Cytospin Preparations Research Articles

Early molecular detection of central nervous system relapse in a child with systemic anaplastic large cell lymphoma: Case report and review of the literature

We report a case of anaplastic large cell lymphoma (ALCL) with central nervous system relapse in an 11-year-old boy. The relapse was suspected on morphologic examination of the cytospin preparations of the cerebrospinal fluid (CSF) with a WBC of 10 cells/microl. CSF relapse was confirmed using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) for abnormal ALK expression or gene structure. The patient developed large intracranial metastases, despite systemic, and intrathecal chemotherapy. This case demonstrates the feasibility of detecting ALCL in paucicellular CSF specimens and suggests that even low CSF involvement can herald massive parenchymal disease.

The association of bloody nipple discharge with breast pathology

It is believed that bloody spontaneous nipple discharge (SND) portends a greater chance of malignancy than nonbloody discharge, and that cytologic evaluation of SND assists in treatment planning. Our aims were to assess (1) the pathology of women with/without SND who require diagnostic breast surgery, (2) whether bloody SND is associated with a different spectrum of pathologic findings than nonbloody, and (3) whether SND cytology is influenced by pathologic findings. One hundred seventy-five women who underwent breast operation were enrolled. Cytologic evaluation was performed on Papanicolaou-stained cytospin preparations of SND. Papilloma and hyperplasia (both without atypia) were more frequent in breasts with than without SND; breast cancer was more common in breasts without SND (P <.001 for all). All cases of breast cancer with SND, but only 33% without, contained a papilloma or papillary features. Seventy-five percent of cancers presenting with SND were nonbloody. Papilloma was the most common diagnosis in breasts with bloody SND and was more common (P=.017) than in breasts without, whereas hyperplasia was the most common diagnosis in breasts with nonbloody SND and was more common (P=.031) than in breasts with bloody SND. SND cytology was not significantly influenced by pathology. Breast cancer can present as unilateral nonbloody SND, indicating the importance of surgical intervention. Papilloma was more common in women with SND than without and most often presented as bloody SND.

How long is urinary cytology abnormal after flexible cystoscopy?

To examine the urinary cytological changes caused by flexible cystoscopy and provide clinical guidelines for a reliable time interval for urinary cytological examination after flexible cystoscopy. Forty-eight patients attending for flexible cystoscopy were recruited into the study. Each patient was asked to provide eight urine samples before, immediately after and at 1, 2, 7, 14 and 28 days after cystoscopy. Cytospin preparations of the urine samples were made and slides stained using the Papanicolaou stain. Cytology was analysed while unaware of sample origin, by three different cytopathologists. The cytological changes were characterized by a striking increase in cellularity immediately after flexible cystoscopy, mostly accounted for by urothelial cells. Consistent morphological changes included the formation of 'columnar' cells, papillary clusters, increased nucleo-cytoplasmic ratio and nuclear atypia. These changes were transient, with most disappearing within a day of flexible cystoscopy. There are cytological changes, on voided urine cytology, after flexible cystoscopy but they were transient, and urine sent more than a day after flexible cystoscopy should be free from artefactual change caused by instrumentation. These results suggest that clinicians sending urine for cytological analysis should provide information about the nature and timing of any endoscopy so as to avoid false-positive interpretations of urine cytology by the cytopathologist.

Modulation of FAK, Akt, and p53 by stress release of the Fibroblast-Populated collagen matrix 1,2

Background Fibroblast survival in a three-dimensional collagen matrix is dependent in part upon the rigid anchorage of the matrix to tissue culture plastic. We hypothesized that focal adhesion kinase (FAK) and protein kinase B (Akt) would be activated and that the p53 level would be low in the rigidly anchored (attached) collagen matrix; loss of anchorage (detachment) was hypothesized to have the opposite effects. Materials and methods Human foreskin fibroblasts were cultured in attached bovine collagen matrices for 48 h before detachment as free-floating matrices. At various time points postrelease, matrix lysates were blotted for the proteins of interest, and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end label assay was performed on both whole matrices and cytospin preparations. Irradiated monolayer fibroblasts were used as positive controls for the amount of p53 protein. Results Terminal deoxynucleotidyltransferase-mediated dUTP nick-end label positivity in attached versus detached matrices (at 24 h post detachment) was 0.7 ± 03 versus 5.3 ± 1.7% ( P < 0.05, unpaired t test). FAK and Akt were phosphorylated (activated) in the attached matrix; there was a near complete of loss of both activated forms within 4 h of matrix detachment. Irradiated monolayer fibroblasts had increased levels of p53, mdm2, and p21. In contrast, the p53, mdm2, and p21 levels were just at the level of detection in the attached matrix, but were induced 5- to 10-fold within 2–4 h after matrix detachment. Conclusions FAK and Akt are activated in the attached fibroblast-populated collagen matrix whereas the p53 level is relatively low; matrix detachment downregulates FAK and Akt activity and induces p53. The state of mechanical anchorage of the collagen matrix regulates the survival of embedded fibroblasts through a mechanism which may involve FAK.

Significance of psammoma bodies in serous cavity fluid: a cytopathologic analysis.

Psammoma bodies (PBs) are encountered only rarely in body cavity fluids (BCF). Although to the authors' knowledge their presence in certain neoplasms (e.g., those of the thyroid, ovary, lung, brain, etc.) is established, their significance in BCF has not been well defined. Diagnoses concerning 3335 BCF samples were reviewed from the cytopathology files for the presence of PBs over an 8-year period. Cytologic preparations included cytospin preparations and Millipore filters stained with the Papanicolaou stain. Clinicopathologic correlation was performed on any subsequent surgical studies. Of the 3335 BCF samples studies (2444 pleural samples, 688 peritoneal samples, and 203 pericardial samples), PBs were noted in 123 cases (3.7%). Of these 123 cases, 112 were the peritoneal fluid (91%), 10 were the pleural fluid (8.1%), and 1 was in the pericardial fluid (0.81%). All 11 cases of pleural and pericardial effusions with PBs were malignant (carcinomas of the thyroid, lung, and ovary) compared with 62 of 112 peritoneal fluid samples (55.4%) (carcinoma of the ovary and uterus and mesothelioma). Nine of the remaining 50 cases of cytologically benign peritoneal fluids with PBs detected on follow-up tissue biopsy demonstrated peritoneal metastases from ovarian or endometrial carcinoma. Therefore, 41 of 112 cases of peritoneal fluid with PBs remained benign even after clinical follow-up and/or tissue biopsy (36.6%) and demonstrated ovarian cystadenoma/cystadenofibroma, papillary mesothelial hyperplasia, endosalpingiosis, endometriosis, and other miscellaneous benign diagnoses. PBs in BCF is a rare finding. Although in the authors' experience their presence in pleural and pericardial effusions signifies carcinomatous involvement, in the current study, peritoneal fluids with PBs were found to be benign in a significant number of cases (36.6%). In the latter scenario and in the absence of an obvious malignancy, attempts should be made to rule out the above-mentioned benign lesions.

Automated detection of immunofluorescently labeled cytomegalovirus-infected cells in isolated peripheral blood leukocytes using decision tree analysis.

Cytomegalovirus (CMV) infection continues to be a major problem for immunocompromised patients. Detection of viral antigens in leukocytes (antigenemia assay) is widely used for the diagnosis of CMV infection and for guiding antiviral therapy. The antigenemia technique, contingent upon the manual microscopic analysis of rare cells, is a laborious task that is subject to human error. In this study, we combine automated microscopy with artificial intelligence for reliable detection of fluorescently labeled CMV-infected cells. Cytospin preparations of peripheral blood leukocytes were immunofluorescently labeled for the CMV lower matrix phosphoprotein (pp65) and scanned in the Rare Event Imaging System (REIS), a fully automated image cytometer. The REIS detected potential positive objects and digitally recorded 49 measured cellular features for each identified case. The measurement data of these objects were analyzed by the See5 decision tree (DT) algorithm to ascertain whether they were true-positive detections. The DT was built from the measurement data of 2,047 true- and 2,028 false-positive detections, collected from 32 patient samples. By designating misclassifications of false-negatives three times more costly, the 10-fold cross-validation sensitivity, specificity, and misclassification error of the assay was 94.3%, 56.2%, and 25%, respectively. The method was also validated using an independent test set of 21 patient samples, in which similar results were obtained. To our knowledge, this study represents the first attempt to improve the accuracy of rare event image cytometry through the implementation of artificial intelligence methodology. Results suggest that cost-sensitive decision tree analysis of digitally measured cellular features vastly improves the performance of rare event image cytometry.

Immunological phenotype of freshly isolated human microvascular endothelial cells from human term placenta

Objectives: It is known that endothelial cells may play an important role in the pathogenesis in angiogenetic events as well as in disturbances of pregnancy like preeclampsia, intrauterine growth retardation. In order to examine pathophysiological mechanisms, particularly, in the endothelium of placenta, we isolated human placental microvascular endothelial cells . Methods: Fine minced specimens from fresh placenta cotyledons were washed three times in phosphate buffered saline, then incubated overnight in 1.2 units dispase II per ml Puck's solution at 4°C. Afterwards specimens were gently pressed by using syringes. Supernatant cells were plated in tissue culture flasks that had been coated in 1% gelatine and cultured in endothelial cell basal medium supplemented with 20% normal human serum, 5 ng epidermal growth factor per ml, 2 mM L-glutamine, 2mM L-arginine, 100µg streptomycin per ml,100 U penicillin per ml, 250 ng amphotericin B per ml.Primary cultures, if necessary, were purified using immunomagnetic beads recognizing CD 31 according to the manufacturer's protocol. Immunohistochemical procedures as well as flow cytometer analysis were done in order to characterize the phenotype and to assess the purity of the obtained placental microvascular endothelial cells. Results: Confluent monolayers of placental endothelial cells showed the typical cobblestone morphology. Cytospin preparation and flow cytometer analysis revealed positive immunostaining with Ulex europaeus 1 lectin, von Willebrand factor, PECAM-1 (CD31), VE-cadherin, CD34, thrombomodulin. Using anti-fibroblast antibodies did not show any staining. Glut -1 staining was dependent on endothelial cell origin (maternal versus fetal) Stimulation of placental microvascular endothelial cells with TNF-alpha 200 U per ml for 4 hours resulted in expression of E-Selectin-1 as a further evidence of endothelial cells. Conclusions: Our method is suitable to isolate purified microvascular placental (maternal and fetal) endothelial cells as assessed morphologically, by immunohistochemical phenotype and functionally (cytokine-induced E-Selectin-expression). These microvascular endothelial cells are different from endothelial cells deriving large vessels such as umbilical veins and therefore are important for further investigation of endothelial cell related disturbances in pregnancy.

Interleukin-5 mediates peritoneal eosinophilia induced by the F1 cell wall fraction of Histoplasma capsulatum.

An alkali-insoluble fraction 1 (F1), which contains mainly ss-glucan isolated from the cell wall of Histoplasma capsulatum, induces eosinophil recruitment into the peritoneal cavity of mice. The present study was carried out to determine the participation of interleukin-5 (IL-5) in this process. Inbred C57BL/6 male mice weighing 15-20 g were treated ip with 100 microg of anti-IL-5 monoclonal antibody (TRFK-5, N=7) or an isotype-matched antibody (N=7), followed by 300 microg F1 in 1 ml PBS ip 24 h later. Controls (N=5) received only 1 ml PBS. Two days later, cells from the peritoneal cavity were harvested by injection of 3 ml PBS and total cell counts were determined using diluting fluid in a Neubauer chamber. Differential counts were performed using Rosenfeld-stained cytospin preparations. The F1 injection induced significant (P<0.01) leukocyte recruitment into the peritoneal cavity (8.4 x 10(6) cells/ml) when compared with PBS alone (5.5 x 10(6) cells/ml). Moreover, F1 selectively (P<0.01) induced eosinophil recruitment (1 x 10(6) cells/ml) when compared to the control group (0.07 x 10(6) cells/ml). Treatment with TRFK-5 significantly (P<0.01) inhibited eosinophil recruitment (0.18 x 10(6) cells/ml) by F1 without affecting recruitment of mononuclear cells or neutrophils. We conclude that the F1 fraction of the cell wall of H. capsulatum induces peritoneal eosinophilia by an IL-5-dependent mechanism. Depletion of this cytokine does not have effect on the recruitment of other cell types induced by F1.

Identification and characterization of intestinal peyer's patch interferon-α producing (plasmacytoid) dendritic cells

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-α (IFN-α) producing cells in blood. In this study, C57BL/10 (B10;H2 b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 μg/d; i.p.; 10 days) that expands DC selectively in vivo. Putative pDC (CD11c +B220 +) were identified in the subepithelial dome and in interfollicular regions of intestinal Peyer's patches (PP) from both normal and Flt3L-treated animals. Freshly-isolated, immunobead-purified CD11c + DC from PP were flow-sorted to obtain lineage − (CD11b −CD19 −) CD11c + B220 + DC (purity > 96%). Flow cytometric analysis revealed that these sorted PPpDC were negative for surface markers associated with myeloid DC (CD11b) and expressed only low levels of the “lymphoid-related” DC marker CD8αα +. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of naı̈ve allogeneic (C3H; H2 k) T-cell proliferation. Cytospin preparations of sorted CD11c +B220 + cells revealed plasmacytoid morphology similar to that of human pDC. Immunocytochemistry and enzyme immunoassay revealed that, within 24-hour culture with Herpes simplex virus (10 p.f.u./cell), a subpopulation of stimulated (but not unstimulated) CD11c +B220 + DC produced and secreted IFN-α. This novel DC subset may play important roles in innate and adaptive immune responses of the gut and in the regulation of mucosal immune reactions.

Phagocytosis of Candida albicans by metastatic and non metastatic human breast cancer cell lines in vitro

Experiments were carried out to investigate the kinetic characteristics of phagocytosis of candida by metastatic (MCF-7 and ZR-75-1) and nonmetastatic (HCC70) breast cancer cell (BCC) lines. Cancer cells were mixed with candida at a ratio of 1:10 and attachment/phagocytosis were examined using cytospin preparations stained with either Giemsa or tannic acid and May-Grünwald Giemsa and by using flow cytometry. A high attachment of candida to cancer cells (29–39%) was detected as early as 10 min. Following attachment, cancer cells phagocytized yeast. The phagocytic activity of MCF-7 and ZR-75 cells was significantly higher (58–61%) than that of HCC70 cells (26%). A similar trend was observed with respect to the phagocytic index. Phagocytosis of candida by tumor cells was inhibited significantly by both cytochalasin B (50%) and by lowering temperature to 4 °C (66%). Phagocytosis was not associated with oxidative burst in any cell lines used. In conclusion, metastatic breast cancer cells possess phagocytic activity which may explain their invasive property.

Evaluation of bronchoalveolar cells in pulmonary paracoccidioidomycosis.

To investigate the local immune response, the cellular infiltrate and cytokine levels were analysed in bronchoalveolar lavage (BAL) from patients with pulmonary paracoccidioidomycosis. The group consisted of 19 patients aged 34-65 yrs. The diagnosis was confirmed by demonstration of the fungus in the sputum or BAL fluid and by serological tests. Cytospin preparations showed an increased number of lymphocytes and neutrophils in BAL. A higher number of CD8+ lymphocytes were observed in BAL compared with peripheral blood. Alveolar macrophages (AM) expressed approximately three-fold more major histocompatibility class II, intercellular adhesion molecule-1 and B7-2 molecules on their surfaces than their circulating counterparts, indicating that they had differentiated into activated macrophages inside the lungs. Cultured AM produced higher levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and macrophage inflammatory protein (MIP)-1alpha than peripheral blood monocytes. BAL fluid contained low but detectable amounts of IL-6, TNF-alpha and MIP-1alpha, and specific antibodies to Paracoccidioides brasiliensis, mainly of the immunoglobulin G2 isotype. As macrophage inflammatory protein-1alpha was shown to selectively attract CD8+ T-cells and this population was elevated in bronchoalveolar lavage, the data suggest that, besides macrophages, CD8+ T-cells may have an important role in the pathogenesis of pulmonary paracoccidioidomycosis.

Antineutrophil cytoplasmic antibodies in patients with Graves’ disease: association of antimyeloperoxidase autoantibodies with propylthiouracil therapy

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitic disorders have been reported in patients with Graves’ disease during propylthiouracil (PTU) therapy. To investigate whether ANCA are found in serum samples from patients with Graves’ disease, and whether PTU therapy is associated with ANCA positivity, levels of serum ANCA were examined in Graves’-disease patients receiving either PTU (n = 49) or 1-methyl-2-mercaptoimidazole (methimazole, MMI) (n = 50), and in untreated Graves’-disease patients (n = 32) by enzyme-linked immunosorbent assay (ELISA). Serum samples from patients with Hashimoto’s thyroiditis (n = 46) were also analyzed. Antimyeloperoxidase (MPO) autoantibodies (MPO-ANCA) were present in 10 (20.4%) of 49 Graves’-disease patients receiving PTU therapy, whereas MPO-ANCA were not detected in Graves’-disease patients receiving MMI, in untreated Graves’-disease patients, or in Hashimoto’s thyroiditis patients. The MPO-ANCA-positive sera showed a perinuclear staining pattern which was detected by indirect immunofluorescence microscopy using a human polymorphonuclear leukocyte–cytospin preparation. Furthermore, Western blot analysis revealed that MPO-ANCA in the Graves’-disease patients, as well as MPO-ANCA in patients with idiopathic pauci-immune necrotizing and crescentic glomerulonephritis, recognize the 105-kD protein of native MPO. These results indicate that MPO-ANCA in Graves’-disease patients are strongly associated with PTU therapy, and not simply related to the autoimmune thyroid disease. This study also suggests that the presence of MPO-ANCA alone may not be sufficient for the development of vasculitic disorders.

Detection and Prognostic Value of Cytokeratin Positive Tumor Cells in Bone Marrow of Patients With Renal Cell Carcinoma

Several investigations indicate the prognostic value of disseminated cytokeratin positive tumor cells in bone marrow of patients with carcinoma of different origin. In this study we evaluated the prognostic significance of epithelial cells in bone marrow of patients with renal cell carcinoma (RCC). Aspiration of bone marrow was performed preoperatively in 335 patients with RCC between 1990 and 1998. A total of 287 patients fulfilled all study inclusion (eg M0 R0 tumor stage) and exclusion (eg second malignancy during followup) criteria for the final analysis. Cytospin preparations were made after density gradient centrifugation of bone marrow samples and incubated with monoclonal antibodies directed against cytokeratin 18 (CK2) and pan-cytokeratin. Staining was performed using the alkaline phosphatase-anti-alkaline phosphatase method and 256 samples were evaluated. RESULTS In 25% (63 of 256) of the patients cytokeratin positive (CK+) cells were detected in bone marrow. Tumor progression (defined as tumor associated death, local recurrence or new metastases) was present in 12% (31 of 256) during the followup period (median 40 months), and 14% (9 of 63) with CK+ cells and 11% (22 of 193) with negative bone marrow status exhibited tumor progression. Survival analysis (log-rank test) showed no significant difference between the CK+ and cytokeratin negative group. The detection of CK+ cells was not an independent prognostic parameter in multivariate analysis (Cox regression model). These results indicate that the immunocytochemical detection of disseminated cytokeratin positive tumor cells in the bone marrow of patients with RCC has no prognostic significance.

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RSV-induced bronchiolitis but not upper respiratory tract infection is accompanied by an increased nasal IL-18 response.

The aim of this study was to investigate potential differences in the local nasal immune response between bronchiolitis and upper respiratory tract infection induced by respiratory syncytial virus (RSV). Nasal brush samples were obtained from 14 infants with RSV bronchiolitis and from 8 infants with RSV upper respiratory tract infection. The samples were taken during infection (acute phase) and 2–4 weeks later (convalescent phase). Cytospin preparations were stained immunohistochemically for T cells, macrophages, and eosinophils. Staining also took place for intercellular adhesion molecule‐1 (ICAM‐1), T‐helper 1 (Th1)‐like (interleukin‐12 [IL‐12], interferon‐γ [IFN‐γ]), Th2‐like (IL‐4, IL‐10), and proinflammatory cytokines (IL‐6, IL‐8, IL‐18). During both RSV‐induced bronchiolitis and upper respiratory tract infection, cellular inflammation was observed. This was characterised by an increase in the numbers of nasal macrophages, which tended to be higher in bronchiolitis than in upper respiratory tract infection. Numbers of T lymphocytes and ICAM‐1 positive cells increased during both bronchiolitis and upper respiratory tract infection. There were no differences between numbers in the groups. Interestingly, a distinct nasal proinflammatory cytokine response was observed in RSV‐induced bronchiolitis. This is characterised by an increase in the number of IL‐18 positive cells. This increase is specific for bronchiolitis, as a similar increase could not be detected in RSV‐induced upper respiratory tract infection. Numbers of IL‐6 and IL‐12 positive cells were higher in both bronchiolitis and upper respiratory tract infection, and there were no differences between the groups. By contrast, the number of IL‐8, IFN‐γ, IL‐4, and IL‐10‐positive cells remained constant. In conclusion, clear differences were found in nasal immune responses of children with RSV‐induced upper respiratory tract infection or bronchiolitis. The induction of a strong IL‐18 response was typical for bronchiolitis, as this could not be observed in RSV‐induced upper respiratory tract infection, and could explain the eosinophilia that is observed frequently during bronchiolitis. J. Med. Virol. 71:290–297, 2003. © 2003 Wiley‐Liss, Inc.

Thin-Layer Preparations of Dithiothreitol-Treated Bronchial Washing Specimens

To evaluate the combined effect of dithiothreitol (DTT) treatment and ThinPrep (TP) (Cytyc Corp, Boxborough, Massachusetts, U.S.A.) processing on bronchial washing specimens. A total of 431 bronchial washing specimens were initially treated with 0.05% DTT in a 30% methanol solution. After centrifugation, 1 TP slide and 2-4 conventional cytospin or smear preparations (CPs) were prepared. The reports of both preparations were compared in all cases. All 48 abnormal cases and 52 consecutive negative cases were also compared for cellular composition, distribution of the cells, ease of interpretation and overall preparation quality. Screening time was recorded for 20 of the cases. The diagnostic accuracy of one TP slide appeared comparable to that of 2-4 CPs. The TP slide was assessed to be equal or superior in overall quality to CP in 85% of 100 cases of paired specimens. The cleaner background and smaller cellular area of TP slides significantly reduced the screening time. Mucolysis and specimen homogenization were not always optimal, occasionally resulting in uneven subsampling and poorly cellular TPs. However, in general, TP slides were considered superior to CPs in overall quality. Improvement in specimen quality and reduced screening time have to be balanced against the high cost of consumables with the TP technique.

Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei: A Sensitive Method for 1p and 19q Deletion Analysis in Paraffin-Embedded Oligodendroglial Tumor Specimens

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP—Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P = .001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.

HOPE fixation of cytospin preparations of human cells for in situ hybridization and immunocytochemistry.

In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPES-glutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.

Mammary Pseudoangiomatous Stromal Hyperplasia

To describe and reevaluate the fine needle aspiration cytology findings of pseudoangiomatous stromal hyperplasia (PASH) of the breast, with histologic, immunohistochemical and ultrastructural correlation. The authors reviewed the clinical features, fine needle aspiration cytology, histology and immunohistochemical results in all cases of mammary PASH encountered at Pamela Youde Nethersole Eastern Hospital, Hong Kong, during the 4-year period from January 1998 to May 2002. Ultrastructural examination was carried out in a selected example. The findings were compared with those in the literature. Four cases of PASH of the breast were encountered during the study period. The ages of the patients ranged from 34 to 56 years. One of them was a male presenting with gynecomastia. Only 2 cases had fine needle aspiration biopsies with a satisfactory cellular yield available for review. The cytospin preparations were of moderate cellularity and showed cohesive clusters of bland-looking ductal cells in a background of single, naked nuclei and some spindle cells containing fine chromatin and a discernible amount of cytoplasm. Occasional ductal cell clusters assuming a "staghorn" pattern, a feature commonly seen in fibroadenoma, were noted. Besides, there were scantly, loose and hypocellular stromal tissue fragments that contained spindle cells and occasional paired, elongated nuclei embedded in a fibrillary matrix. Histologic examination of the excisional biopsies confirmed the presence of PASH. It was characterized by many slitlike spaces rimmed by CD34-positive myofibroblasts/fibroblasts in a focally hyalinized stroma. Sometimes, ill-formed, fusiform aggregates of fibroblasts were also observed. Their fibroblastic nature was confirmed by electron microscopy. Fine needle aspiration cytology of PASH closely resembles that of fibroadenoma. Though subtle differences do exist, a definitive diagnosis is unlikely on the basis of the cytologic examination alone. PASH needs to be distinguished from borderline lesions, such as phyllodes tumor, and more sinister conditions, which sometimes have a similar cytologic appearance.

Disseminated Tumor Cells in Bone Marrow of Patients with Transitional Cell Carcinoma: Immunocytochemical Detection and Correlation with Established Prognostic Indicators

Previous investigations have demonstrated the prognostic value of disseminated cytokeratin positive cells in bone marrow of patients with breast, gastric, colon and prostate cancer. We evaluated the potential of an immunocytochemical assay, using a monoclonal antibody against cytokeratin 18 (CK 18), for the detection of disseminated tumor cells in bone marrow aspirates of patients with transitional cell carcinoma. Bone marrow aspiration was performed preoperatively on 128 patients with transitional cell carcinoma of various stages and on 27 controls with nonmalignant disease. Cytospin preparations of mononuclear bone marrow cells were incubated with a monoclonal anti-CK 18 antibody and stained using the alkaline phosphatase anti-alkaline phosphatase technique. Of the patients with transitional cell carcinoma 29.7% and none of the controls had a CK 18 positive bone marrow result. A significant correlation between the incidence of CK 18 positive cells in bone marrow and invasive transitional cell carcinoma (p <0.01), lymph node involvement (p <0.01), medium/high grade transitional cell carcinoma (p <0.01) and tumor progression in recurrent transitional cell carcinoma (p <0.05) was demonstrated. Furthermore, the mean number of CK 18 positive cells in bone marrow aspirates of patients with stage M+ and/or N+ disease was nearly 3 times as high as that of patients without clinically evident metastatic disease (10.4 versus 3.8 CK 18 positive cells per patient). A significant correlation between the incidence of CK 18 positive bone marrow results in patients with transitional cell carcinoma and established risk factors could be demonstrated in our study. Further prospective followup studies should be performed to determine the prognostic value of these findings.