Detection of environmental contamination with feline and canine parvoviruses: new perspectives and challenges.

AIMS: To develop a protocol for environmental sampling to detect parvoviruses of dogs and cats in the environment. METHODS AND RESULTS: Environmental contamination was carried out using different dilutions of parvovirus-contaminated materials; further field samplings were performed in areas in which clinical cases of parvovirus infections were present. Sterile cotton swabs and sponges for microbial surface sampling were used. Viruses were detected in these samples with different methods: conventional PCR, nested PCR and real-time PCR, detecting viral DNA; virus isolation, detecting infectious virus; and a commercial rapid enzyme immunoassay, detecting viral antigen. No substantial differences were observed in the two sampling methods, although the sponge was more convenient for sampling rough surfaces. Molecular assays were the most sensitive methods, identifying even very low amounts of viral DNA (up to 10 copies of viral DNA/10 µl of sample). Virus isolation and the rapid test detected the viruses only at the highest viral concentrations, both in the experimental setting and field conditions. CONCLUSIONS: Environmental sampling and molecular protocols were effective in detecting environmental contamination with parvoviruses. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol will be useful to identify possible sources of infection and to assess the efficacy of disinfection protocols in the environment.

AgNOR, p16 and human papillomavirus in low-grade squamous intra-epithelial lesions of the uterine cervix. Preliminary report.

It has been shown that infection with high-risk human papillomaviruses (HR-HPV) is related to the development of cervical cancer. The persistence of the virus in intra-epithelial lesions of cervix uteri (SILs) is the basis for the application of HPV testing for screening and management of patients. Most infections by HR-HPVs resolve spontaneously, however, and do not progress to dysplasia or cancer. p16INK4a is a useful biomarker of cervical intra-epithelial neoplasia and could be a marker for the progression of low-grade squamous intra-epithelial lesions (LSILs) to high-grade squamous intra-epithelial lesions (HSILs), because it correlates independently with increasing SIL grade. We conducted a preliminary histological study of 28 patients diagnosed with LSIL, HSIL or nondysplastic epithelium (NDE) from whom 28 biopsies of uterine cervix and 28 endocervical brushed biopsies were taken. Argyrophilic nucleolar organizer region (AgNOR) and p16INK4a assays were performed on the biopsies, and endocervical brushings were used for HPV typing. The high risk HPV group showed that the number of patients with AgNOR areas greater than 3.3 µm(2) and with expression of p16INK4a were statistically greater than the number of lower risk patients. None of the biopsies of LR-HPV carriers expressed p16 and AgNOR areas> 3.3 µm(2) simultaneously. Four LSILs and the NDE of this group expressed neither of the two markers. If the correlation between AgNOR areas and p16INK4a is good, we may be able to develop a low cost simple technology for studying patients infected with HR-HPV and diagnosed with LSIL of uncertain behavior.

Usefulness of ploidy, AgNOR and immunocytochemistry for differentiating benign and malignant cells in serous effusions.

OBJECTIVE: The objective of this study was to establish the value of different markers in differentiating reactive mesothelial cells from metastatic adenocarcinomatous cells in serous effusions (SE). METHODS: Forty-five SE were processed for morphological examination (Papanicolaou stain), assessment of ploidy, AgNOR counting and immunocytochemical assay of carcinoembryonic antigen (CEA), epithelial membrane antigens (EMA), Ber-EP4 and Leu-M1. Ploidy was established in an image-analyser in smears stained by the Feulgen stain method. AgNOR dots were counted in the smears stained with the silver nitrate assay for non-histone proteins present in the nucleolar organizer region. CEA, EMA, Ber-EP4 and Leu-M1 were evaluated by immunocytochemistry using the streptavidin-biotin complex method. RESULTS: All the smears with positive cytology were aneuploid. Three false negatives by morphological studies were aneuploid, with AgNOR values in two of them corresponding to the neoplastic group. CEA and Leu-M1 showed a low specificity; EMA and Ber-EP4 showed moderate sensitivity. CONCLUSIONS: The assessment of ploidy and the study of AgNOR were better methods than immunocytochemistry for distinguishing between reactive mesothelial cells and adenocarcinomatous cells in serous fluid.

Fine-needle aspiration of a follicular dendritic-cell tumor: report of a case and review of the literature.

Follicular dendritic-cell tumors (FDCT) are rare neoplasms, well-characterized in surgical pathology material. There are, however, few cytopathology reports. We describe the fine-needle aspiration (FNA) findings of a histologically confirmed FDCT. Conventional smears and a cell block showed large spindle to oval neoplastic cells admixed with small mature lymphocytes. The neoplastic cells were present mainly in small syncytial clusters. Immunostains for CD21 and CD35, performed on the cell block, were positive in the neoplastic cells. The diagnosis was fully confirmed by the presence of typical immunohistochemical and ultrastructural features on the surgically removed tumor. The differential diagnosis of FDCT is broad and includes other tumors characterized by an admixture of large neoplastic cells and small mature lymphocytes, such as thymomas, lymphoepithelioma-like carcinomas, and interdigitating dendritic-cell tumors. It may not be possible to diagnose FDCT based on FNA material without the use of immunocytochemical and electron microscopic studies. Certain cytomorphological characteristics, however, might suggest its diagnosis and allow the practicing cytopathologist to perform confirmatory studies.

The cytology of extraskeletal Ewing sarcoma.

BACKGROUND: Extraskeletal Ewing sarcoma (EES) shares histologic, immunohistochemical, and molecular findings with ES of bone. The authors' goal in conducting this study was to examine the cytomorphologic features of EES. In addition, immunocytostaining for CD99/O13 was performed in all cases, and cytogenetic and molecular data were available in about half of the cases. METHODS: The authors studied 20 aspiration cases, all with histopathologic confirmation, and also conducted immunohistochemistry and/or molecular studies. RESULTS: All cases had cellular smears with many single cells and focal clustering. Numerous naked nuclei and focal crush artifacts were seen. Mitosis and necrosis were rare. Four cases had cytoplasmic vacuoles. Five cases showed nuclear molding. Seventeen cases (85%) exhibited small cells with scanty cytoplasm and nuclei with fine chromatin and small nucleoli, representing the so-called typical variant. One case (5%) revealed cells with abundant cytoplasm, large nuclei, and large eosinophilic nucleoli, an example of the atypical or large cell variant. Two cases (10%) had features in between, with cells showing a fair amount of cytoplasm and medium-sized nucleoli, representing the intermediate variant. Nuclear grooves, described as common in the latter, were rare. In all cases, in either cytologic or corresponding histologic material, CD99/O13 immunocytostaining showed strong membranous reactivity. In addition, cytogenetic and/or molecular evidence of ES specific chromosomal translocation was demonstrated in histologic or cytologic material in 10 cases. CONCLUSIONS: EES shows cytologic features similar to ES of bone, with a spectrum of changes ranging from the typical appearance in a majority of cases to intermediate and atypical variants in a minority of cases. CD99/O13 immunocytostaining and/or molecular studies, particularly in the intermediate and atypical variants, may help in establishing a definitive fine-needle aspiration diagnosis, thus avoiding an open surgical biopsy.

Carcinoma renal variedad sarcomatoide: hematoxicilina-eosina, MIB ploidía celular como factores pronósticos / Sarcomatoid renal cell carcinoma: H-E, MIB-1, celular ploidy as prognostic factors

En nuestro servicio se realizaron entre 1987 y 1997, 115 nefrectomías por carcinoma renal, de las cuales 7 (6 por ciento) presentaban la variedad histológica sarcomatoide. En 6 casos, el tumor había sobrepasado la cápsula renal, en el restante estaba confinado al parénquima renal; éste fue uno de los cuales en los que no se observó progresión de la enfermedad luego del tratamiento quirúrgico. Llevamos a cabo estudios de morfología celular con hematoxilina-eosina, inmunohistoquímica (MIB) y ploidía celular por medio de citometría de flujo, comparando en forma retrospectiva la evolución clínica de este tip de tumor y la utilidad de los métodos empleados como factores pronósticos de progresión de la enfermedad

Carcinoma renal variedad sarcomatoide: hematoxicilina-eosina, MIB ploidía celular como factores pronósticos / Sarcomatoid renal cell carcinoma: H-E, MIB-1, celular ploidy as prognostic factors

En nuestro servicio se realizaron entre 1987 y 1997, 115 nefrectomías por carcinoma renal, de las cuales 7 (6 por ciento) presentaban la variedad histológica sarcomatoide. En 6 casos, el tumor había sobrepasado la cápsula renal, en el restante estaba confinado al parénquima renal; éste fue uno de los cuales en los que no se observó progresión de la enfermedad luego del tratamiento quirúrgico. Llevamos a cabo estudios de morfología celular con hematoxilina-eosina, inmunohistoquímica (MIB) y ploidía celular por medio de citometría de flujo, comparando en forma retrospectiva la evolución clínica de este tip de tumor y la utilidad de los métodos empleados como factores pronósticos de progresión de la enfermedad(AU)

Functional adrenal cortical tumors in childhood: a study of ploidy, p53-protein and nucleolar organizer regions (AgNORs) as prognostic markers.

Prognostic markers in pediatric adrenal cortical tumors are difficult to define. We determined the ploidy, immunostaining of p53-protein and number of nucleolar organizer regions (AgNORs) in 16 such tumors and related them to clinical outcome, tumor weight (TW) and histologic Weiss' criteria. Eleven females and 5 males aged 0.4 to 15.6 years were followed for 8.7 years; 10 presented Cushing's and 6 virilization syndrome. Diploid (n = 4, x TW = 269 g, range: 17-800 g) and near-diploid tumors (n = 3, x TW = 55 g, range: 20-85 g) had good outcome, Weiss' criteria were 0-7, and p53 reactivity was negative in all. Among the aneuploid tumors (n = 9, x TW = 298 g, range: 7-1000 g), 6 had good outcome, 2 presented metastasis and 1 was lost to follow-up; Weiss' criteria were 2-8 and p53 reactivity was positive in 3 tumors (2 of them of malignant evolution). AgNORs number was not different in cases of good or poor outcome (3.65 +/- 1.9 vs 2.83 +/- 1.1). Our findings indicate that diploid and near-diploid cases had always a good outcome regardless of tumor weight. In aneuploid cases, tumor weights < 100 g had good outcome, while those > 750 g had poor prognosis. Malignant tumors were aneuploid and had reactivity to p53-protein. Good outcome in aneuploid tumors < 100 g is probably due to early treatment. The expression of p53-protein appears as a promising marker of poor prognosis. Weiss' criteria and AgNORs were not useful in the present series.

Fine needle aspiration for flow cytometry immunophenotyping of non Hodgkin lymphoma.

The aim was to evaluate the usefulness of lymph node biopsies obtained by fine needle aspiration (FNA) for immunophenotyping of non Hodgkin lymphoma (NHL). Seventeen superficial and deep lymph node samples were fractioned for conventional cytological examination and immunophenotyping studies. Out of ten NHL, nine were readily detected by flow cytometry (FC), while failure on the remaining case was due to selective loss of large cell population, which is liable to occur with this procedure. A single case, which proved negative for all markers employed, was finally diagnosed by immunohistochemistry as germ cell tumor. The other six cases, presenting lymphoid population without phenotypic abnormalities, were diagnosed by cytology and/or histology as Hodgkin disease or hyperplasic disorders. To conclude, FC immunophenotyping seems to improve the efficacy of FNA in NHL diagnosis, whereas for Hodgkin disease and hyperplasic disorders, classic morphological criteria are more useful for differential diagnosis. Although FNA for FC immunophenotyping cannot replace histopathological examination for NHL diagnosis, it proves to be a useful tool for staging and follow up, making surgical procedures for sample collection unnecessary.

Fine needle aspiration for flow cytometry immunophenotyping of non Hodgkin lymphoma.

The aim was to evaluate the usefulness of lymph node biopsies obtained by fine needle aspiration (FNA) for immunophenotyping of non Hodgkin lymphoma (NHL). Seventeen superficial and deep lymph node samples were fractioned for conventional cytological examination and immunophenotyping studies. Out of ten NHL, nine were readily detected by flow cytometry (FC), while failure on the remaining case was due to selective loss of large cell population, which is liable to occur with this procedure. A single case, which proved negative for all markers employed, was finally diagnosed by immunohistochemistry as germ cell tumor. The other six cases, presenting lymphoid population without phenotypic abnormalities, were diagnosed by cytology and/or histology as Hodgkin disease or hyperplasic disorders. To conclude, FC immunophenotyping seems to improve the efficacy of FNA in NHL diagnosis, whereas for Hodgkin disease and hyperplasic disorders, classic morphological criteria are more useful for differential diagnosis. Although FNA for FC immunophenotyping cannot replace histopathological examination for NHL diagnosis, it proves to be a useful tool for staging and follow up, making surgical procedures for sample collection unnecessary.

[Fine-needle aspiration puncture of abdominal masses using echographic monitoring. Cytological diagnosis and experience with biological markers]. / Puncion aspiración con aguja fina de masas abdominales bajo control ecográfico. Diagnóstico citológico y experiencia con marcadores biológicos.

43 fine needle aspiration biopsies of abdominal masses were performed under echographic guide. Cytologic diagnosis was done with routine techniques. Tumor, lineage and origin biologic markers were detected by immunocytochemical methods. Sensibility, specificity and accuracy of this procedure were evaluated. Advantages of the method are discussed.

Puncion aspiración con aguja fina de masas abdominales bajo control ecográfico. Diagnóstico citológico y experiencia con marcadores biológicos. / [Fine-needle aspiration puncture of abdominal masses using echographic monitoring. Cytological diagnosis and experience with biological markers]

43 fine needle aspiration biopsies of abdominal masses were performed under echographic guide. Cytologic diagnosis was done with routine techniques. Tumor, lineage and origin biologic markers were detected by immunocytochemical methods. Sensibility, specificity and accuracy of this procedure were evaluated. Advantages of the method are discussed.