin, sectioned, and mounted on glass slides. Light microscopic examination of these sections stained with hematoxylin and eosin and with other histologic stains provides information useful in investigative and diagnostic hematology. Morphologic interpretation of the characteristics of cultured cells is within the capability of pathologists and clinical hematologists. The slides provide a permanent record of growth in vitro.